INCREASING SPACE-TIME-YIELD, CARBON-CONVERSION-EFFICIENCY AND CARBON SUBSTRATE FLEXIBILITY IN THE PRODUCTION OF FINE CHEMICALS

Abstract

Increasing space-time-yield, carbon-conversion-efficiency and carbon substrate flexibility in the production of fine chemicals The inventors of the current invention have found a surprising positive effect of increased cAMP levels and/or manipulating the PTS system on the space-time-yield, carbon-conversion-efficiency and carbon substrate flexibility of fine chemical production of a host organism. This was achieved by de-regulating adenylate cyclase cyaa by deleting the C-terminal regulatory region leading to increased cAMP levels or deleting the Crr protein activity (carbohydrate repression resistance) which regulates the carbohydrate utilization system. Both lead to increased 2-fucosyllactoe and 6-sialyllactose production (human milk oligosaccharides) and increase carbohydrate usage.

Claims

1-15. (canceled)

16. Method to increase the carbon substrate flexibility of the production of and/or to increase the carbon-conversion-efficiency of and/or to increase the space-time-yield of one or more fine chemicals produced by a host organism suitable for the production of one or more fine chemicals including the steps of increasing the Adenosine 3′,5′-cyclic monophosphate (cAMP, CAS Number: 60-92-4) levels of the host organism compared to the non-modified host organisms, maintaining the host organism in a setting allowing it to grow, growing the host organisms in the presence of substrates and under conditions suitable for the production of one or more fine chemicals and optionally separating one or more fine chemicals from the host organism or remainder thereof.

17. Method according to claim 16, wherein the cAMP level of the host organism is increased by a. Inactivating the regulatory activity found in a wildtype adenylate cyclase, and/or b. generating a mutated adenylate cyclase lacking the regulatory activity found in a wildtype adenylate cyclase, and/or c. introduction into the host organism of a mutated adenylate cyclase lacking the regulatory activity found in a wildtype adenylate cyclase.

18. Method according to claim 16 wherein the cAMP level of the host organism is increased in an inducible manner and the increase is compared to the host organisms without induction.

19. Method according to claim 17, wherein the mutated adenylate cyclase is introduced by introduction of a transgene.

20. Method according to claim 17, wherein the mutated adenylate cyclase or the adenylate cyclase with inactivated regulatory activity has a deletion compared to wildtype form of the adenylate cyclase of the host organisms.

21. Method according claim 20, wherein the deletion is removing the regulatory part of the adenylate cyclase without disrupting the part producing cAMP.

22. Method according to claim 20, wherein the deletion is a deletion of the regulatory part of the protein that corresponds to C-terminal part of the adenylate cyclase encoded by an Escherichia coli cyaA gene, preferably that part that corresponds to the C-terminal part of the CyaA protein as provided in SEQ ID NOS:19 or 20, or an adenylate cyclase protein of at least 80% sequence identity to positions 1 to 412.

23. The method according to claim 16, wherein the method includes the step of supplying the host organism with a carbon source, wherein the carbon source is a complex or a defined carbon source or combinations thereof.

24. The method according to claim 16, wherein the host organism is a genetically modified microorganism cell and wherein preferably the one or more fine chemical is one or more oligosaccharide and wherein the method includes before the growth of the genetically modified microorganism the step of inactivating or removing in the genetically modified microorganism the Crr protein or the endogenous protein(s) corresponding to the Crr protein in E. coli (SEQ ID NO: 26).

25. Modified host cell suitable for the production of a fine chemical wherein the host cell is able to grow on glycerol and/or glucose and/or maltose and/or fructose and/or sucrose, preferably sucrose, glycerol, glucose and/or fructose, wherein the modified host cell comprises an adenylate cyclase with inactivated or absent regulatory activity, that has adenylate cyclase activity, and wherein the host organism has increased cAMP level compared to a non-modified host cell, wherein the non-modified host cell is unable to grow substantially on glycerol and/or glucose and/or maltose and/or fructose and/or sucrose.

26. Modified host cell of claim 25, wherein at least one adenylate cyclase protein corresponding to the protein encoded by the cyaA gene of Escherichia coli is lacking a regulatory activity, preferably lacking the part that corresponds to C-terminal part of the CyaA protein as provided in SEQ ID NOS:19 or 20, or an adenylate cyclase protein of at least 80% sequence identity to positions 1 to 412.

27. Modified host cell of any of claim 25, wherein the host cell is a genetically modified microorganism for an enhanced production of oligosaccharides, wherein said genetically modified microorganism is capable to produce oligosaccharides, wherein said genetically modified microorganism comprises functional genes coding for a PTS carbohydrate utilization system, wherein in said genetically modified microorganism the abundance and/or activity of the Crr protein (SEQ ID NO: 26), of variants thereof or of endogenous protein corresponding to the Crr protein in said microorganism is decreased, and wherein the space-time-yield, carbon substrate flexibility or carbon-conversion-efficiency of oligosaccharide production by the genetically modified microorganism is increased compared to a control with unaltered abundance and/or activity of the Crr protein (SEQ ID NO: 26), of variants thereof or of endogenous protein(s) corresponding to the Crr protein.

28. Modified host cell of claim 25, wherein the host cell is a genetically modified microorganisms and the gene encoding the Crr protein, variants thereof or the endogenous protein(s) corresponding to the Crr protein in said microorganism is attenuated or deleted in said genetically modified microorganism.

29. Claim 16 wherein at least one fine chemical is a human milk oligosaccharide.

30. Claim 16 wherein space-time-yield, carbon substrate flexibility and/or carbonconversion-efficiency of the production of one or more fine chemicals, preferably one or more oligosaccharides, is increased by at least 20% compared to the controls.

15. Use of a. an adenylate cyclase protein with inactive regulatory domain and functional catalytic domain to produce cAMP as defined in claim 16; and/or b. inactivation and/or the reduction in abundance of the Crr protein or the endogenous protein corresponding to the Crr protein in E. coli (SEQ ID NO: 26) to increase in a host cell the carbon substrate flexibility of the production of and/or to increase the carbon-conversion-efficiency of and/or to increase the space-time-yield of one or more human milk oligosaccharides. with unaltered abundance and/or activity of the Crr protein (SEQ ID NO: 26), of variants thereof or of endogenous protein(s) corresponding to the Crr protein.

13. Modified host cell of any of claims 10 to 12, wherein the host cell is a genetically modified microorganisms and the gene encoding the Crr protein, variants thereof or the endogenous protein(s) corresponding to the Crr protein in said microorganism is attenuated or deleted in said genetically modified microorganism.

14. Any of the preceding claims wherein at least one fine chemical is a human milk oligosaccharide.

15. Any of the preceding claims wherein space-time-yield, carbon substrate flexibility and/or carbon-conversion-efficiency of the production of one or more fine chemicals, preferably one or more oligosaccharides, is increased by at least 20% compared to the controls.

Description

DESCRIPTION OF FIGURES

[0218] FIG. 1 shows a graphical display of the different lengths of the various DNA protein sequences useful in the methods and host cells of the inventions.

[0219] FIG. 2,

[0220] Part 1) is showing the alignment of the DNA sequences of SEQ ID NO: 1 to 8 and 10, showing the length of the different shortened cyaA DNA sequences compared to the longest variant of the full-length gene

[0221] Part 2) is showing the alignment of the protein sequences of SEQ ID NO: 11 to 18 and 20, showing the length of the different shortened CyaA protein sequences compared to the longest variant of the full-length protein. In comparison the slightly shorter full-length wildtype protein of SEQ ID NO: 19 has only one GEQSMI motif instead of the duplicate GEQSMIGEQSMI (underlined in FIG. 2 part 2) of the 854-variant of the full-length adenylate cyclase.

[0222] FIG. 3 depicts an exemplary construct to create a 2′FL producing E. coli strain

[0223] FIG. 4

[0224] A depicts the first construct introduced to create a 6′-SL producing E. coli strain. The top picture is the construct in the strain without altered CyaA, the bottom is the one in the strain with de-regulated CyaA;

[0225] B: depicts the second construct used to create a 6′-SL producing E. coli strain. The top picture is the construct in the strain without altered CyaA, the bottom is the one in the strain with de-regulated CyaA.

[0226] FIG. 5 depicts the crr locus after deletion of the bulk of the crr gene as explained in the examples below in detail.

FURTHER EMBODIMENTS

[0227] I. Method for the increase of space-time-yield of one or more fine chemicals in a host organism, the carbon-conversion-efficiency of the production of one or more fine chemicals by a host organism and/or carbon substrate flexibility of the production of one or more fine chemicals by a host organism by providing a de-regulated adenylate cyclase protein and/or inactivation and/or reduction in abundance of the Crr protein (SEQ ID NO: 26), of variants thereof or of the endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26 in the host organism, wherein the space-time-yield, carbon-conversion-efficiency and/or carbon substrate flexibility are increased in the modified host organism compared to the non-modified host organism. [0228] II. Method to increase the carbon substrate flexibility of the production of one or more fine chemicals by a host organism, wherein the cAMP levels in the host organism is increased compared to the non-modified host organisms. [0229] III. Method to increase the carbon-conversion-efficiency of the production of one or more fine chemicals by a host organism, wherein the cAMP levels in the host organism is increased compared to the non-modified host organisms. [0230] 1. Method for the increase of space-time-yield of one or more fine chemicals produced by a host organism suitable for the production of one or more fine chemicals including the steps of increasing the Adenosine 3′,5′-cyclic monophosphate (cAMP, CAS Number: 60-92-4) levels of the host organism compared to the non-modified host organisms, maintaining the host organism in a setting allowing it to grow, growing the host organisms in the presence of substrates and under conditions suitable for the production of one or more fine chemicals and optionally separating one or more fine chemicals from the host organism or remainder thereof. [0231] 2. Method to increase the carbon substrate flexibility of the production of one or more fine chemicals by a host organism suitable for the production of one or more fine chemicals, including the steps of increasing the cAMP levels in the host organism compared to the non-modified host organisms, maintaining the host organism in a setting allowing it to grow, growing the host organisms in the presence of substrates and under conditions suitable for the production of one or more fine chemicals and optionally separating one or more fine chemicals from the host organism or remainder thereof. [0232] 3. Method to increase the carbon-conversion-efficiency of the production of one or more fine chemicals by a host organism suitable for the production of one or more fine chemicals, including the steps of increasing the cAMP levels in the host organism compared to the non-modified host organisms, maintaining the host organism in a setting allowing it to grow, growing the host organisms in the presence of substrates and under conditions suitable for the production of one or more fine chemicals and optionally separating one or more fine chemicals from the host organism or remainder thereof. [0233] 4. Method according to any of the preceding embodiments, wherein the cAMP level of the host organism is increased by [0234] a. Inactivating the regulatory activity found in a wildtype adenylate cyclase, and/or [0235] b. generating a mutated adenylate cyclase lacking the regulatory activity found in a wildtype adenylate cyclase, and/or [0236] c. introduction into the host organism of a mutated adenylate cyclase lacking the regulatory activity found in a wildtype adenylate cyclase; and/or [0237] d. reduction of the activity of the enzyme with the activity of a 3′,5′ cAMP phosphodiesterase (EC 3.1.4.53); and/or [0238] e. use of adenylate cyclase toxin of Bordetella pertussis or the adenylate cyclase domain of it, or a variant thereof; and/or [0239] f. inactivation and/or reduction in abundance of the Crr protein (SEQ ID NO: 26), of variants thereof or of the endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26. [0240] 5. Method according to any of the preceding embodiments wherein the cAMP level of the host organism is increased in an inducible manner and the increase is compared to the host organisms without induction. [0241] 6. Method according to any of the preceding embodiments, wherein the mutated adenylate cyclase is introduced by introduction of a transgene. [0242] 7. Method according to any of the preceding embodiments, wherein the mutated adenylate cyclase or the adenylate cyclase with inactivated regulatory activity has a deletion compared to the wildtype form of the adenylate cyclase of the host organisms. [0243] 8. Method according embodiment 7, wherein the deletion is removing the regulatory part of the adenylate cyclase without disrupting the part producing cAMP. [0244] 9. Method according to embodiment 7 or 8, wherein the deletion is a deletion of the regulatory part of the protein that corresponds to C-terminal part of the adenylate cyclase encoded by an Escherichia coli cyaA gene, preferably that corresponds to C-terminal part of the cyaA pro-tein as provided in SEQ ID NOS:19 or 20, or an adenylate cyclase protein of at least 80% sequence identity to positions 1 to 412 preferably to positions 1 to 420 of the protein sequence provided as SEQ ID NO 19; and preferably the deletion is a deletion of the regulatory part of the protein that that corresponds to the part of the Escherichia coli adenylate cyclase that is subsequent to position 420, 450, 558, 582, 585, 653, 709, 736 or 776 of the protein sequence supplied in SEQ ID Nos: 19 or 20 more preferably subsequent to position 558, 582, 585, 653, 709, 736 or 776 of the protein sequence supplied in SEQ ID Nos: 19 or 20, and most preferably a deletion of the amino acids that correspond to the amino acids at the position 777 and following of SEQ ID NO 19 or 20. [0245] 10. The method according to any of the preceding embodiments, wherein the method includes the step of supplying the host organism with a carbon source, wherein the carbon source is a complex or a defined carbon source or combinations thereof. [0246] 11. Modified host cell suitable for the production of a fine chemical wherein the host cell is able to grow on glycerol and/or glucose and/or maltose and/or fructose and/or sucrose, preferably sucrose, glycerol, glucose and/or fructose, wherein the modified host cell has an adenylate cyclase with inactivated or absent regulatory activity, that has adenylate cyclase activity, and/or inactivation and/or reduction in abundance of the Crr protein (SEQ ID NO: 26), of variants thereof or of the endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26, and wherein the host organism has increased cAMP level compared to a non-modified host cell, wherein the non-modified host cell is unable to grow substantially on glycerol and/or glucose and/or maltose and/or fructose and/or sucrose. [0247] 12. Modified host cell of embodiment 11, wherein at least one adenylate cyclase protein corresponding to the protein encoded by the cyaA gene of Escherichia coli is lacking a regulatory activity, preferably lacking the part that corresponds to C-terminal part of the cyaA protein as provided in SEQ ID NOS:19 or 20, or an adenylate cyclase protein of at least 80% sequence identity to positions 1 to 412 more preferably an adenylate cyclase protein of at least 80% sequence identity to positions 1 to 420, of the protein sequence provided as SEQ ID NO 19 or 20, and preferably lacking the part of the adenylate cyclase that corresponds to the Escherichia coli adenylate cyclase part that is subsequent to position 420, 450, 558, 585, 653, 709, 736 or 776, more preferably 450, 558, 585, 653, 709 or 736 of the protein sequence supplied in SEQ ID Nos: 19 or 20 even more preferably subsequent to position 558, 582, 585, 653, 709, 736 or 776 of the protein sequence supplied in SEQ ID Nos: 19 or 20, and most preferably a deletion of the amino acids that correspond to the amino acids at the position 777 and following of SEQ ID NO 19 or 20. [0248] 13. Any of the preceding embodiments, wherein the host cell is a bacterial of fungal host cell, preferably a bacterial cell, more preferably a bacterial cell, even more preferably a gram-negative bacterial cell, most preferably an Escherichia coli cell [0249] 14. Use of de-regulated adenylate cyclase and/or inactivation and/or reduction in abundance of the Crr protein (SEQ ID NO: 26), of variants thereof or of the endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26 for increasing space-time-yield, carbon substrate flexibility and/or carbon-conversion-efficiency of the production of one or more fine chemical by a host organism. [0250] 15. Any of the preceding embodiments wherein at least one fine chemical is a human milk oligosaccharide, preferably a neutral or sialylated HMO, more preferably 2′-fucosyllactose (2′-FL), 3′-fucosyllactose (3′-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), difucosyllactose (2,3-DFL) or 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL) or the method of any of the preceding embodiments, wherein the method includes supplying the host organism with a carbon source, wherein the carbon source is one or more of the following: a complex or a defined carbon source, preferably glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, dextrin, lactose, gluconate, more preferably glycerol, glucose or mannose, and even more preferably glucose or glycerol. [0251] 16. A method for the production of an oligosaccharide by conversion of a source of carbon in a fermentative process comprising the following steps: [0252] Culturing a microorganism genetically modified for the production of oligosaccharides in an appropriate culture medium comprising at least one source of carbon [0253] Recovering the human milk oligosaccharide from the culture medium, wherein said genetically modified microorganism comprises functional genes coding for a PTS carbohydrate utilization system and wherein in said genetically modified microorganism the abundance of the Crr protein (SEQ ID NO: 26), of variants thereof or of the endogenous protein corresponding to the Crr protein of SEQ ID NO: 26 is decreased and/or a deregulated adenylate cyclase as defined in any of the previous embodiments is present in the microorganism. [0254] 17. Any of the preceding embodiments, wherein the source of carbon is selected among the group consisting of glycerol, monosaccharides and disaccharides [0255] 18. Any of the preceding embodiments wherein the levels of Adenosine 3′,5′-cyclic mono-phosphate (cAMP, CAS Number: 60-92-4) are increased compared to a microorganism without alteration of the Crr protein (SEQ ID NO: 26), of variants thereof or of the endogenous protein corresponding to the Crr protein of SEQ ID NO: 26. [0256] 19. Genetically modified microorganism for an enhanced production of fine chemicals wherein said genetically modified microorganism is capable to produce human milk oligosaccharides wherein said genetically modified microorganism comprises functional genes coding for a PTS carbohydrate utilization system and wherein in said genetically modified microorganism the expression of the Crr protein is decreased, preferably at least substantially decreased. [0257] 20. A microorganism according to embodiment 19 wherein the gene encoding the Crr protein is attenuated or deleted in said genetically modified microorganism. [0258] 21. A microorganism according to any of the preceding embodiments, wherein the microorganism is selected among the group consisting of Enterobacteriaceae.

EXAMPLES

[0259] In the examples given below, methods well known in the art were used to construct E. coli strains containing replicating vectors and/or various chromosomal deletions, and substitutions using homologous recombination well described by Datsenko & Wanner, (2000) for Escherichia coli. In the same manner, the use of plasmids or vectors to express or over-express one or several genes in a recombinant microorganism are well known by the man skilled in the art.

[0260] Methods

[0261] Introduction of a DNA construct or vector into a host cell can be performed using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE-Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are known in the art (see, e.g., Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds) Chapter 9, 1987; Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989; and Campbell et al, Curr. Genet. 16:53-56, 1989, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods). The expression of heterologous polypeptide in Trichoderma is described in U.S. Pat. Nos. 6,022,725; 6,268,328; 7,262,041; WO 2005/001036; Harkki et al., Enzyme Microb. Technol. 13:227-233, 1991; Harkki et al, Bio Technol 7:596-603, 1989; EP 244,234; EP 215,594; and Nevalainen et al, “The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes,” in Molecular Industri-al Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation and expression methods). Reference is also made to Cao et al, (Sd. 9:991-1001, 2000; EP 238023; and Yelton et al, Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods) for transformation of Aspergillus strains. The introduced nucleic acids may be integrated into chromosomal DNA or maintained as extrachromosomal replicating sequences.

[0262] Examples with increased cAMP and deregulated adenylate cyclase activity [0263] 1. Creation of shortened cyaA DNA constructs [0264] Shortened DNA cyaA constructs were prepared by generating synthetic DNA constructs with homology for integration and introducing TAA stop codons into the coding sequence of the cyaA gene by gene synthesis. These genetic constructs were then introduced into the genome of the E. coli strain by homologous recombination as described Wang J, et al. 2006, Mol. Biotechnol., 32, 43 [0265] 2. Strain construction [0266] Genetically modified microorganisms with enhanced production of oligosaccharides (e.g. HMOs) are disclosed in patent applications published as WO 2016/008602, WO2013/182206, EP2379708, U.S. Pat. No. 9,944,965, WO2012/112777, WO2001/04341 and US2005019874. All of these disclosures are herein incorporated by reference.

2′-FL Producing Microorganism

[0267] An E coli strain 2′-FL overproducing strain was constructed as follows: In the well characterized E. coli strain JM109, an artificial operon was constructed containing the following genetic elements: a PTAC promoter, an artificial ribosomal binding site (RBS), the fucT2 gene (derived from Helicobacter pylori strain 26695, Wang et al, Mol. microbiol. 1999, 31 1265-1274)), an artificial ribosomal binding site, the gmd gene (de-rived from E. coli K12), the wcaG gene with its authentic ribosomal binding site (derived from E. coli K12), an artificial ribosomal binding site (RBS), the manC gene (derived from E. coli K12) with an adapted codon usage), an artificial ribosomal binding site (RBS), the manB gene (derived from E. coli K12, with an adapted codon usage) and a transcriptional terminator rrnBT1 derived from the 16s rRNA locus of E. coli, using the well-known lambda red technology (e.g. described by Datsenko I and Wanner B. PNAS, 2000 97 (12) 6640-6645, Wang J, et al. 2006, Mol. Biotechnol., 32, 43). The artificial operon was integrated in into the fuc locus of E. coli in which the genes including fuc I and K were deleted. An exemplary construct for creating a 2′FL producing strain is shown as SEQ ID NO: 21.

[0268] The truncated adenylate cyclase gene sequences of SEQ ID NO: 1 to 8 were introduced via homologous recombination using the lambda-red technology into the Escherichia coli host cells. An exemplary construct for creating a 2′FL producing strain is shown as SEQ ID NO: 21.

6′-SL Producing Microorganism

[0269] An E coli strain strain overproducing 6′-SL was constructed as follows: In the well characterized E coli strain W3110, the genes lacZ gene coding for the beta galactosidase LacZ and the lacA gene coding for the acetyltransferase LacA, the genes coding for the nan genes nanAETK were deleted in that all coding sequence was deleted suing the well-known lambda red technology (e.g. described by Datsenko I and Wanner B. PNAS, 2000 97 (12) 6640-6645, Wang J, et al. 2006, Mol. Biotechnol., 32, 43), while the lacI allele was replaced by the known laclq allele. An artificial operon (see SEQ ID NO: 22) was integrated immediately adjacent to the atoB gene of the strain W3110. The artificial operon contained the following genetic elements, a PTAC promoter, an artificial ribosomal binding site (RBS), the St6 gene (derived from Photobacterium spp. ISH 224), an artificial ribosomal binding site, the neuA gene (derived from Campylobacter jejuni ATCC 43438), an artificial ribosomal binding site (RBS), the zeocin resistance genes and a transcriptional terminator rrnBT1 derived from the 16s rRNA locus of E. coli. In addition, an artificial operon was integrated immediately adjacent to the fabl gene. The artificial operon contained the PTAC promoter, an artificial ribosomal binding site (RBS), the neuB gene (derived from Campylobacter jejuni ATCC 43438, see SEQ ID NO: 23), an artificial ribosomal binding site, the neuC gene (derived from Campylobacter jejuni ATCC 43438, see SEQ ID NO: 24), an artificial ribosomal binding site (RBS), the chloramphenicol resistance cassette (CAT) and a transcriptional terminator rrnB derived from the 16s rRNA locus of E. coli. [0270] This 6′-SL producing strain will be called GN488. [0271] Another E coli strain with the designation GN782 was constructed based on the Strain GN488. The resistance genes zeocin and CAT were deleted from the artificial operon of genome of the strain GN488 again using the lambda red technology. In addition, the cyaA was changed in that a stop codon was introduced at codon 582 resulting in a translated protein which has a length of 581 amino acids. [0272] 3. De-regulated adenylate cyclase: Space-time yield in the production of HMO [0273] Fermentation system and procedure [0274] Fermentation conditions: [0275] A fermentation medium was chosen based on the described examples of E. coli fermentation and can be found in: (Riesenberg et al. (1991), Journal of Biotechnology 20, 17-27, D. J. Korz, et al. 1995), J. Biotechnol., 39 pp. 59-65, Biener, R. et al. 2010, Journal of Biotechnology 146(1-2), pp. 45-53. Specifically the medium was altered for the production of oligosaccharides based on lactose in that lactose was added in different concentrations ranging from 20-100 g/l dependent on the experiment. [0276] For analysing strain performance in regard to carbon-conversion-efficiency as well as space-time-yield the following systems were used: AMBR® 250 system and 4 l Biostat® fermenters (both from Sartorius AG, Otto-Brenner-Str. 20, D-37079 Göttingen, Germany). Generally speaking, fermentations were typically conducted under the following regime: A seed culture was grown from a frozen stock. The seed culture was inoculated into the respective fermentation system (AMBR or Biostat) before its carbon content was fully utilized. Alternatively, the main culture was started directly from the frozen stock. The fermentation in the fermentation system was conducted in a fed batch mode, i. e. that a fermentation undergoes two stages—the initial one in which a batched amount of carbon source is being utilized, and the following one in which the carbon source is fed throughout the fermentation under conditions where no or only low amounts of carbon source will accumulate in the fermentation broth. [0277] The seed culture (minimal medium with 10 ml/L trace element solution and 65 g/L glycerol) is inoculated with 1 ml WCB culture (stored in a frozen state). [0278] The seed culture is transferred to the main culture in that an inoculation volume ratio between 1 and 10% are applied. [0279] The main fermentation medium consists of the following media composition: Minimal medium: citric acid 1.1 g/L, glycerol 10.8 g/L, KH2PO4 15.5 g/L, (NH4)2SO4 4.6 g/L, Na2SO4 3 g/L, MgSO4*7H2O 1.5 g/L, thiamine 0.02 g/L, Vitamin B12 0.0001 g/L, 0.5 mM IPTG. The Trace element solution consist of: Na2-EDTA*2H2O 4 g/L, CaSO4*2H2O 1 g/L, ZnSO4*7H2O 0.3 g/L, FeSO4*7H2O 3.7 g/L, MnSO4*H2O 0.2 g/L, CuSO4*5H2O 0.15 g/L, Na2MoO4*2H2O 0.04 g/L, Na2SeO4 0.04 g/L. The trace metal solution is applied at an amount of 30 ml/l of fermentation medium. [0280] After inoculation the fermentation is started and when the measured CTR is exceeding 40 mmol/Lh, the feeding of carbon source such as glycerol (86% w/w concentration) or glucose (60% w/w concentration) is initiated. Carbon source feed rates may vary between 2-8 g/I carbon source per litre of initial fermentation broth volume per hour. Care is taken that carbon source does not accumulate throughout the fermentation process. In the main fermentation stage, the dissolved oxygen concentration (pO2) is controlled at >20% by controlling agitation as well as gas addition. pH is maintained at values ranging from 6.1 to 6.9 and more specifically at pH 6.7 using the base NH4OH in a solution of 15% NH4OH aq. Results in both fermentation systems in regard to the parameters mentioned (carbon-conversion-efficiency and space-time-yield) were found to be fully superimposable and can be understood fully interchangeable. [0281] Surprisingly the cAMP overproduction cells with the truncated cyaA gene resulting in a functional, de-regulated CyaA protein did grow and produce 2′-Fucosyllactose (2′-FL) well on glycerol. In contrast to this, the a cyaA deletion mutant (from the Keio collection, Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko K A, Tomita M, Wanner B L, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2: 2006 0008) having no functional adenylate cyclase was found to be unable to grow on glycerol The unmodified E. coli cells with an adenylate cyclase with a regulatory part are growing more slowly than in the host cells with the de-regulated adenylate cyclase and hence increased cAMP production, and 2′-FL production is lower in the unmodified cells, carbon-conversion efficiency and space/time yield are also decreased in comparison to the host cells with the de-regulated adenylate cyclase and hence increased cAMP production. [0282] 2′-FL

TABLE-US-00007 TABLE 2A Carbon-conversion-efficiency in 2′-FL production. FL is the abbreviation for full-length Carbon-conversion- efficiency (CCE) g 2′-FL/g carbon source Protein ending with 2′-FL (relative (relative values Protein AA number values in %) in %) FL cyaA 854 100 100 cyaA420 420 108 110 cyaA450 450 121 114 cyaA585 585 123 119 cyaA558 558 119 123 cyaA653 653 125 122 cyaA709 709 128 116 cyaA736 736 112 117 cyaA776 776 126 124 [0283] Typically, when the BioStat® and the AMBR® vessels were used, the carbon source was added continuously or in repeated additions. In principle a typical amount of glucose or glycerol can be added once at the start of the main culture, which is advantageous when e.g. shaking flask are used for the fermentation. [0284] The space-time-yield was increased when glucose or glycerol was used as a carbon source for the strains with the de-regulated cyaA gene and hence increased cAMP levels.

TABLE-US-00008 TABLE 2B Space-time-yield in 2′-FL production Space-time- Space-time- yield on glucose yield on glycerol relative values relative values [%] to wildtype [%] to wildtype (=100%) (=100%) cyaA854 (wt) 100 100 cyaA585 140 116 [0285] Similar results were achieved with the E. coli strain producing 6′-Sialyllactose instead of 2′-FL, for these strains see example 1 and 2 above. [0286] 4. Increased carbon source flexibility of 2′-FL producing strains [0287] Carbon sources are batched into the medium as well as fed during the feed phase ranging from 2 h- to 100 h. The carbon sources are applied either in a pure fashion (e.g. glycerol) or diluted in water (glycerol as well as other carbon sources). The feed rate of the carbon source is adapted to the stirring and aeration conditions of the fermenter. [0288] In the course of the fermentation, samples were taken and analyzed by isocratic HPLC elution method. [0289] Carbon source flexibility analysis for 2′-FL production was performed using the following media composition: [0290] 20 mL of medium (10 g/L of the respective carbon source, 5 g/L lactose, 1 g/L (NH.sub.4).sub.2H-citrate, 2 g/L Na.sub.2SO.sub.4, 2.68 g/L (NH.sub.4).sub.2SO.sub.4, 0.5 g/L NH.sub.4Cl, 14.6 g/L K.sub.2HPO.sub.4, 4 g/L NaH.sub.2PO.sub.4*H.sub.2O, 0.5 g/L MgSO.sub.4*7H.sub.2O, 10 g/mL MnSO.sub.4, 3 mL trace metal solution consisting of 8.0 g/L Na.sub.2-EDTA*2H.sub.2O, 1 g/L CaSO.sub.4*2H.sub.2O, 0.3 g/L ZnSO.sub.4*7H2O, 7.4 g/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.2, 0.2 g/L MnSO.sub.4*H.sub.2O, 0.15 g/L CuSO.sub.4*5H.sub.2O, 0.04 g/L Na.sub.2MoO.sub.4*2H.sub.2O, 0.04 g/L Na.sub.2SeO.sub.4, 10 mg/L thiamine*HCl, 0.1 mg/L vitamin B12, 1 mM IPTG, pH 7.0) in a 100 mL baffled shake flask were inoculated with an overnight culture of a 2′-FL producing strain as in example 2 (in the above described medium without lactose and IPTG) to a start OD of 0.5 and incubated for 24 hours in the above described medium including lactose and IPTG as given above at 200 rpm at 37° C. Samples were taken and analysed for carbon utilization and product formation. [0291] Carbon sources were chosen from the following list: [0292] Glucose, Glycerol, Mannose, Fructose,
Table 3: Relative carbon conversion rates for different carbon sources

TABLE-US-00009 TABLE 3 Relative carbon conversion rates for different carbon sources Carbon- Carbon- Carbon- Carbon- conversion- conversion- conversion- conversion- efficiency efficiency efficiency efficiency (g Fucose/ (g Fucose/ (g Fucose/ (g Fucose/ g fructose) g mannose) g glucose) g glycerol) relative to relative to relative to relative to wildtype wildtype wildtype wildtype (=100%) (=100%) (=100%) (=100%) cyaA854 (wt) 100 100 100 100 cyaA585 76 112 133 147 [0293] 5. 6′-sialyllactose (6′-SL) producing strains [0294] Strains GN488 and strain GN782 of example 2 were grown in a Biostat® vessel containing the medium as described in example 3.

TABLE-US-00010 TABLE 4 Increased carbon-conversion-efficiency and space-time-yield in the production of 6′-SL Carbon-conversion- efficiency (CCE) g 6SL/g carbon source space-time- Relevant Carbon relative values yield relative strain genotype source [%] values [%] GN488 cyaA848 glycerol 100 100 GN782 cyaA 582 glycerol 138 133 stop [0295] The results showed that the surprising effects on carbon-conversion-efficiency and spacetime-yield are transferable to other HMO producing strains and the broad applicability of the de-regulated adenylate cyclase to increase cAMP levels since yet another version of the de-regulated CyaA protein corresponding to the amino acids 1 to 581 of the full-length CyaA protein with 848 amino acids (SEQ ID NO: 19) was successfully used. Furthermore, when the strain holding the cyaA585 version of the protein (SEQ ID NO:14) was tested, the space-time-yield of 6′-SL was similarly increased over the strain with an unmodified CyaA protein. [0296] 6. cAMP feeding experiments [0297] An E. coli strain of the Keio collection (Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko K A, Tomita M, Wanner B L, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2: 2006 0008) with a deletion of the cyaA gene shows the normal poor growth on glycerol as carbon source. This strain is grown in the presence of glycerol and cAMP and the growth of the deletion strain is improved. The 2′-FL producing host cells with a shortened adenylate cyclase of examples 1 and 2 above shows increased 2′-FL production on medium containing glycerol compared to the cells with an unmodified cyaA gene only. If the latter are supplied with cAMP, the production of 2′-FL is increased.

[0298] Examples with Altered cAMP Signalling and PTS

Example 7: Construction of a Strain Overproducing 2′-FL

[0299] An E coli strain overproducing 2′-FL with wildtype adenylate cyclase and wildtype crr gene was constructed as described in example 2 above.

[0300] Construction of an Overproducing Strain Carrying a Deletion in the Crr Gene

[0301] An E. coli strain 2′-FL overproducing strain carrying a deletion in the crr gene was constructed as follows: The well-known method described by Datsenko I and Wanner B. PNAS, 2000 97 (12) 6640-6645, Wang J, et al. 2006, Mol. Biotechnol., 32, 43A was used to replace the intact full length crr gene in the 2′FL producing strain with a genetic construct consisting of 50 bp of the 5′ coding region of the crr beginning with the transcriptional start site, a resulting FRT site from the FLP recombination event, and 50 bp of the crr gene ending with the TAA sequence of the translational stop codon. The resulting gene (SEQ ID NO: 29) therefore is not coding for an active crr protein since it is lacking 410 bp of its coding region.

[0302] The deletion of the crr gene was confirmed using the primers given in SEQ ID NO 3 & 4.

Example 8: Construction of a Strain Producing 6′SL Having a Deletion in the Crr Gene

[0303] The strain GN488 overproducing 6′-SL was created as described in example 2 above and used for further modifications. In this strain, the deletion of the crr gene (SEQ ID NO:1) in Escherichia coli strains was made by P1 viral transduction followed by selection on kanamycin containing agar plates.

[0304] A P1 lysate was made of the delta crr strain (JW2410/b2417) crr::kan) from the Keio collection (Baba et al. 2006, Mol Syst Biol. 2:2006.0008). The crr:Kan P1 lysate was used to transduce the strains described in examples 1 and 2 and the transductants were selected on agar plates containing kanamycin. Colonies were screened by PCR using primers selective for the upstream and downstream region of crr to confirm the deletion of crr. A colony with the expected bandsize indicating the correct deletion of the crr gene.

[0305] The deletion of the crr gene (SEQ ID NO:1) in Escherichia coli strains was made by P1 viral transduction (Miller, J. H. 1992. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) followed by selection on kanamycin-citrate containing agar plates.

[0306] A P1 lysate was made of strain (JW2410/b2417) (delta crr::kan(FRT)) from the Keio collection (Baba et al. 2006, Mol. Syst. Biol. 2:2006.0008). The delta crr:Kan P1 lysate was used to transduce the strains described in example 1 & 2 (2′-FL and 6′-SL strains, respectively) and the transductants were selected on agar plates containing kanamycin-citrate. Colonies were screened by PCR using primers Crr ver.F (SEQ ID NO: 27) and Crr ver.R (SEQ ID NO: 28) to confirm the deletion of crr. One correct colony was selected and designated as Ec 6′-SL delta crr.

Example 9: Increased Space-Time Yield in the Production of HMO

[0307] Fermentation conditions, system and procedures were as described above under example 3 above.

TABLE-US-00011 TABLE 5 Space/time yield of 2′-FL production with wildtype (wt) crr or crr functional gene deletion (delta crr) Carbon space-time- Relevant source yield (relative strain genotype applied values [%]) N8_2 Crr wt Glucose 100 N16_1 Delta crr Glucose 146 N8_2 crr wt Glycerol 100 N16_1 Delta crr Glycerol 227

[0308] Typically, when the BioStat® and the AMBR® vessels were used, the carbon source was added continuously or in repeated additions. In principle a typical amount of glucose or glycerol can be added once at the start of the main culture, which is advantageous when e.g. shaking flask are used for the fermentation.

Example 10: Increased Carbon Source Flexibility of Modified Strains Producing 2′FL

[0309] Carbon sources are batched into the medium as well as fed during the feed phase ranging from 2 h- to 100 h. The carbon sources are applied either in a pure fashion (e.g. glycerol) or diluted in water (glycerol as well as other carbon sources). The feed rate of the carbon source is adapted to the stirring and aeration conditions of the fermenter.

[0310] In the course of the fermentation, samples were taken and analysed by isocratic HPLC elution method.

[0311] Carbon source flexibility analysis was performed using the following media composition:

[0312] Carbon sources were chosen from the following list:

[0313] Glucose, glycerol, mannose, fructose

[0314] 20 mL of medium (10 g/L of the respective carbon source, 5 g/L lactose, 1 g/L (NH.sub.4).sub.2H-citrate; 2 g/L Na.sub.2SO.sub.4, 2.68 g/L (NH.sub.4).sub.2SO.sub.4, 0.5 g/L NH.sub.4Cl, 14.6 g/L K.sub.2HPO.sub.4, 4 g/L NaH.sub.2PO.sub.4*H.sub.2O, 0.5 g/L MgSO.sub.4*7H.sub.2O, 10 g/mL MnSO.sub.4, 3 mL trace metal solution consisting of 8.0 g/L Na.sub.2-EDTA*2H.sub.2O, 1 g/L CaSO.sub.4*2H.sub.2O, 0.3 g/L ZnSO.sub.4*7H.sub.2O, 7.4 g/L (NH.sub.4).sub.2Fe(SO.sub.4).sub.2, 0.2 g/L MnSO.sub.4*H.sub.2O, 0.15 g/L CuSO.sub.4*5H.sub.2O, 0.04 g/L Na2MoO.sub.4*2H.sub.2O, 0.04 g/L Na.sub.2SeO.sub.4, 10 mg/L thiamin*HCl, 0.1 mg/L vitamin B12, 1 mM IPTG, pH 7.0) in a 100 mL baffled shake flask were inoculated with an overnight culture (grown on the above described medium without lactose and IPTG) of a 2′-FL producing strain as in example 1 to a start OD of 0.5 and incubated for 24 hours in the above described medium including lactose and IPTG as given above at 200 rpm at 37° C. Samples were taken and analyzed for carbon utilization and product formation. Similarly, the 2′-FL producing strain with crr deletion was cultured sampled and analyzed.

TABLE-US-00012 TABLE 6 Carbon-conversion-efficiency and carbon substrate flexibility for 2′-FL producing strains with wt crr or crr functional gene deletion (delta crr) Carbon- Carbon- Carbon- Carbon- conversion- conversion- conversion- conversion- efficiency efficiency efficiency efficiency (g Fucose/ (g Fucose/ (g Fucose/ (g Fucose/ g fructose) g mannose) g glucose) g glycerol) relative to relative to relative to relative to wildtype wildtype wildtype wildtype (=100%) (=100%) (=100%) (=100%) Crr wt 100 100 100 100 delta 175 116 133 166 crr