NZ755558A - In vivo synthesis of sialylated compounds - Google Patents
In vivo synthesis of sialylated compoundsInfo
- Publication number
- NZ755558A NZ755558A NZ755558A NZ75555817A NZ755558A NZ 755558 A NZ755558 A NZ 755558A NZ 755558 A NZ755558 A NZ 755558A NZ 75555817 A NZ75555817 A NZ 75555817A NZ 755558 A NZ755558 A NZ 755558A
- Authority
- NZ
- New Zealand
- Prior art keywords
- microorganism
- sialylated
- seq
- acetylglucosamine
- acetyl
- Prior art date
Links
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Abstract
The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered microorganisms. The present invention describes engineered microorganisms able to synthesize sialylated compounds via an intracellular biosynthesis route. These microorganisms can dephosphorylate N-acetylglucosamine-6-phosphate to N- acetylglucosamine and convert the N-acetylglucosamine to N- acetylmannosamine. These microorganisms also have the ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the present invention provides a method for the large scale in vivosynthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose, N-acetyllactosamine and/or an aglycon, wherein said microorganism intracellularly dephosphorylates N-acetylglucosamine-6-phosphate to N-acetylglucosamine, converts N-acetylglucosamine to N- acetylmannosamine and convert the latter further to N-acetyl- neuraminate. sialylated compounds via an intracellular biosynthesis route. These microorganisms can dephosphorylate N-acetylglucosamine-6-phosphate to N- acetylglucosamine and convert the N-acetylglucosamine to N- acetylmannosamine. These microorganisms also have the ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the present invention provides a method for the large scale in vivosynthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose, N-acetyllactosamine and/or an aglycon, wherein said microorganism intracellularly dephosphorylates N-acetylglucosamine-6-phosphate to N-acetylglucosamine, converts N-acetylglucosamine to N- acetylmannosamine and convert the latter further to N-acetyl- neuraminate.
Description
INTERNATIONAL APPLICATION PUBLISHED UNDER THK PATENT COOPERATION TREATY (PCT)
(12)
(19) World Intellectual Property
llIIlIIlllIlIlllIlIllIllIllIIIIIIIIIIIIIIIIIIIIllIlIllIlIllIlIllIlIllIIlIlllIIIIIIIIIIIIIIIIIII
Organuation
International Burem&
(10) International Publication Number
(43) International Publication Date
WO 201S/122225 A1
I I PC) I P C T
05 July 2010 (05.07 201(I)
(51) International Patent Claasification: (71) Applicant: LNBIOSK N.V. /BE]; 41, Teclmologiepark
C12P 19/26 (2006 01) C12&V 9/88 (2006 01) 0&naarde 3, 9052 Gent (BE).
C/2P 19/18 (2006 C12/V 9/12 (2006
01) 01)
(72) Inventors; and
C12&V15/52 (2006.01) C12iV 9/88 (2006 01)
(71) Applicanm (fi&n US i&ni&/: BEAUPREZ,,loeri [BE/BE];
C12/V 9/16 (2006 01) C12/V 1/21 (2006 01)
Frmd&rijkiaan 10, 8450 Bredene (BE) COUSSKMENT,
C12&V 9/98 (2006 01) C12&V1/19 (2006 01)
Pieter Jan dhondtstraat 9050 (ientbnivge
[BE/BE]; 29,
C12/V
9/18 (2006 01)
VAN HERPE, Dries Melan»treat
(BE). [BE/BE]; 215,
(21) International Application Number: 9032 Wondelgem (BE). PETERS, (*crt [BE/BE]; Hen-
VER-
ri Dutmntk&an 20 bus 203, 9000 (ient (BE)
CAUTKREN, Annelies [BE/BE]; Eedstraat 19, 9810 Eke
International 1&iling Date:
(22)
(BE).
26 Dcccmbcr 2017 (26.12.2017)
(74) Agent: SAELENS, Claire; Tcchnologicpark Zw&jnaardc 3
lriling Ell lbll
(25) I.anguage:
bus 41, 9052 Gent (BE).
Publication I.miguagc: I/n lish
(26)
Designated States ianie»» nrhernive inr(/carer/, /ar even:
(gl)
kin&/ nariana/ r&vr&ilahlaf. AE, ACj, AL, AM,
(30) Priority Data: nf
pm/ac/inn
I'.P
t( 01916 5 27 December 2016 12.2016) AO, AT, AU,AZ, BA, 13B, BG, 13H, 13N, 13R, BW, 13Y, BZ,
Dl-:,
CA, CH, CL, CN, CO, CR, CU, CZ, DJ, DK, DM, DO,
Title: IN VIVO SYNTIB',SIS OF SIALYLATED COMPOUNDS
(54)
Fig. 3:
6csjalyllactose,
Lactose
Sialyiatediactose 3'-sialyilactose,
3',Eadisialyllactose
Sialic acid
Disialyllacto-N-tetraose„
saccharide
Sialylated
Disialyllacto-N-neotetraose
CMP-Slalic
acid
Sjalyl 1 d
cult-
protein
Sialylated protein
- - . -
Sialyfatedhpid
Siaiylated ganglioside
Siafylated
aglycan
Abstract: Thc uivcntion is in thc tccluuctd tield of synthetic and nmtabolic Morc thc
(57) prcscnt biology cnginccnng. panicularly,
uivcntion is ui thc tccluuc:d iield of fi:micntauon of mctabolically cn inccrcd microorga&usm». Tlm uivmition dcscribcs
prcscnt prcscnt
miguiccrcd microorganisms able to synthcnrc»ralylatai compo&uids via tui intraccliular biosynthc»is route. Thcsc microor anism» can
N- N-
dcphosphorylatc N-acctylglucosamincpho»phatc to acctylglucosaminc and convert thc N-acctylglucosaminc to acctylman-
no»amuic These microorgaiusm» also have thc ability to corn crt N-acctylmannosaminc to N-acetyl-ncuramiimtc, Fut&hcrmorc, thc
invention a method for tim scale m riva sialylatcd a microorgaiusm ui a
prcscnt provides large synthc»is of compoiuid&, cultunng
urec or such but not lnuucd to lactoNbiosc, N-acctyilactosamuic and/or
medium, opuonally compnsing:ui cxogmious prccur. a», lect&&sc,
an aglvcon, wllcrcui said microorgailis&11 uitrt&cclhilarlv dcphosphotylt&tcs N-acctytgluco»aululcphosphate to N-acetyl i&&co»attune,
couvcru N-acctylglucosamiiic to acctylmanno. amuic and convert thc lath:r liunhcr to N-acetyl- ncuramiimtc.
[C&niii mind &&n nevi
page/
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Declarations under Rule 4.17:
of'rhe
us ti& rlie idennrp uivenror (Rule 4.l 7(rft
invenrorsr'nP (Rule 4.(7(reft
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In vivo of
synthesis sialylated compounds
The present invention is in the technical field of synthetic and metabolic engineering.
biology
More particularly, the present invention is in the technical field of fermentation of metabolically
engineered microorganisms. The present invention describes engineered micro-organisms able
via an intracellular route. micro-
to synthesize sialylated compounds biosynthesis These
organisms can dephosphorylate N-acetylglucosaminephosphate to N-acetyl glucosamine and
convert the N-acetylglucosamine to N-acetylmannosamine. These micro-organisms also
havethe ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the
present invention provides a method for the scale in vivo synthesis of sialylated
large
compounds, culturing a microorganism in a culture medium, optionally comprising an
exogenous precursor such but not limited to lactose, lacto-N-biose, N-acetyllactosamine
and/or an wherein said microorganism intracellularly dephosphorylates
aglycon,
converts to
acetylglucosaminephosphate to N-acetylglucosamine, N-acetylglucosamine
acetylmannosamine and convert the latter further to N-acetyl-neuraminate.
Background
Sialylated compounds such as sialic acid and sialylated oligosaccharides have gained attention
the last because of their broad For sialic acid is considered as
years, application range. example,
an anti-viral precursor. Sialylated oligosaccharides form an essential of human milk and are
part
anti-adhesive
ascribed and immunomodulatory properties; others described them to be
involved in brain development. Sialylation, in of lipids or are used in
general, proteins, aglycons
anti-cancer
medicine and in the treatment of neurological diseases.
Sialic acid is a general term used to describe a large family of acidic sugars that are
predominantly found on the cell surface of eukaryotic cells. The most common sialic acid is
nine-carbon
acetylneuraminic acid or Neu5Ac, an acidic sugar that undergoes several
modifications to generate the members of the sialic acid family. As seen in e.g. Fig. 1 of
yy02008097366, the diversity of the sialic acid family is represented with over 50 known
cell-surface
members. Sialic acid represents a large family of carbohydrates that are derived
from an acidic, nine-carbon parent compound called N-acetylneuraminic acid or Neu5Ac.
Neu5Ac is often decorated with acetyl, phosphate, methyl, sulfate and lactyl groups, which are
described to be required for desirable cell signalling and cell adhesion events mediated sialic
acid.
Sialic acids and sialylated compounds are common in higher eukaryotic organisms which
UDP-N-
produce them in a conserved biosynthetic route. This route starts from endogenic
UDP-N-
acetylglucosamine which is converted to sialic acid through the action of a
2-epimerase N-acylmannosamine
acetylglucosamine (hydrolysing) (EC 3.2.1.183), a kinase (EC
Neu5AcP
2.7.1.60), a N-acylneuraminatephosphate synthase (EC 2.5.1.57) and a
3.1.3.29). This sialic acid can be activated and transferred to the
phosphatase (EC subsequently
CMP-sialic
40 desired acceptor via a acid synthase 2.7.7.43) and e.g. a sialyltransferase.
Efforts have been made to express this biosynthetic route in other eukaryotic organisms,
whereas prokaryotic systems were not reported. The was functionally expressed in
pathway
yeast (P/ch/a pastor/s) and plant (Arabidops/s thaliana) to produce sialylated N-glycans.
However, large scale production of sialylated oligosaccharides was never reported. The
functional overexpression of eukaryotic in systems remains a daunting task
genes prokaryotic
without certain outcome due to the lack of specific chaperones, faulty enzyme folding and
missing cell organelles. On top of that remains the huge energy requirement of the pathway and
the depletion of intercellulair UDP-GlcNAc (UDP-N-acetylglucosamine), necessary for cell
growth.
Processes based on enzymatic, chemical as well as fermentative production of sialylated
compounds exist. However, all of them have significant disadvantages. For instance, chemical
synthesis requires many sequential chemical steps and enzymatic synthesis requires expensive
whereas the fermentative process is still under development. Nonetheless,
precursors, heavy
the latter has the highest industrial production potential.
One of described fermentative production process uses a biosynthesis route that originates
type
from prokaryotes like Campylobacter jejuni that naturally produces sialic acid or sialylated
UDP-N-acetylglucosamine
compounds. This biosynthesis route starts from endogenous which
cells use for their cell wall. This is converted to ly-acetylmannosamine and N-acetylneuraminate
the action of an UDP-N-acetylglucosamine epimerase named and a sialic
by (generally neuC)
acid synthase (generally named neuB).
of this prokaryotic biosynthesis route, Priem et al. 2002, 235-
Using only part (Glycobi ology 12,
240) describe the use of living bacterial cells to produce sialyloligosaccharides. In this method,
cells of Escherichia coll
sialyllactose was directly produced by growing metabolically engineered
strains which overexpressed the Ne/sser/a mening/t/d/s genes for alpha-2,3-sialyltransferase and
for CMP-Neu5Ac synthase, these strains were further devoid of beta-galactosidase and
acetylneuraminic acid (Neu5Ac) aldolase activities. These microorganisms were grown at high
cell density with glycerol as the carbon and energy source, while exogenous lactose and Neu5Ac
were supplied as precursors for sialyllactose synthesis. During the growth, lactose and Neu5Ac
were internalized the induction of the expression of an E. col/ galactoside and an exogenous
NeuSAc permease. Lactose and NeuSAc accumulate in the cytoplasm where NeuSAc was then
converted into CMP-Neu5Ac to be further transferred on lactose to form sialyllactose. Large
scale of
production sialyloligosaccharides this microbiological method requires important
amounts of Neu5Ac as a precursor.
Another microbial system was developed for production of sialyloligosaccharides without the
need of an exogenous of sialic acid. WO2007101862 describes such method for
supply
producing sialylated oligosaccharides with microorganisms comprising heterologous genes
encoding a CMP-NeuSAc synthetase, a sialic acid synthase, an UDP-GlcNAcphosphate
a wherein the for sialic acid
epimerase and sialyltransferase, and endogenous genes coding
aldolase and for ManNAc kinase have been deleted or inactivated. The use of
(NanA) (NanK)
40 this route intensive for cell.
prokaryotic biosynthesis is very energy the Furthermore, the
described route for producing the sialylated oligosaccharides competes for the UDP-GlcNAc
which is essential for the cells own synthesis. Building on this et al.
peptidoglycan concept, Kang
have created a production host that does not use a sialic acid synthase, but the endogenous
sialic acid aldolase, which has a less favourable chemical equilibrium (Metabolic engineering 14,
2012, 623-629).
cali
EP1484406 describes the production of Neu5Ac using E. overexpressing
acetylglucosamine 2-epimerase and Neu5Ac synthase, but needs N-acetylglucosamine (GlcNAc)
as external precursor. In the described method, GlcNAc needs to be used as such. Therefore,
the cells in EP1484406 need to be disrupted such that the GlcNAc can be used directly the
GlcNAcepimerase.
As described by Lundgren et al. (Org. Biomol. Chem., 2007, 5, 1903 1909)
intact cells will convert the incoming GlcNAc to N-acetylglucosaminephosphate (GlcNAcP)
which GlcNAcP
will be used by the cell for cell growth. This is not available intercellular and
can therefore not be used for the GlcNAcepimerase which needs a non-phosphorylated
GlcNAc for epimerisation to ManNAc. This explains permeabilization of the cells of
GlcNAcP
EP1484406 is necessary. As explained Lundgren et al., the can be used for making
Neu5Ac but this requires another synthesis LIDP-GlcNAc as an
pathway comprising
intermediate, which is described above in WO2007101862. The resulting pathway further
increases demand compared to the one described in the latter patent because
energy
uridylation of GlcNAc requires an extra ATP.
et 201-214)
Deng al. (Metabolic Engineering 7 (2005), describes the production of GlcNAc via
GlcNAcP
intracellular production of which is then efficiently dephosphorylated and secreted
into the medium as GlcNAc. According to et this
Deng al., dephosphorylation happens upon
export, more specifically in the periplasm of Escherlchia coll. The extracellular produced GlcNAc
described in this method, is not available for intracellular conversion. This method to produce
GlcNAc requires a two-phase fed batch process, l.e. a cell growth phase followed a GlcNAc
production which is induced after the culture had reached a cell to
phase only high density,
minimize inhibitory effects of phosphorylated amino sugars.
Others have attempted the same heterologously expressing phosphatases and encountered
the problem of reduced growth and strong metabolic burden (Lee and Oh, Metabolic
engineering, 2015, 143-150). The main reason for said reduction in growth/biomass formation
is the non-specificity of the phosphatase that is introduced, which dephosphorylates other
essential compounds. Such modifications hence lead to reduced fitness and
phosphorylated
lower specific productivity. It furthermore leads to selective pressure to mutate the production
during production, which reduces the overall process stability.
pathway
The of sialic acid the formation of
production pathways and sialylated oligosacharides require
level of GlcNAcP) and nucleotide intermediates. It is
high phosphorylated (e.g. pathway
commonly understood that such formation leads to aspecific degradation of these
intermediates activation of aspecific phosphatases, which in turn leads to reduced fitness. In
order to circumvent the effect of the expression of metabolic pathways on the growth of the
production hosts, it is standard to use inducible expression systems. In this method first biomass
formed and later in the the activated for
is production process production pathway is
instance IPTG. This was others for the production of sialic acid and sialylated
applied by
40 Priem et al. 235-240; et
oligosaccharides (WO2007101862; Glycobiology 12, 2002, Kang al.,
Metabolic engineering 14, 2012, 623-629; Yang et al., Metabolic engineering 43, 2017, 21-28).
from losing productivity and titer, another downside in the use of inducible systems is the
Apart
excretion of intermediate pathway metabolites such as GlcNAc and ManNAc. This leads to the
requirement of extra downstream processing steps for the purification, hence a higher
production cost in the production of sialic acid, sialyllactose or other sialylated compounds.
The methods for producing sialylated compounds, discussed hereabove, are still insufficient in
meeting the large demand of the biotechnological, pharmaceutical and medical industries. A
metabolic that successfully overcomes the problems referred to above,
engineering approach
would represent a significant and long awaited advance in the field.
Summary
we have been able to create a production that does not require induction,
Surprisingly, pathway
and does not require a UDP-GlcNAc epimerase, but allows constitutive expression which also
allows better tuning of the metabolic production and reducing
pathway improving byproduct
formation during the production process.
According to one embodiment of the present invention, there is provided a method for
sialylated compound production with microorganisms which does not require induction.
According to a further embodiment of the present invention, there is provided a production
pathway that does not require a UDP-GlcNAc epimerase, and comprising modulating expression
of phosphatase which does not a metabolic burden to the cell as was shown in
pose previously
the art. Said further embodiment of the present invention provides also an increased sialylated
compound production modulating the expression of phosphatase.
In another further embodiment, the above method, when combined with the constitutive
expression of the of the metabolic also allows better tuning of the metabolic
genes pathway,
pathway reducing byproduct formation during the production process.
Description
describes an efficient alternative
The present invention economical, more and biosynthesis
route for the production of sialylated compounds micro-organisms.
using
The present invention provides a method of producing sialylated compounds by fermentative
growth of microorganisms.
In particular, the invention relates to a method for the production of sialylated compounds,
wherein the method comprises culturing a microorganism in a culture medium. The
converts reactions: N-acetylglucosaminephosphate
microorganism intracellularly following
to N-acetylglucosamine, /V-acetylglucosamine to N-acetylmannosamine, and
N-acetyl-neuraminate.
acetylmannosamine to Furthermore, this microorganism is unable to: i)
convert N-acetylglucosamineP to glucosamineP, /i) convert N-acetylglucosamine to
N-acetyl-neuraminate N-acetyl-mannosamine.
acetylglucosamineP, and /ii) convert to
Preferably, the conversion of N-acetylglucosaminephosphate to IV-acetylglucosamine is
obtained action an intracellularly In another
the of expressed phosphatase. preferred
embodiment the N-acetylglucosamine is converted to IV-acetylmannosamine an
intracellularly /V-acetylmannosamine In an alternative
expressed epimerase. preferred
embodiment the N-acetylmannosamine is converted an intracellular expressed sialic acid
N-acetyl-neuraminate.
synthase to Even more preferably, the microorganism comprises all
three enzymes such that the microorganism converts N-acetylglucosaminephosphate to
acetylglucosamine by action of an intracellularly expressed phosphatase, /i) the
acetylglucosamine to N-acetylmannosamine an intracellularly expressed
acetylmannosamine and/ii) the N-acetylmannosamine to N-acetyl-neuraminate
epimerase;
an intracellular expressed sialic acid synthase.
Preferably, the microorganism used in the method of the invention is unable to produce
following enzymes a N-acetylglycosaminephosphate deacetylase,/i) a N-acetylglucosamine
N-acetylneuraminate
kinase, and i/i) a aldolase.
The present invention also provides a microorganism which expresses a phosphatase to
N-acetylglucosaminephosphate to N-acetylglucosamine a
dephosphorylate (EC 3.1.3.), /i)
GlcNAc 2-epimerase to convert N-acetylglucosamine (GlcNAc) to N-acetylmannosamine
and a sialic acid synthetase to synthesise N-acetyl-neuraminate
(manNac) (EC 5.1.3.8), /ii)
(Neu5Ac) from N-acetylmannosamine (ManNAc) (EC 2.5.1.56). Furthermore, this
microorganism is unable to: convert N-acetylglucosamineP to glucosamineP, convert
i) /i)
N-acetyl-glucosamine to N-acetyl-glucosamineP, and iii) convert N-acetyl-neuraminate to
acetyl-mannosamine.
In one aspect, the invention provides a micro-organism that is enabled to catalyse the following
reactions: the intracellular conversion of N-acetylglucosaminephosphate to
acetylglucosamine, the intracellular conversion of N-acetylglucosamine to
acetylmannosamine the intracellular conversion of N-acetylmannosamine to sialic acid.
and,
It is generally accepted that N-acetylglucosaminephosphate is naturally efficiently excreted
out of the cell and meanwhile dephosphorylated phosphatases in the periplasm
by (see p. 212,
et Metabolic 7 201-214). without
second column, Deng a/., Engineering Therefore, the
(2005),
present invention, this excreted product would be unavailable for conversion to sialic acid.
re-internalization N-
Furthermore, occurs through transport proteins which phosphorylate the
acetylglucosamine.
N-acetylglucosamineepimerase
The use of an intracellular ensures lower energy (ATP)
consumption than the classical prokaryotic route UDP-N-acetylglucosamine). This enables
(via
a more efficient production of sialic acid, sialylated oligosaccharides and/or sialylated products
with a healthier and more efficient strain. optimizing expression levels, the unfavourable
chemical equilibrium is overcome and no need of large amounts of free N-acetylglucosamine
are necessary, as is in literature. Indeed, in the art, this enzyme is solely used in enzymatic
reactions which use concentrations of N-acetylglucosamine to produce
high
acetylmannosamine. It would be hence logical that the use of an epimerase would require
large
amounts of intracellular formed GlcNAc which is shown to be released in the medium (see Deng
as described however, the present invention has proven this can be avoided. Another
supra),
of the invention over that substrates
advantage present enzymatic methods, is inexpensive can
be used in the present invention, as for example a monosaccharide such as for example
glucose,
40 galactose or fructose, a disaccharide such as for example sucrose or maltose or a polyol, such
as, but not limited to, glycerol. This enables an economic production method fermentation.
N-acetylglucosaminephosphate
Different phosphatases (EC 3.1.3.) that convert into
acetylglucosamine are described in the art and can be used in the present invention.
HAD-like
Phosphatases from the HAD superfamily and the family are described in the art.
Examples from these families can be found in the enzymes expressed from genes inhX,
yqaB,
or from Escher/ch/a co/i. One that catalyzes this
yn/C, yb/V, yldA, cof phosphatase
ybjl, ylgL
reaction is identified in Blastocladl ella emerson//. Phosphatases are generally aspecific and the
activity is generally not related to the family or structure. Other examples can thus be found in
well-
all phosphatase families. Specific phosphatases are easily identified and screened
known methods as described Fahs et al. Chem. Biol., 2016, 11 2944-2961).
by (ACS (11),
Preferably, the phosphatase of the present invention is a HAD-alike phosphatase. A HAD-alike
phosphatase as defined herein refers to phosphatase which comprises:
any polypeptide
one or more of the following motifs as defined below:
Motif 1: ID NO: or
hDxDx[TV] (SEQ 73),
Motif 2: x(1-2) ID NOs:
[GSTDE][DSEN]x(1-2)[hP] [DGTS] (SEQ 74, 75, 76, 77)
wherein h means a hydrophobic amino acid I, L, M, F, V, P, G) and x can be
any distinct amino acid.
HAD-alike
In another preferred embodiment, polypeptides typically have in increasing order of
preference at least 82 83 85 88 89 90 92
80%, 81%, %, %, 84%, %, 86%, 87%, %, %, %, 91%,
93 94 95 96 97 98 or 99 % overall sequence identity to any one of the
%, %, %, %, %, %,
represented SEQ ID NOs: 43,44, 45, 47, 48, 50, 51, 52, 54, 55 or 57. Preferably,
polypeptides by
those polypeptides also comprise at least one of the above identified Motifs. More preferably,
they comprise both motifs.
The overall determined
sequence identity is using a global alignment algorithm, such as the
Needleman Wunsch algorithm in the GAP Wisconsin Package,
program (GCG Accelrys),
with default with of mature
preferably parameters and preferably sequences proteins (i.e.
without taking into account secretion signals or transit peptides). Compared to overall sequence
the sequence identity will be when conserved domains or motifs
identity, generally higher only
are considered.
In a preferred embodiment, the HAD-alike comprises one of SEQ ID NOs: 43
polypeptide any
55 or 57.
,44, 45, 47, 48, 50, 51, 52, 54,
another is chosen from the HAD or
In preferred embodiment, the phosphatase superfamily the
HAD-like phosphatase family. More preferably, the phosphatase is chosen from the
group
enzymes expressed the or from
comprising: i) by genes yqaB inhX, yni C, ybi V, yl dA, ybj l, yigL cof
Escher/ch/a co/i, ii) the phosphatase of Blastocladiella emerson/i and ///) other phosphatase
families.
Examples of N-acetyl-D-glucosmineepimerase 5.1.3.8) can be found in prokaryotes and
are in like for Acaryochloris
eukaryotes. Examples for prokaryotes found cyanobacteria example
marina, Anabaena var/nb/ lls, Anabaena marina, Nostoc punctiforme, Acaryochloris species,
Anabaena Nostoc species and Synechocystis species. are also found in Bacteroides
species, They
40 species like for example Bactero/des ovatus and Bactero/des theta/otaom/cron and in
N-acetyl-D-glucosmine
Capnocytophaga canimorsus and Mobiluncus mul/er/s. In eukaryotics,
WO 2018)122225 PCTIEP2017)084593
epimerase is found in Glycin max, Mus musculus, Homo sapiens, Rattus norvegicus, Bos Taurus,
Sus Canis lupus. Preferably, in the method and microorganism of the present invention,
sero/a,
N-acetylmannosamineepimerase
is chosen from the group comprising i)
acetylmannosamineepimerase from cyanobacteria, more in particular from Acaryochloris
marina, Anabaena variabilis, Anabaena marina, Nostoc Acaryochloris
punctiforme, species,
Anabaena species, Nostoc species and Synechocystis species; N-acetylmannosamine
epimerase from Bacteroides species, more in particular from Bacteroides ovatus, Bacteroides
N-acetyl-D-
thetaiotaomicron, Capnocytophaga canimorsus and Mobiluncus mulieris; iii)
glucosmineepimerase from Glycin Mus musculus, Homo Rattus norvegi Bos
max, sapiens, cus,
Taurus, Sus sero/a or Canis lupus.
N-acetyl neuraminate synthase called sialic acid synthase in the 2.5.1.56) activity
(also art) (EC
is found in several prokaryotic organisms like for example Streptococcus agalatiae, Bacillus
subtilis, Legionella Idiomarina loihiensis, Moritella viscosa,
pneumophilla, Campylobacterjejuni,
Aliivibrio salmonicida, Escherichia coli, Methanocaldococcus jannaschi, Clostridium sordellii,
Butyrivibrio proteoclasticus, Micromonas commoda or Iyeisseria meningitis. Preferably, in the
N-acetyl
method and microorganism of the invention, the sialic acid (or neuraminate) synthase
Streptococcus Bacillus
is chosen from the group comprising: sialic acid synthase from agalatiae,
subtilis, Legionella pneumophilla, Campylobacterj uni, Idiomarina loihiensis, Mori tella viscosa,
Aliivibrio salmonicida, Escherichia coli, Methanocaldococcus jannaschi, Clostridium sordellii,
vibrio
Butyri proteoclasticus, Micromonas commoda or Neisseria meningitis.
In one preferred aspect, any one or more of the phosphatase, N-acetylmannosamine epimerase
and sialic acid synthase is overexpressed in the microorganism. In an alternative preferred
one or more of the N-acetylmannosamine epimerase and sialic acid
aspect, any phosphatase,
synthase is introduced and expressed in the microorganism.
In another the microorganism lacks the genes encoding for following enzymes a
aspect, i)
acetylglycosaminephosphate deacetylase, ii) a N-acetylglucosamine kinase, and iii) a
acetylneuraminate aldolase. In another preferred aspect, the genes encoding for following
enzymes i) a N-acetylglycosaminephosphate deacetylase, ii) a N-acetylglucosamine kinase,
and a N-acetylneuraminate aldolase are reduced in said are deleted
iii) activity, preferably genes
knocked-out,
or in the microorganism.
In another preferred the microorganism further encodes a protein that facilitates uptake
aspect,
of lactose and lacks that metabolize lactose. Methods
enzymes to produce microorganisms
which resist lactose killing and the resulting microorganisms are described in yy02016/075243
which is herein incorporated reference.
In a preferred aspect the microorganisms and used in the method the invention also
of, of,
CMP-sialic
express a acid synthase (EC 2.7.7.43) and a sialyltransferase (EC 2.4.99.1) in order to
activate the sialic acid and transfer it to a desired compound.
N-acetylglucosaminephosphate is obtained introducing a
In a preferred aspect, the by
glucosamine-phosphate N-acetyltransferase (EC 2.3.1.4) which uses intracellular glucosamine-
40 6-phosphate as a substrate. In most micro-organisms, glucosaminephosphate is naturally
present in the cell, but the intracellular production can be elevated expressing a
glutamine:D-fructosephosphate
aminotransferase without inhibition, obtained either
through protein engineering or by screening natural enzymes, such as present in gram positive
bacteria et Metabolic Engineering 7 201-214).
(Deng a/., (2005),
N-acetylglucosamine
In the present invention, the expression of the genes to convert
phosphate to N-acetyl-neuraminate or sialic acid are optimized in a that enables
intracellular of N-acetylglucosaminephosphate, prevents toxic
dephosphorylation
accumulation of N-acetylglucosaminephosphate and prevents excretion of
N-acetylmannosamine.
acetylglucosamine and/or Said optimization is the result of the use of
constitutive expression of the genes of the production pathway. In a preferred embodiment,
the present invention prevents the excretion of at least 10%, 20%, 30%, 35%, 40%, 45%, 50%,
or 60% of the formed N-acetylglucosamine and/or N-acetylmannosamine. In a further preferred
embodiment, the microorganism produces less extracellular N-acetylglucosamine and/or
acetylmannosamine than sialylated compound. More preferably, the microorganism produces
less than 2% extracellular N-acetylglucosamine and/or
50%, 40%, 30%, 20%, 10%, 5%,
acetylmannosamine than sialylated compound. In another preferred embodiment of the
present invention the microorganism produces or more than
equal 50%, 60%, 70%, 80%, 90%,
95%, 98% extracellular sialylated compound on total extracellular carbohydrate.
In a particular aspect, the invention relates to a method for synthesis of sialylated compounds,
without any exogenous sialic acid addition to the culture medium.
The sialylated compound can be N-acetylneuramic acid, a sialylated oligosaccharide, a sialylated
lipid, sialylated glycolipids (such as, but not limited to gangliosides, ceramides), a sialylated
protein or a sialylated
aglycon.
A sialylated oligosaccharide is a charged sialic acid containing oligosaccharide, /.e, an
oligosaccharide having a sialic acid residue. It has an acidic nature. Some examples are 3-SL (3-
6-SL or
sialyllactose), 3-sialyllactosamine, (6-sialyllactose acetylneuraminate alfa 2,6
beta 6-sialyllactosamine, oligosaccharides 6-sialyllactose,
galactosyl 1,4 Glucose), comprising
alfa-2,36al -1,3GalNac -1,36ala-1,46al
S66 hexasaccharide (NeuSAc beta beta beta -1,4Gal),
sialylated tetrasaccharide (NeuSAc-alfa-2,3Gal beta -1,4GlcNAc beta -146lcNAc),
alfa-2,3Gal -1,4GlcNAc -1,3Gal
pentasaccharide LSTD (NeuSAc beta beta beta -1,4Glc), sialylated
lacto-N-triose, sialylated lacto-N-tetraose, sialyllacto-N-neotetraose, monosialyllacto-N-
hexaose, disialyllacto-N-hexaose monosialyllacto-N-neohexaose monosialyllacto-N-
I, I,
neohexaose disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II,
sialyllacto-N-tetraose disialyllacto-N-hexaose sialyllacto-IV-tetraose 3-sialyl
a, I, b,
fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-
sialyllacto-N-fucohexaose disialyllacto-N-fucopentaose
octaose (sialyl Lea), II,
monofucosyldisialyllacto-N-tetraose and oligosaccharides bearing one or several sialic acid
residu(s), including but not limited to: oligosaccharide moieties of the gangliosides selected
from GM3 (3sialyllactose, NeuSAca-2,3Gal beta-4Glc) and oligosaccharides comprising the GM3
Neu5Aca-2,8Neu5Aca-2,3Gal beta -1,4Glc GT3 (Neu5Aca-2,8Neu5Aca-2,8Neu5Aca-
motif, GD3
2,3Gal beta -1,4Glc); GM2 GalNAc beta -1,4(Neu5Aca-2,3)Gal beta -1,4Glc, GM1 Gal beta—
40 beta beta -1,4Glc, GDla Neu5Aca-2,3Gal -1,3GalNAc beta
1,3GalNAc -1,4(Neu5Aca-2,3)Gal beta
beta-
-1,4(Neu5Aca-2,3)Gal beta -1,4Glc 6Tla Neu5Aca-2,8Neu5Aca-2,36al beta -1,3GalNAc
1,4(Neu5Aca-2,3)Gal beta -1,46lc GD2 6alNAc beta -1,4(Neu5Aca-2,8Neu5Aca2,3)Gal beta-
-1,4Glc
1,46lc 6T2 GspalNAc beta -1,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3}Gal beta 6Dlb,
-1,3GalNAc -1,4Glc NeuSAca-2,3Gal
Gal beta beta -1,4(NeuSAca-2,8NeuSAca2,3)Gal beta GTlb
beta -1,3GalNAc beta -1,4(Neu5Aca-2,8Neu5Aca2,3)Gal beta-1,4Glc GQ1b Neu5Aca-
-1,3GalNAc -1,4Glc
2,8Neu5Aca-2,3Gal beta beta -1,4(Neu5Aca-2,8Neu5Aca2,3)Gal beta GT1c
Gal beta -1,3GalNAc beta -1,4(NeuSAca-2,8Neu5Aca-2,8Neu5Aca2,3)Gal beta -1,4Glc GQlc,
Neu5Aca-2,3Gal beta -1,36alNAc beta -1,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gal beta-
1,46lc GPlc Neu5Aca-2,8Neu5Aca-2,3Gal beta -1,3GalNAc beta -1,4(Neu5Aca-2,8Neu5Aca-
-1,4Glc Neu5Aca-2,3Gal beta—
2,8Neu5Aca2,3)Gal beta GDla beta -1,3(NeuSAca-2,6)GalNAc
1,4Gal beta -1,46lc Fucosyl-GM1 Fuca-1,2Gal beta -1,36alNAc beta -1,4(NeuSAca-2,3)Gal beta
-1,4Glc; all of which be extended to the production of the corresponding gangliosides
may by
reacting the above oligosaccharide moieties with ceramide or synthetizing the above
oligosaccharides on a ceramide.
The term micro-organism or organism or cell as indicated above refers to a microorganism
chosen from the list a bacterium, a or a refers to a or animal
comprising yeast, fungus, or, plant
cell. The latter bacterium preferably belongs to the phylum of the Proteo bacteria or the phylum
of the Firmicutes or the of the Cyanobacteria or the Deinococcus-Thermus. The
phylum phylum
latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family
Enterobacteriaceae, Escherichia coll.
preferably to the species The latter bacterium preferably
relates to any strain belonging to the species Escherichia coll such as but not limited to
Escherichia cali Escherichia coll Escherichia co/i Escherichia coll Escherichia coll
B, C, W, K12,
Nissle. More specifically, the latter term relates to cultivated Escherlchia coll strains designated
as E. coll K12 strains which are well-adapted to the laboratory environment, unlike wild
and,
strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coll
type
K12 strains are K12 Wild W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100,
type,
JM101, NZN111 and AA200. Hence, the present invention specifically relates to a mutated
and/or transformed Escherichia coll strain as indicated above wherein said E. coll strain is a K12
strain. More specifically, the present invention relates to a mutated and/or transformed
Escherichia cali strain as indicated above wherein said K12 strain is E. coll MG1655. The latter
bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably
Lactobacilliales, with members such as Lactobacillus lactls, Leuconostoc mesenteroides, or
Bacillales with members such as from the species Bacillus, Bacillus subtilis or, B.
amyloliquefaciens. The latter Bacterium to the Actinobacteria,
belonging phylum preferably
belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum
or C. afermentans, or belonging to the family of the of the Streptomycetaceae with members
griseus 5. The latter of
5treptomyces or fradlae. yeast preferably belongs to the phylum the
Ascomycota or the of the Basidiomycota or the of the Deuteromycota or the
phylum phylum
phylum of the Zygomycetes. The latter yeast belongs preferably to the genus 5accharomyces,
Pichia, Hansenula, Kluyveromyces, Yarrowia or5tarmerella. The latter fungus belongs preferably
to the genus Rhizopus, Dictyostelium, Penicilllum, Mucor or Aspergillus.
The culture medium for the host can
40 production optionally comprise an exogenous precursor
or this precursor can be produced the strain itself, such as a glycan like for example lactose,
lacto-N-triose, lacto-N-tetraose, lacto-N-neotetraose;
lactosamine, an oligosaccharide; a
peptide; a lipid or an aglycon. In one particular aspect, the process of the invention is based on
tri-saccharide,
the active uptake of an exogenous precursor, such as for example a mono, di or
acta-N-
more particularly an exogenous precursor selected from lactose, N-acetyllactosamine, l
biose, galactose, beta-galactoside, and alpha-galactoside such as but not limited to globotriose
(Gal-alpha-1,4Gal-beta-1,4Glc),
while the microorganisms are growing on an inexpensive carbon
substrate, such as a disaccharide such as sucrose or maltose. Moreover, these microorganisms
are also able to on fructose or The expression exogenous precursor is
grow glucose, glycerol.
intended to denote a compound involved in the biosynthetic pathway of the product according
to the invention that is internalized by the microorganism.
lacto-N-
In one aspect, the invention provides for method for production of sialylated forms of
lacto-N-tetraose lacto-N-neotetraose.
triose, or Any one of these three molecules are
synthetized the micro-organism via the activity of a galactosyltransferase (EC 2.4.1.38),
originating from the Homo sapiens, Bos taurus, Mus mulatta,
preferably group comprising
Gallus gallus, Danio rerio, Helicobacter pylori and Haemophilus ducrey and/or a
acetylglucosaminyltransferase 2.4.1.90) from the
(EC preferably originating group comprising
Bos Taurus, Homo Sapiens and Mus Musculus. To enhance the formation of these
oligosaccharides the coding for UDP hydrolase and galactosephosphate
genes sugar
uridylyltransferase are lacking, reducing in activity or knocked out in the microorganism.
In another aspect a method for producing a sialylated oligosaccharide is provided in which the
method comprises culturing a microorganism as described above and wherein the
microorganism produces activated N-acetylneuraminate as donor substrate for a
internally,
sialyltransferase; and wherein the method further comprises culturing the microorganism in a
culture medium which comprises an exogenous precursor selected from the consisting of
group
lactose, N-acetyllactosamine, lacto-N-biose, galactose, beta-galactoside, and alpha-galactoside
such as but not limited to globotriose (Gal-alpha-1,4Gal-beta-1,4Glc)galactose. The exogenous
precursor is actively taken into the microorganism and the exogenous precursor is the
acceptor substrate for the sialytransferase for the sialylated oligosaccharide.
producing
In a further aspect, the method according to the invention provides for the production of
3sialyllactose or 6sialyllactose. In this method the microorganism is cultivated at high cell
density on a carbon substrate, such as glucose or glycerol, and fed with lactose. The lactose is
internalized the lactose permease and sialylated the recombinant sialyltransferase
by by using
CMP- N-acetyl-neuraminate
the endogenously generated from N-acetylglucosamine.
The microorganism or cell of the invention is capable to grow on a monosaccharide,
disaccharide, oligosaccharide, polysaccharide, a complex medium or a mixture thereof
polyol,
as the main carbon source. With the term main is meant the most important carbon source for
biomass formation, carbon dioxide and/or by-products formation as acids and/or
(such
alcohols, such as acetate, lactate, and/or ethanol), i.e. 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95,
99 % of all the required carbon is derived from the above-indicated carbon source. In one
embodiment invention, said carbon source the sole source for said
of the is carbon organism,
i.e. 100% of all the required carbon is derived from the above-indicated carbon source.
40 In a further preferred embodiment, the microorganism or cell of the invention is a
using split
metabolism having a production pathway and a biomass pathway as described in
WO2012/007481, which is herein incorporated by reference. Said organism can, for example,
fructosephosphate
be genetically modified to accumulate by altering the genes selected from
the phosphoglucoisomerase phosphofructokinase fructosephosphate aldolase
gene, gene,
gene, fructose isomerase gene, and/or fructose;PEP phosphotransferase gene.
With the term monosaccharide is meant a sugar that is not decomposable into simpler sugars
is classed as either an aldose or ketose, and contains one or more
by hydrolysis, hydroxyl groups
molecule. Examples are glucose, fructose, galactose, mannose, ribose and/or arabinose.
With the term disaccharide is meant a that is composed of two monosaccharides that are
sugar
chemically bound. Examples are maltose, sucrose, lactose, trehalose, cellobiose and/or
chitobiose.
With the term oligosaccharide is meant a sugar that is composed of three to ten
monosaccharides that are chemically bound. Examples are maltotriose, fructo-oligosaccharides,
galacto-oligosaccharides, mannan oligosaccharides, isomaltooligosaccharide, human milk
oligosaccharides and/or glucooligosaccharides.
With the term polyol is meant an alcohol containing multiple hydroxyl groups. For example
glycerol, sorbitol, or mannitol.
With the term complex medium is meant a medium for which the exact constitution is not
determined. Examples are molasses, corn or extract.
steep liquor, peptone, tryptone yeast
Production of sialylated compounds can be increased adding precursors to the medium, such
as N-acetylglusosamine, N-acetylmannosamine, glutamine, glutamate,
phosphoenolpyruvate
and/or pyruvate.
The sialylated compounds produced in the method of the invention as described above be
recovered using various methods, or a combination thereof, known in the art. Depending on
the produced sialylated the compound is available in the extracellular fraction or
compound,
retained in the cells. When the produced sialylated compound is retained in the cells, the
sialylated compound will first be released from the cells cell disruption. on
by Again depending
the produced sialylated compound, the cells may be separated from the extracellular fraction.
In the other case, cells are disrupted without first separation from the extracellular fraction,
wherein cells are disrupted techniques such as, but not limited to, heating, freeze thawing
and/or shear stress through sonication, mixing and/or French press. The extracellular and/or
intracellular fraction may be separated from the cells and/or cell debris centrifugation,
filtration, microfiltration, and nanofiltration. Flocculating agents may be used to aid in product
separation. The sialylated compounds in the extracellular or intracellular fraction may be
extracted ion ultra-or nanofiltration or electrodialysis, such as
by exchange, chromatography
size exclusion, ion chromatography and simulated moving bed. Another example of filtering the
sialylated compounds from liquid phase is filtration using a deep bed filter with cotton and
activated carbon where after the is carbon
or carbon filter, permeate passed through a polisher
micro-
followed a 0.2 micron microfiltration membrane system to remove color,
by e.g.
organisms and suspended carbon particles. Thereafter the sialylated compound may be
concentrated in a vacuum evaporator to obtain a concentrate. The concentrate can be
40 precipitated and/or dried through heat and/or lyophilization to obtain
drying, spray drying high
sialylated compound. An amorphous form powder can then be obtained. This amorphous
purity
powder may further be crystallised to obtain crystalline sialylated compound.
WO 201II/122225
In exemplary embodiment, sialylated compounds may be isolated from the culture medium
methods known in the art for fermentations. For cells be removed from the
using example, may
culture medium by centrifugation, filtration, flocculation, decantation, or the like. Then, the
ion-
sialylated compounds may be isolated from the extracellular fraction using methods such as
exchange. A further purification of said sialylated compounds be accomplished, for
example, nanofiltration or ultrafiltration or ion exchange to remove remaining DNA,
by any
protein, LPS (endotoxins), or other impurity.
In another exemplary embodiment, sialyllactose may be isolated from the culture medium using
methods known in the art for fermentations. For example, cells may be removed from the
culture medium centrifugation, filtration, flocculation, decantation, or the like. Then, the
sialyllactose be isolated from the extracellular fraction methods such as ion-
may using
exchange. A further purification of said sialyllactose be accomplished, for example,
may by
nanofiltration or ultrafiltration or ion exchange to remove remaining LPS
any DNA, protein,
(endotoxins), or other impurity. Another purification and formulation step is accomplished
crystallization or precipitation of the product. Another formulation is to or
step spray dry
lyophilize sialyllactose.
The sialylated compound may contain a counter ion, such as, a monovalent ion, such as a
proton, sodium ion, potassium, a divalent ion, such as calcium magnesium, iron, or, a trivalent
ion such as or a combination of ions.
iron,
N-acetyl
Throughout the disclosure of the present disclosure the term sialic acid, neuraminate
and N-acetyl neuraminic acid are used interchangeably.
As used herein, the term intracellular or intracellularly in e.g, intracellularly converting,
intracellularly production, intracellularly expressed, intracellular formed must be understood to
within of The extracellular understood to
mean the cell the microorganism. term must be mean
outside of the cell.
Further definitions used throughout the present specification
Homologue(s)
"Homologues" of
a protein encompass peptides, oligopeptides, polypeptides, proteins and
enzymes having amino acid substitutions, deletions and/or insertions relative to the
unmodified protein in question and similar biological and functional activity as the
having
unmodified protein from which they are derived.
A deletion refers to removal of one or more amino acids from a protein.
An insertion refers to one or more amino acid residues being introduced into a predetermined
site in a protein. Insertions comprise N-terminal and/or C-terminal fusions as well as
intra-sequence
insertions of single or multiple amino acids. Generally, insertions within the
amino acid sequence will be smaller than or C-terminal fusions, of the order of about 1 to
residues. of or C-terminal fusion or include
Examples proteins peptides the binding
two-hybrid
domain or activation domain of a transcriptional activator as used in the yeast
coat proteins, (histidine)tag, glutathione transferase-tag, protein
system, phage A,
maltose-binding c-myc
40 protein, dihydrofolate reductase, Tag»100 epitope, epitope, FLAG(R)-
epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV
epitope.
A substitution refers to replacement of amino acids of the protein with other amino acids
having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity,
-sheet
to form or break a-helical structures or beta structures). Amino acid
propensity
substitutions are typically of single residues, but may be clustered depending upon functional
constraints placed the and from 1 to 10 amino acids; insertions
upon polypeptide may range
will usually be of the order of about 1 to 10 amino acid residues. The amino acid substitutions
are preferably conservative amino acid substitutions. Conservative substitution tables are well
known in the art for example Creighton Proteins. W.H. Freeman and
(see (1984) Company
and Table 1 below).
(Eds)
Table 1: Examples of conserved amino acid substitutions
Residue Conservative Residue Conservative
Substitutions Substitutions
Ala Ser Leu lie; Val
Arg Lys Lys Arg; Gln
Asn His Met lie
Gin; Leu;
Glu Phe
Asp Met; Leu; Tyr
Gln Ser
As, Thr; Gly
Ser Thr Ser; Val
Gly Pro Tyr Trp; Phe
His Asn; Gln Val lie; Leu
lie Leu; Val
Amino acid substitutions, deletions and/or insertions readily be made
may using peptide
synthetic techniques well known in the art, such as solid phase peptide synthesis and the like,
or recombinant DNA manipulation. Methods for the manipulation of DNA sequences to
produce substitution, insertion or deletion variants of a protein are well known in the art. For
techniques for making substitution mutations at predetermined sites in DNA are well
example,
known to those skilled in the art and include M13 mutagenesis, Gen in vitro mutagenesis
Cleveland, QuickChange Site Directed mutagenesis San PCR-
(USB, OH), (Stratagene, Diego, CA),
mediated site-directed other site- directed
mutagenesis or mutagenesis protocols.
Derivatives
"Derivatives"
include peptides, oligopeptides, polypeptides which may, compared to the amino
acid sequence of the naturally-occurring form of the protein, such as the protein of interest,
substitutions amino acids with non-naturally amino acid or
comprise of occurring residues,
"Derivatives"
additions of non-naturally occurring amino acid residues. of a protein also
which occurring altered
encompass peptides, oligopeptides, polypeptides comprise naturally
non-
(glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or
naturally altered amino acid residues compared to the amino acid sequence of a naturally-
non-amino
occurring form of the polypeptide. A derivative may also comprise one or more acid
substituents or additions compared to the amino acid sequence from which it is derived, for
example a reporter molecule or other covalently or non-covalently bound to the amino
ligand,
non-
acid sequence, such as a reporter molecule which is bound to facilitate its detection, and
naturally occurring amino acid residues relative to the amino acid sequence of a naturally-
occurring protein. Furthermore, "derivatives" also include fusions of the naturally-occurring
form of the protein with peptides such as FLAG, HIS6 or thioredoxin (for a review of
tagging
523-533,
tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 2003).
Orthologue(s)/Paralogue(s)
Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral
relationships of genes. Paralogues are genes within the same species that have originated
through duplication of an ancestral orthologues are from different organisms that
gene; genes
have originated through speciation, and are also derived from a common ancestral gene.
Domain, Motif/Consensus sequence/Signature
"domain"
The term refers to a set of amino acids conserved at specific positions along an
alignment of sequences of evolutionarily related proteins. While amino acids at other positions
can vary between homologues, amino acids that are highly conserved at specific positions
indicate amino acids that are essential in the structure, stability or function of a protein.
likely
Identified their high degree of conservation in aligned sequences of a family of protein
can be used as identifiers to determine if in question belongs
homologues, they any polypeptide
to a previously identified polypeptide family.
"motif "consensus "signature"
The term or sequence" or refers to a short conserved region in
the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of
domains, but also include of the domain, or be located outside of conserved
may only part
domain (if all of the amino acids of the motif fall outside of a defined domain).
Specialist databases exist for the identification of domains, for example, SMART (Schultz et al.
5857-5864; 242-
(1998) Proc. Natl. Acad. Sci. USA 95, Letunic et al. (2002) Nucleic Acids Res 30,
InterPro (Mulder et Nucl. Acids. Res. 315-318), Prosite (Bucher and Bairoch
244), al., (2003) 31,
(1994), A generalized profile syntax for biomolecular sequences motifs and its function in
automatic sequence interpretation. ISMB-94; Proceedings 2nd International Conference on
(In)
Intelligent Molecular Altman Searls
Systems for Biology. R., Brutlag D., Karp P., Lathrop R., D.,
Eds., AAAI Press, Menlo Park; Hulo et Nucl. Acids. Res. 32:D134-D137, or
pp53-61, al., (2004)),
276-280
Pfam (Bateman et al., Nucleic Acids Research 30(1): (2002)). A set of tools for in silica
analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of
in-depth
Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for protein knowledge
and analysis, Nucleic Acids
Res. Domains or motifs also identified routine
31:3784-3788(2003)). may be using techniques,
such as sequence alignment.
Methods for the alignment of sequences for comparison are well known in the art, such
methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of
443-453)
40 Needleman and Wunsch ((1970) J Mol Biol 48; to find the global (i.e. spanning the
complete sequences) alignment of two sequences that maximizes the number of matches and
403-
minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215:
calculates percent sequence identity and performs a statistical analysis of the similarity
between the two sequences. The software for performing BLAST analysis is publicly available
through the National Centre for Biotechnology Information (NCBI). Homologues may readily be
identified for the ClustalW multiple sequence alignment algorithm (version
using, example,
1.83), with the default pairwise alignment parameters, and a scoring method in percentage.
Global percentages of similarity and identity may also be determined using one of the methods
available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul
;4:29. MatGAT: an application that generates similarity/identity matrices protein or
using
DNA sequences.). Minor manual editing be performed to optimise alignment between
conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of
using full-length sequences for the identification of homologues, specific domains may also be
used. The sequence identity values be determined over the entire nucleic acid or amino
acid sequence or over selected domains or conserved motif(s), using the programs mentioned
Smith-Waterman
above using the default parameters. For local alignments, the algorithm is
particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1);195-7).
Reciprocal BLAST
Typically, this involves a first BLAST involving BLASTing a sequence (for example using
query
of the sequences listed in Table A of the Examples section) against sequence database,
any any
such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values)
are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using
standard default values) when starting from a protein sequence. The BLAST results may
be filtered. The full-length sequences of either the filtered results or non-filtered
optionally
results are then BLASTed back (second BLAST) against sequences from the organism from which
the sequence is derived. The results of the first and second BLASTs are then compared. A
query
identified hit the first is from from
paralogue is if a high-ranking from blast the same species as
which the sequence is derived, a BLAST back then ideally results in the sequence
query query
amongst the highest hits; an orthologue is identified if a high-ranking hit in the first BLAST is not
from the same species as from which the sequence is derived, and results
query preferably upon
BLAST back in the query sequence being among the highest hits.
High-ranking hits are those having a low E-value. The lower the E-value, the more significant
the score in other words the lower chance the hit was
(or the that found chance).
Computation of the E-value is well known in the art. In addition to E-values, comparisons are
also scored percentage identity. Percentage identity refers to the number of identical
nucleotides (or amino acids) between the two compared nucleic acid (or
polypeptide)
sequences over a particular length. In the case of families, ClustalW be used, followed
large may
a neighbour tree, to visualize clustering of related and to identify
by joining help genes
orthologues and paralogues.
Construct
40 Additional regulatory elements may include transcriptional as well as translational enhancers.
Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable
for use in the invention. An intron sequence also be added to the 5 untranslated
performing may
region or in the coding sequence to increase the amount of the mature message that
(UTR)
accumulates in the cytosol, as described in the definitions section. Other control sequences
(besides enhancer, silencer, intron sequences, 3UTR and/or 5UTR be
promoter, regions) may
protein and/or RNA stabilizing elements. Such sequences would be known or may readily be
obtained a person skilled in the art.
The genetic constructs of the invention further include an of replication sequence
may origin
that is required for maintenance and/or replication in a specific cell One example is when
type.
a genetic construct is required to be maintained in a bacterial cell as an episomal genetic
element plasmid or cosmid molecule).
(e.g.
For the detection of the successful transfer of the nucleic acid sequences as used in the methods
of the invention and/or selection of transgenic microorganisms comprising these nucleic acids,
it is advantageous to use marker reporter Therefore, the genetic construct
genes (or genes).
optionally comprise a selectable marker gene. The marker genes be removed or
may may
excised from the transgenic cell once are no needed. Techniques for marker removal
they longer
are known in the art, useful techniques are described above in the definitions section.
Regulatory element/Control sequence/Promoter
element", "control "promoter"
The terms "regulatory sequence" and are all used
interchangeably herein and are to be taken in a broad context to refer to nucleic acid
regulatory
sequences capable of effecting expression of the sequences to which they are ligated. The term
"promoter"
refers to a nucleic acid control sequence located upstream from the
typically
transcriptional start of a gene and which is involved in recognising and binding of RNA
and other thereby directing transcription of an linked nucleic
polymerase proteins, operably
acid. Encompassed the aforementioned terms are transcriptional regulatory sequences
derived from a classical eukaryotic genomic (including the TATA box which is required for
gene
accurate with or without and additional
transcription initiation, a CCAAT box sequence)
elements upstream activating enhancers and silencers) which alter
regulatory (i.e. sequences,
tissue-specific
gene expression in response to developmental and/or external stimuli, or in a
manner. Also included within the term is a transcriptional regulatory sequence of a classical
-35 -10
prokaryotic gene, in which case it may include a box sequence and/or box transcriptional
element"
sequences. The term "regulatory also encompasses a synthetic fusion
regulatory
molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule
in a cell, tissue or organ.
Constitutive promoter
A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but
not necessarily all, phases of growth and development and under most environmental
conditions, in at least one cell, tissue or organ.
Transgenic/Transgene/Recombinant
"transgenic", "transgene" "recombinant"
For the purposes of the invention, or means with
regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a
vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid
40 sequences, expression cassettes or vectors according to the invention, all those constructions
brought about recombinant methods in which either
the nucleic acid sequences encoding proteins useful in the methods of the invention, or
genetic control sequence(s) which is operably linked with the nucleic acid sequence
according to the invention, for example a promoter, or
a) and b) are not located in their natural genetic environment or have been modified by
recombinant methods, it being possible for the modification to take the form of, for example, a
substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The
natural genetic environment is understood as meaning the natural genomic or chromosomal
locus in the original microorganism or the presence in a genomic library. In the case of a genomic
library, the natural genetic environment of the nucleic acid sequence is preferably retained, at
least in The environment flanks the nucleic acid sequence at least on one side and has a
part.
sequence length of at least 50 preferably at least 500 especially preferably at least 1000
bp, bp,
most at least 5000 A naturally occurring expression cassette for example the
preferably bp.
naturally occurring combination of the natural promoter of the nucleic acid sequences with the
corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the
present invention, as defined above becomes a transgenic expression cassette when this
expression cassette is modified non-natural, synthetic artificial" methods such for
by as,
example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350
or WO 00/15815.
A transgenic microorganism for the of the invention is thus understood as
purposes meaning,
as above, that the nucleic acids used in the method of the invention are not present in, or
originating from, the genome of said microorganism, or are present in the genome of said
microorganism but not at their natural locus in the genome of said microorganism, it being
possible for the nucleic acids to be expressed homologously or heterologously. However, as
mentioned, transgenic also means that, while the nucleic acids according to the invention or
used in the inventive method are at their natural position in the genome of a microorganism,
the sequence has been modified with regard to the natural sequence, and/or that the regulatory
sequences of the natural sequences have been modified. Transgenic is preferably understood
as meaning the expression of the nucleic acids according to the invention at an unnatural locus
in the i.e. homologous preferably, heterologous expression of the nucleic acids
genome, or,
takes place. Preferred transgenic microorganism are mentioned herein.
"isolated
It shall further be noted that in the context of the present invention, the term nucleic
acid" "isolated
or polypeptide" may in some instances be considered as a synonym for a
"recombinant acid" "recombinant
nucleic or a polypeptide", respectively and refers to a nucleic
acid or polypeptide that is not located in its natural genetic environment and/or that has been
modified recombinant methods.
Modulation
WO 2I/18/122225 PCT/EP2il17/084593
"modulation"
The term means in relation to expression or gene expression, a process in which
the expression level is changed said expression in comparison to the control
by gene
microorganism, the expression level may be increased or decreased. The original, unmodulated
expression may be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA with
subsequent translation. For the of this invention, the original unmodulated expression
purposes
also be absence of expression. The term "modulating the activity" shall mean
may any any
change of the expression of the inventive nucleic acid sequences or encoded proteins, which
leads to increased production yield and/or increased growth of the microorganisms. The
expression can increase from zero (absence or immeasurable expression) to a certain
amount, or can decrease from a certain amount to immeasurable small amounts or zero.
Expression
"expression" "gene expression"
The term or means the transcription of a specific gene or
"expression" "gene
specific or specific genetic construct. The term or expression" in
genes
particular means the transcription of a gene or genes or genetic construct into structural RNA
or mRNA with or without subsequent translation of the latter into a protein. The
(rRNA, tRNA)
process includes transcription of DNA and processing of the resulting mRNA product.
Increased expression/overexpression
"increased expression" "overexpression"
The term or as used herein means any form of
expression that is additional to the original wild-type expression level. For the of this
purposes
invention, the original wild-type expression level might also be zero, i.e. absence of expression
or immeasurable expression.
Methods for increasing expression of genes or gene products are well documented in the art
and include, for overexpression driven promoters, the use of
example, by appropriate
transcription enhancers or translation enhancers. Isolated nucleic acids which serve as
promoter or enhancer elements be introduced in an position
may appropriate (typically
upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a
nucleic acid encoding the of interest. For endogenous promoters be
polypeptide example, may
altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, US 5,565,350; Zarling et
al., W09322443), or isolated promoters be introduced into a microorganism cell in the
proper orientation and distance from a gene of the present invention so as to control the
expression of the gene.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region
3-end
at the of a polynucleotide coding region. The polyadenylation region can be derived from
T-DNA.
the natural gene, from a variety of other microorganism genes, or from
Moreover, the present invention relates to the following specific embodiments:
1. Method for the production of sialylated compounds, the method comprising:
culturing a microorganism in a culture medium, said culture medium optionally comprising an
40 exogenous precursor,
N-acetylglucosaminephosphate
wherein said microorganism intracellularly converts to
acetylglucosamine, said N-acetylglucosamine to N-acetylmannosamine and said
N-acetyl-neuraminate;
acetylmannosamine to and
wherein said microorganism is unable to convert N-acetylglucosamineP to glucosamine-
6-P, convert N-acetyl-glucosamine to N-acetyl-glucosamineP, and convert N-acetyl-
ii) iii)
N-acetyl-mannosamine.
neuraminate to
2. The method according to embodiment 1 wherein:
said conversion of N-acetylglucosaminephosphate to N-acetylglucosamine is obtained
i) by
the action of an intracellularly expressed phosphatase,
said N-acetylglucosamine to N-acetylmannosamine conversion is performed an
ii) by
intracellularly expressed N-acetylmannosamine and
epimerase;
iii) intracellular expressed sialic acid synthase converts said N-acetylmannosamine to N-acetyl-
neuraminate.
3. The method according to any one of embodiment 1 or 2 wherein said organism is unable
to produce following enzymes a N-acetylglycosaminephosphate deacetylase, a
i) ii)
acetylglucosamine kinase, and iii) a N-acetylneuraminate aldolase.
4. The method according to one of embodiment 1 to wherein all said conversions are
any 3,
catalysed enzymes encoded constitutively expressed genes.
by by
The embodiment wherein
. method according to 2 the phosphatase is chosen from the
HAD-like
HAD superfamily or the phosphatase family, preferably said phosphatase is chosen
from the comprising: enzymes expressed the genes inhX,
group i) by ygaB, yniC, ybi V, yidA, ybj I,
yigt or cof from Escherichia co/i, ii] the phosphatase of Blastocladiella emersonii and iii) other
I-IAD-alike
phosphatase families, more preferably said phosphatase is a phosphatase
polypeptide as defined in the claims.
6. The method according to one of the embodiments 4 or wherein the
any 2, 3, 5,
acetylmannosamineepimerase is chosen from the group comprising
acetylmannosamineepimerase from cyanobacteria, more in particular from Acoryochloris
marina, Anobaena variabilis, Anabaena Nostoc
marina, punctiforme, Acaryochloris species,
Anabaena Nostoc species and Synechocystis N-acetylmannosamine
species, species; ii)
epimerase from Bacteroides species, more in particular from Bacteroides ovatus, Bacteroides
thetaiotaomicron, canimorsus and Mobiluncus mulieris/ N-acetyl-D-
Capnocytophaga iii)
glucosmineepimerase
from 6/ycin max, Mus musculus, Homo sapiens, Rattus norvegi cus, Bos
Taurus, Sus scrofa or Canis lupus.
7. The method one the embodiments wherein sialic
according to any of 2, 3, 4, 5 or 6, the
acid synthase is chosen from the comprising; sialic acid synthase from Streptococcus
group
agalatiae, Bacillussubtilis, Eegione))a pneumophilla, Campylobacterjejuni, ldiomarina loihiensis,
Moritella viscosa, Aliivibrio salmonicida, Escherichia co/i, Methanocaldococcus jannaschi,
Neisseria
Clostridium sordellii, Butyrivibrio proteoclasticus, Micromonas commoda or
meningitis.
8. The method according to any one of the preceding embodiments, wherein said
sialylated compound is selected from the consisting of N-acetylneuramic acid, sialylated
group
oligosaccharide, sialylated lipids, sialylated protein, sialylated aglycon.
9. The method according to the previous embodiment, wherein said sialylated compound
is a sialylated oligosaccharide.
. The method according to embodiment wherein said sialylated oligosaccharide is
sialyllactose, one of 3-SL or 6-SL.
preferably any
11. The method according to embodiment wherein said sialylated oligosaccharide is
lacto-N-tetraose.
disialyl
12. The method according to embodiment wherein said sialylated compound is
acetylneuraminic acid.
13. The method according to any one of embodiment 1 to 10 wherein said sialylated
compound is a sialylated lacto-N-triose, lacto-N-tetraose or a lacto-N-neotetraose, and wherein
said microorganism further comprises the activity of a galactosyltransferase (EC 2.4.1.38),
said galactosyltransferase originates from the Homo Bos
preferably group comprising sapiens,
taurus, Mus mulatta, Gallus gallus, Dan/a rerio, Helicobacter pylori and Haemophilus ducrey;
and/or said microorganism comprises the activity of a N-acetylglucosaminyltransferase
2 4.1 90), preferably said N acetylglucosaminyltransferase originates from the group comprising
Bos taurus, Homo sapiens and Mus musculus.
14. The method according to embodiment 13 wherein said microorganism is unable to
express the coding for UDP sugar hydrolase and galactosephosphate
genes
uridylyltransferase.
. The method according to one of embodiments 1 to wherein said microorganism
any 14,
produces less than 50%, 40%, 30%, 20%, 10%, 5%, 2% extracellular N-acetylglucosamine and/or
N-acetylmannosamine than sialylated compound and/or said micro-organism produces
equal
or more than 50%, 60%, 70%, 80%, 90%, 95%, 98% sialylated compound on total carbohydrate
16. A method for producing a sialylated oligosaccharide, comprising;
culturing method of one embodiments to 14
a) a microorganism according to the any of 1 7,
and and wherein said microorganism produces internally, activated N-acetylneuraminate as
donor substrate for a sialyltransferase; and
culturing said microorganism in a culture medium an exogenous precursor
b) comprising
lacto-N-biose,
selected from the group consisting of lactose, N-acetyllactosamine, galactose,
beta-galactoside, and alpha-galactoside such as but not limited to globotriose (Gal-alpha-
wherein active into of said
1,4Gal-beta-1,4Glc)galactose, uptake the microorganism exogenous
precursor occurs and wherein said exogenous precursor is the acceptor substrate for said
sialytransferase for producing the sialylated oligosaccharide.
17. The method according to embodiment wherein one or more of said
2, any phosphatase,
N-acetylmannosamine
epimerase and sialic acid synthase is overexpressed in the
microorganism.
18. The method according to embodiment 2, wherein any one or more of said phosphatase,
N-acetylmannosamine epimerase and sialic acid synthase is introduced and expressed in the
microorganism.
19. The method according to embodiment wherein said microorganism lacks the genes
encoding for following enzymes a N-acetylglycosaminephosphate deacetylase, a
/) /i)
acetylglucosamine kinase, and/ll) a N-acetylneuraminate aldolase.
. The method according to embodiment wherein in said microorganism the
3, genes
encoding for following enzymes a N-acetylglycosaminephosphate deacetylase, a
i) ii)
N-acetylneuraminate
acetylglucosamine kinase, and i//) a aldolase are reduced in activity,
preferably said genes are deleted or knocked-out.
21. The method according to one of the embodiments 1 to wherein said
any 20,
microorganism further encodes a protein that facilitates uptake of lactose and lacks enzymes
that metabolize lactose.
22. The method according to any one of embodiments 1 to 21, wherein said microorganism
Escher/chio col/ Escherichia co/i
is a bacteria, preferably an strain, more preferably an strain
which is a K12 strain, even more preferably the Escherichia coli K12 strain is Escherichla co//
MG1655.
23. The method according to any one of embodiments 1 to 21, wherein said microorganism
is a yeast.
24. The method according to any one of embodiments 1 to 23, wherein the exogenous
precursor is chosen from the comprising lactose, galactose, beta-galactoside, and alpha-
group
galactoside, such as globotriose (Gal-alpha-1,4Gal-beta-1,4Glc).
. A microorganism for the production of sialylated compounds, said microorganism
intracellularly converts N-acetylglucosaminephosphate to N-acetylglucosamine, said
acetylglucosamine to N-acetylmannosamine and said N-acetylmannosamine to N-acetyl-
neuraminate; and
is unable to convert N-acetylglucosamineP to glucosamineP, convert N-acetyl-
/) ii)
N-acetyl-glucosamineP, and convert N-acetyl-neuraminate to N-acetyl-
glucosamine to /ll)
mannosamine.
26. A microorganism for the production of a sialylated compound, said microorganism
defined in one of embodiments 2 to 24.
being any
27. A cell culture medium comprising lactose as precursor and the microorganism of any
one of embodiments 25 or 26.
28. method of embodiments for of
The according to one 1 to 24, the production
3sialyllactose or 6sialyllactose, wherein the microorganism is cultivated at high cell density on a
carbon substrate, such as glucose or and fed with lactose which is internalized the
glycerol,
lactose permease and sialylated said recombinant sialyltransferase the CMP- N-acetyl-
by using
neuraminate endogenously generated from N-acetylglucosamine.
29. The method according to any one of embodiments 1 to 24, wherein said sialylated
compound is isolated from said culture medium means of a unit operation selected from the
group centrifugation, filtration, microfiltration, ultrafiltration, nanofiltration, ion exchange,
electrodialysis, chromatography, simulated moving bed, evaporation, precipitation,
crystallization, lyophilization and/or
spray drying
. A sialylated compound produced according to the method described in one of
embodiments 1 to 24, wherein said sialylated compound is purified by centrifugation and/or
filtration, ion-exchange, concentration through evaporation or nanofiltration, formulation
through crystallization or spraydrying or lyophilization.
31. A sialylated compound produced according to the method described in any one of
embodiments 1 to wherein said sialylated compound is added to food formulation, feed
formulation, pharmaceutical formulation, cosmetic formulation, or agrochemical formulation.
32. The method according to one of embodiments 1 to wherein said culture medium
any 24,
comprises any one or more of the following: a monosaccharide, disaccharide, oligosaccharide,
polysaccharide, polyol, a complex medium as the main carbon source.
33. The method according to embodiment 32, wherein said main carbon source provides at
least 99% or'00% of all
%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,
required carbon for the growth of said microorganism.
34. The method according to embodiment wherein said monosaccharide is chosen from
the comprising glucose, fructose, galactose, mannose, ribose or arabinose.
group
. The method according to embodiment wherein said disaccharide is chosen from the
group comprising maltose, sucrose, lactose, trehalose, cellobiose or chitobiose.
36. The method according to embodiment wherein said oligosaccharide is chosen from
the group comprising maltotriose, fructo-oligosaccharides, galacto-oligosaccharides, mannan
oligosaccharides, isomaltooligosaccharide or glucooligosaccharides.
37. The method according to embodiment 32, wherein said polyol is chosen from the group
comprising
glycerol.
38. The method embodiment wherein said is chosen
according to 32, complex medium
from the molasses, corn or extract.
group comprising steep liquor, peptone, tryptone yeast
In a preferred aspect, the present invention relates to the following preferred specific
embodiments:
1. A method for the production of a sialylated compound in a microorganism, the method
comprising;
culturing a microorganism in a culture medium, said culture medium optionally comprising an
exogenous precursor,
wherein said microorganism comprises at least one nucleic acid encoding a phosphatase, at
least one nucleic acid encoding an N-acetylmannosamine epimerase; and at least one nucleic
acid encoding a sialic acid synthase, and
wherein said microorganism is unable to/) convert N-acetylglucosamineP to glucosamine
convert N-acetyl-glucosamine to N-acetyl-glucosamineP, and convert N-acetyl-
P, /I) ///)
N-acetyl-mannosamine;
neuraminate to and
modulating expression in said microorganism of a nucleic acid encoding a HAD-alike
phosphatase wherein said HAD-alike phosphatase comprises;
polypeptide, polypeptide
at least one of the following motifs:
Motif 1: hDxDx[TV] ID NO: or
(SEQ 73),
Motif 2: [GSTDE][DSEN]x(1-2)[hP] x(1-2) [DGTS] (SEQ ID NOs: 74, 75, 76, 77)
wherein h means a hydrophobic amino acid and x can be
(A, I, L, M, F, V, P, G)
any distinct amino acid;
or a homologue or derivative of one of ID NOs:
any SEQ 43,44, 45, 47, 48, 50, 51, 52, 54,
55 or 57 having at least 80 81 82 83 84 85 86 87 88 89 90
%, %, %, %, %, %, %, %, %, %, %,
91 92 93 94 95 96 97 98 ol 99 % overall sequence identity to said
%, %, %, %, %, %, %, %,
polypeptide.
2. The method according to preferred embodiment wherein said HAD-alike
1, polypeptide
comprises any one of SEQ ID NOs: 43,44, 45, 47, 48, 50, 51, 52, 54, 55, 57.
3. Method according to preferred embodiment wherein said modulated expression is
effected nucleic acid HAD-alike
introducing and expressing in a microorganism a encoding a
polypeptide.
4. Method according to preferred embodiment 1, wherein said modulated expression is
effected the action of a constitutive promoter.
5. The method according to any one of the preceding preferred embodiments, wherein
said sialylated compound is selected from the consisting of N-acetylneuramic acid,
group
sialylated oligosaccharide, sialylated lipids, sialylated protein, sialylated aglycon.
6. The method according to the previous preferred embodiment, wherein said sialylated
compound is a sialylated oligosaccharide.
7. The method according to preferred embodiment wherein said sialylated
oligosaccharide is sialyllactose.
8. The method according to preferred embodiment wherein said sialylated
lacto-N-tetraose.
oligosaccharide is disialyl
9. The method according to preferred embodiment wherein said sialylated compound
is N-acetylneuraminic acid.
. The method according to any one of preferred embodiment 1 to 9 wherein said
sialylated compound is a sialylated lacto-N-triose, lacto-N-tetraose or a lacto-N-neotetraose,
and wherein said microorganism further comprises the activity of a galactosyltransferase (EC
2.4.1.38), preferably said galactosyltransferase originates from the group comprising Homo
Bos taurus, Mus mu)atta, Gal)us Danio rerio, Helicobacter and
sapiens, ga))us, pylori
Haemophilus ducrey; and/or said microorganism comprises the activity of a
acetylglucosaminyltransferase (86 2.4.1.90), preferably said N-acetylglucosaminyltransferase
originates from the Bos taurus, Homo sapiens and Mus musculus.
group comprising
11. The method according to preferred embodiment 12 wherein said microorganism is
unable to express the genes coding for UDP sugar hydrolase and galactosephosphate
uridylyltransferase.
12. The method according to any one of preferred embodiments 1 to 13, wherein said
microorganism produces less than 50/o, 40/o, 30/o, 20/o, 10/o, 5/o, 2/o extracellular
acetylglucosamine and/or N-acetylmannosamine than sialylated compound and/or said micro-
organism produces equal or more than 50/., 60/., 70/o, 80/., 90/., 95/., 98/. sialylated
compound on total carbohydrate
13. A method for producing a sialylated oligosaccharide, comprising:
a) culturing a microorganism according to the method of any one of preferred embodiments 1
to 12, and wherein said microorganism produces internally, activated N-acetylneuraminate as
donor substrate for a sialyltransferase; and
b) culturing said microorganism in a culture medium comprising an exogenous precursor
selected from the consisting of lactose, N-acetyllactosamine, lacto-N-biose, galactose,
group
(Gal-alpha-
beta-galactoside, and alpha-galactoside such as but not limited to globotriose
1,4Gal-beta-1,4Glc)galactose, wherein active uptake into the microorganism of said exogenous
precursor occurs and wherein said exogenous precursor is the acceptor substrate for said
sialytransferase for producing the sialylated oligosaccharide.
14. The method according to preferred embodiment 1, wherein any one or more of said
acetylmannosamine epimerase and sialic acid synthase is overexpressed in the microorganism.
. The method according to preferred embodiment 1, wherein any one or more of said
acetylmannosamine epimerase and sialic acid synthase is introduced and expressed in the
microorganism.
16. The method according to preferred embodiment wherein said microorganism lacks the
genes encoding for following enzymes l) a N-acetylglycosaminephosphate deacetylase, ll) a
N-acetylglucosamine kinase, andlli) a ly-acetylneuraminate aldolase.
17. The method according to preferred embodiment 1, wherein in said microorganism the
genes encoding for following enzymes a N-acetylglycosaminephosphate deacetylase, li) a
N-acetylglucosamine N-acetylneuraminate aldolase are reduced
kinase, and ill) a in activity,
preferably said are deleted or knocked-out.
genes
18. The method according to any one of the preferred embodiments 1 to 17, wherein said
microorganism further encodes a protein that facilitates uptake of lactose and lacks enzymes
that metabolize lactose.
19. The method according to any one of preferred embodiments 1 to 18, wherein said
microorganism is a bacterium, an Escherichia co/i strain, more an
preferably preferably
Escheri chio Escherichia
co/i strain which is a K12 strain, even more preferably the co/i K12 strain
is Escheri chio co/i MG1655.
20. The method according to one of preferred embodiments 1 to wherein said
any 18,
microorganism is a yeast.
21. The method according to one of preferred embodiments 1 to wherein the
any 20,
exogenous precursor is chosen from the lactose, galactose, beta-galactoside,
group comprising
(Gal-alpha-1,4Gal-beta-1,4Glc).
and alpha-galactoside, such as globotriose
22. Microorganism, obtainable a method according to one of claims 1 to 21, wherein said
by any
microorganism comprises a recombinant nucleic acid encoding a HAD-alike
polypeptide.
23. A microorganism for the production of sialylated compounds wherein said microorganism
comprises at least one nucleic acid encoding a at least one nucleic acid encoding
phosphatase,
N-acetylmannosamine
an epimerase; and at least one nucleic acid encoding a sialic acid
N-acetylglucosamineP
synthase, and wherein said microorganism is unable to/) convert to
glucosamineP, i/) convert N-acetyl-glucosamine to N-acetyl-glucosamineP, and l//) convert
N-acetyl-neuraminate to N-acetyl-mannosamine; characterised in that said microorganism
HAD-alike
comprises a modulated expression of a nucleic acid encoding a phosphatase
as defined in preferred embodiment 1.
polypeptide
24. Construct comprising:
nucleic acid encoding a HAD-alike as defined in preferred embodiment
polypeptide
1 or
one or more control sequences capable of driving expression of the nucleic acid
(ii)
sequence of and optionally
(i);
a transcription termination sequence.
(iii)
. Construct according to preferred embodiment 24, wherein one of said control sequences is
a constitutive promoter.
26. construct to embodiment 24 or method for
Use of a according preferred 25 in a producing
sialylated compounds.
27. A sialylated compound produced according to the method described in any one of preferred
embodiments 1 to wherein said sialylated compound is added to food formulation, feed
formulation, pharmaceutical formulation, cosmetic formulation, or agrochemical formulation.
28. A microorganism for the production of a sialylated compound, said microorganism being
in of embodiments 21.
defined any one 2 to
29. A cell culture medium comprising lactose as precursor and the microorganism of any
one of embodiments 23 or 28.
. The method according to one of embodiments 1 to for the production of
3sialyllactose or 6sialyllactose, wherein the microorganism is cultivated at high cell density on a
carbon substrate, such as glucose or glycerol or sucrose, and fed with lactose which is
internalized by the lactose permease and sialylated by said recombinant sialyltransferase using
the CMP- N-acetyl-neuraminate endogenously generated from N-acetylglucosamine.
31. The method according to any one of embodiments 1 to 21, wherein said sialylated
compound is isolated from said culture medium means of a unit operation selected from the
centrifugation, filtration, microfiltration, ultrafiltration, nanofiltration, ion
group exchange,
electrodialysis, chromatography, simulated moving bed, evaporation, precipitation,
crystallization, lyophilization and/or spray drying
32. A sialylated compound produced according to the method described in one of
wherein
embodiments 1 to 21, said sialylated compound is purified by centrifugation and/or
filtration, ion-exchange, concentration through evaporation or nanofiltration, formulation
through crystallization or or lyophilization.
spraydrying
33. A sialylated compound produced according to the method described in any one of
embodiments 1 to wherein said sialylated compound is added to food formulation, feed
formulation, pharmaceutical formulation, cosmetic formulation, or agrochemical formulation.
34. The method according to any one of embodiments 1 to 21, wherein said culture medium
comprises any one or more of the following: a monosaccharide, disaccharide, oligosaccharide,
a complex medium as the main carbon source.
polysaccharide, polyol,
. The method according to embodiment 34, wherein said main carbon source provides at
least 99% or 100% of all
%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,
required carbon for the growth of said microorganism.
36. The method according to embodiment wherein said monosaccharide is chosen from
the group comprising glucose, fructose, galactose, mannose, ribose or arabinose.
37. The method according to embodiment wherein said disaccharide is chosen from the
group comprising maltose, sucrose, lactose, trehalose, cellobiose or chitobiose.
38. The method according to embodiment wherein said oligosaccharide is chosen from
the group comprising maltotriose, fructo-oligosaccharides, galacto-oligosaccharides, mannan
oligosaccharides, isomaltooligosaccharide or glucooligosaccharides.
39. The embodiment wherein is chosen from
method according to 34, said polyol the group
comprising glycerol.
40. The method according to embodiment 34, wherein said complex medium is chosen
from the molasses, corn or extract.
group comprising steep liquor, peptone, tryptone yeast
The following drawings and examples will serve as further illustration and clarification of the
present invention and are not intended to be limiting.
Brief of the
description drawings
Fig. 1 shows an exemplary pathway as used in example 2 for the production of sialic acid
according to the present invention. 1A shows the without all KO and
Fig. pathway
knock-out
overexpression signs. Fig. 1B shows the pathway as used in example 2 with the
indicated with a cross and overexpression with an arrow next to the indicated enzyme.
upgoing
2 shows the production results of the Escherichia co//strain capable of sialic acid
Fig. producing
as described in example 2.
3 shows examples of different sialylated compounds which can be produced in the method
Fig.
of the present invention.
Fig. 4 shows the optical density and sialic acid production of strains supplemented with the
indicated phosphatases.
shows the rates of strains with the indicated phosphatases.
Fig. growth supplemented
Fig. 6 shows the parts of an alignment of the phosphatases tested in the examples.
Exam le 1: Materials and methods
Method and materials Escherichia co/i
Media
Three different media were used, namely a rich Luria Broth a minimal medium for shake
(LB),
flask trace
(MMsf) and a minimal medium for fermentation (MMf). Both minimal media use a
element mix.
Trace element mix consisted of 3.6 FeCI2.4H20, 5 CaCI2.2H20, 1.3 MnCI2.2H20, 0.38
g/L g/L g/L
CuCI2.2H20, 0.5 CoCI2.6H20, 0.94 ZnCI2, 0.0311 H3B04, 0.4 Na2EDTA.2H20
g/L g/L g/L g/L g/L
and 1.01 thiamine.HCI. The molybdate solution contained 0.967 Na2Mo04.2H20. The
g/L g/L
selenium solution contained 42 Se02.
Broth medium
The Luria (LB) consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium),
0.5 % yeast extract (Difco) and 0.5% sodium chloride (VWR, Leuven, Belgium).
Luria Broth plates consisted of the LB media, with 12 Erembodegem,
agar (LBA) agar (Difco,
added.
Belgium)
Minimal medium for shake flask experiments (MMsf) contained 2.00 NH4CI, 5.00
g/L g/L
2.993 KH2P04, 7.315 K2HP04, 8.372 MOPS, 0.5 NaCI, 0.5
(NH4)2504, g/L g/L g/L g/L g/L
carbon source chosen not limited fructose,
MgS04.7H20. A from, but to glucose, maltose,
glycerol and maltotriose, was used. The concentration was default 15 but this was subject
g/L,
to on the experiment. 1 trace element 100 molybdate
change depending mL/L mix, PL/L
solution, and 1 mL/L selenium solution. The medium was set to a of 7 with 1M KOH.
Depending on the experiment lactose could be added as a precursor.
The minimal medium for fermentations contained 6.75 NH4CI, 1.25 (NH4)2S04, 1.15
g/L g/L g/L
KH2P04 or KH2P04 7.31 KH2P04
(low phosphate medium) 2.93 and g/L (high phosphate
medium), 0.5 NaCI, 0.5 MgS04.7H20, a carbon source including but not limited to
g/L g/L
sucrose, fructose, maltose, glycerol and maltotriose, 1 mL/L trace element mix, 100
glucose,
PCT/EP2//17/084593
PL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described
above.
Complex medium, e.g. LB, was sterilized by autoclaving (121 21) and minimal medium (MMsf
and MMf) filtration (0.22 Sartorius). If necessary the medium was made selective
by pm by
an antibiotic ampicillin chloramphenicol carbenicillin
adding (e.g. (100mg/L), (20 mg/L),
spectinomycin and/or kanamycin
(100mg/L), (40mg/L) (50mg/L)).
Strains
Escheri eh/a co/i MG1655 [lambda, rph-1] was obtained from Coli Genetic Stock Center
F, (US),
Strain/8
CGSC 7740 in March 2007. Mutant strains were constructed using the homologous
recombination, as described Datsenko and Wanner (PNAS 97 6640-6645).
by (2000),
Plasmids
pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked
chloramphenicol resistance (contains an FRT-flanked kanamycin resistance
(cat) gene), pKD4
and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof.
(kan) gene),
R. Cunin Universiteit Brussel, Belgium in 2007).
(Vrije
Plasmid pCX-CjneuB was constructed using Gibson assembly. The gene Cjneu81 was expressed
the expression vector as described Aerts et. al Life Sci. 2011, No. 10-19).
using by (Eng. 11, 1,
Plasmid pCX-CjneuB-NmneuA-Pdbst was constructed using Gibson assembly. The genes
neuB1, NmneuA and Pdbst were expressed the expression vector as described Aerts
Cj using by
et. al (Eng. Life Sci. 2011, 11, No. 10-19).
Plasmids for phosphatase expression were constructed Golden Gate assembly. The
using
phosphatases (EcAphA, EcCof, EcHisB, EcOtsB, EcSurE, EcYaed, EcYcjU, EcYedP, EcYfbT, EcyidA,
EcYigB, EcYihX, EcYniC, EcYqaB, EcYrbL and PsMupP) were expressed using promoters apFAB87
and apFAB346 and UTRs gene10 SD2-junction HisHA and UTR1
AATTCGCCGGAGGGATATTAAAAtgaatggaaaattgAAACATCTTAATCATGCTAAGGAGGTTTTCTAATG
(SEQ ID NO: 41). All promoters and UTRs except UTR1 are described by Mutalik et. al (Nat.
Methods 2013, No. 354-360). Also phosphatases EcSerB, EcYbhA,
, EcAppA, EcGph, EcNagD,
ScDOG1 BsAraL are
EcYbiV, EcYbjL, EcYfbR, EcYieH, EcYjgL, Ec EcYrfG, EcYbiU, and
YjjG,
expressed the same promoters and UTRs.
using
pBR322-NmneuB
Plasmid was constructed using a pBR322 vector via Golden Gate assembly.
The promoter and UTR used for the expression of NmNeuB are promoter apFAB299 and UTR
SD2-junction pSC101-NmneuA-Pdbst
galE BCD12. Plasmid was constructed using a pSC101
vector via Golden Gate assembly. The promoters and UTRs used for the expression of NmneuA
UTR SD2-junction The UTRs for
are promoter apFAB37 and galE BCD18. promoters and used
SD2-junction
the expression of Pdbst are promoter apFAB339 and UTR galE BCD12. All
promoters and UTRs are described Mutalik et. al Methods 2013, No. 354-360).
by (Nat. 10,
Plasmids were maintained in the host E. coll DH5alpha phi80d/acZde/taM15, delta(/acZYA-
mk'), th/-1,
argE) U169, deoR, recA1, endA1, hsdR17(rk, phoA, supE44, lambda, gyr496, re/A1).
40 Bought from lnvitrogen.
Gene disruptions
Gene disruptions as well as introductions were performed the technique published
gene using
6640-6645).
by Datsenko and Wanner (PNAS 97 (2000), This technique is based on antibiotic
selection after homologous recombination performed lambda Red recombinase. Subsequent
catalysis of a recombinase ensures removal of the antibiotic selection cassette in the
flippase
final production strain.
In Table A the necessary primers for the construction of the disruption cassette are listed.
gene
Table A; Lists of primers to construct disruption cassette for the target gene.
Gene target Fw primer Rv primer
' ''
~ ~ ~ ~ ~ ~
nagABCDE CGCTTAAAGATGCCTAATCCGCCAACGG GGCGTTTGTCATCAGAGCCAACCACGT
CTTACATTTTACTTATTGAGGTGAATAGT CCGCAGACGTGGTTGCTATCATATGAAT
GTAGGCTGGAGCTGCFTC ID NO: ATCCTCCTTAG ID NO:
(SEQ (SEQ 4)
I J I l k
~ ~ ~ ~ s ~ ~
I I k 4 I k l 4 I I I 4 I I l I I I I
I I J k I J k I l I I I 4 I I J 4
'' '
~ ~ ~ ~ ~
For the genomic integration of the necessary genes into the production hosts genome based on
the same technique used for the disruption, discussed before, with specific alterations to
gene
the disruption cassette. Between a homology site and the FRT site of the disruption cassette,
the to be integrated constructed is located. This allows for elegant integration of the
constructed in the region dictated the homology sites.
this workf a direct disruption and genomic integration is possible. Primers that
Using low, gene
were used for target integration are at specific sites are listed in Table B.
Table B: Primers used for genomic integration
GTGTAGGCI GGAGCTGCTTC TTAAGCGACTTCATTCACC ID
(SEQ (SEQ
ID NO: 9) NO: 10)
nanATEK CATGGCGGTAATGCGCCGCCAGTA CCAACAACAAGCACTGGATAAAGCG
AATCAACATGAAATGCCGCTGGCTC AGTCTGCGTCGCCTGGTTCAGTTCAC
CGTGTAGGCTGGAGCTGCTTC TTAAGCGACTTCATTCACC ID
(SEQ (SEQ
ID NO: NO;
11) 12)
'' ' '
s ~ ~
iacZYA GCGCAACGCAATTAATGTGAGTTAG GCTGAACTTGTAGGCCTGATAAGCG
CTCACTCATTAGGCACCCCAGGCTT CAGCGTATCAGGCAATTTTTATAATC
GTGTAGGCTGGAGCTGCTTC TTAAGCGACTTCATTCACC ID
(SEQ (SEQ
ID NO: 15) NO: 16)
atpl-g/dB CAAAAAGCGGTCAAATTATACGGTG ATAACGTGGCTTTTTTTGGTAAGCAG
CGCCCCCGTGATTTCAAACAATAAG AAAATAAGTCATTAGTGAAAATATCT
GTGTAGGCTGGAGCTGCTTC (SEQ TAAGCGACTTCATTCACC ID
(SEQ
ID NO: NO:
17) 18)
Clones carrying the temperature sensitive pKD46 helper plasmid were grown in 10 mL LB media
with ampicillin and L-arabinose at 30 to an of 0.6. The cells were
(100 mg/L) (10 mM) ODsoonm
ice-cold
made electro competent sequential washing, once with 50 mL, and once with 1mL
deionized water. the cells were resuspended in 50 of ice-cold water. 10-100
Next, pL Finally, ng
of disruption/integration cassette was added to 50 of the washed cell solution for
electroporation. Electroporation was performed a Gene Pulser (trademark of BioRad)
using (600
Ohm 25 PFD, and 250 V).
After electroporation, cells were resuscitated in 1 mL LB media for 1 h at 37 and finally plated
onto LB-agar of of select
out containing 25 chloramphenicol or 50 kanamycin to
mg/L mg/L
antibiotic resistant transformants. The selected mutants were verified PCR with primers
upstream and downstream of the modified region and were subsequently grown on LB-agar at
42 for the loss of the pKD46 helper plasmid. The mutants were finally tested for ampicillin
sensitivity.
The selected mutants (chloramphenicol or kanamycin resistant) were transformed with pCP20
which ampicillin and chloramphenicol resistant that shows temperature-
plasmid, is an plasmid
sensitive replication and thermal induction of FLP synthesis. The ampicillin-resistant
transformants were selected at 30 after which a few were colony purified in LB at 42 and
then tested for loss of all antibiotic resistances and thus also of the FLP helper plasmid. The gene
disruptions and/or integration are checked with control primers and sequenced. These
gene
primers are listed in Table C.
Table C: Primers to validate either gene disruption and/or genomic integration for specific gene
targets.
Gene targets Fw primer Rv primer
lacZYA CAGGTTTCCCGACTGGAAAG TGTGCGTCGTTGGGCTGATG
(SEQ (SEQ
ID NO; 19) ID NO; 20)
nagABCDE CGCTTGTCATTGTTGGATGC GCTGACAAAGTGCGATTTGTTC
(SEQ (SEQ
ID NO; ID NO;
21) 22)
nanATEK GTCGCCCTGTAATTCGTAAC CTTTCGGTCAGACCACCAAC ID
(SEQ (SEQ
ID NO; 23) NO; 24)
manXYZ
ACGCCTCTGATTTGGCAAAG (SEQ AGCCAGTGCGCTTAATAACC (SEQ ID
ID NO; 25) NO; 26)
atpl-gidB GCTGAACAGCAATCCACTTG (SEQ TGAACGATATGGTGAGCTGG (SEQ
ID NO; ID NO;
27) 28)
Heterolo ous and homolo ous ex ression
Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically
synthetized with one of the following companies: DNA2.0, Gen9 or IDT.
Escherl co/I native as the E, coll K-12
eh/a genes, e.g., phosphatases, were picked from MG1655
genome. The origin of other genes are indicated in the relevant table.
Expression could be further facilitated the codon to the codon of the
by optimizing usage usage
expression host. Gene were optimized using the tools of the supplier.
Cultivation conditions
A preculture of 96well microtiter plate experiments was started from single colony on a LB plate,
in 175 and was incubated for Bh at 37 C on an orbital shaker at 800 rpm. This culture was
as inoculum for 96well microtiter with 175 medium 300x.
used a plate, MMsf diluting
PL by
These cultures in turn, were used as a preculture for the final experiment in a 96well
plate, again
by diluting 300x. The 96well plate can either be microtiter plate, with a culture volume of 175
or a 24well deepwell plate with a culture volume of 3mL.
A preculture for shake flask experiments was started from a single colony on a LB-plate, in 5 mL
medium incubated for at orbital shaker From this
LB and was 8 h 37 on an at 200 rpm. culture,
1 mL was transferred to 100 mL minimal medium in a 500 mL shake flask and incubated
(MMsf)
at 37 on an orbital shaker at 200 rpm. This setup is used for shake flask experiments.
A shake flask experiment for 16h could also be used as an inoculum for a bioreactor. 4%
grown
Dcu-B
of this cell solution was to inoculate a 2L Biostat with a 4 L working volume, controlled by
MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culturing condition
were set to 37 800 rpm stirring, and a gas flow rate of 1.5 L/min. The pH was controlled at 7
0.5 M H2504 and 25% NH4OH. The exhaust was cooled. 10% solution of silicone
using gas
antifoaming agent was added when foaming raised during the fermentation (approximately 10
6L). The use of an inducer is not required as all genes are constitutively expressed.
Material and methods Saccharom ces cerev/sae
Media
Strains are grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD
or CSM drop-out CSM-Ura) containing 6.7 Yeast Nitrogen Base without amino acids
CSM) (SD
(YNB w/o AA, Difco), 20 agar (Difco) (solid cultures), 22 glucose monohydrate or 20
g/L g/L g/L
lactose and 0.79 CSM or 0.77 CSM-Ura Biomedicals).
g/L g/L (MP
Strains
cerev/s/ae et 14:115-32)
Saccharomyces BY4742 created by Bachmann al. (Yeast (1998) was
used available in the Euroscarf culture collection. All mutant strains were created
homologous recombination or plasmid transformation the method of Gietz 11:355-
using (Yeast
360, 1995). Kluyveromyces marx/anus lact/s is available at the LMG culture collection (Ghent,
Belgium).
Plasmids
sia 70 for
Yeast expression plasmid p2a 2p GFA1 (Chan 2013 (Plasmid (2013) 2-17)) was used
expression of foreign genes in Saccharomyces cerev/sae. This plasmid contains an ampicillin
resistance and a bacterial origin of replication to allow for selection and maintenance in E.
gene
coll. The plasmid further contains the yeast ori and the Ura3 selection marker for selection
and maintenance in yeast. Finally, the plasmid can contain a beta-galactosidase expression
cassette. Next, this plasmid also contains a /y-acetylglucosamineepimerase (for example from
Bactero/des ovatus and a sialic acid synthase example from un/
(BoAGE)) (for Campylobacterj ej
fructoseP-aminotransferase
(CjneuB)). Finally, it also contains the from Saccharomyces
cerev/s/ae, ScGFA1.
sia is based sia modified in that
Yeast expression plasmid p2a 2p glmS on p2a 2p but a way
also (fructoseP-aminotransferase from Escherl eh/a coll is expressed.
9/mS
Yeast expression plasmids p2a sia~lmS phospha is based on p2a sia~lmS but
2p 2p
modified in a that also EcAphA ID NO; EcCof ID NO: EcHisB ID NO:
way (SEQ 42), (SEQ 43), (SEQ
44), EcOtsB (SEQ ID NO: 45), EcSurE (SEQ ID NO: 46), EcYaed (SEQ ID NO: 47), EcYcjU (SEQ ID
NO: EcYedP ID NO: EcYfbT ID NO: EcYidA ID NO: EcYigB
48), (SEQ 49), (SEQ 50), (SEQ 51), (SEQ
NO: EcYihX ID NO: NO: ID NO:
ID 52), (SEQ 53), EcYniC (SEQ ID 54), EcYqaB (SEQ 55), EcYrbL (SEQ
ID NO: PsMupP ID NO: ID NO: EcGph ID NO: EcSerB
56), (SEQ 57), EcAppA (SEQ 58), (SEQ 59),
(SEQ ID NO: 60), EcNagD (SEQ ID NO: 61), EcYbhA (SEQ ID NO: 62), Ecybiy (SEQ ID NO: 63), EcYbjL
ID NO: EcyfbR ID NO: EcYieH ID NO: EcYjgL ID NO: Ec
(SEQ 64), (SEQ 65), (SEQ 66), (SEQ 67), YjjG
(SEQ ID NO; 68), EcYrfG (SEQ ID NO: 69), EcYbiU (SEQ ID NO: 70), ScDOG1 (SEQ ID NO: 71) and
BsAraL (SEQ ID NO: 72) are expressed.
SL-glmS
Yeast expression plasmid p2a 2p is based on p2a 2p sia but modified in a way that
also KILAC12 (lactose permease from Kluyveromyces NmneuA (CMP-sialic acid synthase
/act/s),
Ne/sser/a Pdbst Photabacterium
from mening/t/des) and (sialyltransferase damselae) are
expressed.
Plasmids were maintained in the host E. coli DHSalpha phi80d/acZdeltaM15, delta(/acZYA-
mk"), th/-1,
argF)U169, deoR, recA1, endA1, hsdR17(rk, supE44, lambda, re/A1).
phoA, gyrA96,
Bought from lnvitrogen.
Gene ex ression romoters
Genes are expressed using synthetic constitutive promoters, as described in by Blazeck
(Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).
Heterolo ous and homolo ous ex ression
Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically
synthetized with one of the following companies: DNA2.0, Gen9 or IDT
Expression could be further facilitated optimizing the codon usage to the codon usage of the
expression host. Gene were optimized the tools of the
using supplier.
Cultivations conditions
In strains were initially on SD CSM plates to obtain single colonies. These
general, yeast grown
2-3 30'C.
plates were grown for days at
Starting from a single a preculture was grown over night in 5 mL at 30 shaking at
colony,
flask inoculated with of
200rpm. Subsequent 500 mL shake experiments were 2% this
preculture, in 100 mL media. These shake flasks were incubated at 30 with an orbital shaking
of 200 rpm. The use of an inducer is not required as all genes are constitutively expressed.
Material and methods Bacillus subt///s
Media
Two different media are used, namely a rich Luria Broth (LB), a minimal medium for shake flask
(MMsf). The minimal medium uses a trace element mix.
Trace element mix consisted of 0.735 CaCI2.2H20, 0.1 MnCI2.2H20, 0.033
g/L g/L g/L
CuCI2.2H20, 0.06 CoCI2.6H20, 0.17 ZnCI2, XX H3B04, XX Na2EDTA.2H20 and 0.06
g/L g/L
g/L g/L
Na2Mo04. The Fe-citrate solution contained Na-Citrate
0. 135 FeCI3.6H20, 1 (Hoch
g/L g/L g/L
1973 PMC1212887).
The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium),
0.5% yeast extract (Difco) and 0.5% sodium chloride (VYVR, Leuven, Belgium).
Luria Broth agar (LBA) plates consisted of the LB media, with 12 agar (Difco, Erembodegem,
Belgium) added.
Minimal medium for shake flask experiments (MMsf) contains 2 (NH4)2S04, 7.5 KH2P04,
g/L g/L
17.5 K2HP04, 1.25 Na-Citrate, 0.25 MgS04.7H20, from 10 to
g/L g/L g/L 0.05g/L tryptophan, up
glucose or another carbon source including but not limited to glucose, fructose, maltose,
glycerol and maltotriose, 10 mL/L trace element mix, and 10 mL/L Fe-citrate solution. The
medium was set to a of 7 with 1M KOH.
Complex medium, e.g. LB, was sterilized autoclaving (121 and minimal medium
by 21)
(MMsf) by filtration (0.22 pm Sartorius). If necessary, the medium was made selective by adding
an antibiotic zeocin
(e.g. (20mg/L)).
Strains
Bacillus subtilis 168, available at Bacillus Genetic Stock Center (Ohio, USA).
Plasmids and ene overex ression
Plasmids for gene deletion via Cre/lox are constructed as described yan et al. /k
by (Appl
environm microbial, 2008, p5556-5562).
sept
Expression vectors can be found at Mobitec (Germany), or at ATCC (ATCCa number 87056). The
ScGNA1 and CjneuB are cloned in these expression vectors. A suitable promoter
genes Bsg/mS,
for expression can be derived from the part repository (iGem): sequence id: BBa K143012,
BBa K823000, BBa K823002 or BBa K823003. Cloning can be performed Gibson
using
Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
Plasmids are maintained in the host E. co/i DHSalpha 80dlacZdeltaM15, delta(/acZYA-
(F, phi
argF)U169, deoR, recA1, endA1, hsdR17(rk, mk'), phoA, supE44, lambda, thi-l, gyrA96, re/A1).
Bought from lnvitrogen.
~GN H
of is done via homologous recombination with linear DNA and transformation
Disrupting genes
183-191).
via the electroporation as described Xue et al. microb. Meth. 34 (1999) The
by (2
method of knock-outs is described Liu et al. (Metab. Engine. 24 61-69). This
gene by (2014)
method uses 1000bp homologies up- and downstream of the target gene. The homologies to
be used in this invention, are listed in table D. After the modification, the mutants are verified
and downstream of modified These are in
using primers upstream the region. primers given
table E. Next, the modification is confirmed at LGC Genomics
by sequencing (performed (LGC
group, Germany)).
Table D
Gene to be Upstream Downstream
homology homology
disrupted
nagA-nagB Gactgcaagatttcggcctgggcggacgggaat Aaggaacatgctgacttatgaatatcaataaaca
cgtcagttttgtaatttctgtatcaatgattttcat atcgcctattccgatttactatcagattatggagca
ggtctcttcctcaagtccgagccggtcgtattgct attaaaaacccaaattaagaacggagagctgcag
tgccctgctcccagagttcaagattcatgacaat ccggatatgcctcttccttctgagcgcgaatatgcc
cgtgattcgtttattgcttctgaccgcgccagcgc gaacaattcgggatcagccggatgacagttcgcc
caaatagcgtcatcacattgataatgccaaggcc aggcgctttctaatttagttaatgaaggcttgctct
cctgatctcaagaaggtgctcaattaattccgga atcgcctgaaagggcggggcacctttgtcagcaa
gcgtttcccacaagagtatcctgatcctcctgccg gccaaaaatggaacaagcacttcaagggctgaca
tatttcaacgcaatcatcggcaacaaggcgatgc agctttaccgaggatatgaaaagccgcgggatga
cctcttttcacaagctctagcgctgtttcgctttttc
caccgggcagcaggctcattgattatcagcttatt
cgacgccgctttttcctgtgatcagcacgccga gattcaactgaggagctcgcggctatattaggctg
accatatatatcgacaagaacgccatgaattgct cgggcacccctcctctatccataaaatcactcggg
gtggtaggcgccagcctgctctcaaggaagttgg tgcggctggcaaatgatattccgatggcgattgag
ttaaacggcttgacagtcttgtcgttttcagcggc tcctcacatattccgtttgagcttgcgggtgaattg
gatctgaggacaggcaccccatttttctcggagg aacgaatcgcattttcagtcgtcgatctatgatcat
cgtcaatcagctcctgcgggatgggcatatctct attgaaaggtacaacagcataccgatttcccgtgc
agaaagaataatagctggtgttacatcagtgcac aaaacaggagcttgagccaagcgctgccaccacg
agagaatccattcgctgctttttctcctcttcagga
gaagaagcgaatattcttggtattcaaaagggag
agctgttcaaagaaagaaagctctgtttttccga cgcctgtcctattaattaaacgaacaacatatctgc
gaagctgcacgcgctccctcgggtaatatgtaaa agaacggaactgcttttgagcatgcaaaatccgta
atatccggcaatttcaatacctggtcttgataggt tacagaggcgaccgttatacatttgtccactatatg
gatcgtctttcataaaaaaagcctccaacccttttt
cactcattgtaatcgggcggttaattccttcttctc
cgctgattaattccaaattgaactgttccattacg aaggattggagacatggcgaaaatcaaactggtc
tcttttgtgcgaacctttgccacgatatgttcctcc
tggtgccggacgatatgtttcttttttcgtcttgaac
tgttccgggctgccccgagcttgctcacaatactt ttccagatcggtgatttcgttttgccgttaaaactgt
tcattttatcactttcgggcttgaacctaaaacag cttccactataatgtaccaataataaacagactgc
attttataaaaggggggaaaacacctcagctggt ggttcaagatgatcccagcggaattcagctgtgtc
ctagatcactagtctgaaaaagagtaaaataaa cccgctcttcacttgctcccgttttccgagctcttca
ggtattcaaattccagaaaggcggatcatet ttggtatatacgtta ID NO:
(SEQ 34)
ID NO:
(SEQ 33)
gamA Tggcggacatggaataaatcacaaacgacaaa Gtgacaccccctcaaagagatagacaagcaccat
atttgttatgaccaatttatgatacttgtcattacga
gatgacgccggcaagaatagagttaatcaaata
gagcacgggcgcaacgaacaagaaagaaaact atttagcaccgcccttatcaaactgtcaatattaat
caaccggttctgtaattccggtcagcatagatgt ttctgaaaatttgttataaaagaaggatacaaatc
gagcgccgcagaaatcatcacgccggagatcat tttcatattgggagggcaaatggtattatggtctca
ttttttcttttccggacgcgcggtatggataatggc atgaaaaagaacggattgcatacagaatgggga
aagagcaacggccggcagacagaaaatcatgt gaatgaaatgacagctttatattctgttatcaagtt
aagggaaatcccccatcataaagcgcccggctg taaaatcattgagttaattaaatcgggcaaatatc
tcgggtctcccgcgaaaaaccttgtcaggtcgcc aggcgaatgatcagctgccgacggagagtgagtt
ggttacggtgttgcctgttgatgggtctgtgtattc ttgcgaacaatatgatgtcagcagaacaactgtga
tcccatcataaaatagaaaggcgtataaaaaat gactggctctgcagcagctagagcttgagggatat
attaaaagaattcaaggaaaagggacatttgtat
atgatgcaggccaaaaggaatcagcaaacgat
agatcgttgcataaaagaacaggccgactgttg cggcggccaaaatacaaacgccgattccgcataa
aatcggcaattaaactgctggctgcgttaattcc gattacgagctttgcagaacaaatgagaggacttc
gttttggatcagcggccaaacgaatgagaaaat gttctgaatcaaaagtgcttgagcttgtggtgattc
gacgccgatcaccaatgaactgacggaagtaat ctgccgatcattccatcgccgagcttttgaaaatga
gatcgggacaaagcgttttccagagaaaaatcc aagagaatgaacctgtcaacaagcttgtcagagt
aaggaccggatgcagctcgattgatgaaaatcg cagatacgccgagggggaacctttgcagtatcat
cttatataaataggcggcgagaagcccgataat acctcatatattccctggaaggcggcaccggggct
gattcctccgaaaacccccatatcaatcaggtgc
ggcgcaggaggaatgcaccggctcgctgtttgaat
tcggctccttcatacggaggctgaaggccgagta tgttaaggacaaaatacaatattgaaatcagcag
attttcccatattgtcgagggtgacggttaaaatt gggcacggaatcgatcgaaccgattttaacggat
aagtatccgatgacagcggcaagtccggctaca gaaacgatcagcggacacttattaaccaatgtcg
ccccca gagcgcctgcgtttttatcagaatcccttacctatg
ccttctccgccggctaatccgatcgcga
cggcgaaaatcagcggaaggttatcgaatacaa ataaaaatgaagaagtggtggaatatgcgcaaat
cgccgcccgcatcctttataatagggatgttcagt tattacacggggagaccgaacgaaattcaccgta
gaacagtcatatcattcataaagcaatgtgttttaa
aaatccttgtctccgaaacggagcaaaagacct
gaagggaatggtggttctatgtttttatttacgaat
gctgccggcaggacggcaaccggagtcatcaac
gcgcggccaagctgctgcagaatttgaaatgcct ggaaaagtgctgtggggagcagt (SEQ ID NO:
ttttaaacatgacagtctccttttattgtg 36)
(SEQ
ID NO:
Table E
Heterolo ous and homolo ous ex ression
Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically
synthetized with one of the following companies: DNA2.0, Gen9 or IDT.
Expression could be further facilitated by optimizing the codon usage to the codon usage of the
expression host. Gene were optimized using the tools of the supplier.
Cultivations conditions
A preculture, from a single colony on a LB-plate, in 5 mL LB medium was incubated for 8 h at 37
on an orbital shaker at 200 rpm. From this culture, 1 mL was transferred to 100 mL minimal
medium in a 500 mL shake flask and incubated at 37 C on an orbital shaker at 200
(MMsf) rpm.
This setup is used for shake flask experiments. The use of an inducer is not required as all genes
are constitutively expressed.
Anal tical methods
i d it
~Oti
Cell density of the culture was frequently monitored measuring optical density at 600 nm
Nanophotometer NP80, Cell was obtained
(Implen Westburg, Belgium). dry weight by
centrifugation (10 min, 5000 Legend X1R Thermo Scientific, Belgium) of 20 reactor broth in
g, g
pre-dried
and weighted falcons. The pellets were subsequently washed once with 20 mL
solution and dried at 70 to a constant weight. To be able to convert
physiological (9 g/L NaCI)
ODeoonm measurements to biomass concentrations, a correlation curve of the OD6oonm to the
biomass concentration was made.
Measurement of cell dr wei ht
From a broth sample, 4 x 10 was transferred to centrifuge tubes, the cells were spun down
4 5 and the cells were washed twice with 0.9% NaCI solution. The centrifuge
(5000g, min),
tubes containing the cell pellets were dried in an oven at 70 for 48 h until constant weight.
cell dry weight was obtained gravimetrically; the tubes were cooled in a desiccator prior to
weighing.
Li uid chromato ra h
The concentration of carbohydrates like, but not limited to, glucose, fructose and lactose were
determined with a Waters UPLC H-class system with an ELSD detector, a
Acquity using Acquity
UPLC BEH amide, 130 1.7 2.1 mm x 50 mm heated at 35 using a 75/25
A, pm,
acetonitrile/water solution with 0.2% triethylamine (0.130 mL/min) as mobile phase.
Sialyllactose was quantified on the same machine, with the same column. The eluent however
was modified to 75/25 acetonitrile/water solution with 1% formic acid. The flow rate was set to
0.130 mL/min and the column temperature to 35
Sialic acid was quantified on the same machine, the REZEX ROA column x 7.8 mm
using (300 ID).
The eluent is 0.08% acetic acid in water. The flow rate was set to 0.5 mL/min and the column
temperature to 65 C. GlcNAc and ManNAc were also measured using this method.
Growth rate measurement
The maximal growth rate was calculated based on the observed optical densities at
(pMax)
600nm using the R package grofit.
Exam le 2: roduction of sialic acid in Escherlchla coll
A first example provides an Escherl chio co/i strain capable of producing ly-acetylneuraminate
(sialic acid) (see figure 18).
A strain capable of accumulating glucosaminephosphate using sucrose as a carbon source
was further engineered to allow for N-acetylneuraminate production. The base strain
overexpresses a sucrose phosphorylase from Bifidobacterium adolescentis a
(BaSP),
fructoseP-aminotransferase
fructokinase from Zymomonas mabilis (Zmfrk), a mutant
"54, 419-429
as described Deng et al. (Biochimie 88, To allow for gene sia lie
by (2006))).
(EcglmS
acid the nanATEK and manXYZ were BaSP
production operons nagABCDE, disrupted. and Zmfrk
were introduced at the location of nagABCDE and was introduced at the location of
EcglmS
nanATEK. modifications were described in are based on
These done as example 1 and the
principle of Datsenko & Wanner (PNAS USA 97, 6640-6645
(2000)).
In this strain, the biosynthetic for sialic acid as described in this invention,
pathway producing
was implemented overexpressing a glucosamineP-aminotransferase from Saccharomyces
cerev/s/ae a N-acetylglucosamineepimerase from Bacteroides ovatus and
(ScGNA1), (BoAGE)
a sialic acid synthase from Campylobacterjejun/ (CjneuB). 5cGNA1 and BoAGE were expressed
on locations nagABCDE and manXYZ, respectively. CjneuB was expressed the
using high copy
plasmid pCX-CjneuB.
The strain was cultured as described in example 1 (materials and methods). Briefly, a SmL LB
preculture was inoculated and overnight at 37 This culture was used as inoculum in a
grown
shake flask experiment with 100mL medium which contains sucrose and was made as
10g/L
described in example 1. Regular samples were taken and analyzed as described in example 1.
The evolutions of the concentrations of biomass, sucrose and sialic acid are easily followed and
N-acetylneuraminate
an end concentration of 0.22g/L was produced extracellularly, as can be
seen in figure 2.
The same organism also produces N-acetylneuraminate based on maltose or
glucose, glycerol
as carbon source.
6-sial Escherichia
Exam le 3: roduction of llactose in cali
Another example according to present invention is the use of the method and strains for the
6-sialyllactose.
production of
The strain of example 3 is a daughter strain of the strain used in example 2. The strain is further
Escher/ch/a co/i
modified by overexpressing a lactose permease Eclacy from (as described and
demonstrated in example 1 of yyO 2016/075243 which is here also incorporated reference),
CMP-sialic
a acid synthethase from Ne/sser/a menlng/tides (NmneuA) and a sialyltransferase
from Photobacterium damselae On of that lacZ is disrupted.
(Pdbst). top
NmneuA
The genes and Pdbst, are expressed from a plasmid, together with CjneuB. This plasmid
is pCX-CjneuB-NmneuA-Pdbst, and is made as described in example 1.
Said strain is inoculated of 5ml described in
as a preculture consisting LB medium as example 1.
37'C
After growing overnight at in an incubator. 1% of this preculture is inoculated in a shake
flask containing 100ml medium containing sucrose as carbon source and 10
(MMsf) 10g/I
37'C.
lactose as precursor. The strain is for 300h at
grown
6-sialyllactose.
This strain produces quantities of
Exam le 4: roduction of sialic acid in 5accharom ces cerevisiae usin heterolo ous
fructoseP-aminotransferase
Another use of in form of 5accharomyces
example provides an eukaryotic organism, the
cerev/sae, for the invention. This method utilizing the of the invention shall be obtained
pathway
N-acetylglucosamineepimerase
in 5accharomyces cerevlsi ae by introducing and expressing a
(for example from Bactero/des ovatus (BoAGE)) and a sialic acid synthase (for example from
Campylobacterj ej un/ neuB)).
As starting point, a strain with increased metabolic flux towards N-acetylglucosamine
is needed. This achieved fructoseP-aminotransferase
phosphate is overexpressing the
"Szf}.
mutant from Escher/ch/o col/
(Ecglm5
To create a N-acetylneuraminate producing 5accharomyces cerevislae according to this
invention, the genes are introduced via a 2-micron plasmid (Chan 2013 (Plasmid 70 (2013}
17)) and the genes are expressed using synthetic constitutive promoters (Blazeck 2012
and Bioengineering, Vol. No. as also described in example 1. The
(Biotechnology 109, 11))
specific plasmid used in this embodiment is p2a 2p sia glmS. This plasmid is introduced into
Saccharomyces cerevisae using the transformation technique described Gietz and Woods
PMID 12073338) and a mutant strain is obtained
(2002,
Said strain is capable of converting fructosephosphate into glucosaminephosphate,
N-acetylglucosaminephosphate.
followed by glucosaminephosphate conversion in This
acetylglucosaminephosphate moiety is further converted to N-acetylglucosamine, said
N-acetylmannosamine N-acetylmannosamine
acetylglucosamine into and finally this is
converted into N-acetylneuraminate.
A preculture of said strain is made in SmL of the synthetic defined medium SD-CSM containing
'C
22 glucose and grown at as described in example 1. This preculture is inoculated in
'C.
100mL medium in a shakeflask with sucrose as sole carbon source and at
10g/L grown
Regular samples are taken and the production of N-acetylneuraminate is measured as described
in example 1. This strain and method produces quantities of N-acetylneuraminate.
The same organism also produces N-acetylneuraminate based on glucose, maltose or glycerol
as carbon source.
6-sial cerevlsiae
Exam le 5: roduction of llactose in Saccharom ces
Another example provides use of an eukaryotic organism, in the form of Saccharomyces
cerevisae, for the invention. This method utilizing the of the invention shall be obtained
pathway
N-acetylglucosamineepimerase
in Saccharomyces cerevislae by introducing and expressing a
(for example from Bacteroides ovatus (BoAGE)) and a sialic acid synthase (for example from
Campylobacterj neuB)).
ej (Cj
On top of that, further modifications are made in order to produce 6sialyllactose. These
CMP-sialic
modifications comprise the addition of a lactose permease, a acid synthase and a
sialyltransferase. The preferred lactose permease is the KILAC12 gene from Kluyveromyces lactls
CMP-sialic
(). The preferred acid synthase and the sialyltransferase are
respectively NmneuA from Neisseria meningitides and Pdbst from Photobacterl um damselae, as
also described in example 3.
As starting a strain with increased metabolic flux towards N-acetylglucosamine
point,
needed. This is achieved the fructoseP-aminotransferase
phosphate is by overexpressing
mutant from Escherl eh/a coll (Ecglm5*54).
N-acetylneuraminate
To create a producing Saccharomyces cerevisiae according to this
invention, the genes are introduced via a 2-micron plasmid (Chan 2013 (Plasmid 70 (2013)
and the are expressed synthetic constitutive promoters (Blazeck 2012
17)) genes using
(Biotechnology and Bioengineering, Vol. No. as also described in example 1. The
109, 11))
used in this embodiment is sia This introduced into
specific plasmid p2a 2p glmS. plasmid is
Saccharomyces cerevlsae using the transformation technique described Gietz and Woods
and a mutant strain is obtained
(2002)
fructosephosphate
Said strain is capable of converting into glucosaminephosphate, said
glucosaminephosphate into N-acetylglucosaminephosphate, said N-acetylglucosamine
N-acetylmannosamine
phosphate into N-acetylglucosamine, said N-acetylglucosamine into and
finally said N-acetylmannosamine into N-acetylneuraminate. Said N-acetylmannosamine is then
converted to CMP-sialic acid and transferred to lactose to obtain 6sialyllactose.
SD-CSM
A preculture of said strain is made in SmL of the synthetic defined medium containing
'C
22 glucose and grown at as described in example 1. This preculture is inoculated in
'C.
100mL medium in a shakeflask with 10g/L sucrose as sole carbon source and grown at
N-acetylneuraminate
Regular samples are taken and the production of is measured as described
in example 1. This strain and method produces quantities of 6sialyllactose.
The same organism also produces N-acetylneuraminate based on maltose or
glucose, glycerol
as carbon source.
Saccharom cerev/siae
Exam le 6: roduction of sialic acid in ces usin autolo ous
fructoseP-aminotransferase
Another example provides use of an eukaryotic organism, in the form of Saccharomyces
cerevisae, for the invention. This method utilizing the of the invention shall be obtained
pathway
in Saccharomyces cerevisiae introducing and a N-acetylglucosamineepimerase
by expressing
Bacteroides ovatus
(for example from (BoAGE)) and a sialic acid synthase (for example from
Campy/obacterjejuni (CjneuB)).
As starting a strain with increased metabolic flux towards N-acetylglucosamine
point,
fructoseP-
phosphate is needed. This is achieved overexpressing the native
aminotransferase ScGFA1.
To create a N-acetylneuraminate producing Saccharomyces cerevisiae according to this
via 2-micron 70
invention, the genes are introduced a plasmid (Chan 2013 (Plasmid (2013)
and the genes are expressed using synthetic constitutive promoters (Blazeck 2012
17))
and Vol. No. as also described in example 1. The
(Biotechnology Bioengineering, 109, 11))
specific plasmid used in this embodiment is p2a sia GFA1. This plasmid is introduced into
Saccharomyces cerevisae the transformation technique described Gietz and Woods
using by
(2002) and a mutant strain is obtained
strain is fructosephosphate into said
Said capable of converting glucosaminephosphate,
glucosaminephosphate into N-acetylglucosaminephosphate, said N-acetylglucosamine
into N-acetylglucosamine, said N-acetylglucosamine into N-acetylmannosamine and
phosphate
N-acetylneuraminate.
finally said N-acetylmannosamine into
A preculture of said strain is made in 5mL of the synthetic defined medium SD-CSM containing
22 glucose and grown at 30 C as described in example 1. This preculture is inoculated in
100mL medium in a shakeflask with sucrose as sole carbon source and at 30 C.
10g/L grown
Regular samples are taken and the production of /V-acetylneuraminate is measured as described
in example 1. This strain and method produces quantities of N-acetylneuraminate.
N-acetylneuraminate
The same organism also produces based on glucose, maltose or glycerol
40 as carbon source.
Exam le 7: roduction of sial llactoses and othersial lated cpm ounds
In an alternative embodiment of example the sialyltransferase is changed to another
sialyltransferase with different activity. This can be an alpha-2,3-sialyltransferase alpha-2,6-
sialyltransferase, an alpha-2,8-sialyltransferase or a combination thereof. These
sialyltransferases are widely available in nature and well annotated.
In this production of different sialyllactoses like for example 6-sialyllactose, 3-sialyllactose
way,
or a mixture thereof can be obtained.
The strains are cultivated as stated in example 1 and example 3.
The pathways created in examples 2 to 7 can also be combined with other pathways for the
synthesis of larger oligosaccharides, sialyl-lacto-N-triose, sialyllacto-N-tetraose,
e.g.
disialyllactose-N-tetraose, sialyllacto-N-neotetraose, disialyllactose-N-neotetraose.
and To this
end, the transferases to synthetize these glycosidic bonds are co-expressed with the pathway
CMP-sialic
genes to form acid and the transferase (as described above) to sialylate said
oligosaccharide.
Examples of such sialyltransferases are ST6Gall, ST6Galll, ST3Gall until Vl, ST6GalNAc I until Vl
and ST85ia I until as described Datta (Current 2009, 483-498) and
Vl, by Drug Targets, 10,
Harduin-Lepers 727-737).
(Biochimie 83 (2001) Further examples originating from marine
organisms are described yamamoto (Mar. Drugs 2010, 2781-2794).
by 8,
lacto-N-neotetraose
Exam le 8: roduction of sial lated
The aim of this experiment was to demonstrate the functionality of presented invention of the
production of other sialylated oligosaccharides, in this case sialyltated lacto-N-neotetraose.
lacto-N-neotetraose strain described in
A producing was developed following the protocol
example 1. For production, the expression of a N-acetylglucosaminyltransferase and a
galactosyltransferase are needed, this is achieved introduction of the NmlgtA and
by genes
NmlgtB respectively, both from Ne/sser/a mening/tides. Next, the lactose importer Ec/acy from
Escherl chio co// is described and demonstrated in example 1 of which is
here also incorporated reference). Finally, the genes ushA and galT are knocked out. In this
a lacto-N-neotetraose strain is obtained.
way, producing
To be able to grow on lactose and produce N-acetylglucosaminephosphate, a sucrose
from Bifldobacterium ado/escent/s a fructokinase from Zymomonas
phosphorylase (BaSP),
fructoseP-aminotransferase
mobilis and a mutant (EcglmS*54, as described Deng et
(frk) by
al (Biochimie 419-429 were overexpressed as described in example 1.
88, (2006)))
In this strain, the method for producing sialic acid as described in this invention, was
implemented overexpressing a glucosamineP-aminotransferase from Saccharomyces
N-acetylglucosamineepimerase ovatus
cerevisiae (ScGNA1), a from Bacteroides (BoAGE) and
a sialic acid synthase from Campy/obacterjejuni ScGNA1 and BoAGE are expressed on
(Cjneug).
locations and manXYZ, is from pCX-CjneuB-
nagABCDE respectively. CjneuB expressed plasmid
NmneuA-Pdbst.
WO 2II18/122225 PCT/EP2il17/084593
lacto-N-neotetraose
Sialylation of the moiety is performed by the conversion of sialic acid to
CMP-salic acid a CMP sialic acid synthethase, NmneuA from Neisseria meningtides,
by e.g.
Photobacterium damselae.
subsequently followed by a sialyl transferase, e.g. Pdbst, from These
genes (NmneuA and Pdbst) are expressed from the high plasmid pCX-Cjneug-NmneuA-
copy
Pdbst.
The strain is cultured as described in example 1 (materials and methods). Briefly, a 5mL LB
37'C.
preculture is inoculated and grown overnight at This culture was used as inoculum in a
shake flask experiment with 100mL medium which contains 10g/L sucrose as carbon and energy
source, 10g/L lactose as precursor and was made according to the description in example 1.
lacto-N-
Regular samples are taken and analyzed. This strain produces quantities of sialylated
neotetraose.
Alternative glycosyltransferases are possible. If Ecyygbo (from Escherichia coli 055rH7) is
expressed instead of Nmfgtg for production of sialylated lacto-N-tetraose is obtained.
example,
Exam le 9; Production of sialic acid with Bacillus subtifis
N-acetylneuraminate
In another embodiment, this invention can be used for production of in
Bacigus subtifis, another bacterial production host.
N-acetylneuraminate
A producing strain is obtained through this invention by starting with a
strain, capable of overproducing glucosaminephosphate intracellularly. For this, the native
fructoseP-aminotransferase
(BsglmS) is overexpressed. The following enzymatic activities are
disrupted knocking out the and N-acetylglucosaminephosphate
by genes nagA, nagB gamA:
deacetylase and glucosaminephosphate isomerase.
In this strain, the method for producing sialic acid as described in this invention, is implemented
glucosamineP-aminotransferase Saccharomyces cerevisiae
by overexpressing a from
(ScGNA1), a N-acetylglucosamineepimerase from Bacteroides ovatus (BoAGE) and a sialic
acid synthase from Campylobacterjejuni (CjneuB). These genes are introduced via a plasmid, as
described in examplel.
The strain is cultured as described in example 1 (materials and methods). Briefly, a SmL LB
preculture is inoculated and grown overnight at 30 This culture is used as inoculum in a shake
flask with medium which contains sucrose to
experiment 100mL 10g/L and is made according
the description in example 1. This strain produces quantities of N-acetylneuraminic acid.
6-sialyllactose
Example 10: Fermentations of producing strain with no excretion of
GlcNAc, ManNAc or sialic acid
Another example according to the present invention provides use of the method and strains for
the production of 6-sialyllactose.
An Escherichia coli strain capable of accumulating glucosaminephosphate using sucrose as a
N-acetylneuraminate
carbon source was further engineered to allow for production. This base
strain overexpresses a sucrose phosphorylase from Bifidobacterium adolescentis a
(BaSP),
fructokinase from Zymomonas mobilis a mutant fructoseP-aminotransferase
(Zmfrk),
419-429 6-
(EcglmS"54, as described Deng et ak (Biochimie 88, (2006)). To allow for
40 sialyllactose production the operons nagABCDE, nanATEK and manXYZ were disrupted. BaSP
EcglmS"54
and Zmfrk were introduced at the location of nagABCDE, was introduced at the
location of nanATEK. These modifications were done as described in example 1 and are based
6640-6645
on the principle of Datsenko & Wanner (PNAS USA 97, (2000)).
In this strain, the biosynthetic pathway for producing 6-sialyllactose as described in this
invention, was implemented overexpressing a glucosamineP-aminotransferase from
5accharomyces cerevisiae (ScGNA1), a N-acetylglucosamineepimerase from Bacteroides
ovatus (BoAGE) and a sialic acid synthase from Neisseria meningitides (IymneuB). ScGNA1 and
BoAGE were expressed on locations nagABCDE and manXYZ, respectively. NmNeuB was
pBR322-NmNeuB.
expressed using the high copy plasmid The strain is further modified by
overexpressing a lactose permease EclacY from Escherichia coli (as described and demonstrated
in example 1 of which is here also incorporated reference), a CMP-sialic
acid synthethase from Neisseria meningitides (NmNeuA) and a sialyltransferase from
Photobacterium damselae On of lacZ is disrupted. NmNeuA and Pdbst were
(Pdbst). top that,
pSC101-NmneuA-Pdbst.
expressed using the low plasmid
copy
The strain was cultured in a bioreactor as described in example 1 (materials and methods).
Briefly, a 5mL LB preculture was inoculated and grown overnight at 37 This culture was used
as inoculum in a shake flask experiment with 500mL medium which contains 10g/L sucrose and
was made as described in example 1. This culture was used as inoculum in a 2L bioreactor
experiment. Regular samples were taken and analyzed as described in example 1. The final
concentration of 6-sialyllactose was 30.5 g/L. No extracellular GlcNAc, ManNAc and sialic acid
was detected during the fermentation and in the final broth.
6-sialyllactose
The same organism also produces based on glucose, maltose or glycerol as
carbon source.
Exam le 11: Effect of hos hatase on rowth and roduction of sialic acid
A further example provides growth results and sialic acid production of several Escherichia coll
N-acetylneuraminate
strains capable of producing (sialic acid) wherein the strains are
expressing an extra phosphatase as indicated hereunder.
strain mutant fructoseP-aminotransferase "54,
The base overexpresses a as
(EcglmS
419-429
described et al. (Biochimie a glucosamineP-aminotransferase
by Deng 88, (2006)),
from 5accharomyces cerevisiae N-acetylglucosamineepimerase from Bacteroides
(ScGNA1), a
ovatus (BoAGE) and a sialic acid synthase from Campylobacterj ejuni (CjneuB). To allow for gene
sialic acid production the operons nagABCDE and nanATEK. The lac YZA was replaced
operon by
only a single gene operon, the native lacY, which is required for the production of sialyllactose
as described in example 10. These modifications were done as described in example 1 and are
based on the principle of Datsenko & Wanner (PNAS USA 97, 6640-6645 (2000)).
This strain with different for
base was then supplemented phosphatase bearing plasmids
comparing the effect of the phosphatase on growth and sialic acid production. The base strain
was used as blank in the comparison. These plasmids consisted besides the and
of, phosphatase
40 a promoter driving expression of the phosphatase, a pSC101 ori and a spectomycin resistance
marker. The following phosphatases were expressed: EcAphA (SEQ ID NO: 42), EcCof (SEQ ID
NO: 43), EcHisB (SEQ ID NO: 44), EcOtsB (SEQ ID NO: 45), EcSurE (SEQ ID NO: 46), EcYaed (SEQ
ID NO: EcYcjU ID NO: EcYedP ID NO; EcYfbT ID NO: EcYidA
47), (SEQ 48), (SEQ 49), (SEQ 50), (SEQ
ID NO; 51), EcYigB (SEQ ID NO: 52), EcYihX (SEQ ID NO: 53), EcYniC (SEQ ID NO; 54), EcYqaB (SEQ
ID NO; EcYrbL (SEQ ID NO: 56) and PsMupP (SEQ ID NO; 57). Other phosphatases that are
55),
expressed are ID NO: ID NO: EcSerB ID NO:
EcAppA (SEQ 58), EcGph (SEQ 59), (SEQ 60), EcNagD
ID NO: EcYbhA ID NO: EcYbiV ID NO: ID NO: EcYfbR
(SEQ 61), (SEQ 62), (SEQ 63), (SEQ 64),
EcYbjL
(SEQ ID NO: 65), EcYieH (SEQ ID NO: 66), EcYjgL (SEQ ID NO: 67), Ec YjjG (SEQ ID NO: 68), EcYrfG
ID NO: EcYbiU ID NO: ScDOG1 ID NO: 71) and BsAraL ID NO: 72).
(SEQ 69), (SEQ 70), (SEQ (SEQ
first
In a experiment a subset of the above described strains was used. In a second experiment
a second subset of the above described strains were tested.
Each strain was cultured as described in example 1 (materials and methods). the
Briefly,
workflow consists of 3 growth steps: first growth on LB, followed growth on MMsf with 15
and finally a MMsf. The first is performed in
g/L glycerol, growth stage using 15g/L glycerol step
a 96well plate, using 175 LB per well, and incubated overnight at 37 The second step is
37'C.
performed in a 96well 175 medium, incubated for 24 h at The final growth
plate using pL
step was performed in;/j in a 96well plate using 175 medium, incubated at 37 to determine
the pMax for the first experiment (see figure 5) and//j in a 24well deepwell plates using 3 mL
do determine sialic acid production and optical densities for the second experiment (see figure
Reference table for Figure 4 and 5:
label phosphatase SEQ ID NO Promotor
blank NA NA NA
EcAphA apFAB346
42 apFAB87
EcAphA
EcCof 43 apFAB87
EcCof 43 apFAB346
EcHisB 44 apFAB346
EcOtsB 45 apFAB346
EcSurE 46 apFAB346
EcSurE 46 apFAB87
EcYaed 47 apFAB346
EcYaed 47 apFAB87
EcYcjU 48 apFAB87
12 EcYedP 49 apFAB87
EcYfbT 50 apFAB87
14 EcYidA 51 apFAB346
EcYidA
51 apFAB87
16 52 apFAB346
EcYigB
17 EcYihX 53 apFAB346
EcYihX
18 53 apFAB87
19 EcYniC 54 apFAB346
EcYniC 54 apFAB87
21 EcYqaB 55 apFAB87
22 EcYqaB 55 apFAB346
Based on figures 4 and 5 enabling strains to better than the blank strain
phosphatases grow (no
crippled growth) and producing more sialic acid than the blank strain, can be chosen.
Based on the above, it was found that phosphatases at least Motif 1 and Motif 2
comprising
provide a strain which is not crippled and produces more sialic acid than the blank strain.
Exam le 12: Identification of further se uences related to the hos hatases used in the
methods of the invention
Sequences related to SEQ ID NOs; 43, 44, 45, 47, 48, 49, 50, 51, 52, 54, 55 and 57
(polypeptides)
were identified amongst those maintained in the Entrez Nucleotides database at the National
Center for Biotechnology Information (NCBI) using database sequence search tools, such as the
Basic Local Alignment Tool (Altschul et al. 1 Mol. Biol. 215:403-410; and Altschul
(BLAST) (1990)
et al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used to find regions of local
similarity between sequences nucleic acid or sequences to sequence
by comparing polypeptide
databases and calculating the statistical significance of matches. The output of the analysis
was viewed by pairwise comparison, and ranked according to the probability score (E-value),
where the score reflect the probability that a particular alignment occurs chance (the lower
the E-value, the more significant the In addition to E-values, comparisons were also scored
hit).
percentage identity. Percentage identity refers to the number of identical amino acids
between the two compared sequences over a particular length. In some instances,
polypeptide
the default parameters may be adjusted to modify the stringency of the search. For example
the E-value be increased to show less stringent matches. This short nearly exact
may way,
matches may be identified.
Table 1A to 1K provides a list of homologue sequences related to SEQ ID NO:
polypeptide 43,
44, 45, 47, 48, 50, 51, 52, 54, 55 and 57, respectively.
Table 1A: Examples of polypeptides related to Ec Cof (SEQ ID NO; 43), showing sequence identity
to SEQ ID 43:
short identifier ID NO
% identity (matgat) genbank SEQ
Shigella flexneri WP 095762248.1 78
99,6
WP 095785299.1 79
99,3 Shigella boydii
98,2 Escherichia fergusonii WP 024256925.1 80
Staphylococcus aureus WP 094409981.1 81
89,3
Escherichia albertii WP 000113024.1
89 82
Citrobacter amalonaticus WP 046476411.1 83
81,6
Salmonella enterica WP 023234244.1
81,6 84
80,5 Escherichia coli WP 088543831.1 85
WO 2I/18/122225 PCT/EP2il17/084593
Table 1B: Examples of polypeptides related to Ec HisB (SEQ ID NO: 44), showing sequence
identity to SEQ ID 44;
% identity (matgat) short genbank identifier SEQ ID NO
K-315
99,4 Shigella flexneri EIQ21345.1 86
99,2 Escherichia albertii WP 059217413.1
98,9 Shigella flexneri WP 094085559.1
98,6 Shigella sonnei WP 077125326.1
Escherichia coli WP 088129012.1 90
98,6
98 Shigella dysenteriae WP 000080078.1
98 Escherichia marmotae WP 038355110.1
Salmonella bongori WP 000080052.1 93
94,6
Table 1C: Examples of related to Ec OtsB ID NO: showing sequence
polypeptides (SEQ 45),
identity to SEQ ID 45:
SEQ ID NO
% identity (matgat) short genbank identifier
99,6 Shigella sonnei WP 077124555.1
Escherichia coli WP 032172688.1
99,6
99,2 Shigella flexneri WP 064198868.1
Escherichia albertii WP 059227241.1
85,7
83,1 Escherichia fergusonii WP 000165652.1
Table 1D: of related Ec Yaed NO:
Examples polypeptides to (SEQ ID 47), showing sequence
identity to SEQ ID 47:
% identity (matgat) short genbank identifier NO
Escherichia fergusonii WP 001140180.1
99,5
sonnei WP 047565591.1 100
99,5 Shigella
99 Escherichia coli WP 061103769.1
Escherichia albertii WP 001140171.1 102
95,8
93,2 Kluyvera intermedia WP 047371746.1
Citrobacter koseri WP 047458784.1
93,2
Kosakonia arachidis 090122712.1 105
89 WP
Kluyvera cryocrescensWP 061282459.1
85,9
Leclercia WP 039030283.1 107
85,9 adecarboxylata
Table 1E: of related to ID
Examples polypeptides Ec Yc)UB (SEQ NO; 48), showing sequence
identity to SEQ ID NO: 48:
ID NO
% identity short genbank identifier SEQ
(matgat)
99,5 Shigella sonnei WP 094313132.1
Escherichia coli WP 000775764.1
97,7
95,4 Escherichia coli WP 032302947.1
92,7 Shigella flexneri OUZ88260.1
PCT/EP2tl17/084593
Table 1F: Examples of related to Ec YfbT ID NO: showing sequence
polypeptides (SEQ 50),
identity to SEQ ID NO; 50:
SEQ ID NO
Io identity (matgat) short genbank identifier
Shigella sonnei WP 094323443.1
99,1
87,5 Citro bacter werkmanii N BRC 105721 GAL43238.1
Citrobacter freundii KGZ33467.1
86,6
86,6 Citrobacter amalonaticus Y19 AKE59306.1
85,6 Salmonella enterica WP 080095242.1
Escherichia fergusonii WP 001203376.1
85,6
Salmonella enterica subsp. enterica serovar 118
Hadar KKD79316.1
85,6
Table 1G; Examples of related to Ec YidA ID NO: showing sequence
polypeptides (SEQ 51),
identity to SEQ ID NO; 51:
SEQ ID
Io identity (matgat) short genbank identifier NO
Escherichia coli WP 053263719.1
99,6
99,3 Escherichia fergusonii WP 000985562.1
99,3 Shigella sonnei WP 094337696.1
Trabulsiella guamensis WP 038161262.1
94,4
Citrobacter amalonaticus 061075826.1 123
94,1 WP
93,7 Klebsiella pneumoniae WP 048288968.1
Trabulsiella odontotermitis 054178096.1
93,3 WP
90 Enterobacter kobei WP 088221256.1
Table 1H: Examples of polypeptides related to Ec YigB (SEQ ID NO: 52), showing sequence
identity to SEQ ID NO: 52:
Io identity short genbank identifier SEQ ID NO
(matgat)
99,6 Shigella sonnei WP 094322240.1
Shigella sonnei WP 052962467.1
93,7
87 Salmonella enterica WP 079797638.1
85,7 Citrobacter braakii WP 080625916.1
Enterobacter hormaechei WP 047737367.1 131
81,9
81,1 Lelliottia amnigena WP 059180726.1
80,3 Leclercia adecarboxylata WP 039031210.1
Table ll: Examples of polypeptides related to Ec YniC ID NO: showing sequence identity
(SEQ 54),
to SEQ ID NO: 54:
SEQ ID NO
Io identity short genbank identifier
(matgat)
Shigella flexneri 1235-66 EIQ75633.1 134
85,6
85,1 Kosakonia sacchari WP 074780431.1
85,1 Enterobacter mori WP 089599104.1
Lelliottia amnigena WP 064325804.1
84,7
84,7 Enterobacter sp. 638 WP 012017112.1
Kosakonia radicincitans WP 071920671.1
84,2
Salmonella enterica subsp. enterica serovar 140
2010K-2159
84,2 Newport str. CDC AKD18194.1
Table 1J: Examples of related to Ec YqaB ID NO: showing sequence
polypeptides (SEQ 55),
identity to ID NO: 55:
SEQ ID NO
% identity (matgat) short genbank identifier
Shigella flexneri K-315 EIQ18779.1
97,9
059215906.1 142
93,6 Escherichia albertii WP
Salmonella enterica WP 079949947.1
88,3
85,6 Kluyvera intermedia WP 085006827.1
85,1 Trabulsiella odontotermitis WP 054177678.1
Yokenella regensburgei WP 006817298.1
84,6
84,6 Raoultella terrigena WP 045857711.1
Klebsiella pneumoniae WP 064190334.1
83,5
Table 1K: Examples of related to Ps ID NO: showing sequence
polypeptides MupP (SEQ 57),
NO: 57:
identity to SEQ ID
SEQ ID NO
% identity (matgat) short genbank identifier
Pseudomonas putida WP 062573193.1
94,6 group
94,6 Pseudomonas sp. GM84 WP 008090372.1
93,3 Pseudomonas entomophila
92,4 Pseudomonas vranovensis WP 028943668.1
83,9 Pseudomonas cannabina WP 055000929.1
Pseudomonas monteilii WP 060480519.1
93,3
Sequences have been tentatively assembled and publicly disclosed research institutions, such
as The Institute for Genomic Research (TIGR; beginning with TA). The Eukaryotic Gene Orthologs
database may be used to identify such related sequences, either keyword search or
(EGO) by by
using the BLAST algorithm with the nucleic acid sequence or polypeptide sequence of interest.
Special nucleic acid sequence databases have been created for particular organisms, such as
Joint Institute.
the Genome
13: Identification of domains motifs in
Example and comprised polypeptide sequences
useful in the methods of invention
performing the
The Integrated Resource of Protein Families, Domains and Sites database is an
(InterPro)
text- sequence-
integrated interface for the commonly used signature databases for and based
searches. The lnterPro database combines these databases, which use different methodologies
and degrees of biological information about well-characterized proteins to derive
varying
SWISS-PROT,
protein signatures. Collaborating databases include PROSITE, TrEMBL, PRINTS,
ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence
alignments and hidden Markov models covering common protein domains and families.
many
Pfam is hosted at the Sanger Institute server in the United Kingdom. Interpro is hosted at the
European Bioinformatics Institute in the United Kingdom.
results of the InterPro scan of the polypeptide sequences as represented by SEQ ID NOs:
43, 44, 45, 47, 48, 49, 50, 51, 52, 54 and 55 are presented in Table 2.
Table 2: InterPro scan results (major accession numbers) of the polypeptide sequence as
represented ID NOs: 54 and 55.
by SEQ 43, 44, 45, 47, 48, 49, 50, 51, 52,
Alignment of the tested was done and 6 shows of the
phosphatase polypeptides figure part
alignment. Motif 1 and motif 2 are indicated with boxes. Alignment was made using
clustalomega.
Example 14: Effect of phosphatase on growth and production of sialic acid in
5accharomyces cerevisiae
A further example of sialic acid production of several saccharomyces cerev/siae strains capable
of producing N-acetylneuraminate (sialic acid) wherein the strains are expressing an extra
indicated hereunder.
phosphatase as
The strain used here is derived from the strain described in example 4. To enhance growth and
production of sialic acid in Saccharomyces cerevisiae according to this invention, the
phosphatase are introduced via a 2-micron plasmid 2013 {Plasmid 70 2-17))
genes (Chan (2013)
and the genes are expressed using synthetic constitutive promoters (Blazeck 2012
(Biotechnology and Bioengineering, Vol. No. as also described in example 1. The
109, 11))
used in this embodiment is This based on
specific plasmids p2a 2p sia~lmS-phospha. plasmid
the plasmid sia~lmS plasmid is described in example 1. It is introduced into
p2a 2p
Saccharomyces cerevisae using the transformation technique described by Gietz and Woods
(2002, PMID 12073338) and a mutant strain is obtained. The effect of phosphatase expression
on and production of sialic acid of these mutants are evaluated as described in example
growth
Example 15: Effect of phosphatase on growth and production of sialic acid in Bacillus
subtills
In another embodiment, this invention can be used to enhance growth and production of sialic
acid in Bacillus subtilis, yet another bacterial production host.
The strain used here is derived from the strain described in example 9. Additionally to the
alterations described in example 9, phosphatase genes EcAphA (SEQ ID NO: 42), EcCof (SEQ ID
NO: EcHisB ID NO: EcOtsB ID NO: EcSurE ID NO: EcYaed
43), (SEQ 44), (SEQ 45), (SEQ 46), (SEQ
ID NO: 47), EcYcjU (SEQ ID NO: 48), EcYedP (SEQ ID NO: 49), EcYfbT (SEQ ID NO: 50), EcYidA (SEQ
ID NO: EcYigB ID NO: EcYihx ID NO: EcYniC ID NO: EcYqaB
51), (SEQ 52), (SEQ 53), (SEQ 54), (SEQ
ID NO: 55), EcYrbL (SEQ ID NO: 56), PsMupP (SEQ ID NO: 57), EcAppA (SEQ ID NO: 58), EcGph
ID NO: EcSerB ID NO: EcNagD ID NO: EcYbhA ID NO:
(SEQ 59), (SEQ 60), (SEQ 61), (SEQ 62),
EcybiV (SEQ ID NO: 63), (SEQ ID NO: 64), EcyfbR (SEQ ID NO: 65), EcYieH (SEQ ID NO; 66),
EcYbjL
EcYjgL (SEQ ID NO: Ec (SEQ ID NO: EcYrfG (SEQ ID NO: EcYbiU (SEQ ID NO:
67), YjjG 68), 69), 70),
ScDOG1 ID NO: and BsAraL ID NO: are overexpressed on a as
(SEQ 71) (SEQ 72) plasmid,
described in example 1. Subsequently, this plasmid is introduced in Bacillus subtl lls. The effect
of phosphatase expression on growth and production of sialic acid of the created mutants are
evaluated as described in example 11.
PCT/EP20 1 7/084593
Claims (23)
1. A method for the production of a sialylated compound in a microorganism, the method comprising: culturing a microorganism in a culture medium, said culture medium optionally comprising an exogenous precursor, wherein said microorganism comprises at least one nucleic acid encoding a phosphatase, at least one nucleic acid encoding an N-acetylmannosamine epimerase; and at least one nucleic acid encoding a sialic acid synthase, and wherein said microorganism is unable convert N-acetylglucosamineP to glucosamineP, ii to if ) N-acetyl-glucosamineP, N-acetyl-neuraminate convert N-acetyl-glucosamine to and iii convert to N-acetyl-mannosamine; and HAD-alike modulating expression in said microorganism of a nucleic acid encoding a phosphatase wherein said HAD-alike phosphatase polypeptide comprises: polypeptide, at least one of the following motifs: Motif 1: hDxDx[TV] ID NO: or (SEQ 73), Motif 2: [GSTDE][DSEN]x(1-2) [hP]x(1-2)[DGTS] (SEQ ID NOs: 74, 75, 76, 77) wherein h means a amino acid and x can be hydrophobic (A, I, L, M, F, V, P, G) any distinct amino acid; or a homologue or derivative of one of ID NOs: 55 or any SEQ 43,44, 45, 47, 48, 50, 51, 52, 54, 57 having at least 80 %, 81%, 82 %, 83 %, 84 %, 85 %, 86%, 87 %, 88 %, 89 %, 90 %, 91%, 92 %, or'9 94 97 overall said 93 %, %, 95 %, 96 %, %, 98 %, % sequence identity to polypeptide. The method claim wherein HAD-alike
2. according to said polypeptide comprises any one of SEQ ID NOs: 55 or 57. 43,44, 45, 47, 48, 50, 51, 52, 54,
3. Method according to claim 1, wherein said modulated expression is effected by introducing and in a microorganism a nucleic acid encoding a HAD-alike expressing polypeptide.
4. Method according to claim 1, wherein said modulated expression is effected by the action of a constitutive promoter.
5. Method according to any one of the preceding claims, wherein said sialylated compound is selected from the consisting of N-acetylneuramic acid, sialylated oligosaccharide, sialylated group lipid, sialylated protein, sialylated aglycon.
6. Method according to the previous claim, wherein said sialylated compound is a sialylated oligosaccharide.
7. Method according to claim wherein said sialylated oligosaccharide is sialyllactose. lacto-N-tetraose.
8. Method according to claim 6, wherein said sialylated oligosaccharide is disialyl
9. Method according to claim wherein said sialylated compound is N-acetylneuraminic acid.
10. Method according to one of claim 1 to 9 wherein said sialylated compound is a sialylated lacto-N-triose, lacto-N-tetraose lacto-N-neotetraose, or a and wherein said microorganism further comprises the activity of a galactosyltransferase 2.4.1.38).
11. Method according to claim 10 wherein said microorganism is unable to express the genes coding for UDP hydrolase and galactosephosphate uridylyltransferase. sugar
12. Method according to any one of claims 1 to 11, wherein said microorganism produces less than 2% extracellular N-acetylglucosamine than sialylated compound. 50%, 40%, 30%, 20%, 10%, 5%,
13. Method for producing a sialylated oligosaccharide, comprising: culturing a microorganism according to the method of one of claims 1 to and wherein said a) any 12, N-acetylneuraminate microorganism produces internally, activated as donor substrate for a sialyltransferase; and b) culturing said microorganism in a culture medium comprising an exogenous precursor selected beta- from the consisting of lactose, N-acetyllactosamine, lacto-N-biose, galactose, group (Gal-alpha-1,4Gal- beta- galactoside, and alpha-galactoside such as but not limited to globotriose 1,4Glc)galactose, wherein active uptake into the microorganism of said exogenous precursor occurs and wherein said exogenous precursor is the acceptor substrate for said sialytransferase for producing the sialylated oligosaccharide.
14. Method claim wherein one of said N-acetylmannosamine according to 1, any or more epimerase and sialic acid synthase is overexpressed in the microorganism.
15. Method according to claim wherein one or more of said N-acetylmannosamine epimerase 1, any and sialic acid synthase is introduced and expressed in the microorganism.
16. Method according to any one of claims 1 to 15, wherein said microorganism is a bacterium, preferably an Escherich/a col/ strain, more preferably an Escherichia co/i strain which is a K12 strain, Escher/ch/a co/i strain is Escher/chio co/i MG1655. even more preferably the K12
17. Method according to any one of claims 1 to 15, wherein said microorganism is a yeast.
18. Microorganism, obtainable a method according to one of claims 1 to wherein said by any 17, HAD-alike microorganism comprises a recombinant nucleic acid encoding a polypeptide.
19. A microorganism for the production of sialylated compounds wherein said microorganism comprises at least one nucleic acid encoding a phosphatase, at least one nucleic acid encoding an least nucleic acid sialic acid acetylmannosamine epimerase; and at one encoding a synthase, and wherein said microorganism is unable to/) convert IV-acetylglucosamineP to glucosamineP, //) convert N-acetyl-glucosamine to N-acetyl-glucosamineP, and/ii) convert N-acetyl-neuraminate to N-acetyl-mannosamine; characterised in that said microorganism comprises a modulated expression of a nucleic acid encoding a HAD-alike phosphatase polypeptide as defined in claim 1 or 2.
20. Construct comprising: HAD-alike nucleic acid encoding a polypeptide as defined in claim 1 or 2; one or more control sequences capable of driving expression of the nucleic acid sequence of (ii) (a); and optionally a transcription termination sequence. (iii)
21. Construct according to claim 20, wherein one of said control sequences is a constitutive promoter.
22. Use of a construct according to claim 20 or 21 in a method for producing sialylated compounds.
23. A sialylated compound produced according to the method described in one of claims 1 to any 17, wherein said sialylated compound is added to food formulation, feed formulation, pharmaceutical formulation, cosmetic formulation, or agrochemical formulation.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP16206916.5 | 2016-12-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| NZ755558A true NZ755558A (en) |
Family
ID=
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