Increased space-time yield, carbon conversion efficiency, and carbon substrate adaptability in producing fine chemicals

Abstract

Increased space-time yield, carbon conversion efficiency, and carbon substrate flexibility in fine chemical production. The present inventors have found that increasing cAMP levels and / or manipulating the PTS system can have a surprising positive effect on the space-time yield, carbon conversion efficiency, and carbon substrate flexibility of fine chemical production in the host organism. This was achieved by deregulating adenylate cyclase cyaa through deletion of the C-terminal regulatory region, which leads to increased cAMP levels, or by deleting the Crr protein activity that regulates the carbohydrate utilization system (carbohydrate repression resistance). Both resulted in increased production of 2-fucosyllactose and 6-sialyllactose (human milk oligosaccharides), enhancing carbohydrate utilization.

Description

[Technical Field]

[0001] The present inventors have found that increasing cAMP levels has a surprising positive effect on the space-time yield, carbon conversion efficiency, and carbon substrate flexibility of fine chemical production by a host organism. In addition, the present inventors have found that adenylate cyclase activity, which is not endogenously regulated and is therefore always active in cAMP production, is beneficial to the space-time yield and carbon substrate flexibility of fine chemical production by a host organism.

[0002] Furthermore, the inventors of the present invention have also found that reducing the expression of the crr gene or a variant thereof and / or inactivating or reducing the Crr protein or a variant thereof has surprising effects on the carbon conversion efficiency, carbon substrate adaptability, and space / time of prokaryotic oligosaccharide production. [Background technology]

[0003] Crr proteins are part of the PTS carbohydrate utilization system of pathogenic bacteria and are also linked to cAMP levels within microbial cells.

[0004] It is known from the state of the art that a reduction in the expression of PTS carbohydrate utilization system (PTS system) proteins has an effect on the production of certain compounds other than oligosaccharides.

[0005] Flores et al. (Nature Biotechnology (1996), Volume 14, pages 620-623) describe pathway engineering for the production of aromatic compounds in Escherichia coli. Theoretical analysis of the pathway involved in the production of aromatic compounds in E. coli suggests that the yield of this compound is limited by the availability of phosphoenolpyruvate (PEP). This compound is one of the key building blocks in several biosynthetic pathways and is also the donor utilized within the PTS system for glucose internalization. Two molecules of PEP are generated from one mole of glucose via the glycolytic pathway. However, if one mole of PEP is subsequently used by the PTS system during glucose transport, only one mole of PEP remains available for other metabolic reactions per mole of glucose consumed. Flores et al. found that when an E. coli strain lacking ptsH, ptsI, and crr genes was grown in a minimal medium utilizing glucose as the sole carbon source in a fermentor, a heterogeneous population of PTS-glucose+ revertants could be detected after 2 days. Such revertants were able to transport glucose through GalP, while in the cytoplasm, glucose was phosphorylated by glucokinase using ATP. Summary of the Invention

[0006] A further aspect of the present invention relates to the combination of endogenously unregulated adenylate cyclase activity and reduced expression of the crr gene or variants thereof, and / or inactivation or reduction of the Crr protein or variants thereof, and the effect this has on carbon conversion efficiency, carbon substrate adaptability, and space / time in oligosaccharide production by prokaryotic host organisms when combined in a host cell.

[0007] Space-time yield is defined as the rate of product formation per unit time. It can be related to the space or amount of reaction mixture or fermentation, defined by volume or weight. Typical definitions include the weight of product, e.g., grams, produced per unit volume (e.g., liter) or unit weight (e.g., kg) of fermentation broth per unit of time (e.g., hour).

[0008] Increasing the space-time yield of a given fine chemical as a product means increasing the rate of product formation, defined by its volume or weight, over time in a given reaction space, thereby improving the productivity of that particular product. Increasing the space-time yield in a given period of time means that a larger amount of fine chemical product can be produced using the same equipment. Increasing the space-time yield also means that the same amount of fine chemical can be produced in a shorter time in a given equipment.

[0009] Carbon conversion efficiency is known as the ratio of specific product formation expressed as the amount per constant amount of carbon source consumed. Carbon conversion efficiency can be related to a molar ratio, for example, the number of moles of product produced per constant number of moles of carbon source consumed. Carbon conversion efficiency can also be described as the ratio of functional moieties in the final molecule per molecule of product. In a preferred definition, carbon conversion efficiency according to the present invention is defined as the weight of a specific product produced per constant weight of carbon source used in the process. This calculation method can be advantageous because carbon conversion efficiencies using different carbon sources with different molecular weights (e.g., maltose, glucose, mannose, glycerol, sucrose, gluconate) are directly comparable.

[0010] Additionally, carbon conversion efficiency in the production of fine chemicals is increased by the methods and in the host cells of the invention. Increasing host cell cAMP increases the percentage of carbon atoms fed to the cell that go into the desired fine chemical products, and therefore less carbon is lost to unwanted side reactions or carbon dioxide from cellular respiration. On the way to a more climate-friendly economy, it is desirable to reduce carbon-to-carbon dioxide losses.

[0011] Preferably, the carbon conversion efficiency and / or space-time yield is increased by 1, 2, 3 percent, more preferably 4, 5, 6, 7, 8, 9, or 10 percent compared to a control, i.e., an unmodified cell that retains only adenylate cyclase under normal regulation.

[0012] More preferably, the carbon conversion efficiency and / or space / time yield is improved by a factor of 1.1, 1.2, 1.25, 1.3, 1.4, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0013] Also part of the present invention is a method for increasing 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 are increased compared to the unmodified host organism.

[0014] Carbon substrate adaptability is defined by the ability of a host cell to use two or more specific carbon sources. Representative carbon sources suitable for fine chemical production strains can be found in E. coli and Salmonella: Cellular and Molecular Biology ASM press 1996. As used throughout this specification, increased carbon substrate adaptability is a characteristic in which an engineered host cell grows on a carbon source that an unengineered host cell cannot grow on, or grows on a carbon source substantially better than a control, which may be a wild-type cell or an unengineered host cell.

[0015] The carbon source is added to the medium all at once and / or fed in a feeding stage. Typical fine chemical production periods are 24 to 100 hours.

[0016] The cAMP level of the host organism is preferably the intracellular cAMP level, more preferably the cytoplasmic cAMP level, of the host organism. The cAMP level can be determined by several methods known in the art, for example, using a cAMP-specific antibody that can be used with a range of detection methods, including luciferase-based assays. Commercially available kits for measuring cAMP levels in cells, tissues, and biological samples (e.g., Sigma-Aldrich CA200 cAMP enzyme immunoassay kit) are available. Other methods for determining cAMP can be found in Crasnier 1990, Journal of General Microbiology 136: 1825-1831; Guidi-Rontani et al. 1981 J. Bacteriology 148:753-761; or J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2012 909:14-21.

[0017] In one embodiment, cAMP levels are increased by exogenously adding cAMP and / or by introducing or reintroducing cAMP into the host cell. In another embodiment, cAMP levels in a host organism are increased by inactivating a regulatory activity found in wild-type adenylate cyclase and / or by introducing a mutant adenylate cyclase lacking the regulatory activity found in wild-type adenylate cyclase. In another embodiment, cAMP levels can be increased by reducing the activity of enzymes with 3',5' cAMP phosphodiesterase (EC 3.1.4.53) activity, and optionally other diesterases, such as enzymes in the enzyme classes EC 3.1.4.17 or EC 3.1.4.16, when acting on 3,5 cAMP. Reduction of activity can be achieved, for example, by gene knockout, antisense or RNAi technology, introduction of activity-reducing or activity-inhibiting mutations, or by inhibitors. An example of a 3',5' cAMP phosphodiesterase is that encoded by the E. coli gene cpdA. Another method of increasing intracellular cAMP levels is by using the adenylate cyclase domain of the adenylate cyclase toxin of Bordetella pertussis, or the entire adenylate cyclase toxin protein.

[0018] The method of the present invention is a method for increasing the space-time yield of one or more fine chemicals produced by a host organism, as well as increasing the carbon substrate adaptability and carbon conversion efficiency of the production of one or more fine chemicals by a host organism, compared to an unmodified host organism, comprising the steps of providing a host organism capable of producing one or more fine chemicals; increasing the adenosine 3',5'-cyclic monophosphate (cAMP, CAS number: 60-92-4) level of the host organism; maintaining the host organism in conditions that allow its growth; growing the host organism in the presence of substrates and nutrients under conditions suitable for the production of one or more fine chemicals; and optionally isolating the one or more fine chemicals from the host organism or the remainder thereof, wherein the host organism is suitable for producing the one or more fine chemicals in unmodified and modified forms.

[0019] In one embodiment, the cAMP level of the host organism is increased in an inducible manner, relative to a host organism that has not been so induced. For example, methods of inducer-dependent gene expression using the inducer isopropyl β-d-1-thiogalactopyranoside (IPTG) are known in the art.

[0020] In a preferred embodiment, increasing cAMP levels can be achieved by providing in a host cell an adenylate cyclase protein in which the regulatory domain is inactivated, inhibited, or missing (referred to herein as an inactivated regulatory domain or inactivated regulatory portion) and a functional catalytic domain for producing cAMP. An inactivated regulatory domain can be inactive due to the presence of an inhibitor, due to an inactivating mutation, or due to deletion of all or part of the regulatory domain of the adenylate cyclase protein. Absence of all or part of the regulatory domain of the adenylate cyclase protein can be achieved by any number of means, for example, as demonstrated in numerous ways in the present invention, by introducing a copy of a truncated adenylate cyclase gene, by altering the adenylate cyclase mRNA, by prematurely terminating protein translation of the transcript, or by removing all or part of the regulatory domain after translation.

[0021] The enzyme adenylate cyclase is also called 3',5'-cyclic AMP synthetase, adenyl cyclase, adenylyl cyclase, or ATP pyrophosphate lyase.

[0022] International patent application WO 98 / 29538 discloses the adenylate cyclase gene of Ashbya gossypii and its use in microorganisms to produce fine chemicals, such as riboflavin. Furthermore, the application discloses that riboflavin production by the fungus Ashbya gossypii grown on glucose-containing media is increased when the endogenous adenylate cyclase gene is disrupted in the adenosine 3',5'-cyclic monophosphate (3',5'-cyclic AMP or cAMP, CAS number: 60-92-4)-producing portion. It also discloses that increasing cAMP levels by adding cAMP has a negative effect on riboflavin production in the disrupted strain.

[0023] Altering the activity of adenylate cyclase has been shown to affect the uptake of carbon sources using the so-called phosphotransferase system (PTS) or other mechanisms affected by mutations in the cyaA gene, which encodes adenylate cyclase. Mutations in cyaA have been shown to cause inability to utilize carbon sources such as lactose, maltose, arabinose, mannitol, or glycerol, as well as poor fermentation and slow growth on glucose, fructose, and galactose (Perlman R, et al. 1969 Biochemical and Biophysical Research Communications 37(1), pp. 151-157).

[0024] It has not previously been shown that the production of fine chemicals, especially oligosaccharides, is positively affected by alterations in the cyaA gene that increase the synthesis of cAMP.

[0025] As mentioned above, the inactivation of the regulatory activity found in wild-type adenylate cyclase can be achieved in several ways, for example, by using inhibitors, or by inactivating mutations, or by deleting all or part of the regulatory domain of the wild-type adenylate cyclase protein, or, for example, by partially altering or deleting the mRNA encoding adenylate cyclase, the mRNA translation of adenylate cyclase in the host organism, or by mutating or deleting the gene sequence encoding the regulatory portion of adenylate cyclase. For example, CRISPR / CAS technology (Wang, HH. (2013), Mol. Syst. Biol. 9 (1): 641) can be used to specifically remove or replace the part of the gene sequence of adenylate cyclase involved in the regulatory portion of the adenylate cyclase protein in a non-functional manner. An international patent application published as WO2011102305 discloses that specific mutations to leucine at position 432 of the cyaA gene of Escherichia coli are useful in producing amino acids. Reddy et al. (Analytical Biochemistry 231, 282-286 (1995)) and Crasnier et al. (J. Gen. Microbiol. 1990;136:1825-31, Mol. Gen. Genet. 1994;243:409-16) disclose that the catalytic domain of Escherichia coli adenylate cyclase is located in the N-terminal part of the protein, and that deletion of the C-terminal part may increase adenylate cyclase activity or prevent negative regulation by effectors. Lindner (Biochem. J. (2008), 415, 449-454) discloses the results of a detailed examination of residues located in the catalytic portion of E. coli adenylate cyclase, including amino acid positions 1-412.

[0026] Preferably, a regulatory portion or domain is defined as a portion of a protein with adenylate cyclase activity that is not directly involved in the production of cAMP, but that controls the activity of the cAMP-generating portion that contains the active site.

[0027] Adenylate cyclase-producing moieties useful in the methods and host cells of the present invention are proteins or portions thereof having the enzymatic activity of EC 4.6.1.1 and the ability to produce adenosine 3',5'-cyclic monophosphate (cAMP).

[0028] Two adenylate cyclase protein variants and their encoding genes have been found in E. coli cells. One is a widely occurring protein with a length of 848 amino acids (SEQ ID NO:19, encoded by the nucleotide sequence shown as SEQ ID NO:9), and the other is a variant of this full-length protein with a six-amino acid overlap, thus having 854 amino acids (SEQ ID NO:20, encoded by the nucleotide sequence shown as SEQ ID NO:10). In the longer variant, the amino acid motif GEQSMI is present as a duplication (see Figure 2, part 2, underlined stretch of amino acids), while the 848-amino acid variant contains this motif only once. This motif is part of the PFAM domain PF01295 found in adenylate cyclase. The present invention discloses that deregulated versions of either of these two variants of E. coli adenylate cyclase result in increased space / time yield, carbon conversion efficiency, and carbon source adaptability.

[0029] In the context of the present invention, the cyaA gene of E. coli is understood to be any of the genes set forth in SEQ ID NO: 9 or 10, or the protein sequence of SEQ ID NO: 19 or 20, or a DNA encoding a protein having 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, more preferably at least 95%, at least 97%, at least 98%, or at least 99% identity to the full length of any one of SEQ ID NO: 19 or 20, most preferably a DNA encoding a protein having adenylate cyclase activity, i.e., activity of EC 4.6.1.1.

[0030] Truncated adenylate cyclase proteins in which the regulatory portion is reduced or inactivated but which still retain cAMP forming activity are useful in the methods and host cells of the invention.

[0031] Particularly useful in the methods and host cells of the invention are adenylate cyclase proteins corresponding to the protein encoded by the cyaA gene of E. coli, but lacking regulatory activity, and preferably lacking a portion corresponding to the C-terminal portion of the CyaA protein set forth in SEQ ID NO: 19 or 20, or which have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more similar to positions 1 to 412 of the protein sequence set forth as SEQ ID NO: 19 or 20, and more preferably positions 1 to 420 of the protein set forth as SEQ ID NO: 19 or 208. or an adenylate cyclase protein having 98% or more sequence identity thereto, preferably lacking the portion of E. coli adenylate cyclase following position 420, 450, 558, 585, 653, 709, 736, or 776, more preferably following position 450, 558, 585, 653, 709, or 736, and even more preferably following position 558, 582, 585, 653, 709, 736, or 776 of the protein sequence provided in SEQ ID NO: 19 or 20. Following a given position is understood as all amino acids found in the protein of interest following the amino acid corresponding to the given position in SEQ ID NO: 19 or 20.

[0032] A list of representative truncated adenylate cyclase proteins and genes is shown in Table 1.

[0033] [Table 1]

[0034] The truncated proteins cyaA653, cyaA709, cyaA736, and cyaA776 (SEQ ID NOs: 15-18) contain the duplicated GEQSMI motif as found in the full-length 854 amino acid version (SEQ ID NO: 20). The other truncated versions lack the motif altogether. The beneficial effects in the methods and host cells of the invention were found to be independent of the presence of single or duplicated GEQSMI motifs, as detailed in the Examples section below.

[0035] In a preferred embodiment, the method of the present invention is a method for increasing the space-time yield of one or more fine chemicals produced by a host organism, as well as increasing the carbon substrate adaptability and carbon conversion efficiency of the production of one or more fine chemicals by the host organism, comprising the steps of providing a host organism capable of producing one or more fine chemicals, providing a deregulated adenylate cyclase capable of producing cAMP in the host organism, maintaining the host organism in conditions that allow its growth, growing the host organism in the presence of substrates and nutrients under conditions suitable for the production of one or more fine chemicals, and optionally separating the one or more fine chemicals from the host organism or the remainder thereof.

[0036] In one embodiment, a deregulated adenylate cyclase protein useful in the methods and host cells of the invention is an enzyme with adenylate cyclase activity that does not contain the regulatory moieties found in the wild-type adenylate cyclase protein of the host cell. Preferably, the protein is the adenylate cyclase protein of the host cell, or a variant or portion thereof, which is an active adenylate cyclase enzyme but is not subject to at least some of the regulatory mechanisms that are present in the unmodified adenylate cyclase of said host cell, and corresponds to the E. coli adenylate cyclase as set forth in SEQ ID NO: 19 or 20. Preferably, the deregulated adenylate cyclase useful in the methods and host cells of the present invention is an adenylate cyclase protein that lacks a portion corresponding to the C-terminal portion of the CyaA protein set forth in SEQ ID NO: 19 or 20, or that has at least 80% sequence identity with positions 1 to 412 of the protein sequence set forth in SEQ ID NO: 19 or 20, more preferably at least 80% sequence identity with positions 1 to 420. More preferably, the deregulated adenylate cyclase protein lacks a portion of an adenylate cyclase corresponding to the portion of E. coli adenylate cyclase following position 420, 450, 558, 585, 653, 709, 736 or 776 of the protein sequence provided in SEQ ID NO: 19 or 20, more preferably following position 450, 558, 585, 653, 709 or 736, even more preferably following position 558, 582, 585, 653, 709, 736 or 776 of the protein sequence provided in SEQ ID NO: 19 or 20, and most preferably lacks amino acids corresponding to amino acids at position 777 onwards of SEQ ID NO: 19 or 20. In another preferred embodiment, the deregulated adenylate cyclase protein is a portion of the endogenous adenylate cyclase of the host organism corresponding to any of the sequences set forth in SEQ ID NOs: 11 to 18, more preferably any of the sequences set forth as SEQ ID NOs: 11 to 18, or a fusion protein encoded by any of the sequences set forth in SEQ ID NOs: 1 to 8 or variants thereof, and comprising a protein having a tag and the deregulated adenylate cyclase.In one embodiment, amino acid sequences having one to several amino acid changes compared to the sequences of SEQ ID NOS: 11 to 18 are also included, so long as they have adenylate cyclase activity that is not subject to the regulation of said activity found in the unmodified CyaA protein of a host cell corresponding to the protein of SEQ ID NOS: 19 or 20. Preferably, deregulated adenylate cyclase causes an increase in the cAMP level of an increasing host cell. Preferably, such variants of amino acid sequences do not contain a substitution of an L-lysine residue with an L-glutamine in the adenylate cyclase portion (position corresponding to position 432 of the sequence disclosed as SEQ ID NOS: 2 in the international application published as WO2011102305).

[0037] Engineered host cells harboring a deregulated adenylate cyclase protein can be achieved by several means, including mutation and selection, recombinant methods such as introduction of a truncated cyaA gene, and gene editing methods such as CRISPR / CAS.

[0038] The host cell of the invention or host cell useful in the methods of the invention is preferably a bacterial or fungal host cell, more preferably a bacterial cell selected from the group consisting of gram-positive and gram-negative bacteria, or a yeast cell, and even more preferably the host cell is selected from the group consisting of Bacillus, Clostridium, Enterobacteriaceae, Enterococcus, Erwinia, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Mycoplasma, or the like. plasma, Pasteurella, Rhodobacter, Rhodoseudomonas, Salmonella, Staphylococcus, Streptococcus, Vibrio, and Xanthomonas, or yeast cells of the genera Pichia, Kluveromyces, or Saccharomyces, and even more preferably Escherichia coli cells, Corynebacterium sp. cells, or Saccharomyces sp. cells.

[0039] In one embodiment, the host cell of the invention is a bacterial or fungal host cell, preferably a bacterial cell, preferably a cell that utilizes cAMP to regulate a cellular pathway, more preferably a cell that harbors a functional adenylate cyclase, more preferably a Proteobacterium, a Gammaproteobacterium, a bacterium of the Enterobacteriaceae family, even more preferably a bacterium of the genus Escherichia, and even more preferably a bacterium of the species Escherichia.

[0040] The fine chemicals of the present invention are biochemicals containing two or more sugar units. Preferably, the fine chemicals are biochemicals produced by genetically modified organisms. More preferably, the fine chemicals of the present invention comprise or consist of one or more oligosaccharides. Even more preferably, the fine chemicals produced by the host cells and methods of the present invention comprise or consist of human milk oligosaccharides (HMOs), even more preferably neutral or sialylated HMOs, even more preferably fucosylated or sialylated HMOs, and even more preferably, the fine chemicals are 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 2'-fucosyllactose (2'-FL), difucosyllactose (2,3-DFL), 3'-fucosyllactose (3'-FL), lacto-N-triose, lacto-N-tetraose (LNT), or lacto-N-neotetraose (LNnT). Examples of human breast milk oligosaccharides can be found in Ninonuevo MR et al. (2006). J. Agric. Food Chem. 54:7471-7480, Bode L (2009) Nutr. Rev. 67:183-191, Bode L (2012) Glycobiology 22:1147-1162, Bode L (2015) Early Hum. Dev. 91:619-622. In a most preferred embodiment, the fine chemical of the present invention is 2'-FL or 6'-SL.

[0041] Terms and Meanings Unless otherwise specified, the terms used herein should be understood to follow conventional usage by those skilled in the relevant art. In addition to the definitions of terms provided herein, definitions of common terms in molecular biology can also be found in Rieger et al., 1991 Glossary of genetics: classical and molecular, 5th Ed., Berlin: Springer-Verlag; and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols (a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.) (1998 Supplement).

[0042] As used herein and in the claims, it should be understood that "a" or "an" can mean one or more, depending on the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell is available. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0043] In the description of the present invention, genes and proteins are identified using the names of the corresponding genes in E. coli, but unless otherwise specified, the use of these names has a more general meaning according to the present invention and encompasses all corresponding genes and proteins in other organisms, particularly microorganisms.

[0044] Standard techniques for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, etc., for cloning, DNA isolation, amplification and purification, as well as various separation techniques, are well known and commonly used by those skilled in the art.Many standard techniques are described in M. Green & J. Sambrook (2012) Molecular Cloning: a laboratory manual, 4th Edition, Cold Spring Harbor Laboratory Press, CSH, New York; Ausubel et al., Current Protocols in Molecular Biology, Wiley Online Library; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY; Wu (eds.) 1993 Meth. Enzymol. 218, Part I; Wu (eds.) 1979 Meth. Enzymol. 68; Wu et al., (eds.) 1983 Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) 1980 Meth. Enzymol. 65; Miller (eds.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Old and Primrose, These techniques are described in 1981 Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (ed.), 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.), 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender, 1979 Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York.

[0045] Unless otherwise specified herein, the abbreviations and nomenclature, when used, are considered to be standard in the art and commonly used in professional journals such as those cited herein.

[0046] The terms "essentially," "about," "approximately," "substantially," and the like, in association with an attribute or value, also specifically define the attribute or value exactly, respectively. The term "substantially," in the context of the same functional activity or substantially the same function, refers to a difference in function, preferably within 20%, more preferably within 10%, and most preferably within 5% or less, compared to the reference function. In the context of a formulation or composition, the term "substantially" (e.g., "a composition consisting essentially of compound X") can be used herein to refer to a formulation or composition that substantially contains the reference compound having a given effect, and does not contain additional compounds having such effect, or contains the maximum amount of such compound that does not exhibit a measurable or relevant effect. The term "about," in the context of a given numerical value or range, specifically relates to a value or range that is within 20%, within 10%, or within 5% of the given value or range. As used herein, the term "comprising" also encompasses the term "consisting of."

[0047] The term "isolated" means that a material is substantially free from at least one other component that naturally accompanies it in its original environment. For example, a naturally-occurring polynucleotide, polypeptide, or enzyme present in a living animal is not isolated, but the same polynucleotide, polypeptide, or enzyme separated from some or all of the coexisting materials in the natural system is isolated. As a further example, an isolated nucleic acid (e.g., a DNA or RNA molecule) is one that is not immediately contiguous with 5' or 3' flanking sequences that are normally immediately contiguous with it when present in the naturally occurring genome of the organism from which it originates. Such a polynucleotide could be part of a vector, integrated into the genome of a cell of an unrelated genetic background (or into the genome of a cell with an essentially similar genetic background, but at a location different from where it naturally occurs), or produced by PCR amplification or restriction enzyme digestion; alternatively, RNA molecules produced by in vitro transcription, and / or such polynucleotides, polypeptides, or enzymes, can be part of a composition and, further, isolated, such vector or composition is not part of its natural environment.

[0048] "Purified" means that the material is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, or at least about 98% or 99% pure. Preferably, "purified" means that the material is in a 100% pure state.

[0049] A "synthetic" or "artificial" compound is produced by in vitro chemical or enzymatic synthesis. This term includes, but is not limited to, variant nucleic acids made with optimal codon usage for a host organism, such as a yeast cell host or other expression host of choice, or variant protein sequences with amino acid modifications (e.g., substitutions) compared to the wild-type protein sequence, for example, to optimize the properties of the polypeptide.

[0050] The term "non-naturally occurring" refers to a (poly)nucleotide, amino acid, (poly)peptide, enzyme, protein, cell, organism, or other substance that is not found in its original environment or source, although it may have originally been derived from its original environment or source and subsequently reproduced by other means. Such a non-naturally occurring (poly)nucleotide, amino acid, (poly)peptide, enzyme, protein, cell, organism, or other substance may be structurally and / or functionally similar to or identical to its naturally occurring counterpart.

[0051] The terms "native" (or "wild-type" or "endogenous") cell or organism and "native" (or wild-type or endogenous) polynucleotide or polypeptide refer to a cell or organism as found in nature, and a polynucleotide or polypeptide of interest as found in a cell in its natural form and genetic environment (i.e., without any human intervention), respectively. In one aspect, a wild-type adenylate cyclase is to be understood as a protein that has adenylate cyclase activity (EC 46.1.1), includes its normal regulatory portion or domain, and is subject to regulation as found in nature.

[0052] "Homologous" refers to genes, polypeptides, polynucleotides that have a high degree of similarity, e.g., in position, structure, function, or characteristics, but not necessarily a high degree of sequence identity. "Homologous" should not be used interchangeably with "endogenous" or as the opposite of "heterologous" (see below).

[0053] The term "heterologous" (or exogenous or foreign or recombinant) polypeptide is defined herein as follows: (a) A polypeptide that is not native to the host cell. Such heterologous polypeptide protein sequences are synthetic, non-naturally occurring, "man-made" protein sequences; (b) a polypeptide native to the host cell, but in which structural modifications (e.g., deletions, substitutions, and / or insertions) have been made to alter the native polypeptide; or (c) A polypeptide native to a host cell whose expression is quantitatively altered or whose expression is from a genomic location different from that of the native host cell as a result of manipulation of the host cell's DNA by recombinant DNA techniques (e.g., a stronger promoter).

[0054] The above descriptions (b) and (c) refer to a sequence that is in its native form but is not naturally expressed by the cells used for production. The produced polypeptide is therefore more accurately defined as a "recombinantly expressed endogenous polypeptide," which does not contradict the above definition, but reflects the special situation in which the protein sequence is not synthesized or engineered, but is the manner in which the polypeptide molecule is produced.

[0055] Similarly, the term "heterologous" (or exogenous or foreign or recombinant) polynucleotide means: (a) a polynucleotide that is not native to the host cell; (b) a polynucleotide native to the host cell, but in which structural modifications (e.g., deletions, substitutions, and / or insertions) have been made to alter the native polynucleotide; or (c) a polynucleotide native to a host cell, the expression of which has been quantitatively altered as a result of the manipulation of regulatory elements of the polynucleotide by recombinant DNA techniques (e.g., a stronger promoter); or (d) A polynucleotide that is native to a host cell but is not integrated into its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques.

[0056] With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterize that the two or more polynucleotide sequences or two or more amino acid sequences do not naturally occur in specific combination with each other.

[0057] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and tail) and intervening sequences (introns) between individual coding segments (exons).

[0058] The term "gene" refers to a segment of DNA that contains the genetic information that is passed from parent to offspring and contributes to the phenotype of an organism. The influence of a gene on the form and function of an organism is mediated through transcription into RNA (tRNA, rRNA, mRNA, non-coding RNA) and, in the case of mRNA, through translation into peptides and proteins.

[0059] The terms "polynucleotide," "nucleic acid sequence," "nucleotide sequence," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein and refer to a polymeric, unbranched form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or a combination of both.

[0060] For nucleotide sequences (e.g., consensus sequences), IUPAC nucleotide nomenclature (Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1984). "Nomenclature for Incompletely Specified Bases in Nucleic Acid Sequences") was used, including the following nucleotide and nucleotide ambiguity definitions that are important to the present invention: A, adenine; C, cytosine; G, guanine; T, thymine; K, guanine or thymine; R, adenine or guanine; W, adenine or thymine; M, adenine or cytosine; Y, cytosine or thymine; D, not cytosine; N, any nucleotide.

[0061] Additionally, the notation "N(3-5)" means that the indicated consensus position can have any of 3 to 5 (N) nucleotides. For example, the consensus sequence "AWN(4-6)" represents three possible variants: AWNNNN, AWNNNNN, and AWNNNNNN, containing either 4, 5, or 6 nucleotides at the termini.

[0062] The term "hybridization," as defined herein, refers to the process by which substantially complementary nucleotide sequences anneal to each other. The hybridization process can occur entirely in solution, i.e., both complementary nucleic acids are in solution. The hybridization process can also occur when one complementary nucleic acid is immobilized on a matrix such as magnetic beads, Sepharose beads, or any other resin. The hybridization process can also occur when one complementary nucleic acid is immobilized on a solid support such as a nitrocellulose membrane or nylon membrane, or on a siliceous glass support, for example, by photolithography (the latter known as a nucleic acid array, microarray, or nucleic acid chip). To allow hybridization to occur, nucleic acid molecules are generally thermally or chemically denatured to melt the double strand into two single strands and / or to remove hairpins or other secondary structures from single-stranded nucleic acids.

[0063] The term "stringency" refers to the conditions under which hybridization occurs. Hybridization stringency is affected by conditions such as temperature, salt concentration, ionic strength, and hybridization buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. Moderate stringency conditions are when the temperature is 20°C lower than Tm, and high stringency conditions are when the temperature is 10°C lower than Tm. High stringency hybridization conditions are typically used to isolate hybridizable sequences with high sequence similarity to the target nucleic acid sequence. However, due to the degeneracy of the genetic code, nucleic acids may encode substantially identical polypeptides despite diverging sequences. Therefore, moderate stringency hybridization conditions may be necessary in some cases to identify such nucleic acid molecules.

[0064] "Tm" is the temperature at which 50% of a target sequence hybridizes to a perfectly matched probe at a defined ionic strength and pH. Tm depends on the solution conditions and the base composition and length of the probe. For example, longer sequences hybridize specifically at higher temperatures. Maximum hybridization rates are achieved at temperatures approximately 16°C to as much as 32°C below Tm. The presence of monovalent cations in the hybridization solution reduces the electrostatic repulsion between two nucleic acid strands, thereby facilitating hybrid formation; this effect is observed at sodium concentrations up to 0.4M (although this effect is negligible at higher concentrations). Formamide lowers the melting temperatures of DNA-DNA and DNA-RNA duplexes by 0.6-0.7°C per 1% formamide; the addition of 50% formamide allows hybridization to occur at 30-45°C, but the hybridization rate will be slower. Base pair mismatches reduce the hybridization rate and the thermal stability of the duplex. On average, for large probes, T decreases by about 1°C per 1% of base mismatches. Depending on the type of hybrid, T can be calculated using the following equation: DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984): Tm = 81.5°C + 16.6 × log[Na+]a + 0.41 × %[G / Cb] - 500 × [Lc] - 1 - 0.61 × % formamide DNA-RNA or RNA-RNA hybrids: Tm=79.8+18.5(log10[Na+]a)+0.58(%G / Cb)+11.8(%G / Cb)2-820 / Lc Oligo-DNA or oligo-RNA hybrids: For less than 20 nucleotides: Tm=2(ln) For 20-35 nucleotides: Tm = 22 + 1.46 (ln) For a or other monovalent cations, it is only accurate in the range of 0.01 to 0.4M. b Accurate for %GC only in the range of 30%–75%. c L = length of the duplex in base pairs. d Oligo: oligonucleotide; ln: effective length of primer = 2 × (number of G / C) + (number of A / T).

[0065] Nonspecific binding can be controlled using any one of a number of known techniques, such as blocking the membrane with a protein-containing solution, adding heterologous RNA, DNA, and SDS to the hybridization buffer, and treating with RNase. For unrelated probes, a series of hybridizations can be performed by varying one of the following: (i) gradually decreasing the annealing temperature (e.g., from 68°C to 42°C) or (ii) gradually decreasing the formamide concentration (e.g., from 50% to 0%). Those skilled in the art are aware of various parameters that can be altered during hybridization and will maintain or change stringency conditions.

[0066] In addition to hybridization conditions, hybridization specificity typically also depends on post-hybridization washing. To remove background from nonspecific hybridization, samples are washed with a dilute salt solution. Important factors for such washing include the ionic strength and temperature of the final washing solution: the lower the salt concentration and the higher the washing temperature, the higher the stringency of the wash. Washing conditions are typically performed at or below the hybridization stringency. Positive hybridization results in a signal at least twice the background signal. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as described above. Higher or lower stringency conditions can also be selected. Those skilled in the art are aware of various parameters that can be changed during washing and will maintain or change the stringency conditions.

[0067] For example, typical high stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 1xSSC at 65°C or in 1xSSC and 50% formamide at 42°C, followed by washing in 0.3xSSC at 65°C. Examples of moderate stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 4xSSC at 50°C or in 6xSSC and 50% formamide at 40°C, followed by washing in 2xSSC at 50°C. The hybrid length is the expected length for the hybridizing nucleic acid. When hybridizing nucleic acids of known sequence, the hybrid length can be determined by aligning the sequences and identifying conserved regions as described herein. 1x SSC is 0.15M NaCl and 15mM sodium citrate; hybridization and wash solutions may further contain 5x Denhardt's reagent, 0.5-1.0% SDS, 100µg / mL denatured, fragmented salmon sperm DNA, and 0.5% sodium pyrophosphate. Another example of high stringency conditions is hybridization in 0.1x SSC containing 0.1% SDS and optionally 5x Denhardt's reagent, 100µg / mL denatured, fragmented salmon sperm DNA, and 0.5% sodium pyrophosphate at 65°C, followed by washing in 0.3x SSC at 65°C.

[0068] For purposes of defining stringency levels, reference may be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989 and annually revised).

[0069] "Recombinant" (or transgenic), with respect to a cell or organism, means that the cell or organism contains an exogenous polynucleotide introduced by means of genetic techniques, and with respect to the polynucleotide, means either: (a) the sequence of a polynucleotide or a portion thereof; or (b) one or more gene control sequences (e.g., promoters) operably linked to the polynucleotide; or (c) Both (a) and (b) means all those constructs resulting from genetic / recombinant DNA techniques in which the genes are not located in or modified in their wild-type genetic environment.

[0070] It will be further noted that the terms "isolated nucleic acid" or "isolated polypeptide" can in some instances be considered synonymous with "recombinant nucleic acid" or "recombinant polypeptide," respectively, and refer to a nucleic acid or polypeptide that is not located in its natural genetic or cellular environment and / or that has been modified by recombinant methods, respectively. An isolated nucleic acid sequence or isolated nucleic acid molecule is one that is not in its native environment or in proximity to its native nucleic acid, but is physically and functionally linked to other nucleic acid sequences or molecules and is found as part of a nucleic acid construct, vector sequence, or chromosome. Typically, isolated nucleic acids are obtained in laboratory conditions by isolating RNA from cells and converting it into copy DNA (cDNA).

[0071] The term "control sequence" is defined herein to include all sequences that affect the expression of a polynucleotide, including, but not limited to, the expression of a polynucleotide encoding a polypeptide. Each control sequence may be native or foreign to the polynucleotide, or to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, 5'-UTR, ribosome binding site (RBS, Shine-Dalgarno sequence), 3'-UTR, signal peptide sequence, or transcription terminator. At a minimum, control sequences include a promoter and transcription start and stop signals.

[0072] The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0073] A "parent" (or "reference" or "template") nucleic acid, protein, enzyme, or organism (also referred to as a "parent nucleic acid," "reference nucleic acid," "template nucleic acid," "parent protein," "reference protein," "template protein," "parent enzyme," "reference enzyme," "template enzyme," "parent organism," "reference organism," or "template organism") is the starting point for introducing changes (e.g., by introducing one or more nucleic acid or amino acid substitutions) that result in a "variant" of the parent. Thus, terms such as "enzyme variant" or "sequence variant" or "variant protein" are used to distinguish a modified or variant sequence, protein, enzyme, or organism from the parent sequence, protein, enzyme, or organism from which the respective variant sequence, protein, enzyme, or organism is derived. Thus, a parent sequence, protein, enzyme, or organism includes a wild-type sequence, protein, enzyme, or organism, as well as variants of the wild-type sequence, protein, enzyme, or organism that are used to develop further variants. Variant proteins or enzymes differ to a certain extent in their amino acid sequence from the parent proteins or enzymes; however, variants maintain at least the functional properties (e.g., enzymatic properties) of their respective parents. In one embodiment, the enzymatic properties are improved in the variant enzymes compared to their respective parent enzymes. In one embodiment, the variant enzymes have at least the same enzymatic activity as their respective parent enzymes, or the variant enzymes have increased enzymatic activity as compared to their respective parent enzymes.

[0074] In describing variants (mutants), the nomenclature described as follows is used: Abbreviations for single amino acids used within the present invention follow the commonly accepted IUPAC one-letter or three-letter amino acid abbreviations. Although the following definitions describe variants in the context of amino acid changes, nucleic acids can be similarly modified (e.g., by nucleotide substitution, deletion, and / or insertion).

[0075] A "substitution" is described by giving the original amino acid followed by the number of its position in the amino acid sequence, followed by the substituted amino acid. For example, a substitution of histidine at position 120 with alanine would be designated "His120Ala" or "H120A."

[0076] A "deletion" is the original amino acid followed by the number of its position in the amino acid sequence, followed by * Thus, the deletion of glycine at position 150 is described as "Gly150 * " or "G150 * Alternatively, the deletion may be indicated by, for example, "deletion of D183 and G184."

[0077] An "insertion" is described by giving the original amino acid followed by the number of the position in the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, an insertion of a lysine at position 180 next to a glycine would be designated "Gly180GlyLys" or "G180GK." If more than one amino acid residue is inserted (e.g., a Lys and an Ala after Gly180), this can be shown as Gly180GlyLysAla or G180GKA.

[0078] If the substitution and insertion occur at the same position, this can be designated as S99SD+S99A or for short S99AD.

[0079] It is clear that degeneracy in nomenclature occurs when an amino acid residue identical to an existing amino acid residue is inserted, e.g., if a glycine is inserted after the glycine in the above example, this would be indicated by G180GG.

[0080] Variants containing multiple changes are separated by "+", e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple changes can be separated by spaces or commas (e.g., R170Y G195E or R170Y, G195E, respectively).

[0081] Where different changes can be introduced at one position, the different changes are separated by commas, e.g., "Arg170Tyr, Glu" denotes the substitution of arginine at position 170 with tyrosine or glutamic acid. Alternatively, the different changes or optional substitutions can be indicated in parentheses (e.g., Arg170[Tyr, Gly] or Arg170{Tyr, Gly} or, in short, R170[Y,G] or R170{Y,G}).

[0082] Variants can contain one or more alterations, any of the same type, e.g., all substitutions or a combination of substitutions, deletions, and / or insertions. Alterations can be introduced into nucleic acid or amino acid sequences.

[0083] In one embodiment, the deregulated adenylate cyclase variant (mutant) contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 or more changes and has adenylate cyclase activity.

[0084] Deregulated adenylate cyclase variants (mutants) include nucleic acids and polypeptides that have about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1-10 or 10-20, respectively, and have adenylate cyclase activity, preferably without the regulatory portion of wild-type adenylate cyclase or which are inactive, down-regulated, or absent.

[0085] For the substitution of an amino acid in a base sequence selected from any of the sequences of SEQ ID NO: 1-10 or 26, regardless of the presence of that amino acid in others of these sequences, the following conditions apply, where letters indicate L-amino acids using common abbreviations and numbers in parentheses indicate the priority of the substitution (higher numbers indicate higher priority): A can be replaced by any amino acid selected from S(1), C(0), G(0), T(0) or V(0). C can be replaced by A(0). D can be replaced by any amino acid selected from E(2), N(1), Q(0) or S(0). E can be replaced by any amino acid selected from D(2), Q(2), K(1), H(0), N(0), R(0) or S(0). F can be replaced by any amino acid selected from Y(3), W(1), I(0), L(0) or M(0). G can be replaced by any amino acid selected from A (0), N (0) or S (0). H can be replaced by any amino acid selected from Y (2), N (1), E (0), Q (0) or R (0). I can be replaced by any amino acid selected from V (3), L (2), M (1) or F (0). K can be replaced by any amino acid selected from R (2), E (1), Q (1), N (0) or S (0). L can be replaced by any amino acid selected from I (2), M (2), V (1) or F (0). M can be replaced by any amino acid selected from L (2), I (1), V (1), F (0) or Q (0). N can be replaced by any amino acid selected from D (1), H (1), S (1), E (0), G (0), K (0), Q (0), R (0) or T (0). Q can be replaced by any amino acid selected from E (2), K (1), R (1), D (0), H (0), M (0), N (0) or S (0).R can be replaced by any amino acid selected from K(2), Q(1), E(0), H(0) or N(0). S can be replaced by any amino acid selected from A(1), N(1), T(1), D(0), E(0), G(0), K(0) or Q(0). T can be replaced by any amino acid selected from S(1), A(0), N(0) or V(0). V can be replaced by any amino acid selected from I(3), L(1), M(1), A(0) or T(0). W can be replaced by any amino acid selected from Y(2) or F(1). Y can be replaced by any amino acid selected from F(3), H(2) or W(2).

[0086] Nucleic acids and polypeptides can be modified to include tags or domains. Tags can be used for a variety of purposes, including detection, purification, solubilization, or immobilization, and can include, for example, biotin, fluorophores, epitopes, mating factors, or regulatory sequences. Domains can be of any size, can provide a desired function (e.g., conferring increased stability, solubility, activity, simplifying purification), and can include, for example, binding domains, signal sequences, promoter sequences, regulatory sequences, N-terminal extensions, or C30-terminal extensions. Combinations of tags and / or domains can also be used.

[0087] The term "fusion protein" refers to two or more polypeptides linked together by any means known in the art, including chemical synthesis or splicing of encoding nucleic acids by recombinant genetic engineering.

[0088] Gene editing Gene editing or genome editing can be performed by using various techniques such as "gene shuffling" or "directed evolution," which consists of repeated DNA shuffling followed by appropriate screening and / or selection, in which DNA is inserted, replaced, or removed from the genome, to generate mutants of nucleic acids or portions thereof that encode proteins with altered biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Patent Nos. 5,811,238 and 6,395,547), or by "T-DNA activation" tagging, in which the resulting transgenic organisms exhibit dominant phenotypes due to altered expression of genes adjacent to the introduced promoter (Hayashi et al. Science (1992) 1350-1353), or "TILLING" (Targeted Induced Local Lesions Induced in the Genome). TILLING refers to a type of genetic engineering that can be obtained by TILLING (Genomes) and is a mutagenesis technique useful for creating and / or identifying nucleic acids that encode proteins with altered expression and / or activity. TILLING also allows for the selection of organisms that carry such mutations. Methods for TILLING are well known in the art (reviewed by McCallum et al., (2000) Nat Biotechnol 18: 455-457; Stemple (2004) Nat Rev Genet 5(2): 145-50). Another technique uses artificially engineered nucleases, such as zinc finger nucleases, transcription activator-like effector nucleases (TALENs), CRISPR / Cas systems, and engineered meganucleases (such as engineered homing endonucleases) (Esvelt, KM.; Wang, HH. (2013), Mol Syst Biol 9 (1): 641; Tan, WS. et al. (2012), Adv Genet 80: 37-97; Puchta, H.; Fauser, F. (2013), Int. J. Dev. Biol 57: 629-637).

[0089] "Enzyme activity" refers to at least one catalytic function exerted by an enzyme. In one embodiment, enzyme activity is expressed as units per milligram of enzyme (specific activity) or units of molecules of substrate converted per minute per molecule of enzyme (molecular activity). In the case of adenylate cyclase activity, molecular enzyme activity can be understood as the number of cAMP molecules produced per minute per molecule of adenylate cyclase or adenylate cyclase-containing portion of a protein.

[0090] The alignment of sequences is preferably carried out using the Needleman and Wunsch algorithm. Needleman and Wunsch algorithm: Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-453. This algorithm is implemented, for example, in the "NEEDLE" program, which performs a global alignment of two sequences. The NEEDLE program is, for example, included in the European Molecular Biology Open Software Suite (EMBOSS), a collection of various programs: The European Molecular Biology Open Software Suite (EMBOSS), Trends in Genetics 16 (6), 276 (2000).

[0091] Enzyme variants (mutants) can be defined by their sequence identity when compared with the parent enzyme. Sequence identity is usually provided as "% sequence identity" or "% identity." In the first step, to determine the percent identity between two amino acid sequences, a pairwise sequence alignment is made between the two sequences, where the two sequences are aligned over their entire length (i.e., pairwise global alignment). The alignment is generated using a program that implements the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443-453), preferably by using the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with the program's default parameters (gapopen=10.0, gapextend=0.5, and matrix=EBLOSUM62). For the purposes of the present invention, the preferred alignment is the one that allows the highest sequence identity to be determined.

[0092] The following examples are intended to illustrate two types of nucleotide sequences, but the same calculations apply to protein sequences: Seq A: AAGATACTG Length: 9 bases Seq B: GATCTGA Length: 7 bases.

[0093] Therefore, the relatively short sequence is sequence B.

[0094] Generating a pairwise global alignment showing both sequences over their full length yields: TIFF0007793519000002.tif16146

[0095] The "|" symbol in the alignment indicates identical residues (meaning bases for DNA or amino acids for proteins). The number of identical residues is six.

[0096] The "-" symbol in the alignment indicates a gap. The number of gaps introduced by the alignment within Seq B is 1. The number of gaps introduced by the alignment at the edge of Seq B is 2, and at the edge of Seq A is 1.

[0097] The alignment length, showing sequences aligned over their entire length, is 10.

[0098] The generation of pairwise alignments showing a relatively short sequence over its entire length in accordance with the present invention results in the following: TIFF0007793519000003.tif16146

[0099] Generating a pairwise alignment showing sequence A over its entire length according to the present invention results in the following: TIFF0007793519000004.tif16146

[0100] Generating a pairwise alignment showing sequence B over its entire length according to the present invention results in the following: TIFF0007793519000005.tif16146

[0101] The alignment length showing the shorter sequence over its entire length is 8 (there is one gap, which is included in the alignment length of the shorter sequence).

[0102] Therefore, the alignment length showing Seq A over its entire length would be 9 (meaning that Seq A is a sequence of the present invention).

[0103] Therefore, the alignment length showing Seq B over its entire length would be 8 (meaning that Seq B is a sequence of the present invention).

[0104] In a second step, after aligning the two sequences, an identity value is determined from the alignment. To this end, the following calculation of percent identity is applied according to this description: % identity = (identical residues / length of the alignment region representing the shorter sequence over its entire length) * 100. That is, the sequence identity associated with the comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region representing the shorter sequence over its entire length. This value is multiplied by 100 to give the "% identity." Following the example provided above, the % identity is: (6 / 8) * 100 = 75%.

[0105] Gene editing A number of techniques are known for targeted modifications in the genome of an organism. The technique known as CRIPR or CRISPR / CAS is the most widely known: CRISPR (clustered regularly interspaced short palindromic repeats) technology can be used to modify the genome of a target organism, for example, to introduce any given DNA fragment into almost any site in the genome, to replace a portion of the genome with a desired sequence, or to precisely delete a given region in the genome of a target organism, allowing for unprecedented precision in genome engineering.

[0106] The CRISPR system was initially identified as an adaptive defense mechanism in bacteria belonging to the genus Streptococcus (WO 2007 / 025097). These bacterial CRISPR systems rely on a guide RNA (gRNA) in a complex with a cleavage protein to direct the degradation of complementary sequences present in invading viral DNA. The application of CRISPR systems for genetic manipulation in various eukaryotic organisms has been demonstrated (WO 2013 / 141680; WO 2013 / 176772; WO 2014 / 093595). Cas9, the first protein identified in the CRISPR / Cas system, is a large monomeric DNA nuclease that is guided to DNA target sequences flanked by a protospacer adjacent motif (PAM) sequence motif by a complex of two non-coding RNAs: the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA). Synthetic RNA chimeras (single guide RNAs or sgRNAs) created by fusing crRNA with tracrRNA have also been shown to be equally functional (WO 2013 / 176772). CRISPR systems from other sources containing DNA nucleases different from Cas9 (such as Cpf1, C2c1p, or C2c3p) have been described to have the same functionality (WO 2016 / 0205711, WO 2016 / 205749). Other authors describe systems in which the nuclease is guided by a DNA molecule rather than an RNA molecule. Such a system is, for example, the AGO system as disclosed in U.S. Patent Application Publication No. 2016 / 0046963.

[0107] Several research groups have found that CRISPR cutting properties can be used to disrupt target regions in the genome of almost any organism with unprecedented ease.It has now become clear that by providing a repair template, it is possible to edit genomes with almost any desired sequence at almost any site, making CRISPR a powerful gene editing tool (WO 2014 / 150624, WO 2014 / 204728).The repair template is called donor nucleic acid, and contains sequences complementary to the target region at the 3' and 5' ends, which allows homologous recombination in each template after introducing double-strand breaks in the target nucleic acid by each nuclease.

[0108] The main limitation of selecting target region in a given genome is the necessity of the presence of PAM sequence motifs near the region where CRISPR-associated nuclease introduces double-strand break.However, various CRISPR systems recognize different PAM sequence motifs.This allows the most suitable CRISPR system to be selected for each target region.In addition, the AGO system does not require any PAM sequence motifs.

[0109] This technology can be applied to alter gene expression in any organism, for example, by replacing the upstream promoter of a target gene with one of different strength or specificity. Other methods disclosed in the prior art describe fusing an activating or repressing transcription factor to a nuclease, a minus-CRISPR nuclease protein. Such fusion proteins can be expressed in a target organism together with one or more guide nucleic acids that guide the transcription factor portion of the fusion protein to any desired promoter in the target organism (WO 2014 / 099744; WO 2014 / 099750). Gene knockout can be easily achieved by introducing point mutations or deletions into the respective target gene, for example, by introducing non-homologous end-joining (NHEJ), which normally leads to gene disruption (WO 2013 / 176772).

[0110] Modified Organisms The term "recombinant organism" refers to a eukaryotic organism (yeast, fungus, algae, plant, animal) or prokaryotic microorganism (e.g., bacterium) that has been genetically altered, modified, or engineered, thereby exhibiting an altered, modified, or different genotype when compared to the wild-type organism from which it is derived. Preferably, a "recombinant organism" comprises an exogenous nucleic acid. The terms "recombinant organism," "genetically modified organism (GMO)," and "transgenic organism" are used interchangeably herein. The exogenous nucleic acid can be located on an extrachromosomal piece of DNA (such as a plasmid) or can be integrated into the chromosomal DNA of the organism. Recombinant is understood to mean that the nucleic acid used is not present in or derived from the genome of the organism, or is present in the genome of the organism but not at its natural locus in the genome of the organism, and is capable of being expressed under the control of one or more endogenous and / or exogenous control elements.

[0111] "Host cell" The host cell, also referred to as the host organism, can be any cell selected from a bacterial cell, a yeast cell, a fungus, an algae, or a cyanobacterial cell, a non-human animal or mammalian cell, or a plant cell. Those skilled in the art are familiar with the genetic elements that must be present on the genetic construct in order to successfully transform, select, and propagate a host cell containing the sequence of interest.

[0112] In one embodiment, host cell or host organism are used interchangeably. Typical host cells are Gram-positive bacteria such as Bacillus and Streptomyces. Useful Gram-positive bacteria include, but are not limited to, Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus iautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus Prokaryotic organisms include Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Most preferably, the prokaryotic organism is a Bacillus cell, preferably a Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, or Bacillus lentus bacillus cell.Some other preferred bacteria include strains of the order Actinomycetales, preferably of the genus Streptomyces, preferably Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include strains of the genus Myxococcus, such as M. virescens.

[0113] Further exemplary host cells are Gram-negative: Escherichia coli, Pseudomonas, preferred Gram-negative bacteria are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).

[0114] Further exemplary host cells are fungi such as Aspergillus, Fusarium, and Tricoderma. The microorganism may be a fungal cell. As used herein, "fungi" includes fungi of the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and Deuteromycotina, and all mitosporic fungi. Representative groups of the Ascomycota include, for example, the genera Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts listed below. Examples of the Basidiomycota include mushrooms, rusts, and smuts. Representative groups of the Chytridiomycota include, for example, Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of the phylum Oomycota include, for example, Saprolegniomycetous aquatic fungi (water molds), such as Achlya. Examples of vegetative spore-forming fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of the phylum Zygomycota include, for example, Rhizopus and Mucor.

[0115] Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, such as the genera Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, and the like. resii, Myrothecium verrucana (IFO 6113), Verticillium alboatrum, Verticillium dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli or Dressclera halodes.

[0116] Other preferred fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, such as the genera Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (formerly called Polyporus), such as T. versicolor (e.g. PR4 28-A).

[0117] Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, such as the genus Rhizopus or Mucor, in particular Mucor hiemalis.

[0118] Further exemplary host cells are yeasts, such as Pichia species or Saccharomyces species. Fungal host cells can be yeast cells. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). Ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is composed of four subfamilies: Schizosaccharomycoideae (e.g., Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., Kluyveromyces, Pichia, and Saccharomyces). Basidiospore-forming yeasts include the genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungi Imperfecti are divided into two families: Sporobolomycetaceae (eg, Sporobolomyces and Bullera) and Cryptococcaceae (eg, Candida).

[0119] Typical host cells are also eukaryotic, such as non-human animals, non-human mammals, birds, reptiles, insects, plants, yeast, fungi or plants.

[0120] Preferably, the host organism according to the present invention may be a Gram-positive or Gram-negative prokaryotic microorganism.

[0121] Useful Gram-positive prokaryotic microorganisms include, but are not limited to, Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus iautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus Prokaryotic organisms include Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Most preferably, the prokaryotic organism is a Bacillus cell, preferably a Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, or Bacillus lentus bacillus cell.Some other preferred bacteria include strains of the order Actinomycetales, preferably of the genus Streptomyces, preferably Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include strains of the genus Myxococcus, such as M. virescens.

[0122] Further exemplary prokaryotes are gram-negative: Escherichia coli, Pseudomonas; preferred gram-negative prokaryotic microorganisms are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).

[0123] Most preferably, the prokaryotic microorganism is Escherichia coli.

[0124] The term "monosaccharide" preferably refers to a sugar of 5 to 9 carbon atoms, which is an aldose (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), a ketose (e.g., D-fructose, D-sorbose, D-tagatose, etc.), a deoxysugar (e.g., L-rhamnose, L-fucose, etc.), a deoxyaminosugar (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), a uronic acid, a ketoaldonic acid (e.g., sialic acid), or an equivalent.

[0125] The term "oligosaccharide" preferably refers to a sugar polymer containing at least three monosaccharide units (see above). Oligosaccharides can have monosaccharide units with a linear or branched structure linked to each other by interglycosidic bonds. Examples include, but are not limited to, maltodextrin, cellodextrin, human milk oligosaccharides, fructooligosaccharides, and galactooligosaccharides. Preferably, the oligosaccharides are human milk oligosaccharides (HMOs).

[0126] The term "human milk oligosaccharide" or "HMO" preferably refers to complex carbohydrates found in human breast milk (Urashima et al.: Milk Oligosaccharides. Nova Science Publishers, 2011). HMOs have a core structure of a lactose unit at the reducing end, which can be extended by one or more β-N-acetyl-lactosaminyl and / or one or more β-lacto-N-biosyl units, and this core structure can be substituted by αL-fucopyranosyl and / or α-N-acetyl-neuraminyl (sialyl) moieties. In this regard, non-acidic (or neutral) HMOs lack sialyl residues, while acidic HMOs have at least one sialyl residue in their structure.

[0127] Non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose (LNTri, GlcNAc(β1-3)Gal(β1-4)Glc), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH), and lacto-N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3'-FL), difucosyllactose (2,3-DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFH-III), and lacto-N-fucopentaose III (LNFH-III). Fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose I (FpLNnH I), fucosyl-para-lacto-N-hexaose II (F-pLNnH II), and fucosyl-lacto-N-neohexaose (FLNnH). Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II), and disialyl-lacto-N-tetraose (DSLNT). Examples of human milk oligosaccharides can also be found in Ninonuevo MR et al. (2006). J. Agric. Food Chem. 54:7471-7480, Bode L (2009) Nutr. Rev. 67:183-191, Bode L (2012) Glyco-biology 22:1147-1162, Bode L (2015) Early Hum. Dev. 91:619-622.

[0128] More preferably, the HMO is a neutral or acidic HMO.

[0129] Even more preferably, the oligosaccharides are 2'-fucosyllactose (2'-FL), 6'-sialyllactose (6'-SL) and / or lacto-N-tetraose (LNT).

[0130] The terms "increase", "improve" or "enhance" in the context of enzyme activity, cAMP levels or fine chemical production, carbon conversion efficiency, space-time yield or growth, or carbon source flexibility are interchangeable and will mean, in the applied sense, an increase of at least 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% or more compared to a control, such as, but not limited to, an unmodified host organism.

[0131] The terms "decrease", "reduction" or "decrease" in the context of gene expression or protein presence or protein abundance or inactivation are interchangeable and, within the meaning of the present application, refer to a reduction of at least 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 94%, 95% or 98% or more compared to a control as defined herein.

[0132] The term "enhanced production of oligosaccharides" refers to enhanced oligosaccharide productivity and / or enhanced oligosaccharide titer and / or enhanced carbon conversion efficiency compared to the parent strain. Microbial production of oligosaccharides in a culture medium can be clearly recorded by standard analytical means known to those skilled in the art. Several genetically modified microorganisms with enhanced oligosaccharide (e.g., HMO) production are disclosed in patent applications published under the names WO 2016 / 008602, WO 2013 / 182206, EP 2379708, US 9944965, WO 2012 / 112777, WO 2001 / 04341, and US 2005019874 for Escherichia coli strains. All of these disclosures are incorporated herein by reference.

[0133] Furthermore, the inventors have surprisingly found that the carbon conversion efficiency, carbon substrate adaptability, and space / time for the production of oligosaccharides by prokaryotes can be increased by manipulating the PTS system to prevent the Crr protein, or a protein of said prokaryote corresponding to the Crr protein, by engaging the PTS either by reducing or preventing expression of the crr gene (SEQ ID NO: 25) or a mutant thereof, or by inactivating or reducing the Crr protein (SEQ ID NO: 26) or a mutant thereof. A host organism carrying such an inactivated or reduced protein of the Crr family or in which expression of a gene of the crr gene family is reduced or prevented is, in one embodiment, a prokaryotic microorganism.

[0134] In one aspect of the invention, increased carbon substrate adaptation is characteristic of the engineered microorganism growing on carbon, and the unmodified microorganism is unable to grow or grows substantially better on the carbon source than a control, which can be a wild-type cell or a genetically engineered microorganism without modifications for adenylate cyclase activity and / or modifications for the gene or protein corresponding to the crr gene (SEQ ID NO: 25) or Crr protein (SEQ ID NO: 26), respectively.

[0135] In one embodiment, a method of the invention is for increasing the space-time yield of one or more fine chemicals, preferably one or more oligosaccharides, produced by a genetically modified microorganism, and / or for increasing the carbon substrate adaptability and / or carbon conversion efficiency of the production of one or more fine chemicals, preferably one or more oligosaccharides, by a genetically modified microorganism, compared to a microorganism without a modification associated with a gene or protein corresponding to the crr gene (SEQ ID NO: 25) or the Crr protein (SEQ ID NO: 26), the method comprising the steps of: providing a microorganism capable of producing one or more fine chemicals; increasing cyclic adenosine 3',5'-monophosphate (cAMP, CAS No. 60-92-4) levels in the microorganism by inactivation or absence of a Crr protein or an endogenous protein corresponding to the Crr protein (SEQ ID NO: 26) in E. coli; maintaining the modified microorganism in an environment that allows the modified microorganism to grow; growing the modified microorganism in the presence of substrates and nutrients under conditions suitable for the production of the one or more fine chemicals; and optionally separating the one or more fine chemicals from the modified microorganism or a remainder thereof. In one embodiment, the modified microorganism is suitable for producing said one or more fine chemicals in unmodified and modified forms.

[0136] In one embodiment, the mutant (variant) CRR protein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 or more modifications compared to an unmodified Crr protein or a protein corresponding to a Crr protein, and the abundance, activity and / or lifespan of the variant is reduced compared to the unmodified CRR protein family member of the microorganism.

[0137] Variants include nucleic acids and polypeptides having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 25 or 26, respectively.

[0138] The term "genetically modified microorganism" refers to a prokaryotic microorganism (e.g., a bacterium) that has been genetically altered, modified, or engineered to exhibit an altered, modified, or different genotype compared to the wild-type organism from which it was derived. "Genetically modified microorganism," "recombinant microorganism," and "transgenic microorganism" are used interchangeably herein. The exogenous nucleic acid in the genetically modified microorganism can be located on an extrachromosomal piece of DNA (such as a plasmid) or can be integrated into the chromosomal DNA of the organism.

[0139] The genetically modified microorganism according to the invention can be a Gram-positive or Gram-negative prokaryotic microorganism.

[0140] Gram-positive prokaryotic microorganisms useful for generating the genetically modified microorganisms of the invention and those useful in the methods of the invention include, but are not limited to, Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus iautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus Prokaryotic organisms include Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Most preferably, the prokaryotic organism is a Bacillus cell, preferably a Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, or Bacillus lentus bacillus cell.Some other preferred bacteria include strains of the order Actinomycetales, preferably of the genus Streptomyces, preferably Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include strains of the genus Myxococcus, such as M. virescens.

[0141] Further exemplary prokaryotes useful for making the genetically modified microorganisms of the invention and useful in the methods of the invention are Gram-negative: Escherichia coli, Pseudomonas, with preferred Gram-negative prokaryotic microorganisms being Escherichia coli and Pseudomonas, preferably Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).

[0142] Most preferably, the prokaryotic microorganism useful for producing the genetically modified microorganisms of the present invention and useful in the methods of the present invention is Escherichia coli.

[0143] The PTS carbohydrate utilization system (PTS) is a well-characterized carbohydrate transport system utilized by microorganisms such as bacteria. See Postma et al. 1993 (Postma PW, Lengeler JW, Jacobson G R. Phosphoenolpyruvate: carbohydrate phosphotransferase systems of bacteria. Microbiol Rev. 1993 September; 57(3): 543-94.) and Tchieu et al. 2001 (Tchieu JH, Norris V, Edwards JS, Saier MH Jr. The complete phosphotransferase system in Escherichia coli. J Mol Microbiol Biotechno. 2001 July; 3(3): 329-46), which are incorporated herein by reference in their entireties. Exemplary bacteria containing a PTS include those from the genera Bacillus, Clostridium, Enterobacteriaceae, Enterococcus, Erwinia, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Mycoplasma, Pasteurella, Rhodobacter, Rhodopseudomonas, Salmonella, Staphylococcus, Streptococcus, Vibrio, and Xanthomonas.Representative species include Escherichia coli, Salmonella typhimurium, Staphylococcus camosus, Bacillus subtilis, Mycoplasma capricolum, Enterococcus faecalis, Staphylococcus aureus, Streptococcus salivarius, Streptococcus mutans, Klebsiella pneumoniae, Staphylococcus carnosus, Streptococcus sanguis, and Rhodobacter capsulatus. These include: Vibrio capsulatus, Vibrio alginolyticus, Erwinia chrysanthemi, Xanthomonas campestris, Lactococcus lactis, Lactobacillus casei, Rhodoseudomonas sphaeroides, Erwinia carotovora, Pasteurella multocida, and Clostridium acetobutylicum.

[0144] Surprisingly, the inventors have found for the first time that reducing the abundance of Crr proteins results in an increase in the space-time yield, carbon substrate adaptability or carbon conversion efficiency of oligosaccharides produced by modified, preferably genetically modified, microorganisms.

[0145] Modified microorganisms, preferably genetically modified microorganisms, having microorganisms with reduced or absent abundance of Crr proteins can be achieved by many means, such as gene knockout or partial or complete deletion, reduction of crr gene expression including antisense or RNAi approaches, or other recombinant methods such as gene editing methods like CRISPR / CAS, or isolation of Crr proteins with aberrant binding partners, e.g., antibodies.

[0146] In one embodiment, the manipulation, preferably the reduction or complete elimination of Crr protein levels, is performed in an inducible manner, and the increase in space-time yield, carbon substrate adaptability, and / or carbon conversion efficiency is compared to the genetically modified microorganism without such induction. Methods for inducer-dependent gene expression, e.g., with the inducer isopropyl β-d-1-thiogalactopyranoside (IPTG), are known in the art.

[0147] In a preferred embodiment, the method of the present invention is a method for increasing the space-time yield of one or more fine chemicals produced by a microorganism, and increasing the carbon substrate adaptability and carbon conversion efficiency of the production of one or more fine chemicals by a microorganism, the method comprising the steps of providing a microorganism capable of producing one or more fine chemicals, inactivating or down-regulating in the microorganism a genetic locus of a gene corresponding to SEQ ID NO: 25 or a variant thereof, or inactivating or removing a protein corresponding to the Crr protein encoded by SEQ ID NO: 25 or a variant thereof, maintaining the genetically modified microorganism in an environment that allows the genetically modified microorganism to grow, growing the genetically modified microorganism in the presence of substrates and nutrients and under conditions suitable for the production of one or more fine chemicals, and optionally separating the one or more fine chemicals from the genetically modified microorganism or the remainder thereof.

[0148] The activity of a Crr protein, its mutant (variant), or a protein corresponding to a Crr protein in a microorganism should be understood as the normal biological function of the Crr protein or its mutant (variant), or a protein corresponding to a Crr protein. This can include, for example, kinase activity, since Crr proteins are known to contain a kinase domain. Inactivation should be understood in that the activity is not present at the same normal level, but is substantially lower or completely absent. Normal levels of abundance of these proteins of interest are required for normal biological function. If the abundance of the proteins of interest is substantially reduced, the biological function, and therefore the overall activity, will be reduced. If the protein of interest is absent, for example, because the gene encoding it is nonfunctional, partially deleted, completely deleted, knocked out, or its expression is prevented, the biological function will sooner or later disappear.

[0149] In a preferred aspect of the invention, host cells useful in the methods and uses of the invention have a deregulated adenylate cyclase of the invention in combination with reduced expression of the crr gene or variants thereof and / or inactivation or reduction of the Crr protein or variants thereof, relative to carbon conversion efficiency, carbon substrate suitability and space / time of prokaryotic production of oligosaccharides.

[0150] In one embodiment, the method of the invention comprises the step of inactivating or removing a Crr protein or an endogenous protein corresponding to the Crr protein in E. coli (SEQ ID NO: 26) as defined herein in the genetically modified microorganism prior to propagation of the genetically modified microorganism. The inactivation or removal of the CRR protein family member can be carried out before, simultaneously with, or after the deregulated adenylate cyclase is first present in the microorganism, i.e., before, simultaneously with, or after any of the following actions: (a) inactivating a regulatory activity found in wild-type adenylate cyclase in the host organism, and / or (b) generating a mutant adenylate cyclase in a host organism that lacks the regulatory activity found in wild-type adenylate cyclase; and / or (c) Introduction into a host organism of a mutant adenylate cyclase that lacks the regulatory activity found in wild-type adenylate cyclase.

[0151] Another preferred embodiment of the present invention is a composition comprising one or more host cells comprising a deregulated abundance and / or activity of adenylate cyclase and / or Crr protein (SEQ ID NO: 26), a mutant (variant) thereof, or an endogenous protein corresponding to the Crr protein in one or more microorganisms, which is reduced compared to a control host cell, i.e., a host cell having wild-type levels and activity of wild-type adenylate cyclase and / or Crr protein (SEQ ID NO: 26), a mutant (variant) thereof, or an endogenous protein corresponding to the Crr protein in said microorganism. In a more preferred embodiment, the composition of the present invention further comprises one or more fine chemicals, preferably one or more human milk oligosaccharides.

[0152] Preferably, the host cell or genetically modified microorganism producing 2'-fucosyllactose (2'-FL) of the invention and useful in the method of the invention is an Escherichia coli strain and comprises at least: -1,2-fucosyltransferase enzyme, and A means of providing a fucose moiety and lactose to a fucosyltransferase enzyme suitable for the production of -2′-FL.

[0153] Preferably, the host cell or genetically modified microorganism producing the 6'-sialyllactose (6'-SL) of the present invention and useful in the method of the present invention is an Escherichia coli strain and comprises at least: a sialyltransferase enzyme, and A means of providing a sialic acid moiety and lactose to a sialyltransferase enzyme suitable for the production of -6'-SL.

[0154] Preferably, the host cell or genetically modified microorganism producing lacto-N-tetraose (LNT) of the invention and useful in the methods of the invention is an E. coli strain and comprises at least: -β1,3-galactosyltransferase enzyme, and - A means of providing nucleotide-activated galactose and LNT2 to a β1,3-galactosyltransferase enzyme suitable for the production of LNT.

[0155] Cultivation of host cells or microorganisms often requires that the cells be cultured in a medium containing a carbon source, a nitrogen source, and sources of various nutrients required for the growth of the cells, including, but not limited to, amino acids, vitamins, minerals, etc. The fermentation medium can be a minimal medium as described in WO 98 / 37179, or the fermentation medium can be a complex medium containing complex nitrogen and carbon sources, where the complex nitrogen source can be partially hydrolyzed, as described in WO 2004 / 003216.

[0156] That is, the fermentation medium contains components necessary for the growth of the cultured microorganism or host cell. In one embodiment, the fermentation medium contains one or more components selected from the group consisting of a nitrogen source, a phosphorus source, a sulfur source, and salts, and optionally one or more additional components selected from the group consisting of micronutrients such as vitamins, amino acids, minerals, and trace elements. In one embodiment, the fermentation medium also contains a carbon source. Such components are generally well known in the art (see, e.g., Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, 1989 Cold Spring Harbor, NY; Talbot, Molecular and Cellular Biology of Filamentous Fungi: A Practical Approach, Oxford University Press, 2001; Kinghom and Turner, Applied Molecular Genetics of Filamentous Fungi, Cambridge University Press, 1992; and Bacillus (Biotechnology Handbooks) by Colin R. Harwood, Plenum Press, 1989). Culture conditions for a given cell type can also be found in the scientific literature and / or from cell sources such as the American Type Culture Collection (ATCC) and the Fungal Genetics Stock Center.

[0157] As nitrogen sources, inorganic and organic nitrogen compounds can be used, both individually and in combination. Suitable organic nitrogen sources include, but are not limited to, protein-containing substances such as extracts derived from microbial, animal, or plant cells, including, but not limited to, plant protein preparations, soybean flour, corn flour, pea flour, corn gluten, cotton flour, peanut flour, potato flour, meat and casein, gelatin, whey, fish meal, yeast protein, yeast extract, tryptone, peptone, bactotryptone, bactopeptone, waste products derived from the processing of microbial cells, plants, meat, or animal parts, and combinations thereof. Inorganic nitrogen sources include, but are not limited to, ammonium, nitrate, and nitrite, and combinations thereof. In one embodiment, the fermentation medium contains a nitrogen source, wherein the nitrogen source is a complex nitrogen source, a defined nitrogen source, or a combination thereof. In one embodiment, the complex nitrogen source is selected from the group consisting of plant proteins, including but not limited to potato protein, soy protein, corn protein, peanut protein, cotton protein, and / or pea protein, casein, tryptone, peptone, and yeast extract, and combinations thereof. In one embodiment, the defined nitrogen source is selected from the group consisting of ammonia, ammonium, ammonium salts (e.g., ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, ammonium acetate), urea, nitric acid, nitrates, nitrite, and amino acids (including but not limited to glutamic acid), and combinations thereof.

[0158] In one embodiment, the fermentation medium further comprises at least one carbon source. The carbon source can be a complex carbon source, a defined carbon source, or a combination thereof. Various sugars and sugar-containing substances are suitable carbon sources, and sugars may be present at various stages of polymerization. Complex carbon sources include, but are not limited to, molasses, corn steep liquor, cane sugar, dextrin, starch, starch hydrolysates, and cellulose hydrolysates, as well as combinations thereof. Defined carbon sources include, but are not limited to, carbohydrates, organic acids, and alcohols. In one embodiment, defined carbon sources include, but are not limited to, glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, lactose, gluconate, acetate, propionate, lactic acid, formate, malate, citric acid, fumarate, glycerol, inositol, mannitol, and sorbitol, as well as combinations thereof. In one embodiment, the defined carbon source is provided in the form of a syrup, which may contain up to 20%, up to 10%, or up to 5% impurities. In one embodiment, the carbon source is sugar beet syrup, sugar cane syrup, or corn syrup (including but not limited to high fructose corn syrup). Complex carbon sources include, but are not limited to, molasses, corn steep liquor, dextrin, and starch, or combinations thereof. In a preferred embodiment, the defined carbon source includes, but is not limited to, glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, dextrin, lactose, gluconate, or combinations thereof.

[0159] In another preferred embodiment, one carbon source or carbon sources is sucrose, and with this carbon source the methods of the invention and the host cells or genetically modified microorganisms of the invention offer even greater advantages compared to organisms and methods known in the art.

[0160] In one embodiment, the fermentation medium also includes a phosphorus source, including but not limited to, phosphate, and / or a sulfur source, including but not limited to, sulfate. In one embodiment, the fermentation medium also includes a salt. In one embodiment, the fermentation medium includes one or more inorganic salts, including but not limited to, alkali metal salts, alkaline earth metal salts, phosphates, and sulfates. In one embodiment, the one or more salts include, but are not limited to, NaCl, KH2PO4, MgSO4, CaCl2, FeCl3, MgCl2, MnCl2, ZnSO4, Na2MoO4, and CuSO4. In one embodiment, the fermentation medium also includes one or more vitamins, including but not limited to, thiamine chloride, biotin, and vitamin B12. In one embodiment, the fermentation medium also includes trace elements, including but not limited to, Fe, Mg, Mn, Co, and Ni. In one embodiment, the fermentation medium includes one or more salt cations selected from the group consisting of Na, K, Ca, Mg, Mn, Fe, Co, Cu, and Ni. In one embodiment, the fermentation medium includes one or more divalent or trivalent cations, including but not limited to Ca and Mg.

[0161] In one embodiment, the fermentation medium also includes an antifoaming agent.

[0162] In one embodiment, the fermentation medium also contains a selection agent, including but not limited to, an antibiotic (including but not limited to, ampicillin, tetracycline, kanamycin, hygromycin, bleomycin, chloramphenicol, streptomycin, or phleomycin) or a herbicide to which the selectable marker of the cells provides resistance.

[0163] Fermentation can be carried out as a batch, repeated batch, fed-batch, repeated fed-batch, or continuous fermentation process. In a fed-batch process, before the start of fermentation, no or only a portion of a compound containing one or more structural and / or catalytic elements, such as a carbon source or nitrogen source, is added to the medium, and all or the remaining portion of the compound containing one or more structural and / or catalytic elements, respectively, is supplied during the fermentation process. The compounds selected for supply can be supplied to the fermentation process together or separately from each other. In a repeated fed-batch or continuous fermentation process, a complete starting medium is additionally supplied during fermentation. The starting medium can be supplied together with or separately from the feed. In a repeated fed-batch process, a portion of the fermentation broth containing biomass is removed at regular time intervals, while in a continuous process, a portion of the fermentation broth is continuously removed. Thereby, the fermentation process is replenished with a portion of fresh medium corresponding to the amount of fermentation broth removed.

[0164] Many cell cultures incorporate a carbon source, such as glucose, as a substrate feed in the cell culture during fermentation. Thus, in one embodiment, the method for culturing a microorganism includes a feed comprising a carbon source. The carbon source-containing feed can include a defined carbon source or a complex carbon source, or a mixture thereof, as described in detail herein.

[0165] Fermentation time, pH, conductivity, temperature, or other specific fermentation conditions can be adapted according to standard conditions known in the art, hi one embodiment, the fermentation conditions are adjusted to obtain maximum yield of the protein of interest.

[0166] In one embodiment, the temperature of the fermentation broth during fermentation is between 30°C and 45°C.

[0167] In one embodiment, the pH of the fermentation medium is adjusted to pH 6.5-9.

[0168] In one embodiment, the conductivity of the fermentation medium, after pH adjustment, is between 0.1 and 100 mS / cm.

[0169] In one embodiment, the fermentation time is from 1 to 200 hours.

[0170] In one embodiment, the fermentation is carried out while stirring and / or shaking the fermentation medium, hi one embodiment, the fermentation is carried out while stirring the fermentation medium at 50 to 2000 rpm.

[0171] In one embodiment, oxygen is added to the fermentation medium during cultivation, including but not limited to by stirring and / or agitation or by aeration (including but not limited to aeration with 0-3 bar air or oxygen). In one embodiment, the fermentation is carried out under oxygen saturation.

[0172] In one embodiment, the fermentation medium and methods using the fermentation medium are for industrial scale fermentation, hi one embodiment, the fermentation medium of the present description may be useful for any fermentation having at least 20 liters, at least 50 liters, at least 300 liters, or at least 1000 liters of culture medium.

[0173] In one embodiment, the fermentation method is for the production of a protein of interest in relatively high yields, including, but not limited to, a protein of interest expressed in an amount of at least 2 g protein (dry matter) / kg raw fermentation medium, at least 3 g protein (dry matter) / kg raw fermentation medium, at least 5 g protein (dry matter) / kg raw fermentation medium, at least 10 g protein (dry matter) / kg raw fermentation medium, or at least 20 g protein (dry matter) / kg raw fermentation medium.

[0174] In preferred embodiments, the space-time yield, carbon substrate adaptability, and / or carbon conversion efficiency in producing one or more fine chemicals, preferably one or more oligosaccharides, is increased by at least 20%, 30%, 40%, 50%, 60%, 65%, or 70% compared to a control, i.e., a host cell in which cAMP levels are not significantly altered and which has an adenylate cyclase that is subject to regulatory activity and / or which has an unaltered abundance and / or activity of Crr protein (SEQ ID NO: 26), a variant thereof, or an endogenous protein(s) corresponding to the Crr protein.

[0175] Preferably, the increase in cAMP levels is understood to be at least 5%, preferably at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, compared to levels in an unmodified host cell, e.g., a host cell having only adenylate cyclase under normal regulation and no deregulated adenylate cyclase, and / or having the normal crr gene locus in E. coli or the normal locus and corresponding protein of the endogenous gene corresponding to the crr gene at wild-type levels of abundance or activity. For example, an engineered microorganism (engineered to reduce CRR protein levels) has its cAMP levels compared to the cAMP levels of an unmodified microorganism. In another preferred embodiment, the cAMP level of a host organism capable of producing one or more fine chemicals, preferably one or more oligosaccharides, is increased by 1.1, 1.2, 1.25, 1.3, 1.4, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to the normal level of the host organism. The cAMP level of the host organism is understood to be preferably the intracellular cAMP level, and more preferably the cytoplasmic cAMP level, of the host organism. The cAMP level can be determined as disclosed herein above.

[0176] A further preferred embodiment is to use a deregulated adenylate cyclase and / or to inactivate and / or reduce the abundance of Crr protein (SEQ ID NO: 26), a variant thereof, or endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26 in order to increase the space-time yield, carbon substrate adaptability, and / or carbon conversion efficiency in the production of one or more fine chemicals by a host organism according to the invention.

[0177] Further embodiments relate to methods of the invention or host cells of the invention, wherein the activity and / or abundance of the Crr protein (SEQ ID NO: 26), a variant thereof, or endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26 is reduced by more than 15% or 20%, more preferably 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 94%, 95%, or 98% compared to a control, i.e., a cell having wild-type levels of activity and / or abundance of the endogenous protein(s) corresponding to the Crr protein (SEQ ID NO: 26), a variant thereof, or the Crr protein of SEQ ID NO: 26. [Brief explanation of the drawings]

[0178] [Figure 1] FIG. 1 shows a diagrammatic representation of various DNA protein sequences of different lengths useful in the methods and host cells of the present invention. [Figure 2-1] Part 1) shows an alignment of the DNA sequences of SEQ ID NOs: 1-8 and 10. The lengths of the different truncated cyaA DNA sequences are shown compared to the longest variant of the full-length gene. Part 2) shows an alignment of the protein sequences of SEQ ID NOs: 11-18 and 20. The lengths of the different truncated CyaA protein sequences are shown compared to the longest variant of the full-length protein. In comparison, the slightly shorter full-length wild-type protein of SEQ ID NO: 19 has only one GEQSMI motif instead of the duplicated GEQSMIGEQSMI of the 854 variant of full-length adenylate cyclase (underlined in Figure 2, part 2). [Figure 2-2] (Continued from Figure 2) [Figure 2-3] (Continued from Figure 2) [Figure 2-4] (Continued from Figure 2) [Figure 2-5] (Continued from Figure 2) [Figure 2-6] (Continued from Figure 2) [Figure 2-7] (Continued from Figure 2) [Figure 2-8] (Continued from Figure 2) [Figure 2-9] (Continued from Figure 2) [Figure 2-10] (Continued from Figure 2) [Figure 3] FIG. 1 depicts representative constructs for creating 2′FL-producing E. coli strains. [Figure 4A] A shows the first construct introduced to create a 6'-SL-producing E. coli strain. The top diagram shows the construct in a strain without an engineered yaA, and the bottom diagram shows the construct in a strain with a deregulated CyaA. [Figure 4B] B is a diagram depicting the second construct used to create a 6'-SL-producing E. coli strain: the top diagram is the construct in a strain without an engineered CyaA, and the bottom diagram is the construct in a strain with a deregulated CyaA. [Figure 5] FIG. 1 is a diagram depicting the crr locus after deletion of most of the crr gene, as described in detail in the Examples below. DETAILED DESCRIPTION OF THE INVENTION

[0179] I. A method for increasing the 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 the host organism, and / or the carbon substrate adaptability for the production of one or more fine chemicals by the host organism by providing a deregulated adenylate cyclase protein and / or inactivating and / or reducing the abundance of Crr protein (SEQ ID NO: 26), a variant thereof, or an 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 adaptability are increased in the modified host organism compared to an unmodified host organism.

[0180] II. A method for increasing the carbon substrate adaptability of the production of one or more fine chemicals by a host organism, wherein cAMP levels in the host organism are increased compared to an unmodified host organism.

[0181] III. A method for increasing the carbon conversion efficiency of the production of one or more fine chemicals by a host organism, wherein cAMP levels in the host organism are increased compared to an unmodified host organism.

[0182] 1. A method for increasing 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, comprising the steps of increasing the adenosine 3',5'-cyclic monophosphate (cAMP, CAS number: 60-92-4) level of the host organism compared to an unmodified host organism, maintaining the host organism under conditions that allow its growth, growing the host organism in the presence of a substrate under conditions suitable for the production of one or more fine chemicals, and optionally separating the one or more fine chemicals from the host organism or the remainder thereof.

[0183] 2. A method for increasing the carbon substrate adaptability for the production of one or more fine chemicals by a host organism suitable for the production of one or more fine chemicals, comprising the steps of increasing cAMP levels in the host organism compared to an unmodified host organism, maintaining the host organism under conditions that allow its growth, growing the host organism in the presence of a substrate under conditions suitable for the production of one or more fine chemicals, and optionally isolating the one or more fine chemicals from the host organism or the remainder thereof.

[0184] 3. A method for increasing the carbon conversion efficiency in the production of one or more fine chemicals by a host organism suitable for the production of one or more fine chemicals, comprising the steps of increasing the cAMP level in the host organism compared to an unmodified host organism, maintaining the host organism in conditions that allow its growth, growing the host organism in the presence of a substrate under conditions suitable for the production of one or more fine chemicals, and optionally separating the one or more fine chemicals from the host organism or the remainder thereof.

[0185] 4. The cAMP level of the host organism is a. inactivating the regulatory activity found in wild-type adenylate cyclase, and / or b. generating mutant adenylate cyclases that lack the regulatory activity found in wild-type adenylate cyclases; and / or c. introducing into the host organism a mutant adenylate cyclase that lacks the regulatory activity found in wild-type adenylate cyclase; and / or d. reducing the activity of an enzyme having the activity of 3',5' cAMP phosphodiesterase (EC 3.1.4.53); and / or e. using the adenylate cyclase toxin of Bordetella pertussis, or its adenylate cyclase domain, or a variant thereof; and / or f. Inactivating and / or reducing the abundance of Crr protein (SEQ ID NO: 26), a variant thereof, or the endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26 10. The method of any preceding embodiment, wherein the number of cells in the blood is increased by:

[0186] 5. The method of any of the preceding embodiments, wherein the cAMP levels of the host organism are increased in an inducible manner, the increase being relative to an uninduced host organism.

[0187] 6. The method of any of the preceding embodiments, wherein the mutant adenylate cyclase is introduced by introduction of a transgene.

[0188] 7. The method of any of the preceding embodiments, wherein the mutant adenylate cyclase or the adenylate cyclase with inactivated regulatory activity has a deletion compared to a wild-type form of adenylate cyclase of the host organism.

[0189] 8. The method of embodiment 7, wherein the deletion removes a regulatory portion of adenylate cyclase without destroying the portion that produces cAMP.

[0190] 9. The deletion is a deletion of a regulatory part of a protein corresponding to the C-terminal part of the adenylate cyclase encoded by the E. coli cyaA gene, preferably corresponding to the C-terminal part of the cyaA protein shown in SEQ ID NO: 19 or 20, or an adenylate cyclase protein having at least 80% sequence identity with positions 1 to 412, preferably positions 1 to 420, of the protein sequence shown as SEQ ID NO: 19, preferably the deletion is a deletion of a regulatory part of a protein corresponding to the C-terminal part of the cyaA protein shown in SEQ ID NO: 19 or 20 9. The method of embodiment 7 or 8, wherein the deletion is in a regulatory part of the protein corresponding to the part of E. coli adenylate cyclase following position 420, 450, 558, 582, 585, 653, 709, 736 or 776 of the protein sequence provided in SEQ ID NO: 19 or 20, more preferably following position 558, 582, 585, 653, 709, 736 or 776 of the protein sequence provided in SEQ ID NO: 19 or 20, and most preferably a deletion of amino acids corresponding to amino acids from position 777 onwards of SEQ ID NO: 19 or 20.

[0191] 10. The method of any of the preceding embodiments, comprising providing a carbon source to the host organism, wherein the carbon source is a complex, or defined carbon source, or a combination thereof.

[0192] 11. A modified host cell suitable for the production of fine chemicals, wherein the host cell is capable of growing 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 adenylate cyclase activity, an adenylate cyclase with inactivated or absent regulatory activity, and / or a Crr protein (SEQ ID NO: 26), a variant thereof, or an inactivated and / or reduced abundance of endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26, and wherein the host organism has increased cAMP levels compared to an unmodified host cell, wherein the unmodified host cell is substantially unable to grow on glycerol and / or glucose and / or maltose and / or fructose and / or sucrose.

[0193] 12. At least one adenylate cyclase protein corresponding to the protein encoded by the cyaA gene of Escherichia coli lacks regulatory activity, preferably lacking a portion corresponding to the C-terminal portion of the cyaA protein set forth in SEQ ID NO: 19 or 20, or an adenylate cyclase protein having at least 80% sequence identity with positions 1 to 412 of the protein set forth in SEQ ID NO: 19 or 20, more preferably an adenylate cyclase protein having at least 80% sequence identity with positions 1 to 420, preferably a protein provided in SEQ ID NO: 19 or 20. 12. The modified host cell of embodiment 11, wherein the modified host cell lacks a portion of an adenylate cyclase corresponding to the portion of E. coli adenylate cyclase following position 420, 450, 558, 585, 653, 709, 736, or 776 of the protein sequence provided in SEQ ID NO: 19 or 20, more preferably following position 450, 558, 585, 653, 709, or 736 of the sequence, even more preferably following position 558, 582, 585, 653, 709, 736, or 776 of the protein sequence provided in SEQ ID NO: 19 or 20, and most preferably comprising a deletion of amino acids corresponding to amino acids at position 777 and beyond of SEQ ID NO: 19 or 20.

[0194] 13. Any of the preceding embodiments, wherein the host cell is a bacterial or fungal host cell, preferably a bacterial cell, more preferably a bacterial cell, even more preferably a Gram-negative cell, most preferably an E. coli cell.

[0195] 14. Use of deregulated adenylate cyclase and / or inactivation and / or reduction in abundance of Crr protein (SEQ ID NO: 26), a variant thereof, or endogenous protein(s) corresponding to the Crr protein of SEQ ID NO: 26 to increase the space-time yield, carbon substrate adaptability, and / or carbon conversion efficiency of the production of one or more fine chemicals by a host organism.

[0196] 15. Any of the preceding embodiments, wherein the at least one fine chemical is a human breast 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 a method according to any of the preceding embodiments, comprising the step of providing a carbon source to the host organism, wherein the carbon source is one or more of the following: a complex or 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.

[0197] 16. A method for producing oligosaccharides by conversion of a carbon source in a fermentation process, comprising: Cultivating the genetically modified microorganism to produce oligosaccharides in a suitable culture medium containing at least one carbon source; recovering human breast milk oligosaccharides from the culture medium; the genetically modified microorganism comprises a functional gene encoding a PTS carbohydrate utilization system; and The method, wherein the genetically modified microorganism has a reduced abundance of Crr protein (SEQ ID NO: 26), a variant thereof, or an endogenous protein corresponding to the Crr protein of SEQ ID NO: 26, and / or a deregulated adenylate cyclase as defined in any of the previous embodiments is present in the microorganism.

[0198] 17. Any of the preceding embodiments, wherein the carbon source is selected from the group consisting of glycerol, monosaccharides, and disaccharides.

[0199] 18. Any of the preceding embodiments, wherein the level of adenosine 3',5'-cyclic monophosphate (cAMP, CAS Number: 60-92-4) is increased compared to a microorganism that does not have an alteration in the Crr protein (SEQ ID NO: 26), a variant thereof, or an endogenous protein corresponding to the Crr protein of SEQ ID NO: 26.

[0200] 19. A genetically modified microorganism for enhanced production of fine chemicals, the microorganism having the ability to produce human breast milk oligosaccharides and comprising a functional gene encoding a PTS carbohydrate utilization system, wherein expression of a Crr protein is reduced, preferably at least substantially reduced, in the genetically modified microorganism.

[0201] 20. The microorganism described in embodiment 19, wherein the gene encoding the Crr protein is attenuated or deleted in the genetically modified microorganism.

[0202] 21. The microorganism of any of the preceding embodiments, wherein the microorganism is selected from the group consisting of Enterobacteriaceae.

[0203] [Example] In the examples below, E. coli strains containing replicating vectors and / or various chromosomal deletions and substitutions were constructed using homologous recombination, as fully described for E. coli by Datsenko and Wanner (2000), using methods well known in the art. Similarly, the use of plasmids or vectors to express or overexpress one or several genes in recombinant microorganisms is also well known to those skilled in the art.

[0204] method Introduction of a DNA construct or vector into a host cell can be accomplished using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection- or DEAE-dextrin-mediated transfection, or transfection using recombinant phage viruses), incubation with calcium phosphate DNA precipitates, 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, each of which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods).Expression of heterologous polypeptides in Trichoderma, particularly with respect to transformation and expression methods, 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 Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY pp. 129-130, each of which is hereby incorporated by reference in its entirety. 148, 1992. For transformation of Aspergillus strains, see also Cao et al. (Sd. 9:991-1001, 2000; EP 238023; and Yelton et al., Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984, each of which is hereby incorporated by reference in its entirety, particularly with regard to transformation methods. The introduced nucleic acid can be integrated into chromosomal DNA or can be maintained as an extrachromosomal replicating sequence.

[0205] Examples of cAMP increase and deregulation of adenylate cyclase activity 1. Creation of a truncated cyaA DNA construct Shortened DNA cyaA constructs were prepared by generating synthetic DNA constructs with homology for integration and by introducing a TAA stop codon into the coding sequence of the cyaA gene by gene synthesis. These gene constructs were then introduced into the genome of E. coli strains by homologous recombination as described in Wang J, et al. 2006, Mol. Biotechnol., 32, 43.

[0206] 2. Strain Construction Genetically modified microorganisms with enhanced production of oligosaccharides (e.g., HMOs) are disclosed in patent applications published as WO 2016 / 008602, WO 2013 / 182206, EP 2379708, U.S. Pat. No. 9,944,965, WO 2012 / 112777, WO 2001 / 04341, and US 2005019874, all of the disclosures of which are incorporated herein by reference.

[0207] 2'-FL producing microorganisms An E. coli strain overproducing 2'-FL was constructed as follows. That is, in the well-characterized E. coli strain JM109, an artificial operon containing the following genetic elements: a PTAC promoter, an artificial ribosome binding site (RBS), the fucT2 gene (derived from H. pylori strain 26695; Wang et al., Mol. Microbiol. 1999, 31 1265-1274), an artificial ribosome binding site, the gmd gene (derived from E. coli K12), the wcaG gene (derived from E. coli K12) with its authentic ribosome binding site, an artificial ribosome binding site (RBS), the manC gene (derived from E. coli K12) with optimized codon usage, an artificial ribosome binding site (RBS), the manB gene (derived from E. coli K12) with optimized codon usage, and the transcription terminator rrnBT1 derived from the 16s rRNA locus of E. coli was transformed using well-known lambda red technology (e.g., Datsenko et al., J. Microbiol. 1999, 31 1265-1274). and Wanner B. PNAS, 2000 97 (12) 6640-6645, Wang J, et al. 2006, Mol. Biotechnol., 32, 43. The artificial operon was integrated into the fuc locus of E. coli, in which genes including fuc I and K had been deleted.

[0208] A representative construct for creating a 2'FL producing strain is presented as SEQ ID NO: 21. The truncated adenylate cyclase gene sequences of SEQ ID NOs: 1-8 were introduced into E. coli host cells by homologous recombination using lambda Red technology. A representative construct for creating a 2'FL producing strain is presented as SEQ ID NO: 21.

[0209] 6'-SL producing microorganisms An E. coli strain overproducing 6'-SL was constructed as follows: In the well-characterized E. coli strain W3110, the lacZ gene encoding the β-galactosidase LacZ, the lacA gene encoding the acetyltransferase LacA, and the nan gene encoding the nanAETK gene were deleted using the well-known lambda Red technology (e.g., Datsenko I and Wanner B. PNAS, 2000 97 (12) 6640-6645; Wang J, et al. 2006, Mol. Biotechnol., 32, 43), with the lacI allele replaced by the known lacIq allele. An artificial operon (see SEQ ID NO: 22) was inserted immediately adjacent to the atoB gene of the W3110 strain. The artificial operon contained the following genetic elements: a PTAC promoter, an artificial ribosome binding site (RBS), the St6 gene (derived from Photobacterium spp. ISH224), an artificial ribosome binding site, the neuA gene (derived from Campylobacter jejuni ATCC43438), an artificial ribosome binding site (RBS), a zeocin resistance gene, and the transcription terminator rrnBT1 derived from the 16s rRNA locus of E. coli. Additionally, the artificial operon was inserted immediately adjacent to the fabI gene. The artificial operon contained a PTAC promoter, an artificial ribosome binding site (RBS), the neuB gene (derived from Campylobacter jejuni ATCC 43438, see SEQ ID NO: 23), an artificial ribosome binding site, the neuC gene (derived from Campylobacter jejuni ATCC 43438, see SEQ ID NO: 24), an artificial ribosome binding site (RBS), a chloramphenicol resistance cassette (CAT), and a transcription terminator rrnB derived from the 16s rRNA locus of E. coli. This 6'-SL producing strain was designated GN488.

[0210] Another E. coli strain, designated GN782, was constructed based on the GN488 strain. The resistance genes Zeocin and CAT were deleted from the artificial operon in the GN488 strain genome, again using lambda Red technology. In addition, cyaA was modified so that a stop codon was introduced at codon 582, resulting in a translated protein with a length of 581 amino acids.

[0211] 3. Deregulated adenylate cyclase: space-time yield in HMO production Fermentation system and procedure Fermentation conditions: The fermentation medium was selected based on the described examples of E. coli fermentation and can be found in (Riesenberg et al. (1991), Journal of Biotechnology 20, 17-27, DJ Korz, et al. 1995), J. Biotechnol., 39 pp. 59-65, Biener, R. et al. 2010, Journal of Biotechnology 146(1-2), pp. 45-53. In particular, the medium for producing oligosaccharides was based on lactose, since lactose was added at different concentrations ranging from 20 to 100 g / l depending on the experiment.

[0212] When analyzing the performance of the strains in terms of carbon conversion efficiency and space-time yield, the following systems were used: an AMBR® 250 system and a 4 L Biostat® fermenter (both manufactured by Sartorius AG, Otto-Brenner-Str. 20, D-37079 Göttingen, Germany). In general terms, fermentations were typically carried out as follows: seed cultures were grown from frozen stocks. The seed cultures were inoculated into the respective fermentation systems (AMBR or Biostat) and then allowed to fully utilize their carbon content. Alternatively, the main cultures were started directly from frozen stocks. Fermentations in the fermentation systems were carried out in a fed-batch mode, i.e., in a two-stage mode (a first stage in which a batch amount of carbon source was utilized, and a subsequent stage in which the carbon source was supplied throughout the fermentation under fermentation conditions in which no or only small amounts of carbon source accumulated in the fermentation broth).

[0213] A seed culture (minimal medium containing 10 ml / L of trace element solution and 65 g / L of glycerol) is inoculated with 1 ml of WCB culture (stored frozen).

[0214] Transfer the seed culture to the main culture so that an inoculation volume ratio of 1-10% is applied.

[0215] The main fermentation medium consisted of the following medium composition: Minimal medium: 1.1 g / L citric acid, 10.8 g / L glycerol, 15.5 g / L KH2PO4, 4.6 g / L (NH4)2SO4, 3 g / L Na2SO4, 1.5 g / L MgSO4*7H2O, 0.02 g / L thiamine, 0.0001 g / L vitamin B12, 0.5 mM IPTG. The trace element solution is composed of 4g / L Na2-EDTA*2H2O, 1g / L CaSO4*2H2O, 0.3g / L ZnSO4*7H2O, 3.7g / L FeSO4*7H2O, 0.2g / L MnSO4*H2O, 0.15g / L CuSO4*5H2O, 0.04g / L Na2MoO4*2H2O, and 0.04g / L Na2SeO4. The trace metal solution is applied in an amount of 30ml / L in the fermentation medium.

[0216] After inoculation, fermentation is initiated, and when the measured CTR exceeds 40 mmol / Lh, the feeding of a carbon source, such as glycerol (86% w / w concentration) or glucose (60% w / w concentration), is initiated. The carbon source feed rate can vary between 2 and 8 g / L of carbon source per liter of initial fermentation broth volume per hour. It is important to ensure that the carbon source does not accumulate throughout the fermentation process. During the main fermentation phase, the dissolved oxygen concentration (pO2) is controlled to >20% by controlling agitation and gas addition. The pH is maintained between 6.1 and 6.9, more specifically, at 6.7, using base NH4OH in 15% aqueous NH4OH solution. The results in both fermentation systems were found to be completely superimposable for the described parameters (carbon conversion efficiency and space-time yield) and can be considered completely interchangeable.

[0217] Surprisingly, cAMP-overproducing cells carrying a truncated cyaA gene, resulting in a functional, deregulated CyaA protein, grew reliably on glycerol and efficiently produced 2'-fucosyllactose (2'-FL). In contrast, a cyaA deletion mutant lacking a functional adenylate cyclase (from the Keio collection, Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2: 2006 0008) was found to be unable to grow on glycerol. Unmodified E. coli cells with adenylate cyclase containing a regulatory moiety grow more slowly than host cells in which adenylate cyclase is deregulated, thereby increasing cAMP production, and the unmodified cells also produce less 2'-FL, and the carbon conversion efficiency and space / time yield are also reduced compared to host cells in which adenylate cyclase is deregulated, thereby increasing cAMP production.

[0218] 2'-FL

[0219] [Table 2]

[0220] Generally, when BioStat® and AMB® vessels were used, the carbon source was added continuously or repeatedly. In principle, a representative amount of glucose or glycerol can be added once at the beginning of the main culture, which is advantageous, for example, when shake flasks are used for fermentation. When glucose or glycerol was used as the carbon source for strains with a deregulated cyaA gene and therefore also increased cAMP levels, the space-time yield increased.

[0221] [Table 3]

[0222] Similar results were achieved with an E. coli strain that produces 6'-sialyllactose instead of 2'-FL, see Examples 1 and 2 above.

[0223] 4. Increasing carbon source adaptability of 2'-FL-producing strains The carbon source was batch-dosed into the medium and fed in the feed phase for periods ranging from 2 to 100 hours. The carbon source was applied in pure form (e.g., glycerol) or diluted in water (glycerol and other carbon sources). The carbon source feed rate was adjusted to suit the agitation and aeration conditions of the fermenter.

[0224] Samples were taken during the course of the fermentation and analyzed by isocratic HPLC elution.

[0225] Carbon source adaptability analysis for 2'-FL production was carried out using the following medium composition: Dispense 20 mL of medium (10 g / L of each carbon source, 5 g / L lactose, 1 g / L (NH)2H-citric acid, 2 g / L Na2SO4, 2.68 g / L (NH4)2SO4, 0.5 g / L NH4Cl, 14.6 g / L K2HPO4, 4 g / L NaH2PO4*H2O, 0.5 g / L MgSO4*7H2O, 10 g / mL MnSO4, and 3 mL of trace metal solution (8.0 g / L Na2-EDTA*2H2O, 1 g / L CaSO4*2H2O, 0.3 g / L ZnSO4*7H2O, 7.4 g / L (NH4)2Fe(SO4)2, 0.2 g / L MnSO4*H2O, 0.15 g / L A medium (consisting of 0.04 g / L CuSO4*5H2O, 0.04 g / L Na2MoO4*2H2O, 0.04 g / L Na2SeO4, 10 mg / L thiamine*HCl, 0.1 mg / L vitamin B12, 1 mM IPTG, pH 7.0) was inoculated with an overnight culture of the 2'-FL producing strain (in the above medium without lactose and IPTG) as in Example 2 at a starting OD of 0.5 and incubated at 200 rpm, 37°C for 24 hours in the above medium with lactose and IPTG as above. Samples were taken and analyzed for carbon utilization and product formation. The carbon source was selected from the following list: Glucose, glycerol, mannose, fructose,

[0226] [Table 4]

[0227] 5. 6'-sialyllactose (6'-SL) producing strain Strains GN488 and GN782 from Example 2 were grown in Biostat® vessels containing medium as described in Example 3.

[0228] [Table 5]

[0229] The results demonstrate the broad applicability of deregulated adenylate cyclase for increasing cAMP levels, as the surprising effects on carbon conversion efficiency and space-time yield hold true for other HMO-producing strains and yet another version of the deregulated CyaA protein, corresponding to amino acids 1-581 of the full-length 848-amino acid CyaA protein (SEQ ID NO: 19), was successfully used. Furthermore, when a strain harboring the cyaA585 version of the protein (SEQ ID NO: 14) was tested, the space-time yield of 6'-SL was similarly increased relative to a strain with the unmodified CyaA protein.

[0230] 6. cAMP Feed Experiment E. coli strains from the Keio collection (Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2: 2006 0008) lacking the cyaA gene exhibit poor growth on glycerol as a carbon source. The strains grow in the presence of glycerol and cAMP, and growth of the deleted strains is improved. The 2'-FL-producing host cells with the truncated adenylate cyclase from Examples 1 and 2 above exhibit increased 2'-FL production on media containing glycerol compared to cells with only the unmodified cyaA gene. If cells with only the unmodified cyaA gene are supplied with cAMP, 2'-FL production increases.

[0231] Examples of altered cAMP signaling and PTS [Example 7] Construction of a strain overproducing 2'-FL A 2'-FL overproducing E. coli strain harboring wild-type adenylate cyclase and wild-type crr genes was constructed as described in Example 2 above.

[0232] Construction of an overproducing strain with a deletion of the crr gene A 2'-FL-overproducing strain of E. coli with a deletion of the crr gene was constructed as follows: Using 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, the native full-length crr gene in the 2'FL-producing strain was replaced with a gene construct consisting of the 50-bp 5' coding region of crr starting from the transcription start site, an FRT site obtained from an FLP recombination event, and the crr gene (50 bp) ending with the TAA sequence of the translation termination codon. The resulting gene (SEQ ID NO: 29) therefore lacks 410 bp of its coding region and therefore does not encode an active Crr protein. Deletion of the crr gene was confirmed using primers shown in SEQ ID NOs: 3 and 4.

[0233] [Example 8] Construction of a 6'SL-producing strain with a deletion of the crr gene The 6'-SL overproducing strain GN488 was created as described above in Example 2 and used for further engineering, in which deletion of the crr gene (SEQ ID NO: 1) in the E. coli strain was achieved by P1 viral transduction followed by selection on kanamycin-containing agar plates.

[0234] P1 lysate was prepared from 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 transductants were selected on agar plates containing kanamycin. Colonies were screened by PCR using primers selective for the upstream and downstream regions of crr to confirm the deletion of crr. Colonies with the expected band size indicate correct deletion of the crr gene.

[0235] Deletion of the crr gene (SEQ ID NO: 1) in E. coli strains was achieved by P1 viral transduction (Miller, JH 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, NY) followed by selection on kanamycin-citrate containing agar plates.

[0236] P1 lysate was prepared from 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 Examples 1 and 2 (2'-FL and 6'-SL strains, respectively), and 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 Ec 6'-SL delta crr.

[0237] [Example 9] Increasing space-time yield in HMO production The fermentation conditions, system, and procedures were as described above in Example 3 above.

[0238] [Table 6]

[0239] Generally, when using BioStat® and AMB® vessels, the carbon source was added continuously or repeatedly. In principle, a representative amount of glucose or glycerol can be added once at the beginning of the main culture, which is advantageous, for example, when shake flasks are used for the fermentation.

[0240] [Example 10] Increased carbon source adaptability of the engineered strain producing 2′FL The carbon source was batch-dosed into the medium and fed in the feed phase for periods ranging from 2 to 100 hours. The carbon source was applied in pure form (e.g., glycerol) or diluted in water (glycerol and other carbon sources). The carbon source feed rate was adjusted to suit the agitation and aeration conditions of the fermenter.

[0241] During the course of the fermentation, samples were taken and analyzed by isocratic HPLC elution.

[0242] Carbon source adaptability assays were performed using the following medium composition: The carbon source was selected from the following list: Glucose, glycerol, mannose, fructose Dispense 20 mL of medium (10 g / L of each carbon source, 5 g / L lactose, 1 g / L (NH)2H-citric acid, 2 g / L Na2SO4, 2.68 g / L (NH4)2SO4, 0.5 g / L NH4Cl, 14.6 g / L K2HPO4, 4 g / L NaH2PO4*H2O, 0.5 g / L MgSO4*7H2O, 10 g / mL MnSO4, and 3 mL of trace metal solution (8.0 g / L Na2-EDTA*2H2O, 1 g / L CaSO4*2H2O, 0.3 g / L ZnSO4*7H2O, 7.4 g / L (NH4)2Fe(SO4)2, 0.2 g / L MnSO4*H2O, 0.15 g / L A 2'-FL production strain containing the crr deletion was inoculated with an overnight culture of the 2'-FL production strain (in the above medium without lactose and IPTG) at a starting OD of 0.5, as in Example 1, at 200 rpm and 37°C for 24 hours. Samples were taken and analyzed for carbon utilization and product formation. Similarly, a 2'-FL production strain with the crr deletion was cultured, sampled, and analyzed.

[0243] [Table 7]

Claims

1. 1. A method for increasing the carbon substrate tolerance of production and / or increasing the carbon conversion efficiency and / or increasing the space-time yield of one or more human milk oligosaccharides produced by a host organism suitable for the production of one or more human milk oligosaccharides, comprising the steps of increasing adenosine 3',5'-cyclic monophosphate (cAMP, CAS number: 60-92-4) levels in the host organism compared to an unmodified host organism, maintaining the host organism under conditions that allow its growth, growing the host organism in the presence of a substrate under conditions suitable for the production of one or more human milk oligosaccharides, and optionally isolating the one or more human milk oligosaccharides from the host organism or the remainder thereof. wherein the cAMP level of the host organism is (a) providing in a host cell an adenylate cyclase protein having an inactive regulatory domain and a functional catalytic domain for producing cAMP, wherein the regulatory domain is inactive due to the presence of an inhibitor, or due to an inactivating mutation, or due to deletion of all or part of the regulatory domain of the adenylate cyclase protein; and / or (b) inactivating or eliminating the Crr protein or the endogenous protein corresponding to the Crr protein (SEQ ID NO: 26) in E. coli; is increased by A method wherein the host organism is a prokaryotic microorganism, the adenylate cyclase is CyaA, and the human milk oligosaccharides comprise at least one of 2'-FL and 6'-SL.

2. 2. The method of claim 1, wherein the cAMP level of the host organism is increased in an inducible manner, the increase being relative to an uninduced host organism.

3. 3. The method of claim 1, wherein the mutant adenylate cyclase is introduced by introducing a transgene.

4. 4. The method of claim 2 or 3, wherein the mutant adenylate cyclase, or the adenylate cyclase whose regulatory activity has been inactivated, has a deletion compared to the wild-type form of adenylate cyclase of the host organism.

5. 5. The method of claim 4, wherein the deletion removes a regulatory portion of adenylate cyclase without destroying the portion that produces cAMP.

6. 6. The method of claim 4 or 5, wherein the deletion is a deletion of a regulatory portion of a protein corresponding to the C-terminal portion of adenylate cyclase encoded by the E. coli cyaA gene.

7. 7. The method of claim 4, 5 or 6, wherein the deletion is a deletion of a portion of the CyaA protein corresponding to the C-terminal portion thereof as set forth in SEQ ID NO: 19 or 20, or an adenylate cyclase protein having at least 90% sequence identity with positions 1 to 412 of SEQ ID NO: 19 or 20.

8. 8. The method of any one of claims 1 to 7, wherein the method comprises providing a carbon source to the host organism, the carbon source being a complex or defined carbon source, or a combination thereof.

9. 9. The method of any one of claims 1 to 8, wherein the host organism is a genetically modified microbial cell, and the method comprises a step of inactivating or removing, in the genetically modified microorganism, a Crr protein or an endogenous protein(s) corresponding to the Crr protein in Escherichia coli (E. coli) (SEQ ID NO: 26) prior to propagation of the genetically modified microorganism.

10. 1. A modified host cell suitable for the production of human milk oligosaccharides, comprising: the host cell is capable of growing on glycerol and / or glucose and / or maltose and / or fructose and / or sucrose, The modified host cell comprises an adenylate cyclase having adenylate cyclase activity and inactivated or absent regulatory activity; and the host cell has increased cAMP levels compared to unmodified host cells, and the unmodified host cells are substantially unable to grow on glycerol and / or glucose and / or maltose and / or fructose and / or sucrose; In a host cell, an adenylate cyclase protein is provided having an inactive regulatory domain and a functional catalytic domain for producing cAMP, wherein the regulatory domain is inactive due to the presence of an inhibitor, or due to an inactivating mutation, or due to deletion of all or part of the regulatory domain of the adenylate cyclase protein; the host cell is a prokaryotic microorganism, the adenylate cyclase is CyaA, and the human milk oligosaccharides include at least one of 2'-FL and 6'-SL; Modified host cells.

11. 11. The modified host cell of claim 10, wherein at least one adenylate cyclase protein corresponding to the protein encoded by the cyaA gene of E. coli lacks regulatory activity.

12. 12. The modified host cell of claim 10, wherein at least one adenylate cyclase protein corresponding to a protein encoded by the cyaA gene of Escherichia coli lacks a portion corresponding to the C-terminal portion of the CyaA protein set forth in SEQ ID NO: 19 or 20, or is an adenylate cyclase protein having at least 90% sequence identity with positions 1 to 412 of SEQ ID NO: 19 or 20.

13. 13. The modified host cell of any one of claims 10, 11 or 12, wherein the host cell is a microorganism that has been genetically modified to enhance the production of oligosaccharides, wherein the genetically modified microorganism has the ability to produce oligosaccharides, wherein the genetically modified microorganism comprises a functional gene encoding a PTS carbohydrate utilization system, wherein the abundance and / or activity of a Crr protein (SEQ ID NO: 26), a variant thereof, or an endogenous protein corresponding to a Crr protein in the microorganism is reduced in the genetically modified microorganism, and wherein the space-time yield, carbon substrate adaptability, or carbon conversion efficiency of oligosaccharide production by the genetically modified microorganism is increased compared to a control in which the abundance and / or activity of the Crr protein (SEQ ID NO: 26), a variant thereof, or an endogenous protein(s) corresponding to a Crr protein is / are not modified.

14. 14. The modified host cell of any one of claims 10 to 13, wherein the host cell is a genetically modified microorganism and expression of a gene encoding a Crr protein, a variant thereof, or an endogenous protein(s) corresponding to a Crr protein in said microorganism is attenuated or deleted in said genetically modified microorganism.

15. 15. The modified host cell of any one of claims 10 to 14, wherein the glycerol and / or glucose and / or maltose and / or fructose and / or sucrose is sucrose, glycerol, glucose, and / or fructose.

16. To increase the carbon substrate adaptability of the production of one or more human milk oligosaccharides in a host prokaryotic microbial cell, and / or to increase the carbon conversion efficiency and / or to increase the space-time yield. (a) an adenylate cyclase protein having an inactivating regulatory domain and a functional catalytic domain for producing cAMP as defined in any one of claims 1 to 14, and / or (b) inactivating and / or reducing the abundance of Crr proteins or endogenous proteins corresponding to Crr proteins (SEQ ID NO: 26) in E. coli; 10. The use of claim 9, wherein the adenylate cyclase is CyaA and the human milk oligosaccharides include at least one of 2'-FL and 6'-SL.

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