Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CSNR-DEFICIENT ACTINOBACTERIA FOR THE PRODUCTION
OF AN ENZYME HAVING CHITOSANASE ACTIVITY
TECHNOLOGICAL FIELD
This application relates to cells for the production of a chitosanase as well
as
methods using these cells for the production of a chitosanase.
BACKGROUND
Chitosanases are enzymes hydrolysing chitosan, a 13-1,4 linked D-glucosamine
bio-
polymer. Chitosan oligosaccharides have numerous emerging applications and
chitosanases can be used for industrial enzymatic hydrolysis of chitosan.
These
extracellular enzymes, produced by many organisms including fungi and
bacteria,
are well studied at the biochemical and enzymatic level but very few works
were
dedicated to the regulation of their gene expression.
Chitosan, a partly N-deacetylated form of chitin, is naturally found in the
cell walls of
fungi, especially in Zygomycetes (Mucor sp., Rhizopus sp.), and in the green
algae
Chlorophyceae (Chlorella sp.). Chitosan, is a polysaccharide made of 13-1,4-
linked D-
glucosamine (GIcN) units with a variable content of N-acetyl-D-glucosamine
(GIcNAc) units. Chitosan is produced at industrial scale by alkaline
deacetylation of
chitin, originating mainly from crustacean shells. This polysaccharide, almost
unique
among natural polymers for its amino groups that remain positively charged in
mild
acidic solutions, is the subject of numerous works oriented towards its
numerous
emerging applications in medicine, agriculture, dietetics, environment
protection and
several other fields. Chitosan is also a valuable source of GIcN, a
neutraceutical
used as a therapeutic agent in osteoarthritis. Many properties of chitosan,
especially
in biological applications are dependent on its molecular weight, i.e. on its
degree of
polymerization. The very short derivatives of chitosan - the chito-
oligosaccharides are
of particular interest, due to their increased solubility in aqueous solutions
and their
specific biological activities. To obtain chitosan chain of varying degrees of
polymerization, several chemical and physical techniques were investigated.
Enzymatic techniques with either free or immobilized chitinase or chitosanase
enzymes are also intensively studied. Chitosanase production has been found in
many microorganisms, bacteria or fungi. The enzymes so far characterized at
the
primary sequence level belong to seven families of glycoside hydrolases: GH3,
GH5,
GH7, GH8, GH46, GH75 and GH80. While these enzymes are endo-hydrolases,
their mechanism could potentially be transformed into exo-type by protein
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engineering as shown for the GH46 chitosanase from Bacillus circulans MH-K1.
Chitosan can be also hydrolyzed by enzymes acting by an exo-mechanism
generating GIcN monomers. The chitosanases from Streptomyces have been widely
studied in various aspects of structure-function relationships. Usually, these
chitosanases are produced in the heterologous host Streptomyces lividans via
the
multi-copy vector pFD666. However, very few works have been dedicated to the
regulation of chitosanase gene expression in the native and/or heterologous
hosts.
Most studies were limited to the follow up of chitosanase production in
various culture
media. An efficient production of CsnN106 or CsnN174 chitosanases in
Streptomyces lividans TK24 is strictly dependent on the addition of chitosan
or its
derivatives to the culture medium indicating that these foreign genes are
still
subjected to some kind of chitosan-dependent regulation in the heterologous
host.
However, the addition of chitosan as a component in any culture medium is not
without problems due to the well known anti-microbial properties of this
polysaccharide which can slow down the bacterial growth. Here, it is shown
that the
expression of the heterologous gene csnN106 in S. lividans is regulated at the
transcriptional level. This led us to engineer a new expression system which
does not
require anymore the presence of chitosan or its derivatives as inducers of
enzyme
production.
Microbiological studies and the analysis of sequenced genomes showed that
chitosanases are widespread among filamentous fungi and Gram-positive
bacteria,
particularly in bacilli and actinobacteria. In Streptomyces, well-studied
chitosanases
belong to glycoside hydrolase families GH2, GH5, and GH46. Putative
chitosanases
from these families, as well as from GH75 (characterized only from fungal
organisms)
are found in many recently sequenced actinomycete genomes (CaZy database).
Streptomyces lividans is an actinomycete isolated from soil, commonly used as
heterologous host for production of proteins in an extracellular mode,
including the
well-studied chitosanase from Streptomyces sp. N174 (CsnN174). Until the
publication of the genome sequence of S. coelicolor A3(2) and, more recently,
of the
S. lividans genomic contigs (GenBank accession no. ACEY010000), these two
closely related species were thought to be devoid of chitosanase activity
because
they grew very poorly on media with chitosan and no chitosanase activity was
detected in their cultures. However, genes encoding putative chitosanases of
the
GH46 family are present in both genomes: S000677 (csnA) and SCO2024 (csnB) in
Streptomyces coelicolor A3(2) and the almost identical genes SSPG_06922
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(genomic coordinate 7.62 Mb) and SSPG_05520 (genomic coordinate 6.14 Mb) in S.
lividans TK24. The biochemical properties of CsnA from S. coelicolor A3(2)
have
been studied in detail recently. In vivo studies performed with S. lividans
TK24 have
shown that CsnA is produced at a very low level (in the range of milliunits
per ml),
explaining the lack of chitosanase detection by earlier, less-sensitive
techniques.
Despite this low expression level, the deletion of csnA resulted in increased
sensitivity to the antimicrobial effect of chitosan. While there are numerous
reports on
biochemical properties of chitosanases, knowledge about the regulation of
chitosanase gene expression is very scarce. In contrast, the genetic
regulation of the
degradation of chitin, the N-acetylated form of chitosan, has been extensively
studied
in Streptomyces. Members of this genus play an important part in chitin
degradation
in soil and produce a wide array of chitinases and chitin-binding proteins.
The
regulation of chitinase (chi) gene expression in Streptomyces is rather
complex, and
as many as four different mechanisms have been identified, some of them linked
to
more general phenomena such as carbon catabolite repression, antibiotic
production,
and morphogenesis through the chitin-derived monomer N-acetyl-D-glucosamine
(GIcNAc). The Cpb1 regulator controls the expression of the chiA gene in S.
lividans.
The two-component system ChiS/ChiR participates to the genetic regulation of
chiC
gene of S. coelicolor. Reg1, the negative regulator of a-amylase genes in S.
lividans,
seems also to be involved in the genetic regulation of chitinase genes.
Finally DasR,
a member of the HutC/GntR subfamily, regulates the expression of some
chitinase
genes through interaction with the dre motif in S. coelicolor. DasR also has a
more
global effect on other genes involved in GIcNAc metabolism.
It would be highly desirable to be provided with an expression system for a
chitosanase which is not dependant on the presence of chitosan in the culture
medium. It would be desirable to be provided with an expression system which
would
allow for the expression of endogenous as well as exogenous chitosanase. It
would
also be highly desirable to be provided with an expression system for a
chitosanase
which limits or avoids the production of protease in the culture medium. It
would
further be desirable, for pharmaceutical applications, to be provided with an
expression system for a chitosanase which can be cultured in a defined medium.
BRIEF SUMMARY
The present application concerns the use of a genetically modified
actinobacterium
host for the production of a chitosanase in the absence of chitosan.
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In a first aspect, the present application provides a genetically modified
actinobacterium cell for the production of an enzyme having chitosanase
activity, said
genetically modified actinobacterium cell having a reduced activity of a
native CsnR
polypeptide when compared to the activity of said native CsnR polypeptide in a
native actinobacterium cell. The CsnR polypeptide may be encoded by a csnR
gene
or one of its ortholog. In an embodiment, the actinobacterium cell is a
Streptomyces,
such as, for example, a Steptomyces lividans. In another embodiment, the
enzyme
has an exo-chitosanase activity, such as those represented in the glycoside
hydrolase (GH) 2 family. In another embodiment, the enzyme has an endo-
chitosanase activity, such as those represented in the the glycoside hydrolase
(GH)
5, 8 or 46 family. In still another embodiment, the enzyme further comprises
at least
one additional enzymatic activity: a beta-1,4-glucanase activity (encompassing
cellulose activity) and/or a licheninase activity. In still another
embodiment, the
enzyme is exogenous to the genetically modified actinobacterium cell. In yet a
further
embodiment, the enzyme is encoded by a nucleic acid vector, such as, for
example,
an integratable vector. In yet another embodiment, the enzyme is endogenous to
the
genetically modified actinobacterium cell. In another embodiment, an open-
reading
frame of a csnR gene is disrupted in the actinobacterium host. In still
another
embodiment, a fragment of the csnR gene is deleted in the actinobacterium
host. In
yet another embodiment, an exogenous nucleic acid molecule is inserted in the
open-reading frame of the csnR gene in the actinobacterium host. In another
embodiment, a complete csnR gene is deleted in the actinobacterium host.
In a second aspect, the present application provides a method for producing an
enzyme having chitosanase activity. Broadly the method comprises (i) placing
the
genetically modified actinobacterium cell as described herein in a culture
medium
devoid of chitosan, chitosan fragments or chitosan derivatives and (ii)
culturing the
genetically modified actinobacterium cell under conditions suitable for the
production
of the chitosanase. In an embodiment, the method further comprises (iii)
purifying the
chitosanase from the culture medium. In another embodiment, the culture medium
comprises malt extract, KH2PO4, K2HPO4, (NH4)2804 and MgSO4. In another
embodiment, the culture medium consists of malt extract, KH2PO4, K2HPO4,
(NH4)2804 and MgSO4.
In a third aspect, the present application provides a method of reducing the
molecular
weight of a chitosan molecule. Broadly the method comprises contacting the
enzyme
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produced by the method described herein with said chitosan molecule under
conditions sufficient to allow the cleavage of said chitosan molecule by said
enzyme.
In a fourth aspect, the present application provides a method of producing a
low-
molecular weight chitosan. Broadly, the method comprises contacting the enzyme
produced by the method as described herein with a chitosan molecule under
conditions sufficient to allow the cleavage of said chitosan molecule by said
enzyme
into said low molecular weight chitosan.
In a fifth aspect, the present application provides a method of producing a
chitosan
oligosaccharide. Broadly, the method comprises contacting the enzyme produced
by
the method described herein with a chitosan molecule under conditions
sufficient to
allow the cleavage of said chitosan molecule by said enzyme into said chitosan
oligosaccharide.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the nature of the invention, reference will
now be
made to the accompanying drawings, showing by way of illustration, a preferred
embodiment thereof, and in which:
Figure 1. Primer extension analysis of csnN106 transcripts. The apparent 5'
terminus
for the csnN106 transcript was identified by annealing a radiolabeled primer
complementary to the mRNA of csnN106 and extension with reverse transcriptase.
40 pg of total RNA, from GIcN-chitosan oligomers induced S. lividans TK24(pHPr-
WT), were used for extension reaction. The same primer was used for DNA
sequencing reactions with the pHPr-WT plasmid. (-0: primer extension product;
(*):
apparent transcription start site. Vertical arrows: palindromic sequence.
Figure 2. Promoter regions characterized in this work. (A) Fragment of the
promoter
region of csnN106 gene variants. Pr-WT: native promoter region, the putative -
35
and -10 boxes are indicated in blue. Pr-Ph: a construct in which the native
promoter
has been replaced by a double promoter from Streptomyces ghanaensis phage 119,
the respective -35 and -10 boxes are over and underlined. Low case letters
indicate
nucleotide changes between Pr-WT and Pr-PH. (*): start points of
transcription.
Arrows: inverted repeats of the palindromic box. (B) Alignment of palindromic
sequences present in the promoter regions of chitosanase genes in
actinomycetes.
Nucleotides are numbered relative to the center of symmetry. In the consensus
sequence, nucleotides are coloured according to their importance for DNA-
protein
interaction established by equilibrium competition experiments (Dubeau et al.,
2005):
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red: nucleotides critical for interaction; green: nucleotides moderately
important for
interaction; black: nucleotides without apparent effect on interaction. (1):
base pairs
mutated in the Pr-Pa construct. GH: glycoside hydrolase family.
Figure 3. Effect of csnR deletion on DNA-protein interaction at the csnN106
gene
operator. Gel retardation experiment was set up combining 0.1 nM double strand
oligonucleotide probe covering the palindromic box of csnN106 with 10 pg of
crude
protein extracts from S. lividans TK24 strain (WT) or the csnR deleted strain
(AcsnR)
cultivated in medium with 0.125% G1cN and 0.375% chitosan oligomers for the
time
(hours) indicated. P: probe only; T+: control reaction with 2 pg of partially
purified
protein from Kitasatospora sp. N106 (Dubeau etal., 2005).
Figure 4. Chitosanase activity and relative purity assessment and assay of
protease
levels. (A) chitosanase activity; (B) protease activity; (C) SDS-PAGE of
proteins in
culture supernatants.The upper-table aligns the genotype of each strain and
lists the
type of medium for the corresponding columns in graphs (A) and (B) and wells
of (C).
WT = wild type; A = AcsnR mutant host; M = mutated palindromic box; Multi =
chitosanase genes introduced on a multi-copy vector. Culture media: Me = malt
extract medium; Ch = chitosan flakes medium; 01 = medium with G1cN and
chitosan
oligomers. All determinations have been done after 72 h of culture. Data and
error
bars (A and B) are the mean of culture duplicates. *** P 5_ 0.001, ** P 0.01,
* P
0.05 from one-way ANOVA with Bonferroni's post test (GraphPad PrismTM version
5.00). (C) 20 pl of culture supernatants were loaded on a 12% SDS-PAGE gel.
PageRulerTM prestained protein ladder (0.5 pl; Fermentas) was used as
standard.
After electrophoresis, proteins were stained with Coomassie brilliant blue.
Chitosanase migrates as a 26.5 kDa band.
Figure 5. Alignment of palindromic sequences found upstream of genes encoding
chitosanases or ROK family regulator genes in actinomycetes and LOGO
representation of consensus sequence. Complementary bases in palindromes bases
are shown in blue. "Pos." (position) indicates the distance in bp from the
central
nucleotide of the palindromic sequence to the start codon of the associated
gene. K.
sp. N106 = Kitasatospora sp. N106; S. sp. N174 = Streptomyces sp. N174.
Figure 6. Purification of CsnR. Protein samples from each stage of the CsnR
purification were analyzed by 10% SDS-PAGE and visualized after silver nitrate
staining. M = PageRulerTM prestained molecular mass protein ladder
(Fermentas);
S = soluble fraction of cell lysate obtained from a culture of E. coli Rosetta-
gami 2
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(DE3) (pLysS) (pGEX-csnR) induced with 0.1 mM IPTG; (-) = purification attempt
without previous treatment of the soluble fraction of cell lysate; (+) =
purification steps
with a previous 2 mM ATP and 5 mM MgC12 treatment of the soluble fraction of
cell
lysate; E = eluate collected from the glutathione-Sepharose 4B resin following
a 4-h
incubation with PreScissionTM protease; F = 20 pl of the size exclusion
chromatography fraction with the highest GroEL contamination; P = 20 pl of
pooled
size exclusion chromatography fractions with purified CsnR.
Figure 7. DNase I footprinting analysis of the CsnR binding site to csnA and
csnR
promoters. (A) A 298-bp labeled probe (csnA-IR) and a 256-bp labeled probe
(csnR-
IR), both including the entire intergenic regions upstream from csnA and csnR,
respectively, were subjected to partial DNase I digestion in the presence (+)
or
absence (-) of -0.5 nmol of purified CsnR. Vertical arrows correspond to the
palindromic sequence shown in panel B. (B) Partial intergenic region sequences
upstream of csnA and csnR. Boxes correspond to the protected region in panel
A.
Arrows correspond to the palindromic sequence. Boldface gtg represents the
translation initiation codon. **, transcription initiation site as determined
by primer
extension. The -35 and -10 boxes of the deduced promoter sequence are shown in
italic.
Figure 8. Determination of dissociation constant (KD). Various concentrations
of
labeled csnA-WT or csnR-WT probe (0.1 nM to 1.5 nM) and 1 pl of purified CsnR
were used in electrophoretic mobility shift reactions. Data were collected
from bands
intensities analysis using lmageQuantTM software (version 5.2). KD
calculations were
done using the Michaelis-Menten non-linear fit (least squares) GraphPad
PrismTM
version 5.03 for Windows.
Figure 9. Effect of saccharides on the interaction between CsnR and the csnA-
WT
operator. The indicated saccharides were added (500 nM) to binding reaction
mixtures containing -8.5 pmol of CsnR and 0.03 nM csnA-WT probe. Free and
complexed DNA fragments were separated by 6% polyacrylamide gel
electrophoresis and visualized by PhosphorlmagerTM.
Figure 10. Chitosan dimer IC50 determination. Electrophoretic mobility shift
reactions
were done with -8.5 pmol of CsnR, pre-incubated with (GIcN)2 at various
concentrations (0.00075 mM to 5 mM) for 15 min on ice before the addition of
labelled csnA-WT probe (0.03 nM). Two specific shifts were observed and
considered in the IC50 determination.
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Figure 11. S. lividans TK24 gene cluster led by csnR. Black arrows cover the
coding
sequence of each gene and are numbered according to the annotation in S.
lividans
genome. Gene symbols attributed in this study are shown in italics. The
vertical
arrow shows the position of CsnR palindromic operator. The stem-loop indicates
the
position of a putative transcriptional terminator. The length of each
intergenic
segment is given in brackets. (*) indicates segment sequenced in the current
work.
Figure 12. Sequence alignment of the sequenced intergenic region between
SSPG_04872 (csnR) and SSPG_04871 (csnE) and the published intergenic region
between SCO2657 (csnR homologue) and SCO2658 (csnE homologue). Green
highlight correspond to the stop codon of SSPG_04872 and SCO2657 and the
translation initiation codon of SSPG 4871 and SCO2658. Yellow highlight
correspond to direct repeats. Underlined base pairs correspond to the putative
ribosome binding site.
Figure 13. RT-PCR expression profiling of putative chitosanase genes belonging
to
families GH2 (SAV_1223), GH46 (SAV_2015 and SAV_6161), and GH75
(SAV_1288 and SAV_1850) in Streptomyces avermitilis grown in the absence (-)
or
presence (+) of chitosan oligosaccharides. Expression of the SAV_4958 (rpsl)
gene
was used as an internal control. Asterisks indicate chitosanase genes with the
CsnR
box.
DETAILED DESCRIPTION
Definitions
"Actinobacterium cell" or "Actinobacteria". As used herein, the terms
"Actinobacterium cell", "Actinobacteria" or "Actinomycete" are used
interchangeably
to refer bacteria of the Actinobacteria class. This class includes, but is not
limited to
the following subclasses (and orders): Acidimicrobidae (Acidimicrobiales),
Coriobacteridae (Coriobacteriales), Nitriliruptoridae (Nitriliruptorales,
Euzebyales),
Rubrobacteridae (Rubrobacterales, Solirubrobacterales, Thermoleophilales), and
Actinobacteridae, (Bifidobacteriales or Actinomycetales). Specific
genera of
actinobacteria include, but are not limited to, Streptomyces (such as, for
example,
Streptomyces lividans), Amycolatopsis, Catenulispora, Kitasatospora,
Verrucosispora, Micromonospora, Thermobispora, Salinispora, Streptosporangium,
Actinoplanes, Nocardiopsis, Stackebrandtia, and Saccharopolyspora.
In the context of this application, an actinobacterium cell is also understood
to
express, in its native state (e.g. when it is not genetically engineered), the
csnR gene
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(or one of its ortholog), its corresponding transcript and polypeptide. As
such, a
"native" actinobacterium cell is understood to refer to a wild-type, non-
genetically
engineered bacteria expressing the csnR gene (or its ortholog) and producing
the
corresponding polypeptide (CsnR for example). Native actinobacteria include,
but are
not limited to Streptomyces (such as, for example, Streptomyces lividans),
Amycolatopsis, Catenulispora, Kitasatospora, Verrucosispora, Micromonospora,
The rmobispora, Salinispora, Streptosporangium, Actinoplanes, Nocardiopsis,
and
Stackebrandtia, Saccharopolyspora.
When an actinobacterium cell is qualified as being "genetically engineered",
it is
understood to mean that it has been manipulated to either add a specific
exogenous
nucleic acid molecule and/or removed a specific endogenous nucleic acid
molecule.
The manipulation did not occur in nature and is the results of in vitro
manipulations of
the actinobacterium cell. In an embodiment, the genetic manipulations is
limited to
the cnsR gene (or its ortholog), its corresponding transcript or its
corresponding
polypeptide and are intended to either reduce the expression of the gene,
reduce the
expression and/or stability of the transcript, reduce the expression and/or
stability of
the polypeptide or reduce the functionality of the polypeptide. In one
embodiment, the
open-reading frame of the csnR gene (or its ortholog) is disrupted
specifically by the
introduction of an exogenous nucleic acid molecule.
"Antisense oligonucleotide". This term is understood to mean an
oligonucleotide
which is wholly or partially complementary to, and can hybridize with, a
target nucleic
acid (either DNA or RNA) having the sequence the csnR gene (or its ortholog)
or its
corresponding transcript. For example, an antisense nucleic acid or
oligonucleotide
comprising 10, 15 or 20 nucleotides can be sufficient to lower or inhibit
expression of
the csnR gene (or its ortholog). Alternatively, an antisense nucleic acid or
oligonucleotide can be complementary to 5' or 3' untranslated regions, or can
overlap
the translation initiation codon (5' untranslated and translated regions) of
the csnR
gene (or its ortholog). In another embodiment, the antisense nucleic acid is
wholly or
partially complementary to, and can hybridize with, a target nucleic acid that
encodes
a polypeptide from the csnR gene (or its ortholog). As non-limiting examples,
antisense oligonucleotides may be targeted to hybridize to the following
regions:
mRNA cap region; translation initiation site; translational termination site;
transcription initiation site; transcription termination site; polyadenylation
signal; 3'
untranslated region; 5' untranslated region; 5' coding region; mid coding
region; 3'
coding region; DNA replication initiation and elongation sites. Preferably,
the
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complementary oligonucleotide is designed to hybridize to the most unique 5'
sequence of the csnR gene (or its ortholog), including any of about 15-35
nucleotides
spanning the 5' coding sequence.
"Chitosan". As used herein, chitosan (or a "chitosan molecule") is understood
to
mean a polysaccharide obtained by N-deacetylation of chitin. In industrial
scale
procedures, chitosan is obtained from chitin by alkali treatment of crustacean
shells.
Chitosan is also present in nature in the cell walls of some fungi and algae
and in
insects. Chitosan is mainly composed of 6-1,4-linked D-glucosamine units with
a
variable content of N-acetyl-D-glucosamine units. The percentage of N-acetyl-D-
glucosamine units is defined as the degree of N-acetylation of chitosan
("DA"), while
the percentage of D-glucosamine units is also called the degree of
deacetylation
("DDA") of chitosan. Most commercial preparations of chitosan are
characterized by
DDA values between 70 and 99%.
Chitosan is unique among polysaccharides because it carries amino groups which
are positively charged in mildly acidic aqueous solution (pH < 6.2). Most
biological
properties of chitosan result from the presence of these positively charged
groups.
The amino groups can also be coupled to various chemical groups, resulting in
a
large family of chitosan derivatives.
Since chitosan is a large molecule, its size can be reduced to provide
chitosan
"fragments". Such fragments includes, but are not limited to low molecular
weight
chitosan (usually between 5 and 100 kDa) or chitosan oligosaccharides (usually
between 0.4 and 5-10 kDa). Such fragments can be obtained through chemical
cleavage, but more preferably through the enzymatic action of a chitosanase.
"Chitosanase activity". As used herein, the term "chitosanase activity" or
"chitosanase" is intended to refer to the ability of a glycoside hydrolase to
cleave a
chitosan molecule. In an embodiment, the chitosanase is secreted
extracellularly by
the native host. The chitosanase can be derived from various organisms, but in
an
embodiment, the chitosanase is of bacterial origin. In another embodiment, the
expression of the contemplated chitosanase is regulated by CsnR (or a
polypeptide
encoded by a csnR gene ortholog). The contemplated chitosanase possesses an
operator recognized by the CsnR polypeptide or a polypeptide encoded by a csnR
gene ortholog and such recognition leads to the reduced expression of the
chitosanase-encoding gene.
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The contemplated enzymes can be divided into two groups based on their
biochemical activity: "exo-chitosanase" and "endo-chitosanase". The enzyme
having
exo-chitosanase activity (also referred to as exo-1,4-beta-D-glucosaminidase)
are
known to act specifically on chitosan and chitosan oligosaccharides and do not
hydrolyze a- or 11-glucosides, galactosides, N-acetylglucosaminides including
substrates such as colloidal chitin, cellulose, carboxymethylcellulose or
cello-
oligosaccharides. Known enzymes having exo-chitosanase activity whose
expression
is regulated by CsnR or a polypeptide encoded by a csnR ortholog belong to the
GH2 family of glycoside hydrolase Exemplary exo-chitosanases include, but are
not
limited to CsxA (or AorCsx from Amycolatopsis orientalis) and SAV1223
(Streptomyces avermitilis).
On the other hand, the enzyme having endo-chitosanase activity is known to
mediate
the endohydrolysis of beta-1,4-linkages between residues in a partly
acetylated
chitosan. The endo-chitosanase includes, but is not limited to, the enzymes
belonging to the following families of glycoside hydrolase: GH5, GH8, GH46,
GH75,
GH80. In some embodiment, the enzyme having endo-chitosanase activity can also
present additional enzymatic activity, such as, for example, cellulase and/or
licheninase activity. Exemplary endo-chitosanases include, but are not limited
to
CsnN106 (Kitasatospora sp. N106; formerly known as Nocardioides sp. N106),
CsnN174 (Streptomyces sp. N174), CsnA (or SC00677 from Streptomyces
coelicolor A3(2)), CsxA (or SAV1223 from Streptomyces avermitilis), SAV1850
(Streptomyces avermitilis), SAV2015 (Streptomyces avermitilis), SCAB_86311 (or
SscGH5 from Streptomyces scabies 87.22), SGR_1341 (or SgrGH5 from
Streptomyces griseus IF013350), SSDG_05015 (or SprGH5 from Streptomyces
pristinaespiralis ATCC 25486), CsnA (or SliCsn or SSPG_06922 from Streptomyces
lividans), and/or AA4GH8 (or = SSMG_06552 from Streptomyces sp. AA4).
"csnR gene". This term is understood to mean a gene encoding a negative
transcriptional regulator of the ROK family mediating its effect on the
chitosanase
gene expression of actinobacteria. In actinobacteria, this transcription
factor interacts
with the operator of the chitosanase-encoding gene and negatively impacts its
transcription. The presence of chitosan in the culture medium of the
actibacterium
cell lessens the affinity of the transcription factor for the operator of the
chitosanase-
encoding gene and facilitates its transcription (and ultimately its
expression). The
csnR gene has been described specifically in Streptomyces lividans (SliROK or
SSPG_04872).
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However, the csnR gene is not limited to the one described in S. lividans and
also
encompasses all csnR gene orthologs. In the context of this application, a
"cnsR
gene ortholog" is understood to be a gene in a different species that evolved
from a
common ancestral gene by speciation. In the context of this application, a
csnR
ortholog retains the same function, e.g. it can act as a transcription factor
for
regulating the expression of chitosanase-encoding genes. Known csnR ortholog
include, but are not limited to those described in Streptomyces coelicolor
A3(2)
(SCO2657), Streptomyces avermitilis (SAV5384), Streptomyces scabies 87.22
(SCAB_59491), Streptomyces griseus IF013350 (SgrROK or SGR_4874),
Streptomyces pristinaespiralis ATCC 25486 (SprROK or SSDG_02817), and
Streptomyces sp. AA4 (AA4ROK or SSMG_00813). In an embodiment, the degree of
identity of csnR gene ortholog with respect to the csnR gene is at least 60%,
65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% (when determined on the
entire open-reading frame of the csnR gene).
As used herein, a "transcript" of the csnR gene (or its ortholog) refers to
nucleic acid
molecules (most likely mRNA) directed from the csnR gene and encoding the CsnR
polypeptide (or the polypeptide encoded by a csnR ortholog). The transcript
can be a
nucleic acid molecule of transient expression.
"CsnR polypeptide". As used herein, the CsnR polypeptide is understood to
refer to
the polypeptide encoded by the csnR gene or its ortholog. As indicated above,
the
"wild-type" or "native" CsnR polypeptide is a transcription factor that can
bind to the
operator of a chitosanase-encoding gene to modulate (e.g. decrease or repress)
its
expression. In an embodiment, the CsnR polypeptide (or the polypeptide encoded
by
a csnR gene ortholog) can bind to the consensus operator sequence of presented
in
SEQ ID NO:25 and/or the consensus sequence presented in SEQ ID NO:88. In
another embodiment, the CsnR polypeptide can bind to any one of the operator
sequences presented in SEQ ID NO: 14 to 24 as well as SEQ ID NO: 74 to 87. In
yet
another embodiment, the CsnR polypeptide is capable of repressing the
expression
of the chitosanase-encoded gene located downstream of the operator to which it
binds. In an embodiment, the CsnR polypeptide (or the polypeptide encodeded by
a
csnR gene ortholog) binds to an operator located at the most at 100 base pairs
upstream from the transcription start site. In another embodiment, the CsnR
polypeptide or the polypeptide encoded by a csnR gene ortholog) is at least as
50%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the
polypeptide encoded by the csnR gene of Streptomyces lividans (SliROK or
CA 02767534 2012-02-09
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SSPG_04872) (when alignment is performed on the entire length of the compared
polypeptides).
A "dominant-negative" CsnR polypeptide is a modified (e.g. non-native) CsnR
polypeptide that binds to the operator provided above (any one of those
presented in
SEQ ID NO: 14 to 25 or 74 to 88) but cannot repress as efficiently the
expression of
the chitosanase-encoding gene located downstream of the operator as the native
CsnR polypeptide. Preferably the affinity of the dominant-negative CsnR
polypeptide
for the operator is higher than the affinity of the native CsnR polypeptide
for the same
operator, which will result in the effective displacement (or competition) of
the native
CsnR polypeptide from the operator of the chitosanase-encoding gene.
"Identity", as known in the art, is a relationship between two or more
polypeptide
sequences, as determined by comparing the sequences. In the art, identity also
means the degree of sequence relatedness between polypeptide/polynucleotide
sequences, as the case may be, as determined by the match between strings of
such
sequences. Identity and similarity can be readily calculated by known methods.
The
percentage of identity is determined over a specific portion of the nucleic
acid/amino
acid sequence of the csnR gene (or its ortholog) or CsnR polypeptide (or the
polypeptide encoded by a csnR gene ortholog), usually the entire length of the
polypeptide sequence. In order to determine the percentage of identity between
any
amino acid sequences, various tools are known to those skilled in the art. For
example, one can use the Protein Blast with the blastp algorithm, a software
which is
freely accessible through the NCBI's web site
(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=bla
stp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome).
"Endogenous". In the context of this application, an element which is
endogenous to
an organism is understood to mean that such element is natively provided in
the
organism. For example, an enzyme having chitosanase activity which is
considered
endogenous to an actinobacterium cell is considered to have been natively
produced
by such actinobacterium cell and is not the result of a genetic modification
by man.
As an another example, a nucleic acid molecule which is considered to be
endogenous to an actinobacterium cell is considered to have been natively
produced
by such actinobacterium cell and was not introduced by genetic means from man
into
the actinobacterium cell.
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"Exogenous". In the context of this application, an element which is exogenous
to an
organism is understood to mean that such element is not natively provided in
the
organism. For example, an enzyme having chitosanase activity which is
considered
exogenous to an actinobacterium cell is considered not to have been natively
produced by such actinobacterium cell. An enzyme that is exogenous was
introduced into the actinobacterium cell, most likely through means of genetic
modification. As another example, a nucleic acid molecule which is considered
to be
exogenous to an actinobacterium cell is considered not to have been natively
produced by such actinobacterium cell and was introduced (by genetic means)
into
the actinobacterium cell.
"Nucleic acid vector". As used herein, a "nucleic acid vector" or
"vector" is
understood to be a nucleic acid molecule which was provided at one point in
isolated
form and which is used to transfer a nucleic acid molecule from an organism to
another. Vectors can be derived from bacterial plasmids or chromosomal
segments
or mobile genetic elements as well as bacteriophages.
In one embodiment, the transferred nucleic acid molecule can encode a
polypeptide
(such as, for example, the CsnR polypeptide or a polypeptide encoded by a csnR
gene ortholog)). Optionally, the sequence of the nucleic acid can be optimized
for
codon usage and recognition depending on the host cell that is considered for
expression of the chitosanase gene and protein. More specifically, the vector
can
comprise a promoter sequence, preferably located upstream of the nucleic acid
encoding the chitosanase. In an embodiment, the promoter sequence can be the
native promoter of a chitosanase-encoding gene (or a portion thereof such as,
for
example, the operator, the ribosome-binding sequence, as well as the
transcription
termination sequence preventing transcription from an upstream gene). In
another
embodiment, the vector can also comprise a selection marker to facilitate the
identification of host cells carrying the vector and/or a signal peptide
sequence
directing an efficient secretion into the culture medium. Optionally, the
vector can
further comprise a fusion peptide or protein or tag, operatively linked to the
coding-
sequence of the chitosanase.
In another embodiment, the nucleic acid molecule can be provided to achieve
the
disruption of the csnR gene open-reading frame. In such embodiment, it may be
advantageous to provide a vector that is capable of being integrated (e.g.
integretable) into the bacterial host genome. In an embodiment, the
integration is
CA 02767534 2012-02-09
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specific to the csnR gene and can even lead to a deletion in the coding
sequence of
the gene. Such method is provided in Dubeau et al. (2009).
"microRNA" or "miRNA". This term is understood as a short ribonucleic acid
(RNA)
molecule found in eukaryotic cells capable of mediating gene silencing. A
microRNA
molecule has at least 15, 20 or even 22 oligonucleotides. On average, a miRNA
has
22 oligonucleotides. miRNAs are post-transcriptional regulators that bind to
complementary sequences on target messenger RNA transcripts (mRNAs), usually
resulting in translational repression or target degradation and gene
silencing. miRNA
can be designed to specifically silence the csnR gene (or its ortholog), favor
the
degradation of its transcript and/or repress the translation of its
transcript.
"Operator". As used herein, an "operator" is located immediately upstream of a
transcription start site of an open-reading frame and specifically binds a
transcription
factor which will modulate gene expression of the downstream open-reading
frame
(and even the entire operon in some embodiments). In the context of this
application,
the csnR operator binds CsnR (or the polypeptide encoded by a csnR gene
ortholog). The binding of CsnR (or the polypeptide encoded by a csnR gene
ortholog)
to the operator diminishes and even represses the expression of the downstream
located chitosanase gene. In an embodiment, the consensus sequence of the csnR
operator is SEQ ID NO: 25. In an embodiment, the consensus sequence of the
csnR
operator is SEQ ID NO: 88. In still another embodiment, the specific sequence
of the
csnR operator is any one of SEQ ID NO: 14 to 24 and SEQ ID NO: 77 to 87.
"Ribozymes". A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme
or
catalytic RNA) is an RNA molecule that catalyzes a chemical reaction.
Ribozymes
can play an important role as enzymes which target defined RNA sequences.
Ribozymes can be genetically engineered to specifically cleave a transcript of
a csni?
gene (or its ortholog).
"RNA interference" or "RNAi" is a post-transcriptional gene silencing process
that is
induced by a microRNA (miRNA) or a double-stranded RNA (or dsRNA (a small
interfering RNA; siRNA)), and has been used to modulate gene expression.
Generally, RNAi is being performed by contacting cells with a double stranded
siRNA
or a small hairpin RNA (shRNA). However, manipulation of RNA outside of cells
is
tedious due to the sensitivity of RNA to degradation. It is thus also
encompassed
herein a deoxyribonucleic acid (DNA) compositions encoding small interfering
RNA
(siRNA) molecules, or intermediate siRNA molecules (such as shRNA), comprising
CA 02767534 2012-02-09
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one strand of a siRNA. Accordingly, it is herewith provided an isolated DNA
molecule, which includes an expressible template nucleotide sequence of at
least
about 16 nucleotides encoding an intermediate siRNA, which, when a component
of
a siRNA, mediates RNA interference (RNAi) of a target RNA. It is also
contemplated
"Small interfering RNA" or "siRNA" refers to any nucleic acid molecule capable
of
mediating RNA interference "RNAi" or gene silencing. For example, siRNA can be
double stranded RNA molecules from about 10 to about 30 nucleotides long that
are
named for their ability to specifically interfere with protein expression. In
one
embodiment, siRNAs are 12-28 nucleotides long, more preferably 15-25
nucleotides
long, even more preferably 19-23 nucleotides long and most preferably 21-23
nucleotides long. Therefore preferred siRNA are 12, 13, 14, 15, 16, 17, 18,
19, 20,
21, 22, 23, 24, 25, 26, 27, 28 nucleotides in length. As used herein, siRNA
molecules
need not to be limited to those molecules containing only RNA, but further
encompass chemically modified nucleotides and non-nucleotides. siRNA are
designed to decrease expression of the csnR gene (or its ortholog) in a
actinobacterium cell by RNA interference. siRNAs comprise a sense region and
an
antisense region wherein the antisense region comprises a sequence
complementary to an mRNA sequence for the csnR gene (or its ortholog) and the
sense region comprises a sequence complementary to the antisense sequence of
the gene's mRNA. A siRNA molecule can be assembled from two nucleic acid
fragments wherein one fragment comprises the sense region and the second
fragment comprises the antisense region of siRNA molecule. The sense region
and
antisense region can also be covalently connected via a linker molecule. The
linker
molecule can be a polynucleotide linker or a non-polynucleotide linker.
"Triplex oligonucleotides". This expression is understood to mean
oligonucleotides
which will bind to duplex nucleic acid (i.e., DNA:DNA or DNA:RNA), to form a
stable
triple helix containing or triplex nucleic acid. Such triplex oligonucleotides
can inhibit
transcription and/or expression of the csnR gene (or its ortholog). Triplex
oligonucleotides are constructed using the base-pairing rules of triple helix
formation
and the nucleotide sequence of the csnR gene (or its ortholog).
CA 02767534 2012-02-09
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Optimized cells for the production of an enzyme having chitosanase activity
The present application provides an expression system for producing an enzyme
having chitosanase activity. Such expression system is based on the use of
actinobacteria for the production of the enzyme.
As known in the art, actinobacteria have been shown useful for the production
of
chitosanase, either of exogenous or endogenous nature. However, and unlike
other
host cells of non-actinobacterial origin, the production of chitosanase in
actinobacteria has been shown to be strictly dependant on the presence of
chitosan
in the growth medium. As indicated above, the use of chitosan during
fermentation is
not trivial and impose severe limitations. As shown herein, the dependence on
chitosan has been shown to be in part associated with CsnR, a transcription
factor
which represses the expression of chitosanase-encoding genes. In the presence
of
chitosan, the affinity of CsnR for the promoter of the chitosan-enconding
genes
lessens and, ultimately expression of the chitosanase is permitted or
augmented. As
also shown herein, a reduction in CsnR activity (e.g. for example via a
genetic
alteration of in the open-reading frame of the csnR gene) resulted in a
derepression
in the expression of the chitosanase-encoding gene, an absence of dependence
towards chitosan in the culture medium for expressing a chitosanase and an
increase
expression in chitosanase in actinobacteria.
As such, it is contemplated that a genetically modified actinobacterium cell
(whose
activity in CsnR would be reduced) could successfully be used for the
production of
an enzyme having chitosanase activity. In an embodiment, the genetically
modified
actinobacterium cell possesses a reduced amount of a native (e.g. functional)
CsnR
polypeptide when compared to a corresponding wild-type (e.g. native)
actinobacterium. In an embodiment, the genetically modified actinobacterium
cell
does not express any detectable amount of a native (e.g. functional) CsnR
polypeptide and/or of a transcript of a csnR gene.
Such reduced or abolished activity of the CsnR polypeptide can be obtained by
modifying the csnR gene or its surrounding regions. For example, the 5' and/or
3'
untranslated regions of the csnR gene can be modified in such a way that the
expression of the csnR gene is reduced or abolished.
Alternatively, it is also possible to disrupt the open-reading frame of the
csnR gene
directly to mediate this effect. The disruption of the csnR open-reading
frame, even
by a single nucleotide, will provide mutations which will either introduce a
stop codon
CA 02767534 2012-02-09
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prematurely or code for a non-functional CsnR polypeptide (or fragment
thereof). In
an embodiment, the disruption can include the deletion and/or the addition of
at least
one nucleotide in the csnR gene open-reading frame. The disruption can include
the
deletion and/or the addition of a fragment of in the csnR gene open-reading
frame. In
an embodiment, this fragment is at least 700 base pairs, at least 728 base
pairs or at
least 774 base pairs. The disruption can also concern the deletion of the
entire csnR
gene's open-reading frame. The genetic disruption of the csnR gene can be
mediated through specific integration (and optionally the subsequent specific
removal) of a nucleic acid vector (for example an integrating vector).
It is also possible to reduce or abolish the activity or expression of the
CsnR
polypeptide by reducing the amount and/or the stability of the transcripts of
the csnR
gene. This may be achieved by providing a nucleic acid tool specific for the
transcript
of the csnR gene which will reduce the stability of the transcript and
ultimately limit
the amount of the transcript. Such nucleic acid tools include, but are not
limited to,
antisense oligonucleotides, small interfering oligonucleotides, ribozymes,
oligonucleotides capable of forming triplex oligonucleotides and double
stranded
RNA.
In yet another embodiment, it is possible to reduce or abolish CsnR activity
in the
actinobacteria by introducing a dominant-negative CsnR polypeptide in the cell
host.
Such dominant negative CsnR polypeptide can reduce the repression at the
chitosanase-encoding gene's operator and as such facilitate the expression of
the
chitosanase gene.
The genetically modified cell described herewith can be useful for the
production of
an enzyme which is either endogenous or exogenous to the cell. As shown in
Examples I and III, exogenous enzymes have bee successfully produced in a
genetically modified actinobacterium cell as described herein. As shown in
Example
II, endogenous enzymes have bee successfully produced in a genetically
modified
actinobacterium cell as described herein.
When an exogenous chitosanase is expressed in the genetically modified
bacterium,
it can be provided on a nucleic acid vector to be introduced into the
bacterial host.
The vector encoding the chitosanase can either multiply independently in the
bacterial host (in the form of a plasmid for example) or can be integrated
into the
host's genome for increased genetic stability. In an embodiment, the nucleic
acid
vector comprises not only the chitosanase-encoding gene but also the promoter
CA 02767534 2012-02-09
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associated thereto (including the operator which is specifically recognized by
CsnR
or a polypeptide encoded by a csnR gene ortholog), especially the ribosome-
binding
sequence and, optionally, a transcription termination sequence preventing
transcription from an upstream gene.
Methods for producing the enzyme having chitosanase activity
The present application provides optimized methods and processes for producing
chitosanase from an actinobacterium cell. In order to do so, the
actinobacterium cell
described above is first placed in a culture medium in the absence of (devoid
of)
chitosan, chitosan fragments or chitosan derivatives. As such, no exogenous
chitosan, chitosan fragments or chitosan derivatives have been added to the
culture
medium nor can chitosan, chitosan fragments or chitosan derivatives can be
detected in the culture medium.
The actinobacterium is then cultured in conditions favorable for chitosanase
gene
expression, and, ultimately, chitosanase production. Such conditions can
include
temperature control, shaking, etc.
Even though the production of a chitosanase by the genetically modified
actinobacterium has been observed in various media, it is possible to
successfully
optimize the culture medium used to obtain superior results results. One of
the
culture medium that can be used comprises malt extract, KH2PO4, K2HPO4,
(NH4)2SO4 and MgSO4. The malt extract can be present at a concentration (w/v)
between 0.5 and 2 (preferably 2%). The KH2PO4 can be provided at a
concentration
(w/v) between 0.1% and 0.4% (preferably 0.4%). The K2HPO4 can be provided at a
concentration (w/v) between 0.5% and 2.2% (preferably 2.2%). The (NH4)2SO4 can
be provided at a concentration (w/v) between 0.1% and 0.6% (preferably 0.56%).
The MgSO4 can be provided at a concentration (w/v) between 0.02% and 0.125%
(preferably 0.125%). The pH of the resulting medium is ideally between 6.5 and
7.0
(preferably 6.9). In some embodiment, the culture medium can also comprise a
selection marker (such as for example an antibiotic) to selectively propagate
the
genetically modified actibacterium cell.
During the fermentation process, it is possible to monitor chitosanase
activity in the
supernatant to identify when the enzyme is released from the actinobacterium
cell. It
is also possible to monitor for the presence of protease(s) in the supernatant
to
identify is contaminants are present and, ultimately, if the fermentation
should be
stopped to prevent the degradation of the chitosanase.
CA 02767534 2012-02-09
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The methods/processes described herewith provide a chitosanase end-product
which is of relatively high purity (at least 80%, 85%, 90% or 95% purity) and
can be
used without further purification in an industrial process. However, for some
applications, it may be necessary to further purify the chitosanase from the
fermentation broth. Such purification can include, but is not limited to,
filtration,
dialysis, precipitation, affinity-purification (antibody-based or tag-based)
and
chromatography. In an embodiment, the chitosanase is purified using cation-
exhange
chromatography.
Optionally, before placing the genetically modified actinobacterium cell in a
culture
medium, it may be advisable to apply a selective pressure on the initial
actinobacterium cell population used to select a genetically modified
actinobacterium
cell having a reduced Csn polypeptide activity. Such selective pressure may be
associated with a specific genetic trait (such as for example an integration
or a
deletion in the csnR gene). It may also be necessary, to optimize production,
to
continue to add the selective pressure during the fermentation process.
Methods of using the enzyme having chitosanase activity
The present application also provides methods of cleaving chitosan molecules
to
generate either low molecular weight chitosan (usually between 5 and 100 kDa)
or
chitosan oligosaccharides (usually between 0.4 and 5-10 kDa). The methods
presented herein use the chitosanase produced by the genetically engineered
actinobacterium cell described herein for reducing the molecular weight of the
chitosan. The method presented herein should be conducted under conditions
allowing enzymatic activity of the chitosanase. Such condition can include,
but are
not limited to, temperature, pH, reaction medium, presence of substrate,
absence of
inhibitors, etc. The method can also optionally comprise a step for the
recuperation
and purification of the products of the enzymatic reaction (e.g. LMWC or
CHOS).
The chitosanase does not need to be purified in order to be used in the
method. For
example, a sample of a culture medium that was previously cultured with the
genetically engineered actinobacterium cell capable of expressing (and
preferably
secreting) a chitosanase, such as the one described herein, can be used.
However,
the chitosanase may be purified, in part, in order to be included in the
method.
Purification means that may be used include, but are not limited to,
centrifugation,
precipitation, filtration, dialysis, solvent extraction, electrophoresis,
lyophilization
and/or chromatography (such as, for example, cation-exchange chromatography).
CA 02767534 2012-02-09
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The method is preferably conducted at a temperature optimal for the
chitosanase,
usually between 30 C to 40 C (for reactions lasting between 24 and 120 hours)
or
between 40 C and 60 C (for reactions lasting between 10 min and 6 hours).
Higher
temperatures facilitate the dissolution of chitosan in aqueous solutions.
However,
mixtures of chitosan molecules, especially concentrated mixtures of chitosans
of
lower molecular weight are subjected to the Maillard's reaction at high
temperatures,
resulting in brownish, chemically altered products which are inadequate for
many
applications. The occurrence of this reaction sets the upper temperature
limits for
enzymatic hydrolysis of chitosan at 70 C to 75 C for reaction times between 10
min
and 6 h and 55 C to 60 C for reaction times between 24 hours and 48 hours.
The chitosanase can cleave a variety of different chitosan molecules. Chitosan
is a
linear polysaccharide composed of randomly distributed 3-(1-4)-linked D-
glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit).
Typically, the chitosan molecules are defined by their length as well as their
degree
of deacetylation or DDA. Chitosan which are commonly used in the industry
usually
have a DDA of more than 50%, and usually 70% or more.
The chitosanase retains its enzymatic activity over a relatively large pH
range, e.g.
between 3.8 and 6.5. The method is preferably performed at a pH between 4.4
and
5.5, the optimal pH of the chitosanase.
The present invention will be more readily understood by referring to the
following
examples which are given to illustrate the invention rather than to limit its
scope.
EXAMPLE I - CSNR-K.-0. CELLS FOR THE EXPRESSION OF EXOGENOUS
CHITOSANASE
In this example, a study on the genetic regulation of a heterologous
chitosanase
gene (csnN106) in Streptomyces lividans is provided. Two S. lividans strains
were
used for induction experiments: the wild type strain and its mutant (AcsnR),
harbouring an in-frame deletion of the csnR gene, encoding a negative
transcriptional
regulator. Comparison of chitosanase levels in various media indicated that
CsnR
regulates negatively the expression of the heterologous chitosanase gene
csnN106.
Using the AcsnR host and a mutated csnN106 gene with a modified transcription
operator, substantial levels of chitosanase could be produced in the absence
of
chitosan, using inexpensive medium components. Furthermore, chitosanase
production was of higher quality as lower levels of extracellular protease and
protein
contaminants were observed. This new chitosanase production system is of
interest
CA 02767534 2012-02-09
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for biotechnology as only common media components are used and enzyme of high
degree of purity is obtained directly in the culture supernatant.
Material and methods
Bacterial strains and general culture conditions. E. coli strain DH5a TM
(Invitrogen) was
used for cloning experiments and DNA propagation. E. coli DH5aTmwa5 grown on
Luria-Bertani broth supplemented with 500 pg/ml hygromycin (Hm) or 50 pg/ml
kanamycin (Km). Standard methods were used for E. coli transformation, plasmid
isolation and DNA manipulation. Streptomyces lividans TK24 and S. lividans
AcsnR
(Dubeau et al., 2009) were used as hosts for chitosanase genes. Preparation of
S.
lividans protoplasts and transformation using rapid small-scale procedure and
R5
regeneration medium were performed as described previously (Kieser et al.,
2000).
After DNA transfer, hygromycin or kanamycin-resistant colonies were selected
after
addition of 5 mg Hm or Km to 2.5 ml of soft agar overlay. Transformants were
chosen
following two subsequent cycles of purification on solid yeast/malt extract
(YME)
medium (Kieser et al., 2000) with 250 pg/m1 Hm or Km. Sporulation was obtained
by
heavy inoculation of SLM3 agar medium plates (Dewitt, 1985). Spores were
collected
with glass beads and stored in 20% glycerol at -20 C.
Gel mobility shift assay. 108 spores of S. lividans TK24 or S. lividans AcsnR
were
inoculated into 50 ml of Tryptic soy broth (TSB, Difco) and grown for 64 h at
30 C
with shaking. Cultures were centrifuged, the mycelial pellets were washed with
sterile
0.9% saline and suspended in two volumes of saline. Then, 1 mpv (equivalent of
1 ml
of pellet volume) was added to 100 ml of induction medium. Induction medium is
a
modified M14 medium (M14M) (Page et al., 1996) composed of 0.1% KH2PO4,
0.55% K2HPO4, 0.14% (NH4)2SO4, 0.1% of trace elements solution (2 g/L
CoC12.7H20, 5 g/L FeSO4.7H20, 1.6 g/L MnSO4.H20, 1.4 g/L ZnSO4.7H20), pH 6.9.
Before use, 0.03% Mg504, 0.03% CaCl2, 0.125% GIcN and 0.375% chitosan
oligomers (1:1 dimer-trimer mix) was added to the M14M. Cultures were
incubated at
C with shaking. Every 24 h, 10 ml of culture were collected and centrifuged
and
pellets were kept frozen at -80 C. Pellets were melted on ice, washed with
cold
30 extraction buffer (50 mM Tris, 60 mM NaCl, 5% glycerol, 1 mM EDTA, 1 mM
DL-
dithiothreitol (DTT), pH 8.0) and suspended in 1 ml of extraction buffer
containing a
protease inhibitor cocktail (CompleteTM; Roche Molecular Biochemicals). The
bacterial cells were then disrupted by sonication with one treatment of 40 s
at 40%
amplitude (Vibra-CeIITM, 130 Watt 20 kHz, Sonics and materials inc., USA).
Total
CA 02767534 2012-02-09
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protein extracts were centrifuged at 3000 g for 10 min at 4 C. Supernatants
were
then frozen and stored at -80 C until used.
The double-stranded csnN106 palindromic probe (MP12F) was prepared by
complementary oligonucleotide annealing and end-labeling with hi-321'1AT P
(PerkinElmer) and T4 polynucleotide kinase as described by Dubeau et al.
(2009).
DNA binding reactions (24 pl) contained 10 mM HEPES (pH 7.9), 10% glycerol,
0.2
mM EDTA, 0.5 mM PMSF, 0.25 mM DTT, 1 pg poly(dl-dC), 150 mM KCI, 0.1 nM of
labeled probe and 10 pg of protein crude extract. The reaction mixtures were
incubated at room temperature for 15 min and then subjected to electrophoresis
in a
pre-run gel of 6% polyacrylamide (10 mM Tris, 80 mM glycine, 0.4 mM EDTA, pH
8.3). The gel was dried and viewed with a PhosphorimagerTM (Molecular
Dynamics).
Vector construction. The csnN106 gene fragment (GenBank accession number
L40408.1) was amplified by PCR reaction using FwcsnN106 and RvcsnN106 primers
(Table 1) and plasmid pCSN106-2 as template (Masson et al., 1995). The
amplified
Sphl - HindlIl fragment was cloned into the vector pFDES (Lacombe-Harvey et
al.,
2009) digested with the same enzymes, giving plasmids pFDES-csnN106. The
promoter region of csnN106 (Pr-WT) was PCR-amplified with primers FwPr-WT and
RvPr-WT. Purified PCR fragment was cloned between BamHI and Sphl restriction
sites of pFDES-csnN106 generating pFPr-WT. A mutated version of Pr-WT with two
base-pairs substitutions in the palindromic operator (Pr-Pa) was obtained with
the
PCR-directed mutagenesis method (Ho etal., 1989) using SEQ.1, Rv1Pr-Pa, Fw2Pr-
Pa and RvcsnN106 as primers (Table 1) and the pFPr-WT plasmid as DNA template.
The mutated PCR product was digested with BamHI and Sphl and cloned into
pFDES-csnN106 generating pFPr-Pa. The phage-type version of csnN106 promoter
(Pr-Ph) was obtained by annealing two short DNA segments:
5'ATCCTGACGGCCCGTCCGCCCAGCGGTACGAGGGCCCCGACCGGAGTTCCGGTCGGGGCCT
TTCGCATGACCGCGCGGGCAAACATGGCGCTTGACCTTGATGAGGCGGCGTGAGCTACAATC
AATATCTAGTTAGGAAACTTTCCTAACTCTCCTCATGGGTCCGGAGACCCGCATG3' (SEQ ID
NO: 1) and
FCGGGTCTCCGGACCCATGAGGAGAGTTAGGAAAGTTTCCTAACTAGATATTGATTGTAGCTCACGCC
GCCTCATCAAGGTCAAGCGCCATGTTTGCCCGCGCGGTCATGCGAAAGGCCCCGACCGGAACTCCGGTC
GGGGCCCTCGTACCGCTGGGCGGACGGGCCGTCAG3I (SEQ ID NO: 2).
CA 02767534 2012-02-09
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Table 1. Oligonucleotides used in this study
SEQ ID
Aim of primers Name Sequence (5'--3')
NO.:
For csnN106 coding FwcsnN1 06 CCGGAGACCCGCATGCCCCGGAC 3
region cloning* RvcsnN1 06 CGGTGCGCCAAGCTTGCGTTCGG 4
FwPr-WT GTCTGCGCGGATCCTGACGGCCC 5
For Pr-WT cloning*
RvPr-WT GTCCGGGGCATGCGGGTCTCCGG 6
SEQ.1 ACAACTTCGTCGCGCACATCCA 7
FOR-directed mutagenesis Rw1Pr-Pa ATGAGGAGAGTTCGGACAGTTTC 8
for Pr-Pa cloning** Fw2Pr-Pa GAAACTGTCCGAACTCTCCTCAT
RvcsnN106 TGAGGTCGAAGTTCTTGGCGTT 10
Presence verification of SEQ.1 ACAACTTCGTCGCGCACATCCA 11
pFDES derivatives
T7 promoter TTAATACGACTCACTATAGGG 12
into hosts
For Primer extension PE-csnN106 TGGGGTGCTTGAGACGCAT 13
*Bold nucleotides correspond to restriction site
**Bold nucleotide correspond to mutated nucleotide
Plasmids were introduced into S. lividans strains by transformation and
selection with
kanamycin for pFDES derivatives carrying neoS as resistance gene. The presence
of
pFDES derivatives were verified by FOR using primers in Table1.
Results
Transcription startpoint mapping by primer extension. 108 spores of S.
lividans
TK24(pHPr-WT) strain were inoculated into 50 ml of TSB with 50 pg/ml Hm and
grown for 64 h at 30 C with shaking. Chitosanase gene expression was obtained
in
M14 M medium with GIcN and chitosan oligomers as described for gel mobility
shift
assay. After 14 h, four culture samples of 10 ml each were collected and mixed
immediately with stop solution (0.2 volumes of ethanol-equilibrated phenol,
95:5).
Samples were centrifuged for 10 min at 4 C. Bacterial pellets were frozen at -
80 C
until lysis. Total RNA extraction was carried out using the Qiagen RNeasy0
Mini Kit
(Qiagen) with the following modifications. Cell disruption was achieved by
sonication
with two 30 s burst at 35% amplitude separated with a 15 s cooling period,
followed
by two phenol-chloroform extractions and one chloroform extraction for cell
debris
elimination. The on-column DNase treatment was done with the RNase-free DNase
set (Qiagen). RNA purity and concentration were assessed in a NanoDropTm1000
CA 02767534 2012-02-09
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spectrophotometer (Thermo Scientific). RNA quality was verified by
electrophoresis
on agarose gel in lx MOPS electrophoresis buffer with 0.22 M formaldehyde.
20 pmoles of PE-csnN106 primer (Table 1) were end-labeled with (Y-32PIATP
(PerkinElmer) and 20 units of T4 polynucleotide kinase, then purified on a G-
25
column (GE Healthcare). Total RNA (40 rig) was hybridized with the end-labeled
primer (0.5 pmole) in the presence of 10 mM Tris-HCI pH 8.6, 300 mM NaCI and 1
mM EDTA, in a volume of 22 pl by incubation at 95 C for 5 min, then 55 C for
90
min. RNA/primer mix was then precipitated with 200 pl ammonium acetate 1 M and
200 pl isopropanol. The pellet was washed with 70% Et0H, dried and suspended
in
10 pl of 10 mM Tris-HCI (pH 8.6), reverse transcriptase buffer (lx, Promega),
10 mM
OTT, 1 mM dNTPs, 1 pg actinomycine D, 5 units of AMV reverse transcriptase
(Promega) and 20 units of RNAsin (Promega) for a total volume of 20 pl. The
reaction mixture was incubated at 45 C for 60 min and stopped with formamide
dye.
A sequencing reaction was performed with the end-labeled primer and the
ALFexpress TmAutoCycle TM Sequencing Kit (Amersham Biosciences)
using
manufacturer's recommendations. The primer extension sample and the sequence
reactions were heated 5 min at 95 C just before loading on a 6% polyacrylamide
sequencing gel. The gel was run, dried, visualised and analyzed by a
PhosphorimagerTM and the lmageQuantTM Version 5.2 software (Molecular
Dynamics).
Chitosanase production experiments. 109 spores of S. lividans strains (WT +
pFPr-
WT, AcsnR + pFPr-Pa) were inoculated into 50 ml of TSB supplemented with 50
pg/ml Km (WT + pFPr-WT and AcsnR + pFPr-Pa) and grown for 64 h at 30 C with
shaking. Three types of culture were tested. First, a rich, malt extract-based
medium
(4x M14M without microelements, 0.12% MgSO4, 2% malt extract) was directly
inoculated with a portion of the pre-culture in TSB corresponding to an
inoculation
proportion of 4 mpv/100 ml. Second, 100 ml of chitosan medium (M14 M, 0.03%
MgSO4, 0.03% CaCl2, 0.2% malt extract, 0.8% chitosan flakes (Sigma), 0.2%
GIcN)
was inoculated with 1 mpv of saline washed pre-culture. Third, 100 ml of
GIcN/chitosan oligomer medium (M14 M, 0.03% MgSO4, 0.03% CaCl2, 0.125% GIcN
and 0.375% chitosan oligomers) was inoculated with 1 mpv of saline washed pre-
culture. For each WT + pFPr-WT and AcsnR + pFPr-Pa flasks, 50 pg/ml Km was
added. Cultures were done in duplicate and incubated at 30 C with shaking. 10
ml
samples were collected every 24 h. Chitosanase and protease activities and
total
protein concentration were determined in supernatants.
CA 02767534 2012-02-09
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Biochemical procedures. Chitosanase activity was measured using the dyed
substrate sRBB-C. Briefly, 50 pl of appropriately diluted culture supernatant
were
added to 950 pl of soluble Remazol Brilliant Blue chitosan (5 mg/ml in 0.1 M
Na-
acetate buffer pH 4.5) and the mixture was incubated for 60 min at 37 C.
Reaction
was stopped with 500 pl of 1.2 N NaOH and cooled on ice for 20 min. After
centrifugation, the optical density of supernatant was read at 595 nm and
converted
into chitosanase activity as described (Zitouni etal., 2010). Protein
concentration was
estimated by the method of Bradford, with bovine serum albumin as standard.
Protease activity was determined with azocasein (Aretz et al., 1989).
By primer extension, we determined the start site for mRNA transcribed from
csnN106 (Figure 1 and Figure 2A), defining the probable -35 and -10 boxes of
the
promoter of csnN106 as TTGCGC and TTCAAT with a spacer of 18 nucleotides
(shown in blue on Figure 2A). To test another promoter, described as a
"strong"
promoter by Labes et al. (1997), the original -35 and -10 boxes of csnN106
gene
were substituted with the two tandemly arrayed and overlapping promoters of
the
Streptomyces ghanaensis phage 119, taking the respective transcription start
sites as
reference (Figure 2A).
A palindromic sequence overlaps the transcriptional start site of csnN106
(Figure
2A). Highly similar sequences are also present upstream from the coding
sequences
of chitosanase genes found in other genomes of actinomycetes, displaying a
clear
consensus (Figure 2B). Previous gel retardation experiments have shown an
interaction between a protein present in partially purified cell extract from
Kitasatospora sp. N106 and a short DNA segment including the palindromic
sequence (Dubeau et al., 2005). Competition tests with mutated
oligonucleotides
allowed determining the bases which were critical for the interaction with the
regulatory protein in vitro (Figure 2B) (Dubeau etal., 2005). Two most
important base
pairs in the right half of the palindromic sequence were mutated (while
keeping intact
the original -10 and -35 promoter boxes) and introduced upstream from the
csnN106
coding sequence, resulting in a third version of this heterologous gene. These
three
genes were introduced in two hosts: Streptomyces lividans TK24 (the host used
so
far in most works involving actinobacterial chitosanase studies) and a mutant
harbouring an in-frame deletion in csnR gene (AcsnR, formerly described as
A2657 h
by Dubeau et al. 2007). The csnR gene (SSPG_04872, according to GenBank
annotation) is coding for a putative transcriptional regulator of the
endogenous
CA 02767534 2012-02-09
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chitosanase gene a protein belonging to the ROK family created by Titgemeyer
et al.
(1994).
Crude extracts prepared from the cells of both strains cultivated in the
presence of
chitosan oligosaccharides (a mixture of GIcN and chitosan oligomer) were used
in gel
retardation experiments using a 32P-labelled oligonucleotide including the
palindromic
sequence from csnN106 as a probe. A shift in mobility was observed with the
extract
from the wild type strain but not with AcsnR mutant (Figure 3). The CsnR
protein
from S. lividans binds then efficiently the palindromic sequence of the
heterologous
csnN106 gene.
Chitosanase production in the absence of chitosan or derivatives. In previous
work,
efficient production of chitosanase by either native or recombinant
actinobacterial
strains was strictly dependent on the addition of chitosan or derivatives
(GIcN or
chitooligosaccharides) in the culture media. Testing various concentrations of
malt
extract, salt formulations and methods of inoculation allowed obtaining
routinely
activities in the range of 10- 12 units per ml and, in the best case, up to 24
units per
ml. Protease activity was also highly dependent on medium composition and type
of
inoculum. Addition of magnesium ions was found to be essential to promote
efficient
chitosanase production (and low level of protease), while the microelements of
the
M14 M medium could be omitted.
In previous work, chitosanase production was performed with S. lividans TK24
harbouring csn genes originating from various bacterial species cloned in
multicopy
plasmids. To compare the new gene/host combination with the former ones, we
cloned the csnN106 gene (with a wild type operator) into the multicopy vector
pFDES
and introduced it in the wild type host. In parallel, the same plasmid but
with the
mutated operator has been introduced into the AcsnR host. Chitosanase
production
by these two strains has been measured. Three media formulations were tested:
a
medium containing malt extract as main nutrient source, a medium with chitosan
flakes and GIcN, often used in our previous work, and a medium with more
expensive components, GIcN and chitosan oligomers, used in basic research for
the
induction of chitosanase gene expression. On Figure 4, only the 72 h time
point is
presented, as chitosanase level was maximal around this time point and then
remained stable or slightly decreased. The culture in medium with chitosan
flakes
and GIcN gives the best chitosanase level for the strain keeping intact both
partners
of the regulatory interaction (Figure 4A). However, cultures in media with
chitosan
gave much higher levels of extracellular proteases (Figure 4B). Furthermore,
the
CA 02767534 2012-02-09
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analysis of total extracellular proteins by SDS-PAGE revealed that there were
less
contaminant proteins in the malt extract medium than in the chitosan flakes
medium
(Figure 4C). The AcsnR host seems to be particularly useful for the
inexpensive
production of almost pure chitosanase in stable, low-protease conditions.
Discussion
The results presented herein are dedicated to the genetic regulation of a
heterologous chitosanase gene in S. lividans. It was shown that CsnR regulates
negatively the expression of csnN106 gene. Deletion of csnR or mutations in
the
operator sequence of csnN106 resulted in the derepression of expression in the
absence of inducer molecules. However, even in the derepressed gene/host
combination, some residual induction by chitosan derivatives was still
observed. This
could be due to a regulator responding directly to the presence of chitosan or
indirectly, through a stress pathway resulting from the interaction between
chitosan
and the cell. A complex transcriptomic response has been observed after
contact
with chitosan in cells of Staphylococcus aureus and Saccharomyces cerevisiae.
One
usual way to change the genetic regulation of a given gene is done by promoter
replacement. In our earlier work, testing three different promoters from
Streptomycetes did not led to the improvement of chitosanase production. As
shown
herein, the -35 and -10 boxes from csnN106 promoter sequence were replaced
while
conserving all the remaining segments. Despite the use of a promoter
considered as
strong, this substitution did not result in better chitosanase production. For
reasons
that remain unclear, the chitosanase expression was less efficient for a total
of four
different hybrid gene constructions when the protein coding sequence of Csn
was
separated from its native upstream segment. This could result from a lower
stability
of mRNAs transcribed from these hybrid genes, but this remains to be
investigated.
Masson et al. (1993) optimized a chitosanase production medium for the CsnN174
production in the heterologous host S. lividans. Masson showed that the
addition of
malt extract to the chitosan medium was beneficial for enzyme production. Our
media
formulations were based on malt extract in our attempts to produce chitosanase
with
the new gene/host combination in the absence of chitosan. It was shown that
equivalent, and sometimes higher chitosanase levels can be obtained without
the
addition of chitosan to the culture medium. Interestingly, the new medium/host
combination resulted in much lower levels of contaminant proteins in the
supernatant.
Finally, in earlier culture media formulations including chitosan flakes, a
raise of
extracellular protease activity at later culture stage could often result in a
rapid loss of
CA 02767534 2012-02-09
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chitosanase activity (Masson et aL, 1993). The new medium/host combination
provides a substantial improvement, as protease levels are much lower,
resulting in
stable chitosanase production.
The chitosanase production system based on a new medium/host combination was
shown to be at least as efficient as the former one without the necessity to
include
chitosan or derivatives into the culture medium. Extensive optimization of
culture
parameters will probably lead to much higher chitosanase activities. For
biotechnology, the new host will be of interest for large scale chitosanase
production
as only inexpensive media components can be used.
EXAMPLE II¨ CSNR-K.-0. CELLS FOR THE EXPRESSION OF ENDOGENOUS
CHITOSANASE
A palindromic sequence is present in the intergenic region preceding the
chitosanase
gene csnA (SSPG_06922) of Streptomyces lividans TK24. This sequence was also
found in front of putative chitosanase genes in several other actinomycete
genomes
and upstream genes encoding putative transcriptional regulators of the ROK
family,
including csnR (SSPG_04872) in S. lividans. The latter was examined as a
possible
transcriptional regulator (CsnR) of chitosanase gene expression. In vitro,
purified
CsnR bound strongly to the palindromic sequences of the csnA and csnR genes
(equilibrium dissociation constant [KD] = 0.032 and 0.040 nM, respectively).
Binding
was impaired in the presence of chitosan oligosaccharides and d-glucosamine,
and
chitosan dimer was found to be the best effector, as determined by an
equilibrium
competition experiment and 50% inhibitory concentration (IC50) determination,
while
glucose, N-acetyl-glucosamine, and galactosamine had no effect. In vivo,
comparison of the S. lividans wild type and ACsnR strains using 6-lactamase
reporter
genes showed that CsnR represses the expression of csnA and of its own gene,
which was confirmed by quantitative PCR (qPCR). CsnR is localized at the
beginning
of a gene cluster, possibly an operon, the organization of which is conserved
through
many actinomycete genomes. The CsnR-mediated chitosanase regulation
mechanism seems to be widespread among actinonnycetes.
Materials and methods
Bacterial strains, media, and culture conditions. Escherichia coli DH5aTM
(Invitrogen)
was used as the host for cloning and DNA propagation. The methylase-negative
mutant E. coil strain ET12567, containing the nontransmissible pUZ8002 plasmid
was used as the donor in intergeneric conjugation with the S. lividans
recipient. E.
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cot/ Rosetta-gami 2 (DE3) (pLysS) (Novagen) was used for recombinant CsnR
production. E. coli strains were grown on Luria-Bertani (LB) broth
supplemented,
when necessary, with 100 pg/ml ampicillin (Ap), 34 pg/ml chloramphenicol (Cm),
500
pg/ml hygromycin (Hm), 50 pg/ml kanamycin (Km), 100 pg/ml spectinomycin (Sm),
or
12.5 pg/ml tetracycline (Tet). Standard methods were used for E. coli
transformation,
plasmid isolation, and DNA manipulation. S. lividans TK24 (Dubeau et al.,
2009) and
the isogenic S. lividans AcsnR strain (formerly the A2657h strain) (Kieser et
al.,
2000) were used as recipients for transformation or conjugation. They were
also
used in quantitative PCR (qPCR) assays, while Streptomyces avermitilis MA-4680
was used in reverse transcription (RT)-PCR experiments.
DNA transformation with S. lividans protoplasts, using a rapid small-scale
procedure
and R5 regeneration medium, was performed as described previously (Kieser et
al.,
2000). Hm-resistant colonies were selected after DNA transfer by addition of 5
mg
Hm to 2.5 ml of soft agar overlay. Transformants were chosen following two
subsequent cycles of purification on solid yeast-malt extract (YME) medium
(27) with
250 pg/ml Hm. Intergeneric conjugation was done with S. lividans AcsnR spores
following a known protocol (Kieser et al., 2000). Approximately 5 x 107 spores
of the
S. lividans AcsnR strain and 5 x 107 cells of E. coli ET12567(pUZ8002)
carrying the
appropriate plasmid were combined for conjugation. The mixed bacteria were
spread
on SLM3 agar plates supplemented with 10 mM MgC12. Plates were overlaid with 1
ml of sterile water, including 5 mg Sm and 0.5 mg nalidixic acid per plate.
Exconjugants were purified on solid YME medium with 200 pg/m1 Sm and 25 pg/ml
nalidixic acid. Sporulation was obtained by heavy inoculation of plates with
SLM3
agar medium. Spores were collected with glass beads and stored in 20% glycerol
at
-20 C.
Production and purification of CsnR. The coding sequence of csnR was PCR
amplified from S. lividans genomic DNA with the primers EcoRl-csnR and Xhol-
csnR
(see Table 2). The PCR product was digested with EcoRI and Xhol and ligated
into
pGEX-6P-1 vector (GE Healthcare) digested with the same enzymes, generating
pGEX-csnR. This plasmid was used to produce the recombinant CsnR tagged with
glutathione S-transferase (GST) at the N terminus. For protein expression, the
plasmid was transformed into E. coli Rosetta-gami 2 (DE3) (pLysS).
Transformants
were selected on LB agar medium with Ap, Cm, and Tet. For production of CsnR,
the
transformant was grown in 1.25 liters of LB medium with Ap, Cm, and Tet
inoculated
1:20 with overnight culture. Cultures were grown at 37 C until the optical
density at
CA 02767534 2012-02-09
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600 nm (0D600) reached 0.4 to 0.6. Then 0.1 mM IPTG (isopropyl-p-d-
thiogalactopyranoside) was added, and cultures were further incubated for 4 h
at
room temperature with shaking. Bacteria were recovered by centrifugation, and
pellets were kept frozen at -80 C. For protein extraction, pellets were thawed
for 15
min on ice and suspended in a total volume of 250 ml of phosphate-buffered
saline.
Then 1 mg/ml lysozyme was added, and the suspension was incubated for 30 min
on
ice. The suspension was treated by son ication with six rounds of 10-s bursts
at 45%
amplitude (130W, 20 kHz) (Vibra-Cell; Sonics and Materials, Inc.) separated by
10-s
cooling periods on ice. The lysate was centrifuged for 20 min at 10,000 x g at
4 C.
The supernatant (soluble crude extract) was incubated for 1 h at room
temperature
with 2 mM ATP and 5 mM MgC12. All further steps were done at 4 C with cold
solutions and centrifugation steps of 1 min at 500 x g. The total volume of
soluble
crude extract (250 ml) was mixed with 1 ml of a 50% suspension of glutathione
Sepharose 4B, divided into 50-ml aliquots, and incubated for 1 h with slight
agitation.
The suspensions were centrifuged, and the supernatants were transferred for a
second round of binding with fresh resin. Pelleted resin was washed four times
with
1.4 ml of phosphate-buffered saline (PBS) and two times with cleavage buffer
(50
mM Tris-CI, 150 mM NaCI, 1 mM EDTA, 1 mM dl-dithiothreitol [DTT] [pH 7.0]).
For
each wash, pelleted resin was incubated 10 min with slight agitation and
centrifuged.
For each resin aliquot, 16 pl of PreScission protease (GE Healthcare) and 400
pl of
cleavage buffer were added to cleave the GST tag from CsnR. Suspensions were
pooled and incubated for 4 h at 4 C. The suspension was centrifuged, and the
supernatant was saved. The resin pellet was suspended in 600 pl of cleavage
buffer,
incubated for 10 min, and centrifuged. Both supernatants were pooled and
divided
into three fractions for size exclusion chromatography. Approximately 500 pl
of
partially purified CsnR was loaded onto a SuperdexTM 200 10/300 GL column (GE
Healthcare), with a mixture of 137 mM NaCI, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM
KH2PO4, 5% glycerol, 0.5% Tween 20, and 1 mM DTT (pH 7.4) as the elution
buffer.
After SOS-PAGE analysis, purified fractions were pooled, aliquoted, and frozen
at
-80 C until use. Identification of the contaminant protein from E. coli was
performed
by the Proteomics platform of the Quebec Genomics Center, Quebec, Canada.
CA 02767534 2012-02-09
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Table 2. PCR primers used in this example
SEQ ID
Aim of primers Name Sequence (5'--43')
NO:
For GST-CsnR EcoRl-csnR GCGGTCGAATTCCAGGTGTGGACA 26
production Xhol-csnR ATTCCGGGCCTCGAGAAGCTCC 27
DF-csnR CCTGCCATGCGTGTCCA 28
In DNase 1 footprinting DF-csnA CGGAAGGGGTGCCTCAC 29
UF-csnA ACAACTTCGTCGCGCACATCCA 30
BamHI-IR-csnA GGAGCAGCCGGATCCCTGACGGA 31
For IR-csnA cloning
SphI-IR-csnA AGGGGTGCCGCATGCAATCTCCA 32
BamH1-1R-csnR GCACCAGCAAGGATCCCCGCCCG 33
For IR-csnR cloning
Sph1-1 R-csnR TGCGTGTCCGCATGCGCCTCTCG 34
Fw1-csnAMM AATACGACACCAGATGGACGGC 35
For PCR-directed
Rv1-csnAMM CCGGGCACTGATCGGACAGTTTC 36
mutagenesis for IR-
Fw2-csnAMM GAAACTGTCCGATCAGTGCCCGG 37
csnAMM cloning
Rv2-csnAMM TTGTCCTCCACCTTCCAGTCCTT 38
In complementation
and DNAse I Xbal-csnRC TCCGCCGTCTAGAACCAGCAA 39
footprinting
In complementation EcoRl-csnRC CGAGGGCCGGAATTCTGGATAT 40
Underlined bases pairs in oligonucleotide sequence correspond to
restriction sites or mutated bases pairs from original sequence.
DNase I footprinting. To obtain end-labeled DNA probes, 30 pmol of the
downward
primers DF-csnR and DF-csnA (see Table 2) was end labeled with [Y-32P)ATP
(3,000
Ciimmol) (PerkinElmer) and 20 U of T4 polynucleotide kinase and then purified
on a
G-25 column (GE Healthcare). Approximately 20 pmol of the end-labeled primers
was used in 50-hl PCRs. For csnR, pMP302-A2657h (Dubeau et al., 2009) was used
as the template with the Xbal-csnRC primer. For csnA, pFDES-csnA was used as
the
template with UF-csnA primer. The end-labeled probes from PCR products were
purified with the High PureTM PCR product purification kit (Roche). DNA
binding
reaction mixtures (100 pl) contained 20 mM potassium phosphate buffer (pH
6.8), 5
mM MgC12, 150 mM KCI, 1 mM P-mercaptoethanol, 20% glycerol, 0.5 pg poly(dl-
dC),
approximately 20,000 cpm of end-labeled DNA probe, and ¨0.5 nmol of purified
CsnR. After 20 min of incubation at room temperature, 30 U of DNase 1 (Roche)
was
added to the reaction mixtures. After 90 s (60 s for reactions without
protein),
CA 02767534 2012-02-09
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reactions were stopped by addition of 15 mM EDTA (pH 7.9). DNA fragments were
extracted by phenol-chloroform and precipitated with 0.1 pg/pl yeast tRNA, 0.3
M
sodium acetate (pH 5.2), and 2 volumes of isopropanol. Precipitated DNA was
washed once with 70% ethanol, dried, and suspended in formamide loading
buffer.
Sequence reactions were done with end-labeled primers and DNA templates used
in
PCRs for probe labeling, and the ALFexpress AutoCycleTM sequencing kit
(Amersham Biosciences) according to the manufacturer's recommendations.
Samples and sequence reaction mixtures were heated for 5 min at 95 C just
before
being loaded onto a 6% polyacrylamide sequencing gel. The gel was run, dried,
visualized, and analyzed by a PhosphorlmagerTM with lmageouantTM version 5.2
software (Molecular Dynamics).
EMSA. For the electrophoretic mobility shift assay (EMSA), pairs of
complementary
oligonucleotides were annealed, generating double-stranded oligonucleotides
csnA-
WT (wild type), csnA-M1, csnA-M2, csnA-MM, and csnR-WT (see Table 3). Fifty
picomoles of double-stranded oligonucleotide was end labeled with [v-32PIATP
(3,000
Ci/mmol) and 20 U of T4 polynucleotide kinase and purified on a G-25 column.
DNA
binding reaction mixtures (24 pl) contained 10 mM HEPES (pH 7.9), 10%
glycerol,
0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.25 mM DTT, 1 pg
poly(dl-dC), and 150 mM KCI. For equilibrium dissociation constant (KD)
determination, various concentrations of labeled csnA-WT or csnR-WT probe (0.1
nM
to 1.5 nM) and ¨8.5 pmol of purified CsnR were used. For the determination of
the
50% inhibitory concentration (IC50) of the DNA competitors, 0.03 nM labeled
csnA-
WT probe and ¨8.5 pmol of CsnR were used with various concentrations of
competitor double-stranded oligonucleotide (0.1 to 125 nM). For the sugar
binding
assay, ¨8.5 pmol of CsnR was preincubated with glucose, GIcNAc, galactosamine,
GIcN, or chitosan oligosaccharides (GIcN)2 to (GIcN)5 at various
concentrations
(0.00075 to 75 mM) in the binding reaction mixture for 15 min on ice before
the
addition of labeled csnA-WT probe (0.03 nM). Reaction mixtures were incubated
at
room temperature for 15 min with the labeled probe and then subjected to
electrophoresis at 4 C in a prerun gel (15 min) of 6% polyacrylamide (10 mM
Tris, 80
mM glycine, 0.4 mM EDTA [pH 8.3]). Following electrophoresis, gels were dried,
and
band intensities were visualized with a PhosphorlmagerTM and estimated with
lmageQuantTM software (version 5.2). All determinations were done in
triplicate. KD
calculations were done with the Michaelis-Menten nonlinear fit (least
squares), and
CA 02767534 2012-02-09
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the one-site log 1050 nonlinear fit (least squares) was used for 1050
calculations
(GraphPad PrismTM version 5.03 for Windows; GraphPad, San Diego, CA).
Table 3. Double-stranded oligonucleotides used as probe or competitor DNA in
EMSA experiments
SEQ ID
Name Sequence (5'--3')
NO:
csn R-WT CTCCAGCCAACAGGAAACTTTCCTAACAGA 41
csnA-WT CCTCTTCTGGTAGGAAACTTTCCTATCAGT 42
csnA-M1 CCTCTTCTGGTAGGAAACTGTCCTATCAGT 43
csnA-M2 CCTCTTCTGGTAGGAAACTTTCCGATCAGT 44
csnA-MM CCTCTTCTGGTAGGAAACTGTCCGATCAGT 45
Genetic complementation of the S. lividans LcsnR strain. The csnR gene coding
sequence together with its complete upstream (211-bp) and downstream (106-bp)
intergenic regions were PCR amplified from genomic DNA of S. lividans TK24
with
primers Xbal-csnRC and EcoRl-csnRC (see Table 2). The PCR product was
digested and introduced between the Xbal and EcoRI restriction sites of the
integrative, conjugative vector pSET152m (Laing etal., 2006), generating
pSETmC.
Complementation plasmid was introduced into the S. lividans AcsnR strain by
intergeneric conjugation. pSET152m vector was used as a negative control.
Successful integration of vectors was confirmed by PCR analysis.
qPCR and endpoint RT-PCR. For quantitative PCR (qPCR) analysis, 2 x 108 spores
of the S. lividans TK24, AcsnR, AcsnR + pSETmC, or AcsnR + pSET152m strain
were inoculated into 50 ml TSB medium. The cultures were incubated at 30 C for
approximately 64 h with shaking. The cultures were centrifuged and then washed
with sterile 0.9% saline, and the pellets were suspended in 2 volumes of
saline. A
total of 1.5 ml of this suspension was used to inoculate 50 ml of M14M (see
above)
either with mannitol or with 1:3 GIcN-chitosan oligomers. The experiment was
done
in triplicate. Cultures were incubated at 30 C with shaking. After 14 h, 10 ml
of each
culture were collected and mixed immediately with stop solution (0.2 volume of
95:5
ethanol-phenol). Samples were centrifuged for 10 min at 4 C. Bacterial pellets
were
frozen at -80 C until lysis. For RT-PCR experiments, culture conditions were
identical to those for qRT-PCR experiments, except that S. avermitilis MA-4680
was
grown for 24 h in M14M supplemented with 1% mannitol or 0.2% GIcN and 0.8%
chitosan oligomers.
CA 02767534 2012-02-09
- 35 -
For qPCR and RT-PCR experiments, total RNA extraction was carried out with the
Qiagen RNeasyTM minikit (Qiagen), with the following modifications. Cell
disruption
was achieved by sonication with two 30-s bursts at 35% amplitude separated by
a
15-s cooling period. Sonication was followed by two phenol-chloroform
extractions
and one chloroform extraction for elimination of cell debris. The on-column
DNase
treatment was done with the RNase-free DNase set (Qiagen). Additionally,
another
DNase digestion was done after RNA elution with the Turbo DNAfreeTM kit
(Ambion).
RNA purity and concentration were assessed in a NanoDropTM 1000
spectrophotometer (Thermo Scientific). RNA quality was verified by
electrophoresis
on agarose gel in MOPS (morpholinepropanesulfonic acid) electrophoresis buffer
with 0.22 M formaldehyde. Reverse transcription was performed on 2 pg of total
RNA
with the first-strand cDNA synthesis kit (GE Healthcare) and 72% G+C-rich
random
hexamers.
Quantitative PCRs were performed in an Mx3000PTM real-time FOR system
(Stratagene). PCR mixtures (20 pl) contained 2 pl of 20x diluted template
cDNA, 250
nM the appropriate primer (see Table 4), and a SYBRTM green FOR mix. The PCR
conditions were 95 C for 3 min, followed by 40 cycles at 95 C for 15 s, 60 C
for 45 s,
and 72 C for 15 s. An additional dissociation step (95 C for 1 min, 60 C for
30 s, and
95 C for 30 s) was added to assess nonspecific amplification. PCRs were run in
triplicate. The absence of genomic DNA was verified by using samples in which
the
reverse transcriptase was omitted from the cDNA synthesis reaction. The gyrA
and
rrn genes of S. lividans (encoding gyrase A and 16S rRNA, respectively) were
used
as internal controls for relative quantification. Efficiencies of all primer
pairs were
verified. Raw data were transformed into threshold cycle (CT) values. Relative
gene
expression was calculated by the comparative CT method (Pfaffl, 2001) for each
strain incubated in the GIcN-chitosan oligomer medium compared to the mannitol
medium.
Table 4. Set of primers used for qRT-PCR experiments
Expected
Gene SEQ
Primer sequence (5'¨>3') fragment
symbol/annotation ID NO:
size (bp)
ACCGGTACATCGAGGACATCGG 46
csnA 139
AGATAGGGCGCGAGGACGTT 47
csnR GGTCGAGTACGAGAACGACGTGAA 96 48
CA 02767534 2012-02-09
- 36 -
Expected
Gene SEQ
Primer sequence (5'¨>3') fragment
symbol/annotation ID NO:
size (bp)
TGGTTCCACAGCAGGACGAAGT 49
GAACTACCACGGCTACGAGACC 50
csnE 182
TGTTGCGGTACTTCTCCAGCTTCT 51
CCTCCTACTACCTGCGCTACTACT 52
csnH 112
ATCTGCAGCAGTTGCCGTTCCAT 53
GCCGAGAACTCGACCACGAAGT 54
csnB 136
TGTAGCGCTCGACCAGCATGA 55
ACCACCATCGCGAAGCTCAA 56
SPPG 04866 139
GCATCTCCTTCTGCATCTTCTCGT 57
GCGACGACCGCAAGACCAAGCTGAT 58
gyrA 84
TGACGACGATGTCCTCCTCGGCGAT 59
TCTGGGCCGATACTGACGCTGAGGA 60
rm 105
ATGTTGCCCACACCTAGTGCCCACC 61
For RT-PCR experiments with S. avermitilis MA-480, an endpoint FOR followed
reverse transcription reactions. FOR mixtures (20 pl) contained 1 pl of 10x
diluted
template of cDNA, 2 pM each primer (see Table 5), 10% dimethyl sulfoxide
(DMSO),
lx ThermoPol buffer, and 250 pM deoxynucleoside triphosphates (dNTPs). The PCR
conditions were 95 C for 3 min, followed by 35 cycles at 95 C for 30 s, 58.5
to
66.4 C (depending on the set of primers used) and 72 C for 30 s, with a final
elongation step at 72 for 10 min. The rpsl gene of S. avermitilis (encoding
the 30S
ribosomal protein S9) was used as an internal control.
Table 5. Sets of primers used for PCRs in the RT-PCR experiment.
Expected
Gene SEQ
Primer sequence (5'¨>3') fragment
symbol/annotation ID NO:
size (bp)
TTCCAGGTGCCGTGGTGGTA 62
SAV_1223 266
AGCCAGTCGATCCAGCCCAT 63
CACCAGCTTCAGCAGCATCCG 64
SAV 2015 245
AGCCGATGTGGTAGCTGTCCC 65
GCCATGAAACGTGCCGCTCT 66
SAV6191 229
_
GCCAGTCCAGGGTGGAGTTCT 67
CA 02767534 2012-02-09
- 37 -
Expected
Gene SEQ
Primer sequence (5'¨>3') fragment
symbol/annotation ID NO:
size (bp)
AATGCCGAGACCCTGCCGTA 68
SAV 1288 284
ACGTGGTTCTCGATGGGCGA 69
TTCCACCAGTCCGACGGCAA 70
SAV1850 299
_
ATGGGCGAGACCTGCGAGTT 71
GCAGGAAGTCAACGAGCCCTTC 72
rps1 290
CTTGCTGTACTGCGGGGCCTT 73
Results
Identification of a candidate gene regulating chitosanase expression.
Palindromic
sequences of similar lengths and sharing a high level of identity have been
previously
described in the upstream segments of several endo- and exochitosanase genes
from actinomycetes. By EMSA experiments, a DNA-protein interaction between a
protein present in partially purified extracts from Kitasatospora sp. N106 and
a
double-stranded oligonucleotide probe covering the palindromic sequence was
characterized. A BLAST search with this sequence as the query returned
numerous
hits, mostly from intergenic regions of actinomycete genomes. Their partial
listing
was used in an alignment (Fig. 5) and yielded the AGGAAA(G/C)TTTCCTA (SEQ ID
NO: 88) consensus.
The palindromic sequence was found in front of two categories of genes: those
encoding studied or putative chitosanases from various families (including the
csnA
gene, SSPG_06922, from S. lividans) and genes encoding putative
transcriptional
regulators, all belonging to the ROK family established by Titgemeyer etal.
(1994):
among these are S. lividans gene SSPG_04872, localized at map coordinate 5.42
Mb at a 2.2-Mb distance from csnA. The protein encoded by this gene as a
possible
candidate for a transcriptional regulator of chitosanase gene expression
(CsnR) was
examined.
Purification of CsnR. CsnR was overproduced with a GST tag. The majority of
the
recombinant protein was detected in inclusion bodies. Attempts to renaturate
the
insoluble protein were not successful as precipitation during dialysis
occurred.
Purification was then attempted with the soluble portion of the lysate. A
major protein
contaminant (-60 kDa) copurified with GST-CsnR (Fig. 6). This protein was
identified
by partial sequencing as the chaperone GroEL, known to contaminate several
CA 02767534 2012-02-09
- 38 -
recombinant proteins from E. coli during purification. The soluble lysate was
incubated with 2 mM ATP and 5 mM MgC12 before the affinity purification step.
This
additional step was helpful in eliminating the contaminant (Fig. 6). After an
additional
size exclusion chromatography step, essentially pure CsnR was obtained, as
shown
by SOS-PAGE followed by silver nitrate staining (Fig. 6).
CsnR binds in vitro to the palindromic sequences upstream of csnA and csnR. As
determined by DNase I footprinting, CsnR binds asymmetrically to the
palindromic
box found in the promoter region of csnA, covering 15 nucleotides upstream and
12
nucleotides downstream from the palindrome axis (Fig. 7). CsnR binds in a
similar
way to the palindromic box in the promoter region of its own gene, covering 17
nucleotides upstream and 12 nucleotides downstream from the axis (Fig. 7). As
determined by primer extension, the protected region superimposes to the
transcriptional start site in csnR gene (Fig. 7B). Despite several attempts,
the
transcription start site of csnA could not be determined.
Oligonucleotide probes corresponding to the longest protected segment (17-1-
12)
were used to characterize the CsnR-DNA interaction by EMSA. The KO values were
0.032 nM (standard error [SE] = 0.009) and 0.040 nM (SE = 0.008) for the
operators
of csnA and csnR, respectively (see Fig. 8). It appears that CsnR binds to the
operators of the chitosanase gene as well as its own gene with similar
affinity.
On the basis of our previous experiments performed in vitro with partly
purified
protein extracts from Kitasatospora sp. N106 and mutated oligonucleotides
representing the operator of the chitosanase N106 gene, it was hypothesized
that
nucleotides at the -2 and +2 positions were critical for binding, while
positions -7,
-6, +6, and +7 were of moderate importance. Accordingly, annealed double-
stranded
oligonucleotides corresponding to the CsnR target sequence mutated by
transversion
at positions +2 and/or +6 of the palindrome were used in equilibrium
competition
experiments against a labeled csnA-WT probe (Table 6). The effect of mutations
on
binding was estimated from IC50s. The mutation at the +2 position was
particularly
deleterious for binding (Table 6). Mutation at the +6 position had a lesser
effect, and
the double mutation seemed to bring a cooperative effect. The doubly mutated
oligonucleotide lost most of its affinity for the CsnR protein. This suggests
a similarity
between the DNA binding mechanism of CsnR from S. lividans and that of the
putative chitosanase gene regulator from Kitasatospora sp. N106.
CA 02767534 2012-02-09
- 39 -
Table 6. Effect of mutations in the operator sequence on CsnR binding
evaluated by
equilibrium competition experiments
Sequence (5'--33)(a) IC50(b) __ SE of
Name
-6 -2 0+2 +6 (nM) logIC50(b)
csnA-WT CCTCTTCTGGTAGGAAACTTTCCTATCAGT 1.3 0.11
csnA-M1 CCTCTTCTGGTAGGAAACTGTCCTATCAGT 52.1 0.13
csnA-M2 CCTCTTCTGGTAGGAAACTTTCCGATCAGT 2.7 0.08
csnA-MM CCTCTTCTGGTAGGAAACTGTCCGATCAGT 105 0.33
aMutated nucleotides are in boldface.
b1050 and standard error of log 1050 values were determined using
Graph-Pad Prism software from data compilation of three independent
experiments.
DNA binding by CsnR is sensitive to the presence of chitosan oligomers.
Equilibrium
competition experiments were also used to determine the ability of various
carbohydrates to interfere with DNA binding of CsnR to operator sequence of
csnA.
At first, EMSA experiments with CsnR showed that 500 nM GIcN, chitosan dimer,
and chitosan pentamer strongly affected the gel shift pattern, while glucose
and
GIcNAc had no effect (Fig. 9). Then detailed 1050 determinations revealed that
the
chitosan dimer had the strongest effect on the displacement of CsnR from its
target,
having the lowest IC50 (18.2 nM; SE of log 1050 = 0.05), compared to the
chitosan
monomer, GIcN (977 nM; SE of log IC50 = 0.05). For higher oligomers, the I050
increased progressively with their length: 30.6 nM (SE of log I050 = 0.08) for
trimer,
37.3 nM (SE of log 1050 = 0.08) for tetramer, and 154 nM (SE of log IC50 =
0.080) for
pentamer. CsnR seems to bind specifically the products of chitosan degradation
by
chitosanases, as the other tested sugars (glucose, galactosamine, and GIcNAc)
do
not interfere with CsnR binding to the csnA-WT probe even at the maximal
tested
concentration of 75 mM in binding reactions. When undersaturating
concentrations of
chitosan oligosaccharides were present in the binding reaction mixtures, a
band of
intermediate mobility appeared in EMSA gels (Fig. 9 and see Fig. 10),
reflecting the
progressive disassembly of the multimeric complex of CsnR with its DNA target.
CsnR regulates negatively the transcription of csnA and of a gene cluster led
by
csnR. Close examination of the genomic sequence of S. lividans reveals that
csnR is
localized in a gene cluster composed of six genes (Table 7 and see Fig. 11).
The
functions putatively assigned to these genes indicate that the cluster could
be
CA 02767534 2012-02-09
- 40 -
dedicated to sugar transport and metabolism (Table 7). The intergenic regions
between these genes are very short (the longest region of 106 bp being
localized
between csnR and csnE), while a much larger region containing a possible
transcription terminator consisting of a 14-bp inverted repeat, separates csnK
from
the following gene, SSPG_04866 (see Fig. 11).
Table 7. Components of csnR-led gene cluster in S. lividans.
Gene annotation Gene symbol Putative function
SSPG_04872 csnR ROK-family transcriptional regulator
SSPG_04871* csnE Secreted sugar binding protein
SSPG_04870* csnF Sugar transport system permease
SSPG_04869* csnG Sugar transport membrane protein
SSPG_04868 csnH Glycoside hydrolase, family GH4
SSPG_04867 csnK Sugar kinase
'Names were adopted as described by Bertram et al. (2004).
bThese three genes determine a putative ABC transporter.
To get more insight into the regulatory mechanism of CsnR, the transcript
abundance
of various genes was evaluated by qPCR in both wild-type and AcsnR strains
(Table
8). A mutant strain in which the deletion has been complemented by a wild-type
copy
of the csnR gene (including its entire 211-bp upstream IR) on an integrative
vector
(S. lividans AcsnR + pSETmC) was examined (Table 8). Data were collected from
cultures growing in control medium with mannitol or in M14M with chitosan-
derived
carbon sources. There was no detectable csnR expression in either the AcsnR
strain
or the control complementation strain (S. lividans AcsnR + pSET152m strain),
confirming the deletion genotype. It was found that the expression of csnR
itself was
induced more than 100x by chitosan-derived saccharides (Table 8), but,
surprisingly,
the induction ratio was very low in the complementation strain when csnR was
introduced in a different genomic location with the integrative plasmid
pSETmC. This
appeared to be due to unexpectedly high csnR transcript abundance in the
mannitol
medium (Table 8). In other words, CsnR failed to autorepress transcription
when its
own gene (including its operator) was carried by the integrated pSETmC vector.
Table 8. Effect of the AcsnR mutation and its complementation on transcript
abundance patterns of chitosan-related genes in S. lividans strains.
Results are shown as mean SE relative abundance and induction ratioa
Tested genes
S. lividans
csnA csnB csnR
csnE csnH SSPG 04866
strains
M SE M SE M SE M
SE M SE M SE
C(a) 0.096 0.0032 0.023 0.0017 0.0055 0.0022 0.0011 , 0.000033 0.26
0.0029 0.097 0.0049
TK24 1(a) 4.2 0.67 0.070 0.0054 0.86 0.35 1.6
0.36 2.0 0.23 0.35 0.063
R(b) 43 3.0 156 1398
78 3.6
0
C 9.4 0.86 0.16 0.019 1.5
0.17 1.5 0.14 0.58 0.079 0
1.)
..,
AcsnR I 7.0 0.51 0.30 0.022 ND 2.3
0.39 2.4 0.26 0.60 0.020 0,
..,
R 0.74 1.9 1.5
1.7 1.0 .p.
0.
.
1.)
0
C 0.13 0.0073 0.034 0.0068
0.14 0.018 0.0016 0.000088
0.025 0.0022 0.15 0.023
1.)
AcsnR +
1
I 3.0 0.29 0.054 0.0046 0.42 0.14
1.2 0.20 2.5 0.17 0.36 0.048 0
1.)
i
pSETmC
_______________________________________________________________________________
_______________________________________________ 0
R 23 1.6 3.0 734
100 2.4 ko
C 1.6 0.26 0.067 ' 0.0050 0.96
0.089 2.0 0.27 0.11 0.019
AcsnR +
I 0.77 0.15 0.026 0.0041 ND 1.4
0.077 2.8 . 0.15 0.056 0.017
pSET152m
R 0.47 0.40 1.4
1.4 0.51
aThe values shown are expressed as the relative transcript abundance in the
control medium or in induction medium normalized to the transcript
abundance of the gyrA gene. Similar values were obtained after normalization
to the expression level of rm (data not shown). The values shown are
means SEs of three independent cultures with a culture time of 14 h.
Induction ratios represent the ratio of transcript abundance in the induction
medium to that in control medium. ND, not determined. b R (induction ratio) is
the ratio of transcript abundance in the induction medium to that in
control medium.
CA 02767534 2012-02-09
- 42 -
For the chitosanase gene, csnA, a 43-fold induction ratio was observed (Table
8).
Complete derepression of csnA expression was observed in the AcsnR strain, and
repression was restored by complementation (Table 8). A similar CsnR-dependent
expression pattern was observed for genes csnE and csnH localized inside the
cluster (Table 8). It is thus probable that this cluster forms a polycistronic
transcription unit negatively regulated by CsnR.
A much higher induction ratio was however observed for csnE than for csnR and
csnH (Table 8). The intergenic region between csnR and csnE was sequenced and
it
was observed that it includes four direct repeats (see Fig. 12), a possible
site of a
regulatory interaction.
Transcript abundance of SSPG_04866, the gene following csnK, did not follow a
CsnR-dependent pattern (Table 8). SSPG_04866 putatively encodes a secreted
protein of unknown function and does not seem to belong functionally to the
csnR to -
K gene cluster. An extensive inverted repeat localized in the IR following
csnK could
function as a transcription terminator (see Fig. 11).
While this work provides evidence that CsnR is the transcriptional repressor
of the
chitosanase gene csnA, the uniform expression pattern observed for its
homolog,
csnB (SSPG_05520), indicated that csnB is not regulated by CsnR (Table 8).
This
was somewhat expected, as the palindromic sequence recognized by CsnR was not
found in the genomic environment of csnB.
The CsnR-mediated regulatory mechanism is widespread in actinobacteria. After
the
identification of the csnR to -K gene cluster in S. lividans, a bioinformatic
search was
performed to establish if similar gene clusters are present in other fully or
partly
assembled genomes. So far, orthologs of CsnR with no less than 46% identity
have
been found in 23 genomes of actinobacteria, and the presence of a highly
similar
gene cluster of six genes has been confirmed in 12 genomes, including
actinobacteria other than Streptomycetes, such as Saccharopolyspora erythraea
NRRL2338, Streptosporangium roseum DSM43021, and Kribbella flavida
D5M17836. Table 9 shows the cluster annotation in some streptomycete species
in
which a palindromic box corresponding to the CsnR consensus presented in Fig.
5 is
present upstream of the gene cluster. All the putative chitosanase genes
belonging to
well-established glycoside hydrolase (GH) families are also listed (Table 9).
While the
distributions of members of various GH families differ among the analyzed
species, it
is noteworthy that each genome includes at least one putative chitosanase gene
provided with a CsnR box.
Table 9. CsnR gene clusters and putative chitosanase genes in sequenced
Streptomyces genomes
Confirmed') or putative endo and exo-chitosanase genes
Species CsnR cluster
GH2 GH5
GH46 GH75
and strain components(a)
_______________________________________________________________________________
__________
Gene Box Gene
Box Gene Box Gene Box
Streptomyces SSPG 04872:
SSPG 06922(c) +
SSPG 00778 -
lividans TK24 SSPG 04867
SSPG 05520 -
Streptomyces SCO2657 :
SC00677(c) +
SC07070
-
coelicolor A3(2) SCO2662
SCO2024 - 0
Streptomyces
.
SAV5384 :
SAV2015 + SAV1850 +
_
"
..3
avermitilis MA- SAV1223(c) +
_ _
0,
,
..3
SAV5379 _
SAV_6191 - SAV 1288
_
.A.
w
4680
o.) 0.
'
IV
,
_______________________________________________________________________________
_____________________________________________________ 0
Streptomyces
SCAB 59491 :
1.)
1
scabies SCAB 86311 +
SCAB 83781 - 0
1.)
'
SCAB 59441
0
87.22
ko
Streptomyces
SGR 4874:
griseus SGR 1341m +
SGR 1238 -
SGR 4869
IFO 13350
SSEG 04515:
Streptomyces
SSEG 04514
SSEG 02093 -
sviceus
SSEG 10562 -
SSEG 09506:
SSEG 10482 +
ATCC 29083
SSEG 09503(b)
Confirmed M or putative endo and exo-chitosanase genes
Species CsnR cluster
GH2 GH5
GH46 GH75
and strain components(a)
_______________________________________________________________________________
__________
Gene Box Gene Box
Gene Box Gene Box
Streptomyces
SCLAV 1826:
clavuligerus SCLAV 5580 + SCLAV 4996 SCLAV 5034
SCLAV 1831
ATCC 27064
Streptomyces
SSDG 02817:
SSDG 00156
pristinaespiralis SSDG 05015 +
SSDG 04141 -
SSDG 02822
SSDG 03879
ATCC 25486
0
1.)
'All clusters begin with a csnR ortholog having the palindromic box in the
upstream segment.
bThese six genes form an uninterrupted cluster even if their numbers do not
follow each other.
bThese genes encode enzymes the identities of which as chitosanases have been
confirmed by biochemical studies. 1.)
0
1.)
0
1.)
0
CA 02767534 2012-02-09
- 45 -
Among the analyzed species, S. avermitilis stands out for its highest number
of
putative chitosanase genes, belonging to three different families. The
transcriptional
behavior of all these putative chitosanases in the absence or the presence of
chitosan oligomers was thus compared by endpoint RT-PCR. Induction with
chitosan-derived oligosaccharides was observed only for the three putative
chitosanase genes having a CsnR-type operator (Fig. 13).
Our data indicate that the regulatory mechanism mediated by CsnR is an
evolutionary ancient mechanism of chitosanase gene regulation present in many
actinobacteria and not limited to the GH46 family in which it was discovered,
but
extends to other chitosanase families as well.
Discussion
This work describes the identification and characterization of CsnR, a novel
chitosanase gene regulator in bacteria and also the first characterized
transcriptional
regulator of the ROK family in actinobacteria. DNase footprinting and EMSA
experiments demonstrated that CsnR binds directly to the palindromic box found
upstream from the csnA and csnR gene cluster. This binding target is different
from
the operators characterized for other transcriptional regulators belonging to
the ROK
family. The CsnR box is tightly organized around the symmetry axis, the
positions -2
and +2 being most critical for binding. In contrast, the operator consensus
sequences
of NagC and Mlc of E. coli and XylR of Firmicutes are essentially composed of
two
NT-rich inverted repeats separated by a 5- to 9-bp spacer with strictly
conserved
positions 6 and 5. Equilibrium competition experiments showed that (GIcN)2
is the
preferential CsnR ligand molecule. This dimer is the major product obtained
from
chitosan hydrolysis by endochitosanases. Oligosaccharide products resulting
from
the hydrolysis of polymers catalyzed by endohydrolases were often described as
the
effectors for the transcriptional regulation of glycoside hydrolase genes in
actinomycetes. Cellopentaose is the inducer of CebR, the repressor of cell in
Streptomyces reticuli. Maltopentaose is the inducer of MaIR, the repressor of
genes
coding for a-amylase in Streptomyces coelicolor A3(2). Direct estimation of
transcript
abundance with qPCR (Table 8) showed that CsnR is subject to autorepression.
It
was previously showed that a system controlled by negative autoregulation
offers the
advantage of faster response to the presence of inducer molecules over a
system
controlled by an open loop regulator. Also, negative autoregulation ensures a
more
homogenous distribution of the steady-state level of the repressor between
cells in a
population. In S. lividans, S. coelicolor A3(2), and several other
actinobacteria, CsnR
CA 02767534 2012-02-09
- 46 -
is localized at the beginning of a gene cluster (csnREFGHK) including an ABC
transporter, a glycoside hydrolase, and a sugar kinase. Previously, close
orthologs of
csnEFG in the S. coelicolor A3(2) genome (SCO2658 to SCO2660; localized on the
SC6D10.01 cosmid) have been described and found by in silico analysis of
carbohydrate uptake systems. Trehalose, maltose, and lactose were cited as
possible substrates for this uptake system. Our study indicates that the
transcription
of genes localized in this cluster is induced by chitosan oligosaccharides and
that
they share a negative regulatory mechanism with the chitosanase gene, csnA. It
is
suggested that this cluster represents an operon-like structure involved in
the uptake,
transport and intracellular metabolism of oligosaccharides resulting from the
hydrolysis of chitosan (or N-deacetylated segments of chitin) by chitosanases.
As
shown in Table 9, gene clusters highly similar to csnREFGHK of S. lividans
were
found in several other actinobacterial genomes.
EXAMPLE III ¨ OPTIMIZATION OF CHITOSANASE PRODUCTION
Spores of S. lividans AcsnR strain harboring a heterologous chitosanase gene
(as
described in Example I) were inoculated into tryptic soy broth (ratio of 2x109
spores
per 100 ml of broth) and incubated for 64 h at 30 C with shaking (250 rpm) to
obtain
a dense pre-culture. A small volume (50 ml) of this pre-culture was
centrifuged (10
min at 3000xg) in order to measure the pellet volume equivalents. This dense
pre-
culture was used to inoculate directly the chitosanase production medium in a
ratio of
four ml of pellet volume equivalents per 100 ml of chitosanase production
medium.
This culture was incubated (30 C, 300 rpm) for further 72 ¨ 96h. Chitosanase
and
protease activities as well as total protein concentration were determined (as
described in Example I) in culture supernatant.
The chitosanase production medium contained, for 1000 ml, 20 g of malt
extract, 4 g
KH2F04, 22 g K2HPO4, 5.6 g (NH4)2SO4 and distilled H2O. The pH was adjusted to
6.9 and the volume to 975 ml. The chitosanase production medium was autoclaved
for 15 min. Before use, 25 ml of MgSO4 5% in distilled water (0.22 pm filtered
sterilized) was aseptically added.
CA 02767534 2012-02-09
- 47 -
Table 10. Chitosanase production characterization in function of time.
Time (h) Chitosanase Protease Total proteins
(U/m1) (U/m1) (pg/ml)
24 0.6 nd* nm
48 4.1 nd nm
72 14.9 nd 421.9
96 25.1 6.3 623.2
(*)nd: activity not detected (under detection limit)
(**)nm: not measure
REFERENCES
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While the invention has been described in connection with specific embodiments
thereof, it will be understood that it is capable of further modifications and
this
application is intended to cover any variations, uses, or adaptations of the
invention
following, in general, the principles of the invention and including such
departures
from the present disclosure as come within known or customary practice within
the
art to which the invention pertains and as may be applied to the essential
features
hereinbefore set forth, and as follows in the scope of the appended claims.
