Genetically engineered bacteria resistant to massive cell lysis

Abstract

The present invention relates generally to biotechnology techniques, and specifically to genetically modified bacteria of the genus Methylobacillus having improved properties that are particularly useful in large-scale methanol fermentation. More specifically, the present invention provides bacteria of the genus Methylobacillus that have been modified to reduce exopolysaccharide (EPS) production compared to an otherwise identical bacterium lacking the modification. The present invention further provides methods for producing biochemical compounds using the genetically modified bacteria of the present invention.

Description

[Technical field]

[0001] The present invention relates generally to biotechnology techniques, and in particular to genetically modified bacteria that are resistant to extensive cell lysis, making them particularly useful in large-scale fermentation. More specifically, the present invention provides bacteria that have been modified to reduce the expression and / or activity of an endogenous polypeptide having polyphosphate kinase activity compared to an otherwise identical bacterium lacking the modification. The present invention further provides methods for producing biochemical compounds using the genetically modified bacteria of the present invention. [Background technology]

[0002] Biotechnological chemical production by microbial fermentation is a more sustainable and environmentally friendly alternative to petroleum and other fossil fuel-based production. Several sustainable fermentation substrates, mainly C6 and C5 carbohydrates such as glucose and xylose, have been used commercially for chemical production. Organisms commonly used in such fermentations are Escherichia coli, Bacillus subtilis and Corynebacterium species, among others.

[0003] Although glucose-based is sustainable, shifting all chemical production to glucose-based fermentation would require vast amounts of arable land just to produce enough feedstock. One way to avoid this and still achieve sustainable chemical production is to replace glucose with a less land-intensive feedstock. Methanol is a possible candidate for this. Methanol can be produced from carbon dioxide and hydrogen, so it does not require land. Methanol can be easily stored and transported, and it is more energy dense than glucose.

[0004] There are many bacteria, such as Bacillus methanolicus, Methylobacterium extorquens, Methylobacillus glycogenes and Methylobacillus flagellatus, that can utilize methanol as the sole energy and carbon source. Methanol-utilizing bacteria, i.e., methylotrophic bacteria, are commonly found in harsh natural environments or within / closely associated with plants where access to nutrients is poor. Adaptation to such conditions results in several desirable properties in industrial fermentation microorganisms, such as the ability to grow rapidly in minimal media and tolerance to fluctuating nutrient and oxygen supplies. A specific example is the ability to store reserve phosphate in the form of insoluble polyphosphate granules, which is facilitated by the enzyme polyphosphate kinase (ppK). This allows the organism to take up more phosphate than is needed for growth when phosphate is available and to utilize the reserve when it is in short supply. Members of the genus Methylobacillus possess many such favorable qualities and are promising candidates for use in industrial methanol fermentation.

[0005] However, some environmental adaptations can result in undesirable phenotypes in industrial biotechnology contexts. Programmed cell death and other cell lysis mechanisms are examples of such adaptations. It is often advantageous for the population as a whole if a large proportion of individual bacteria lyse when nutrients are scarce, the population is too large, or in response to other stressors. This allows a small proportion of surviving cells to be provided with nutrients, allowing them to survive when a larger population could potentially be eliminated entirely. Programmed cell death in unicellular organisms is a poorly understood phenomenon that has only recently been accepted as a reality by the scientific community, as it requires a degree of "selflessness" from the unicellular organism. The specific trigger is unknown, but it has been linked to quorum sensing mechanisms. Such behavior is highly undesirable in industrial bioreactors, where cell densities must reach extremely high values. In many bioprocesses, the majority of product is formed in the stationary phase after maximum cell density is reached and carbon begins to flow towards product formation rather than biomass. Thus, extensive cell lysis, which occurs in the early stages of a bioprocess in many organisms, such as Methylobacillus species, significantly reduces the achievable product yield and titer. Summary of the Invention

[0006] The aim of the present invention is to overcome the drawbacks in bioprocessing related to extensive cell lysis, especially during the early stages of the process. This is achieved by the inventors who have engineered genetically modified bacteria that are resistant to extensive cell lysis.

[0007] Based on a model bacterium of the genus Methylobacillus, we observed an unexpected and complete elimination of sudden massive cell lysis by reducing the expression and / or activity of an endogenous polypeptide with polyphosphate kinase (PPK) activity. Although neither the gene nor the encoded protein have been previously linked to cell lysis or programmed cell death in microorganisms, reducing (e.g., disrupting) its function in bacteria that contain an endogenous polyphosphate kinase that would otherwise lead to lysis prevented cell lysis under all tested conditions. This significantly improves their utility in chemical production, since they can be cultured to higher cell densities, remain metabolically active, and produce more product for longer periods of time.

[0008] Furthermore, the inventors have observed that reducing (eg, disrupting) acyl homoserine lactone (AHL) synthase function prevents lysis under certain conditions (carbon limitation).

[0009] Based on the above discoveries, in a first aspect the present invention provides a genetically engineered bacterium that has been modified to reduce the expression and / or activity of an endogenous polypeptide having polyphosphate kinase activity compared to an otherwise identical bacterium lacking the modification.

[0010] The present invention further provides a method for the production of a biochemical compound comprising culturing the bacteria of the invention under suitable culture conditions.

[0011] The present invention can be summarized by the following clauses.

[0012] 1. A genetically engineered bacterium that has been modified to reduce the expression and / or activity of an endogenous polypeptide having polyphosphate kinase activity, relative to an otherwise identical bacterium lacking said modification.

[0013] 2. The bacterium of clause 1, which has been modified to reduce expression of an endogenous polypeptide having polyphosphate kinase activity relative to an otherwise identical bacterium lacking said modification.

[0014] 3. The bacterium according to clause 1 or 2, wherein the expression level of an endogenous polypeptide having polyphosphate kinase activity is reduced by at least 50%, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%, compared to an otherwise identical bacterium lacking the modification.

[0015] 4. The bacterium according to any one of clauses 1 to 3, wherein the endogenous gene encoding the polypeptide having polyphosphate kinase activity is inactivated.

[0016] 5. The bacterium according to clause 4, wherein the endogenous gene encoding the polypeptide having polyphosphate kinase activity is inactivated by deletion of part or the entire gene sequence.

[0017] 6. The bacterium according to clause 3 or 4, wherein the endogenous gene encoding the polypeptide having polyphosphate kinase activity has been inactivated by introducing into or expressing in the bacterium a rare-cutting endonuclease capable of selectively inactivating the endogenous gene encoding the polypeptide by DNA cleavage.

[0018] 7. The bacterium described in clause 6, wherein the rare-cutting endonuclease is a transcription activator-like effector (TALE) nuclease, a meganuclease, a zinc finger nuclease (ZFN) or an RNA-guided endonuclease.

[0019] 8. The bacterium according to clause 7, wherein the RNA-guided endonuclease is a catalytically inactive Cas9 protein.

[0020] 9. The bacterium of clause 8, comprising (e.g. expressing) a single guide RNA (sgRNA) that specifically hybridizes (e.g. binds) under cellular conditions to genomic DNA encoding said polypeptide.

[0021] 10. The bacterium according to any one of clauses 1 to 3, wherein expression of the endogenous polypeptide having polyphosphate kinase activity is reduced (e.g. inhibited) by transcriptional and / or translational repression of the endogenous gene encoding the polypeptide.

[0022] 11. The bacterium according to any one of clauses 1 to 3, wherein expression of the endogenous polypeptide having polyphosphate kinase activity is reduced (e.g. inhibited) by introducing into or expressing in the bacterium an inhibitory nucleic acid molecule that specifically hybridizes (e.g. binds) under cellular conditions to cellular mRNA and / or genomic DNA encoding said polypeptide.

[0023] 12. The bacterium according to clause 11, wherein the inhibitory nucleic acid molecule is an antisense oligonucleotide, a ribozyme or an interfering RNA (RNAi) molecule.

[0024] 14. The bacterium according to clause 12, wherein the interfering RNA molecule is a microRNA (miRNA), a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

[0025] 15. The bacterium according to clause 1, which has been modified to reduce the activity of an endogenous polypeptide having polyphosphate kinase activity relative to an otherwise identical microorganism lacking said modification.

[0026] 16. The bacterium according to clause 15, wherein the activity of the polypeptide is reduced by at least one active site mutation resulting in reduced or lost activity.

[0027] 17. The bacterium according to clause 16, wherein at least one active site mutation is a non-conservative amino acid substitution.

[0028] 18. The bacterium according to any one of clauses 1 to 17, which has been further modified to reduce the expression and / or activity of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity compared to an otherwise identical bacterium not having said modification.

[0029] 19. The bacterium described in any one of clauses 1 to 18, which has been modified to reduce expression of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity, compared to an otherwise identical bacterium not having the modification.

[0030] 20. The bacterium according to clause 18 or 19, wherein the expression level of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity is reduced by at least 50%, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%, compared to an otherwise identical bacterium lacking the modification.

[0031] 21. The bacterium described in any one of clauses 18 to 20, wherein the endogenous gene encoding the polypeptide having acyl homoserine lactone (AHL) synthase activity is inactivated.

[0032] 22. The bacterium according to clause 21, wherein the endogenous gene encoding the polypeptide having acyl homoserine lactone (AHL) synthase activity is inactivated by deletion of part or the entire gene sequence.

[0033] 23. The bacterium according to clause 21 or 22, wherein the endogenous gene encoding the polypeptide having acyl homoserine lactone (AHL) synthase activity has been inactivated by introducing into or expressing in the bacterium a rare-cutting endonuclease capable of selectively inactivating the endogenous gene encoding the polypeptide by DNA cleavage.

[0034] 24. The bacterium described in clause 23, wherein the rare-cutting endonuclease is a transcription activator-like effector (TALE) nuclease, a meganuclease, a zinc finger nuclease (ZFN) or an RNA-guided endonuclease.

[0035] 25. The bacterium according to clause 24, wherein the RNA-guided endonuclease is a catalytically inactive Cas9 protein.

[0036] 26. The bacterium according to clause 25, comprising (e.g. expressing) a single guide RNA (sgRNA) that specifically hybridizes (e.g. binds) under cellular conditions to genomic DNA encoding said polypeptide.

[0037] 27. The bacterium described in clause 18 or 19, wherein expression of the endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity is reduced (e.g. inhibited) by transcriptional and / or translational repression of the endogenous gene encoding the polypeptide.

[0038] 28. The bacterium described in clause 18 or 19, wherein expression of the endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity is reduced (e.g. inhibited) by introducing into or expressing in the bacterium an inhibitory nucleic acid molecule that specifically hybridizes (e.g. binds) under cellular conditions to cellular mRNA and / or genomic DNA encoding the polypeptide.

[0039] 29. The bacterium according to clause 28, wherein the inhibitory nucleic acid molecule is an antisense oligonucleotide, a ribozyme or an interfering RNA (RNAi) molecule.

[0040] 30. The bacterium according to clause 29, wherein the interfering RNA molecule is a microRNA (miRNA), a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

[0041] 31. The bacterium according to clause 18, which has been modified to reduce the activity of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity compared to an otherwise identical microorganism lacking said modification.

[0042] 32. The bacterium according to clause 31, wherein the activity of the polypeptide is reduced by at least one active site mutation resulting in reduced or lost activity.

[0043] 33. The bacterium according to clause 32, wherein at least one active site mutation is a non-conservative amino acid substitution.

[0044] 34. The bacterium according to any one of clauses 1 to 33, which is a mesophilic methylotrophic bacterium.

[0045] 35. The bacterium according to any one of clauses 1 to 34, wherein said bacterium belongs to the genus Methylobacillus, Methylobacterium or Methylorubrum, preferably Methylobacillus or Methylorubrum.

[0046] 36. Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus caricics, Methylobacillus methilovorans, Methylobacillus species, Methylobacterium extroquens, Methylobacterium organophilum and Methylorubrum extroquens. 36. The bacterium according to any one of clauses 1 to 35, selected from the group consisting of Bacillus subtilis, Bacillus oryzae.

[0047] 37. The bacterium according to any one of clauses 1 to 36, which is of the genus Methylobacillus.

[0048] 38. The bacterium according to any one of clauses 1 to 37, selected from Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus platensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus calycis, Methylobacillus methylovorans and Methylobacillus species.

[0049] 39. The bacterium described in any one of clauses 1 to 38, wherein the bacterium is Methylobacillus flagellatus.

[0050] 40. The bacterium described in any one of clauses 1 to 38, wherein the bacterium is Methylobacillus glycogenes.

[0051] 41. The bacterium according to any one of clauses 37 to 40, which has been modified to reduce production of exopolysaccharide (EPS) compared to an otherwise identical bacterium lacking said modification.

[0052] 42. The bacterium according to clause 41, which has been modified to reduce the expression and / or activity of at least one endogenous polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium, compared to an otherwise identical bacterium lacking said modification.

[0053] 43. The bacterium according to clause 41 or 42, which has been modified to reduce expression of at least one endogenous polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium compared to an otherwise identical bacterium lacking said modification.

[0054] 44. A bacterium according to any one of clauses 41 to 43, which has been modified to reduce expression of at least two endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium, compared to an otherwise identical bacterium not having said modification.

[0055] 45. A bacterium according to any one of clauses 41 to 44, which has been modified to reduce expression of at least three endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium, compared to an otherwise identical bacterium not having said modification.

[0056] 46. ​​A bacterium according to any one of clauses 41 to 45, which has been modified to reduce expression of at least four endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium, compared to an otherwise identical bacterium not having said modification.

[0057] 47. A bacterium according to any one of clauses 41 to 46, which has been modified to reduce expression of at least five endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium, compared to an otherwise identical bacterium not having said modification.

[0058] 48. A bacterium according to any one of clauses 41 to 47, which has been modified to reduce expression of at least six endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium, compared to an otherwise identical bacterium not having said modification.

[0059] 49. A bacterium described in any one of clauses 1 to 8, which has been modified to reduce expression of all endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium, compared to an otherwise identical bacterium lacking said modification.

[0060] 50. A bacterium according to any one of clauses 42 to 49, wherein at least one polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is selected from the group consisting of a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 27 to 64, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 27 to 64 and having the same functional properties as the reference polypeptide.

[0061] 51. The bacterium according to any one of clauses 42 to 50, wherein at least one polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is selected from the group consisting of a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 27 to 52, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 27 to 52 and having the same functional properties as the reference polypeptide.

[0062] 52. The bacterium according to any one of clauses 42 to 50, wherein at least one polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is selected from the group consisting of a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 53 to 64, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 53 to 64 and having the same functional properties as the reference polypeptide.

[0063] 53. The bacterium according to any one of clauses 42 to 52, wherein at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting of a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 65 to 112, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 65 to 112 and having the same functional properties as the reference polypeptide.

[0064] 54. The bacterium according to any one of clauses 42 to 53, wherein at least one polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is selected from the group consisting of a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 65 to 86, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 65 to 86 and having the same functional properties as the reference polypeptide.

[0065] 55. The bacterium according to any one of clauses 42 to 53, wherein at least one polypeptide involved in the production of exopolysaccharides (EPS) in said bacterium is selected from the group consisting of a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 87 to 112, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 87 to 112 and having the same functional properties as the reference polypeptide.

[0066] 56. The bacterium according to any one of clauses 42 to 55, wherein at least one polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is selected from the group consisting of polypeptides having peptidyl-prolyl cis-trans isomerase activity, polypeptides capable of functioning as polysaccharide exporters, polypeptides that function as chain length determining proteins, and polypeptides having protein tyrosine kinase activity.

[0067] 57. The bacterium according to clause 56, wherein the polypeptide having peptidyl-prolyl cis-trans isomerase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 32, the polypeptide capable of functioning as a polysaccharide exporter comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 33, the polypeptide functioning as a chain length determining protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 34, and the polypeptide having protein tyrosine kinase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 35.

[0068] 58. The bacterium according to clause 56, wherein the polypeptide having peptidyl-prolyl cis-trans isomerase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 68, the polypeptide capable of functioning as a polysaccharide exporter comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 69, the polypeptide functioning as a chain length determining protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 70, and the polypeptide having protein tyrosine kinase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 71.

[0069] 59. A bacterium described in any one of clauses 42 to 58, wherein the expression of at least one polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is reduced by at least 50%, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%, compared to an otherwise identical bacterium not having the modification.

[0070] 60. A bacterium according to any one of clauses 42 to 59, in which the expression of at least one polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is eliminated compared to an otherwise identical bacterium not having the modification.

[0071] 61. The bacterium according to any one of clauses 42 to 60, wherein an endogenous gene encoding the polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is inactivated.

[0072] 62. A bacterium described in any one of clauses 42 to 61, wherein an endogenous gene encoding the polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is inactivated by deletion of part or the entire gene sequence.

[0073] 63. A bacterium according to any one of clauses 42 to 62, wherein an endogenous gene encoding said polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium has been inactivated by introducing into or expressing in the bacterium a respective rare-cutting endonuclease capable of selectively inactivating the endogenous gene encoding said polypeptide by DNA cleavage.

[0074] 64. The bacterium described in clause 63, wherein the rare-cutting endonuclease is selected from the group consisting of transcription activator-like effector (TALE) nucleases, meganucleases, zinc finger nucleases (ZFNs) and RNA-guided endonucleases.

[0075] 65. The bacterium according to clause 64, wherein the RNA-guided endonuclease is a catalytically inactive Cas9 protein.

[0076] 66. The bacterium according to clause 65, comprising (e.g. expressing) at least one single guide RNA (sgRNA) that specifically hybridizes (e.g. binds) to genomic DNA encoding the enzyme under cellular conditions.

[0077] 67. A bacterium described in any one of clauses 42 to 61, wherein expression of a polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is reduced (e.g. inhibited) by transcriptional and / or translational repression of an endogenous gene encoding the polypeptide.

[0078] 68. A bacterium described in any one of clauses 42 to 61, wherein expression of a polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is reduced (e.g. inhibited) by introducing into or expressing in the bacterium at least one inhibitory nucleic acid molecule that specifically hybridizes (e.g. binds) under cellular conditions to cellular mRNA and / or genomic DNA encoding said polypeptide.

[0079] 69. The bacterium according to clause 68, wherein the inhibitory nucleic acid molecule is an antisense oligonucleotide, a ribozyme or an interfering RNA (RNAi) molecule.

[0080] 70. The bacterium according to clause 69, wherein the interfering RNA molecule is a microRNA (miRNA), a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

[0081] 71. A bacterium described in any one of clauses 42 to 70, wherein one or more endogenous polypeptides involved in the production of exopolysaccharides (EPS) in the bacterium are encoded by one or more genes contained in a first EPS gene cluster and / or a second EPS gene cluster.

[0082] 72. The bacterium according to clause 71, wherein the first EPS gene cluster comprises genes defined by or orthologous to an open reading frame (ORF) present in SEQ ID NO: 200 or 203.

[0083] 73. The bacterium according to clause 71 or 72, wherein the first EPS gene cluster comprises the genes epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS or their orthologues.

[0084] 74. A bacterium according to any one of clauses 71 to 73, wherein the first EPS gene cluster comprises the genes epsD, epsE, epsF and epsG or their orthologues.

[0085] 75. The bacterium according to any one of clauses 71 to 74, wherein the first EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 200 or 203.

[0086] 76. The bacterium according to any one of clauses 71 to 74, wherein the first EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 200.

[0087] 77. A bacterium according to any one of clauses 71 to 76, wherein at least one gene in the first EPS gene cluster is inactivated, for example by deletion of part or the entire gene sequence.

[0088] 78. The bacterium according to any one of clauses 71 to 77, wherein at least one gene selected from epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS or their orthologues is inactivated, for example by deletion of part or the entire gene sequence.

[0089] 79. The bacterium according to any one of clauses 71 to 78, wherein at least one gene selected from epsD, epsE, epsF and epsG or an orthologue thereof is inactivated, for example by deletion of part or the entire gene sequence.

[0090] 80. The bacterium according to any one of clauses 71 to 79, wherein the genes epsD, epsE, epsF and epsG or their orthologues are inactivated, for example by deletion of part or the entire gene sequence.

[0091] 81. The bacterium according to clause 79 or 80, wherein the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 32, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 33, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 34, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 35.

[0092] 82. The bacterium according to clause 81, wherein the gene epsD comprises a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 118, the gene epsE comprises a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 119, the gene epsF comprises a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 120, and the gene epsG comprises a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 121.

[0093] 83. The bacterium according to clause 79 or 80, wherein the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 68, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 69, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 70, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 71.

[0094] 84. The bacterium according to clause 83, wherein the epsD gene comprises a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 154, the gene epsE comprises a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 155, the gene epsF comprises a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 156, and the gene epsG comprises a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 157.

[0095] 85. A bacterium described in any one of clauses 71 to 84, wherein the first EPS gene cluster is inactivated.

[0096] 86. A bacterium according to any one of clauses 71 to 85, wherein the first EPS gene cluster is inactivated by deletion of part or the entire sequence of said cluster.

[0097] 87. A bacterium described in any one of clauses 71 to 85, wherein the first EPS gene cluster is inactivated by modification of the promoter and / or ribosome binding site region (e.g., by introducing at least one mutation therein) resulting in the absence of gene expression.

[0098] 88. The bacterium according to any one of clauses 71 to 87, wherein the second EPS gene cluster is defined by or contains genes orthologous to an open reading frame (ORF) present in SEQ ID NO: 201 or 205.

[0099] 89. A bacterium described in any one of clauses 71 to 88, wherein the second EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity to the nucleotide sequence of 201 or 205.

[0100] 90. A bacterium described in any one of clauses 71 to 88, wherein the second EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity to the nucleotide sequence of 201.

[0101] 91. A bacterium according to any one of clauses 71 to 90, wherein at least one gene in the second EPS gene cluster is inactivated, for example by deletion of part or the entire gene sequence.

[0102] 92. A bacterium described in any one of clauses 71 to 91, wherein the second EPS gene cluster is inactivated.

[0103] 93. A bacterium according to any one of clauses 71 to 92, wherein the second EPS gene cluster is inactivated by deletion of part or the entire sequence of said cluster.

[0104] 94. A bacterium described in any one of clauses 71 to 92, wherein the second EPS gene cluster has been inactivated by modification of the promoter and / or ribosome binding site region (e.g., by introducing at least one mutation therein) resulting in the absence of gene expression.

[0105] 95. A bacterium according to any one of clauses 40 to 92, wherein all endogenous genes encoding enzymes involved in the production of exopolysaccharide (EPS) in said bacterium are inactivated, e.g. deleted.

[0106] 96. A method for producing a biochemical compound, comprising culturing a bacterium described in any one of clauses 1 to 95 under suitable culture conditions.

[0107] 97. A method according to clause 96, comprising culturing the bacterium according to any one of clauses 40 to 95 under suitable culture conditions in a medium containing reduced one-carbon compounds, such as methanol, or multi-carbon compounds not containing carbon-carbon bonds, such as dimethyl ether and dimethylamine.

[0108] 98. The method of clause 97, wherein the medium comprises methanol.

[0109] 99. The method according to any one of clauses 96 to 98, wherein the culturing is carried out in a bioreactor.

[0110] 100. The method according to any one of clauses 96 to 99, wherein the biochemical compound is selected from organic acids, amino acids, fatty acids and derivatives thereof.

[0111] 101. A method for producing biomass, comprising culturing a bacterium according to any one of clauses 1 to 95 under suitable culture conditions. [Brief description of the drawings]

[0112] [Figure 1]Fluorescence images of M. flagellatus cells stained with DAPI (wild-type cells (left) and ppK-deficient cells (right)) at 400x magnification. Arrows point to polyphosphate granules stained yellow by DAPI staining. [Diagram 2] Plots of the fermentation biomass-producing bioprocess using wild-type (top) and ppK-deficient Methylobacillus flagellatus (bottom). [Diagram 3] Plot of the fermentative GABA-producing bioprocess using wild-type (top) and ppK-deficient (bottom) Methylobacillus flagellatus expressing the glutamate dehydrogenase enzyme. [Figure 4] Screen capture of antiSMASH algorithm output using known N-acyl homoserine lactone-producing clusters from other organisms. [Diagram 5] Fermentation biomass-producing bioprocess plots using wild-type (top) and AHL synthase-deficient (bottom) Methylobacillus flagellatus. [Figure 6] Sugar content of hydrolyzed M. flagellatus shake flask supernatants. Dark grey bars represent total glucose (in mg / L) and light grey bars represent total monosaccharides. Error bars are standard deviations of technical replicates. [Figure 7] Measured viscosity of bioreactor broth produced by different strains of Methylobacillus flagellatus. [Figure 8] Photographs of wild-type M. flagellatus and M. glycogenes cultures after 24 hours of incubation (from left: wild-type ABME5 and 6, then a single cluster disruption in M. flagellatus, and finally a double disruption). [Figure 9] Plot of antifoam addition (y-axis) in two representative bioreactor fermentations of ABME6 and ABME131 against fermentation time (x-axis). [Figure 10] Phase contrast microscopy images of bioreactor fermentation broth of wild-type Methylobacillus flagellatus (left) versus the dual disruption strain ABME131 (right). [Figure 11] Improved growth of EPS-free ABME131 compared to the parent strain.

[0113] The present invention will now be described in more detail below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0114] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of biochemistry, genetics, and microbiology.

[0115] Although any method and material similar or equivalent to those described herein can be used to carry out or test the present invention, the preferred method and material are described herein.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will take precedence.Furthermore, the materials, methods and examples are merely illustrative and are not intended to be limiting unless otherwise specified.

[0116] The practice of the present invention employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology and recombinant DNA which are within the skill of the art and are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, 3rd ed. (Sambrook et al., 2001, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Mullis et al., U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins, eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); "Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984); "The series, Methods In ENZYMOLOGY" (J. Abelson and M. Simon, editors, Academic Press, Inc., New York), specifically the 154th and 155th editions (Wu et al., eds.) and the 185th edition, "Gene Expression Technology" (D.See "Gene Transfer Vectors For Mammalian Cells" edited by J. H. Miller and M. P. Calos, Cold Spring Harbor Laboratory, 1987.

[0117] The bacteria of the present invention As indicated above, the present invention is based on the unexpected and surprising discovery that certain drawbacks in bioprocessing, in particular extensive cell lysis, can be overcome by reducing the expression and / or activity of an endogenous polypeptide having polyphosphate kinase activity in bacteria.

[0118] Thus, in a first aspect, the present invention provides a genetically engineered bacterium that has been modified to reduce the expression and / or activity of an endogenous polypeptide having polyphosphate kinase activity compared to an otherwise identical bacterium lacking the modification.

[0119] A "polypeptide having polyphosphate kinase activity" reacts with ATP + (phosphate) n <=>ADP+(phosphate) n+1 Polyphosphate kinase (PPK), encoded by the ppk gene, is highly conserved in many bacteria, including methylotrophs such as Methylobacillus flagellatus and Methylobacillus glycogenes, and plays an important role in the ability of bacteria to adapt to nutritional emergencies and environmental stresses. Non-limiting examples of endogenous polypeptides with polyphosphate kinase activity are provided in SEQ ID NOs: 1, 3, 5, 7, 9, and 11.

[0120] According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0121] According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 2.

[0122] According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0123] According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 4. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 4.

[0124] According to some embodiments, the endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. According to some embodiments, the endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. According to some embodiments, the endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 5.

[0125] According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 6. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 6. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 6.

[0126] According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0127] According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8.

[0128] According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.

[0129] According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10.

[0130] According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. According to some embodiments, an endogenous polypeptide having polyphosphate kinase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 11.

[0131] According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12. According to some embodiments, the endogenous gene encoding a polypeptide having polyphosphate kinase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12.

[0132] According to some embodiments, the bacteria of the invention may be modified to reduce expression of an endogenous polypeptide having polyphosphate kinase activity compared to an otherwise identical bacterium lacking the modification.

[0133] According to some embodiments, the bacteria of the invention may be modified to reduce expression of an endogenous polypeptide having polyphosphate kinase activity compared to an otherwise identical bacterium lacking the modification, for example the expression level of an endogenous polypeptide having polyphosphate kinase activity may be reduced by at least 50%, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%, compared to an otherwise identical bacterium lacking the modification.

[0134] According to some embodiments, the bacterium of the invention may be modified to reduce the expression level of an endogenous gene encoding said endogenous polypeptide having polyphosphate kinase activity compared to an otherwise identical bacterium lacking said modification, for example the expression level of the endogenous gene may be reduced by at least 50%, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% compared to an otherwise identical bacterium lacking said modification.

[0135] According to some embodiments, the endogenous gene encoding said polypeptide having polyphosphate kinase activity is inactivated, such as by deletion of part or the entire gene sequence.

[0136] According to some embodiments, the endogenous gene encoding the polypeptide having polyphosphate kinase activity is inactivated by introducing into or expressing in the microorganism a rare-cutting endonuclease capable of selectively inactivating the endogenous gene encoding the enzyme by DNA cleavage, preferably by double-strand breaks. The rare-cutting endonuclease used according to the invention to inactivate the endogenous gene may be, for example, a transcription activator-like effector (TALE) nuclease, a meganuclease, a zinc finger nuclease (ZFN) or an RNA-guided endonuclease.

[0137] One method for inactivating endogenous genes encoding polypeptides with polyphosphate kinase activity is to use the CRISPRi system. The CRISPRi system was developed as a tool for targeted suppression of gene expression or blocking targeted locations on the genome. The CRISPRi system consists of a catalytically inactive or "dead" Cas9 protein (dCas9) and a guide RNA that defines the binding site of dCas9 to DNA.

[0138] Thus, according to some embodiments, an endogenous gene encoding a polypeptide having polyphosphate kinase activity is inactivated by introducing into or expressing in the bacterium an RNA-guided endonuclease, such as a catalytically inactivated Cas9 protein, and a single guide RNA (sgRNA) that specifically hybridizes (e.g., binds) to the genomic DNA encoding said polypeptide under cellular conditions.

[0139] According to some embodiments, expression of said endogenous polypeptide having polyphosphate kinase activity is reduced by inhibition.

[0140] The inhibition of the expression of said endogenous polypeptide may be achieved by any suitable means known in the art.For example, the expression may be inhibited by gene silencing techniques involving the use of inhibitory nucleic acid molecules such as antisense oligonucleotides, ribozymes or interfering RNA (RNAi) molecules such as microRNA (miRNA), small interfering RNA (siRNA) or small hairpin RNA (shRNA).

[0141] According to some embodiments, expression of the endogenous polypeptide having polyphosphate kinase activity is reduced (e.g., inhibited) by transcriptional and / or translational repression of the endogenous gene encoding the polypeptide.

[0142] According to some embodiments, expression of a polypeptide having endogenous polyphosphate kinase activity is inhibited by introducing or expressing an inhibitory nucleic acid molecule in the bacterium. For example, the inhibitory nucleic acid molecule may be introduced by an exogenous nucleic acid molecule comprising a nucleotide sequence encoding said inhibitory nucleic acid molecule operably linked to a promoter, such as an inducible promoter, functional to cause production of said inhibitory nucleic acid molecule in the bacterium. Suitably, the inhibitory nucleic acid molecule specifically hybridizes (e.g. binds) under cellular conditions with cellular mRNA and / or genomic DNA encoding the endogenous polypeptide. Depending on the target, transcription of the encoding genomic DNA and / or translation of the encoding mRNA is inhibited.

[0143] According to some embodiments, the inhibitory nucleic acid molecule is an antisense oligonucleotide, a ribozyme or an interfering RNA (RNAi) molecule. Preferably, such a nucleic acid molecule comprises at least 10 consecutive nucleotides of the complement of a cellular mRNA and / or genomic DNA encoding a polypeptide or enzyme of interest (e.g., a cellular mRNA and / or genomic DNA encoding a polypeptide).

[0144] According to some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide. Such an antisense oligonucleotide is a nucleic acid molecule (either DNA or RNA) that specifically hybridizes (e.g., binds) under cellular conditions to cellular mRNA and / or genomic DNA encoding a polypeptide.

[0145] In some embodiments, the inhibitory nucleic acid molecule is a ribozyme, such as a hammerhead ribozyme, which is designed to catalytically cleave an mRNA transcript to prevent translation of a polypeptide.

[0146] According to some embodiments, the inhibitory nucleic acid molecule is an interfering RNA (RNAi) molecule. RNA interference is a biological process in which an RNA molecule inhibits the expression, typically destroying a specific mRNA. Exemplary types of RNAi molecules include microRNA (miRNA), small interfering RNA (siRNA) and small hairpin RNA (shRNA). According to some embodiments, the RNAi molecule is miRNA. According to some embodiments, the RNAi molecule is siRNA. According to some embodiments, the RNAi molecule is shRNA.

[0147] According to some embodiments, the bacteria of the invention have been modified to reduce the activity of an endogenous polypeptide having polyphosphate kinase activity compared to an otherwise identical bacterium lacking the modification.

[0148] The reduction in activity of a polypeptide having polyphosphate kinase activity may be achieved by any suitable means known in the art. For example, the activity may be reduced by introducing one or more mutations into the active site of the polypeptide, which result in reduced or lost activity. Thus, according to some embodiments, the activity of an endogenous polypeptide having polyphosphate kinase activity is reduced by at least one active site mutation, which results in reduced or lost activity. The at least one active site mutation may, for example, be at least one non-conservative amino acid substitution.

[0149] By way of example, when the activity of an endogenous polypeptide having polyphosphate kinase activity is reduced in Methylobacillus flagellatus, the at least one active site mutation may occur at any one of positions R379, S384, F492, P511, R568, R625, Q679, H439 and H458 in the amino acid sequence set forth in SEQ ID NO: 1 which forms part of the active site. In the case of an orthologous polypeptide having polyphosphate kinase activity, the at least one active site mutation may be at a position corresponding to any one of positions R379, S384, F492, P511, R568, R625, Q678, H439 and H458 in the amino acid sequence set forth in SEQ ID NO: 1.

[0150] As another example, when the activity of an endogenous polypeptide having polyphosphate kinase activity is reduced in Methylobacillus glycogenes, the at least one active site mutation may occur at any one of positions R79, S84, F192, P211, R268, R325, Q378, H139 in the amino acid sequence set forth in SEQ ID NO: 3 that forms part of the active site. In the case of an orthologous polypeptide having polyphosphate kinase activity, the at least one active site mutation may be at a position corresponding to any one of positions R79, S84, F192, P211, R268, R325, Q378, H139, H158 in the amino acid sequence set forth in SEQ ID NO: 3.

[0151] As another example, when the activity of an endogenous polypeptide having polyphosphate kinase activity is reduced in Methylobacillus rhizosphaerae, the at least one active site mutation may occur at any one of positions R379, S384, F492, P511, R568, R625, Q678, H439 and H458 in the amino acid sequence set forth in SEQ ID NO: 5 which forms part of the active site. In the case of an orthologous polypeptide having polyphosphate kinase activity, the at least one active site mutation may be at a position corresponding to any one of positions R379, S384, F492, P511, R568, R625, Q678, H439 and H458 in the amino acid sequence set forth in SEQ ID NO: 5.

[0152] As another example, when the activity of an endogenous polypeptide having polyphosphate kinase activity is reduced in Methylobacterium organophilum, the at least one active site mutation may occur at any one of positions R392, S397, F505, P524, R581, R643, H452 and H471 in the amino acid sequence set forth in SEQ ID NO: 7 that forms part of the active site. In the case of an orthologous polypeptide having polyphosphate kinase activity, the at least one active site mutation may be at a position corresponding to any one of positions R392, S397, F505, P524, R581, R643, H452 and H471 in the amino acid sequence set forth in SEQ ID NO: 7.

[0153] As another example, when the activity of an endogenous polypeptide having polyphosphate kinase activity is reduced in Methylorblum extorquens, at least one active site mutation may occur at any one of positions R451, S456, F564, P583, R640, R702, H511 and H530 in the amino acid sequence set forth in SEQ ID NO:9 which forms part of the active site. In the case of an orthologous polypeptide having polyphosphate kinase activity, at least one active site mutation may be at a position corresponding to any one of positions R451, S456, F564, P583, R640, R702, H511 and H530 in the amino acid sequence set forth in SEQ ID NO:9.

[0154] As another example, when the activity of an endogenous polypeptide having polyphosphate kinase activity is reduced in E. coli, at least one active site mutation may occur at any one of positions R375, S380, F488, P507, R564, R621, Q674, H435 and H454 in the amino acid sequence set forth in SEQ ID NO: 11 which forms part of the active site. In the case of an orthologous polypeptide having polyphosphate kinase activity, at least one active site mutation may be at a position corresponding to any one of positions R375, S380, F488, P507, R564, R621, Q674, H435 and H454 in the amino acid sequence set forth in SEQ ID NO: 11.

[0155] The resistance of such bacteria to cell lysis may be further enhanced, particularly under certain conditions such as carbon limitation, by reducing the expression and / or activity of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity.

[0156] Thus, according to some embodiments, the bacteria of the invention may be further modified to reduce expression and / or activity of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity compared to an otherwise identical bacterium lacking said modification.

[0157] A "polypeptide having acyl homoserine lactone (AHL) synthase activity" is a polypeptide (EC 2.3.1.184) that catalyzes the reaction: acyl-[acyl carrier protein] + S-adenosyl-L-methionine <=> [acyl carrier protein] + S-methyl-5'-thioadenosine + N-acyl-L-homoserine lactone. Acyl homoserine lactones (AHLs) are small signaling molecules used by many Gram-negative bacteria to adjust their behavior as a function of their population density. This process, based on biosynthesis and sensing of such molecular signals, also referred to as quorum sensing (QS), controls various gene expressions including growth, virulence, biofilm formation and toxin production. Non-limiting examples of endogenous polypeptides having acyl homoserine lactone (AHL) synthase activity are provided in SEQ ID NOs: 13, 15, 17, 19, 21, 23 and 25.

[0158] According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 13.

[0159] According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14. According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14. According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14.

[0160] According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0161] According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16.

[0162] According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0163] According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 18. According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 18. According to some embodiments, the endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 18.

[0164] According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 19.

[0165] According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 20. According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 20. According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 20.

[0166] According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 21. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 21. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 90%, such as at least 95%, at least 98%, or at least 99%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 21.

[0167] According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 22. According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 22. According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 22.

[0168] According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 23.

[0169] According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 24. According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 24. According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 24.

[0170] According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 25. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 25. According to some embodiments, an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to the amino acid sequence set forth in SEQ ID NO: 25.

[0171] According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 70%, such as at least 75%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 26. According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 80%, such as at least 85%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 26. According to some embodiments, an endogenous gene encoding a polypeptide having acyl homoserine lactone (AHL) synthase activity comprises a nucleic acid sequence having at least 90%, such as at least 95%, sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 26.

[0172] According to some embodiments, the bacteria of the invention may be modified to reduce expression of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity compared to an otherwise identical bacterium lacking the modification.

[0173] According to some embodiments, the bacteria of the invention may be modified to reduce expression of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity compared to an otherwise identical bacterium lacking the modification. The expression level of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity may be reduced by at least 50%, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%, compared to an otherwise identical bacterium lacking the modification.

[0174] According to some embodiments, the bacterium of the invention may be modified to reduce the expression level of an endogenous gene encoding said endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity compared to an otherwise identical bacterium lacking said modification. The expression level of the endogenous gene may be reduced by at least 50%, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%, compared to an otherwise identical bacterium lacking said modification.

[0175] According to some embodiments, the endogenous gene encoding said polypeptide having acyl homoserine lactone (AHL) synthase activity is inactivated, such as by deletion of part or the entire gene sequence.

[0176] According to some embodiments, the endogenous gene encoding the polypeptide having acyl homoserine lactone (AHL) synthase activity is inactivated by introducing into or expressing in the microorganism a rare-cutting endonuclease capable of selectively inactivating the endogenous gene encoding the enzyme by DNA cleavage, preferably by double-strand breaks. The rare-cutting endonuclease used according to the present invention to inactivate the endogenous gene may be, for example, a transcription activator-like effector (TALE) nuclease, a meganuclease, a zinc finger nuclease (ZFN) or an RNA-guided endonuclease.

[0177] One method for inactivating the endogenous gene encoding the polypeptide with acyl homoserine lactone (AHL) synthase activity is to use the CRISPRi system.The CRISPRi system was developed as a tool for targeted suppression of gene expression or blocking targeted positions on the genome.The CRISPRi system consists of a catalytically inactive or "dead" Cas9 protein (dCas9) and a guide RNA that defines the binding site of dCas9 to DNA.

[0178] Thus, according to some embodiments, an endogenous gene encoding the polypeptide having acyl homoserine lactone (AHL) synthase activity is inactivated by introducing into or expressing in the bacterium an RNA-guided endonuclease, such as a catalytically inactive Cas9 protein, and a single guide RNA (sgRNA) that specifically hybridizes (e.g., binds) to the genomic DNA encoding the polypeptide under cellular conditions.

[0179] According to some embodiments, expression of said endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity is reduced by inhibition.

[0180] The inhibition of the expression of said endogenous polypeptide may be achieved by any suitable means known in the art.For example, the expression may be inhibited by gene silencing techniques involving the use of inhibitory nucleic acid molecules such as antisense oligonucleotides, ribozymes or interfering RNA (RNAi) molecules such as microRNA (miRNA), small interfering RNA (siRNA) or small hairpin RNA (shRNA).

[0181] According to some embodiments, expression of the endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity is reduced (e.g., inhibited) by transcriptional and / or translational repression of the endogenous gene encoding the polypeptide.

[0182] According to some embodiments, the expression of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity is inhibited by introducing or expressing an inhibitory nucleic acid molecule in the bacterium. For example, the inhibitory nucleic acid molecule may be introduced by an exogenous nucleic acid molecule comprising a nucleotide sequence encoding the inhibitory nucleic acid molecule operably linked to a promoter, such as an inducible promoter, functional to cause the production of the inhibitory nucleic acid molecule in the bacterium. Suitably, the inhibitory nucleic acid molecule specifically hybridizes (e.g. binds) under cellular conditions with cellular mRNA and / or genomic DNA encoding the endogenous polypeptide. Depending on the target, transcription of the encoding genomic DNA and / or translation of the encoding mRNA is inhibited.

[0183] According to some embodiments, the inhibitory nucleic acid molecule is an antisense oligonucleotide, a ribozyme or an interfering RNA (RNAi) molecule. Preferably, such a nucleic acid molecule comprises at least 10 consecutive nucleotides of the complement of a cellular mRNA and / or genomic DNA encoding a polypeptide or enzyme of interest (e.g., a cellular mRNA and / or genomic DNA encoding a polypeptide).

[0184] According to some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide. Such an antisense oligonucleotide is a nucleic acid molecule (either DNA or RNA) that specifically hybridizes (e.g., binds) under cellular conditions to cellular mRNA and / or genomic DNA encoding a polypeptide.

[0185] In some embodiments, the inhibitory nucleic acid molecule is a ribozyme, such as a hammerhead ribozyme, which is designed to catalytically cleave an mRNA transcript to prevent translation of a polypeptide.

[0186] According to some embodiments, the inhibitory nucleic acid molecule is an interfering RNA (RNAi) molecule. RNA interference is a biological process in which an RNA molecule inhibits the expression, typically destroying a specific mRNA. Exemplary types of RNAi molecules include microRNA (miRNA), small interfering RNA (siRNA) and small hairpin RNA (shRNA). According to some embodiments, the RNAi molecule is miRNA. According to some embodiments, the RNAi molecule is siRNA. According to some embodiments, the RNAi molecule is shRNA.

[0187] According to some embodiments, the bacteria of the invention have been modified to reduce activity of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity compared to an otherwise identical bacterium lacking the modification.

[0188] The reduction in activity of a polypeptide having acyl homoserine lactone (AHL) synthase activity may be achieved by any suitable means known in the art. For example, the activity may be reduced by introducing one or more mutations into the active site of the polypeptide, which result in reduced or lost activity. Thus, according to some embodiments, the activity of an endogenous polypeptide having acyl homoserine lactone (AHL) synthase activity is reduced by at least one active site mutation, which results in reduced or lost activity. The at least one active site mutation may, for example, be at least one non-conservative amino acid substitution.

[0189] Generally, the bacterium referred to herein may be any suitable bacterium, so long as it contains a gene encoding a polypeptide having polyphosphate kinase activity in its genome. The bacterium may be gram-positive or gram-negative. Preferably, the bacterium is a gram-negative bacterium. Non-limiting examples of gram-negative bacteria include species from the genera Methylobacillus, Methylobacterium, Methylorblum, Escherichia, Erwinia, Klebsiella and Citrobacter. Non-limiting examples of gram-positive bacteria include species from the genera Bacillus, Lactococcus, Lactobacillus, Geobacillus, Pediococcus, Moorella, Clostridium, Corynebacterium, Streptomyces, Streptococcus and Cellulomonas.

[0190] According to some embodiments, the bacterium of the present invention is a methylotrophic bacterium, preferably a mesophilic methylotrophic bacterium.

[0191] According to some embodiments, the bacteria of the invention belong to the family Methylophilus or Methylobacterium.

[0192] According to some embodiments, the bacterium of the invention belongs to the family Methylophilaceae.

[0193] According to some embodiments, the bacterium of the invention belongs to the family Methylobacterium.

[0194] According to some embodiments, the bacterium of the present invention belongs to the genus Methylobacillus, Methylobacterium, or Methylorburum.

[0195] According to some embodiments, the bacterium of the present invention belongs to the genus Methylobacillus or Methylobacterium.

[0196] According to some embodiments, the bacterium of the present invention belongs to the genus Methylobacterium or Methylorburum.

[0197] According to some embodiments, the bacterium of the present invention is selected from Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus platensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus calycis, Methylobacillus methylovorans, Methylobacillus species, Methylobacterium extroquens, Methylobacterium organophilum, and Methylolblum extroquens.

[0198] According to some embodiments, the bacterium of the present invention belongs to the genus Methylobacillus.

[0199] According to some embodiments, the bacterium of the present invention is selected from Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus platensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus calycis, Methylobacillus methylovorans, and Methylobacillus species.

[0200] According to some embodiments, the bacterium of the present invention is Methylobacillus flagellatus.

[0201] According to some embodiments, the bacterium of the present invention is Methylobacillus glycogenes.

[0202] According to some embodiments, the bacterium of the present invention is Methylobacillus rhizosphaerae.

[0203] According to some embodiments, the bacteria of the invention are of the family Enterobacteriaceae.

[0204] According to some embodiments, the bacterium of the present invention belongs to the genus Escherichia.

[0205] According to some embodiments, the bacterium of the present invention is E. coli.

[0206] We further investigated general EPS production in Methylobacillus. Based on bioinformatic analysis of the M. flagellatus and M. glycogenes genomes, we identified two putative EPS production gene clusters. Disruption of both EPS clusters resulted in a complete loss of EPS production.

[0207] An unexpected and surprising observation in the engineered M. flagellatus and M. glycogenes strains was their behavior in vegetative cultures. When expression of one or more endogenous polypeptides involved in the production of exopolysaccharides (EPS) in Methylobacillus bacteria was abolished, the cultures exhibited a number of surprising and unexpected properties favorable for the development of methanol-based bioprocesses. Unexpectedly, excessive culture foaming in both shake flasks and bioreactors was reduced. In addition, cell clumping, observed in the unmodified parent strain, was reduced or even absent in the engineered Methylobacillus cultures. Centrifugation and filtration of biomass were also improved when EPS production was eliminated. These combined properties significantly improved the bioprocess, strain handling and downstream processing, making the engineered Methylobacillus bacteria suitable for large-scale methanol fermentation. Removal of EPS synthesis also unexpectedly improved the methanol tolerance of M. flagellatus and other Methylobacillus species.

[0208] Thus, according to some embodiments, the bacteria of the invention that are of the genus Methylobacillus may be further modified to reduce exopolysaccharide (EPS) production compared to an otherwise identical bacterium lacking the modification. More specifically, such engineered bacteria of the genus Methylobacillus may be modified to reduce the expression and / or activity of at least one endogenous polypeptide involved in exopolysaccharide (EPS) production in said bacterium compared to an otherwise identical bacterium lacking the modification.

[0209] According to some embodiments, the bacteria of the invention have been modified to reduce expression of at least one endogenous polypeptide, such as at least two, involved in the production of exopolysaccharide (EPS) in the bacterium, compared to an otherwise identical bacterium lacking the modification.

[0210] According to some embodiments, the bacteria of the invention have been modified to reduce expression of at least three, such as at least four, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking the modification.

[0211] According to some embodiments, the bacteria of the invention have been modified to reduce expression of at least five, such as at least six, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking the modification.

[0212] According to some embodiments, the bacteria of the invention have been modified to reduce expression of at least seven, such as at least eight, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking the modification.

[0213] According to some embodiments, the bacteria of the invention have been modified to reduce expression of at least nine, such as at least ten, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking the modification.

[0214] According to some embodiments, the bacteria of the invention have been modified to reduce expression of at least 11, such as at least 12, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking the modification.

[0215] According to some embodiments, the bacteria of the invention have been modified to reduce expression of at least 13, such as at least 14, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking the modification.

[0216] According to some embodiments, the bacteria of the invention have been modified to reduce expression of at least 15, such as at least 16, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking the modification.

[0217] According to some embodiments, the bacteria of the invention have been modified to reduce expression of all endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking the modification.

[0218] According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 27-64, and b) a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity to any one of SEQ ID NOs: 27-64, and having the same functional properties as the reference polypeptide.

[0219] According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 27-52, and b) a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity to any one of SEQ ID NOs: 27-52, and having the same functional properties as the reference polypeptide.

[0220] According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 53-64, and b) a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity to any one of SEQ ID NOs: 53-64, and having the same functional properties as the reference polypeptide.

[0221] According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 65-112, and b) a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity to any one of SEQ ID NOs: 65-112, and having the same functional properties as the reference polypeptide.

[0222] According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharides (EPS) in the bacterium is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 65-86, and b) a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity to any one of SEQ ID NOs: 65-86, and having the same functional properties as the reference polypeptide.

[0223] According to some embodiments, the at least one enzyme involved in the production of exopolysaccharides (EPS) in the bacterium is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 87-112, and b) a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, sequence identity to any one of SEQ ID NOs: 87-112, and having the same functional properties as the reference polypeptide.

[0224] According to some embodiments, the at least one polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is selected from the group consisting of polypeptides having peptidyl-prolyl cis-trans isomerase activity, polypeptides capable of functioning as polysaccharide exporters, polypeptides functioning as chain length determining proteins, and polypeptides having protein tyrosine kinase activity.

[0225] According to some embodiments, the polypeptide having peptidyl-prolyl cis-trans isomerase activity comprises an amino acid sequence having at least 70%, such as at least 85%, sequence identity to SEQ ID NO:32.

[0226] According to some embodiments, the polypeptide capable of functioning as a polysaccharide exporter comprises an amino acid sequence having at least 70%, such as at least 85%, sequence identity to SEQ ID NO:33.

[0227] According to some embodiments, a polypeptide that functions as a chain length determining protein comprises an amino acid sequence having at least 70%, such as at least 85%, sequence identity to SEQ ID NO:34.

[0228] According to some embodiments, the polypeptide having protein tyrosine kinase activity comprises an amino acid sequence having at least 70%, such as at least 85%, sequence identity to SEQ ID NO:35.

[0229] According to some embodiments, the polypeptide having peptidyl-prolyl cis-trans isomerase activity comprises an amino acid sequence having at least 70%, such as at least 85%, sequence identity to SEQ ID NO:68.

[0230] According to some embodiments, the polypeptide capable of functioning as a polysaccharide exporter comprises an amino acid sequence having at least 70%, such as at least 85%, sequence identity to SEQ ID NO:69.

[0231] According to some embodiments, a polypeptide that functions as a chain length determining protein comprises an amino acid sequence having at least 70%, such as at least 85%, sequence identity to SEQ ID NO:70.

[0232] According to some embodiments, the polypeptide having protein tyrosine kinase activity comprises an amino acid sequence having at least 70%, such as at least 85%, sequence identity to SEQ ID NO:71.

[0233] The expression level of the endogenous polypeptide may be reduced by at least 50%, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%, for example, compared to an otherwise identical bacterium lacking the modification.

[0234] The reduction in expression of the endogenous polypeptide may be achieved by any suitable means known in the art, for example, the expression may be reduced by inactivating an endogenous gene encoding said polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium, such as by deletion of part or the entire gene sequence.

[0235] According to some embodiments, expression of at least one polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is abolished compared to an otherwise identical bacterium lacking the modification.

[0236] According to some embodiments, the endogenous gene encoding the polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is inactivated, such as by deletion of part or the entire gene sequence.

[0237] According to some embodiments, the endogenous gene encoding the polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is inactivated by introducing into or expressing in the bacterium a rare-cutting endonuclease capable of selectively inactivating the endogenous gene encoding the enzyme by DNA cleavage. The rare-cutting endonuclease used according to the invention to inactivate the endogenous gene may be, for example, a transcription activator-like effector (TALE) nuclease, a meganuclease, a zinc finger nuclease (ZFN) or an RNA-guided endonuclease.

[0238] One method for inactivating endogenous genes encoding polypeptides involved in the production of exopolysaccharides (EPS) in bacteria is to use the CRISPRi system. The CRISPRi system was developed as a tool for targeted suppression of gene expression or blocking targeted locations on the genome. The CRISPRi system consists of a catalytically inactive or "dead" Cas9 protein (dCas9) and a guide RNA that defines the binding site of dCas9 to DNA.

[0239] According to some embodiments, expression of an endogenous polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is reduced by inhibition.

[0240] The expression of said polypeptide may be inhibited by any suitable means known in the art.For example, the expression may be inhibited by gene silencing techniques involving the use of inhibitory nucleic acid molecules such as antisense oligonucleotides, ribozymes or interfering RNA (RNAi) molecules such as microRNA (miRNA), small interfering RNA (siRNA) or small hairpin RNA (shRNA).

[0241] According to some embodiments, expression of an endogenous polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is reduced (e.g., inhibited) by transcriptional and / or translational repression of the endogenous gene encoding said polypeptide.

[0242] According to some embodiments, the expression of an endogenous polypeptide involved in the production of exopolysaccharide (EPS) in a bacterium is inhibited by introducing or expressing an inhibitory nucleic acid molecule in the bacterium. For example, the inhibitory nucleic acid molecule may be introduced by an exogenous nucleic acid molecule comprising a nucleotide sequence encoding said inhibitory nucleic acid molecule operably linked to a promoter, such as an inducible promoter, functional to cause the production of said inhibitory nucleic acid molecule in the bacterium. Suitably, the inhibitory nucleic acid molecule specifically hybridizes (e.g. binds) under cellular conditions with cellular mRNA and / or genomic DNA encoding the endogenous polypeptide of interest. Depending on the target, transcription of the encoding genomic DNA and / or translation of the encoding mRNA is inhibited.

[0243] According to some embodiments, the inhibitory nucleic acid molecule is an antisense oligonucleotide, a ribozyme or an interfering RNA (RNAi) molecule. Preferably, such a nucleic acid molecule comprises at least 10 consecutive nucleotides of the complement of a cellular mRNA and / or genomic DNA encoding a polypeptide of interest.

[0244] According to some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide. Such an antisense oligonucleotide is a nucleic acid molecule (either DNA or RNA) that specifically hybridizes (e.g., binds) under cellular conditions to cellular mRNA and / or genomic DNA encoding the enzyme of interest.

[0245] In some embodiments, the inhibitory nucleic acid molecule is a ribozyme, such as a hammerhead ribozyme, which is designed to catalytically cleave an mRNA transcript to prevent translation of a polypeptide of interest.

[0246] According to some embodiments, the inhibitory nucleic acid molecule is an interfering RNA (RNAi) molecule. RNA interference is a biological process in which an RNA molecule inhibits the expression, typically causing the destruction of a specific mRNA. Exemplary types of RNAi molecules include microRNA (miRNA), small interfering RNA (siRNA) and small hairpin RNA (shRNA). According to some embodiments, the RNAi molecule is miRNA. According to some embodiments, the RNAi molecule is siRNA. According to some embodiments, the RNAi molecule is shRNA.

[0247] In some embodiments, the bacteria of the invention have been modified to reduce the function (e.g., activity) of at least one polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical microorganism lacking the modification.

[0248] The reduction in function (e.g. activity) of at least one endogenous polypeptide may be achieved by any suitable means known in the art. For example, if the polypeptide is an enzyme, its activity may be reduced by introducing one or more mutations into the active site of the enzyme that result in reduced or lost activity. Thus, according to some embodiments, the activity of at least one endogenous enzyme involved in the production of exopolysaccharides (EPS) in bacteria is reduced by at least one active site mutation that results in reduced or lost activity. The at least one active site mutation may, for example, be at least one non-conservative amino acid substitution.

[0249] As mentioned above, the present inventors have identified two putative EPS gene clusters in the Methylobacillus genome, and thus, one or more endogenous polypeptides involved in the production of exopolysaccharides (EPS) are encoded by one or more genes contained in the first EPS gene cluster and / or the second EPS gene cluster.

[0250] According to some embodiments, the one or more endogenous polypeptides involved in the production of exopolysaccharides (EPS) in the bacterium are encoded by one or more genes contained in a first EPS gene cluster and / or a second EPS gene cluster.

[0251] According to some embodiments, the first EPS gene cluster comprises genes defined by or orthologous to the open reading frame (ORF) present in SEQ ID NO: 200 or 203.

[0252] According to some embodiments, the first EPS gene cluster comprises the genes epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS or their orthologs.

[0253] According to some embodiments, the first EPS gene cluster comprises the genes epsD, epsE, epsF and epsG or their orthologues.

[0254] According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity, such as at least 55%, to the nucleotide sequence of SEQ ID NO: 200 or 203. According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 60% sequence identity, such as at least 65% to the nucleotide sequence of SEQ ID NO: 200 or 203. According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to the nucleotide sequence of SEQ ID NO: 200 or 203. According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to the nucleotide sequence of SEQ ID NO: 200 or 203. According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 90% sequence identity, such as at least 93%, to the nucleotide sequence of SEQ ID NO: 200 or 203. According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 95%, such as at least 97%, sequence identity to the nucleotide sequence of SEQ ID NO:200 or 203.

[0255] According to some embodiments, the first EPS gene cluster has a sequence similar to that of SEQ ID NO:200, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at %, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0256] According to some embodiments, the first EPS gene cluster has a sequence similar to the nucleotide sequence of SEQ ID NO:203, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 12 %, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0257] According to some embodiments, the second EPS gene cluster comprises genes defined by or orthologous to an open reading frame (ORF) present in SEQ ID NO:201 or 205.

[0258] According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity, such as at least 55%, to the nucleotide sequence of SEQ ID NO:201 or 205. According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 60% sequence identity, such as at least 65% to the nucleotide sequence of SEQ ID NO:201 or 205. According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to the nucleotide sequence of SEQ ID NO:201 or 205. According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to the nucleotide sequence of SEQ ID NO:201 or 205. According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 90% sequence identity, such as at least 85% to the nucleotide sequence of SEQ ID NO:201 or 205.

[0259] According to some embodiments, the second EPS gene cluster has a sequence identical to the 201 nucleotide sequence and is at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, The invention includes nucleotide sequences having at least 50% sequence identity, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0260] According to some embodiments, the second EPS gene cluster comprises 205 nucleotide sequences and at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, The invention includes nucleotide sequences having at least 50% sequence identity, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0261] According to some embodiments, at least one gene in the first EPS gene cluster is inactivated, for example by deletion of part or the entire gene sequence.

[0262] According to some embodiments, the at least one gene is selected from epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS or their orthologues.

[0263] According to some embodiments, the at least one gene is selected from epsD, epsE, epsF and epsG or their orthologues.

[0264] According to some embodiments, the genes epsD, epsE, epsF and epsG or their orthologues are inactivated, for example by deletion of part or the entire gene sequence.

[0265] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, to SEQ ID NO: 32, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, to SEQ ID NO: 33, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, to SEQ ID NO: 34, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, to SEQ ID NO: 35.

[0266] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, with SEQ ID NO: 32, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, with SEQ ID NO: 33, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, with SEQ ID NO: 34, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, with SEQ ID NO: 35.

[0267] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 32, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 33, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 34, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 35.

[0268] According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to SEQ ID NO:118, the gene epsE comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to SEQ ID NO:119, the gene epsF comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to SEQ ID NO:120, and the gene epsG comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to SEQ ID NO:121.

[0269] According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to SEQ ID NO:118, the gene epsE comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to SEQ ID NO:119, the gene epsF comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to SEQ ID NO:120, and the gene epsG comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to SEQ ID NO:121.

[0270] According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 90% sequence identity, such as at least 95% to SEQ ID NO:118, the gene epsE comprises a nucleotide sequence having at least 90% sequence identity, such as at least 95% to SEQ ID NO:119, the gene epsF comprises a nucleotide sequence having at least 90% sequence identity, such as at least 95% to SEQ ID NO:120, and the gene epsG comprises a nucleotide sequence having at least 90% sequence identity, such as at least 95% to SEQ ID NO:121.

[0271] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, to SEQ ID NO: 68, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, to SEQ ID NO: 69, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, to SEQ ID NO: 70, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity, such as at least 75% sequence identity, to SEQ ID NO: 71.

[0272] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, to SEQ ID NO: 68, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, to SEQ ID NO: 69, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, to SEQ ID NO: 70, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, to SEQ ID NO: 71.

[0273] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 68, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 69, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 70, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity, such as at least 95% sequence identity, to SEQ ID NO: 71.

[0274] According to some embodiments, the gene epsD comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to SEQ ID NO:154, the gene epsE comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to SEQ ID NO:155, the gene epsF comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to SEQ ID NO:156, and the gene epsG comprises a nucleotide sequence having at least 70% sequence identity, such as at least 75% to SEQ ID NO:157.

[0275] According to some embodiments, the gene epsD comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to SEQ ID NO:154, the gene epsE comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to SEQ ID NO:155, the gene epsF comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to SEQ ID NO:156, and the gene epsG comprises a nucleotide sequence having at least 80% sequence identity, such as at least 85% to SEQ ID NO:157.

[0276] According to some embodiments, the gene epsD comprises a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:154, the gene epsE comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO:155, the gene epsF comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO:156, and the gene epsG comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO:157.

[0277] According to some embodiments, the genetically engineered bacteria of the present invention have been modified to inactivate the first EPS gene cluster.

[0278] According to some embodiments, the genetically engineered bacteria of the present invention are (further) modified to inactivate the second EPS gene cluster.

[0279] According to some embodiments, the genetically engineered bacteria of the present invention have been modified to inactivate the first EPS gene cluster and the second EPS gene cluster.

[0280] According to some embodiments, the first EPS gene cluster is inactivated by deletion of part or all of the sequence of said cluster.

[0281] According to some embodiments, the first EPS gene cluster is inactivated by deletion of the entire coding sequence of said cluster.

[0282] According to some embodiments, the first EPS gene cluster is inactivated by deletion of the entire sequence of said cluster.

[0283] According to some embodiments, the first EPS gene cluster is inactivated by modification of the promoter and / or ribosome binding site region (e.g., by introducing at least one mutation therein) resulting in the absence of gene expression.

[0284] According to some embodiments, the second EPS gene cluster is inactivated by deletion of part or all of the sequence of said cluster.

[0285] According to some embodiments, the second EPS gene cluster is inactivated by deletion of the entire coding sequence of said cluster.

[0286] According to some embodiments, the second EPS gene cluster is inactivated by deletion of the entire sequence of said cluster.

[0287] According to some embodiments, the second EPS gene cluster is inactivated by modification of the promoter and / or ribosome binding site region (e.g., by introducing at least one mutation therein) resulting in the absence of gene expression.

[0288] According to some embodiments, the genetically engineered bacteria of the invention are modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 207 or 209. According to some embodiments, the genetically engineered bacteria of the invention are modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 207 or 209. According to some embodiments, the genetically engineered bacteria of the invention are modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 207 or 209.

[0289] According to some embodiments, the genetically engineered bacteria of the invention are modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:208 or 210. According to some embodiments, the genetically engineered bacteria of the invention are modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:208 or 210. According to some embodiments, the genetically engineered bacteria of the invention are modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:208 or 210.

[0290] According to some embodiments, the bacteria of the invention are modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:207, and to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:208. According to some embodiments, the bacteria of the invention are modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:207, and to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:208. According to some embodiments, the bacteria of the invention are modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:207, and to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:208.

[0291] According to some embodiments, the bacteria of the invention are modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:209, and to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:210. According to some embodiments, the bacteria of the invention are modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:209, and to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:210. According to some embodiments, the bacteria of the invention are modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:209, and to delete a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO:210.

[0292] Methods of the Invention The invention also provides a method for producing a biochemical compound comprising culturing a bacterium according to the invention under suitable culture conditions, the method optionally further comprising recovering the biochemical compound from the culture medium.

[0293] According to some embodiments, the present invention provides a method for producing GABA or a derivative thereof. In particular, the present invention provides a method for producing GABA or a derivative thereof, comprising culturing the bacterium according to the present invention in a medium. The method may further comprise recovering GABA or a derivative thereof from the medium. According to some embodiments, the GABA derivative is selected from the group consisting of 2-pyrrolidone, N-methyl-2-pyrrolidone and polybutyrolactam.

[0294] The medium used may be any conventional medium suitable for culturing the bacterial cells of interest and may be constructed according to the principles of the prior art. The medium usually contains all the nutrients necessary for the growth and survival of the respective bacteria, such as carbon and nitrogen sources and other inorganic salts. Suitable media, such as minimal or complex media, are available from commercial suppliers or may be prepared according to published recipes, such as the strain catalogue of the American Type Culture Collection (ATCC). Non-limiting standard media well known to those skilled in the art include Luria-Bertani (LB) broth, Sabouraud dextrose (SD) broth, MS broth, yeast peptone dextrose, BMMY, GMMY or yeast malt extract (YM) broth, all of which are commercially available. Non-limiting examples of media suitable for culturing bacterial cells, such as E. coli cells, include minimal and rich media, such as Luria Broth (LB), M9 medium, M17 medium, SA medium, MOPS medium, Terrific Broth and YT.

[0295] The carbon source may be any suitable carbon substrate known in the art, particularly any carbon substrate commonly used in bacterial and / or fermentative cultures. Non-limiting examples of suitable fermentable carbon substrates include carbohydrates (e.g., C5 sugars such as arabinose or xylose or C6 sugars such as glucose), glycerol, glycerin, acetate, dihydroxyacetone, one-carbon sources, methanol, methane, oils, animal fats, animal oils, vegetable oils, fatty acids, lipids, phospholipids, glycerolipids, monoglycerides, diglycerides, triglycerides, renewable carbon sources, polypeptides (e.g., microbial or vegetable proteins or peptides), yeast extract, components from yeast extract, peptones, casamino acids, or any combination of two or more of the above.

[0296] According to some embodiments, the carbon substrate is selected from the group consisting of a C5 sugar (such as arabinose or xylose), a C6 sugar (such as glucose or fructose), lactose, sucrose, glycerol, glycerin, acetate, corn steep liquor, yeast extract, components from yeast extract, peptone, casamino acids, or combinations thereof.

[0297] When the bacterium of the invention is a methylotrophic bacterium, the medium preferably comprises a reduced one-carbon compound, such as methanol or methylamine, or a multi-carbon compound that does not contain a carbon-carbon bond, such as dimethylamine. Thus, according to some embodiments, the medium comprises methanol as a carbon source. The concentration of methanol in the medium may generally range from about 0.5% w / v to about 4% w / v, such as from about 2% w / v to about 4% w / v. According to some embodiments, the concentration of methanol in the medium ranges from about 2.5% w / v to about 3.5% w / v.

[0298] As nitrogen sources, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, natural nitrogen sources such as peptones, soybean hydrolysates, and digested fermentative microorganisms can be used. As minerals, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, etc. can be used.

[0299] Suitably, the bacteria are cultured under suitable conditions for the production of the desired product. Suitable conditions for culturing each bacterium are well known to those skilled in the art. Typically, the bacteria are cultured at a temperature in the range of about 20 to about 45°C, such as about 30 to about 38°C, for example about 37°C. The culture can be preferably carried out at a temperature of about 20 to about 45°C, such as about 30 to 38°C, preferably about 37°C, by shaking or stirring culture, or under aerobic conditions, such as in a bioreactor with aeration. The pH of the culture is usually greater than 5, such as in the range of about 6 to about 8, preferably about 6.5 to about 7.5, more preferably about 6.8 to about 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases and buffers. The culture may be carried out for a period in the range of 10 to 70 hours, preferably in the range of 24 to 60 hours, more preferably in the range of 36 to 50 hours.

[0300] After culturing, solids such as cells can be removed from the medium by centrifugation or membrane filtration. The biochemical compounds can be recovered by conventional methods for isolating and purifying compounds from the medium. Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, chromatographic methods such as ion exchange, e.g., ion exchange chromatography or gel filtration chromatography, and crystallization methods. The method may further include recovering the biochemical compounds from the medium.

[0301] The biochemical compounds produced by the method of the present invention may be any desired compound obtained by culturing the bacteria of the present invention. Non-limiting examples include organic acids, amino acids, fatty acids and their derivatives. Non-limiting examples of organic acids include fumaric acid, glycolic acid, succinic acid, malic acid, malonic acid, lactic acid and their derivatives. Non-limiting examples of amino acids include glutamic acid, lysine, methionine, tryptophan, phenylalanine and their derivatives.

[0302] Generally, when the bacteria of the invention are used in the production of a biochemical compound, the bacteria have the ability to produce said biochemical compound. This means that when the bacteria are cultured in a medium, the bacteria can produce, excrete or secrete the biochemical compound of interest in the medium or in the bacteria, and / or cause its accumulation. A bacterium is considered to have the ability to produce a biochemical compound of interest when it expresses all the enzymes involved in the biosynthetic pathway to obtain the biochemical compound. The bacterium may be inherently capable of producing the biochemical compound of interest, or may be modified to have the ability to produce the biochemical compound of interest, for example by using recombinant DNA techniques. In the latter case, the bacterium is genetically modified to heterologously express the enzymes required for the biosynthesis of the biochemical compound.

[0303] As an example, a bacterium has the ability to produce GABA if the biochemical compound to be produced is GABA or a derivative thereof. A bacterium is considered to have the ability to produce GABA or a derivative thereof if it expresses all of the enzymes involved in the biosynthetic pathway and GABA or a derivative thereof is obtained.

[0304] To produce GABA, most GABA-producing bacteria use the glutamic acid decarboxylase (GAD) enzyme, which converts glutamic acid to GABA by removing CO2. Thus, a bacterium capable of producing GABA preferably contains (e.g. expresses) a polypeptide having glutamic acid decarboxylase (GAD) activity (EC 4.1.1.15; encoded by genes gadA or gadB or their orthologues). The bacterium may inherently have the ability to produce GABA or its derivatives, or may be modified to have the ability to produce GABA or its derivatives, for example by using recombinant DNA techniques. In the latter case, the bacterium is genetically modified to heterologously express a polypeptide having glutamic acid decarboxylase (GAD) activity.

[0305] As used herein, a "polypeptide having glutamic acid decarboxylase (GAD) activity" refers to a polypeptide (EC 4.1.1.15) that catalyzes the reaction: L-glutamic acid <=> 4-aminobutanoic acid + CO(2). Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 211-220 and variants thereof having at least 70% sequence identity thereto, such as at least 80%, at least 85%, at least 90%, at least 95% or at least 97%.

[0306] Thus, according to some embodiments, the bacterium of the invention expresses a polypeptide having glutamic acid decarboxylase (GAD) activity.

[0307] According to some embodiments, the bacterium of the present invention expresses a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 211-220. According to some embodiments, the bacterium of the present invention expresses a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 211-220. According to some embodiments, the bacterium of the present invention expresses a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 211-220.

[0308] In bacteria, GAD enzymes are involved in the acid stress response. When some bacteria are exposed to low pH, they increase the expression of gad genes. This leads to rapid decarboxylation of glutamic acid, consuming intracellular protons and raising the pH. GABA is produced in the process. Since GAD activity is required in an acidic environment, it is optimally active at pH 4.0-5.0, but is inactive at neutral pH 7.0. The lack of GAD activity under normal physiological conditions in the cytosol, especially at neutral pH, prevents continuous GABA production at neutral pH.

[0309] Thus, although the present invention contemplates the use of wild-type polypeptides having glutamic acid decarboxylase (GAD) activity (i.e., glutamic acid decarboxylases that occur naturally in an organism), it may be advantageous to use mutant GAD enzymes, particularly those that are catalytically active at a pH in the range of pH 6 to pH 8.

[0310] Two approaches were used to improve the pH range of GAD enzymes. In one method, a portion of the C-terminus of GAD that physically blocks the catalytic site of GAD when the environmental pH is neutral was removed. By truncating the C-terminus of Lactobacillus brevis and Lactobacillus plantarum GAD enzymes, the pH range of the enzymes was expanded to 4.0–8.0 (Yu et al., 2012). In the second approach, specific sites on the GAD enzyme were mutated by random mutagenesis to obtain mutant enzymes active at neutral pH. By mutating the penultimate His residue (H465) of E. coli GAD, the C-terminus was again unable to block GAD activity at neutral pH (Pennacchieti et al., 2009). The E89Q mutation in E. coli GAD also prevents the loss of activity at neutral pH (Thu Ho et al., 2013). A similar effect was observed in Lactobacillus brevis GAD into which four amino acid changes (T17I, D294G, E312S, Q346H) were introduced.

[0311] Thus, according to some embodiments, the bacterium of the present invention expresses a polypeptide that has glutamic acid decarboxylase (GAD) activity and exhibits catalytic activity at a pH in the range of pH 6 to pH 8, more preferably at pH 7.0.

[0312] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity is a GAD mutant that contains at least one amino acid substitution in its amino acid sequence that renders it active at a pH in the range of pH 6 to pH 8, compared to the wild-type GAD enzyme from which it is derived.

[0313] According to some embodiments, at least one amino acid substitution is at a position corresponding to position 89 and / or position 465 of SEQ ID NO:211. Thus, according to some embodiments, the GAD mutant comprises an amino acid substitution in its amino acid sequence at a position corresponding to position 89 of SEQ ID NO:211. According to some embodiments, the GAD mutant comprises an amino acid substitution in its amino acid sequence at a position corresponding to position 465 of SEQ ID NO:211. According to some embodiments, the GAD mutant comprises two amino acid substitutions in its amino acid sequence at positions corresponding to positions 89 and 465 of SEQ ID NO:211.

[0314] In some embodiments, the polypeptide is a GAD mutant that has 2 to 20 amino acids, such as 2 to 14 amino acids, deleted from the C-terminus compared to the wild-type GAD enzyme from which it is derived.

[0315] According to some embodiments, the polypeptide having glutamic acid decarboxylase (GAD) activity is selected from the group consisting of: a) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO:211 and comprising an amino acid substitution at a position corresponding to position 89 and / or position 465 of SEQ ID NO:211; b) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO:212 and comprising an amino acid substitution at a position corresponding to position 84 and / or position 457 of SEQ ID NO:212; c) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 213 and comprising an amino acid substitution at a position corresponding to position 93 and / or position 467 of SEQ ID NO: 213; d) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO:214 and comprising an amino acid substitution at a position corresponding to position 90 and / or position 467 of SEQ ID NO:214; e) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 215 and comprising an amino acid substitution at a position corresponding to position 85 and / or position 462 of SEQ ID NO: 215; f) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 216 and comprising an amino acid substitution at a position corresponding to position 89 and / or position 465 of SEQ ID NO: 216; g) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 217 and comprising an amino acid substitution at a position corresponding to position 89 and / or position 466 of SEQ ID NO: 217; h) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 218 and comprising an amino acid substitution at a position corresponding to position 89 and / or position 466 of SEQ ID NO: 218; i) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO:219 and comprising an amino acid substitution at a position corresponding to position 89 and / or position 463 of SEQ ID NO:219; j) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO:220 and comprising amino acid substitutions at positions corresponding to positions 17, 91, 94, 213, 346 and / or 468 of SEQ ID NO:220; and k) a polypeptide having glutamic acid decarboxylase (GAD) activity comprising an amino acid sequence having at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 211, 212, 213, 214, 215, 216, 217, 218, 219 or 220, wherein 2 to 20 amino acids, such as 2 to 14 amino acids, are deleted from the C-terminus. is selected from one of the following:

[0316] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:211, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 465 of SEQ ID NO:211. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:211, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 465 of SEQ ID NO:211. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:211, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 465 of SEQ ID NO:211.

[0317] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:212, and comprises an amino acid substitution at a position corresponding to position 84 and / or position 457 of SEQ ID NO:212. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:212, and comprises an amino acid substitution at a position corresponding to position 84 and / or position 457 of SEQ ID NO:212. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:212, and comprises an amino acid substitution at a position corresponding to position 84 and / or position 457 of SEQ ID NO:212.

[0318] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:213, and comprises an amino acid substitution at a position corresponding to position 93 and / or position 467 of SEQ ID NO:213. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:213, and comprises an amino acid substitution at a position corresponding to position 93 and / or position 467 of SEQ ID NO:213. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:213, and comprises an amino acid substitution at a position corresponding to position 93 and / or position 467 of SEQ ID NO:213.

[0319] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:214, and comprises an amino acid substitution at a position corresponding to position 90 and / or position 467 of SEQ ID NO:214. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:214, and comprises an amino acid substitution at a position corresponding to position 90 and / or position 467 of SEQ ID NO:214. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:214, and comprises an amino acid substitution at a position corresponding to position 90 and / or position 467 of SEQ ID NO:214.

[0320] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:215, and comprises an amino acid substitution at a position corresponding to position 85 and / or position 462 of SEQ ID NO:215. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:215, and comprises an amino acid substitution at a position corresponding to position 85 and / or position 462 of SEQ ID NO:215. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:215, and comprises an amino acid substitution at a position corresponding to position 85 and / or position 462 of SEQ ID NO:215.

[0321] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:216, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 465 of SEQ ID NO:216. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:216, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 465 of SEQ ID NO:216. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:216, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 465 of SEQ ID NO:216.

[0322] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:217, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 466 of SEQ ID NO:217. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:217, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 466 of SEQ ID NO:217. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:217, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 466 of SEQ ID NO:217.

[0323] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:218, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 466 of SEQ ID NO:218. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:218, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 466 of SEQ ID NO:218. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:218, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 466 of SEQ ID NO:218.

[0324] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:219, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 463 of SEQ ID NO:219. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:219, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 463 of SEQ ID NO:219. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:219, and comprises an amino acid substitution at a position corresponding to position 89 and / or position 463 of SEQ ID NO:219.

[0325] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:220, and comprises an amino acid substitution at a position corresponding to position 17, position 91, position 94, position 213, position 346, and / or position 468 of SEQ ID NO:220. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:1220, and comprises an amino acid substitution at a position corresponding to position 17, position 91, position 94, position 213, position 346, and / or position 468 of SEQ ID NO:220. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:220 and comprises amino acid substitutions at positions corresponding to position 17, position 91, position 94, position 213, position 346 and / or position 468 of SEQ ID NO:220.

[0326] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219, or 220, where 2-20 amino acids have been deleted from the C-terminus. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219, or 220, where 2-20 amino acids have been deleted from the C-terminus. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219 or 220, wherein 2-20 amino acids have been deleted from the C-terminus.

[0327] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219, or 220, where 2-14 amino acids have been deleted from the C-terminus. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219, or 220, where 2-14 amino acids have been deleted from the C-terminus. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219 or 220, wherein 2-14 amino acids have been deleted from the C-terminus.

[0328] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219 or 220, where 4-10 amino acids have been deleted from the C-terminus. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219 or 220, where 4-10 amino acids have been deleted from the C-terminus. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO:211, 212, 213, 214, 215, 216, 217, 218, 219 or 220, wherein 4-10 amino acids have been deleted from the C-terminus.

[0329] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 221, with the proviso that the amino acid at position 89 is not E, preferably the amino acid at position 89 is Q. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 221, with the proviso that the amino acid at position 89 is not E, preferably the amino acid at position 89 is Q. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 221, with the proviso that the amino acid at position 89 is not E, preferably the amino acid at position 89 is Q.

[0330] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 223, with the proviso that the amino acid at position 84 is not E, preferably the amino acid at position 84 is Q. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 223, with the proviso that the amino acid at position 84 is not E, preferably the amino acid at position 84 is Q. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 223, with the proviso that the amino acid at position 84 is not E, preferably the amino acid at position 84 is Q.

[0331] According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 70% sequence identity, such as at least 75%, to SEQ ID NO: 225, with the proviso that the amino acid at position 17 is not T, the amino acid at position 294 is not D, the amino acid at position 312 is not E, and / or the amino acid at position 346 is not Q, preferably with the proviso that the amino acid at position 17 is I, the amino acid at position 294 is G, the amino acid at position 312 is S, and / or the amino acid at position 346 is H. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 80% sequence identity, such as at least 85%, to SEQ ID NO: 225, with the proviso that the amino acid at position 17 is not T, the amino acid at position 294 is not D, the amino acid at position 213 is not E, and / or the amino acid at position 346 is not Q, preferably with the proviso that the amino acid at position 17 is I, the amino acid at position 294 is G, the amino acid at position 312 is S, and / or the amino acid at position 346 is H. According to some embodiments, a polypeptide having glutamic acid decarboxylase (GAD) activity comprises an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 225, with the proviso that the amino acid at position 17 is not T, the amino acid at position 294 is not D, the amino acid at position 312 is not E, and / or the amino acid at position 346 is not Q, preferably with the proviso that the amino acid at position 17 is I, the amino acid at position 294 is G, the amino acid at position 312 is S, and / or the amino acid at position 346 is H.

[0332] To achieve heterologous expression of an enzyme required for the biosynthesis of a biochemical compound such as GABA, one or more exogenous nucleic acid molecules containing one or more nucleotide sequences encoding said enzymes are introduced into the bacterium. Thus, the genetically modified bacterium may contain one or more exogenous nucleic acid molecules containing one or more nucleotide sequences of interest encoding said enzymes. To promote the expression of said enzymes in the bacterium, the exogenous nucleic acid molecule may contain a suitable regulatory element, such as a promoter functional in bacterial cells, which causes the production of an mRNA molecule and is operably linked to the nucleotide sequence encoding said enzyme.

[0333] Techniques for introducing foreign nucleic acid molecules, such as DNA molecules, into bacterial cells are well known to those of skill in the art and include, inter alia, transformation (eg, heat shock or natural transformation).

[0334] Preferably, the exogenous nucleic acid molecule comprises, from 5' to 3', at least one transcription unit comprising a promoter that is functional in the bacterium to cause the production of an mRNA molecule and is operably linked to a nucleotide sequence of interest encoding the enzyme and a transcription terminator sequence.

[0335] A useful promoter according to the present invention is any known promoter functional in a given host cell to cause the production of an mRNA molecule. Many such promoters are known to those of skill in the art. Such promoters include promoters normally associated with other genes and / or promoters isolated from any bacteria. The use of promoters for protein expression is generally known to those of skill in the art of molecular biology, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. The promoter used may be an inducible one, such as a temperature-inducible promoter (e.g., the pL or pR phage lambda promoters, each of which can be controlled by the temperature-sensitive lambda repressor c1857). The term "inducible" as used in the context of a promoter means that the promoter induces transcription of an operably linked nucleotide sequence only in the presence of a stimulus, such as a change in temperature or the presence of a chemical (a "chemical inducer"). As used herein, "chemical induction" according to the present invention refers to the physical application of an exogenous or endogenous substance (including macromolecules, such as proteins or nucleic acids) to a host cell. This has the effect that the target promoter present in the host cell increases the transcription rate. Alternatively, the promoter used may be constitutive. The term "constitutive" used in the context of a promoter means that the promoter can induce the transcription of an operably linked nucleotide sequence in the absence of a stimulus (heat shock, chemical, etc.).

[0336] Temperature-inducible systems work, for example, by using promoters that are repressed by thermolabile repressors. These repressors are active at lower temperatures, e.g., 30°C, but are inactive at 37°C because they are not folded correctly. Such circuits can therefore be used to directly regulate genes of interest, also by genomic integration of the gene together with the repressor (St-Pierre et al., 2013). Examples of such temperature-inducible expression systems are based on the pL and / or pR λ phage promoters, which are regulated by the thermolabile cI857 repressor. Similar to the genomically integrated DE3 system, expression of the T7 RNA polymerase gene may also be controlled using a temperature-controlled promoter system (Mertens et al., 1995), and expression of the gene of interest can be controlled using the T7 promoter.

[0337] Non-limiting examples of promoters functional in bacteria include both constitutive and inducible promoters such as the T7 promoter, β-lactamase and lactose promoter systems, alkaline phosphatase (phoA) promoter, tryptophan (trp) promoter system, tetracycline promoter, lambda phage promoter, ribosomal protein promoters, and hybrid promoters such as the tac promoter. Other bacterial and synthetic promoters are also suitable.

[0338] In addition to the promoter, the exogenous nucleic acid molecule may further comprise at least one regulatory element selected from 5' untranslated region (5'UTR) and 3' untranslated region (3'UTR).Many such 5'UTR and 3'UTR from prokaryotes and eukaryotes are well known to those skilled in the art.Such regulatory elements include 5'UTR and 3'UTR normally combined with other genes, and / or 5'UTR and 3'UTR isolated from any bacteria.

[0339] The 5'UTR usually contains a ribosome binding site (RBS), also known as the Shine-Dalgarno sequence, usually located 3-10 base pairs upstream from the start codon.

[0340] The exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or vector. Thus, the exogenous nucleic acid molecule may be a vector, such as an expression vector, or a part of such a vector, such as an expression cassette contained by such a vector. Usually, such a vector remains extrachromosomal in the bacterial cell, meaning that it is present outside the genome of the bacterial cell. Alternatively, the exogenous nucleic acid molecule may be stably integrated (e.g., by random or targeted insertion) into the bacterial genome. In particular, the exogenous nucleic acid molecule may be an expression cassette that is stably integrated (e.g., by random or targeted insertion) into the bacterial genome.

[0341] The present invention also provides biochemical compounds obtainable by the methods detailed above.

[0342] The present invention also provides a method for producing biomass comprising culturing a bacterium according to the present invention under suitable culture conditions.

[0343] Details regarding culture conditions such as media and temperature are provided above in relation to the method for producing biochemical compounds and apply mutatis mutandis.

[0344] Certain other definitions As used herein, "biochemical compound" means any carbon-based compound produced by living organisms.

[0345] The term "mesophilic" as used herein in the context of bacteria means that the bacteria grows best at moderate temperatures, with an optimal growth range of 20-45°C.

[0346] The term "methylotrophic" as used herein in the context of bacteria means that the bacteria can use reduced one-carbon compounds, such as methanol, methane, formate or methylamine, as well as multi-carbon compounds that do not contain carbon-carbon bonds, such as dimethyl ether and dimethylamine, as carbon sources for their growth.

[0347] "Polypeptide" and "protein" are used interchangeably herein to mean a polymer of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes D- and L-amino acids and mixtures of D- and L-amino acids.

[0348] "Nucleic acid" or "polynucleotide" are used interchangeably herein to mean a polymer of at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) covalently linked by phosphodiester bonds, regardless of length or base modification.

[0349] "Recombinant" or "non-naturally occurring," e.g., when used with reference to a host cell, nucleic acid, or polypeptide, refers to material that has been modified in a way that would not otherwise occur in nature, or that is identical thereto, but that has been produced or derived from synthetic material and / or by manipulation using recombinant technology, or material that corresponds to the natural or native form of the material. Non-limiting examples include recombinant bacterial cells, particularly those that express genes not present in the native (non-recombinant) form of the cell, or that express native genes that are otherwise expressed at different levels.

[0350] "Heterologous" or "foreign" as used herein in the context of a gene or nucleic acid molecule refers to a gene or nucleic acid molecule (i.e., a DNA or RNA molecule) that does not naturally occur as part of the genome of the bacterium in which it resides, or that is present in one or more locations in the genome that are different from the location(s) in which it naturally resides. Thus, a "heterologous" or "foreign" gene or nucleic acid molecule is not endogenous to the bacterium and has been introduced exogenously into the microorganism. A "heterologous" gene or nucleic acid molecule DNA molecule may be from a different organism, different species, different genus, or different kingdom as the host DNA.

[0351] "Heterologous" as used herein in the context of a polypeptide means that the polypeptide is not normally present in or produced (i.e., expressed) by the host microbial organism and originates from a different organism, different species, different genus, or different kingdom.

[0352] As used herein, the term "ortholog" refers to a gene, the nucleic acid molecule encoded thereby, i.e., mRNA, or the protein encoded thereby that exists in different species but that originates from a common ancestral gene.

[0353] "Reduced expression" of a gene means that the amount of transcript and the amount of polypeptide (e.g., enzyme) encoded by said gene produced by the modified bacterium are reduced, respectively, compared to an otherwise identical bacterium lacking said modification. More specifically, "reduced expression" of a gene means that the amount of transcript and the amount of polypeptide (e.g., enzyme) encoded by said gene produced by the modified bacterium are reduced by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%, respectively, compared to an otherwise identical bacterium lacking said modification. The level of expression of a gene can be determined by well-known methods, including PCR and Southern blotting. The level of gene expression can also be estimated by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting and quantitative RT-PCR. The amount of polypeptide encoded by a gene can be measured by well-known methods, including ELISA, immunohistochemistry or Western blotting.

[0354] Expression of a gene can be reduced by introducing a mutation into the gene in the genome of the bacterium such that the intracellular activity of the polypeptide encoded by the gene is reduced compared to an otherwise identical bacterium not carrying said mutation. Mutations that result in reduced expression of a gene include the substitution of one or more nucleotides to cause an amino acid substitution in the polypeptide encoded by the gene (missense mutation), the introduction of a stop codon (nonsense mutation), the deletion or insertion of nucleotides to cause a frameshift, the insertion of a drug resistance gene, or the deletion of part of or the entire gene (Qiu and Goodman, 1997; Kwon et al., 2000). Expression can also be reduced by modifying expression control sequences such as promoters, Shine-Dalgarno (SD) sequences, etc. Expression of a gene can also be reduced by gene replacement, such as "lambda Red-mediated gene replacement" (Datsenko and Wanner, 2000). Lambda Red-mediated gene replacement is a particularly suitable method for inactivating one or more of the genes described herein.

[0355] "Inactivating", "inactivation" and "inactivated" when used in the context of a gene or gene cluster, means that the gene or gene cluster of interest no longer expresses a functional protein. The modified DNA region can render the gene or gene cluster unable to be naturally expressed by deleting part or the entire sequence of the gene or gene cluster, shifting the reading frame of the gene or gene cluster, introducing missense / nonsense mutations or modifying the regulatory region of the gene or gene cluster, including sequences that control gene expression, such as promoters, enhancers, attenuators, ribosome binding sites, etc. Preferably, the gene or gene cluster of interest is inactivated by deleting part or the entire sequence of the gene or gene cluster, such as by gene replacement. Inactivation may also be achieved by introducing or expressing a rare-cutting endonuclease that can selectively inactivate the gene or gene cluster of interest by DNA cleavage, preferably by double-strand breaks. "Rare-cutting endonucleases" within the context of the present invention include transcription activator-like effector (TALE) nucleases, meganucleases, zinc finger nucleases (ZFNs) and RNA-guided endonucleases.

[0356] The presence or absence of a gene or gene cluster in a bacterial genome can be detected by well-known methods, including PCR and Southern blotting, etc. The level of gene expression can also be estimated by measuring the amount of mRNA transcribed from a gene or gene cluster using a variety of well-known methods, including Northern blotting and quantitative RT-PCR, etc. The amount of a polypeptide encoded by a gene or gene cluster can be measured by well-known methods, including SDS-PAGE followed by immunoblotting assays (Western blotting analysis), etc.

[0357] As used herein, "reduced" expression of a polypeptide (such as a polypeptide described herein), "reducing" or "reducing" it means that expression of the polypeptide in the modified bacterium is reduced compared to expression of the polypeptide in an otherwise identical bacterium (control) that does not have the modification. Expression of the polypeptide in the modified bacterium may be reduced by at least about 10%, preferably at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100%, or any percentage in whole integers between 10% and 100% (e.g., 6%, 7%, 8%, etc.) compared to expression of the polypeptide in an otherwise identical bacterium (control) that does not have the modification. More specifically, "reduced" polypeptide expression, "reducing" it or its "reduction" means that the amount of the polypeptide in the modified bacterium is reduced by at least about 10%, preferably at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% or any whole integer percentage between 10% and 100% (e.g., 6%, 7%, 8%, etc.) compared to the amount of said polypeptide in an otherwise identical bacterium (control) lacking the modification. Expression or amount of the polypeptide in the bacterium can be determined by any suitable means known in the art, including techniques such as ELISA, immunohistochemistry, Western blotting or flow cytometry.

[0358] As used herein, "eliminated" expression of a polypeptide (such as a polypeptide described herein) means that expression of said polypeptide in an altered bacterium is not detectable compared to expression of said polypeptide in an otherwise identical bacterium (control) that does not have the alteration.

[0359] As used herein, "reduced" polypeptide (such as an enzyme described herein) activity, "reducing" thereof or "reduction" thereof means that the catalytic activity of the polypeptide in the modified bacterium is reduced compared to the catalytic activity of the polypeptide in an otherwise identical bacterium (control) that does not have the modification. The activity of the polypeptide in the modified bacterium may be reduced by at least about 10%, preferably at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100%, or any percentage of whole integers between 10% and 100% (e.g., 6%, 7%, 8%, etc.) compared to the expression of the polypeptide in an otherwise identical bacterium (control) that does not have the modification. The activity of the polypeptides in bacteria can be determined by any suitable protein and enzyme activity assays.

[0360] "Expression" includes any step involved in producing a polypeptide (e.g., an encoded enzyme), including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0361] As used herein, a "regulatory region" of a gene or gene cluster refers to a nucleic acid sequence that influences expression of a coding sequence. Regulatory regions are known in the art and include, but are not limited to, promoters, enhancers, transcription terminators, polyadenylation sites, matrix attachment regions and / or other elements that regulate expression of a coding sequence.

[0362] "Substitution" or "substituted" refers to the modification of a polypeptide by replacing one amino acid residue with another, for example, the replacement of a serine residue with a glycine or alanine residue in a polypeptide sequence is an amino acid substitution. When used in reference to a polynucleotide, "substitution" or "substituted" refers to the modification of a polynucleotide by replacing one nucleotide with another, for example, the replacement of a cytosine with a thymine in a polynucleotide sequence is a nucleotide substitution.

[0363] "Conservative substitution", when used in reference to a polypeptide, refers to the replacement of an amino acid residue with a different residue having a similar side chain, and thus typically includes the replacement of an amino acid in a polypeptide with an amino acid in the same or similar class of amino acid.By way of example and not limitation, an amino acid having an aliphatic side chain may be replaced with another aliphatic amino acid, such as alanine, valine, leucine and isoleucine, an amino acid having a hydroxyl side chain is replaced with another amino acid having a hydroxyl side chain, such as serine and threonine, an amino acid having an aromatic side chain is replaced with another amino acid having an aromatic side chain, such as phenylalanine, tyrosine, tryptophan and histidine, an amino acid having a basic side chain is replaced with another amino acid having a basic side chain, such as lysine and arginine, an amino acid having an acidic side chain is replaced with another amino acid having an acidic side chain, such as aspartic acid or glutamic acid, and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0364] "Non-conservative substitutions," when used in reference to a polypeptide, refer to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between groups rather than within a defined group, and affect (a) the structure of the peptide backbone in the area of ​​substitution (e.g., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulk of its side chain. By way of example and not limitation, exemplary non-conservative substitutions may be an acidic amino acid replaced with a basic or aliphatic amino acid, an aromatic amino acid replaced with a small amino acid, and a hydrophilic amino acid replaced with a hydrophobic amino acid.

[0365] As used herein, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded nucleic acid loop into which additional nucleic acid segments can be ligated. Certain vectors can induce the expression of genes operatively linked thereto. Such vectors are referred to herein as "expression vectors." Certain other vectors can facilitate the insertion of foreign nucleic acid molecules into the genome of bacteria. Such vectors are referred to herein as "transformation vectors." In general, vectors of utility in recombinant nucleic acid technology are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably with plasmids, the most commonly used form of vector. Many suitable vectors are known to those of skill in the art and are commercially available.

[0366] As used herein, a "promoter" refers to a sequence of DNA, usually upstream (5') of the coding region of a structural gene, that controls expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors that may be required for initiation of transcription. The choice of promoter depends on the nucleic acid sequence of interest. A suitable "promoter" is generally one that is capable of supporting the initiation of transcription in the bacteria of the invention, resulting in the production of an mRNA molecule.

[0367] As used herein, "operably linked" refers to a juxtaposition in a relationship permitting the components described to function in their intended manner. A control 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. A promoter sequence is "operably linked" to a gene if it is sufficiently proximal to the transcription start site of the gene to regulate transcription of the gene.

[0368] "Percentage of sequence identity" or "sequence identity %" and "identity %" refer to the sequence identity between a nucleotide sequence and a reference nucleotide sequence or between an amino acid sequence and a reference amino acid sequence. Sequence identity can be determined by comparing positions in each sequence that can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between nucleotide or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids, respectively, at positions shared by the nucleotide or amino acid sequences. A variety of alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and can be used with default settings.

[0369] "Reference sequence" or "reference amino acid sequence" refers to a defined sequence that is compared to another sequence. In the context of the present invention, a reference amino acid sequence may be, for example, the amino acid sequence set forth in SEQ ID NO:1.

[0370] As used herein, a "derivative" of GABA refers to a compound derived from GABA by its modification. Non-limiting examples of GABA "derivatives" include 2-pyrrolidone, N-methyl-2-pyrrolidone, and polybutyrolactam (nylon 4).

[0371] As used herein, the term "about" means ±10% of the value of the number with which it is used.

[0372] Where a numerical limitation or range is recited herein, the endpoints are included, and all values ​​and subranges within the numerical limitation or range are specifically included as if they were expressly written out.

[0373] As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more," unless the context clearly dictates otherwise.

[0374] As used herein, the terms "comprising," "including," and "having," as well as grammatical variations thereof, should be construed as specifying the stated features, steps, or components, but not excluding the addition of one or more further features, steps, components, or groups thereof.

[0375] Having generally described the invention, a further understanding can be obtained by reference to specific embodiments, which are provided herein for purposes of illustration only and are not intended to be limiting, unless otherwise specified. EXAMPLES

[0376] Example 1: Identification of the polyphosphate kinase (ppk) gene in Methylobacillus Wild-type Methylobacillus was found to consume much more phosphate than required for biomass formation by standard biomass preparation methods. Polyphosphate was identified as a potential phosphate sink, and the presence of intracellular polyphosphate granules was confirmed by fluorescence microscopy and polyphosphate-specific staining with DAPI fluorescent dye. Synthesis and degradation of polyP in the microorganism was catalyzed by polyphosphate kinase enzyme (ppk). To identify genes that contribute to polyP formation in Methylobacillus, a BLAST search was performed against all available genomes from the genus Methylobacillus using the known ppk gene sequence (SEQ ID NO: 11) from E. coli. This yielded 10 hits (data not shown). The same was repeated for the genus Methylobacterium, yielding 176 hits (data not shown). Sequence alignments using the MAFT algorithm were performed using SnapGene version 6.0 software. Residues forming the active site (Zhu et al., 2005) and necessary for polyphosphate synthesis activity in the E. coli enzyme were identified from the literature (Tzeng and Kornberg, 2000). The alignment revealed that essential amino acids at positions corresponding to R375, S380A, F488A, P507A, R564A, R621A, Q674A in E. coli ppk1 (SEQ ID NO: 11), and active site amino acids corresponding to amino acids H435 and H592 in E. coli (SEQ ID NO: 11) are conserved in more than 95% of the analyzed sequences (data not shown).

[0377] Example 2: Deletion of the polyphosphate kinase gene in Methylobacillus flagellatus The polyphosphate kinase gene (SEQ ID NO:2) of Methylobacillus flagellatus was deleted from the chromosome by homologous recombination of a DNA construct containing a selection marker into the original ppk gene locus. Briefly, a 2000 base pair sequence upstream of the ppk gene and a 2000 base pair sequence downstream of this gene were amplified from genomic DNA by PCR. These fragments were added to an antibiotic resistance cassette containing its own promoter, terminator and ribosome binding site. The resulting linear construct was transformed into a Methylobacillus strain by electroporation. Transformants were successfully identified using a selection step on antibiotic-containing minimal medium agar plates with methanol as carbon source. DNA integration into the correct locus was confirmed by colony PCR. The entire region was amplified from the genome after integration and confirmed by DNA sequencing. To ensure that the polyphosphate gene was not present in the genome, a set of PCR primers was designed to specifically target the gene. Colony PCR utilizing these internal primers did not amplify any fragments, providing further evidence that the ppk gene was no longer present.

[0378] Example 3: Deletion of the polyphosphate kinase gene in Methylobacillus glycogenes The polyphosphate kinase gene (SEQ ID NO: 4) of Methylobacillus glycogenes was deleted from the chromosome by homologous recombination of a DNA construct containing a selection marker into the original ppk gene locus. Briefly, a 2000 base pair sequence upstream of the ppk gene and a 2000 base pair sequence downstream of this gene were amplified from genomic DNA by PCR. These fragments were added to a kanamycin resistance cassette containing its own promoter and terminator, and the linear construct was transformed into a Methylobacillus strain by electroporation. Transformants were successfully identified using a selection step on kanamycin-containing minimal methanol medium agar plates, and DNA integration into the correct locus was confirmed by colony PCR. The entire region was amplified from the genome after integration and confirmed by DNA sequencing. To ensure that the polyphosphate gene was not present in the genome, a set of PCR primers was designed to specifically target the ppk gene. Negative colony PCR utilizing internal primers was used in combination with other evidence as an indication that the ppk gene was no longer present.

[0379] Example 4: Comparison of polyP granules in wild-type and ppK-deficient strains Cultures of M. flagellatus with and without a deletion of the ppK gene (SEQ ID NO:2) were grown in minimal methanol medium in 5 liter bioreactors and shake flasks. After achieving sufficient cell density (OD600: approximately 2), samples from each culture were stained with 40 micromolar 4,6-diamidino-2-phenylindole (DAPI). The staining solution was prepared in phosphate-buffered saline at pH 7. DAPI is often used as a DNA stain, but it can also be used to stain polyphosphate granules when added to samples at higher concentrations (micromolar range). When bound to polyphosphate granules, the emission spectrum of the dye underwent a spectral shift from blue to yellow to higher wavelengths. Samples were excited with ultraviolet (405 nm) light and viewed through a long-pass 430 emission filter. Cells expressing the ppK gene were observed to contain polyphosphate granules, whereas cells in which the ppK gene was deleted did not contain granules (Figure 1).

[0380] Example 5: Comparison of fermentation with and without biomass generation (i.e. ppK destruction) Alternating left-right bioreactor fermentations were performed with Methylobacillus flagellatus strains with and without the ppK gene. A mineral medium containing KH2PO4, Na2HPO4, MgSO4, NH4SO4 and a trace element mixture was used. Methanol concentration was dynamically maintained at 4-5 g / L throughout the fermentation by a feedback loop using an online proprietary methanol sensor. pH was maintained at 7 by automatic addition of ammonium hydroxide, which also served as a nitrogen source. Bioprocess parameters such as optical density, CFU count, total broth dry weight and free phosphate were measured periodically. In the non-ppK deletion bioprocess, rapid cell lysis occurred when the optical density reached approximately 30, which was observed by a drop in optical density, colony forming units (CFU) and a 99% reduction in methanol and ammonium hydroxide feed rates. In the absence of the ppK gene, the optical density reached a higher value of about 45-50, the CFU remained constant upon reaching stationary phase, and no sharp drop in the feed rate was observed, indicating that the cells remained metabolically active during stationary phase. Stationary phase capable of product synthesis could be maintained for several days (Figure 2).

[0381] Example 6: Comparison of GABA production with and without the ppK deletion Alternating left-right bioreactor fermentations were performed using GABA-producing Methylobacillus flagellatus with and without the ppK gene. To enable GABA production in the strain, a glutamate dehydrogenase enzyme (Eco GAD mutant E89Q ΔC14) was expressed from E. coli encoding the protein according to SEQ ID NO: 221 (SEQ ID NO: 222). The expressed E. coli GAD enzyme contained the point mutation E89Q and was truncated at the C-terminus by removing the last 14 amino acids. These modifications were introduced to reduce the pH dependence of the native E. coli GAD enzyme and allow GABA synthesis at neutral pH. The gene was expressed from a plasmid under the control of the IPTG-inducible lacO / trc promoter.

[0382] A mineral medium containing KH2PO4, Na2HPO4, MgSO4, NH4SO4 and a trace element mixture was used. Methanol concentration was dynamically maintained at 4-5 g / L throughout the fermentation by a feedback loop using an online proprietary methanol sensor. pH was maintained at 7 by automatic addition of ammonium hydroxide, which also served as a nitrogen source. Samples from both bioreactors were taken at regular intervals and analyzed for GABA content by HPLC. In the presence of the ppK gene, cell lysis occurred when the culture reached an optical density of approximately 30. This was followed by a surge in GABA production as the expressed GAD enzyme was released into the fermentation broth along with other cellular contents. No further production of GABA was detected after this initial surge. In the case of the ppK deletion strain, GABA production was constant throughout the process and accumulated during the stationary phase (Figure 3).

[0383] Example 7: Bioinformatic analysis of acyl homoserine lactone synthases Programmed cell death has been linked to quorum sensing mechanisms. In Gram-negative bacteria, quorum sensing depends on N-acyl homoserine lactones (AHLs) synthesized by N-acyl homoserine lactone synthases. To explore the presence of quorum sensing-related gene clusters in M. flagellatus, we utilized the antiSMASH 6.0 (Blin et al., 2021) algorithm to predict gene clusters using the entire M. flagellatus genome (NCBI accession number: NC_007947.1) as input. With this algorithm, we attempted to map characteristic gene clusters with known AHL synthesis functions to the analyzed genome (Figure 4). We identified a single homoserine lactone production cluster (sequence number 227) containing a homoserine lactone synthase gene (sequence number 14). We then used BLAST to identify homologs of the M. flagellatus enzyme (SEQ ID NO: 13) in the genomes of Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus rhizosphaerae, Methylobacterium organophilum, Methylolblum extorquense and Methylolblum extorquense_AM1. We identified several candidate AHL synthase proteins in the organisms (SEQ ID NOs: 15, 17, 19, 21, 23 and 25, respectively).

[0384] Example 8: Deletion of the AHL synthase gene in M. flagellatus The AHL synthase gene (SEQ ID NO: 14) of M. flagellatus was deleted from the chromosome by homologous recombination of a DNA construct containing a selection marker into the original AHL gene locus. Briefly, a 2000 base pair sequence upstream of the AHL synthase gene and a 2000 base pair sequence downstream of the gene were amplified from genomic DNA by PCR. These fragments were added to an antibiotic resistance cassette containing its own promoter, terminator and ribosome binding site. The resulting linear construct was transformed into a M. flagellatus strain by electroporation. Transformants were successfully identified using a selection step on antibiotic-containing minimal medium agar plates with methanol as carbon source. DNA integration into the correct locus was confirmed by colony PCR. The entire region was amplified from the genome after integration and confirmed by DNA sequencing. To ensure that the AHL synthase gene was not present in the genome, a set of PCRs was designed to specifically target the gene. Colony PCR utilizing these internal primers did not amplify any fragments, providing further evidence that the AHL synthase gene was no longer present.

[0385] Example 9: Comparison of biomass production with and without AHL synthase deficiency with carbon limitation A staggered bioreactor fermentation was performed with Methylobacillus flagellatus strains with and without the AHL synthase gene (SEQ ID NO: 14). A mineral medium containing KH2PO4, Na2HPO4, MgSO4, NH4SO4 and a trace element mixture was used. Carbon limitation was achieved by feeding methanol through a feedback loop connected to a dissolved oxygen sensor at a rate that maintained dissolved oxygen at 30% throughout the fermentation. pH was maintained at 7 by automatic addition of ammonium hydroxide, which also served as a nitrogen source. Bioprocess parameters such as optical density, CFU count, total broth dry weight and free phosphate were measured periodically. In the wild-type bioprocess, rapid cell lysis occurred once the optical density reached approximately 26, which was observed by a drop in optical density, colony forming units (CFU) and a 99% drop in the feed rates of methanol and ammonium hydroxide. In the absence of the AHL synthase gene, OD and CFU remained constant upon reaching stationary phase. Stationary phase could be maintained, during which product synthesis could take place (Figure 5).

[0386] Example 10: Identification of EPS-producing genes in Methylobacillus flagellatus and Methylobacillus glycogenes Based on bioinformatic analysis, several genomic loci were selected for disruption. The gene locations of two EPS-related gene clusters in M. flagellatus have been published previously (Chistoserdova et al., 2007). To annotate the genes in the first M. flagellatus EPS-producing gene cluster (SEQ ID NOs: 113–138), a custom-written script in the Python programming language was used to compare the homologs of genes from the published M. spp. 12S methanol synthesis cluster (Yoshida et al., 2000) to the M. flagellatus genome by NCBI BLAST protein search. Top hits were selected based on a calculated combined score consisting of E-value, query coverage and sequence identity. The single top hit for each gene was then manually mapped to the genome sequence and annotated. The locations of these top hits were consistent with the published location of the first cluster. Since no obvious homologues were found for each gene, all genes between the most distant homologues were considered as part of the cluster. Most such unannotated genes encoded sugar and polysaccharide-related genes. Using the same procedure, we identified a completely unannotated EPS cluster in Methylobacillus glycogenes (SEQ ID NOs: 151-172).

[0387] A second EPS-related cluster was predicted in the genome sequence of Methylobacillus flagellatus (SEQ ID NOs: 139-150). A BLAST search was performed comparing these genes to the methanol cluster, and no obvious homologs were found. The protein products of each individual gene in this cluster were identified by an NCBI protein BLAST search against all non-redundant protein sequences, and the top hits for each were selected as above. The protein sequences of each gene from this Methylobacillus flagellatus protein cluster were cross-searched in the genome of Methylobacillus glycogenes using NCBI protein BLAST. The top hits were found to form a similar cluster in Methylobacillus glycogenes (SEQ ID NOs: 173-198).

[0388] The open reading frames (ORFs) identified in each EPS-related gene cluster are shown in Tables 1 to 4 below. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0389] For deletion, the ORFs annotated as epsD, epsE, epsF, and epsG (SEQ ID NOs: 32-35 in M. flagellatus and SEQ ID NOs: 68-71 in M. glycogenes) were selected based on their similarity to essential genes for EPS production in Methylobacillus sp. strain 12 (Yoshida et al., 2003). All 12 ORFs in M. flagellatus (SEQ ID NOs: 53-64) and all 26 ORFs in Methylobacillus glycogenes (SEQ ID NOs: 87-112) were selected for deletion because the second EPS cluster had no obvious homologues in other distinct EPS clusters.

[0390] Example 11: DNA Deletion Modified strains of ABME5 and ABME6 were prepared in which either one or both EPS production clusters were deleted. Genome editing was achieved by introducing a linear DNA fragment consisting of a kanamycin or rifampicin resistance cassette flanked by 2 kb-long sequences upstream and downstream of the integration site. The DNA fragments were transformed into ABME5 and ABMR6 by electroporation. Transformants were plated on selective PM7 plates containing 20 g / L agar, 50 μg / mL kanamycin or 5 μg / mL rifampicin for 24–48 h. ABME5-derived strains were incubated at 30°C. ABME6-derived strains were grown at 37°C. Correct clones were confirmed by colony PCR. Strains prepared by DNA deletion are listed in Table 6. [Table 6]

[0391] Example 12: Measurement of total EPS in shake flask cultures before and after EPS gene disruption in Methylobacillus flagellatus To evaluate the effect of deleting the EPS production genes in ABME6, three modified ABME6 strains (ABME80, ABME82, and ABME131) were prepared as described in Example 11 and tested for EPS production.

[0392] Confirmed transformants were grown overnight on PM7 + 50 μg / mL kanamycin agar plates. Single colonies were resuspended in 25 mL of PM7 medium + 25 μg / mL kanamycin in a 250 mL baffled shake flask and incubated overnight at 200 rpm. The next day, 50 mL of PM7 medium + 25 μg / mL kanamycin in a 250 mL baffled shake flask was inoculated with 2.5 mL of the overnight culture. All ABME5-derived strains were grown at 30°C and all ABME6-derived strains were grown at 37°C. These cultures were then cooled on ice, dispensed into 2 mL Eppendorf tubes, and centrifuged at 15000 rpm for 15 min at 4°C in a standard tabletop centrifuge. When a layer of EPS became visible on the cell pellet, it was transferred back into the supernatant and mixed. The culture supernatant was saved for EPS hydrolysis. Hydrolysis was performed by adding half the supernatant volume of 6 M TFA (trifluoroacetic acid) to obtain a final TFA concentration of 3 M. The tubes were placed in a thermoblock heated to 120° C. and incubated for 6 h. After hydrolysis, the liquid (including TFA) was evaporated at 80° C. until completely dry. The dried mass was resuspended in 0.5 M NaOH (same volume as the original culture aliquot) and analyzed for monosaccharides by HPLC. [Table 7]

[0393] After centrifugation, ABME6 produced a visible slime layer above the cell pellet and reached approximately 1 g / L of total monosaccharides after hydrolysis. Single cluster disruption (ABME80 and ABME82) did not have a visible slime layer but still produced approximately 500 mg / L of total sugars. Interestingly, the glucose:total sugar ratio was reversed depending on which gene cluster was disrupted, likely due to the production of two different types of EPS. When both clusters were disrupted (strain ABME131), sugars were below the detection limit.

[0394] Example 13: Measurement of viscosity in reactor cultures of Methylobacillus flagellatus before and after EPS gene disruption To evaluate the change in fermentation broth viscosity before and after disruption of EPS production, the strains were compared in 5 liter bioreactor fermentations under oxygen limitation. Bioreactor seed cultures were always prepared as follows: 25 mL of PM7 medium in a 250 mL baffled shake flask was inoculated with 250 uL of strain cryostock with the stock's OD adjusted to 5 to a starting OD of 0.05. These cultures were incubated overnight at 200 rpm and 37°C. In the morning, 200 mL of PM7 medium in a 2 L baffled shake flask was inoculated with 10% (20 mL) of the overnight culture and grown to an OD of approximately 1 (+ / - 0.2). This culture was then used to inoculate a 2.5 L starting volume of sterile PM3 medium in a 5 L bioreactor (8% inoculum). The pH was maintained at 7 by automatic addition of NH4OH and the temperature was maintained at 37°C.

[0395] For the oxygen-limited process, methanol was automatically fed to maintain the concentration at 3-6 g / L. The reactor aeration and agitation were maintained at maximum, but the oxygen concentration always dropped to 0% during the process. The broth was sampled periodically for OD, mineral and metabolite measurements. Viscosity was measured at the end of the process and the results are summarized in Figure 7. The wild-type M. flagellatus fermentation broth had a high viscosity of 400 centipoise at the time of sampling. A different species of Methylobacillus, namely ABME145, was also measured and produced a similarly viscous broth of 422 centipoise. The dual disruption produced a visually low viscous broth that could be centrifuged and filtered using standard benchtop equipment (something that was not possible with past samples). This assessment was confirmed by a viscometer, which measured 35 cP, or nearly a 10-fold drop in viscosity. A representative sample of E. coli fermentation broth was also measured for comparison.

[0396] Example 14: Measuring the reduction in foaming in shake flasks before and after EPS gene removal in Methylobacillus flagellatus We observed that the EPS-deficient strains exhibited significantly less foaming. To compare the foam formation of ABME6 to the EPS-deficient mutant strains (ABME82 and ABME131), the strains were grown as described in Example 12.

[0397] To measure foam formation, the flasks were removed from the shaker and the liquid was allowed to settle for 5 min. The layer of foam on top of the liquid was measured with a tape measure (Figure 8). Wild-type cultures produced a 0.5-1 cm layer of foam on top of the liquid, and single cluster disruption led to the accumulation of only approximately 1 mm of foam at the liquid meniscus. No foam was visible at all in the double disruption cultures.

[0398] Example 15: Measuring reduction in foaming in bioreactor cultures before and after EPS gene disruption in Methylobacillus flagellatus To evaluate the change in fermentation broth foaming before and after disruption of EPS production, the strains were compared in 5 liter bioreactor fermentations. The amount of foam formation was estimated by the amount of antifoaming agent (SAG5693) added. The fermentations were performed under carbon limitation.

[0399] The reactor was inoculated as described in Example 12. To achieve carbon limitation, 50% methanol was automatically fed to the reactor at a dynamically calculated rate that maintained the pO2 value at approximately 30% at maximum aeration and agitation settings. The added methanol was consumed immediately and maintained below detection levels. Antifoam was automatically added when foam reached a detector placed above the fermentation broth meniscus. ABME6 and ABME131 were tested in this manner. For ABME131, antifoam addition began after 5 hours. Depending on the fermentation time, approximately 60% less antifoam was added for the ABME131 fermentation (Figure 9).

[0400] Example 16: Improved methanol tolerance of EPS-free M. flagellatus Tolerance to high methanol concentrations is an important parameter for efficient bioprocessing. To ensure that methanol tolerance is not reduced in strains unable to produce EPS, growth at increasing methanol concentrations was tested in ABME6 and its EPS-producing derivative strain ABME131.

[0401] The strains were tested in shake flasks as described in Example 13. To test for methanol tolerance, the initial methanol concentration was increased to 4.5%. OD600 was measured after 24 hours.

[0402] Surprisingly, ABME131 had higher methanol tolerance compared to ABME6. For example, at 2.75% initial methanol concentration, EPS-free ABME131 grew to an OD600 of 1.6, while wild-type ABME6 only grew to an OD600 of 0.3. The difference in growth was most pronounced at 2.75%. However, improved growth of ABME131 compared to ABME6 was evident at all methanol concentrations (Figure 11).

[0403] Example 17: Disruption of polyphosphate kinase at the transcriptional level by inserting a terminator A promoter sequence is required for the RNA polymerase to begin transcribing a gene into mRNA. Transcription ends when the RNA polymerase reaches a terminator sequence, which results in its release from the transcription complex. Thus, transcription of a gene can be disrupted by removing the promoter sequence or by inserting a terminator sequence before or within the gene. Both of these interventions are performed in Methylobacillus flagellatus by inserting a terminator sequence, such as the lambda terminator (SEQ ID NO: 230), immediately before the gene. This allows the gene to be successfully transcribed in its entirety after releasing the RNA polymerase. Briefly, a 2000 base pair sequence upstream of the ppk gene and a 2000 base pair sequence within this gene are amplified from genomic DNA by PCR. These fragments are added to an antibiotic resistance cassette with the terminator sequence at the 3' end. The resulting linear construct, containing the first 2000 bp of the ppk gene with the terminator immediately before the start codon, is transformed into a Methylobacillus strain by electroporation. Transformants were successfully identified using a selection step on antibiotic-containing minimal medium agar plates with methanol as the carbon source. DNA integration into the correct locus is confirmed by colony PCR. After integration, the entire region is amplified from the genome and confirmed by DNA sequencing to ensure that the terminator sequence is located directly upstream of the gene on the chromosome.

[0404] Example 18: Disruption of polyphosphate kinase expression at the translational level by mutation of the ribosome binding site A 6-nucleotide ribosome binding site (RBS) complementary to the 3' end of the 16S ribosomal subunit is required for ribosomes to bind to transcribed mRNA molecules and initiate translation. The level of translation is determined by the affinity of the 16S ribosomal subunit for the RBS. Optimal binding affinity in M. flagellatus is achieved with an RBS of "AGGAGA" based on the sequence of the 16S ribosomal subunit (SEQ ID NO: 228). Replacing the RBS with a sequence with lower affinity, such as "ATATAT", reduces expression of the protein. Such a replacement is made in M. flagellatus by homologous recombination. Because the exact RBS of a gene is difficult to predict, the 20 base pairs before the start codon of the ppk gene are replaced with 10 "AT" repeats. Briefly, a 2000 base pair sequence upstream of the ppk gene and a 2000 base pair sequence downstream of this gene are amplified from genomic DNA by PCR. These fragments are added to an antibiotic resistance cassette and a negative selection marker containing its own promoter, terminator and ribosome binding site. The resulting linear construct is transformed by electroporation into a Methylobacillus strain to delete the original ppk. A selection step on antibiotic-containing minimal medium agar plates with methanol as carbon source is used to identify successful transformants. DNA integration into the correct locus is confirmed by colony PCR. After integration, the entire region is amplified from the genome and confirmed by DNA sequencing. A second linear fragment is then prepared by amplifying the ppk sequence from the wild-type M. flagellatus genome by PCR using primers containing 20 "A" repeats before the start codon. The ppk gene containing the "A" repeats is then linked with the 2000 bp upstream and downstream fragments and transformed into a deletion strain. Successful transformants are screened using the negative selection marker, where only cells that have removed the original resistance cassette in favor of the modified sequence survive to form colonies. Finally, the integration of the 20 "AT" repeats is confirmed by DNA sequencing.

[0405] Example 19: Disruption of polyphosphate kinase at the protein function level gene by frameshift mutation Frameshift mutations are caused by the insertion or deletion of any number of nucleotides not divisible by three. As a result, the reading frame shifts, completely changing the amino acid sequence of the translated protein and rendering it non-functional. A frameshift mutation is performed in Methylobacillus flagellatus by first deleting the native ppk sequence and then replacing it with a modified DNA sequence, in which the "T" nucleotide at position 4 (immediately following the stop codon) is removed (SEQ ID NO: 229). Briefly, a 2000 base pair sequence upstream of the ppk gene and a 2000 base pair sequence downstream of this gene are amplified from genomic DNA by PCR. These fragments are added to an antibiotic resistance cassette and a negative selection marker containing its own promoter, terminator and ribosome binding site. The resulting linear construct is transformed by electroporation into a Methylobacillus strain to delete the original ppk. A selection step on antibiotic-containing minimal medium agar plates with methanol as carbon source is used to successfully identify transformants. DNA integration into the correct locus is confirmed by colony PCR. After integration, the entire region is amplified from the genome and confirmed by DNA sequencing. A second linear fragment is then prepared by amplifying the ppk sequence from the wild-type M. flagellatus genome by PCR using primers that eliminate the "T" at position 4 of the ppk gene. The modified ppk gene is then ligated with the 2000 bp upstream and downstream fragments and transformed into a deletion strain. Successful transformants are screened using a negative selection marker, where only cells that have removed the original resistance cassette supporting the modified sequence survive to form colonies. Finally, integration of the 20 "AT" repeats is confirmed by DNA sequencing.

[0406] Example 20: Comparison of fermentation of Methylobacillus strains with and without biomass production (i.e. ppK destruction) Alternating left-right bioreactor fermentations were performed using Methylobacillus strains with and without the ppK gene. A mineral medium containing KH2PO4, Na2HPO4, MgSO4, NH4SO4 and a trace element mixture was used. Methanol concentration was dynamically maintained at 4-5 g / L throughout the fermentation by a feedback loop using an online proprietary methanol sensor. pH was maintained at 7 by automatic addition of ammonium hydroxide, which also served as a nitrogen source. Bioprocess parameters such as optical density, CFU count, total broth dry weight and free phosphate were measured periodically. In the non-ppK deletion bioprocess, rapid cell lysis occurred when the optical density reached approximately 30, which was observed by a drop in optical density, colony forming units (CFU) and a 99% reduction in methanol and ammonium hydroxide feed rates. In the absence of the ppK gene, the optical density reached a higher value of about 45-50, the CFU remained constant upon reaching stationary phase, and no sharp drop in the feed rate was observed, indicating that the cells remained metabolically active during stationary phase. Stationary phase capable of product synthesis could be maintained for several days (Figure 2).

[0407] List of references cited in this specification Pennacchietti E, Lammens TM, Capitani G, Franssen MC, John RA, Bossa F, De Biase D., 2009. "Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH", J Biol Chem. 284(46):31587-96. doi:10.1074 / jbc.M109.049577. Thu Ho, NA, Hou, CY, Kim, WH, Kang, TJ, 2013. "Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness", J. Biosci. Bioeng. https: / / doi.org / 10.1016 / j.jbiosc.2012.09.002. Yu, K., Lin, L., Hu, S., Huang, J., Mei, L., 2012. "C-terminal truncation of glutamate decarboxylase from Lactobacillus brevis CGMCC 1306 extends its activity toward near-neutral pH", Enzyme Microb. Technol. 50, 263-269. https: / / doi.org / 10.1016 / j.enzmictec.2012.01.010. Blin, K. et al. (2021) "antiSMASH 6.0: improving cluster detection and comparison capabilities", Nucleic Acids Res., 49(W1):W29-W35. doi:10.1093 / nar / gkab335. Chistoserdova, L. et al. (2007) "Genome of Methylobacillus flagellatus, Molecular Basis for Obligate Methylotrophy, and Polyphyletic Origin of Methylotrophy", Journal of Bacteriology. American Society for Microbiology Journals, 189(11):4020-4027. Yoshida, T. et al. (2000) "Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S", Applied Microbiology and Biotechnology. doi:10.1007 / s002530000407. Yoshida, T. et al. (2003) "Genes involved in the synthesis of the exopolysaccharide methanolan by the obligate methylotroph Methylobacillus sp. strain 12S", Microbiology. Microbiology Society, pp. 431-444. doi:10.1099 / mic.0.25913-0. Qiu Z and Goodman MF. "The Escherichia coli polB locus is identical to dinA, the structural gene for DNA polymerase II. Characterization of Pol II purified from a polB mutant." J Biol Chem. 1997, 272(13):8611-8617. Kwon DH, Pena JA, Osato MS, Fox JG, Graham DY, Versalovic J. Frameshift mutations in rdxA and metronidazole resistance in North American Helicobacter pylori isolates. J Antimicrob Chemother 2000,46(5):793-796. Datsenko KA, Wanner BL "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products", Proc Natl Acad Sci USA 2000,97:6640-6645. Zhu Y, Huang W, Lee SS, Xu W. "Crystal structure of a polyphosphate kinase and its implications for polyphosphate synthesis." EMBO Rep. 2005;6(7):681-7. Tzeng CM, Kornberg A. "The multiple activities of polyphosphate kinase of Escherichia coli and their subunit structure determined by radiation target analysis." J Biol Chem. 2000;275(6):3977-83.

Claims

1. A genetically engineered bacterium of the genus Methylobacillus that has been modified to reduce the expression level and / or activity of an endogenous polypeptide having polyphosphate kinase activity by at least 70% compared to an otherwise identical bacterium lacking said modification, wherein said endogenous polypeptide comprises an amino acid sequence having at least 75% sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 12.

2. The bacterium described in claim 1, which has been modified to reduce the expression level of an endogenous polypeptide having polyphosphate kinase activity by at least 70% compared to an identical bacterium without said modification.

3. The bacterium of claim 1 , wherein the endogenous gene encoding the polypeptide having polyphosphate kinase activity is inactivated.

4. 2. The bacterium according to claim 1, wherein the endogenous gene encoding the polypeptide having polyphosphate kinase activity is inactivated by deletion of part or the entire gene sequence.

5. The bacterium of claim 1, which has been further modified to reduce the expression and / or activity of an endogenous polypeptide having acylhomoserine lactone (AHL) synthase activity compared to an otherwise identical bacterium lacking said modification.

6. The bacterium of claim 1, which has been modified to reduce expression of an endogenous polypeptide having acyl-homoserine-lactone (AHL) synthase activity, compared to an otherwise identical bacterium lacking said modification.

7. The bacterium of claim 5, wherein the endogenous gene encoding the polypeptide having acylhomoserine lactone (AHL) synthase activity is inactivated.

8. The bacterium according to claim 7, wherein the endogenous gene encoding the polypeptide having acyl-homoserine-lactone (AHL) synthase activity is inactivated by deletion of part or all of the gene sequence.

9. 2. The bacterium of claim 1, which has been modified to reduce exopolysaccharide (EPS) production compared to an otherwise identical bacterium lacking said modification.

10. 10. The bacterium of claim 9, which has been modified to reduce the expression and / or activity of at least one endogenous polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium compared to an otherwise identical bacterium lacking said modification.

11. The bacterium according to any one of claims 1 to 10, which is selected from Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus caricics, and Methylobacillus methilovorans.

12. The bacterium according to any one of claims 1 to 10, wherein the bacterium is Methylobacillus flagellatus or Methylobacillus glycogenes.

13. A method for producing a biochemical compound, comprising culturing a bacterium according to any one of claims 1 to 10 under suitable culture conditions.

14. 14. The method of claim 13, wherein the medium comprises methanol.

15. A method for producing biomass, comprising culturing the bacterium according to any one of claims 1 to 10 under suitable culture conditions.

Images