Genetically engineered Methylobacillus bacteria with improved properties

Abstract

The present invention relates generally to biotechnology, and specifically to genetically modified bacteria of the genus Methylobacillus that have improved properties that make them particularly useful for 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 otherwise identical bacteria without 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, and specifically to genetically modified bacteria of the genus Methylobacillus that have improved properties that make them particularly useful for 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 otherwise identical bacteria without the modification. The present invention further provides methods for producing biochemical compounds using the genetically modified bacteria of the present invention.

[0002] Background of the Invention Methanol fermentation has the potential to replace petroleum-derived chemical production. To achieve this, efficient methylotrophic strains (those that consume one-carbon compounds, such as methanol or methane, or multicarbon compounds without carbon-carbon bonds) must be developed. Several bacteria have been identified that can utilize methanol as a sole carbon source for growth and product formation. Some examples include Bacillus methanolicus, Methylobacterium extorquens, Methylobacillus glycogenes, and Methylobacillus flagellatus. Methylotrophic bacteria can be broadly divided into two groups based on the methanol assimilation pathway. The first step is always the conversion of methanol to formaldehyde, catalyzed by the enzyme methanol dehydrogenase. In the first group, formaldehyde reacts with ribulose monophosphate (RuMP) to form a C6 compound, which is subsequently further metabolized. Members of this group are commonly referred to as "RuMP cycle methylotrophs." The second group, including M. extorquens, assimilates formaldehyde by reacting it with glycine to form serine in a pathway known as the serine cycle. Both groups of methylotrophic bacteria have been used to produce various biochemicals from methanol. However, the RuMP cycle is more energy efficient than the serine cycle and results in faster growth and biomass formation.

[0003] Methylotrophic microorganisms can exist in a wide variety of environments, including the ocean, soil, plant rhizosphere, and even sewage. Because methylotrophic bacteria must be able to survive these diverse and often harsh environments, they often tend to be highly robust and tolerant to various types of stress while growing efficiently with minimal nutrients. Methylobacillus strains that utilize the RuMP cycle possess many desirable traits for biochemical production systems, including rapid and reproducible growth in minimal mineral media and insensitivity to fluctuations in temperature and methanol concentration. Furthermore, these strains are highly amenable to genetic manipulation, unlocking the potential for metabolic engineering of complex pathways.

[0004] Many methylotrophs, including Methylobacillus, produce large amounts of exopolysaccharides (EPS), which confer multiple benefits to the organism in its natural environment, such as protection from desiccation, biofilm scaffolding, and energy storage. In fed-batch fermentation, Methylobacillus biomass can consist of up to 30% EPS.

[0005] EPS production in Methylobacillus in particular has been relatively unstudied. Studies have been conducted on only a single strain, Methylobacillus sp. 12S, an obligate methylotroph isolated from soil, which was found to synthesize a novel EPS called methanolane (Yoshida et al., 2000). The gene cluster responsible for methanolane synthesis in Methylobacillus sp. 12S has been identified and characterized (Yoshida et al., 2003), but no such studies have been conducted on other Methylobacillus species. Based on bioinformatics analysis and sequence homology, EPS gene clusters have been predicted in Methylobacillus flagellatus strain KT ( Chistoserdova et al., 2007 ) and Methylobacillus glycogenes ( Hattori et al., 2020 ), but the genes themselves have not been characterized.

[0006] Summary of the Invention The objective of the present invention is to overcome certain drawbacks in methanol-based bioprocesses. This is achieved by the inventors' engineering of various genetically modified bacteria of the genus Methylobacillus that have reduced production of exopolysaccharides (EPS).

[0007] We investigated general EPS production in Methylobacillus. Based on bioinformatics analysis of the genomes of M. flagellatus and M. glycogenes, we identified two putative EPS-producing gene clusters. Disruption of both EPS clusters completely eliminated EPS production.

[0008] An unexpected and surprising observation in the engineered M. flagellatus and M. glycogenes strains was their behavior in growth cultures. Eliminating expression of one or more endogenous polypeptides involved in exopolysaccharide (EPS) production in Methylobacillus bacteria resulted in cultures exhibiting a number of surprising and unexpected properties beneficial for the development of methanol-based bioprocesses. Unexpectedly, excessive culture foaming was reduced in both shake flasks and bioreactors. Furthermore, while cell clumping was observed in the unmodified parent strain, clumping was reduced or absent in the engineered Methylobacillus cultures. Eliminating EPS production also improved biomass centrifugation and filtration. The combination of these properties significantly improved bioprocessing, strain handling, and downstream processing, making the engineered Methylobacillus bacteria suitable for large-scale methanol fermentation. Elimination of EPS synthesis also unexpectedly improved the methanol tolerance of M. flagellatus and other Methylobacillus species.

[0009] Based on the above findings, in a first aspect, the present invention provides a genetically modified bacterium of the genus Methylobacillus that has been modified so that production of exopolysaccharide (EPS) is reduced compared to an otherwise identical bacterium without the modification.

[0010] The present invention further provides a method for producing a biochemical compound, comprising culturing a bacterium of the invention under suitable culture conditions in a culture medium comprising a reduced one-carbon compound, such as methanol, or a multi-carbon compound without a carbon-carbon bond, such as dimethylamine.

[0011] The present invention can be summarized by the following items: 1. A genetically modified bacterium of the genus Methylobacillus that has been modified to reduce exopolysaccharide (EPS) production compared to an otherwise identical bacterium without the modification.

[0012] 2. The bacterium according to item 1, wherein the expression and / or function (e.g., activity) of at least one endogenous polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium has been modified such that it is reduced compared to an otherwise identical bacterium (reference bacterium) that does not have the modification.

[0013] 3. The bacterium according to item 1 or 2, wherein the expression of at least one endogenous polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium has been modified so as to be reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0014] 4. The bacterium according to any one of items 1 to 3, wherein the expression of at least two endogenous polypeptides involved in the production of exopolysaccharide (EPS) in the bacterium has been modified to be reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0015] 5. The bacterium according to any one of items 1 to 4, wherein the expression of at least three endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium has been modified to be reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0016] 6. The bacterium according to any one of items 1 to 5, wherein the expression of at least four endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium has been modified to be reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0017] 7. The bacterium according to any one of items 1 to 6, wherein the expression of at least five endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium has been modified to be reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0018] 8. The bacterium according to any one of items 1 to 7, wherein the expression of at least six endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium has been modified to be reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0019] 9. The bacterium according to any one of items 1 to 8, wherein the expression of all endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacterium has been modified to be reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0020] 10. The bacterium according to any one of items 2 to 9, wherein at least one polypeptide involved in the production of exopolysaccharide (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: 1 to 38, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 1 to 38 and having the same functional properties as the reference polypeptide.

[0021] 11. The bacterium according to any one of items 2 to 10, wherein at least one polypeptide involved in the production of exopolysaccharide (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: 1 to 26, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1 to 26 and having the same functional properties as the reference polypeptide.

[0022] 12. The bacterium according to any one of items 2 to 10, wherein at least one polypeptide involved in the production of exopolysaccharide (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 to 38, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 27 to 38 and having the same functional properties as the reference polypeptide.

[0023] 13. The bacterium according to any one of items 2 to 12, wherein at least one polypeptide involved in the production of exopolysaccharide (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: 39 to 86, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 39 to 86 and having the same functional properties as the reference polypeptide.

[0024] 14. The bacterium according to any one of items 2 to 13, wherein at least one polypeptide involved in the production of exopolysaccharide (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: 39 to 60, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 39 to 60 and having the same functional properties as the reference polypeptide.

[0025] 15. The bacterium according to any one of items 2 to 13, wherein at least one polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is selected from the group consisting of a) a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 61 to 86, and b) a polypeptide comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 61 to 86 and having the same functional properties as the reference polypeptide.

[0026] 16. The bacterium according to any one of items 2 to 15, wherein 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 acting as polysaccharide exporters; polypeptides acting as chain length determining proteins; and polypeptides having protein tyrosine kinase activity.

[0027] 17. The bacterium described in item 16, 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: 6, the polypeptide capable of acting as a polysaccharide exporter comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7, the polypeptide acting as a chain length determining protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8, and the polypeptide having protein tyrosine kinase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9.

[0028] 18. The bacterium described in item 16, 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: 42, the polypeptide capable of acting as a polysaccharide exporter comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 43, the polypeptide acting as a chain length determining protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 44, and the polypeptide having protein tyrosine kinase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 45.

[0029] 19. The bacterium according to any one of items 2 to 18, 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.

[0030] 20. The bacterium according to any one of items 2 to 19, wherein the expression of at least one polypeptide involved in the production of exopolysaccharide (EPS) in said bacterium is abolished compared to an otherwise identical bacterium.

[0031] 21. The bacterium according to any one of items 2 to 20, wherein an endogenous gene encoding the polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is inactivated.

[0032] 22. The bacterium according to any one of items 2 to 21, wherein the 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.

[0033] 23. The bacterium according to any one of items 2 to 22, wherein the endogenous genes encoding the polypeptides involved in the production of exopolysaccharides (EPS) in the bacterium have been inactivated by introducing into or expressing into the bacterium the respective rare-cutting endonucleases capable of selectively inactivating the endogenous genes encoding the polypeptides by DNA cleavage.

[0034] 24. The bacterium described in item 23, 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.

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

[0036] 26. The bacterium described in item 25, which contains (e.g., expresses) at least one single guide RNA (sgRNA) that specifically hybridizes (e.g., binds) to genomic DNA encoding the enzyme under cellular conditions.

[0037] 27. A bacterium according to any one of items 2 to 21, wherein the 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.

[0038] 28. A bacterium according to any one of items 2 to 21, wherein the expression of a polypeptide involved in the production of exopolysaccharide (EPS) in the 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 to) under cellular conditions cellular mRNA and / or genomic DNA encoding the polypeptide.

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

[0040] 30. The bacterium described in item 29, wherein the interfering RNA molecule is a microRNA (miRNA), a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

[0041] 31. The bacterium according to any one of items 2 to 30, wherein the endogenous polypeptide(s) involved in the production of exopolysaccharide (EPS) in the bacterium are encoded by a gene(s) contained in a first EPS gene cluster and / or a second EPS gene cluster.

[0042] 32. The bacterium described in item 31, wherein the first EPS gene cluster is defined by the open reading frame (ORF) found in SEQ ID NO: 174 or 177 or comprises a gene orthologous thereto.

[0043] 33. The bacterium according to item 31 or 32, wherein the first EPS gene cluster comprises the genes epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS, or orthologues thereof.

[0044] 34. The bacterium according to any one of items 31 to 33, wherein the first EPS gene cluster comprises the genes epsD, epsE, epsF and epsG, or orthologues thereof.

[0045] 35. The bacterium according to any one of items 31 to 34, wherein the first EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 174 or 177.

[0046] 36. The bacterium according to any one of items 31 to 34, wherein the first EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 174.

[0047] 37. The bacterium according to any one of items 31 to 36, wherein at least one gene in the first EPS gene cluster is inactivated, for example by deletion of part or the entire gene sequence.

[0048] 38. The bacterium according to any one of items 31 to 37, wherein at least one gene selected from epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS, or an orthologue thereof, is inactivated, for example by deletion of part or the entire gene sequence.

[0049] 39. The bacterium according to any one of items 31 to 38, 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.

[0050] 40. The bacterium according to any one of items 31 to 39, wherein the genes epsD, epsE, epsF and epsG, or their orthologues, are inactivated, for example by deletion of part or the entire gene sequence.

[0051] 41. A bacterium according to item 39 or 40, wherein the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9.

[0052] 42. The bacterium according to item 41, wherein the gene epsD comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 92, the gene epsE comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 93, the gene epsF comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 94, and the gene epsG comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 95.

[0053] 43. The bacterium according to item 39 or 40, wherein the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 42, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 43, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 44, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 45.

[0054] 44. The bacterium according to item 43, wherein the epsD gene comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 128, the gene epsE comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 129, the gene epsF comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 130, and the gene epsG comprises a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 131.

[0055] 45. The bacterium according to any one of items 31 to 44, wherein the first EPS gene cluster is inactivated.

[0056] 46. ​​The bacterium according to any one of items 31 to 45, wherein the first EPS gene cluster is inactivated by deletion of part or all of the sequence of said cluster.

[0057] 47. The bacterium according to any one of items 31 to 45, wherein the first EPS gene cluster is inactivated by modification of the promoter and / or ribosome binding site region (e.g., introduction of at least one mutation into said region) resulting in the absence of gene expression.

[0058] 48. The bacterium of any one of items 31 to 47, wherein the second EPS gene cluster is defined by an open reading frame (ORF) found in SEQ ID NO: 175 or 179 or comprises a gene orthologous thereto.

[0059] 49. The bacterium of any one of items 31 to 48, wherein the second EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 175 or 179.

[0060] 50. The bacterium according to any one of items 31 to 48, wherein the second EPS gene cluster comprises a nucleotide sequence having at least 50% sequence identity to the nucleotide sequence of SEQ ID NO: 175.

[0061] 51. The bacterium according to any one of items 31 to 50, wherein at least one gene in the second EPS gene cluster is inactivated, for example by deletion of part or the entire gene sequence.

[0062] 52. The bacterium according to any one of items 31 to 51, wherein the second EPS gene cluster is inactivated.

[0063] 53. The bacterium according to any one of items 31 to 52, wherein the second EPS gene cluster is inactivated by deletion of part or all of the sequence of said cluster.

[0064] 54. A bacterium according to any one of items 31 to 52, wherein the second EPS gene cluster is inactivated by modification (e.g., introduction of at least one mutation) of the promoter and / or ribosome binding site region, resulting in the absence of gene expression.

[0065] 55. The bacterium according to any one of items 1 to 52, wherein all endogenous genes encoding enzymes involved in the production of exopolysaccharide (EPS) in said bacterium are inactivated, e.g., deleted.

[0066] 56. The bacterium according to any one of items 1 to 55, wherein the bacterium is selected from Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus caricics, Methylobacillus methilovorans, and Methylobacillus species.

[0067] 57. The bacterium according to any one of items 1 to 56, wherein the bacterium is selected from Methylobacillus flagellatus and Methylobacillus glycogenes.

[0068] 58. The bacterium according to any one of items 1 to 55, wherein the bacterium is Methylobacillus flagellatus.

[0069] 59. The bacterium according to any one of items 1 to 55, wherein the bacterium is Methylobacillus glycogenes.

[0070] 60. A method for producing a biochemical compound, comprising culturing the bacterium according to any one of items 1 to 59 under suitable culture conditions in a culture medium containing a reduced one-carbon compound, such as methanol, or a multi-carbon compound without a carbon-carbon bond, such as dimethylamine.

[0071] 61. The method of item 60, wherein the culture medium contains methanol.

[0072] 62. The method according to item 60 or 61, wherein the culturing is carried out in a bioreactor.

[0073] 63. The method according to any one of items 60 to 62, wherein the biochemical compound is selected from organic acids, amino acids, fatty acids and their derivatives. [Brief explanation of the drawings]

[0074] [Figure 1] FIG. 1 shows the sugar content of hydrolyzed M. flagellatus shake flask supernatants. Dark gray bars represent total glucose in mg / L, and light gray bars represent total monomeric sugars. Error bars are the standard deviation of technical replicates. [Figure 2] FIG. 1 shows the measured viscosity of bioreactor broth produced by various strains of Methylobacillus flagellatus. [Figure 3] Photographs of wild-type M. flagellatus and M. glycogenes cultures after 24 hours of incubation. On the left are wild-type ABME 5 and 6, followed by a single cluster disruption in M. flagellatus, and finally a double disruption. [Figure 4]FIG. 1 shows a plot of antifoam addition (y-axis) during two representative bioreactor fermentations of ABME 6 and ABME 131 against fermentation time (x-axis). [Figure 5] Phase contrast microscopy images of bioreactor fermentation broth of wild-type Methylobacillus flagellatus (left) versus the double disruptant strain ABME 131 (right). [Figure 6] FIG. 1 shows the improved growth of EPS-free ABME 131 compared to the parent strain.

[0075] The present invention will now be described in more detail below.

[0076] Detailed Description of the Invention Unless otherwise 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.

[0077] All methods and materials similar or equivalent to those described herein can be used in carrying out or testing the present invention, and suitable methods and materials 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, shall prevail.Furthermore, unless otherwise specified, materials, methods and examples are merely illustrative and are not intended to be limiting.

[0078] The practice of the present invention will employ, 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 explained fully in the literature.For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA);Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press);Oligonucleotide Synthesis (MJ Gait ed., 1984);Mullis et al. al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harries & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory).

[0079] The bacterium of the present invention As noted above, the present invention is based on the unexpected and surprising discovery that certain drawbacks in methanol-based bioprocesses can be overcome by downregulating exopolysaccharide (EPS) production in bacteria of the genus Methylobacillus.

[0080] Thus, in a first aspect, the present invention provides a genetically modified bacterium of the genus Methylobacillus that has been modified such that its production of exopolysaccharide (EPS) is reduced compared to an otherwise identical bacterium without the modification.

[0081] More specifically, the present invention provides genetically modified bacteria of the genus Methylobacillus, which have been modified such that the expression and / or activity of at least one endogenous polypeptide involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0082] According to some embodiments, the genetically modified bacteria of the invention have been modified such that expression of at least one, e.g., at least two, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0083] According to some embodiments, the genetically modified bacteria of the invention have been modified such that expression of at least three, such as at least four, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0084] According to some embodiments, the genetically modified bacteria of the invention have been modified such that expression of at least five, such as at least six, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0085] According to some embodiments, the genetically modified bacteria of the invention have been modified such that the expression of at least seven, such as at least eight, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0086] According to some embodiments, the genetically modified bacteria of the invention have been modified such that expression of at least nine, such as at least ten, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0087] According to some embodiments, the genetically modified bacteria of the invention have been modified such that the expression of at least 11, such as at least 12, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0088] According to some embodiments, the genetically modified bacteria of the invention have been modified such that expression of at least 13, such as at least 14, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0089] According to some embodiments, the genetically modified bacteria of the invention have been modified such that expression of at least 15, such as at least 16, endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium (reference bacterium) without the modification.

[0090] According to some embodiments, the genetically modified bacteria of the invention have been modified such that expression of all endogenous polypeptides involved in the production of exopolysaccharide (EPS) in said bacteria is reduced compared to an otherwise identical bacterium without the modification (reference bacterium).

[0091] 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: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-38; and b) a polypeptide comprising an amino acid sequence having at least 70%, e.g., 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: 1-38, and having the same functional properties as the reference polypeptide.

[0092] 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: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-26; and b) a polypeptide comprising an amino acid sequence having at least 70%, e.g., 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: 1-26, and having the same functional properties as the reference polypeptide.

[0093] 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: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 27-38; and b) a polypeptide comprising an amino acid sequence having at least 70%, e.g., 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: 27-38, and having the same functional properties as the reference polypeptide.

[0094] 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: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 39-86; and b) a polypeptide comprising an amino acid sequence having at least 70%, e.g., 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: 39-86, and having the same functional properties as the reference polypeptide.

[0095] 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: a) a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 39-60; and b) a polypeptide comprising an amino acid sequence having at least 70%, e.g., 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: 39-60, and having the same functional properties as the reference polypeptide.

[0096] According to some embodiments, 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: 61-86; and b) a polypeptide comprising an amino acid sequence having at least 70%, e.g., 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: 61-86, and having the same functional properties as the reference polypeptide.

[0097] 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 a polypeptide having peptidyl prolyl cis-trans isomerase activity, a polypeptide capable of acting as a polysaccharide efflux transporter; a polypeptide acting as a chain length determining protein; and a polypeptide having protein tyrosine kinase activity.

[0098] According to some embodiments, the polypeptide having peptidyl prolyl cis-trans isomerase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:6.

[0099] According to some embodiments, the polypeptide capable of acting as a polysaccharide efflux transporter comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:7.

[0100] According to some embodiments, the polypeptide that acts as a chain length determining protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:8.

[0101] According to some embodiments, the polypeptide having protein tyrosine kinase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:9.

[0102] According to some embodiments, the polypeptide having peptidyl prolyl cis-trans isomerase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:42.

[0103] According to some embodiments, the polypeptide capable of acting as a polysaccharide efflux transporter comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:43.

[0104] According to some embodiments, the polypeptide that acts as a chain length determining protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:44.

[0105] According to some embodiments, the polypeptide having protein tyrosine kinase activity comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:45.

[0106] The expression level of the endogenous polypeptide may, for example, 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.

[0107] The reduction of expression of endogenous polypeptides can be achieved by any suitable means known in the art, for example, expression can be reduced by inactivating endogenous genes encoding said polypeptides involved in the production of exopolysaccharides (EPS) in bacteria, for example by deletion of part or the entire gene sequence.

[0108] According to some embodiments, expression of at least one polypeptide involved in exopolysaccharide (EPS) production in the bacterium is abolished compared to an otherwise identical bacterium.

[0109] According to some embodiments, the endogenous genes encoding the polypeptides involved in the production of exopolysaccharides (EPS) in bacteria are inactivated, for example by deletion of part or the entire gene sequence.

[0110] According to some embodiments, the endogenous gene encoding the polypeptide involved in exopolysaccharide (EPS) production in the bacterium is inactivated by introducing or expressing a rare-cutting endonuclease into the bacterium, which is capable of selectively inactivating the endogenous gene encoding the enzyme by DNA cleavage. The rare-cutting endonuclease used in accordance with the present invention to inactivate the endogenous gene can be, for example, a transcription activator-like effector (TALE) nuclease, a meganuclease, a zinc finger nuclease (ZFN), or an RNA-guided endonuclease.

[0111] 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 of target locations in the genome. The CRISPRi system consists of a catalytically inactive "dead" Cas9 protein (dCas9) and a guide RNA that defines the binding site of dCas9 on DNA.

[0112] According to some embodiments, the expression of an endogenous polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is reduced by inhibition.

[0113] The expression of the above-mentioned polypeptide can be inhibited by any suitable means known in the art.For example, the expression can be inhibited by gene silencing techniques, which involve the use of inhibitory nucleic acid molecules such as antisense oligonucleotides, ribozymes or interfering RNA (RNAi) molecules, for example, microRNA (miRNA), small interfering RNA (siRNA) or short hairpin RNA (shRNA).

[0114] According to some embodiments, expression of an endogenous polypeptide involved in the production of exopolysaccharide (EPS) in a bacterium is reduced (e.g., inhibited) by transcriptional and / or translational repression of the endogenous gene encoding said polypeptide.

[0115] According to some embodiments, expression of an endogenous polypeptide involved in the production of exopolysaccharides (EPS) in bacteria is inhibited by introducing or expressing an inhibitory nucleic acid molecule into the bacterium. For example, the inhibitory nucleic acid molecule can be introduced by an exogenous nucleic acid molecule comprising a nucleotide sequence encoding the inhibitory nucleic acid molecule operably linked to a promoter, e.g., an inducible promoter, that functions to cause the production of the inhibitory nucleic acid molecule in the bacterium. Preferably, the inhibitory nucleic acid molecule specifically hybridizes (e.g., binds) under cellular conditions to 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.

[0116] 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 contiguous nucleotides of the complement of cellular mRNA and / or genomic DNA encoding the polypeptide of interest.

[0117] According to some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide, which 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.

[0118] According to some embodiments, the inhibitory nucleic acid molecule is a ribozyme, such as a hammerhead ribozyme, which is designed to catalytically cleave mRNA transcripts to prevent translation of the polypeptide of interest.

[0119] 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 expression, typically causing the destruction of a specific mRNA. Exemplary types of RNAi molecules include microRNA (miRNA), small interfering RNA (siRNA), and short 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.

[0120] According to some embodiments, the bacteria of the invention have been modified such that the function (e.g., activity) of at least one polypeptide involved in the production of exopolysaccharide (EPS) in the bacterium is reduced compared to an otherwise identical microorganism (reference bacterium) without the modification.

[0121] The reduction in function (e.g., activity) of at least one endogenous polypeptide can be achieved by any suitable means known in the art. For example, if the polypeptide is an enzyme, the activity can 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 can be, for example, at least one non-conservative amino acid substitution.

[0122] As mentioned above, the present inventors have identified two putative EPS gene clusters in the Methylobacillus genome, and the endogenous polypeptide(s) involved in the production of exopolysaccharides (EPS) are thus encoded by the gene(s) contained in the first EPS gene cluster and / or the second EPS gene cluster.

[0123] According to some embodiments, the endogenous polypeptide(s) involved in the production of exopolysaccharide (EPS) in the bacterium are encoded by a gene(s) contained in a first EPS gene cluster and / or a second EPS gene cluster.

[0124] According to some embodiments, the first EPS gene cluster is defined by the open reading frame (ORF) found in SEQ ID NO: 174 or 177 or includes genes orthologous thereto.

[0125] According to some embodiments, the first EPS gene cluster comprises the genes epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS, or orthologues thereof.

[0126] According to some embodiments, the first EPS gene cluster comprises the genes epsD, epsE, epsF and epsG, or orthologues thereof.

[0127] According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 50%, such as at least 55%, sequence identity to the nucleotide sequence of SEQ ID NO: 174 or 177.

[0128] According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 60%, such as at least 65%, sequence identity to the nucleotide sequence of SEQ ID NO: 174 or 177.

[0129] According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to the nucleotide sequence of SEQ ID NO: 174 or 177.

[0130] According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to the nucleotide sequence of SEQ ID NO: 174 or 177.

[0131] According to some embodiments, the first EPS gene cluster comprises a nucleotide sequence having at least 90%, such as at least 93%, sequence identity to the nucleotide sequence of SEQ ID NO: 174 or 177.

[0132] 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: 174 or 177.

[0133] According to some embodiments, the first EPS gene cluster has at least 50%, e.g., 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 and nucleotide sequences having 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% or at least 99% sequence identity.

[0134] According to some embodiments, the first EPS gene cluster has at least 50%, e.g., 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 and nucleotide sequences having 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% or at least 99% sequence identity.

[0135] According to some embodiments, the second EPS gene cluster is defined by the open reading frame (ORF) found in SEQ ID NO: 175 or 179 or includes genes orthologous thereto.

[0136] According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 50%, such as at least 55%, sequence identity to the nucleotide sequence of SEQ ID NO: 175 or 179.

[0137] According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 60%, such as at least 65%, sequence identity to the nucleotide sequence of SEQ ID NO: 175 or 179.

[0138] According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to the nucleotide sequence of SEQ ID NO: 175 or 179.

[0139] According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to the nucleotide sequence of SEQ ID NO: 175 or 179.

[0140] According to some embodiments, the second EPS gene cluster comprises a nucleotide sequence having at least 90%, such as at least 85%, sequence identity to the nucleotide sequence of SEQ ID NO: 175 or 179.

[0141] According to some embodiments, the second EPS gene cluster has at least 50%, e.g., 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 and nucleotide sequences having 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% or at least 99% sequence identity.

[0142] According to some embodiments, the second EPS gene cluster has at least 50%, e.g., 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 and nucleotide sequences having 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% or at least 99% sequence identity.

[0143] 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.

[0144] According to some embodiments, the at least one gene is selected from epsD, epsE, epsF, epsG, epsB, epsL, epsH, epsI, epsJ and epsS, or an orthologue thereof.

[0145] According to some embodiments, the at least one gene is selected from epsD, epsE, epsF and epsG, or orthologues thereof.

[0146] 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.

[0147] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO:6, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO:7, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO:8, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO:9.

[0148] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 6, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 7, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 8, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 9.

[0149] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 6, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 7, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 8, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 9.

[0150] According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 92, the gene epsE comprises a nucleotide sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 93, the gene epsF comprises a nucleotide sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 94, and the gene epsG comprises a nucleotide sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 95.

[0151] According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 92, the gene epsE comprises a nucleotide sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 93, the gene epsF comprises a nucleotide sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 94, and the gene epsG comprises a nucleotide sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 95.

[0152] According to some embodiments, the epsD gene comprises a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 92, the gene epsE comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 93, the gene epsF comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 94, and the gene epsG comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 95.

[0153] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 42, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 43, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 44, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 45.

[0154] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 42, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 43, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 44, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 45.

[0155] According to some embodiments, the gene epsD encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 42, the gene epsE encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 43, the gene epsF encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 44, and the gene epsG encodes a polypeptide comprising an amino acid sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 45.

[0156] According to some embodiments, the gene epsD comprises a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 128, the gene epsE comprises a nucleotide sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 129, the gene epsF comprises a nucleotide sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 130, and the gene epsG comprises a nucleotide sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 131.

[0157] According to some embodiments, the gene epsD comprises a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 128, the gene epsE comprises a nucleotide sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 129, the gene epsF comprises a nucleotide sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 130, and the gene epsG comprises a nucleotide sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 131.

[0158] 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: 128, the gene epsE comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 129, the gene epsF comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 130, and the gene epsG comprises a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 131.

[0159] According to some embodiments, the genetically modified bacteria of the present invention have been modified to inactivate the first EPS gene cluster.

[0160] According to some embodiments, the genetically modified bacteria of the present invention are (further) modified to inactivate the second EPS gene cluster.

[0161] According to some embodiments, the genetically modified bacteria of the present invention have been modified to inactivate the first EPS gene cluster and the second EPS gene cluster.

[0162] According to some embodiments, the first EPS gene cluster is inactivated by deletion of part or all of the sequence of said cluster.

[0163] According to some embodiments, the first EPS gene cluster is inactivated by deletion of the entire coding sequence of said cluster.

[0164] According to some embodiments, the first EPS gene cluster is inactivated by deletion of the entire sequence of said cluster.

[0165] According to some embodiments, the first EPS gene cluster is inactivated by modifying the promoter and / or ribosome binding site region (e.g., by introducing at least one mutation) resulting in the absence of gene expression.

[0166] According to some embodiments, the second EPS gene cluster is inactivated by deletion of part or all of the sequence of said cluster.

[0167] According to some embodiments, the second EPS gene cluster is inactivated by deletion of the entire coding sequence of said cluster.

[0168] According to some embodiments, the second EPS gene cluster is inactivated by deletion of the entire sequence of said cluster.

[0169] According to some embodiments, the second EPS gene cluster is inactivated by modifying the promoter and / or ribosome binding site region (e.g., by introducing at least one mutation) to cause a lack of gene expression.

[0170] According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 181 or 183. According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 181 or 183. According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 181 or 183.

[0171] According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 182 or 184. According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 182 or 184. According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 182 or 184.

[0172] According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 181 and to delete a nucleotide sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 182. According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 181 and to delete a nucleotide sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 182. According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 181 and to delete a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 182.

[0173] According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 70%, such as at least 75%, sequence identity to SEQ ID NO: 183, and to delete a nucleotide sequence having at least 70%, such as at least 75% sequence identity to SEQ ID NO: 184. According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 80%, such as at least 85%, sequence identity to SEQ ID NO: 183, and to delete a nucleotide sequence having at least 80%, such as at least 85% sequence identity to SEQ ID NO: 184. According to some embodiments, the genetically modified bacteria of the invention have been modified to delete a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 183, and to delete a nucleotide sequence having at least 90%, such as at least 95% sequence identity to SEQ ID NO: 184.

[0174] According to some embodiments, the genetically modified bacteria of the invention have been modified to delete all endogenous genes encoding polypeptides involved in the production of exopolysaccharides (EPS) in said bacteria.

[0175] The bacterium according to the present invention can be produced from any suitable bacterium of the genus Methylobacillus, which is a genus of Gram-negative methylotrophic bacteria.

[0176] According to some embodiments, the bacterium of the present invention is selected from Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis, and Methylobacillus rhizosphaerae.

[0177] According to some embodiments, the bacterium of the present invention is selected from Methylobacillus flagellates and Methylobacillus glycogenes.

[0178] According to some embodiments, the bacterium of the present invention is Methylobacillus flagellatus.

[0179] According to some embodiments, the bacterium of the present invention is Methylobacillus glycogenes.

[0180] Methods of the Invention The present invention also provides a method for producing a biochemical compound, comprising culturing a bacterium according to the present invention under suitable culture conditions in a culture medium comprising a reduced one-carbon compound, such as methanol, or a multi-carbon compound without a carbon-carbon bond, such as dimethylamine.

[0181] The culture medium used can be any conventional medium suitable for culturing the bacterial cells in question and can be constructed according to prior art principles. The medium typically contains all nutrients required for the growth and survival of the respective bacteria, such as carbon and nitrogen sources, as well as other inorganic salts. Suitable media, such as minimal or complex media, are available from commercial suppliers or can be prepared according to published recipes, such as the strain catalogs of the American Type Culture Collection (ATCC). Non-limiting standard media 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, YT, etc.

[0182] The carbon source can be any suitable carbon substrate known in the art, particularly any carbon substrate commonly used in the cultivation and / or fermentation of methylotrophic bacteria. Carbon sources of particular interest are reduced one-carbon compounds, such as methanol, or multi-carbon compounds without carbon-carbon bonds, such as methylamine or dimethylamine. Thus, according to some embodiments, the culture medium includes methanol as a carbon source. The concentration of methanol in the culture medium can generally range from about 0.5% (w / v) to about 4% (w / v), e.g., from about 2% (w / v) to about 4% (w / v). According to some embodiments, the concentration of methanol in the culture medium ranges from about 2.5% (w / v) to about 3.5% (w / v).

[0183] Nitrogen sources that can be used include ammonia, various ammonium salts such as ammonium sulfate, other nitrogen compounds such as amines, natural nitrogen sources such as peptone, soybean hydrolysate, and digests of fermenting microorganisms. Minerals that can be used include potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, etc.

[0184] Preferably, the culture is carried out under aerobic conditions, for example, by shaking culture, stirring culture, or in an aerated bioreactor, at a temperature of about 20 to about 45°C, for example, about 30 to 38°C, preferably about 37°C. The pH of the culture is usually greater than 5, for example, 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 can be carried out for a period of 10 to 70 hours, preferably 24 to 60 hours, more preferably 36 to 50 hours.

[0185] After culturing, solids such as cells can be removed from the culture medium by centrifugation or membrane filtration. The biochemical compounds can be recovered by conventional methods for isolating and purifying chemical compounds from the culture medium. Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, ion exchange, chromatography, such as ion exchange chromatography or gel filtration chromatography, and crystallization. The method can further include recovering the biochemical compounds from the culture medium.

[0186] The biochemical compounds produced by the method of the present invention can 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 fumarate, glycolate, succinate, malate, malonate, lactate, and their derivatives. Non-limiting examples of amino acids include glutamate, lysine, methionine, tryptophan, phenylalanine, and their derivatives.

[0187] The present invention thus provides biochemical compounds obtainable by the methods detailed herein.

[0188] Certain other definitions "Polypeptide" and "protein" are used interchangeably herein to refer to polymers of at least two amino acids covalently joined by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). D- and L-amino acids, and mixtures of D- and L-amino acids, are included in this definition.

[0189] "Nucleic acid" or "polynucleotide" are used interchangeably herein to refer to 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.

[0190] "Recombinant" or "non-naturally occurring," for example, as used in reference to a host cell, nucleic acid, or polypeptide, refers to a substance or a substance that corresponds to a natural or naturally occurring form of that substance, that has been modified in a manner not otherwise found in nature, or that is identical to it, but that has been produced or derived from synthetic material and / or by manipulation using recombinant technology. Non-limiting examples include recombinant bacterial cells, among others, that express genes not found in the native (non-recombinant) form of the cell, or that express native genes that are otherwise expressed at different levels.

[0191] As used herein in the context of a gene or nucleic acid molecule, "heterologous" or "exogenous" 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 is located, or that is found in a different location(s) within the genome than where it occurs naturally. Thus, a "heterologous" or "exogenous" gene or nucleic acid molecule is not endogenous to the bacterium, but is one that has been exogenously introduced into the microorganism. The DNA molecule of a "heterologous" gene or nucleic acid molecule can be from a different organism, different species, different genus, or different kingdom as the host DNA.

[0192] "Heterologous" as used herein in the context of a polypeptide means that the polypeptide is not normally found in or made (i.e., expressed) by the host microbial organism, but is derived from a different organism, a different species, a different genus, or a different kingdom.

[0193] 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 is derived from a common ancestral gene but that exists in different species.

[0194] "Decreased expression" of a gene means that the amount of transcript, respectively, the amount of polypeptide (e.g., enzyme) encoded by the gene, produced by the modified bacterium is reduced compared to an otherwise identical bacterium without the modification. More specifically, "decreased expression" of a gene means that the amount of transcript, respectively, the amount of polypeptide (e.g., enzyme) encoded by the gene, produced by the modified bacterium is reduced by at least 10%, e.g., 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%, compared to an otherwise identical bacterium without the modification. The level of gene expression can be determined by known methods, including PCR, Southern blotting, and the like. Additionally, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of polypeptide encoded by the gene can be measured by known methods, including ELISA, immunohistochemistry, Western blotting, and the like.

[0195] Gene expression can be reduced by introducing a mutation into a gene in the bacterial genome so that the intracellular activity of the polypeptide encoded by the gene is reduced compared to an otherwise identical bacterium lacking the mutation. Mutations that result in reduced gene expression include substitutions of one or more nucleotides (missense mutations) that result in amino acid substitutions in the polypeptide encoded by the gene, introduction of a stop codon (nonsense mutation), deletion or insertion of nucleotides that result in a frameshift, insertion of a drug resistance gene, or deletion of part or the entire gene (Qiu and Goodman, 1997; Kwon et al., 2000). Expression can also be reduced by modifying expression regulatory sequences such as promoters and Shine-Dalgarno (SD) sequences. Gene expression can also be reduced by gene replacement, such as "λ-Red-mediated gene replacement" (Datsenko and Wanner, 2000). λ-Red-mediated gene replacement is a particularly suitable method for inactivating one or more genes described herein.

[0196] "Inactivate," "inactivation," and "inactivated," when used in the context of a gene or gene cluster, mean that the gene or gene cluster in question no longer expresses a functional protein. The altered DNA region may be unable to naturally express the gene or gene cluster due to deletion of 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 deletion of part or the entire sequence of the gene or gene cluster, such as by gene replacement. Inactivation can 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 double-strand breaks. "Rare-cutting endonucleases" in the context of the present invention include transcription activator-like effector (TALE) nucleases, meganucleases, zinc finger nucleases (ZFNs) and RNA-guided endonucleases.

[0197] The presence or absence of a gene or gene cluster in a bacterial genome can be detected by known methods, including PCR, Southern blotting, etc. Additionally, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene or gene cluster using various known methods, including Northern blotting, quantitative RT-PCR, etc. The amount of a polypeptide encoded by a gene or gene cluster can be measured by known methods, including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), etc.

[0198] As used herein, "reduced," "decreased," or "reduction" in expression of a polypeptide (such as an enzyme described herein) means that expression of the polypeptide in the modified bacterium is reduced compared to expression of the polypeptide in an otherwise identical bacterium (control) but without the modification. Expression of the polypeptide in the modified bacterium can 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 integer percentage between 10% and 100% (e.g., 6%, 7%, 8%, etc.) compared to expression of the polypeptide in an otherwise identical bacterium (control) but without the modification. More specifically, "reduced," "decreased," or "reduction" in polypeptide expression 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 integer percentage between 10% and 100% (e.g., 6%, 7%, 8%, etc.), compared to the amount of the polypeptide in an otherwise identical bacterium lacking the modification (control). Polypeptide expression or amount in bacteria can be determined by any suitable means known in the art, including techniques such as ELISA, immunohistochemistry, Western blotting, or flow cytometry.

[0199] As used herein, "eliminated" expression of a polypeptide (such as an enzyme described herein) means that expression of said polypeptide in the modified bacterium is not detectable compared to expression of said polypeptide in an otherwise identical bacterium (control) without said modification.

[0200] As used herein, "reduced," "decreased," or "reduction" in the activity of a polypeptide (such as an enzyme described herein) 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) without the modification. The activity of the polypeptide in the bacterial microorganism can 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 integer percentage between 10% and 100% (e.g., 6%, 7%, 8%, etc.) compared to expression of the polypeptide in an otherwise identical bacterium (control) without the modification. The activity of the polypeptide in bacteria can be determined by any suitable protein and enzyme activity assays.

[0201] "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.

[0202] As used herein, a "regulatory region" of a gene or gene cluster refers to a nucleic acid sequence that influences the 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 the expression of a coding sequence.

[0203] "Substitution" or "substituted" refers to the modification of a polypeptide by replacing one amino acid residue with another, e.g., replacing 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, e.g., replacing a cytosine with a thymine in a polynucleotide sequence is a nucleotide substitution.

[0204] " Conservative substitution ", when used in relation to polypeptide, refers to the substitution of an amino acid residue with a different residue having a similar side chain, and therefore generally includes the substitution of an amino acid in a polypeptide with an amino acid of the same or similar class.For example, but not limited to, an amino acid with an aliphatic side chain can be substituted with another aliphatic amino acid, such as alanine, valine, leucine and isoleucine; an amino acid with a hydroxyl side chain can be substituted with another amino acid with a hydroxyl side chain, such as serine and threonine; an amino acid with an aromatic side chain can be substituted with another amino acid with an aromatic side chain, such as phenylalanine, tyrosine, tryptophan and histidine; an amino acid with a basic side chain can be substituted with another amino acid with a basic side chain, such as lysine and arginine; an amino acid with an acidic side chain can be substituted with another amino acid with an acidic side chain, such as aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid can be substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0205] "Non-conservative substitution," when used in reference to a polypeptide, refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions can use amino acids between, rather than within, a defined group and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., serine for glycine), (b) the charge or hydrophobicity, or (c) the bulkiness of the side chain. By way of example, and not limitation, exemplary non-conservative substitutions can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0206] As used herein, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been 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 are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Certain other vectors are capable of facilitating the insertion of exogenous nucleic acid molecules into the genome of bacteria. Such vectors are referred to herein as "transformation vectors." Generally, vectors useful in recombinant nucleic acid techniques are often in the form of plasmids. Because the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. Many suitable vectors are known to those of skill in the art and are commercially available.

[0207] As used herein, "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. Suitable "promoters" are generally those that are capable of supporting the initiation of transcription in the bacteria of the invention, resulting in the production of mRNA molecules.

[0208] As used herein, "operably linked" refers to a juxtaposition in which the components described are in a relationship permitting them 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" when it is sufficiently close to the transcription start site of a gene to regulate transcription of the gene.

[0209] "Percentage of sequence identity," "% sequence identity," and "percent 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 purposes. 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. Identity between two sequences can be calculated using various alignment algorithms and / or programs, including FASTA or BLAST, which are available as part of the GCG Sequence Analysis Package (University of Wisconsin-Madison, Wisconsin) and can be used with default settings.

[0210] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is used.

[0211] Where numerical limits or ranges are set forth herein, the endpoints are included, and all values ​​and subranges within the numerical limits or ranges are specifically included as if expressly written out.

[0212] As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more," unless the context clearly dictates otherwise.

[0213] As used herein, the words "comprising," "including," "having," and grammatical variations thereof are deemed to specify the stated features, steps, or components, but do not exclude the addition of one or more additional features, steps, components, or groups thereof.

[0214] Having generally described the invention, a further understanding can be obtained by reference to certain specific embodiments, which are presented herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

[0215] Example Example 1: Identification of EPS-producing genes in Methylobacillus flagellatus and Methylobacillus glycogenes Based on bioinformatics analysis, several genomic loci were selected for disruption. The genetic locations of two EPS-related gene clusters in Methylobacillus flagellatus have been previously published (Chistoserdova et al., 2007). To annotate the genes in the first Methylobacillus flagellatus EPS-producing gene cluster (SEQ ID NOs: 87–112), homologs of genes from the published Methylobacillus species 12S methanolane synthesis cluster (Yoshida et al., 2000) were compared to the Methylobacillus flagellatus genome by NCBI BLAST protein search using a custom script written in the Python programming language. 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 located and annotated in the genome sequence. The locations of these top hits matched the published locations of the first cluster. Because no clear homologs were found for each gene, all genes between the most distant homologs were considered part of the cluster. Most of these 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: 125-146).

[0216] A second EPS-related cluster was predicted in Methylobacillus flagellatus (SEQ ID NOs: 113-124). A BLAST search comparing these genes to the methanol cluster revealed no obvious homologs. The protein products of individual genes within this cluster were identified by an NCBI protein BLAST search against all nonredundant protein sequences, and the top hits for each were selected as described above. The protein sequences of each gene from this Methylobacillus flagellatus protein cluster were cross-searched with the Methylobacillus glycogenes genome using NCBI protein BLAST. The top hits were found to form a similar cluster in Methylobacillus glycogenes (SEQ ID NOs: 147-172).

[0217] The open reading frames (ORFs) identified in each EPS-related gene cluster are shown in Tables 1 to 4 below.

[0218] [Table 1-1] [Table 1-2]

[0219] [Table 2]

[0220] [Table 3]

[0221] [Table 4-1] [Table 4-2]

[0222] [Table 5]

[0223] ORFs annotated as epsD, epsE, epsF, and epsG (SEQ ID NOS: 6-9 in M. flagellatus and SEQ ID NOS: 42-45 in M. glycogenes) were selected for deletion based on their similarity to essential EPS production genes in Methylobacillus sp. strain 12 (Yoshida et al., 2003). Because the second EPS cluster had no obvious homologs in other characterized EPS clusters, all 12 ORFs in M. flagellatus (SEQ ID NOS: 27-38) and all 26 ORFs in Methylobacillus glycogenes (SEQ ID NOS: 61-86) were selected for deletion.

[0224] Example 2: DNA deletion We generated modified strains of ABME5 and ABME6 lacking one or both EPS production clusters. 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 ABME6 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 hours. ABME5-derived strains were incubated at 30°C, while ABME6-derived strains were grown at 37°C. Correct clones were confirmed by colony PCR. The strains generated by DNA deletion are listed in Table 6.

[0225] [Table 6-1] [Table 6-2]

[0226] Example 3: Measurement of total EPS in shake flask cultures before and after EPS gene disruption in Methylobacillus flagellatus To evaluate the effect of deletion of EPS production genes in ABME 6, three modified ABME 6 strains (ABME 80, ABME 82, and ABME 131) were generated as described in Example 3 and tested for EPS production.

[0227] Confirmed transformants were grown overnight on PM7 + 50 μg / mL kanamycin agar plates. Single colonies were resuspended in 25 mL PM7 medium + 25 μg / mL kanamycin in a 250 mL baffled shake flask and incubated overnight at 200 rpm. The following day, 2.5 mL of the overnight culture was inoculated into 50 mL PM7 medium + 25 μg / mL kanamycin in a 250 mL baffled shake flask. All ABME 5-derived strains were grown at 30°C, and all ABME 6-derived strains were grown at 37°C. The cultures were then cooled on ice, aliquoted into 2 mL Eppendorf tubes, and centrifuged at 15,000 rpm for 15 minutes in a standard tabletop centrifuge at 4°C. When a layer of EPS was visible above the cell pellet, it was added 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 bring the final TFA concentration to 3 M. The tubes were placed in a thermoblock heated to 120°C and incubated for 6 hours. After hydrolysis, the liquid (containing TFA) was evaporated to complete dryness at 80°C. The dried mass was resuspended in 0.5 M NaOH (same volume as the original culture aliquot) and analyzed by HPLC for monomeric sugars.

[0228] [Table 7]

[0229] ABME 6 produced a visible slime layer above the cell pellet after centrifugation, and total monomeric sugars after hydrolysis reached approximately 1 g / L. Single cluster disruption (ABME 80 and ABME 82) did not have a visible slime layer but still produced approximately 500 mg / L of total sugars. Interestingly, the ratio of glucose to total sugars was reversed depending on the disrupted gene cluster, most likely due to the two different types of EPS produced. When both clusters were disrupted (strain ABME 131), sugars were below the detection limit.

[0230] Example 4: 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, 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 μL of strain cryostock, adjusted to a stock OD of 5, resulting in a starting OD of 0.05. The culture was 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). The culture was then used to inoculate a 5 L bioreactor (8% inoculum) with 2.5 L of sterile PM3 medium starting volume. The pH was maintained at 7 by automatic addition of NH4OH, and the temperature was maintained at 37°C.

[0231] For the oxygen-limited process, methanol was automatically fed to maintain a concentration of 3–6 g / L. Maximum aeration and agitation of the reactor were maintained, but the oxygen concentration consistently dropped to 0% throughout the process. Broth samples were periodically sampled for OD, mineral, and metabolite measurements. Viscosity measurements were performed at the end of the process, and the results are summarized in Figure 2. Wild-type M. flagellatus fermentation broth was highly viscous at 400 centipoise at the time of sampling. Similar measurements of another Methylobacillus species, ABME 145, yielded a similarly viscous broth of 422 centipoise. The double disruption yielded a significantly less viscous broth, allowing for centrifugation and filtration using standard benchtop equipment, which was not possible with the previous sample. A viscometer confirmed this assessment, measuring a 35 cP, or nearly 10-fold, decrease in viscosity. A representative sample of Escherichia coli fermentation broth was also measured for comparison.

[0232] Example 5: Measurement of foam reduction in shake flasks before and after EPS gene removal in Methylobacillus flagellates It was observed that the EPS-deficient strain exhibited significantly less foaming. To compare foam formation between ABME 6 and the EPS-deficient mutant strains (ABME 82 and ABME 131), the strains were cultured as described in Example 3.

[0233] To measure foam formation, the flasks were removed from the shaker and the liquid was allowed to settle for 5 minutes. The foam layer above the liquid was measured with a tape measure (Figure 3). Wild-type cultures produced a 0.5-1 cm foam layer above the liquid, whereas single-cluster disruption resulted in only approximately 1 mm of foam accumulating at the liquid meniscus. No foam was observed in double-disruption cultures.

[0234] Example 6: Measurement of foam reduction in bioreactor cultures before and after EPS gene disruption in Methylobacillus flagellatus To evaluate the change in foaming of the fermentation broth 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 antifoam agent (SAG 5693) added. Fermentations were performed under carbon limitation.

[0235] The reactor was inoculated as described in Example 5. To achieve carbon limitation, 50% methanol was automatically fed into the reactor at a rate dynamically calculated to maintain a pO2 value of approximately 30% at the highest 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 meniscus of the fermentation broth. ABME 6 and ABME 131 were tested in this manner. For ABME 131, antifoam addition began after 5 hours. Approximately 60% less antifoam was added during the ABME 131 fermentation, depending on the fermentation time (Figure 4).

[0236] Example 7: Microscopic images of Methylobacillus flagellatus cell aggregates before and after EPS gene disruption To assess changes in cell aggregation before and after disruption of EPS production, strains were cultivated in 5-liter bioreactor fermentations under oxygen limitation as described in Example 3.

[0237] Broth samples were observed by phase-contrast microscopy using a Zeiss Axioplan 2 microscope with a Zeiss Ph2 Plan-NEOFLUAR 40x objective (NA 0.75) and recorded with an sCMOS camera. ABME 6 cells formed large clumps and were immobilized in the EPS matrix (Figure 5). For the double-disrupted strain ABME 131, cells were individually suspended in solution, with minimal clumping.

[0238] Example 8: Improved methanol tolerance of EPS-free M. flagellates Tolerance to high methanol concentrations is an important parameter for efficient bioprocessing. To confirm that methanol tolerance is not reduced in strains unable to produce EPS, growth of ABME 6 and its EPS-producing derivative, ABME 131, in increasing concentrations of methanol was tested.

[0239] Strains were tested in shake flasks as described in Example 4. To test for methanol tolerance, the initial methanol concentration was increased to 4.5%. OD600 was measured after 24 hours.

[0240] Surprisingly, the methanol tolerance of ABME 131 was higher than that of ABME 6. For example, at an initial methanol concentration of 2.75%, EPS-free ABME 131 grew to an OD of 1.6, whereas wild-type ABME 6 only grew to an OD of 0.3. The difference in growth was most pronounced at 2.75%. However, the improved growth of ABME 131 compared to ABME 6 was evident at all methanol concentrations (Figure 6).

[0241] List of References Cited herein 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. Hattori, M. et al. (2020) Methylobacillus glycogenes JCM 2850, whole genome shotgun sequencing p - Nucleotide - NCBI. 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. Qui 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 U S A 2000, 97:6640-6645.

Claims

1. A genetically modified bacterium of the genus Methylobacillus that has been modified to inactivate the EPS gene cluster defined by or containing orthologous genes in the open reading frame (ORF) found in SEQ ID NO: 175 or 179.

2. The bacterium of claim 1, wherein the additional EPS gene cluster is inactivated and the additional EPS gene cluster is defined by or comprises orthologous genes of an open reading frame (ORF) found in SEQ ID NO: 174 or 177.

3. 3. The bacterium of claim 2, wherein at least one gene in the additional EPS gene cluster is inactivated, for example by deletion of part or the entire gene sequence.

4. 4. The bacterium of claim 2 or 3, wherein the additional EPS gene cluster comprises the genes epsD, epsE, epsF and epsG, or orthologues thereof.

5. 5. The bacterium according to claim 4, wherein the genes epsD, epsE, epsF and epsG, or their orthologues, are inactivated, for example by deletion of part or the entire gene sequence.

6. The bacterium according to claim 4 or 5, wherein the epsD gene encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6, the epsE gene encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7, the epsF gene encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and the epsG gene encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9, or the epsD gene encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:42, the epsE gene encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:43, the epsF gene encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:44, and the epsG gene encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

45.

7. A bacterium described in any one of claims 1 to 6, in which the additional EPS gene cluster is inactivated.

8. A bacterium according to any one of claims 1 to 7, wherein the EPS gene cluster is inactivated.

9. A bacterium as described in claim 7 or 8, wherein the additional EPS gene cluster and / or the EPS gene cluster has been inactivated by deletion of part or all of the sequence of the cluster or by modification of the promoter and / or ribosome binding site region, for example by introduction of at least one mutation into said region, which results in the absence of gene expression.

10. 10. The bacterium according to any one of claims 1 to 9, wherein the bacterium is Methylobacillus flagellatus.

11. 10. The bacterium according to any one of claims 1 to 9, wherein the bacterium is Methylobacillus glycogenes.

12. 12. A method for producing a biochemical compound, comprising culturing the bacterium of any one of claims 1 to 11 under suitable culture conditions in a culture medium containing a reduced one-carbon compound or a multi-carbon compound that does not have a carbon-carbon bond.

13. 13. The method of claim 12, wherein the reduced one-carbon compound is methanol.

14. 14. The method of claim 12 or 13, wherein the multi-carbon compound is dimethylamine.

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