NZ625199B2 - Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides - Google Patents
Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides Download PDFInfo
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- NZ625199B2 NZ625199B2 NZ625199A NZ62519912A NZ625199B2 NZ 625199 B2 NZ625199 B2 NZ 625199B2 NZ 625199 A NZ625199 A NZ 625199A NZ 62519912 A NZ62519912 A NZ 62519912A NZ 625199 B2 NZ625199 B2 NZ 625199B2
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- RPKLZQLYODPWTM-KBMWBBLPSA-N cholanoic acid Chemical compound C1CC2CCCC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@@H](CCC(O)=O)C)[C@@]1(C)CC2 RPKLZQLYODPWTM-KBMWBBLPSA-N 0.000 title claims abstract description 70
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- LQEBEXMHBLQMDB-UHFFFAOYSA-N [[5-(2-amino-6-oxo-3H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] (3,4,5-trihydroxy-6-methyloxan-2-yl) hydrogen phosphate Chemical compound OC1C(O)C(O)C(C)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C3=C(C(N=C(N)N3)=O)N=C2)O1 LQEBEXMHBLQMDB-UHFFFAOYSA-N 0.000 claims abstract description 37
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
Abstract
Disclosed is the use of a modified expression of the transcriptional regulators; the aerobic respiration control protein ArcA and the isocitrate lyase regulator IclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl that code for a phosphomannomutase, a mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase and GDP-fucose synthase, respectively. Further disclosed is a process for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising: genetically changing the transcriptional regulators, the aerobic respiration control protein ArcA, and the isocitrate lyase regulator IclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl. Further disclosed is a mutated and/or transformed bacterium in which the regulators ArcA and IclR, in combination with the genes encoding for the enzymes phosphoglucose isomerase and phosphofructokinase, are knocked out or are rendered less functional. Further disclosed is the use of a modified expression of the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase regulator IclR, to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gada, gadC, gadE, gadX, gadWand/or slp. hat code for a phosphomannomutase, a mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase and GDP-fucose synthase, respectively. Further disclosed is a process for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising: genetically changing the transcriptional regulators, the aerobic respiration control protein ArcA, and the isocitrate lyase regulator IclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl. Further disclosed is a mutated and/or transformed bacterium in which the regulators ArcA and IclR, in combination with the genes encoding for the enzymes phosphoglucose isomerase and phosphofructokinase, are knocked out or are rendered less functional. Further disclosed is the use of a modified expression of the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase regulator IclR, to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gada, gadC, gadE, gadX, gadWand/or slp.
Description
MUTANT RGANISMS TO SYNTHESIZE COLANIC ACID, MANNOSYLATED
AND/OR LATED OLIGOSACCHARIDES.
FIELD OF THE INVENTION
The present invention relates to mutated and/or transformed microorganisms for the
synthesis of various compounds. More specifically, the present invention discloses
microorganisms mutated in the genes encoding for the regulators ArcA and lclR. The
latter mutations result in a significant upregulation of the genes that are part of the colanic
acid operon. Hence, said microorganisms are useful for the synthesis of any compound
being part of the colanic acid pathway such as GDP-fucose, GDP-mannose and colanic
acid, and/or, can be further used — starting from GDP—fucose as a precursor — to
synthesize fucosylated oligosaccharides or - ng from GDP~mannose as a precursor—
to synthesize mannosylated oligosaccharides. In addition, mutations in the genes coding
for the transcriptional regulators ArcA and lclR lead to an acid resistance phenotype in the
ntial growth phase ng the sis of pH sensitive molecules and organic
acids.
OUND OF THE INVENTION
The genes arcA encoding for the aerobic respiration control protein and ic/R encoding the
2O rate lyase regulator are known to regulate the central carbon metabolism. ArcA is a
global transcriptional regulator that regulates a wide variety of genes, while lclR is a local
riptional regulator that regulates the glyoxylate y. ArcA is known to regulate
the central carbon metabolism in response to oxygen deprivation and has no connection
with lclR other than that it also regulates the glyoxylate pathway (24, 28, 29, 37, 38). In
an earlier study the combined effect of AicIRAarcA mutant strains on the central carbon
metabolism has been observed. Increased fluxes were shown in the tricarboxylic acid
(TCA) cycle and late pathway and an interesting and surprising phenotype
appeared when both genes where knocked out, namely the double mutant strain formed
biomass with a yield that approached the maximal theoretical yield (4, 39).
Some compounds, such as GDP-fucose, are in high . The latter compound is
indeed a precursor of fucosylated oligosaccharides such as llactose,
fucosyllactoNbiose and lewis X oligosaccharides, or, of fucosylated proteins. These
sugars are components of human mother milk in which they have anti-inflammatory and
prebiotic effects and/or have applications in therapeutics as nutraceutical, anti—
inflammatory agent or tic, in addition, fucosylated proteins find applications in the
pharmaceutics (5, 8, 27). However, an ent method to produce the latter high-value
nds is still .
In addition GDP-mannose is also an intermediate of the pathway towards GDP-fucose.
Interrupting the pathway prematurely leads to the accumulation of this compound, which is
a precursor of ylated oligosacharides. These oligosaccharides find for example
applications in the ent of gram—negative bacterial infections, in addition, GDP-
mannose is important for the humanization of protein glycosylations, which is ial for
the production of certain therapeutic proteins (18, 30). Mannosylated oligosaccharides
and mannosylated glycoconjugates are also used for drug targeting, for instance
mannosylated antivirals can specifically target the liver and kidneys (7).
The present invention provides microorganisms which are genetically d in such a
manner that they can efficiently produce the latter compounds.
Moreover, the sis of pH sensitive molecules, such as — but not limited to -—
glucosamine, and organic acids, such as —— but not limited to — pyruvic acid, succinic acid,
adipic, sialic acid, sialylated oligosaccharides... are preferably produced at low pH, either
to stabilize the product or for downstream processing reasons (4, 12, 40). Therefore,
strains that can grow at low pH are cial for these production processes. E. coli is an
organism that can adapt easily to various conditions, for instance it can easily adapt to the
2O harsh pH conditions in the stomach, which is about pH 2 (14). Nonetheless, E. coli does
not seem to grow at these conditions, but induces its acid resistance mechanisms in the
nary phase (40). During this phase the cell does not multiply anymore and therefore
s productivity. Up to now, no solution was found to this problem. However, in the
present invention, a genetically engineered microorganism is provided that can induce
acid resistance mechanisms in the exponential growth phase, which is the phase that is
mostly used for production of organic acids and pH instable products.
Y OF THE INVENTION
in one aspect of the present invention, there is provided a use of a modified expression of
the transcriptional regulators; the aerobic respiration l protein ArcA and the
isocitrate lyase regulator lclR, to upregulate at least one of the genes of the colanic acid
operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fol that code for
a phosphomannomutase, a mannose-l-phosphate guanylyltransferase, GDP-mannose
4,6-dehydratase and GDP-fucose synthase, respectively.
[followed by page 2a]
In a further aspect of the present invention, there is provided a s for the synthesis
of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising:
genetically changing the transcriptional regulators, the aerobic ation control protein
ArcA, and the isocitrate lyase regulator lclR, to upregulate at least one of the genes of the
colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl.
In a further aspect of the present ion, there is provided a mutated and/or
transformed bacterium in which the regulators ArcA and lclR, in combination with the
genes encoding for the enzymes phosphoglucose isomerase and phosphofructokinase,
are knocked out or are rendered less functional.
In a r aspect of the present invention, there is provided a use of a modified
expression of the transcriptional regulators the aerobic respiration control protein ArcA
and the isocitrate lyase tor lclR, to upregulate at least one of the following acid
resistance related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadW
and/or slp.
in a further aspect of the present invention, there is provided a process for the synthesis
of acids, sialic acid, sialylated oligosaccharides or glucosamine comprising genetically
changing the transcriptional regulators the aerobic respiration control protein ArcA and the
rate lyase regulator lclR to upregulate at least one of the following acid resistance
related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, d/or slp.
BRIEF PTION OF FIGURES
Figure 1: Relative gene sion pattern of the wild type, the AicIR and AarcA mutant
strain to the AaroAAic/R mutant strain of genes involved in colanic acid thesis in
batch fermentation conditions. The genes involved in colanic acid biosynthesis are
presented in Figures 3 and 4.
[followed by page 3]
WO 2013087884
Figure 2: Gene expression pattern of the coianic acid operon of the wild type, the Aic/R
and AarcA mutant strain in chemostat fermentation conditions relative to the AaroAAic/R
mutant strain.
Figure 3: The gene organisation of the colanic acid operon and an overview of the
function of these genes:
Gene: Function:
Colanic acid polymerase
gmm nnose hydroiase
wcal Glycosyitransferase
Mannose~1—phosphate guanyiyltransferase
Glycosyltransterase
ted protein in colanic acid biosynthesis
Figure 4: The colanic acid biosynthesis pathway.
Figure 5: Regulatory network of the colanic acid . This network was constructed
with Pathway tools v 13.0.
Figure 6: Effect of the AarcAAic/R mutations on the GDP—fucose biosynthesis route.
Figure 7: Overview of the genetic modifications needed to enhance fucosyllactose and
fucosylated accharides production starting from glucose as a substrate.
Figure 8: Starting from sucrose, fucosylated sugar derivates such as fucosyllactose and
more specifically 1,2-fucosyllactose are produced. The strain is ed to force the cell
to produce frucose-B-phosphate which is an intermediate in the synthesis of GDP-fucose.
Glucose or glucose-t-phosphate (if the starting enzyme is either a sucrase or a sucrose
phosphorylase) is then fed to the central carbon metabolism via the pentose phosphate
pathway.
Figure 9: Overview of the c modifications needed to enhance fucosyllactose and
fucosylated oligosaccharides production starting from glucose as a substrate in a split
metabolism.
Figure 10: Detail of the pentose phosphate y flux in a strain in which the genes
coding for phosphoglucose isomerase and phosphofructokinase are d out.
Figure 11: Starting from sucrose, mannosylated sugar derivates are ed. The strain
is modified to force the cell to produce frucose-G-phosphate which is an intermediate in
the synthesis of GDP—fucose. Glucose or glucose—i—phosphate (if the starting enzyme is
either a sucrase or a sucrose phosphorylase) is then fed to the central carbon metabolism
via the pentose phosphate pathway.
Figure 12: Gene sion pattern acid resistance related genes of the wild type, the
Aid]? and AarcA mutant strain in batch culturing ions relative to the AaroAAic/R
mutant .
Figure 13: LC MSMS analysis chromatograms of culture broth and a 2-fucosyllactose
standard. A. LC MSMS analysis of the standard, B. LC MSMS analysis of a sample of the
culture broth of a mutant strain expressing a H. pylori fucosyltransferase, C. LC MSMS
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analysis of a sample of the e broth of a mutant strain expressing a H. pylori
fucosyltransferase.
Figure 14: LC MSMS analysis mass spectrum from the chromatograms shown in Figure
13 of culture broth and a 2-fucosyllactose standard. A. Mass (m/z) of the rd, B.
Mass (m/z) of the sample of the culturing broth of a mutant strain expressing a H. pylori
fucosyltransferase, C. Mass (m/z) of the sample of the ing broth of a mutant strain
expressing a H. pylori fucosyltransferase.
Figure 15: The sequence of the artificial hybrid promoter as given by SEQ ID N° 6 (the
combination of the native and an artificial promoter) that was cloned in front of the colanic
acid operon.
DESCRIPTION OF INVENTION
The present ion provides microorganisms such as Enterobacteriaceae which are
genetically changed in such a manner that they can ntly produce compounds which
are part of the colanic acid pathway. A particular compound of interest is GDP-fucose
which is used as a sor to synthesize fucosylated oligosaccharides. The latter have
health-promoting effects as indicated above but there is no efficient production method
available to produce said compounds.
The present invention thus provides for the usage of a d and/or ormed
microorganism comprising a genetic change leading to a modified expression and/or
activity of the transcriptional regulators the aerobic respiration control protein ArcA and the
isocitrate lyase regulator lclR to upregulate at least one of the genes of the colanic acid
operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fa! that code for
a phosphomannomutase, a mannose-t-phosphate guanylyltransferase, GDP-mannose
4,6—dehydratase and GDP-fucose synthase, respectively. The latter operon may also
comprise the genes cpsG, cpsB, gmd, fcl and wza. In addition the expression of the gene
rcsA is increased. This gene is a transcriptional regulator of the colanic acid operon.
Enhanced expression of this gene increases transcription of the colanic acid operon (13,
36).
Hence the present invention relates to the usage of a mutated and/or transformed
microorganism comprising a genetic change leading to a modified sion and/or
activity of the transcriptional regulator, the aerobic ation control n, ArcA and
the isocitrate lyase regulator lclR to upregulate the riptional regulator of the coianic
acid operon, rcsA, which in turn upregulates at least one of the genes of the colanic acid
Hence, the present invention relates to a mutated and/or ormed microorganism such
as ~but not limited to Enterobacteriaceae such as an Escherichia coli (E. coli) strain
comprising a genetic change leading to a ed expression of the transcriptional
regulators: the aerobic respiration control protein ArcA and the isocitrate lyase regulator
lclR.
A mutated and/or transformed microorganism such as E. coli as used here can be
ed by any method known to the person skilled in the art, including but not d to
UV mutagenesis and chemical mutagenesis. A preferred manner to obtain the latter
microorganism is by disrupting (knocking-out) the genes (arcA and fell?) ng for the
proteins ArcA and lclR, or, by replacing the nous promoters of said genes by
artificial promoters or replacing the endogenous ribosome binding site by an artificial
ribosome binding site. The term ‘artificial promoters’ s to heterologous or non-natural
or in siiico designed promoters with known expression th, these promoters can be
derived from ies as described by Alper et al. (2005), Hammer 92‘ al. (2006), or De
Mey et al. (2007) (3, 11, 15). The term logous promoter refers to any promoter that
does not naturally occur in front of the gene. The term ‘artificial promoter’ may also refer to
promoters with DNA sequences that are combinations of the native (autologous) promoter
sequence with parts of different ogous or heterologous) promoter sequences as for
example shown further in the examples. ces of such cial promoters’ can be
found in databases such as for example partsregistry.org (6). The term ‘artificial ribosome
binding site’ relates to heterologous or non—natural or in silico designed ribosome binding
sites with known or measurable translation rates, these libraries can be derived from
libraries or designed via algorithms as described by Salis at al (2009) (26). Hence, the
present invention specifically relates to a mutated and/or transformed microorganism as
indicated above wherein said genetic change is disrupting the genes encoding for ArcA
and lclR, or, reducing or eliminating the function of ArcA and lclR via mutations in the
coding sequence of the genes coding for ArcA and lclR , or, is replacing the endogenous
promoters of the genes encoding for ArcA and lclR by artificial promoters; or, is replacing
the endogenous ribosome binding site by an artificial me binding site. It is further
clear that the mutant and/or transformant according to the present invention
may r
comprise additional genetic changes in one or more other genes within its genome as is
also described further. The term microorganism specifically relates to a bacterium, more
specifically a bacterium belonging to the family of Enterobacteriaceae. The latter
bacterium preferably relates to any strain belonging to the species Escherichia coli such
W0 20131087884
as but not limited to ichia coli B, Escherichia coli C, Escherichia coli W, Escherichia
coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated
Escherichia coli strains — designated as E. coli K12 s — which are well-adapted to the
laboratory environment, and, unlike wild type strains, have lost their y to thrive in the
intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110,
MG1655, M182, MC1000, M01060, M01061, MC4100, JM101, NZN111 and AA200.
Hence, the present invention specifically relates to a mutated and/or transformed
Escherichia coli strain as indicated above wherein said E. coli strain is a K12 strain. More
specifically, the present invention relates to a mutated and/or transformed Escherichia coli
strain as indicated above wherein said K12 strain is E. coli M61655.
The terms ‘leading to a modified expression or activity’ tes that the above described
mutations/transformations affects the transcription and/or translation and/or post-
translational modification of said genes (arcA and iciR) into the transcriptional regulator
proteins of the t invention (ArcA and IclR) in such a way that the latter transcription
has significantly decreased or has even been completely abolished compared to a wild
type strain, which has not been mutated or transformed with regard to both particular
genes of the t invention. Hence, the present invention relates to a mutated and/or
transformed microorganism such as an Escherichia coli strain as indicated above n
said modified expression is a decreased expression, and, to a mutated and/or
transformed microorganism such as an Escherichia coli strain as indicated above wherein
said sed expression is an hed sion.
The terms ‘upregulating at least one of the genes of the colanic acid operon’ indicates that
the expression of at least 1, 2, 3, 4,..., or all of the genes belonging to the colanic acid
operon are significantly (= P > 0.05) upregulated when compared to the expression of said
genes within a corresponding wild type rganism which is cultivated under the same
conditions as the mutated and/0r transformed microorganism. The genes which belong to
the colanic acid operon are wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd,
fcl, gmm, wcal, cpsB, cpsG, wcaJ, wsz, wcaK, wcaL and wcaM as indicated in Fig. 3
and/or as described in (35). Furthermore, the gene rcsA, coding for the transcriptional
regulator of the c acid operon is lated (13, 36). More specifically the terms
‘upregulating at least one of the genes of the colanic acid operon’ or the transcriptional
regulator of the colanic acid operon indicates that at least one of the genes of the colanic
acid operon is 6 to 8 times upregulated in comparison to the expression of the genes of
the colanic acid operon in the corresponding wild type microorganism. in addition the
present invention relates to upregulating genes of the colanic acid operon as described
above by replacing the native promoter by an ‘artificial er‘. More specifically, the
present invention relates to a combination of the sequence of the native promoter with
sequences of other artificial promoter sequences. The ation of the sequence of the
native promoter with the sequence of other artificial promoter sequences is more
cally the replacement of the sigma factor binding site of the native promoter with a
stronger sigma factor binding site. Sigma factors, such as but not limited to sigma70,
sigmaS, sigma24,..., are described (41) , ts of RNA polymerase that determine the
affinity for promoter ces and the transcription rate of genes. The present invention
provides microorganisms which are cally changed in such a manner that they can
efficiently produce compounds which are part of the colanic acid pathway. The terms
‘compounds which are part of the colanic acid pathway’ refer to all compounds as
indicated on Figure 4 ng from fructose-G-P and resulting in extracellular colanic acid.
More specifically the latter terms refer to the nds mannose—6—P, mannose-i-P,
GDP-mannose, GDPdehydro-Bdeoxy—mannose, GDP-fucose and colanic acid. Hence
the present invention specifically relates to the usage as indicated for the synthesis of
colanic acid and/or for the synthesis of cose. As GDP-fucose is a precursor for
fucosylated oligosaccharides such as fucosyllactose, fucosyllactoNbiose and lewis X
accharide or fucosylated proteins, and as these sugars have therapeutical,
nutraceutical, anti-inflammatory and prebiotic effects, the t invention specifically
relates to the usage as described above for the sis of fucosylated oligosaccharides.
In other words, the present invention relates to a process for the synthesis of colanic acid
and/or GDP-fucose and/or fucosylated oligosaccharides comprising genetically ng
the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate
lyase regulator lclR to upregulate at least one of the genes of the c acid operon,
wherein said operon comprises the genes cpsG, cpsB, gmd and fc/ or genes cpsG, cpsB,
gmd, fcl and wza. More specifically, the present invention relates to a process as
described wherein the mutations for ArcA and lclR are applied in combination with at least
one mutation that enhances the production of fucosylated compounds. in order to
efficiently produce fucosylated oligosaccharides (see Figures 1, 2 and 5-10), the above
described ons in arcA and iclR can be applied in combination with other mutations
which further enhance the production of fucosylated compounds. Some of these — non-
limiting- other mutations are: a) the deletion of wcaJ from the colanic operon, stopping the
initiation of the colanic acid thesis and thus allowing the accumulation of GDP—
fucose; b) the introduction of a fucosyltransferase to link fucose with different acceptor
molecules such as e; c) for the accumulation of the precursor of the GDP-fucose
biosynthetic pathway and additional to the deletion of wcaJ, at least one of the following
colanic acid operon genes that do not code for cose biosynthesis is knocked out:
gmm, wcaA, wcaB, wcaC, wcaD, wan, wan, wcal, wcaJ, wcaK, wcaL, wzx, wza, wzb,
wzc, and/or, wcaM; d) for the production of fucosyllactose, lacZ coding for [3-
galactosidase, is knocked out to avoid lactose degradation; e) to accumulate the
precursor fructose and fructose-G-phosphate, a sucrose orylase or invertase is
introduced; f) because fructose-S-phosphate is easily degraded in the glycolysis, the
glycolysis has to be interrupted in order to steer all fructose-B-phosphate in the direction of
GDP—fucose and the genes pgi, pka and pka (coding for glucose—G—phosphate
isomerase and phosphofructokinase A and B) are thus d out; g) ng protein
ation by knocking out a protease coded by a gene such as Ion; h) By constitutively
expressing a lactose permease, subpopulations are avoided in the production process
which are common for e induced gene expression systems (19). ln other words, the
present invention s to a process as described above for the synthesis of fucosylated
oligosaccharides wherein said at least one on that enhance the production of
fucosylated compounds is: the deletion of the wcaJ gene, and/or, ng-out the colanic
acid operon genes gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK,
wcaL, wzx, wza, wzb, wzc, and/or, wcaM, and/or, knocking-out 1802, and/or, introducing a
sucrose phosporylase or invertase, and/or, knocking out the genes pgi, pka and ,0ka,
and/or, knocking out the gene Ion, and/or introducing a fucosyltransferase, and/or a
lactose permease. The term ducing a fucosyltransferase’ relates to upregulating or
heterologous expression of fucosyltransferases which are within, but not limited to the
enzymes in enzyme classes classes EC2.4.1.65, 2.4.1.68, 2.4.1.69, 2.4.1.152, 2.4.1.214,
and/or 2.4.1.221 and/or the glycosyltransferase families GT1, GT2, GT1O GT11, GT23,
GT37, GT65, GT68, and/or GT74 and/or originating from but not limited to Helicobacter
pylori, Campylobacterjejuni, ste/Iium discoideum, Mus musculus, Homo sapiens,
and these fucosyltransferases catalyse the formation of cr(1,2), a(1,3), d(1,4), or a(1,6)
bonds on other sugars such as but not limited to galactose, e, lactoNbiose,
lactoNtetraose, lactosamine, lactoNtetraose, lactoses, disialyllactoses, or fucosylated
proteins, or fucosylated fatty acids, or fucosylated aglycons such as, but not limited to,
antivirals, antibiotics,... .
The present invention provides for the usage of a mutated and/or transformed
microorganism comprising a genetic change g to a modified sion and/or
activity of the transcriptional tors the aerobic respiration control n ArcA and the
isocitrate lyase regulator IclR to upregulate at least one of the genes of the colanic acid
operon, wherein said operon comprises the genes cpsG and cpsB, coding for
phosphomannomutase and mannose-1—phosphate guanylyltransferase, which are needed
for the biosynthesis of GDP—mannose. As GDP-mannose is a precursor for mannosyllated
W0 2013l087884
oligosaccharides and mannosylated glycoconjugates. These oligosaccharides and
onjugates find for example applications in the treatment of gram-negative bacterial
infections, in addition, GDP-mannose is important for the humanization of protein
glycosylations, which is essential for the tion of certain therapeutic proteins (18,
). Mannosylated oligosaccharides and mannosylated glycoconjugates are also used for
drug targeting, for instance mannosylated antivirals can specifically target the liver and
kidneys (7). In order to efficiently produce mannosylated oligosaccharides (see Figures 1,
2, 5, 6 and 11), the above described mutations in arcA and is”? can be applied in
combination with other mutations which r enhance the production of mannosylated
compounds. Some of these — non—limiting- other mutations are: a) the gene gmd of the
colanic acid operon is d, , b) n the gene gmm coding for GDP-
mannose hydrolase is deleted, and/or, c) wherein the colanic acid operon genes that do
not code for GDP—mannose biosynthesis reactions, the genes gmm, wcaA, wcaB, wcaC,
wcaD, wcaE, wcaF, weal, wcaJ, wcaK, wcaL, fol, gmd, wzx, wza, wzb and/or, wcaM, are
deleted, and/or, d) wherein a gene encoding for a sucrose phosphorylase or an invertase
is introduced, and/or, e) wherein the the genes pgi, pka and pka, coding for
oglucose isomerase, phosphofructokinase A and ofructokinase B
respectively, are deleted, , f) knocking out the gene Ion encoding for a se,
and/or f) wherein a gene encoding for a mannosyltransferase is introduced. In other
words, the present invention relates to a process as described above for the synthesis of
colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides for the synthesis of
GDP—mannose and/or for the synthesis of mannosylated oligosaccharides. The present
invention further relates to said process wherein the genes cpsG and cpsB of the c
acid operon are lated and wherein: a) the gene gmd of the colanic acid operon is
d, and/or, b) wherein the gene gmm is deleted, and/or c) wherein the colanic acid
operon genes fcl, gmd, gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK,
wcaL, wzx, wza, wzb, wzc, and/or, wcaM are knocked out and/or, d) wherein a gene
encoding for a sucrose orylase or an invertase is introduced, and/or, 9) wherein
the the genes pgi, pka and pka are deleted, and/or, f) knocking out the gene Ion, and/or
9) wherein a gene encoding for a mannosyltransferase is uced. The term
‘introducing a mannosyltransferase’ relates to lating or heterologous expression of
mannosyltransferases which are within, but not limited to the enzymes in enzyme classes
EC 2.4.1.32, 2.4.1.827, 2.4.1.844, 2.4.1.48, 2.4.1.54, 2.4.1.57, 2.4.1.83, 2.4.1.109,
2.4.1.110, 2.4.1.119, 2.4.1.130, 2.4.1.131, 132, 2.4.1.142, 2.4.1.199, 2.4.1.217,
2.4.1.232, 2.4.1.246, 2.4.1.251, 2.4.1.252, 2.4.1.257, 2.4.1.258, 2.4.1.259, 2.4.1.260,
2.4.1.265, and/or 2.4.1.270 and/or the glycosyltransferase families GT1, GT2, GT4, GT15,
GT22, GT32, GT33, GT39, GT50 and/or GT58 and/or originating from but not limited to
Helicobacter pylori, Campy/obacter , Dictyostellium disco/deum, Mus musculus,
Homo sapiens, and these mannosyltransferases catalyse the formation of d(1,2),
a(1,3), , or o(1,6) bonds on other sugars such as but not d to galactose, N-
acetylglucosamine, Rhamnose, lactose, biose, lactoNtetraose, iactosamine,
lactoNtetraose, sialyllactoses, disialyllactoses, or ylated proteins, or mannosylated
fatty acids, or mannosylated aglycons such as, but not limited to, antivirals, antibiotics,... .
The term ‘heterologous expression’ relates to the expression of genes that are not
naturally present in the production host, genes which can be synthesized chemically or be
picked up from their natural host via PCR, genes which can be codon optimized for the
production host or in which point mutation can be added to enhance enzyme activity or
expression. Expressing heterologous and/or native genes can either be done on the
chromosome, artificial chromosomes or plasmids and transcription can be controlled via
inducible, constitutive, native or artificial promoters and translation can be controlled via
native or artificial ribosome binding sites.
Consequently, the present invention further relates to mutated and/or transformed
organisms in which the tors ArcA and IclR as be above, in combination with
the genes encoding for the enzymes phosphoglucose isomerase and
phosphofructokinase, are knocked out or are rendered less functional. More specifically,
the present invention relates to the latter organisms wherein the enzyme phosphoglucose
isomerase is d by the gene pgi and wherein the enzyme phosphofructokinase is
encoded by the gene(s) pka and/or pka.
The terms ‘genes which are rendered less—functional or nctional’ refer to the well-
known technologies for a skilled person such as the usage of siRNA, RNAi, miRNA,
asRNA, nggenes, knocking-out genes, oson mutagenesis, etc... which are
used to change the genes in such a way that they are less able (i.e. statistically
significantly ‘less able’ compared to a functional wild-type gene) or completely unable
(such as knocked-out genes) to e functional final products. The term ‘(gene) knock
out’ thus refers to a gene which is rendered non~functional. The term ‘deleted gene’ or
‘gene deletion’ also refers to a gene which is rendered non-functional.
The present invention further relates to a mutated and/or transformed organism as
described in the latter aph wherein said organism is further transformed with a gene
encoding for a sucrose phosphorylase.
The present ion also relates to a mutated and/ortransformed organism as described
above wherein, in addition, the activity and/or expression of the gene encoding for a
lactose se is made constitutive and/or increased. Said ty can be increased by
over-expressing said gene and/or by transforming said organisms with a gene ng
for a lactose permease.
The t invention further s to any mutated and/or transformed organism as
described above wherein at least one of the following genes is knocked out or is rendered
less functional:
a gene ng for a beta-galactosidase, a gene encoding for a glucose—1—phosphate
yltransferase, a gene encoding for a glucose—1—phosphatase, a gene encoding for
phosphogluconate dehydratase, a gene encoding for 2-keto-3~deoxygluconate—6-
phosphate aldolase ,a gene encoding for a glucose-i-phosphate uridyltransferase, a gene
encoding for an UDP-glucose~4-epimerase, a gene encoding for an UDP»
glucosezgalactosephosphate uridyltransferase, a gene encoding for an UDP-
galactopyranose mutase, a gene encoding for an UDP-
galactose:(glucosyl)lipopolysaccharide—1,6-galactosyltransferase, a gene ng for an
UDP—galactosyltransferase, a gene encoding for an UDP-glucosyltransferase, a gene
encoding for an UDP-glucuronate transferase, a gene ng for an UDP-glucose lipid
carrier transferase, a gene encoding for a GDP-mannose hydrolase, a gene encoding for
an UDP-sugar hydrolase, a gene encoding for a mannose-B-phosphate isomerase, a
gene encoding for an UDP—N—acetylglucosamine enoylpyruvoyl erase, a gene
encoding for an UDP-N—acetylglucosamine acetyltransferase, a gene encoding for an
UDP—Nacetylglucosamine—2-epimerase, a gene encoding for an undecaprenyl-phosphate
alfa-N—acetylglucosaminyl transferase, a gene encoding for a glucose-G-phosphate—t—
dehydrogenase, and/or, a gene encoding for a L-glutamine:D-fructose~6-phosphate
aminotransferase, a gene encoding for a mannose-B—phosphate isomerase, a gene
encoding for a sorbitol-e-phosphate dehydrogenase, a gene encoding for a mannitoI—1—
phosphate 5-dehydrogenase, a gene encoding for a allulose-G-phosphate 3-epimerase, a
gene encoding for an invertase, a gene encoding for a maltase, a gene encoding for a
ase, a gene ng for a sugar transporting phosphotransferase, a gene encoding
for a protease, or a gene encoding for a hexokinase. The term ‘at least one’ indicated that
at least 1, but also 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or all 33 genes is (are) knocked out or is (are)
ed less functional.
The present invention further relates also to the usage of a mutated and/or transformed
rganism such as an Escherichia coli strain comprising a genetic change leading to
a modified expression of the transcriptional regulators the aerobic respiration control
W0 2013f087884
protein ArcA and the isocitrate lyase regulator lclR to upregulate at least one of the
following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE,
gadX, gadW and/or slp (17, 22). These genes are normally expressed in stationary phase
conditions; however, the present mutated and/or transformed rganism is able to
enhance the expression of these acid resistance related genes in the exponential growth
phase. Hence, the present invention relates to the usage as described above for the
sis of acids or pH sensitive molecules such as but not limited to glucosamine which
is pH sensitive and should be produced at low pH (12). c acids, such as but not
limited to pyruvic acid, succinic acid, adipic, siaiic acid, sialylated oligosaccharides (e.g.
sialyilactose, sialyl Lewis X sugars,... ), acetylated oligosaccharides ns, chitosans,...
), sulfonated oligosaccharides (heparans and sans)... are preferably produced at
low pH for ream processing purposes (4). in other words, the present invention
relates to a process for the synthesis of acids, sialic acid, sialylated oligosaccharides or
glucosamine comprising genetically changing the transcriptional regulators the aerobic
respiration control protein ArcA and the isocitrate lyase tor chR to upregulate at
least one of the following acid resistance d genes: ydeP, ydeO, hdeA, hdeD, gadB,
gadC, gadE, gadX, gadW and/or s/p.
The present invention will now be illustrated by the following non—limiting examples.
EXAMPLES
A high throughput RT—qPCR screening of the microorganisms of the present invention has
been setup with Biotrove OpenArray® technology. in this experiment the transcription of
1800 genes were measured in 4 strains (wild type, AarcA, AicIR, AarcA Aic/R) in two
conditions (chemostat and batch). The data was processed using a curve fitting toolbox in
R (25, 34) and le Normalization, the error on the data was ated using
Bayesian statistics (20, 21, 31).
Material and s
Strains and plasmids
Escherichia coli M61655 [ ', F”, rph-1] was obtained from the Netherlands Culture
Collection of Bacteria (NCCB). Escherichia coli BL21(DE3) was obtained from Novagen.
Escherichia coli M61655 ackA—pta, poxB, pppc ppc-p37 (10), the single knock-outs
E. coli MG1655 arcA and E. coii MG1655 ic/R and the double knock-out E coli
MG1655 arcA, ic/R were constructed in the Laboratory of Genetics and iology
(MICR) using the method of Datsenko & Wanner (9).
Media
The Luria Broth (LB) medium ted of 1 % tryptone peptone (Difco, Erembodegem,
Belgium), 0.5 % yeast extract (Difco) and 0.5 % sodium chloride (VWR, Leuven, Belgium).
Shake flask medium contained 2 g/I NH4CI, 5 g/l SO4, 2.993 in KH2P04, 7.315 9/!
, 8.372 g/l MOPS, 0.5 g/l NaCl, 0.5 g/l MgSO4-7HZO, 16.5 g/l e-H20, 1 ml/I
n solution, 100 ul/l ate solution, and 1 ml/I selenium solution. The medium
was set to a pH of 7 with 1M KOH.
1O Vitamin solution consisted of 3.6 g/I FeC|2 - 4H20, 5 g/l CaCl2 - 2H20, 1.3 g/l MnClz '
2H20, 0.38 g/l CuC12-2H20, 0.5 g/I CoCIz - 6H20, 0.94 g/l ZHC12, 0.0311 g/I H3804, 0.4 g/I
NagEDTA- 2H20 and 1.01 g/l thiamine - HCl. The molybdate solution contained 0.967 g/I
NazMoO4 - 2H20. The selenium solution contained 42 g/I Seog.
The minimal medium for fermentations contained 6.75 g/I NH4CI, 1.25 g/l (NH4)2SO4, 1.15
g/l KH2PO4, 0.5 g/l NaCl, 0.5 g/l MgSO4-7H20, 16.5 9/1 glucose-H20, 1 mill n
solution, 100 ul/I molybdate solution, and 1 mill selenium solution with the same
composition as described above.
Cultivation conditions
A preculture, from a single colony on a LB-plate, in 5 ml LB medium was incubated during
8 hours at 37 °C on an orbital shaker at 200 rpm. From this culture, 2 ml was transferred
to 100 ml minimal medium in a 500 ml shake flask and incubated for 16 hours at 37 °C on
an orbital shaker at 200 rpm. 4 % inoculum was used in a 2 | Biostat B Plus culture vessel
with 1.5 l working volume (Sartorius Stedim Biotech, Melsungen, Germany). The culture
conditions were: 37 °C, stirring at 800 rpm, and a gas flow rate of 1.5 l/min. Aerobic
conditions were ined by sparging with air, anaerobic conditions were obtained by
flushing the culture with a mixture of 3 % 002 and 97 % of N2. The pH was maintained at
7 with 0.5 M H2804 and 4 M KOH. The exhaust gas was cooled down to 4 °C by an
exhaust cooler (Frigomix 1000, ius Stedim Biotech, Melsungen, Germany). 10 %
solution of silicone antifoaming agent (BDH 331512K, VWR Int Ltd., Poole, England) was
added when foaming raised during the fermentation (approximately 10 pl). The off-gas
was ed with an EL3020 off-gas analyser (ABB Automation GmbH, 60488 Frankfurt
am Main, Germany).
PCT/EP2012l075639
All data was logged with the Sartorius MFCS/win v3.0 system rius Stedim h,
Melsungen, Germany).
All strains were cultivated at least twice and the given standard deviations on yields and
rates are based on at least 10 data points taken during the repeated experiments.
Sampling methodology
The bioreactor contains in its interior a harvest pipe (BD Spinal , 1.2x152 mm
(BDMedical Systems, Franklin Lakes, NJ - USA) connected to a r port, linked
outside to a Masterflex—14 tubing (Cole-Parmer, Antwerpen, Belgium) followed by a
harvest port with a septum for sampling. The other side of this harvest port is connected
1O back to the reactor vessel with a Masterflex-iG tubing. This system is referred to as rapid
sampling loop. During sampling, reactor broth is pumped around in the sampling loop. it
has been estimated that, at a flow rate of 150 ml/min, the r broth needs 0.04 s to
reach the harvest port and 3.2 s to er the reactor. At a p02 level of 50 %, there is
around 3 mg/l of oxygen in the liquid at 37 °C. The p02 level should never drop below 20
% to avoid micro—aerobic conditions. Thus 1.8 mg/l of oxygen may be consumed during
transit through the harvesting loop. Assuming an oxygen uptake rate of 0.4 g oxygen/g
biomass/h (the maximal oxygen uptake rate found at pmax), this gives for 5 g/l biomass, an
oxygen uptake rate of 2 g/l/h or 0.56 mg/l/s, which multiplied by 3.2 s ence time in
the loop) gives 1.8 mg/I oxygen consumption.
In order to quench the metabolism of cells during the sampling, r broth was sucked
through the harvest port in a syringe filled with 62 g stainless steel beads pre—cooled at
-20 °C, to cool down 5 ml broth immediately to 4 °C. Sampling was immediately followed
by cold centrifugation (15000 g, 5 min, 4 °C). During the batch experiments, a sample for
ODSOOnm and RT—qPCR measurements was taken using the rapid sampling loop and the
cold stainless bead sampling method.
RT-gPCR
mRNA was extracted with the RNeasy kit (Qiagen,Venlo, The Netherlands). RNA quality
and quantity was checked with a nanodrop 0 spectrophotometer (Nanodrop
technologies, Wilmingto, USA). The ratios 260:280 (nm) and 2602230 (nm) were n
1.8 and 2 and at least 100ng/pl was needed for further analysis. cDNA was synthesised
with random primers with the RevertAidTM H minus first strand cDNA synthesis kit
(Fermentas, St. Leon-Rot, Germany). Finally, the gene expression of 1800 genes was
ed with the ve OpenArray Real time PCR platform. The primers for the RT—
PCR assay were designed with Primer design tools from the Primer database (23).
W0 20131087884
The on mixture was ed as described in the Biotrove OpenArrayTM Real-Time
qPCR system users’ . In short, a mastermix was made with 26.4pl LightCycler®
DNA Master SYBR® Green i (Roche applied Science), 1.1 pl SYBR GREEN l (100x stock
solution, Sigma , 8.8;” glycerol (Sigma G5150), 5.3ul Pluronic® F68 (10% stock,
lnvitrogen), 2.64m BSA (Sigma A7906), 26.4ul magnesium chloride (25mM stock solution,
supplied in the LightCycler® kit of Roche applied Science ), 21.1pl HiDiTM formamide
(Applied biosystems), and 94.66;.” RNase free sterile water resulting in a 186.4pl
mastermix, which is enough to load 1 OpenArrayTM. For 1 SubArray (each ray is
subdivided in 48 SubArrays on which 1 sample can be loaded) 1.5pl sample (with a
1O concentration of 100ng/pl) was mixed with 3.5pl of mastermind, as a no template control,
water was used as blanc. The sample—mastermix mixture was loaded in a Loader plate
(MatriPlateTM 384-well black low volume polypropylene plate, Biotrove) in a RNase free
hood. A full loader plate was loaded with an AutoLoader (Biotrove) and loader tips onto
the OpenArrays. These OpenArrays were then submerged in OpenArrayTM immersion
fluid in an OpenArrayTM ime qPCR case. The case was sealed with Case sealing
glue and ted in the Case Sealing station, which polymerizes the glue with UV light.
Analytical methods
Cell density of the culture was ntly monitored by measuring optical density at 600
nm (Uvikom 922 spectrophotometer, BRS, Brussel, m). Cell dry weight was
obtained by centrifugation (15 min, 5000 g, GSA rotor, l RC—SB, Goffin Meyvis,
Kapeilen, Belgium) of 20 g reactor broth in pre-dried and weighted falcons. The pellets
were subsequently washed once with 20 ml physioiogical solution (9 9/! NaCl) and dried at
70 °C to a constant weight. To be able to convert ODGOOnm measurements to biomass
concentrations, a correlation curve of the ODSOOnm to the biomass concentration was
made. The concentrations of glucose and organic acids were ined on a Varian
Prostar HPLC system (Varian, Sint—Katelijne-Waver, m), using an Aminex HPX—87H
column (Bio-Rad, Eke, Belgium) heated at 65 °C, equipped with a 1 cm precolumn, using
mM H2804 (0.6 ml/min) as mobile phase. A dual-wave UV—VIS (210 nm and 265 nm)
detector (Varian Prostar 325) and a differential refractive index detector (Merck LaChrom
L—7490, Merck, Leuven, m) was used for peak detection. By dividing the
absorptions of the peaks in both 265 and 210 nm, the peaks could be identified. The
division s in a constant value, typical for a certain compound (formula of Beer-
Lambert).
W0 2013f087884
Glucose, se, sucrose, llactose and glucose-l-phosphate were measured by
HPLC with a Hypercarb column and were detected with an MSMS detector (Antonio et al.,
2007; Nielsen et al., 2006).
Genetic methods
All mutant strains were constructed via the methods described below.
Plasmids were maintained in the host E. coli DH50c (F', <p80d/acZAM15, A(lacZYA-
argF)U169, deoR, recAl, endAl, hst17(rk', mk+), phoA, supE44, A', thi—l, gyrA96,
reIAi).
Plasmids. pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT—
flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT—flanked
kanamycin resistance (kan) gene), and pCPZO (expresses FLP recombinase activity)
plasmids were used for the mutant construction. The plasmid pBluescript (Fermentas, St.
Leon-Rot, Germany) was used to construct the derivates of pKD3 and pKD4 with a
promoter library, or with alleles ng a point mutation.
Mutations. The mutations ted in gene disruption (knock-out, KO). They were
introduced using the concept of ko and Wanner (9). The s for the mutation
strategies are described in Table 1.
Transformants carrying a Red helper plasmid were grown in 10 ml LB media with
ampicillin (100 mg/l) and L—arabinose (10 mM) at 30 °C to an ODSOOnm of 0.6. The cells
were made electrocompetent by g them with 50 ml of ice-cold water, a first time,
and with 1 ml ice-cold water, a second time. Then, the cells were resuspended in 50 pl of
ice-cold water. Electroporation was done with 50 ul of cells and 10—100 ng of linear
double-stranded-DNA product by using a Gene PulserTM d) (600 Q, 25 uFD, and
250 volts).
After electroporation, cells were added to 1 ml LB media incubated 1 h at 37 °C, and
finally spread onto LB—agar containing 25 mg/l of chloramphenicol or 50 mg/l of kanamycin
to select antibiotic resistant transformants. The selected mutants were verified by PCR
with s upstream and downstream of the modified region and were grown in LB—agar
at 42 °C for the loss of the helper plasmid. The mutants were tested for llin
ivity.
Linear double-stranded-DNA. The linear ds-DNA amplicons were obtained by PCR
using pKD3, pKD4 and their derivates as template. The primers used had a part of the
sequence complementary to the template and another part complementary to the side on
the chromosomal DNA where the recombination has to take place (
Table 1). For the K0, the region of homology was designed 50-nt upstream and 50-nt
downstream of the start and stop codon of the gene of interest. For the KI, the
transcriptional starting point (+1) had to be respected. PCR products were PCR-purified,
digested with Dpnl, repurified from an agarose gel, and suspended in elution buffer (5 mM
Tris, pH 8.0).
Elimination of the antibiotic resistance gene. The selected mutants (Chloramphenicol
or kanamycin resistant) were transformed with pCP20 plasmid, which is an ampicillin and
1O chloramphenicol resistant d that shows temperature-sensitive replication and
thermal induction of FLP sis. The ampicillin—resistant transformants were selected
at 30 °C, after which a few were colony purified in LB at 42 °C and then tested for loss of
all antibiotic resistance and of the FLP helper plasmid. The gene knock outs and knock ins
are checked with l s (Fw/Rv-gene-out). These primers are given in Table 1.
Table 1: s used to create E. 00/] M61655 arcA E. coli MG1655 iclR and the
double knock-out E. coli M61655 arcA felt? and all other enetic knock outs and
knock ins
Primer name Sequence
lacZ
FW_LaCZ_Pl CATAATGGATTTCCTTACGCGAAATACGGGCAGACATGGCCTGCCCGGTTATTAgtgta
ggctqgagctgcttc (SEQ ID N° 7)
RV_LacZ“P2 GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTcatatgaa
tatcctccttag (SEQ ID N° 8)
FW_LacZ_out GCGGTTGGAATAATAGCG (SEQ ID No 9)
RV_LacZ_out CAGGTTTCCCGACTGGAAAG (SEQ ID N° 10)
glgC
FW—glgC-Pl ccggttttaagcagcgggaacatctctgaacatacatgtaaaacctgcagtgt
aggctggagctgcttc (SEQ ID N° 11)
RV—glgC—P2 Gtctggcagggacctgcacacggattgtgtgtgttccagagatgataaaaaaggagtta
atgaatatcctccttag (SEQ ID N° 12)
FW—glgC-out Gcgaatatcgggaaatgcagg (SEQ ID N° 13)
RV—glgC—out Cagagattgttttacctgctgg (SEQ ID N° 14)
WO 87884
CATATTTCTGTCACACTCTTTAGTGATTGATAACAAAAGAGGTGCCAGGAgtgtaggct
ggagctgcttc (SEQ ID N° 15)
TAAAAACGTTTAACCAGCGACTCCCCCGCTTCTCGCGGGGGAGTTTTCTGcatatgaat
atcctccttag(SEQ ID N° 16)
GCCACAGGTGCAATTATC (SEQ ID N° 17)
CATTTTCGAAGTCGCCGGGTACG(SEQ ID N° 18)
-Pl GGCGCTACAATCTTCCAAAGTCACAATTCTCAAAATCAGAAGAGTATTGCgtgtaggct
ggagctgcttc (SEQ ID N° 19)
RV-pgi-PZ GGTTGCCGGATGCGGCGTGAACGCCTTATCCGGCCTACATATCGACGATGcatatgaat
atcctccttag (SEQ ID N° 20)
Fw_pgi*out( GGCTCCTCCAACACCGTTAC (SEQ ID N° 21)
TACATATCGGCATCGACCTG (SEQ ID N° 22)
Fw pka p1 GACTTCCGGCAACAGATTTCATTTTGCATTCCAAAGTTCAGAGGTAGTCgtgtaggctg
gagctgcttc(SEQ ID N° 25)
RV-pka P2 GCTTCTGTCATCGGTTTCAGGGTAAAGGAATCTGCCTTTTTCCGAAATCCatatgaata
tcctccttag (SEQ ID N° 26)
Fw-pka-out TAGCGTCCCTGGAAAGGTAAC (SEQ ID N° 27)
RV-pka—out ATCATCCGTCATAG (SEQ ID N° 28)
Fw—pka P1 CACTTTCCGCTGATTCGGTGCCAGACTGAAATCAGCCTATAGGAGGAAATthgtaggc
tggagctgcttc (SEQ ID N° 29)
RV pka P2 GTTGCCGACAGGTTGGTGATGATTCCCCCAATGCTGGGGGAATGTTTTTGcatatgaat
atcctccttag (SEQ ID N° 30)
FW—arcA—Pl thtgaaaaataaaaacggcgctaaaaagcgccgttttttttgacggtggtaaagccga
gtgtaggctggagctgcttc (SEQ ID N° 31)
RV‘—arcA—P2 thcagggacttttgtacttcctgtttcgatttagttggcaatttaggtagcaaaccat
atgaatatcctccttag (SEQ ID N° 32)
Ctgccgaaaatgaaagccagta (SEQ ID N" 33)
Ggaaagtgcatcaagaacgcaa (SEQ ID N° 34)
FW—iclR-Pl Ttgccactcaggtatgatgggcagaatattqcctctgcccgccagaaaaaggtgtaggc
tggagctgcttc (SEQ ID N° 35)
RV—iclR—P2 Gttcaacattaactcatcggatcagttcagtaactattgcattagctaacaataaaaca
tatgaatatcctccttag (SEQ ID N° 36)
FW-iclR—out ngtggaatgagatcttgcga (SEQ ID N° 37)
RV—iclR~out Acttgctcccgacacgctca (SEQ ID N° 38)
TTGCCACTCAGGTATGATGGGCAGAATATTGCCTCTGCCCGCCAGAAAAAGCCgCttac
gctgtg (SEQ ID N° 39)
RV_iclR*P9 CATTAACTCATCGGATCAGTTCAGTAACTATTGCATTAGCTAACAATAAAAag
ccatgacccgggaattac (SEQ ID N° 40)
Rv—iclR— CTATTGCATTAGCTAACAATAAAACTTTTTCTGGCGGGCAGAGG (SEQ ID N° 41)
scarless KO
stap 2
Fw—iclR— CCTCTGCCCGCCAGAAAAAGTTTTATTGTTAGCTAATGCAATAGTTAC (SEQ ID N°
~scarless KO 42)
stap 2
GCCAGCGCGATAATCACCAG (SEQ ID N° 43)
TGAATGTGGAAT‘C (SEQ ID N° 44)
Fw—wcaJ_2- TTTTGATATCGAACCAGACGCTCCATTCGCGGATGTACTCAAGGTCGAACgtgtaggct
Pl ggagctgcttc (SEQ ID N° 45)
Rv—wcaJ_2- TCTATGGTGCAACGCTTTTCAGATATCACCATCATGTTTGCCGGACTATGcatatgaat
P2 atcctccttag (SEQ ID N° 46)
fwwwcaJ_Hl' TCAATATGCCGCTTTGTTAACGAAACCTTTGAACACCGTCAGGAAAACGATTTTGATAT
CGAACCAGACG (SEQ ID N° 47)
Rv_wcaJ_H2' TGACAAATCTAAAAAAGCGCGAGCGAGCGAAAACCAATGCATCGTTAATCTCTATGGTG
CAACGCTTTTC (SEQ :3 N° 48)
Fw_wcaJ_H1' CGCTTTGTTAACGAAACCTTTGAACACCGTCAGGAAAACGATTTTGATATCGAACCAGA
CGCTCCATTCG (SEQ ID N° 49)
CAGTCGTGTCATCTGATTACCTGGCGGAAATTAAACTAAGAGAGAGCTCTgtgtaggct
ggagctgcttc(SEQ ID N° 50)
OMEM0100_RV AGCCTGCCAGCCCTGTTTTTATTAGTGCATTTTGCGCGAGGTCAcatatgaat
—lon—P2 atcctccttag (SEQ ID N° 51)
oMEMOlOl_FW AGCGCAACAGGCATCTGGTG (SEQ ID No 52)
—lon—out
OMEM0102_RV TATATCAGGCCAGCCATCCC (SEQ ID N° 53)
-lon-out
lacZXA:P22—
lacY
Fw_lacZYA_c GCTGAACTTGTAGGCCTGATAAGCGCAGCGTATCAGGCAATTTTTATAATCTTCATTTA
hl AATGGCGCGC (SEQ ID N° 54)
rv_lacZYA_c GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTCGCCTACCT
hl GTGACGGAAG (SEQ ID N° 55)
fw_P221acY- GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTGTGTAGGCT
KI_P1 GGAGCTGCTTC (SEQ ID N° 56)
rv_P221acY— GCTGAACTTGTAGGCCTGATAAGCGCAGCGTATCAGGCAATTTTTATAATCTTAAGCGA
KI CTTCATTCACC (SEQ ID N° 57)
fw_lacZYA_H CGACGCTTGTTCCTGCGCTTTGTTCATGCCGGATGCGGCTAATGTAGATCGCTGAACTT
1 1 GTAGGCCTG (SEQ ID N° 58)
IV lacZYA H CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGC
2" "
TGTG (SEQ ID N° 59)
pka:P22-
BaSP
GACTTCCGGCAACAGATTTCATTTTGCATTCCAAAGTTCAGAGGTAGTCgtgtaggCtg
gagctgcttc (SEQ ID N° 60)
- GTCATCGGTTTCAGGGTAAAGGAATCTGCCTTTTTCCGAAATCaagcttgcat
pCXP22_P2 gcctgcatcc (SEQ ID N° 61)
AGAGGCTATTCGGCTATGAC (SEQ ID N°
CGCCEEEEECGEEEGEGEEG (SEQ ED E
TGATTGTTATACTATTTGCACATTCGTTGGATCACTTCGATGTGCAAGAAGACTTCCGG
CAACAGATTTC (SEQ ID N° 64)
Rv_pka_H2_ AATTGCAGAATTCATGTAGGCCTGATAAGCGAAGCGCATCAGGCATTTTTGCTTCTGTC
ext ATCGGTTTCAG (SEQ ID N° 65)
FW--pka—out CATTTGGCCTGAC (SEQ ID N°
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiiIIIIIIIIIIIIIIIIIIIIIIIIIEEEGEGCECTEEGECCEEEC SEQ ID E
Fw-adhE— ATCGGCATTGCCCAGAAGGGGCCGTTTATGTTGCCAGACAGCGCTACTGAgtgtaggct
pCXP22—PI ggagctgcttc (SEQ ID N° 68)
ATTCGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGAGCATTaagcttgca
pCXP22—P2 tgcctgcatcc (SEQ ID N° 69)
Fw—adhE-Hl' AAGCCGTTATAGTGCCTCAGTTTAAGGATCGGTCAACTAATCCTTAACTGATCGGCATT
GCCCAGAAG (SEQ ID N° 70)
Rv-ddhE-HZ' TTGATTTTCATAGGTTAAGCAAATCATCACCGCACTGACTATACTCTCGTATTCGAGCA
GATGATTTACTAAAAAAG (SEQ ID N° 71)
FW_adhE_out GCGTCAGGCAGTGTTGTATC (SEQ ID N° 72)
RV_adhE_out CTGGAAGTGACGCATTAGAG (SEQ ID No 73)
FW_ldhA_out tgtcattacttacacatcccgc (SEQ ID N° 74)
RV_ldhA_out gcattcaatacgggtattgtgg (SEQ ID N° 75)
Fw-ldhA- CATTGGGGATTATCTGAATCAGCTCCCCTGGAATGCAGGGGAGCGGCAAGQtgtaggct
_Pl gcttc (SEQ ID N° 76)
Rv—ldhA- TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTCTTATGaagCttgca
pCXP22_PZ tgcctgcatcc (SEQ ID N° 77)
Fw-ldhA-Hl' CAATTACAGTTTCTGACTCAGGACTATTTTAAGAATAGAGGATGAAAGGTCATTGGGGA
TTATCTGAATCAG (SEQ ID No 78)
RV-ldhA-HZ' GAATTTTTCAATATCGCCATAGCTTTCAATTAAATTTGAAATTTTGTAAAATATTTTTA
GTAGCTTAAATGTGATTCAAC (SEQ ID N° 79)
Fw—ldhA— TTCACCGCTAAAGCGGTTAC (SEQ ID N° 80)
long homol
Rv-ldhA— CGCGTAATGCGTGGGCTTTC (SEQ ID N° 81)
long homol
:Pl4
pCXPl4_SP_I‘ CCGGCATATGGTATAATAGGG (SEQ ID N° 82)
yegH_rc_pur ACGGCTTGCTGGCCATCA (SEQ ID N° 83)
e rv
fw_Pl4- CGAATATAAGGTGACATTATGGTAATTGAATATTGGCTTTCCAATAATGCTACGGCCCC
CA_KI_tetA. AAGGTCCAA (SEQ ID N° 84)
— AATATTGTCAACCTAAAGAAACTCCTAAAAACCATATTGAATGACACTTATTGGCTTCA
CA_KI_tetA GGGATGAGGCG (SEQ ID N° 85)
fw_Pl4— TCCCGACTACGTGGACCTTG (SEQ ID N° 86)
overl
rv_Pl4- CATATGGTATAATAGGGAAATTTCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGT
CA_KI_overl CGACCTCGGCATTATTGGAAAGCCAATATTC (SEQ ID NO 87)
2012/075639
- GCCGCCATGGAAATTTCCCTATTATACCATATGCCGGCCAAGATGTCAAGAAACTTATA
CA_KI_overl GAATGAAGTAAGTGTCATTCAATATGG (SEQ ID N° 88)
fw_Pl4— AATATTGTCAACCTAAAGAAACTCCTAAAAACCATATTGAATGACACTTACTTCATTCT
CA_KI__H1 ATAAGTTTCTTGAC (SEQ ID N° 89)
rv_Pl 4 — CGAATATAAGGTGACATTATGGTAATTGAATATTGGCTTTCCAATAATGCCGAGGTCGA
CA__KI_H2 CGGATCCCAAGCTTC (SEQ ID N° 90)
ormation. ds were transformed in CaCI2 competent cells using the simplified
procedure of Hanahan (16) or via electroporation as described above.
Calculation methods
uction
Different experiments with ent strains were performed. in total 8 different conditions
were tested. There was variation in the genetic background (WT, ic/R knock-out, arcA
knock-out, and combined ic/R—arcA knock-out) and the mode of fermentation (batch, and
chemostat). Each experiment was repeated twice.
to When running the samples through the BioTrove apparatus, a qPCR curve escences
in function of cycle number) and a melt curve scences in function of the
temperature) is obtained for each sample. Those data were exported from the BioTrove
software and further anaiysed in R. The analysis was divided in two steps: first the qPCR
curves were fitted and Ct values were calculated and in the second step the Ct values
were converted to expression data.
Calculating the qPCR curves
The raw qPCR curve data were extracted from the ve software and imported in R
(1). The curves were fitted to a 5 parameter sigmoidal model, with the R package qPCR
(25, 34). The maximum of the second derivative of those curves was used as Ct value. No
normalisation was applied to the data prior to the curve fitting. However, outliers were
removed. The detection of the outliers was done using the following procedure:
- Fit the model to the data.
- Calculate the residuals (defined as the measured fluorescences minus the modelcalculated
ones).
- Assuming the residuals are normally distributed, calculate the mean and standard
deviation of the residuals.
- Using this mean and standard deviation, the 95 % interval is calculated.
- All data—points for which the residuals fall out of this 95 % interval are considered
as outliers.
- The curve is refitted without the outliers.
- This is repeated until no outliers are detected anymore. Using this procedure, the
data do not have to be ised prior to fitting, neither must the first oints
be removed.
Many curves have to be fitted (1800 genes for one experiment). ore, it is undoable
to manually check each curve and automated methods have to be applied to reject bad
curves. For this ent parameters are ted from the curves: the cycle number
value at which the maximum of the first derivative occurs (D1), the cycle number value at
which the maximum of the second derivative occurs (D2), the minimal fluorescence
, and the maximal fluorescence . Combining the values of those parameters,
the validity of the curve and the extent of expression is assessed. How this is done is
explained in the next section.
Filtering the data
For some gene—experiment combinations. no amplification is detected. This can be due to
a y of reasons:
- Expression is too low and 32 cycles (the number of cycles for all BioTrove arrays
was set to 32) is not enough to detect the expression. in this case, the real Ct
cannot be determined and is somewhere between 32 and infinity.
- No expression. in this case, the real Ct is infinite.
- Technical failures: primers not suitable, wrong loading (it is very difficult to
uniformly load the BioTrove arrays, especially the holes at the sides of the array
are frequently empty), etc. in this case the real Ct can vary between 0 and infinity.
Some genes are genuinely not expressed and setting their Ct value to ing else
than infinity is not t. For genes that are expressed, but for which the expression
value, due to technical es or limitations, are not known, setting the Ct value to infinity
is not correct. Furthermore, using arbitrary values that are outside the range of expression
complicates the calculation routines and visualisation routines. Therefore it was opted to
remove the gene-experiment ations for which no correct expression data was
detected.
An obvious case of gene-experiment pairs for which no sion is detected, are those
for which no curve could be fitted to the qPCR data. Less s cases are detailed
below.
Typically for expressed genes, is that the fluorescence values cover a certain range. Data
points for which this range was not high enough, were discarded, as they pointed to very
poorly fitted curves and generally bad data. The minimal fluorescence range was set to
400 (thus Fmax - Fmin > 400).
In a good amplification curve, the first (D1) and second (D2) derivative are quite close to
each other (see the documentation of the SOD on in the qpcR package(25)).
1O Therefore, all data-points for which the difference between D1 and D2 is larger than an
arbitrary value (7 was used) were discarded.
For each primer-pair, a qPCR experiment was performed without adding DNA. Only water
was added. Normally no expression should be ed in those samples. However,
amplification is detected in water for some primer—pairs. Genes for which the Ct value (as
mentioned before, D2 was used) is more than the Ct value of water minus 5, are
discarded, as it cannot be excluded that the fluorescence comes from the amplification of
the primers and not the added DNA.
Normaiising and calculating the contrasts
Prior to calculating the expression differences, the Ct values have to be ised. As so
many genes were measured (1800), quantile normalisation could be used (33). The 1800
genes measured, were divided over 3 types of arrays, each containing 600 genes.
Quantile normalisation was done for each type of array separately. A table was
constructed where the rows represent the different genes and the columns the different
experiments (T1, see ons 1). Each column was sorted independently (T2) and the
original position of the elements was saved. The values in this new table were replaced
with the mean value over the different rows (T3). And y this table was transformed so
that the ons of the values corresponded again to the original positions (T4).
24 33 33
T1=68 72—68 T3=6 2",:
412 412 99 99
Equations 1: Example of quantile normalisation
Differential expressions were ated with the normalised data. This was done with the
R package limma, which uses a Bayesian approach to calculate the statistical relevances
of the differences (31, 32). Limma was adapted to be able to cope with missing data: the
original limma package discards all expression values from a gene over the different
experiments, when one value in one experiment is not available. This hampers the
analysis when one has many different conditions, as for each gene for which one of the
experimental ions es no expression values, a different contrast matrix has to
1O be generated ng that experimental condition. Therefore the function for fitting the
contrasts was adapted to drop data~points with missing data.
Differential expressions were calculated n Ct values and the mean Ct value for a
certain gene. Thus, the higher the value, the lower the expression. For each gene, plots
were generated showing those differences. However, in those plots, the Ct values were
inversed, so that the higher the value, the higher the expression.
Example 1: Effect of arcA and icIR gene deletions on the gene expression of the
colanic acid thesis
Figures 1 and 2 show the expression n of genes involved in colanic acid
biosynthesis (35). Single arcA or ic/R knock out ons did not affect the expression of
the operon in comparison of the wild type strain in batch and chemostat conditions. The
double mutant strain, AarcAAiclR, however upregulates the genes of the colanic acid
operon 6 to 8 times in ison to the wild type and the single mutant strains in both
tat and batch conditions. Both regulators have thus a surprisingly cooperative
effect on the expression of this operon which is independent from the culturing condition
that is applied. Looking at the regulatory network of this operon, no direct link could be
found between both ArcA and lclR and the ription factor that controls the operon,
RcsA e 5). Only ArcA is connected with RcsA via 3 other transcription factors, which
are all upregulated as well. However the AarcA single gene deletion mutant strain did not
affect the transcription of the operon.
Example 2: Effect of arcA and icIR gene deletions on the gene expression of the
GDP-fucose biosynthesis genes
Figures 4 and 6 show the relationship of the colanic acid operon with GDP-fucose
biosynthesis. in Figure 6 the upregulation of GDP-fucose biosynthesis specific genes is
shown. These mutations thus enhance the biosynthesis of GDP—fucose, which is a
precursor for fucosylated oligosaccharides such as tucosyllactose, fucosyllactoNbiose and
lewis X oligosaccharide or fucosylated proteins. These sugars and proteins, as y
indicated above, have applications in therapeutics as nutraceutical, as components in
human mother milk in which they have anti—inflammatory and prebiotic effects (5, 8, 27).
Example 3: Enhancement of GDP-fucose and fucosylated oligosaccharide
biosynthesis
The mutations AarcAAic/R applied in combination with other mutations enhance the
production of fucosylated compounds. A first, ‘other’ genetic modification that enhances
said production is the on of wcaJ from the colanic operon, stopping the initiation of
the colanic acid biosynthesis and thus the accumulation of GDP-fucose. Further, a
fucosyltransferase has to be introduced to link fucose with ent or molecules
such as lactose. The metabolism is then ered further to accumulate the sor
of the GDP-fucose biosynthetic pathway. These modifications are shown in Figure 7.
onal to wcaJ
, the colanic acid operon genes that do not code for cose
biosynthesis ons are knocked out, such as gmm, wcaA, wcaB, wcaC, wcaD, wcaE,
wcaF, wcal, wcaK, wcaL and/or, wcaM. For the production of fucosyllactose, lacZ coding
for B-galactosidase, is knocked out to avoid lactose degradation and the expression of
lach coding for a lactose permease, is enhanced by means of a strong constitutive
promoter.
Example 4: Enhancement of GDP-fucose and fucosylated oligosaccharide
tion via a split metabolism with sucrose as a substrate
To accumulate the cose precursor fructose and fructose—6-phosphate, a sucrose
phosphorylase or invertase is uced. Because fructosephosphate is easily
degraded in the glycolysis, the glycolysis is interrupted in order to steer all fructose—6-
phosphate in the direction of GDP-fucose. The genes pgi, ,0ka and pka are thus knocked
out, coding for glucosephosphate isomerase and ofructokinase A and B. Finally
a fucosyltransferase is introduced to link fucose to an acceptor molecule.
The growth rate of the wild type strain is somewhat affected when grown on sucrose after
introduction of a sucrose phosphorylase (BaSP) (plasmid with sequence SEQ ID N" 2)
(Table 2), however the introduction of pgi mutations and pka and pka double mutations
led to cant reduction of growth rate, the latter was extremely low (0.02 h"). The
combination of all mutations (Apgi and Apka and Apka) led to the lowest growth rate,
however, the growth rate on both sucrose and glucose was surprisingly similar to that of
the pgi single mutant.
Table 2: sgecific growth rates of the glycolysis knock out strains on a minimal medium
with glucose and sucrose
Strain Growth rate on glucose (h‘1) Growth rate on sucrose (h'1)
(strains transformed with
plasmid containing BaSP)
Wild type
Apgi
ApkaApka
Apg/‘ApkaApka
SEQ ID N° 2: d sequence with sucrose ghosphomlase BaSP
AATTCGGAGGAAACAAAGATGGGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGC
ATGAAAAACAAGGTGCAGCTCATCACTTACGCCGACCGCCTTGGCGACGGCACCATCAAG
TCGATGACCGACATTCTGCGCACCCGCTTCGACGGCGTGTACGACGGCGTTCACATCCTG
TTCACCCCGTTCGACGGCGCCGACGCAGGCTTCGACCCGATCGACCACACCAAG
GTCGACGAACGTCTCGGCAGCTGGGACGACGTCGCCGAACTCTCCAAGACCCACAACATC
ATGGTCGACGCCATCGTCAACCACATGAGTTGGGAATCCAAGCAGTTCCAGGACGTGCTG
GGCGAGGAGTCCGAATACTATCCGATGTTCCTCACCATGAGCTCCGTGTTCCCG
AACGGCGCCACCGAAGAGGACCTGGCCGGCATCTACCGTCCGCGTCCGGGCCTGCCGTTC
ACCCACTACAAGTTCGCCGGCAAGACCCGCCTCGTGTGGGTCAGCTTCACCCCGCAGCAG
GTGGACATCGACACCGATTCCGACAAGGGTTGGGAATACCTCATGTCGATTTTCGACCAG
ATGGCCGCCTCTCACGTCAGCTACATCCGCCTCGACGCCGTCGGCTATGGCGCCAAGGAA
ACCAGCTGCTTCATGACCCCGAAGACCTTCAAGCTGATCTCCCGTCTGCGTGAG
GAAGGCGTCAAGCGCGGTCTGGAAATCCTCATCGAAGTGCACTCCTACTACAAGAAGCAG
GTCGAAATCGCATCCAAGGTGGACCGCGTCTACGACTTCGCCCTGCCTCCGCTGCTGCTG
CACGCGCTGAGCACCGGCCACGTCGAGCCCGTCGCCCACTGGACCGACATACGCCCGAAC
AACGCCGTCACCGTGCTCGATACGCACGACGGCATCGGCGTGATCGACATCGGCTCCGAC
CAGCTCGACCGCTCGCTCAAGGGTCTCGTGCCGGATGAGGACGTGGACAACCTCGTCAAC
ACCATCCACGCCAACACCCACGGCGAATCCCAGGCAGCCACTGGCGCCGCCGCATCCAAT
CTCGACCTCTACCAGGTCAACAGCACCTACTATTCGGCGCTCGGGTGCAACGACCAGCAC
TACATCGCCGCCCGCGCGGTGCAGTTCTTCCTGCCGGGCGTGCCGCAAGTCTACTACGTC
GGCGCGCTCGCCGGCAAGAACGACATGGAGCTGCTGCGTAAGACGAATAACGGCCGCGAC
ATCAATCGCCATTACTACTCCACCGCGGAAATCGACGAGAACCTCAAGCGTCCGGTCGTC
AAGGCCCTGAACGCGCTCGCCAAGTTCCGCAACGAGCTCGACGCGTTCGACGGCACGTTC
TCGTACACCACCGATGACGACACGTCCATCAGCTTCACCTGGCGCGGCGAAACCAGCCAG
GCCACGCTGACGTTCGAGCCGAAGCGCGGTCTCGGTGTGGACAACGCTACGCCGGTCGCC
ATGTTGGAATGGGAGGATTCCGCGGGAGACCACCGTTCGGATGATCTGATCGCCAATCCG
GTCGCCTGACTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCAG
1O GCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAA
TCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTC
CCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGG
TCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAA
GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAA
TCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACG
CCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTT
TGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC
ATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATT
CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCT
CACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGT
TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT
ATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGAC
GCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTAC
TCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCT
GCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCG
AAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGG
GAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCA
ATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAA
CAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTT
CCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATC
ATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGG
_.________________________*__________________________
AGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATT
AAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTT
CATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC
______________________________________________________________
CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCT
TCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTA
CCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGC
PCT/EP20121075639
TTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCAC
TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCT
GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGAT
AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG
ACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA
GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG
GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA
CTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGC
AACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT
GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCT
CGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTG
ATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTC
AGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTG
ACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTT
GTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGAGCTCGATATC
CCGGGCGGCCGCTTCATTTATAAATTTCTTGACATTTTGGAATAGATGTGATATAATGTG
TACATATCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGTCGACCTCG
The flux redirections and mutations for GDP—fucose and fucosyllated oligosaccharide
biosynthesis in a split metabolism are shown in Figure 8, both for a strain expressing a
heterologous ase and sucrose phosphorylase. onal to wcaJ, the colanic acid
operon genes that do not code for GDP-fucose biosynthesis reactions are knocked out,
such as gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaK, wcaL andlor, wcaM.
For the production of fucosyllactose, laCZ, coding for B-galactosidase, is knocked out to
avoid lactose degradation and the expression of lacY, coding for a lactose permease, is
enhanced by means of a strong tutive promoter.
Example 5: Enhancement of cose and fucosylated oligosaccharide
production via a split metabolism with e as substrate
When the genes pgi, pka, and pka are knocked out, carbon, taken up as glucose can
only be metabolised via the pentose ate pathway. Due to the biochemical
ties of this pathway, fructose-G-phosphate is formed (Figures 9 and 10). To form
biomass aldehyde—3—phosphate has to be formed, which is formed by the
transketolase reactions coded by thA and thB in E. coli. This Glyceraldehyde
phosphate is formed together with fructose—6—phosphate from xylulose—5—phosphate and
erythrose-S—phosphate. The latter is in turn formed together with fructose-S—phosphate
from glyceraldehyde—3-phosphate and sedoheptulosephosphate via transaldolase
ons coded by taIA and ta/B. To balance all of these reactions together the flux has to
be distributed between xylulose-5—phosphate and ribose—5-phosphate, as such that from 1
mole glucose, 2/3 mole of xylulosephosphate and 1/3 mole ribose—5-phosphate is
formed. To drive these equilibrium reactions, fructose-S—phosphate is pulled out of the
pentose phosphate pathway by the GDP—fucose and fucosyllacted accharide
thesis pathway. Additional to wcaJ, the colanic acid operon genes that do not code
for GDP—fucose biosynthesis reactions are knocked out, such as gmm, wcaA, wcaB,
wcaC, wcaD, wcaE, wcaF, wcal, wcaK, wcaL and/or, wcaM. For the production of
fucosyllactose, lacZ coding for B~galactosidase, is knocked out to avoid lactose
degradation and the expression of laCY, coding for a lactose permease, is enhanced by
means of a strong constitutive promoter.
Example 6: tative 2-fucosyllactose production with a fucosyltransferase
originating from Helicobacter pylori
The mutant strain in which the genes lacZ, g/gC, agp, pka, pka, pgi, arcA, iclR, wcaJ are
knocked out and lacY was expressed via constitutive expression to ensure sion
under all culturing conditions, was transformed r with a fucosyltransferase
originating from Helicobacter pylori and a e phosphorylase originating from
Bifidobacterium adolescentis, which were also constitutively expressed. The constitutive
promoters originate from the promoter library described by De Mey et al. 2007. This strain
was cultured in a medium as described in the materials and s, however with 30 g/l
of sucrose and 50 g/l of lactose. This resulted in the formation of 2—fucosyllactose as
shown in Figure 13 and 14.
Example 7: tative fucosyllactose production with a fucosyltransferase
originating from Dictyostellium discoideum
The mutant strain in which the genes lacZ, glgC, agp, pka, pka, pgi, arcA, iclR, wcaJ are
knocked out and IacY was expressed via constitutive expression to ensure expression
under all culturing conditions, was transformed further with a fucosyltransferase
originating from Dictyoste/lium deum and a sucrose phosphorylase originating from
Bifidobacterium adolescentis, which were also sed constitutively. The tutive
promoters originate from the promoter library described by De Mey er al. 2007. This strain
was cultured in a medium as described in the materials and methods, however with 30 g/l
of sucrose and 50 g/l of lactose. This resulted in the formation of 2-fucosyllactose as
shown in Figure 13 and 14.
e 8: Enhancement of GDP-mannose and mannosylated oligosaccharide
production via a split metabolism with sucrose as substrate
To late the GDP-mannose precursors fructose and fructose-S-phosphate, a
sucrose phosphorylase or invertase is introduced. Because fructose-G-phosphate is easily
ed in the glycolysis, the glycolysis is interrupted in order to steer all fructose—6—
phosphate in the direction of GDP-fucose. The genes pgi, pka and pka are thus knocked
out, coding for glucosephosphate isomerase and phosphofructokinase A and B. Finally
a yltransferase is introduced to link mannose to an acceptor molecule. To avoid
GDP-mannose ation the genes gmm and gmd have to be knocked out in the
colanic acid operon. in addition, the genes that do not code for GDP—mannose
biosynthesis reactions are d out, such as wcaA, wcaB, wcaC, wcaD, wcaE, wcaF,
wcal, wcaJ, wcaK, wcaL and/or, wcaM.
Example 9: lation of acid resistance related genes
Similar to the colanic acid operon upregulation, acid resistance related genes are also
upregulated in a AaroAA/c/R double mutant strain in comparison to the wild type strain and
the single mutant strains. These genes make a strain more resistant to low pH, which is
beneficial for the production of acids (4) or the production of glucosamine (12) which is not
stable at neutral and high pH. Figure 12 presents the gene expression pattern of these
acid resistance related genes and indicates up to 8 fold expression increase in the double
mutant strain.
Example 10: Fed batch production of 2-fucosyllactose
A mutant strain was constructed via the genetic engineering methodologies described
above with the following genotype:
AlacZYA::P22-la0YAglgCAagpApgiApka-P22-
kaAarcAAiciR::s/AwcaJA/onAadhE—PM-frk + pCXP14~FT_H. pylori (a vector with
sequence SEQ ID N°1). The promoter P22 and P14 originate from the promoter library
ucted by De Mey et a/ (11) and was cloned similar to the methodology described by
Aerts et al (2). “:zsl” marks a scarless gene deletion, thus without a FRT site that s
in the chromosome.
This strain was cultured in a bioreactor as described above in materials and methods, in
the mineral medium with 30 g/l of sucrose and 50 g/l of lactose. After the batch phase the
bioreactor was fed with 500 g/I of sucrose, 50 g/l lactose and 1 g/I of magnesium sulphate
heptahydrate. This led to the accumulation of 27.5 g/l of llactose in the supernatant.
SEQID N° 1: pCXP14-FT_H. pyiori
CGCGTTGGATGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCC
TGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCA
GTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCG
ATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGA
AAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC
CTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGG
TGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTG
1O ACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAA
ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGA
AGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCC
TTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG
GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC
GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTAT
TATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATG
ACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAG
AATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAA
GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTC
GCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA
CGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTC
CCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC
TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTG
GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTA
TCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAG
GTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGA
TTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC
TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA
AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA
AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC
CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGT
AGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCC
TGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGAC
GATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCA
GCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG
CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG
GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGT
TTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTAT
GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC
ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGT
GAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAG
CGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCA
TATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCC
GCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGC
GCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGG
GAGAGCTCGATATCCCGGGCGGCCGCCTTCATTCTATAAGTTTCTTGACATCTTGGCCGG
GTATAATAGGGAAATTTCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGTC
GACCTCGAATTCGGAGGAAACAAAGATGGCCTTTAAAGTTGTTCAGATTTGTGGTGGTCT
GGGCAATCAGATGTTTCAGTATGCATTTGCAAAAAGCCTGCAGAAACATAGCAATACACC
GGTTCTGCTGGATATTACCAGCTTTGATTGGAGCAATCGTAAAATGCAGCTGGAACTGTT
TCCGATTGATCTGCCGTATGCAAGCGAAAAAGAAATTGCAATTGCCAAAATGCAGCATCT
GCCGAAACTGGTTCGTAATGTTCTGAAATGCATGGGTTTTGATCGTGTGAGCCAAGAAAT
TGAATATGAACCGAAACTGCTGAAAACCAGCCGTCTGACCTATTTTTATGGCTA
TTTTCAGGATCCGCGTTATTTTGATGCAATTAGTCCGCTGATCAAACAGACCTTTACCCT
GCCTCCGCCTCCGGAAAATGGTAATAACAAAAAAAAAGAAGAAGAGTATCATCGTAAACT
GGCACTGATTCTGGCAGCAAAAAATAGCGTGTTTGTGCATATTCGTCGCGGTGATTATGT
TGGTATTGGTTGTCAGCTGGGCATCGATTATCAGAAAAAAGCACTGGAATACATGGCAAA
ACGTGTTCCGAATATGGAACTGTTTGTGTTTTGCGAGGACCTGGAATTTACCCAGAATCT
GGATCTGGGCTATCCGTTTATGGATATGACCACCCGTGATAAAGAGGAAGAGGCATATTG
GGATATGCTGCTGATGCAGAGCTGTAAACATGGTATTATTGCCAACAGCACCTATAGTTG
AGCATATCTGATTAATAACCCGGAAAAAATCATTATTGGTCCGAAACATTGGCT
GTTTGGCCATGAAAACATCCTGTGTAAAGAATGGGTGAAAATCGAAAGCCACTTTGAAGT
GAAAAGCCAGAAATATAATGCCTAATAAGAGCTCCCAA
Example 11: Fed batch production of 2-fucosyllactose with a hybrid colanic acid
promoter
A hybrid colanic acid promoter was constructed based on the genome information and the
sequences from the promoter library described by De Mey eta/(11).
AlacZYA::P22—lacYAglgCAagpApgiApka::P22-BaSPApka AarcAAiclesl chaJ Alon
PM—frk AldhA::P14- FT_H. pylori ApromCA:P14
This strain was cultured in a ctor as described above in materials and methods, in
the mineral medium with 30 g/I of sucrose and 20 g/l of lactose. After the batch phase the
bioreactor was fed with 500 g/l of sucrose, 20 Q]! lactose and 1 g/l of magnesium sulphate
heptahydrate. This led to the accumulation of 26 g/l of fucosyllactose in the atant
with nearly stoichiometric conversion of lactose. increasing the lactose feed
concentrations leads further to increased final fucosyllactose titers and stoichiometric
e conversion.
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Claims (20)
- CLAIMS : Use of a modified expression of the transcriptional regulators; the aerobic respiration control n ArcA and the isocitrate lyase regulator lclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon ses the genes cpsG, cpsB, gmd and fc/ that code for a phosphomannomutase, a mannosephosphate guanylyltransferase, GDP- mannose 4,6-dehydratase and GDP-fucose synthase, respectively.
- Use according to claim 1, wherein said upregulation of at least one of the genes of 10 the c acid operon is preceded by the upregulation of the transcriptional regulator of said c acid operon rcsA.
- Use according to claim 1 or 2, wherein the transcriptional regulators, the aerobic respiration control protein ArcA and the isocitrate lyase regulator lclR are expressed by an Escherichia coli , wherein said E. coli strain is specifically a 15 K12 strain or, wherein said K12 strain is more specifically E. coli M61655.
- Use according to any one of claims 1-3, wherein said modified expression results from disrupting the genes encoding for ArcA and chR, replacing the endogenous promoters of the genes encoding for ArcA and lclR by artificial promoters, or replacing the endogenous ribosome binding site by an artificial ribosome binding 20 site.
- Use according to any one of claims 1-4 wherein said modified expression is a decreased expression, or n said sed expression specifically is an hed expression.
- Use ing to any one of claims 1-5, n at least one of the genes of the 25 colanic acid operon are upregulated 6 to 8 times in comparison to the expression of the colanic acid operon in the corresponding wild type microorganism. .
- A process for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising: cally changing the transcriptional regulators, the aerobic respiration control protein ArcA, and the isocitrate lyase regulator lclR, 30 to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl. .
- A process according to claim 7, wherein said genetically changing comprises at least one mutation that enhances the production of fucosylated compounds, selected from the group consisting of: 35 the on of the wcaJ gene; knocking-out the colanic acid operon genes gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc and/or wcaM; knocking-out lacZ; introducing a sucrose phosporyiase or ase; knocking out the genes pgi, pka and pka; knocking out the gene Ion; introducing a fucosyltransferase and/or a lactose se; or combinations thereof.
- A s according to claim 7, for the synthesis of GDP-mannose and/or for the synthesis of mannosylated accharides.
- 10. A process according to claim 9 wherein the genes cpsG and cpsB of the colanic acid operon are upregulated and wherein: a) the gene gmd of the colanic acid operon is deleted, and/or, 1O b) wherein the gene gmm is deleted, and/or c) wherein the colanic acid operon genes fcl, gmd, gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc, and/or, wcaM are knocked out and/or, d) wherein a gene encoding for a sucrose phosphorylase or an invertase is 15 uced, and/or, e) n the the genes pgi, pka and pka are deleted, , f) knocking out the gene Ion, and/or 9) wherein a gene encoding for a mannosyltransferase is uced.
- 11. A mutated and/or transformed bacterium in which the regulators ArcA and chR, in 2O combination with the genes encoding for the enzymes phosphoglucose isomerase and phosphofructokinase, are knocked out or are rendered less functional.
- 12. A mutated and/or transformed bacterium according to claim 11 n the enzyme phosphoglucose isomerase is encoded by the gene pgi and wherein the enzyme phosphofructokinase is encoded by the gene(s) pka and/or pka. 25
- 13. A mutated and/or transformed bacterium according to claim 11 or 12 wherein said organism is further transformed with a gene encoding for a sucrose phosphorylase or invertase.
- 14. A mutated and/or transformed bacterium according to any one of claims 11 to 13 wherein the ty of the gene encoding for a lactose permease is increased. 30
- 15. A mutated and/or ormed bacterium according to any one of claims 11 to 14 wherein at least one of the following genes is knocked out or is rendered less functional: a gene encoding for a beta-galactosidase, a gene encoding for a glucose phosphate adenylyltransferase, a gene encoding for a glucose—1-phosphatase, a 35 gene encoding for phosphogluconate atase, a gene encoding for 2—keto-3— deoxygIuconate-S-phosphate aldoiase ,a gene encoding for a glucose phosphate transferase, a gene encoding for an UDP-glucoseepimerase, a gene encoding for an UDP-glucosezgalactose-t—phosphate uridyltransferase, a gene encoding for an UDPgalactopyranose mutase, a gene ng for an UDP- galactose:(glucosyl)Iipcpolysaccharide-1,6-galactosyltransferase, a gene encoding for an UDP-galactosyltransferase, a gene encoding for an UDP- glucosyltransferase, a gene encoding for an UDP-glucuronate transferase, a gene encoding for an UDP-glucose lipid carrier transferase, a gene encoding for a GDP- mannose hydrolase, a gene encoding for an UDP—sugar hydrolase, a gene encoding for a mannose-B-phosphate isomerase, a gene ng for an UDP-N- 1O acetylglucosamine enoylpyruvoyl transferase, a gene ng for an UDP— Nacetylglucosamine acetyltransferase, a gene encoding for an UDP- Nacetylglucosamine-Z-epimerase, a gene encoding for an undecaprenyl- phosphate alfa—N-acetylglucosaminyl transferase, a gene encoding for a glucose- 6-phosphatedehydrogenase, and/or, a gene encoding for a L-glutaminezD- 15 fructosephosphate aminotransferase, a gene encoding for a mannose-6— phosphate ase, a gene encoding for a ol-G-phosphate dehydrogenase, a gene encoding for a mannitol—‘l-phosphate 5-dehydrogenase, a gene encoding for a allulose-B—phosphate 3-epimerase, a gene encoding for an invertase, a gene incoding for a maltase, a gene encoding for a trehalase, a gene encoding for a 20 sugar transporting phosphotransferase, a gene ng for a protease, or a gene encoding for a hexokinase.
- 16. Use of a modified expression of the transcriptional regulators the aerobic ation control protein ArcA and the isocitrate lyase regulator lclR, to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, 25 hdeA, hdeD, gadB, gadC, gadE, gadX, gadWand/or slp.
- 17. Use ing to claim 16 for the synthesis of acids, sialic acid, sialylated oligosaccharides or glucosamine.
- 18. Use according to claim 16 or 17, wherein said modified expression is a decreased expression, or wherein said decreased expression is an abolished expression. 3O
- 19. A process for the synthesis of acids, sialic acid, sialylated oligosaccharides or glucosamine comprising cally ng the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase regulator chR to upregulate at least one of the ing acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, d/or slp. 35
- 20. A use according to claim 1 or 16, substantially as herein described with reference to any one of the Examples and/or
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11194103.5 | 2011-12-16 | ||
EP11194103 | 2011-12-16 | ||
PCT/EP2012/075639 WO2013087884A1 (en) | 2011-12-16 | 2012-12-14 | Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides |
Publications (2)
Publication Number | Publication Date |
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NZ625199A NZ625199A (en) | 2015-07-31 |
NZ625199B2 true NZ625199B2 (en) | 2015-11-03 |
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