Metabolic engineering for the simultaneous consumption of xylose and glucose for the production of chemicals from second-generation sugars

Abstract

The present disclosure provides methods for genetically modifying microorganisms to produce microorganisms capable of co-consuming xylose and glucose to increase productivity output of a desired chemical product. The present disclosure further provides modified bacteria capable of co-consuming xylose and glucose, and compositions comprising said microorganisms.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 829,398, filed Apr. 4, 2019, the content of which is hereby incorporated by reference in its entirety.

[0002] Description of the Sequence Listing The sequence listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference in this specification. The name of the text file containing the sequence listing is BRSK_021_01WO_ST25.txt. The text file is 222 kb, was created on Apr. 4, 2020, and is electronically submitted.

Background Art

[0003] Background of the Disclosure The production of desired chemicals such as monoethylene glycol, glycolic acid, C3 compounds (such as acetone, isopropanol, and propene), amino acids, and polyols from alternative raw materials such as pentoses is an alternative means to obtain them from petrochemicals.

[0004] The use of xylose, a pentose, is a reason that differentiates the most sustainable chemical projects. Considering that the use of lignocellulosic biomass as a raw material does not require the use of plants that produce food in another way, lignocellulosic biomass is a promising sustainable raw material. Lignocellulosic biomass is even more promising as a sustainable raw material due to its sustainability and global availability. The separation and isolation of lignocellulosic sugars is one option to increase sugar production without increasing land use. Xylose is the main carbon source in hemicellulosic hydrolysates, followed by glucose and arabinose. Xylose typically represents 70 - 80% of the sugars present in hemicellulosic hydrolysates, while glucose accounts for 10 - 20%.

[0005] In Escherichia coli, even trace amounts of glucose completely inhibit xylose uptake (even if xylose is the main sugar), thereby limiting the overall conversion of sugars into the desired chemical substances. In industrial processes, productivity (grams of product per liter per hour) is a key element for ensuring economic viability, and thus, microbial strains need to have the ability to constantly convert primary substrates such as xylose into products at the highest rate. Considering that glucose is present in lignocellulosic hydrolysates, during batch operation, xylose uptake is extended until complete depletion of glucose while reducing productivity. Considering fed-batch or continuous operation, a stream containing both xylose and glucose is constantly fed to the reactor, reinforcing the possible inhibition of glucose, thereby preventing the potential productivity of microorganisms capable of producing one or more products from a common renewable feedstock from being maximized.

[0006] There is a need to maximize the productivity of microorganisms capable of producing the desired product from renewable feedstocks, particularly to maximize the use of multiple carbon sources while minimizing or eliminating the inhibitory activity of carbon sources on the microorganism.

[0007] As described herein, the present disclosure provides methods and compositions for microbial engineering that use mixed sugar streams for the production of desired chemical substances without concern for the typical catabolite repression effects caused by the presence of mixed sugar streams. SUMMARY OF THE INVENTION

[0008] SUMMARY OF THE DISCLOSURE In some embodiments, the present disclosure relates generally to recombinant microorganisms capable of producing fermentation products from a feedstock comprising xylose and glucose, wherein the recombinant microorganisms utilize xylose and glucose simultaneously, and the microorganisms have the following: (a) a deletion or inactivation of a transporter protein from the genome of the microorganism such that a pentose ATP-binding transporter protein is not expressed; (b) one or more endogenous or exogenous nucleic acid sequences operably linked to one or more constitutive promoters and encoding at least one C5 sugar co-transporter, wherein the C5 sugar co-transporter comprises (1) a xylose co-transporter and / or (2) an arabinose co-transporter; (c) one or more of the following endogenous or exogenous nucleic acid sequences: (1) encoding a xylose isomerase operably linked to one or more constitutive promoters and a deletion or inactivation of one or more xylulokinases, and / or (2) encoding a xylose dehydrogenase operably linked to one or more constitutive promoters and a deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases.

[0009] In some embodiments, the fermentation products produced by the microorganisms are one or more molecules comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. In some embodiments, two or more molecules are produced simultaneously.

[0010] In one aspect, the present disclosure relates generally to recombinant Escherichia coli (E. coli) capable of producing fermentation products from a feedstock comprising xylose and glucose, the recombinant microorganism using xylose and glucose simultaneously, the microorganism having: (a) deletion or inactivation of transporter proteins from the genome of the microorganism such that the ATP-binding transporter proteins araFGH and xylFGH are not expressed; (b) one or more endogenous or exogenous nucleic acid sequences operably linked to one or more constitutive promoters, encoding at least one C5 sugar co-transporter, wherein the C5 sugar co-transporter comprises (1) a xylose co-transporter and / or (2) an arabinose co-transporter; (c) one or more of the following: (1) encoding a xylose isomerase operably linked to one or more constitutive promoters and deletion or inactivation of one or more xylulokinases; and / or (2) encoding a xylose dehydrogenase operably linked to one or more constitutive promoters and deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases.

[0011] In one aspect, the present disclosure relates generally to recombinant microorganisms capable of producing monoethylene glycol (MEG) and / or acetone from a feedstock comprising xylose and glucose, the recombinant microorganism using xylose and glucose simultaneously, having: (a) deletion or inactivation of aldA, araFGH, and xylFGH from the genome of the parent microorganism; and (b) expression of one or more of one or more endogenous or exogenous nucleic acid molecules encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, the recombinant microorganism expressing a pathway for MEG and / or acetone production.

[0012] In some embodiments, the recombinant microorganism of claim 5, wherein the microorganism further comprises a deletion or inactivation of glcDEF. In some embodiments, the C5 co-transporter is controlled by the GAPDH promoter of the araFGH locus. In some embodiments, the C5 sugar co-transporter is the xylose co-transporter XylE. In some embodiments, XylE comprises an amino acid sequence comprising SEQ ID NO: 49. In some embodiments, XylE is encoded by a nucleic acid sequence comprising SEQ ID NO: 48. In some embodiments, the xylose co-transporter is endogenous to the microorganism.

[0013] In some embodiments, the C5 sugar co-transporter is the arabinose co-transporter AraE. In some embodiments, AraE comprises an amino acid sequence comprising SEQ ID NO: 47. In some embodiments, AraE is encoded by a nucleic acid sequence comprising SEQ ID NO: 46. In some embodiments, the arabinose co-transporter is endogenous to the microorganism.

[0014] In some embodiments, the uptake of xylose is not sensitive to catabolite repression by other monosaccharides. In some embodiments, the microorganism comprises a functional phosphotransferase system. In some embodiments, the microorganism comprises a native wild-type nucleic acid sequence encoding a cAMP receptor protein (CRP). In some embodiments, CRP comprises an amino acid sequence comprising SEQ ID NO: 10. In some embodiments, CRP is encoded by a nucleic acid sequence comprising SEQ ID NO: 9.

[0015] In some embodiments, constitutive overexpression of the xylose co-transporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, constitutive overexpression of the arabinose co-transporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, continuous xylose import occurs independently of the presence of other sugars in the feedstock.

[0016] In some embodiments, the recombinant microorganism comprises the following (c) to (e): (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding a xylose isomerase and / or a ketohexokinase and / or a fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that catalyzes the conversion of glycolaldehyde to MEG; and (e) a pathway for MEG production involving one or more deletions or inactivations of one or more xylulokinases from the genome of the parental microorganism. In some embodiments, (c) and (d) are present in an operon controlled by the proD promoter. In some embodiments, the proD promoter is encoded by a nucleic acid sequence comprising SEQ ID NO: 53.

[0017] In some embodiments, the xylose isomerase is XylA. In some embodiments, the xylose isomerase is endogenous to the microorganism. In some embodiments, XylA comprises an amino acid sequence comprising SEQ ID NO: 6. In some embodiments, XylA is encoded by a nucleic acid sequence comprising SEQ ID NO: 5.

[0018] In some embodiments, the ketohexokinase is derived from Homo Sapiens. In some embodiments, the ketohexokinase is heterologous to the microorganism. In some embodiments, the ketohexokinase is khk-C. In some embodiments, khk-C comprises an amino acid sequence comprising SEQ ID NO: 12. In some embodiments, khk-C is encoded by a nucleic acid sequence comprising SEQ ID NO: 11.

[0019] In some embodiments, the fructose-bisphosphate aldolase is of human origin. In some embodiments, the fructose-bisphosphate aldolase is heterologous to the microorganism. In some embodiments, the fructose-bisphosphate aldolase is aldoB. In some embodiments, aldoB comprises an amino acid sequence comprising SEQ ID NO: 51. In some embodiments, aldoB is encoded by a nucleic acid sequence comprising SEQ ID NO: 50.

[0020] In some embodiments, the glycoaldehyde reductase is endogenous to the microorganism. In some embodiments, the glycoaldehyde reductase is fucO. In some embodiments, fucO comprises an amino acid sequence comprising SEQ ID NO: 98. In some embodiments, fucO is encoded by a nucleic acid sequence comprising SEQ ID NO: 52.

[0021] In some embodiments, the xylulokinase is XylB. In some embodiments, xylB comprises an amino acid sequence comprising SEQ ID NO: 14. In some embodiments, xylB is encoded by a nucleic acid sequence comprising SEQ ID NO: 13.

[0022] In some embodiments, the recombinant microorganism comprises the following (c)-(e): (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycoaldehyde reductase that catalyzes the conversion of glycoaldehyde to MEG; and (e) a pathway for MEG production involving one or more of deletion or inactivation of one or more xylose isomerase and / or one or more xylulokinase from the genome of the parental microorganism.

[0023] In some embodiments, the xylose dehydrogenase is from Caulobacter crescentus, Burkholderia xenovorans, Haloferax volcanii. In some embodiments, the xylose dehydrogenase is xdh. In some embodiments, xdh comprises an amino acid sequence comprising SEQ ID NO: 16, 17, or 19. In some embodiments, xdh is encoded by a nucleic acid sequence comprising SEQ ID NO: 15, 18, or 97. In some embodiments, the xylose dehydrogenase is heterologous to the microorganism.

[0024] In some embodiments, xylonolactonase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, xylonolactonase is xylC. In some embodiments, xylC comprises an amino acid sequence comprising SEQ ID NO: 55, 57, or 59. In some embodiments, xylC is encoded by a nucleic acid sequence comprising SEQ ID NO: 54, 56, or 58.

[0025] In some embodiments, xylonolactonase is heterologous to the microorganism. In some embodiments, xylonolactonase is endogenous to the microorganism.

[0026] In some embodiments, xylose dehydratase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, xylose dehydratase is xylD. In some embodiments, xylD comprises an amino acid sequence comprising SEQ ID NO: 61, 63, or 65. In some embodiments, xylD is encoded by a nucleic acid sequence comprising SEQ ID NO: 60, 62, or 64. In some embodiments, xylose dehydratase is heterologous to the microorganism. In some embodiments, xylose dehydratase is endogenous to the microorganism.

[0027] In some embodiments, glycoaldehyde reductase is endogenous to the microorganism. In some embodiments, glycoaldehyde reductase is fucO. In some embodiments, fucO comprises an amino acid sequence comprising SEQ ID NO: 98. In some embodiments, fucO is encoded by a nucleic acid sequence comprising SEQ ID NO: 52.

[0028] In some embodiments, glycoaldehyde reductase is heterologous to the microorganism. In some embodiments, xylose isomerase is XylA. In some embodiments, xylA comprises an amino acid sequence comprising SEQ ID NO: 6. In some embodiments, xylA is encoded by a nucleic acid sequence comprising SEQ ID NO: 5.

[0029] In some embodiments, the xylulokinase is XylB. In some embodiments, xylB comprises an amino acid sequence comprising SEQ ID NO: 14. In some embodiments, xylB is encoded by a nucleic acid sequence comprising SEQ ID NO: 13.

[0030] In some embodiments, the recombinant microorganism further comprises a pathway for acetone production involving one or more of the following (f)-(h): (f) expression of at least one exogenous nucleic acid molecule encoding acetoacetyl-CoA thiolase; (g) expression of at least one exogenous nucleic acid molecule encoding acetate:acetoacetyl-CoA transferase; and (h) expression of at least one exogenous nucleic acid molecule encoding acetoacetate decarboxylase that catalyzes the conversion of acetoacetate to acetone.

[0031] In some embodiments, (f), (g), and (h) are present in an operon controlled by the OXB11 promoter. In some embodiments, the OXB11 promoter is encoded by a nucleic acid sequence comprising SEQ ID NO: 78. In some embodiments, the acetoacetyl-CoA thiolase is from Clostridium acetobutylicum. In some embodiments, the acetoacetyl-CoA thiolase comprises an amino acid sequence comprising SEQ ID NO: 67 or 69. In some embodiments, the acetoacetyl-CoA thiolase is encoded by a nucleic acid sequence comprising SEQ ID NO: 66 or 68. In some embodiments, the acetate:acetoacetyl-CoA transferase is AtoDA. In some embodiments, the AtoDA subunit alpha comprises an amino acid sequence comprising SEQ ID NO: 72. In some embodiments, the AtoDA subunit alpha is encoded by a nucleic acid sequence comprising SEQ ID NO: 70. In some embodiments, the AtoDA subunit beta comprises an amino acid sequence comprising SEQ ID NO: 73. In some embodiments, the AtoDA subunit beta is encoded by a nucleic acid sequence comprising SEQ ID NO: 71.

[0032] The recombinant microorganism according to claim 77, wherein the acetoacetate decarboxylase is derived from Clostridium beijerinckii or Clostridium acetobutylicum. In some embodiments, the acetoacetate decarboxylase is Adc. In some embodiments, Adc comprises an amino acid sequence comprising SEQ ID NO: 75 or 77. In some embodiments, Adc is encoded by a nucleic acid sequence comprising SEQ ID NO: 74 or 76.

[0033] In some embodiments, the recombinant microorganism further comprises a pathway for isopropanol production involving one or more of the following (f)-(i): (f) expression of at least one exogenous nucleic acid molecule encoding acetoacetyl-CoA thiolase; (g) expression of at least one exogenous nucleic acid molecule encoding acetate:acetoacetyl-CoA transferase; and (h) expression of at least one exogenous nucleic acid molecule encoding acetoacetate decarboxylase that catalyzes the conversion of acetoacetate to acetone, (i) expression of at least one exogenous nucleic acid molecule encoding alcohol dehydrogenase that catalyzes the conversion of acetone to isopropanol.

[0034] In some embodiments, the present disclosure relates generally to a recombinant microorganism capable of producing glycolic acid from a feedstock comprising xylose and glucose, the recombinant microorganism using xylose and glucose simultaneously and comprising one or more of the following: (a) deletion or inactivation of fucO, yqhD, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, the recombinant microorganism further expressing one or more pathways for the production of glycolic acid.

[0035] In some embodiments, the microorganism further comprises a deletion or inactivation of glcDEF. In some embodiments, the microorganism further comprises a deletion or inactivation of dkgA. In some embodiments, the microorganism further comprises a deletion or inactivation of yahK. In some embodiments, the xylose co-transporter is controlled by the GAPDH promoter of the araFGH locus.

[0036] In some embodiments, the C5 sugar co-transporter is the xylose co-transporter XylE. In some embodiments, XylE comprises an amino acid sequence comprising SEQ ID NO: 49. In some embodiments, XylE is encoded by a nucleic acid sequence comprising SEQ ID NO: 48. In some embodiments, the xylose co-transporter is endogenous to the microorganism.

[0037] In some embodiments, the C5 sugar co-transporter is the arabinose co-transporter AraE. In some embodiments, the arabinose co-transporter is endogenous to the microorganism. In some embodiments, the uptake of xylose is not sensitive to catabolite repression by other monosaccharides. In some embodiments, the microorganism comprises a functional phosphotransferase system.

[0038] In some embodiments, the microorganism comprises a native wild-type nucleic acid sequence encoding the cAMP receptor protein (CRP). In some embodiments, CRP comprises an amino acid sequence comprising SEQ ID NO: 10. In some embodiments, CRP is encoded by a nucleic acid sequence comprising SEQ ID NO: 9. In some embodiments, constitutive overexpression of the xylose co-transporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, constitutive overexpression of the arabinose co-transporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, continuous xylose import occurs independently of the presence of other sugars in the feedstock.

[0039] In some embodiments, the recombinant microorganism comprises the following (c) to (e): (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose isomerase and / or ketohexokinase and / or fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding glycoaldehyde dehydrogenase that catalyzes the conversion of glycoaldehyde to glycolic acid; and (e) a pathway for glycolic acid production involving one or more of deletion or inactivation of one or more xylulokinases from the genome of the parental microorganism. In some embodiments, (c) and (d) are present in an operon controlled by the proD promoter. In some embodiments, the proD promoter is encoded by a nucleic acid sequence comprising SEQ ID NO: 53.

[0040] In some embodiments, the xylose isomerase is XylA. In some embodiments, XylA comprises an amino acid sequence comprising SEQ ID NO: 6. In some embodiments, XylA is encoded by a nucleic acid sequence comprising SEQ ID NO: 5. In some embodiments, the xylose isomerase is endogenous to the microorganism.

[0041] In some embodiments, the ketohexokinase is of human origin. In some embodiments, the ketohexokinase is heterologous to the microorganism. In some embodiments, the ketohexokinase is khk-C. In some embodiments, khk-C comprises an amino acid sequence comprising SEQ ID NO: 12. In some embodiments, khk-C is encoded by a nucleic acid sequence comprising SEQ ID NO: 11.

[0042] In some embodiments, the fructose-bisphosphate aldolase is of human origin. In some embodiments, the fructose-bisphosphate aldolase is aldoB. In some embodiments, aldoB comprises an amino acid sequence comprising SEQ ID NO: 51. In some embodiments, aldoB is encoded by a nucleic acid sequence comprising SEQ ID NO: 50. In some embodiments, the fructose-bisphosphate aldolase is heterologous to the microorganism.

[0043] In some embodiments, the glycolaldehyde dehydrogenase is aldA. In some embodiments, aldA comprises an amino acid sequence comprising SEQ ID NO: 4. In some embodiments, aldA is encoded by a nucleic acid sequence comprising SEQ ID NO: 3. In some embodiments, the glycolaldehyde dehydrogenase is endogenous to the microorganism.

[0044] In some embodiments, the xylulokinase is XylB. In some embodiments, xylB comprises an amino acid sequence comprising SEQ ID NO: 14. In some embodiments, xylB is encoded by a nucleic acid sequence comprising SEQ ID NO: 13.

[0045] In some embodiments, the recombinant microorganism comprises the following (c) to (e): (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) a pathway for glycolic acid production involving one or more of the deletion or inactivation of one or more xylose isomerase and / or one or more xylulokinase from the genome of the parental microorganism. In some embodiments, (c) and (d) are controlled by the proD promoter. In some embodiments, the proD promoter is encoded by a nucleic acid sequence comprising SEQ ID NO: 53.

[0046] In some embodiments, the xylose isomerase is XylA. In some embodiments, XylA comprises an amino acid sequence comprising SEQ ID NO: 6. In some embodiments, XylA is encoded by a nucleic acid sequence comprising SEQ ID NO: 5.

[0047] In some embodiments, the xylulokinase is XylB. In some embodiments, xylB comprises an amino acid sequence comprising SEQ ID NO: 14. In some embodiments, xylB is encoded by a nucleic acid sequence comprising SEQ ID NO: 13.

[0048] In some embodiments, the xylose dehydrogenase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylose dehydrogenase is xdh. In some embodiments, xdh comprises an amino acid sequence comprising SEQ ID NO: 16, 17, or 19. In some embodiments, xdh is encoded by a nucleic acid sequence comprising SEQ ID NO: 15, 18, or 97. In some embodiments, the xylose dehydrogenase is heterologous to the microorganism.

[0049] In some embodiments, the xylonolactonase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylonolactonase is xylC. In some embodiments, xylC comprises an amino acid sequence comprising SEQ ID NO: 55, 57, or 59. In some embodiments, xylC is encoded by a nucleic acid sequence comprising SEQ ID NO: 54, 56, or 58.

[0050] In some embodiments, the xylonolactonase is heterologous to the microorganism. In some embodiments, the xylonolactonase is endogenous to the microorganism.

[0051] In some embodiments, the glycolaldehyde dehydrogenase is aldA. In some embodiments, aldA comprises an amino acid sequence comprising SEQ ID NO: 4. In some embodiments, aldA is encoded by a nucleic acid sequence comprising SEQ ID NO: 3. In some embodiments, the glycolaldehyde dehydrogenase is endogenous to the microorganism.

[0052] In some embodiments, the microorganism further expresses a pathway for the production of glycolic acid involving one or more of the following: (f) expression of at least one endogenous or exogenous nucleic acid molecule encoding isocitrate lyase; and / or (g) expression of at least one endogenous or exogenous nucleic acid molecule encoding glyoxylate reductase. In some embodiments, (f) and (g) are present in an operon controlled by the OXB20 promoter. In some embodiments, the OXB20 promoter is encoded by a nucleic acid sequence comprising SEQ ID NO: 96.

[0053] In some embodiments, the isocitrate lyase is AceA. In some embodiments, AceA comprises an amino acid sequence comprising SEQ ID NO: 90. In some embodiments, AceA is encoded by a nucleic acid sequence comprising SEQ ID NO: 89.

[0054] In some embodiments, the glyoxylate reductase is YcdW. In some embodiments, YcdW comprises an amino acid sequence comprising SEQ ID NO: 92. In some embodiments, YcdW is encoded by a nucleic acid sequence comprising SEQ ID NO: 91.

[0055] In some embodiments, the recombinant microorganism is a species of the genus Clostridium, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa obata (SporomusaIt is obtained from a parent microorganism selected from the group consisting of Clostridium acetobutylicum, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, species of the genus Bacillus, species of the genus Corynebacterium, Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus. In some embodiments, the parent microorganism is Escherichia coli.

[0056] In some embodiments, the present disclosure relates generally to recombinant microorganisms capable of producing fermentation products from a feedstock comprising xylose and glucose, the recombinant microorganisms using xylose and glucose simultaneously, the microorganisms comprising: (a) a deletion or inactivation of a binding transporter protein from the genome of the microorganism such that a pentose ATP transporter protein is not expressed; (b) one or more endogenous or exogenous nucleic acid sequences operably linked to one or more constitutive promoters and encoding at least one C5 sugar co-transporter, wherein the C5 sugar co-transporter comprises (1) a xylose co-transporter and / or (2) an arabinose co-transporter; (c) one or more endogenous or exogenous nucleic acid sequences encoding (1) a xylose isomerase operably linked to one or more constitutive promoters and a deletion or inactivation of one or more xylulokinases, and / or (2) a xylose dehydrogenase operably linked to one or more constitutive promoters and a deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases. In some embodiments, the fermentation product produced by the microorganism is one or more molecules comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. In some embodiments, two or more molecules are produced simultaneously.

[0057] In some embodiments, the present disclosure relates generally to recombinant Escherichia coli capable of producing fermentation products from a feedstock comprising xylose and glucose, the recombinant microorganism using xylose and glucose simultaneously, the microorganism comprising: (a) deletion or inactivation of transporter proteins from the genome of the microorganism such that the ATP-binding transporter proteins araFGH and xylFGH are not expressed; (b) one or more endogenous or exogenous nucleic acid sequences operably linked to one or more constitutive promoters and encoding at least one C5 sugar co-transporter, the C5 sugar co-transporter comprising (1) a xylose co-transporter and / or (2) an arabinose co-transporter; (c) one or more of the following: (1) one or more endogenous or exogenous nucleic acid sequences encoding a xylose isomerase operably linked to one or more constitutive promoters and deletion or inactivation of one or more xylulokinases; and / or (2) one or more endogenous or exogenous nucleic acid sequences encoding a xylose dehydrogenase operably linked to one or more constitutive promoters and deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases.

[0058] In some embodiments, the present disclosure relates generally to recombinant microorganisms capable of producing monoethylene glycol (MEG) and / or acetone from a feedstock comprising xylose and glucose, the recombinant microorganism using xylose and glucose simultaneously, the microorganism comprising: (a) deletion or inactivation of aldA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) one or more of the expression of at least one endogenous or exogenous nucleic acid molecule operably linked to one or more constitutive promoters and encoding a C5 sugar co-transporter; the recombinant microorganism expressing a pathway for MEG and / or acetone production.

[0059] In some embodiments, the microorganism further comprises a deletion or inactivation of glcDEF. In some embodiments, the C5 co-transporter is controlled by the GAPDH promoter of the araFGH locus. In some embodiments, the C5 sugar co-transporter is the xylose co-transporter XylE. In some embodiments, the xylose co-transporter is endogenous to the microorganism. In some embodiments, the C5 sugar co-transporter is the arabinose co-transporter AraE. In some embodiments, the arabinose co-transporter is endogenous to the microorganism. In some embodiments, the uptake of xylose is not sensitive to catabolite repression by other monosaccharides. In some embodiments, the microorganism comprises a functional phosphotransferase system. In some embodiments, the microorganism comprises a native wild-type nucleic acid sequence encoding a cAMP receptor protein (CRP). In some embodiments, the one or more nucleic acid molecules encoding aldA comprise the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, the one or more amino acid sequences encoding aldA comprise the amino acid sequence set forth in SEQ ID NO: 4. The recombinant microorganism according to claim 5, wherein constitutive overexpression of the xylose co-transporter enables continuous transfer of xylose from the feedstock to the microorganism. The recombinant microorganism according to claim 5, wherein constitutive overexpression of the arabinose co-transporter enables continuous transfer of xylose from the feedstock to the microorganism. The recombinant microorganism according to claim 5, wherein continuous xylose transfer occurs independently of the presence of other sugars in the feedstock.

[0060] In some embodiments, the recombinant microorganism comprises the following (c)-(e): (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose isomerase and / or ketohexokinase and / or fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that catalyzes the conversion of glycolaldehyde to MEG; and (e) a pathway for MEG production involving one or more deletions or inactivations of one or more xylulokinases from the genome of the parental microorganism.

[0061] In some embodiments, (c) and (d) are present in an operon controlled by the proD promoter. In some embodiments, the xylose isomerase is XylA. In some embodiments, the xylose isomerase is endogenous to the microorganism. In some embodiments, the ketohexokinase is of human origin. In some embodiments, the ketohexokinase is heterologous to the microorganism. In some embodiments, the fructose-bisphosphate aldolase is of human origin. In some embodiments, the fructose-bisphosphate aldolase is heterologous to the microorganism. In some embodiments, the glycolaldehyde reductase is endogenous to the microorganism. In some embodiments, the glycolaldehyde reductase is fucO. In some embodiments, the xylulokinase is XylB.

[0062] In some embodiments, the recombinant microorganism comprises the following (c)-(e): (c) the expression of one or more endogenous or exogenous nucleic acid sequences encoding a xylose dehydrogenase and / or a xylonolactonase and / or a xylose dehydratase operably linked to one or more constitutive promoters; (d) the expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that catalyzes the conversion of glycolaldehyde to MEG; and (e) a pathway for MEG production involving one or more of a deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases from the genome of the parental microorganism.

[0063] In some embodiments, the xylose dehydrogenase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylose dehydrogenase is heterologous to the microorganism. In some embodiments, the xylonolactonase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylonolactonase is heterologous to the microorganism. In some embodiments, the xylonolactonase is endogenous to the microorganism. In some embodiments, the xylose dehydratase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii.

[0064] In some embodiments, the xylose dehydratase is heterologous to the microorganism. In some embodiments, the xylose dehydratase is endogenous to the microorganism. In some embodiments, the glycolaldehyde reductase is endogenous to the microorganism. In some embodiments, the glycolaldehyde reductase is fucO. In some embodiments, the glycolaldehyde reductase is heterologous to the microorganism. In some embodiments, the xylose isomerase is XylA. In some embodiments, the xylulokinase is XylB.

[0065] In some embodiments, the recombinant microorganism further comprises a pathway for acetone production involving expression of one or more of the following (f)-(h): (f) expression of at least one exogenous nucleic acid molecule encoding acetoacetyl-CoA thiolase; (g) expression of at least one exogenous nucleic acid molecule encoding acetate:acetoacetyl-CoA transferase; and (h) expression of at least one exogenous nucleic acid molecule encoding acetoacetate decarboxylase that catalyzes the conversion of acetoacetate to acetone. In some embodiments, (f), (g), and (h) are present in an operon controlled by the OXB11 promoter.

[0066] In some embodiments, the acetoacetyl-CoA thiolase is from Clostridium acetobutylicum. In some embodiments, the acetate:acetoacetyl-CoA transferase is AtoDA. In some embodiments, the acetoacetate decarboxylase is from Clostridium beijerinckii.

[0067] In some embodiments, the recombinant microorganism further comprises a pathway for isopropanol production involving one or more of the following (f)-(i): (f) expression of at least one exogenous nucleic acid molecule encoding acetoacetyl-CoA thiolase; (g) expression of at least one exogenous nucleic acid molecule encoding acetate:acetoacetyl-CoA transferase; and (h) expression of at least one exogenous nucleic acid molecule encoding acetoacetate decarboxylase that catalyzes the conversion of acetoacetate to acetone, (i) expression of at least one exogenous nucleic acid molecule encoding alcohol dehydrogenase that catalyzes the conversion of acetone to isopropanol.

[0068] In some embodiments, the present disclosure relates generally to recombinant microorganisms capable of producing glycolic acid from feedstocks comprising xylose and glucose, the recombinant microorganisms using xylose and glucose simultaneously and comprising one or more of: (a) deletion or inactivation of fucO, yqhD, araFGH, and xylFGH from the genome of a parent microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, the recombinant microorganisms further expressing one or more pathways for the production of glycolic acid. In some embodiments, the microorganism further comprises deletion or inactivation of glcDEF. In some embodiments, the microorganism further comprises deletion or inactivation of dkgA. In some embodiments, the microorganism further comprises deletion or inactivation of yahK. In some embodiments, the xylose co-transporter is controlled by the GAPDH promoter at the araFGH locus. In some embodiments, the C5 sugar co-transporter is the xylose co-transporter XylE. In some embodiments, the xylose co-transporter is endogenous to the microorganism. In some embodiments, the C5 sugar co-transporter is the arabinose co-transporter AraE. In some embodiments, the arabinose co-transporter is endogenous to the microorganism. In some embodiments, xylose uptake is not sensitive to catabolite repression by other monosaccharides.

[0069] In some embodiments, the microorganism comprises a functional phosphotransferase system. In some embodiments, the microorganism comprises a native wild-type nucleic acid sequence encoding a cAMP receptor protein (CRP). In some embodiments, one or more nucleic acid molecules encoding CRP comprise the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, one or more amino acid sequences encoding CRP comprise the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, constitutive overexpression of the xylose co-transporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, constitutive overexpression of the arabinose co-transporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, continuous xylose import occurs independently of the presence of other sugars in the feedstock.

[0070] In some embodiments, the recombinant microorganism comprises the following (c) to (e): (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose isomerase and / or ketohoxokinase and / or fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) a pathway for glycolic acid production involving one or more of deletion or inactivation of one or more xylulokinases from the genome of the parental microorganism. In some embodiments, (c) and (d) are present in an operon controlled by the proD promoter.

[0071] In some embodiments, the xylose isomerase is XylA. In some embodiments, the xylose isomerase is endogenous to the microorganism. In some embodiments, the ketohoxokinase is of human origin. In some embodiments, the ketohoxokinase is heterologous to the microorganism. In some embodiments, the fructose-bisphosphate aldolase is of human origin. In some embodiments, the fructose-bisphosphate aldolase is heterologous to the microorganism. In some embodiments, the glycolaldehyde dehydrogenase is aldA. In some embodiments, the glycolaldehyde dehydrogenase is endogenous to the microorganism. In some embodiments, the xylulokinase is XylB.

[0072] In some embodiments, the recombinant microorganism comprises the following (c)-(e): (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) a pathway for glycolic acid production involving one or more of the deletion or inactivation of one or more xylose isomerase and / or one or more xylulokinase from the genome of the parental microorganism. In some embodiments, (c) and (d) are controlled by the proD promoter.

[0073] In some embodiments, the xylose isomerase is XylA. In some embodiments, the xylulokinase is XylB. In some embodiments, the xylose dehydrogenase is derived from Caulobacter crescentus, Burkholderia xenovorans, Halorhabdus volcanii. In some embodiments, the xylose dehydrogenase is heterologous to the microorganism. In some embodiments, the xylonolactonase is derived from Caulobacter crescentus, Burkholderia xenovorans, Halorhabdus volcanii. In some embodiments, the xylonolactonase is heterologous to the microorganism. In some embodiments, the xylonolactonase is endogenous to the microorganism. In some embodiments, the glycolaldehyde dehydrogenase is aldA. In some embodiments, the glycolaldehyde dehydrogenase is endogenous to the microorganism.

[0074] In some embodiments, the microorganism further expresses a pathway for the production of glycolic acid involving one or more of the following: (f) expression of at least one endogenous or exogenous nucleic acid molecule encoding isocitrate lyase; and / or (g) expression of at least one endogenous or exogenous nucleic acid molecule encoding glyoxylate reductase. In some embodiments, (f) and (g) are present in an operon controlled by the OXB20 promoter. In some embodiments, the isocitrate lyase is AceA. In some embodiments, the glyoxylate reductase is YcdW.

[0075] In some embodiments, the recombinant microorganism is of the genus Clostridium, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa obata (Sporomusaobtained from a parent microorganism selected from the group consisting of Clostridium acetobutylicum, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus species, Corynebacterium species, Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus. [Invention 1001] A recombinant microorganism capable of producing a fermentation product from a raw material containing xylose and glucose, using xylose and glucose simultaneously, (a) deletion or inactivation of the transporter protein from the genome of the microorganism such that the pentose ATP-binding transporter protein is not expressed; (b) one or more endogenous or exogenous nucleic acid sequences encoding at least one C5 sugar co-transporter operably linked to one or more constitutive promoters, wherein the C5 sugar co-transporter comprises (1) a xylose co-transporter and / or (2) an arabinose co-transporter, the one or more endogenous or exogenous nucleic acid sequences; (c) (1) encoding a xylose isomerase operably linked to one or more constitutive promoters and deletion or inactivation of one or more xylulokinases, and / or (2) encoding a xylose dehydrogenase operably linked to one or more constitutive promoters and deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases, one or more endogenous or exogenous nucleic acid sequences comprising one or more of the above, said recombinant microorganism. [Invention 1002] The recombinant microorganism of Invention 1001, wherein the fermentation product produced by the microorganism is one or more molecules containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. [Invention 1003] The recombinant microorganism of Invention 1002, wherein two or more molecules are produced simultaneously. [Invention 1004] A recombinant Escherichia coli (E. coli) capable of producing a fermentation product from a raw material containing xylose and glucose, the recombinant microorganism using xylose and glucose simultaneously, and the microorganism (a) deletion or inactivation of the transporter proteins araFGH and xylFGH from the genome of the microorganism such that the ATP-binding transporter proteins are not expressed; (b) one or more endogenous or exogenous nucleic acid sequences encoding at least one C5 sugar co-transporter operably linked to one or more constitutive promoters, wherein the C5 sugar co-transporter comprises (1) a xylose co-transporter and / or (2) an arabinose co-transporter, the one or more endogenous or exogenous nucleic acid sequences; (c)(1) A xylose isomerase operably linked to one or more constitutive promoters, and deletion or inactivation of one or more xylulokinases, and / or (2) a xylose dehydrogenase operably linked to one or more constitutive promoters, and deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases, one or more endogenous or exogenous nucleic acid sequences The recombinant Escherichia coli comprising one or more of the above. [Inventive Item 1005] A recombinant microorganism capable of producing monoethylene glycol (MEG) and / or acetone from a raw material containing xylose and glucose, Simultaneously using xylose and glucose, (a) Deletion or inactivation of aldA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) Expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters Including one or more of the above, Expressing a pathway for MEG and / or acetone production, The recombinant microorganism. [Inventive Item 1006] The recombinant microorganism of Inventive Item 1005, further comprising deletion or inactivation of glcDEF. [Inventive Item 1007] The recombinant microorganism of Inventive Item 1005, wherein the C5 co-transporter is controlled by the GAPDH promoter at the araFGH locus. [Inventive Item 1008] The recombinant microorganism of Inventive Item 1005, wherein the C5 sugar co-transporter is the xylose co-transporter XylE. [Inventive Item 1009] The recombinant microorganism of Inventive Item 1005, wherein the XylE comprises an amino acid sequence comprising SEQ ID NO: 49. [Inventive Item 1010] The recombinant microorganism of Inventive Item 1005, wherein the XylE is encoded by a nucleic acid sequence comprising SEQ ID NO: 48. [Inventive Item 1011] The recombinant microorganism of Inventive Item 1005, wherein the xylose co-transporter is endogenous to the microorganism. [Inventive Item 1012] The recombinant microorganism of Inventive Item 1005, wherein the C5 sugar co-transporter is the arabinose co-transporter AraE. [Inventive Item 1013] The recombinant microorganism of Inventive Item 1012, wherein the AraE comprises an amino acid sequence comprising SEQ ID NO: 47. [Inventive Item 1014] The recombinant microorganism of Inventive Item 1012, wherein the AraE is encoded by a nucleic acid sequence comprising SEQ ID NO: 46. [Inventive Item 1015] The recombinant microorganism of the present invention 1005, wherein the arabinose cotransporter is endogenous to the microorganism. [The present invention 1016] The recombinant microorganism of the present invention 1005, wherein the uptake of the xylose is not sensitive to inhibition of catabolism by other monosaccharides. [The present invention 1017] The recombinant microorganism of the present invention 1005, which comprises a functional phosphotransferase system. [The present invention 1018] The recombinant microorganism of the present invention 1005, which comprises a natural wild-type nucleic acid sequence encoding a cAMP receptor protein (CRP). [The present invention 1019] The recombinant microorganism of the present invention 1018, wherein the CRP comprises an amino acid sequence comprising SEQ ID NO: 10. [The present invention 1020] The recombinant microorganism of the present invention 1018, wherein the CRP is encoded by a nucleic acid sequence comprising SEQ ID NO: 9. [The present invention 1021] The recombinant microorganism of the present invention 1005, wherein the constitutive overexpression of the xylose cotransporter enables continuous transfer of xylose from the raw material to the microorganism. [The present invention 1022] The recombinant microorganism of the present invention 1005, wherein the constitutive overexpression of the arabinose cotransporter enables continuous transfer of xylose from the raw material to the microorganism. [The present invention 1023] The recombinant microorganism of the present invention 1005, wherein the continuous xylose transfer occurs independently of the presence of other sugars in the raw material. [The present invention 1024] The following (c) to (e): (c) Expression of one or more endogenous or exogenous nucleic acid sequences encoding a xylose isomerase and / or ketohexokinase and / or fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) Expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that catalyzes the conversion of glycolaldehyde to MEG; and (e) Deletion or inactivation of one or more xylulokinases from the genome of the parental microorganism The recombinant microorganism of the present invention 1005, which comprises a pathway for MEG production involving one or more of the above. [The present invention 1025] The recombinant microorganism of the present invention 1024, wherein (c) and (d) are present in an operon controlled by the proD promoter. [The present invention 1026] The recombinant microorganism of the present invention 1025, wherein the proD promoter is encoded by a nucleic acid sequence comprising SEQ ID NO: 53. [The present invention 1027] The recombinant microorganism of the present invention 1024, wherein the xylose isomerase is XylA. [The present invention 1028] Any recombinant microorganism of the present invention, wherein the xylose isomerase is endogenous to the microorganism. [Invention 1029] The recombinant microorganism of Invention 1027, wherein the XylA comprises an amino acid sequence containing SEQ ID NO: 6. [Invention 1030] The recombinant microorganism of Invention 1027, wherein the XylA is encoded by a nucleic acid sequence containing SEQ ID NO: 5. [Invention 1031] The recombinant microorganism of Invention 1024, wherein the ketohexokinase is derived from Homo Sapiens. [Invention 1032] The recombinant microorganism of Invention 1024, wherein the ketohexokinase is heterologous to the microorganism. [Invention 1033] The recombinant microorganism of Invention 1032, wherein the ketohexokinase is khk-C. [Invention 1034] The recombinant microorganism of Invention 1033, wherein the khk-C comprises an amino acid sequence containing SEQ ID NO: 12. [Invention 1035] The recombinant microorganism of Invention 1033, wherein the khk-C is encoded by a nucleic acid sequence containing SEQ ID NO: 11. [Invention 1036] The recombinant microorganism of Invention 1024, wherein the fructose-bisphosphate aldolase is derived from a human. [Invention 1037] The recombinant microorganism of Invention 1024, wherein the fructose-bisphosphate aldolase is heterologous to the microorganism. [Invention 1038] The recombinant microorganism of Invention 1037, wherein the fructose-bisphosphate aldolase is aldoB. [Invention 1039] The recombinant microorganism of Invention 1038, wherein the aldoB comprises an amino acid sequence containing SEQ ID NO: 51. [Invention 1040] The recombinant microorganism of Invention 1038, wherein the aldoB is encoded by a nucleic acid sequence containing SEQ ID NO: 50. [Invention 1041] The recombinant microorganism of Invention 1024, wherein the glycolaldehyde reductase is endogenous to the microorganism. [Invention 1042] The recombinant microorganism of Invention 1024, wherein the glycolaldehyde reductase is fucO. [Invention 1043] The recombinant microorganism of Invention 1042, wherein the fucO comprises an amino acid sequence containing SEQ ID NO: 98. [Invention 1044] The recombinant microorganism of Invention 1042, wherein the fucO is encoded by a nucleic acid sequence containing SEQ ID NO: 52. [Invention 1045] The recombinant microorganism of Invention 1024, wherein the xylulokinase is XylB. [Invention 1046] The recombinant microorganism of the present invention 1045, wherein the xylB comprises an amino acid sequence comprising SEQ ID NO: 14. [The present invention 1047] The recombinant microorganism of the present invention 1045, wherein the xylB is encoded by a nucleic acid sequence comprising SEQ ID NO: 13. [The present invention 1048] The following (c) to (e): (c) Expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) Expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that catalyzes the conversion of glycolaldehyde to MEG; and (e) Deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases from the genome of the parental microorganism The recombinant microorganism of the present invention 1005, comprising a pathway for MEG production involving one or more of the above. [The present invention 1049] The recombinant microorganism of the present invention 1048, wherein the xylose dehydrogenase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. [The present invention 1050] The recombinant microorganism of the present invention 1049, wherein the xylose dehydrogenase is xdh. [The present invention 1051] The recombinant microorganism of the present invention 1050, wherein the xdh comprises an amino acid sequence comprising SEQ ID NO: 16, 17, or 19. [The present invention 1052] The recombinant microorganism of the present invention 1050, wherein the xdh is encoded by a nucleic acid sequence comprising SEQ ID NO: 15, 18, or 97. [The present invention 1053] The recombinant microorganism of the present invention 1048, wherein the xylose dehydrogenase is heterologous to the microorganism. [The present invention 1054] The recombinant microorganism of the present invention 1048, wherein the xylonolactonase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. [The present invention 1055] The recombinant microorganism of the present invention 1054, wherein the xylonolactonase is xylC. [The present invention 1056] The recombinant microorganism of the present invention 1055, wherein the xylC comprises an amino acid sequence comprising SEQ ID NO: 55, 57, or 59. [The present invention 1057] The recombinant microorganism of the present invention 1055, wherein the xylC is encoded by a nucleic acid sequence containing SEQ ID NO: 54, 56, or 58. [The present invention 1058] The recombinant microorganism of the present invention 1048, wherein the xylonolactonase is heterologous to the microorganism. [The present invention 1059] The recombinant microorganism of the present invention 1048, wherein the xylonolactonase is endogenous to the microorganism. [The present invention 1060] The recombinant microorganism of the present invention 1048, wherein the xylose dehydratase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. [The present invention 1061] The recombinant microorganism of the present invention 1060, wherein the xylose dehydratase is xylD. [The present invention 1062] The recombinant microorganism of the present invention 1061, wherein the xylD contains an amino acid sequence containing SEQ ID NO: 61, 63, or 65. [The present invention 1063] The recombinant microorganism of the present invention 1061, wherein the xylD is encoded by a nucleic acid sequence containing SEQ ID NO: 60, 62, or 64. [The present invention 1064] The recombinant microorganism of the present invention 1048, wherein the xylose dehydratase is heterologous to the microorganism. [The present invention 1065] The recombinant microorganism of the present invention 1048, wherein the xylose dehydratase is endogenous to the microorganism. [The present invention 1066] The recombinant microorganism of the present invention 1048, wherein the glycolaldehyde reductase is endogenous to the microorganism. [The present invention 1067] The recombinant microorganism of the present invention 1048, wherein the glycolaldehyde reductase is fucO. [The present invention 1068] The recombinant microorganism of the present invention 1049, wherein the fucO contains an amino acid sequence containing SEQ ID NO: 98. [The present invention 1069] The recombinant microorganism of the present invention 1049, wherein the fucO is encoded by a nucleic acid sequence containing SEQ ID NO: 52. [The present invention 1070] The recombinant microorganism of the present invention 1048, wherein the glycolaldehyde reductase is heterologous to the microorganism. [The present invention 1071] The recombinant microorganism of the present invention 1048, wherein the xylose isomerase is XylA. [The present invention 1072] The recombinant microorganism of the present invention 1071, wherein the xylA contains an amino acid sequence containing SEQ ID NO: 6. [The present invention 1073] The recombinant microorganism of the present invention 1071, wherein the xylA is encoded by a nucleic acid sequence containing SEQ ID NO: 5. [The present invention 1074] The recombinant microorganism of the present invention 1048, wherein the xylulokinase is XylB. [The present invention 1075] The recombinant microorganism of the present invention 1074, wherein the xylB comprises an amino acid sequence containing SEQ ID NO: 14. [The present invention 1076] The recombinant microorganism of the present invention 1074, wherein the xylB is encoded by a nucleic acid sequence containing SEQ ID NO: 13. [The present invention 1077] The following (f) to (h): (f) Expression of at least one exogenous nucleic acid molecule encoding acetoacetyl-CoA thiolase; (g) Expression of at least one exogenous nucleic acid molecule encoding acetate: acetoacetyl-CoA transferase; and (h) Expression of at least one exogenous nucleic acid molecule encoding acetoacetate decarboxylase that catalyzes the conversion of acetoacetate to acetone The recombinant microorganism of the present invention 1024 or 1048, further comprising a pathway for acetone production involving one or more of the above. [The present invention 1076] The recombinant microorganism of the present invention 1077, wherein (f), (g), and (h) are present in an operon controlled by the OXB11 promoter. [The present invention 1077] The recombinant microorganism of the present invention 1076, wherein the OXB11 promoter is encoded by a nucleic acid sequence containing SEQ ID NO: 78. [The present invention 1078] The recombinant microorganism of the present invention 1077, wherein the acetoacetyl-CoA thiolase is derived from Clostridium acetobutylicum. [The present invention 1079] The recombinant microorganism of the present invention 1078, wherein the acetoacetyl-CoA thiolase comprises an amino acid sequence containing SEQ ID NO: 67 or 69. [The present invention 1080] The recombinant microorganism of the present invention 1078, wherein the acetoacetyl-CoA thiolase is encoded by a nucleic acid sequence containing SEQ ID NO: 66 or 68. [The present invention 1081] The recombinant microorganism of the present invention 1077, wherein the acetate: acetoacetyl-CoA transferase is AtoDA. [The present invention 1082] The recombinant microorganism of the present invention 1081, wherein the AtoDA subunit alpha comprises an amino acid sequence containing SEQ ID NO: 72. [The present invention 1083] The recombinant microorganism of the present invention 1081, wherein the AtoDA subunit alpha is encoded by a nucleic acid sequence containing SEQ ID NO: 70. [The present invention 1084] The recombinant microorganism of the present invention 1083, wherein the AtoDA subunit beta comprises an amino acid sequence containing SEQ ID NO: 73. [The present invention 1085] The recombinant microorganism of the present invention 1083, wherein the AtoDA subunit beta is encoded by a nucleic acid sequence containing SEQ ID NO: 71. [The present invention 1086] The recombinant microorganism of the present invention 1077, wherein the acetoacetate decarboxylase is derived from Clostridium beijerinckii or Clostridium acetobutylicum. [The present invention 1087] The recombinant microorganism of the present invention 1086, wherein the acetoacetate decarboxylase is Adc. [The present invention 1088] The recombinant microorganism of the present invention 1086, wherein the Adc contains an amino acid sequence containing SEQ ID NO: 75 or 77. [The present invention 1089] The recombinant microorganism of the present invention 1088, wherein the Adc is encoded by a nucleic acid sequence containing SEQ ID NO: 74 or 76. [The present invention 1090] The following (f) to (i): (f) Expression of at least one exogenous nucleic acid molecule encoding acetoacetyl-CoA thiolase; (g) Expression of at least one exogenous nucleic acid molecule encoding acetate:acetoacetyl-CoA transferase; and (h) Expression of at least one exogenous nucleic acid molecule encoding acetoacetate decarboxylase that catalyzes the conversion of acetoacetate to acetone; (i) Expression of at least one exogenous nucleic acid molecule encoding alcohol dehydrogenase that catalyzes the conversion of acetone to isopropanol The recombinant microorganism of the present invention 1024 or 1048, further comprising a pathway for isopropanol production involving one or more of the above. [The present invention 1091] A recombinant microorganism capable of producing glycolic acid from a raw material containing xylose and glucose, using xylose and glucose simultaneously, (a) Deletion or inactivation of fucO, yqhD, araFGH, and xylFGH from the genome of the parental microorganism; and (b) Expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters including one or more of the above, further expressing one or more pathways for the production of glycolic acid, said recombinant microorganism. [The present invention 1092] The recombinant microorganism of the present invention 1091, further comprising deletion or inactivation of glcDEF. [The present invention 1093] The recombinant microorganism of the present invention 1091, further comprising deletion or inactivation of dkgA. [The present invention 1094] The recombinant microorganism of the present invention 1091 further comprising the deletion or inactivation of yahK. [The present invention 1095] The recombinant microorganism of the present invention 1091, wherein the xylose co-transporter is controlled by the GAPDH promoter at the araFGH locus. [The present invention 1096] The recombinant microorganism of the present invention 1091, wherein the C5 sugar co-transporter is the xylose co-transporter XylE. [The present invention 1097] The recombinant microorganism of the present invention 1096, wherein the XylE comprises the amino acid sequence comprising SEQ ID NO: 49. [The present invention 1098] The recombinant microorganism of the present invention 1096, wherein the XylE is encoded by the nucleic acid sequence comprising SEQ ID NO: 48. [The present invention 1099] The recombinant microorganism of the present invention 1091, wherein the xylose co-transporter is endogenous to the microorganism. [The present invention 1100] The recombinant microorganism of the present invention 1091, wherein the C5 sugar co-transporter is the arabinose co-transporter AraE. [The present invention 1101] The recombinant microorganism of the present invention 1091, wherein the arabinose co-transporter is endogenous to the microorganism. [The present invention 1102] The recombinant microorganism of the present invention 1091, wherein the uptake of xylose is not sensitive to catabolite repression by other monosaccharides. [The present invention 1103] The recombinant microorganism of the present invention 1091 comprising a functional phosphotransferase system. [The present invention 1104] The recombinant microorganism of the present invention 1091 comprising the native wild-type nucleic acid sequence encoding the cAMP receptor protein (CRP). [The present invention 1105] The recombinant microorganism of the present invention 1104, wherein the CRP comprises the amino acid sequence comprising SEQ ID NO: 10. [The present invention 1106] The recombinant microorganism of the present invention 1104, wherein the CRP is encoded by the nucleic acid sequence comprising SEQ ID NO: 9. [The present invention 1107] The recombinant microorganism of the present invention 1091, wherein the constitutive overexpression of the xylose co-transporter enables the continuous transfer of xylose from the raw material to the microorganism. [The present invention 1108] The recombinant microorganism of the present invention 1091, wherein the constitutive overexpression of the arabinose co-transporter enables the continuous transfer of xylose from the raw material to the microorganism. [The present invention 1109] The recombinant microorganism of the present invention 1091, wherein the continuous xylose transfer occurs independently of the presence of other sugars in the raw material. [The present invention 1110] The following (c) to (e): (c) Expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose isomerase and / or ketohexokinase and / or fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) Expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) Deletion or inactivation of one or more xylulokinases from the genome of the parental microorganism A recombinant microorganism of the invention 1091 comprising a pathway for the production of glycolic acid involving one or more of the above. [The invention 1111] The recombinant microorganism of the invention 1110, wherein (c) and (d) are present in an operon controlled by the proD promoter. [The invention 1112] The recombinant microorganism of the invention 1111, wherein the proD promoter is encoded by a nucleic acid sequence comprising SEQ ID NO: 53. [The invention 1113] The recombinant microorganism of the invention 1110, wherein the xylose isomerase is XylA. [The invention 1114] The recombinant microorganism of the invention 1113, wherein the XylA comprises an amino acid sequence comprising SEQ ID NO: 6. [The invention 1115] The recombinant microorganism of the invention 1113, wherein the XylA is encoded by a nucleic acid sequence comprising SEQ ID NO: 5. [The invention 1116] The recombinant microorganism of the invention 1110, wherein the xylose isomerase is endogenous to the microorganism. [The invention 1117] The recombinant microorganism of the invention 1110, wherein the ketohexokinase is of human origin. [The invention 1118] The recombinant microorganism of the invention 1110, wherein the ketohexokinase is heterologous to the microorganism. [The invention 1119] The recombinant microorganism of the invention 1118, wherein the ketohexokinase is khk-C. [The invention 1120] The recombinant microorganism of the invention 1119, wherein the khk-C comprises an amino acid sequence comprising SEQ ID NO: 12. [The invention 1121] The recombinant microorganism of the invention 1119, wherein the khk-C is encoded by a nucleic acid sequence comprising SEQ ID NO: 11. [The invention 1122] The recombinant microorganism of the invention 1110, wherein the fructose-bisphosphate aldolase is of human origin. [The invention 1123] The recombinant microorganism of the invention 1122, wherein the fructose-bisphosphate aldolase is aldoB. [The invention 1124] The recombinant microorganism of the present invention 1123, wherein the aldoB comprises an amino acid sequence containing SEQ ID NO: 51. [The present invention 1125] The recombinant microorganism of the present invention 1123, wherein the aldoB is encoded by a nucleic acid sequence containing SEQ ID NO: 50. [The present invention 1126] The recombinant microorganism of the present invention 1110, wherein the fructose - bisphosphate aldolase is heterologous to the microorganism. [The present invention 1127] The recombinant microorganism of the present invention 1110, wherein the glycolaldehyde dehydrogenase is aldA. [The present invention 1128] The recombinant microorganism of the present invention 1127, wherein the aldA comprises an amino acid sequence containing SEQ ID NO: 4. [The present invention 1129] The recombinant microorganism of the present invention 1127, wherein the aldA is encoded by a nucleic acid sequence containing SEQ ID NO: 3. [The present invention 1130] The recombinant microorganism of the present invention 1110, wherein the glycolaldehyde dehydrogenase is endogenous to the microorganism. [The present invention 1131] The recombinant microorganism of the present invention 1110, wherein the xylulokinase is XylB. [The present invention 1132] The recombinant microorganism of the present invention 1131, wherein the xylB comprises an amino acid sequence containing SEQ ID NO: 14. [The present invention 1133] The recombinant microorganism of the present invention 1131, wherein the xylB is encoded by a nucleic acid sequence containing SEQ ID NO: 13. [The present invention 1134] The following (c) to (e): (c) Expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) Expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) Deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases from the genome of the parental microorganism The recombinant microorganism of the present invention 1091, comprising a pathway for glycolic acid production involving one or more of the above. [The present invention 1135] The recombinant microorganism of the present invention 1134, wherein (c) and (d) are controlled by the proD promoter. [The present invention 1136] The recombinant microorganism of the present invention 1135, wherein the proD promoter is encoded by a nucleic acid sequence containing SEQ ID NO: 53. [The present invention 1137] The recombinant microorganism of the present invention 1134, wherein the xylose isomerase is XylA. [The present invention 1138] The recombinant microorganism of the present invention 1137, wherein the XylA comprises an amino acid sequence containing SEQ ID NO: 6. [The present invention 1139] The recombinant microorganism of the present invention 1138, wherein the XylA is encoded by a nucleic acid sequence containing SEQ ID NO: 5. [The present invention 1140] The recombinant microorganism of the present invention 1134, wherein the xylulokinase is XylB. [The present invention 1141] The recombinant microorganism of the present invention 1140, wherein the xylB comprises an amino acid sequence containing SEQ ID NO: 14. [The present invention 1142] The recombinant microorganism of the present invention 1140, wherein the xylB is encoded by a nucleic acid sequence containing SEQ ID NO: 13. [The present invention 1143] The recombinant microorganism of the present invention 1134, wherein the xylose dehydrogenase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. [The present invention 1144] The recombinant microorganism of the present invention 1143, wherein the xylose dehydrogenase is xdh. [The present invention 1145] The recombinant microorganism of the present invention 1144, wherein the xdh comprises an amino acid sequence containing SEQ ID NO: 16, 17, or 19. [The present invention 1146] The recombinant microorganism of the present invention 1144, wherein the xdh is encoded by a nucleic acid sequence containing SEQ ID NO: 15, 18, or 97. [The present invention 1147] The recombinant microorganism of the present invention 1134, wherein the xylose dehydrogenase is heterologous to the microorganism. [The present invention 1148] The recombinant microorganism of the present invention 1134, wherein the xylonolactonase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. [The present invention 1149] The recombinant microorganism of the present invention 1148, wherein the xylonolactonase is xylC. [The present invention 1150] The recombinant microorganism of the present invention 1149, wherein the xylC comprises an amino acid sequence containing SEQ ID NO: 55, 57, or 59. [The present invention 1151] The recombinant microorganism of the present invention 1149, wherein the xylC is encoded by a nucleic acid sequence containing SEQ ID NO: 54, 56, or 58. [The present invention 1152] The recombinant microorganism of the present invention 1134, wherein the xylonolactonase is heterologous to the microorganism. [The present invention 1153] The recombinant microorganism of the present invention 1134, wherein the xylonolactonase is endogenous to the microorganism. [The present invention 1154] The recombinant microorganism of the present invention 1134, wherein the glycolaldehyde dehydrogenase is aldA. [The present invention 1155] The recombinant microorganism of the present invention 1154, wherein the aldA comprises an amino acid sequence containing SEQ ID NO: 4. [The present invention 1156] The recombinant microorganism of the present invention 1154, wherein the aldA is encoded by a nucleic acid sequence containing SEQ ID NO: 3. [The present invention 1157] The recombinant microorganism of the present invention 1134, wherein the glycolaldehyde dehydrogenase is endogenous to the microorganism. [The present invention 1158] (f) Expression of at least one endogenous or exogenous nucleic acid molecule encoding isocitrate lyase; and / or (g) Expression of at least one endogenous or exogenous nucleic acid molecule encoding glyoxylate reductase The recombinant microorganism of the present invention 1110 or 1134, which further expresses a pathway for the production of glycolic acid involving one or more of the above. [The present invention 1159] The recombinant microorganism of the present invention 1158, wherein (f) and (g) are present in an operon controlled by the OXB20 promoter. [The present invention 1160] The recombinant microorganism of the present invention 1159, wherein the OXB20 promoter is encoded by a nucleic acid sequence containing SEQ ID NO: 96. [The present invention 1161] The recombinant microorganism of the present invention 1158, wherein the isocitrate lyase is AceA. [The present invention 1162] The recombinant microorganism of the present invention 1161, wherein the AceA comprises an amino acid sequence containing SEQ ID NO: 90. [The present invention 1163] The recombinant microorganism of the present invention 1161, wherein the AceA is encoded by a nucleic acid sequence containing SEQ ID NO: 89. [The present invention 1164] The recombinant microorganism of the present invention 1158, wherein the glyoxylate reductase is YcdW. [The present invention 1165] The recombinant microorganism of the present invention 1164, wherein the YcdW comprises an amino acid sequence containing SEQ ID NO: 92. [The present invention 1166] The recombinant microorganism of the present invention 1164, wherein the YcdW is encoded by a nucleic acid sequence containing SEQ ID NO: 91. [The present invention 1167] Species of the genus Clostridium, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa obata(SporomusaAny recombinant microorganism of the present invention obtained from a parent microorganism selected from the group consisting of Blautia producta, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, species of the genus Bacillus, species of the genus Corynebacterium, Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus. [The present invention 1168] The recombinant microorganism of the present invention 1167, wherein the parent microorganism is Escherichia coli.

Brief Description of the Drawings

[0076] [Figure 1] Shows multiple pathways using xylose and glucose to produce the products contemplated in this specification. [Figure 2] A graph showing the detection of simultaneous use of glucose and xylose in co-consumer strains in a 1:1 ratio culture, while in the parental strain, xylose began to be consumed only after glucose was depleted. [Figure 3]A graph showing co - consuming strains that consume 75% of the initial sugar mixture, while the parental strain consumed only 62% (after 36 hours of cultivation). For a 6:1 ratio of cultivation, both the parental strain and the co - consuming strains completely consumed the initial glucose and xylose with similar profiles of xylose consumption and biomass production. [Figure 4] A graph showing the use of MEG in the strain. The total amount of MEG increased by 12% and the amount of acetone increased by 197%. [Figure 5] A graph showing the detection of simultaneous use of glucose and xylose in the co - consuming strain in a 1:1 ratio of cultivation, while in the parental strain, xylose began to decrease only 18 hours after the depletion of glucose. [Figure 6] A graph showing co - consuming strains that consume 61% of the initial sugar mixture, while the parental strain consumed only 52% (after 36 hours of cultivation). For a 6:1 ratio of cultivation, both the co - consuming strain and the parental strain completely consumed the initial glucose and xylose with similar profiles of xylose consumption and biomass production. [Figure 7] A graph showing that the total amount of MEG increased by 9% and the amount of acetone increased by 119%. **Modes for Carrying Out the Invention**

[0077] **Detailed Description of the Disclosure** The present disclosure generally relates to biotechnology for maximizing the production of desired products from bio - renewable plant raw materials that, due to the inhibitory effects of multiple carbon sources present in a single type of raw material, typically do not have the ability to achieve maximum yields and productivities. The present disclosure describes methods and compositions for reducing or eliminating inhibitory effects resulting from the co - consumption of some monosaccharides that lead to microorganisms that do not function at maximum productivity.

[0078] The following definitions and abbreviations should be used in the interpretation of the present disclosure.

[0079] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "a microorganism" includes reference to one or more microorganisms, and the like.

[0080] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having", "contain", "containing", or any other variation thereof are intended to cover non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may contain other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not to an exclusive "or".

[0081] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", and "oligonucleotide" are used interchangeably. These refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides. Coding or non-coding regions of genes or gene fragments, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. When present, modifications to the nucleotide structure may be imparted before or after polymerization of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling component.

[0082] "Complementary" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by conventional Watson-Crick or other non-conventional means. The percentage of complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Fully complementary" means that all consecutive residues of a nucleic acid sequence hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. For example, sequence identity, such as for the purpose of assessing percentage complementarity, may be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally with default settings). The optimal alignment can be evaluated using any suitable parameters of the selected algorithm, including default parameters.

[0083] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (and to, e.g., an mRNA or other RNA transcript), and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product". When a polynucleotide is obtained from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0084] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, may include modified amino acids, and may include non-amino acids interspersed therein. The terms also encompass modifications, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component, among any other manipulated amino acid polymer. As used herein, the term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and both D- or L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0085] As used herein, the term "about" is used synonymously with the term "approximately". By way of example, the use of the term "about" with respect to an amount indicates a value that deviates slightly from the recited value, e.g., ±0.1% to 10%.

[0086] The term "biologically pure culture" or "substantially pure culture" refers to a culture of the bacterial species described herein that does not contain other bacterial species in an amount sufficient to interfere with the replication of the culture or to be detected by ordinary bacteriological techniques.

[0087] As used herein, "control sequence" refers to an operator, promoter, silencer, or terminator.

[0088] As used herein, "introduced" refers to introduction by modern biotechnology, not natural occurrence.

[0089] As used herein, a "constitutive promoter" is a promoter that is active under most conditions and / or during most stages of development. There are several advantages to using a constitutive promoter in an expression vector used in biotechnology, such as high-level production of a protein used to select transgenic cells or organisms, high-level expression of a reporter protein or scorable marker that allows for easy detection and quantification, high-level production of a transcription factor that is part of a regulatory transcription system, production of a compound that requires ubiquitous activity in an organism, and production of a compound required at all stages of development.

[0090] As used herein, a "non-constitutive promoter" is a promoter that is active under specific conditions, in specific types of cells, and / or during specific stages of development. For example, inducible promoters and promoters under developmental control are non-constitutive promoters.

[0091] As used herein, an "inducible" or "repressible" promoter is a promoter that is under the control of chemical or environmental factors. Examples of environmental conditions that can affect transcription by an inducible promoter include anaerobic conditions, specific chemicals, the presence of light, acidic or basic conditions, and the like.

[0092] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that one function is controlled by the other. For example, a promoter is operably linked to a coding sequence if it can control the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence can be operably linked to the regulatory sequence in a sense or antisense orientation. In another example, the complementary RNA regions of the present disclosure may be bound directly or indirectly to the target mRNA at the 5', or at the 3', or within the target mRNA, or the first complementary region to the target mRNA is at the 5' and its complement is at the 3'.

[0093] As used herein, the term "signal sequence" refers to an amino acid sequence that targets peptides and polypeptides to a cellular location or the extracellular environment. Signal sequences are typically present at the N-terminal portion of a polypeptide and are typically removed enzymatically. Polypeptides having those signal sequences are referred to as full-length and / or unprocessed. Polypeptides from which those signal sequences have been removed are referred to as mature and / or processed.

[0094] As used herein, the term "exogenous" with respect to various molecules, such as polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in, and / or are not produced by, a given yeast, bacterium, organism, microorganism, or cell in nature.

[0095] On the other hand, as used herein, the terms "endogenous" or "native" with respect to various molecules, such as polynucleotides, polypeptides, enzymes, etc., refer to molecules that are normally or naturally found in, and / or are produced by, a given yeast, bacterium, organism, microorganism, or cell in nature.

[0096] As used herein in connection with a modified host cell, the term "heterologous" refers to various molecules, such as polynucleotides, polypeptides, enzymes, etc., where at least one of the following is true: (a) the molecule is foreign ( "exogenous") to the host cell (ie, not naturally found therein), (b) the molecule is naturally found in a given host microorganism or host cell (eg, "endogenous" thereto), but is produced at an unnatural location or in an unnatural amount in the cell, and / or (c) the molecule has a nucleotide or amino acid sequence different from the endogenous nucleotide or amino acid sequence, such that a molecule with a nucleotide or amino acid sequence different from the endogenous nucleotide or amino acid found endogenously is produced in an unnatural amount in the cell (eg, more than is naturally found).

[0097] As used herein in connection with a proenzyme or gene of a first family or species, the term "homolog" refers to a distinct enzyme or gene of a second family or species, which are determined by functional analysis, structural analysis, or genomic analysis to be an enzyme or gene of the second family or species corresponding to the proenzyme or gene of the first family or species. Homologs often have functional, structural, or genomic similarities. Techniques are known that allow the easy cloning of homologs of enzymes or genes using genetic probes and PCR. The identity of a cloned sequence as a homolog can be confirmed by functional assays and / or genomic mapping of the gene.

[0098] A protein has "homology" to, or is "homologous" to, a second protein when the amino acid sequence encoded by the gene has an amino acid sequence similar to the amino acid sequence of the second gene. Alternatively, if two proteins have "similar" amino acid sequences, the protein has homology to the second protein. Thus, the term "homologous protein" is intended to mean that two proteins have similar amino acid sequences. In one particular example, the homology between two proteins indicates their shared ancestor, which is associated with evolution. The terms "homologous sequence" or "homolog" are considered, believed, or known to be functionally related. The functional relationship can be shown by any one of several ways, including but not limited to (a) the degree of sequence identity, and / or (b) the same or similar biological functions. Preferably, both (a) and (b) are shown. The degree of sequence identity may vary, but in one aspect, it is at least 50% (when using standard sequence alignment programs known in the art), at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least aboutOther non-limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Michigan), AlignX, and Vector NTI (Invitrogen, Carlsbad, CA). Similar biological functions can include, but are not limited to: catalyzing the same or similar enzymatic reactions, having the same or similar selectivity for substrates or cofactors, having the same or similar stability, having the same or similar tolerance to various fermentation conditions (temperature, pH, etc.), and / or having the same or similar tolerance to various metabolic substrates, products, by-products, intermediates, etc. The degree of similarity of biological functions can vary, but in one aspect, according to one or more assays known to those of skill in the art for determining a given biological function, it is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%.

[0099] The term "variant" refers to any polypeptide or enzyme described herein. Variants also encompass one or more components of multimers, multimers containing individual components, multimers containing multiples of individual components (e.g., multimers of a reference molecule), chemical degradation products, and biological degradation products. In particular, in a non-limiting aspect, an enzyme can be a "variant" with respect to a reference enzyme due to a change in any portion of the polypeptide sequence encoding the reference enzyme. A variant of a reference enzyme can have at least 10%, at least 30%, at least 50%, at least 80%, at least 90%, at least 100%, at least 105%, at least 110%, at least 120%, at least 130% or more enzyme activity in a standard assay used to measure the enzyme activity of a preparation of the reference enzyme. In some aspects, a variant can also refer to a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, 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 to the full-length enzyme or untreated enzyme of the present disclosure. In some aspects, a variant can also refer to a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, 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 to the mature enzyme or treated enzyme of the present disclosure.

[0100] As used herein, the terms "microorganism" or "microbe" should be construed broadly. These terms, used interchangeably, include, but are not limited to, the two prokaryotic domains, bacteria and archaea.

[0101] As used herein, the terms "isolating," "isolated," "isolated microorganism" and like terms are intended to mean that one or more microorganisms have been separated from at least one of the associated substances in a particular environment (e.g., medium, water, reaction chamber, etc.). Thus, an "isolated microorganism" does not exist in its natural environment; rather, it has been removed from its natural situation and placed in a non-natural existence situation through the various techniques described herein. Thus, an isolated strain or isolated microorganism may exist, for example, as a biologically pure culture or as a spore (or other form of the strain). In an embodiment, the isolated microorganism may be associated with an acceptable carrier, which may be a commercially or industrially acceptable carrier.

[0102] In certain aspects of the present disclosure, an isolated microorganism exists as an "isolated biologically pure culture". One of ordinary skill in the art will understand that an isolated biologically pure culture of a particular microorganism substantially contains no other living organisms and contains only the individual microorganism of interest. The culture can contain the microorganism at various concentrations. The present disclosure describes that an isolated biologically pure microorganism is generally "necessarily different from a substance of low purity or an impure substance". See, e.g., In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microbes), and Parke-Davis & Co. v. H.K. Mulford & Co., 189 F.95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), aff’d in part, rev’d in part, 196 F.496 (2d Cir. 1912), each of which is hereby incorporated by reference. Further, in some aspects, the present disclosure provides certain quantitative concentration measurements or purity limits that must be found within an isolated biologically pure microorganism culture. The presence of these purity values is, in certain aspects, an additional attribute that distinguishes the microorganisms disclosed herein from that microorganism as it exists in its natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limits of vitamin B12 produced by a microorganism), which is hereby incorporated by reference.

[0103] The microorganisms of the present disclosure may include spores and / or vegetative cells. In some embodiments, the microorganisms of the present disclosure include microorganisms in a viable but non-culturable (VBNC) state. As used herein, "spore" or "spores" refers to a structure produced by bacteria and fungi suitable for survival and dispersal. Spores are generally characterized as resting structures, but spores have the ability to differentiate through the process of germination. Germination is the differentiation of a spore into a vegetative cell capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are units of asexual reproduction and may be essential structures in the fungal life cycle. Bacterial spores are usually structures for a survival state that cannot normally contribute to the survival or growth of vegetative cells.

[0104] As used herein, "microbial composition" refers to a composition comprising one or more microorganisms of the present disclosure.

[0105] As used herein, "carrier", "acceptable carrier", "commercially acceptable carrier", or "industrially acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle through which a microorganism can be administered, stored, or transported, and which does not have a harmful effect on the microorganism.

[0106] As used herein, the term "productivity" refers to the yield of a product from a biosynthetic pathway. In one embodiment, productivity can be expressed as the weight percentage of the final product per weight of the starting compound.

[0107] As used herein, the term "thermodynamic maximum yield" refers to the maximum yield of a product obtained from the fermentation of a given feedstock, such as glucose, based on the energy value of the product compared to the feedstock. In normal fermentation, for example, when no additional energy source such as light, hydrogen gas, or methane or electricity is used, the product does not contain more energy than the feedstock. The thermodynamic maximum yield means the yield of the product in which all of the energy and mass from the feedstock are converted into the product. This yield can be calculated and is independent of a specific pathway. If a specific pathway to the product has a lower yield than the thermodynamic maximum yield, the product is most likely to lose mass and be improved or replaced by a more efficient pathway to the product.

[0108] The term "balanced redox" refers to a series of reactions that generate as many redox cofactors as they consume. By designing metabolic pathways and engineering organisms such that the redox cofactors are balanced or nearly balanced, it is often possible to achieve more efficient and higher-yield production of a desired compound. Redox reactions always occur together because two half-reactions occur simultaneously, one an oxidation reaction and the other a reduction reaction. In a redox process, the reducing agent transfers electrons to the oxidizing agent. Thus, in a reaction, the reductant or reducing agent loses electrons and is oxidized, while the oxidant or oxidizing agent gains electrons and is reduced. In one aspect, the redox reaction occurs in a biological system. Biological energy is frequently stored and released by redox reactions. Photosynthesis involves the reduction of carbon dioxide to sugar and the oxidation of water to molecular oxygen. The reverse reaction, respiration, oxidizes sugar to produce carbon dioxide and water. As an intermediate step, reduced carbon compounds are used to reduce nicotinamide adenine dinucleotide (NAD+), which then contributes to the generation of a proton gradient that results in the synthesis of adenosine triphosphate (ATP) and is maintained by the reduction of oxygen. The term "redox state" is used to describe the balance of GSH / GSSG, NAD+ / NADH, and NADP+ / NADPH in biological systems such as cells or tissues. The redox state is reflected in the balance of a set of metabolites (e.g., lactate and pyruvate, beta-hydroxybutyrate, and acetoacetate), and their interconversion depends on these ratios. Abnormal redox states can occur in various detrimental situations such as hypoxia, seizures, and sepsis.

[0109] As used herein, the term "productivity" refers to the total amount of biological product produced per hour, grams of product / (liter per hour).

[0110] As used herein, the terms "substantially free of microorganisms", "substantially free of bacteria", or "substantially free of fungi / yeast" should not be construed to mean the absence of microorganisms / bacteria / fungi / yeast, although this may be preferred in some embodiments. Rather, "substantially free of" should be construed to mean, for example, that a composition substantially free of bacteria is a composition in which any bacteria present in the composition are present in an amount below the limit of detection. In some embodiments, the microorganisms are selected from one or more of bacteria, fungi, yeast, viruses, protists, and algae.

[0111] As used herein, the term "free of microorganisms" refers to either the complete absence of microorganisms or the complete absence of viable microorganisms capable of vegetative growth or reproduction.

[0112] Concurrent consumption of xylose and glucose In an industrial or commercial process, the productivity of microorganisms is an important factor that must be considered when examining the economic viability of large-scale reactions and is often a make-or-break factor. Microbial productivity in this sense is the grams of product produced per liter per hour. In the absence of modified microorganisms, a stream containing both xylose and glucose is always fed to the reaction chamber(s), which is likely to result in the inhibition of the uptake of one or more monosaccharides by at least glucose.

[0113] The mechanism underlying the diauxic shift is carbon catabolite repression (CCR), where the global transcriptional regulator CRP (cAMP receptor protein) plays a central role in regulating the transcriptional activation of catabolic operons for secondary sugars such as xylose, arabinose, and galactose. The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is also involved in the glucose-inducible repression of xylose utilization in E. coli. Xylose can be used by E. coli as the sole carbon and energy source and can be metabolized via the pentose phosphate pathway. Xylose can be imported by two uptake systems, a high-affinity ATP-dependent system and a relatively low-affinity D-xylose:H+ symporter. Unlike arabinose transport, which is mainly transported via a more energy-efficient cotransporter, xylose is mainly transported via a higher energy-cost ATP-dependent transporter even at high sugar concentrations. All genes involved in xylose uptake and catabolism are sensitive to CCR.

[0114] To have an effective bioprocess for converting pentoses into desired chemicals, it is necessary to engineer host microorganisms for the efficient, simultaneous, and rapid use of mixed sugars in order to achieve the yields and productivities required for industrial processes. The present disclosure describes metabolic engineering strategies that efficiently promote the simultaneous consumption of xylose and glucose from lignocellulosic biomass, resulting in access to the full potential of engineered microbial strains for desired chemicals obtained from pathways having D-xylonate or D-xylulose-1P or glycolaldehyde as intermediates.

[0115] Common strategies for engineering sugar co - utilization in Escherichia coli rely on inactivation of PTS components, whether or not they are related to improvement of galP (galactose:H+ symporter) activity and mutagenesis of CRP. However, inactivation of PTS components impairs glucose uptake, and CRP mutants often slow down the potential growth phenotype due to unpredictable changes in the expression of other important genes. These two approaches result in a decrease in productivity, especially under conditions of high sugar concentration and low - cost media.

[0116] The applicant considers it a top priority to develop a metabolic engineering strategy that supports co - consumption of glucose and xylose for the production of a desired chemical substance having D - xylulose - 1P or D - xylonate or glycolaldehyde as an intermediate independent of PTS inactivation and having a deletion of an ATP - binding transporter.

[0117] In some embodiments, promoting the simultaneous consumption of xylose and glucose for the production of a desired chemical substance is based on: (1) constitutive overexpression of an ATP - dependent D - xylose symporter; (2) constitutive expression of genes for the conversion of xylose to D - xylulose - 1P or D - xylonate; (3) deletion of the native pentose (primarily xylose and arabinose) ABC transporter system; and / or deletion of xylose catabolic genes.

[0118] The subject matter described herein is distinguished over the state of the art in that a pathway that involves deletion or inactivation of an ABC transporter and expression of a cotransporter and uses or includes D-xylulose, D-xylonate, or glycolaldehyde as an intermediate not only has a positive impact on sugar co-utilization, but also enhances the overall yield and productivity of the pathway for the production of a desired chemical substance. This improvement is by modulation of the overall metabolism of the microorganism, modification of the ATP availability profile, and promotion of the production of the intermediates D-xylulose-1P, D-xylonate, and / or glycolaldehyde. See Kim et al. (2015. Metabolic Engineering, 30:141-148), Sievert et al. (2017. PNAS, 114(28):7349-7354), Wang et al. (2018. Microbial Cell Factories, 17(12):1-12), and Bai et al. (2016. Metabolic Engineering, 38:285-292).

[0119] The present disclosure includes strategies to overcome catabolite repression of glucose over xylulose that enable the simultaneous consumption of both sugars. Unlike other methods for sugar co-consumption, this strategy is designed and implemented to focus on ensuring efficient xylulose uptake that is not sensitive to catabolite repression by sugars while maintaining efficient glucose uptake by the native PTS system.

[0120] In some embodiments, the method comprises making the following modifications in a target microbial strain: 1(a) overexpressing the native xylose co-transporter XylE operably linked to a constitutive promoter, and / or 1(b) overexpressing the native arabinose co-transporter AraE operably linked to a constitutive promoter; 2(a) overexpressing the native xylose isomerase XylA and the heterologous ketohexokinase khk-C under a constitutive promoter, and deleting or inactivating the native xylulokinase XylB, or 2(b) overexpressing the heterologous xylose dehydrogenase xdh operably linked to a constitutive promoter, and deleting or inactivating the native xylose isomerase XylA and / or deleting the native xylulokinase XylB; and 3, deleting the ATP-binding transporter proteins AraFGH, XylFGH, RbsABC, and AlsABC.

[0121] In some embodiments, the constitutive expression of the xylose and arabinose co-transporters is independent of CRP control and, as a result, enables xylose import independent of other sugars present in the culture broth. In some embodiments, the constitutive expression of the xylose isomerase is independent of CRP control and, as a result, also enables xylose utilization independent of other sugars present in the culture broth. In some embodiments, the expression of ketohexokinase khk-C efficiently converts D-xylose to D-xylose 1-P, an intermediate for the production of a desired chemical. In some embodiments, the deletion of xylulokinase prevents the diversion of carbon from the pathway for chemical production to the native pentose phosphate pathway.

[0122] In some embodiments, the constitutive expression of xylose dehydrogenase is independent of CRP control and, as a result, also enables xylose utilization independent of other sugars present in the culture broth and efficiently converts D-xylose to D-xylonate as an intermediate for the production of the desired chemical. In some embodiments, the deletion of xylulokinase and / or xylose isomerase prevents the conversion of carbon from the pathway for chemical production into the native pentose phosphate pathway. In some embodiments, the deletion of ATP-binding cassette transporters such as the arabinose ABC transporter and the xylose ABC transporter avoids the loss of ATP during sugar import. The net amount of ATP can change the activity of central metabolism in E. coli and potentially increase pathway yields.

[0123] Overexpression of the native xylose cotransporter XylE under a constitutive promoter The D-xylose / proton cotransporter XylE is an ATP-dependent low-affinity transporter that is a member of the major facilitator superfamily (MFS) of transporters encoded by the xylE gene. The transcription of xylE is thought to be controlled by XylR (SEQ ID NO: 7 or SEQ ID NO: 8). XylR is a transcription factor encoded by the xylR gene and positively regulates the transcription of xylose metabolic genes and transporter genes in response to xylose (xylE, xylFGH, and xylAB genes).

[0124] Constitutive overexpression of the xylose cotransporter releases carbon catabolite repression and enables continuous xylose import independent of the sugars present in the culture broth, although glucose uptake still occurs via the PTS components. Thus, both glucose and xylose present in the hydrolyzate can be imported simultaneously by E. coli.

[0125] Overexpression of the native arabinose cotransporter AraE under a constitutive promoter The D - arabinose / proton symporter AraE is an ATP - dependent low - affinity transporter that is a member of the major facilitator superfamily (MFS) of transporters encoded by the araE gene. The transcription of araE is controlled by AraC (SEQ ID NO: 32 and SEQ ID NO: 33) and CRP. AraC is a transcription factor encoded by the araC gene that negatively regulates the transcription of xylose metabolism genes and transporter genes in response to arabinose (xylE, xylFGH, and xylAB genes), and positively regulates the transcription of arabinose metabolism genes and transporter genes in response to arabinose (araE, araFGH, and araBAD genes). The expression of araE is induced by arabinose in the absence of glucose. The AraE transporter is promiscuous and is known to be able to transport xylose and other pentoses.

[0126] Constitutive expression of the promiscuous arabinose symporter releases CCR and enables continuous xylose import independent of the sugars present in the culture broth, although glucose uptake still occurs via the PTS components. Therefore, both glucose and xylose present in the hydrolysate can be imported simultaneously by E. coli.

[0127] Expression of the native xylose isomerase XylA and the heterologous ketohexokinase khk - C under a constitutive promoter, and deletion of the native xylulokinase XylB XylA is an endogenous D-xylose isomerase that catalyzes the conversion of D-xylose to D-xylulose (Figure 1, reaction 5, pathway B). D-xylose isomerase (E.C. 5.3.1.5) catalyzes the natural catabolic reaction of D-xylose in Escherichia coli. The transcription of xylA is controlled by XylR and CRP, and its expression is induced by xylose in the absence of glucose. Ketohexokinase (Figure 1, reaction 6, pathway B) catalyzes the phosphorylation of D-xylulose to D-xylulose-1-P. Ketohexokinase (E.C. 2.7.1.3) is found in various organisms, and khk-C derived from human liver is a promising candidate for activity against xylose. D-xylulose 1-P is an important intermediate for the production of various chemicals.

[0128] XylB is the xylulose kinase (2.7.1.17) encoded by xylB that catalyzes the phosphorylation of D-xylulose (Figure 1, reaction 8, pathway B). This is the second step of the natural xylose degradation pathway that produces D-xylulose-5-phosphate, an intermediate of the pentose phosphate pathway. This reaction competes with the phosphorylation of D-xylulose by khk-C and diverts the flow from D-xylulose-1-P production to the pentose phosphate pathway.

[0129] Constitutive expression of the native xylose isomerase xylA releases CCR and, when associated with constitutive heterologous expression of ketohexokinase khk-C, enables continuous xylose utilization independent of the sugars present in the culture broth and generates D-xylulose 1-P as an intermediate for chemical production. Glucose uptake is still carried out by the PTS system components. Therefore, both glucose and xylose present in the hydrolysate can be used simultaneously by Escherichia coli. Deletion of the xylulose kinase xylB prevents the conversion of carbon from the pathway for chemical production to the native pentose phosphate pathway.

[0130] Expression of the heterologous xylose dehydrogenase xdh under a constitutive promoter and deletion of the native xylose isomerase Xyla and / or deletion of the native xylulokinase XylB The heterologous xylose dehydrogenase, xdh, catalyzes the conversion of D-xylose to D-xylonolactone (Figure 1, reaction 1, pathway A). D-Xylose dehydrogenase (E.C. 1.1.1.175) is found in various organisms, but the xdh from Caulobacter crescentus is a candidate for activity on D-xylose. Since D-xylonolactone can be spontaneously converted to D-xylonic acid, expression of xdh on xylose yields D-xylonic acid, an important intermediate for the production of various chemicals.

[0131] XylA, the D-xylose isomerase (E.C. 5.3.1.5) encoded by xylA, catalyzes the conversion of D-xylose to D-xylulose, an intermediate of the pentose phosphate pathway (Figure 1, reaction 5, pathway A). XylB, the xylulokinase (2.7.1.17) encoded by xylB, catalyzes the phosphorylation of D-xylulose, the second step in the xylose catabolic pathway, to produce D-xylulose-5-phosphate, another intermediate of the pentose phosphate pathway (Figure 1, reaction 8, pathway A). Both reactions compete with xdh and divert the flow from D-xylonic acid production to the pentose phosphate pathway.

[0132] Constitutive heterologous expression of xylose dehydrogenase enables continuous xylose utilization independent of the sugars present in the culture broth and yields D-xylonic acid as an intermediate for the production of the desired chemicals. Glucose uptake is still carried out by the PTS system components. Therefore, both glucose and xylose present in the hydrolysate can be used simultaneously by Escherichia coli. Deletion of the D-xylose isomerase and / or xylulokinase prevents the conversion of carbon from the pathway for chemical production to the native pentose phosphate pathway.

[0133] Deletion of ATP-binding cassette transporter proteins AraFGH, XylFGH, RbsABC, and AlsABC The arabinose ABC transporter AraFGH (E.C. 3.6.3.17, TCDB 3.A.1.2.2) is a high-affinity ATP-driven system encoded by the araFGH genes. AraF is a periplasmic binding protein, AraH is a membrane component, and AraG is the ATP-binding component of this ABC transporter. Transcription of the araFGH operon is controlled by AraC and CRP. araFGH expression is induced by arabinose in the absence of glucose. The AraFGH transporter is promiscuous and is known to transport xylose and other pentoses.

[0134] The xylose ABC transporter XylFGH (E.C. 3.6.3.17, TCDB 3.A.1.2.4) is a high-affinity ATP-driven system encoded by the xylFGH genes. XylF is a periplasmic binding protein, XylH is a membrane component, and XylG is the ATP-binding component of this ABC transporter. Transcription of the xylFGH operon is controlled by XylR and CRP, and its expression is induced by xylose in the absence of glucose.

[0135] The ribose ABC transporter RbsABC (E.C. 3.6.3.17; TCDB 3.A.1.2.1) is a high-affinity ATP-driven system encoded by the rbsABC genes.

[0136] In some embodiments, one or more nucleic acid molecules encoding the RbsB periplasmic binding protein subunit of RbsABC comprise the nucleic acid sequence set forth in SEQ ID NO: 35. In some embodiments, one or more amino acid sequences encoding the RbsB periplasmic binding protein subunit of RbsABC comprise the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, one or more nucleic acid molecules encoding the RbsA ATP-binding subunit of RbsABC comprise the nucleic acid sequence set forth in SEQ ID NO: 34. In some embodiments, one or more amino acid sequences encoding the RbsA ATP-binding subunit of RbsABC comprise the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, one or more nucleic acid molecules encoding the RbsC membrane subunit of RbsABC comprise the nucleic acid sequence set forth in SEQ ID NO: 36. In some embodiments, one or more amino acid sequences encoding the RbsC membrane subunit of RbsABC comprise the amino acid sequence set forth in SEQ ID NO: 39.

[0137] The allose ABC transporter AlsABC (E.C. 3.6.3.17; TCDB 3.A.1.2.6) is an ATP-driven system encoded by the alsABC gene.

[0138] In some embodiments, one or more nucleic acid molecules encoding the alsB periplasmic binding protein subunit of AlsABC comprise the nucleic acid sequence set forth in SEQ ID NO: 41. In some embodiments, one or more amino acid sequences encoding the alsB periplasmic binding protein subunit of AlsABC comprise the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, one or more nucleic acid molecules encoding the alsA ATP-binding subunit of AlsABC comprise the nucleic acid sequence set forth in SEQ ID NO: 40. In some embodiments, one or more amino acid sequences encoding the alsA ATP-binding subunit of AlsABC comprise the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, one or more nucleic acid molecules encoding the alsC membrane subunit of AlsABC comprise the nucleic acid sequence set forth in SEQ ID NO: 42. In some embodiments, one or more amino acid sequences encoding the alsC membrane subunit of AlsABC comprise the amino acid sequence set forth in SEQ ID NO: 45.

[0139] Deletion of ATP-binding cassette transporters such as ribose ABC transporter, allose ABC transporter, arabinose ABC transporter, and xylose ABC transporter, including the preferred xylose transporter in E. coli, releases CCR and enables continuous xylose import in relation to xylE constitutive expression (see Examples 1 and 2). Furthermore, the deletion avoids ATP loss during sugar import. The ATP net can change the activity of central metabolism in E. coli.

[0140] Microorganism As described herein, in some embodiments, the recombinant microorganism is capable of using both xylose and glucose simultaneously.

[0141] As described herein, in some embodiments, the recombinant microorganism is a prokaryotic microorganism. In some embodiments, the prokaryotic microorganism is a bacterium. "Bacteria," or "eubacteria," refers to the domain of prokaryotes. Bacteria include at least 11 distinct groups as follows: (1) two major subdivisions: (1) high G+C groups (Actinomycetes, Mycobacteria, Micrococcus, etc.), (2) low G+C groups (Bacillus, Clostridium, Lactobacillus, Staphylococcus, Streptococcus, Mycoplasma), gram-positive (gram+) bacteria; (2) Proteobacteria, e.g., purple photosynthetic + non-photosynthetic gram-negative bacteria (including the most "common" gram-negative bacteria); (3) cyanobacteria, e.g., oxygen-producing phototrophs; (4) Spirochete and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) green sulfur bacteria; (9) green non-sulfur bacteria (also anaerobic phototrophs); (10) radiation-resistant Micrococcus and related species; (11) Thermotoga and Thermosipho thermophiles.

[0142] "Gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. Genera of gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirillum, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0143] "Gram-positive bacteria" include cocci, non-spore-forming rods, and spore-forming rods. Genera of gram-positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0144] In some embodiments, the microorganisms of the disclosure are fungi.

[0145] In some embodiments, the recombinant microorganism is a eukaryotic microorganism. In some embodiments, the eukaryotic microorganism is yeast. In an exemplary embodiment, the yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma.

[0146] In some embodiments, the recombinant microorganism is a prokaryotic microorganism. In an exemplary embodiment, the prokaryotic microorganism is a member of a genus selected from the group consisting of Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.

[0147] In some embodiments, the microorganisms for use in the methods of the present disclosure can be selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, Metschnikowia, Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.

[0148] In some embodiments, the microorganisms resulting from the methods described herein may be species selected from any of the following genera: Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirillum, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, Fusobacterium, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Micrococcus, Nocardia, Staphylococcus, Streptococcus, Streptomyces, Saccharomyces, Pichia, and Aspergillus.

[0149] In some embodiments, the microorganisms for use in the methods of the present disclosure include Clostridium, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchi, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium mesoxybenzovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus spp., Corynebacterium spp., Yarrowia lipolytica, Schizosaccharomyces stipitis, and Terrisporobacter glycolicus.

[0150] The terms "recombinant microorganism" and "recombinant host cell" are used interchangeably herein and refer to a microorganism that has been genetically modified to express a heterologous enzyme, such as an enzyme contained in a vector, to express or overexpress an endogenous enzyme, or a microorganism having a change in the expression of an endogenous gene, in an integration construct. "Change" means that the expression, level, or activity of a gene, an RNA molecule or equivalent RNA molecule encoding one or more polypeptides or polypeptide subunits, or one or more polypeptides or polypeptide subunits is upregulated or downregulated, such that the expression, level, or activity is more or less than that observed in the absence of the change. For example, the term "alter" can mean "inhibit", but the use of the term "change" is not limited to this definition. It is understood that the terms "recombinant microorganism" and "recombinant host cell" refer not only to a particular recombinant microorganism, but also to the progeny or potential progeny of such a microorganism. Such progeny may not actually be identical to the parent cell due to either mutation or environmental influences, but may be included within the scope of the terms used herein because a particular modification may occur in subsequent generations.

[0151] The cultivation of the microorganism used in the method of the present disclosure can be carried out using any number of processes known in the art for culturing and fermenting a substrate using the microorganism of the present disclosure.

[0152] Fermentation may be carried out in any suitable bioreactor, such as a continuous stirred tank bioreactor, a bubble column bioreactor, an air-lift bioreactor, a fluidized bed bioreactor, a packed bed bioreactor, a photo-bioreactor, an immobilized cell reactor, a trickle bed reactor, a moving bed biofilm reactor, a bubble column, a gas lift fermenter, a hollow fiber membrane type bioreactor, or other membrane reactors. In some embodiments, the bioreactor includes a first growth reactor in which the microorganism is cultured and a second fermentation reactor to which the fermentation broth from the growth reactor is supplied and in which most of the fermentation product is produced. In some embodiments, the bioreactor simultaneously achieves the culture of the microorganism and the production of the fermentation product from a carbon source such as the provided substrate and / or raw material.

[0153] Product In some embodiments, the engineered microorganism of the present disclosure produces a fermentation product from a feedstock comprising xylose and glucose, and the recombinant microorganism uses xylose and glucose simultaneously. In some embodiments, the fermentation product produced by the microorganism comprises one or more molecules containing at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 carbon atoms.

[0154] In some embodiments, the engineered microorganism of the present disclosure has the ability to produce desired chemicals such as monoethylene glycol, glycolic acid, C3 compounds (such as acetone, isopropanol, and propene), amino acids, and polyols. See Koch et al. (International Publication No. WO 2017 / 156166 A1) and McBride et al. (International Publication No. WO 2011 / 022651 A1).

[0155] In some embodiments, the engineered microorganism of the present disclosure has the ability to produce the desired chemical at maximum yield due to the absence of the inhibitory effect of multiple carbon sources present in a single type of feedstock.

[0156] Genetic modification Genetic modifications introduced into one or more of the microorganisms of the present disclosure can alter or inactivate the regulatory sequences of the target gene. In some embodiments, genetic modifications introduced into one or more of the microorganisms of the present disclosure can introduce new traits or phenotypes into the one or more microorganisms. One or more regulatory sequences, including heterologous regulatory sequences and regulatory sequences present within the genomes of animals, plants, fungi, yeast, bacteria, or viruses corresponding to the microorganisms into which the genetic mutations are introduced, may also be inserted. Further, the regulatory sequences may be selected based on the expression levels of the genes in the microbial culture. The genetic variation may be a predetermined genetic variation that is introduced specifically at the target site. In some embodiments, the genetic variation is a nucleic acid sequence introduced into one or more microbial chromosomes. In some embodiments, the genetic variation is a nucleic acid sequence introduced into one or more extrachromosomal nucleic acid sequences. The genetic variation may be a random mutation within the target site. The genetic variation may be an insertion or deletion of one or more nucleotides. In some cases, multiple different genetic variations (e.g., 2, 3, 4, 5, 10, or more) are introduced into one or more of the isolated bacteria. The multiple genetic variations may be of any of the above types, the same type or different types, and any combination. In some cases, multiple different genetic variations are introduced sequentially, such that multiple desired modifications are accumulated within the microorganism, e.g., a first genetic variation after a first isolation step, a second genetic variation after a second isolation step, and they are repeated.

[0157] In some embodiments, the genetic modification is a deletion or inactivation of a target gene or regulatory sequence. In some embodiments, the deletion is a removal of the target gene or a substantial portion of the target gene. In some embodiments, the deletion is a replacement of the target gene or a substantial portion of the target gene. In further embodiments, the deletion results in a complete loss of function of the target gene. In some embodiments, the deletion results in a partial loss of function of the target gene. In some embodiments, the loss of function or partial loss of function is determined by comparing the activity of the modified target gene sequence to the activity of the unmodified target gene sequence. In some embodiments, the inactivation of the target gene is the result of deleting or disrupting one or more regulatory or control sequences operably linked to the target sequence. In some embodiments, the inactivation of the target gene is the result of disrupting the target gene with a heterologous sequence. In some embodiments, the inactivation results in a partial loss of function of the target gene. In some embodiments, the inactivation results in a complete loss of function of the target gene.

[0158] In some embodiments, one or more of the substrates described for the production of the desired chemical substance are biosynthesized from carbon sources (e.g., xylose and glucose).

[0159] Generally, the term "genetic variation" refers to any change introduced into a polynucleotide sequence compared to a reference polynucleotide, such as a reference genome or a portion thereof, or a reference gene or a portion thereof. A genetic variation may also be referred to as a "mutation", and a sequence or organism containing a genetic variation may also be referred to as a "genetic variant" or "mutant". Genetic variations can have a number of effects, such as increasing or decreasing several biological activities, including gene expression, metabolism, and cell signaling. Genetic variations can be introduced specifically at a target site or randomly. A variety of molecular tools and methods are available for introducing genetic variations. For example, genetic variations can be introduced via polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, recombineering, lambda red-mediated recombination, the CRISPR / Cas9 system, chemical mutagenesis, and combinations thereof. Chemical methods for introducing genetic variations include exposure of DNA to chemical mutagens such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitrosourea (ENU), N-methyl-N-nitro-N'-nitrosoguanidine, 4-nitroquinoline N-oxide, diethyl sulfate, benzo[a]pyrene, cyclophosphamide, bleomycin, triethylmelamine, acrylamide monomer, nitrogen mustard, vincristine, diepoxyalkanes (e.g., diepoxybutane), ICR-170, formaldehyde, procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12-dimethylbenz[a]anthracene, chlorambucil, hexamethylphosphoramide, bisulfan, etc. Radiation mutagens include ultraviolet light, gamma irradiation, X-rays, and high-speed neutron bombardment. Genetic variations can also be introduced into nucleic acids, for example, using trimethylpsoralen with ultraviolet light. Random or targeted insertion of mobile DNA elements, such as transposable elements, is another suitable method for generating genetic variations.Genetic mutations can be introduced into nucleic acids during amplification in a cell-free in vitro system using polymerase chain reaction (PCR) techniques such as PCR that are prone to errors. Genetic mutations can be introduced into nucleic acids in vitro using DNA shuffling techniques (such as exon shuffling, domain swapping, etc.).

[0160] Genetic mutations can also be introduced into nucleic acids as a result of defects in DNA repair enzymes in cells. For example, the presence of a mutant gene encoding a mutant DNA repair enzyme in a cell is expected to produce a high frequency of mutations (i.e., about 1 mutation / 100 genes to 1 mutation / 10,000 genes) in the genome of the cell. Examples of genes encoding DNA repair enzymes include, but are not limited to, Mut H, Mut S, Mut L, and Mut U, and their homologs in other species (such as MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, etc.). Exemplary descriptions of various methods for introducing genetic mutations are provided, for example, in Stemple (2004) Nature 5:1-7; Chiang et al. (1993) PCR Methods Appl 2(3):210-217; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751, and U.S. Pat. Nos. 6,033,861, and 6,773,900.

[0161] Genetic mutations introduced into microorganisms can be classified as transgenic, cisgenic, intragenomic, intragenus, intergenus, synthetic, evolutionary, rearranged, or SNP.

[0162] Desired mutations can be introduced using CRISPR / Cas9 (Clustered regularly interspaced short palindromic repeats / CRISPR-associated (Cas) system). CRISPR / Cas9 provides adaptive immunity against viruses and plasmids to bacteria and archaea by guiding the silencing of invading nucleic acids using CRISPR RNA (crRNA). The Cas9 protein (or a functionally equivalent protein and / or variant thereof, i.e., a Cas9-like protein) naturally has DNA endonuclease activity that depends on the association of the protein with two natural or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNA). In some cases, the two molecules covalently bind to form a single molecule (also called single guide RNA (sgRNA)). Thus, Cas9 or a Cas9-like protein associates with a DNA-targeting RNA (this term encompasses both the bimolecular guide RNA construct and the single molecule guide RNA construct) that activates the Cas9 or Cas9-like protein and directs the protein to the target nucleic acid sequence. When the Cas9 or Cas9-like protein retains its natural enzymatic function, it cleaves the target DNA to create a double-strand break, which can result in genomic changes (i.e., editing: deletions, insertions (if a donor polynucleotide is present), substitutions, etc.), thereby altering gene expression. Some variants of Cas9 (which are encompassed by the term Cas9-like) are modified such that they have a reduced DNA cleavage activity (in some cases, they cleave a single strand rather than both strands of the target DNA, and in other cases, they are modified to have a significantly reduced DNA cleavage activity up to the case where they have no DNA cleavage activity). Further exemplary descriptions of the CRISPR system for introducing genetic mutations can be found, for example, in US8795965.

[0163] Oligonucleotide-directed mutagenesis, also called site-specific mutagenesis, typically uses synthetic DNA primers. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so that it can hybridize with the DNA in the gene of interest. The mutation may be a single-base change (point mutation), multiple base changes, deletion, or insertion, or a combination of these. Next, a DNA polymerase that copies the rest of the gene is used to extend the single-stranded primer. The gene copied in this way contains the mutation site and can then be introduced into a host cell as a vector and cloned. Finally, mutants are selected by DNA sequencing to confirm that they contain the desired mutation.

[0164] Genetic mutations can be introduced using PCR, which is error-prone. In this technique, the gene of interest is amplified using DNA polymerase under conditions where the fidelity of sequence replication is insufficient. As a result, the amplification product contains at least one error in the sequence. When the gene is amplified and the resulting reaction product contains one or more changes in the sequence compared to the template molecule, the resulting product is mutagenized compared to the template. Another means of introducing random mutations is to expose cells to chemical mutagens such as nitrosoguanidine or ethyl methanesulfonate (Nestmann, Mutat Res 1975 June;28(3):323-30), and then isolate the vector containing the gene from the host.

[0165] Homologous recombination mutagenesis involves recombination between an exogenous DNA fragment and a target polynucleotide sequence. After a double-strand break occurs, a DNA segment around the 5' end of the break is excised in a process called resection. In the subsequent strand invasion step, the protruding 3' end of the damaged DNA molecule then "invades" a similar or identical undamaged DNA molecule. This method can be used for gene deletion, exon removal, gene addition, and introduction of point mutations. Homologous recombination mutagenesis can be permanent or conditional. Typically, a recombination template is also provided. The recombination template may be a component of another vector, may be contained in a separate vector, or may be provided as a separate polynucleotide. In some embodiments, the recombination template is designed to function as a template for homologous recombination, such as within or near the target sequence nicked or cleaved by a site-specific nuclease. The template polynucleotide may be of any suitable length, such as about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of the polynucleotide containing the target sequence. When optimally aligned, the template polynucleotide may overlap with one or more nucleotides of the target sequence (e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more nucleotides). In some embodiments, when the template sequence and the polynucleotide containing the target sequence are optimally aligned, the closest nucleotide of the template polynucleotide is present within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence. Non-limiting examples of site-specific nucleases useful in methods of homologous recombination include zinc finger nucleases, CRISPR nucleases, TALE nucleases, and meganucleases. For further explanation of the use of such nucleases, see, for example, U.S. Patent No. 8,795,965 and U.S. Patent Application Publication No. 2014 / 0301990.

[0166] The introduction of genetic mutations may be an imperfect process, such that some bacteria in the treated bacterial population carry the desired mutation while others do not. In some cases, it may be desirable to apply a selection pressure to enrich for bacteria carrying the desired genetic mutation. Conventionally, selection for successful genetic variants has involved selection for or against some function conferred or abolished by a genetic mutation, such as when inserting an antibiotic resistance gene or abolishing a metabolic activity that can convert a non-lethal compound into a lethal metabolite. It is also possible to apply a selection pressure based on the polynucleotide sequence itself, such that only the desired genetic mutation needs to be introduced (e.g., no selectable marker is required either). In this case, the selection pressure may include a cleaved genome lacking the genetic mutation to be introduced at the target site, such that the selection is effectively directed against the reference sequence into which the genetic mutation is required to be introduced. Typically, cleavage occurs within 100 nucleotides of the target site (e.g., including cleavage at or within the target site within 75, 50, 25, 10, or fewer nucleotides from the target site). Cleavage can be directed by a site-specific nuclease selected from the group consisting of zinc finger nucleases, CRISPR nucleases, TALE nucleases (TALENs), or meganucleases. Such a process is similar to a process for enhancing homologous recombination at the target site, except that a template for homologous recombination is not provided. As a result, bacteria lacking the desired genetic mutation are more likely to undergo cleavage that results in cell death if left unrepaired. The bacteria that survive the selection can then be isolated and evaluated for improved trait conferral.

[0167] CRISPR nucleases can be used as site-specific nucleases to direct cleavage to a target site. By using Cas9 to kill non-mutated cells, improved selection of mutated microorganisms can be obtained. The microorganisms can then be re-isolated from the tissue. The CRISPR nuclease system used for selection against non-variants can use elements similar to those described above for the introduction of genetic mutations, except that a template for homologous recombination is not provided. Cleavage directed to the target site promotes the death of affected cells.

[0168] Other options are available for specifically inducing cleavage at a target site, such as zinc finger nucleases, TALE nuclease (TALEN) systems, and meganucleases. Zinc finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target a desired DNA sequence, such that the zinc finger nuclease can cleave the unique target sequence. When introduced into cells, ZFNs can be used to edit the target DNA (e.g., the cell's genome) within the cell by inducing double-strand breaks. Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENs can be rapidly engineered to bind to substantially any desired DNA sequence, and when introduced into cells, can be used to edit the target DNA (e.g., the cell's genome) within the cell by inducing double-strand breaks. Meganucleases (homing endonucleases) are endodeoxyribonucleases characterized by large recognition sites (12 - 40 base pair double-stranded DNA sequences). Meganucleases can be used to replace, remove, or modify sequences in a highly targeted manner. The target sequence can be altered by modifying their recognition sequences via protein engineering. Meganucleases can be used to modify all genome types, regardless of whether they are bacterial, plant, or animal, and are generally grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family, and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII.

[0169] In some embodiments, the microorganism is a recombinant microorganism. In some embodiments, the microorganism is genetically modified to produce monoethylene glycol. In some embodiments, the microorganism is genetically modified to produce one or more three-carbon compounds such as acetone, isopropanol, and propene. In some embodiments, the microorganism is genetically modified to co-produce monoethylene glycol and one or more three-carbon compounds. In some embodiments, the microorganism is genetically modified in the biosynthetic pathway of the microorganism to produce one or more of monoethylene glycol, acetone, isopropanol, and propene. See Koch et al. (WO 2017 / 156166 A1), which relates to the state of the art regarding engineering microorganisms to produce one or more of monoethylene glycol, acetone, isopropanol, and propene from sustainable feedstocks.

[0170] In some embodiments, the microorganism is genetically modified by introduction of the xylonate pathway. In some embodiments, the microorganism is genetically modified by introduction of the xylulose phosphate pathway. In some embodiments, the microorganism is genetically modified by introduction of the ribulose phosphate pathway. See Koch et al.

[0171] A recombinant microorganism capable of producing a fermentation product from a feedstock comprising xylose and glucose, using xylose and glucose simultaneously In some embodiments, the recombinant microorganism is as follows: (a) deletion or inactivation of a transporter protein from the genome of the microorganism such that a pentose ATP-binding transporter protein is not expressed; (b) one or more endogenous or exogenous nucleic acid sequences operably linked to one or more constitutive promoters and encoding at least one C5 sugar cotransporter, wherein the C5 sugar cotransporter comprises one or more endogenous or exogenous nucleic acid sequences that (1) comprise a xylose cotransporter and / or (2) comprise an arabinose cotransporter; (c) one or more of the following endogenous or exogenous nucleic acid sequences: (1) encoding a xylose isomerase operably linked to one or more constitutive promoters and deletion or inactivation of one or more xylulokinases, and / or (2) encoding a xylose dehydrogenase operably linked to one or more constitutive promoters and deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases.

[0172] In some embodiments, the C5 sugar cotransporter is a cotransporter protein capable of transporting a 5-carbon sugar. In some embodiments, the 5-carbon sugar can be, but is not limited to, xylose, arabinose, or ribose.

[0173] General production of MEG and / or acetone In some embodiments, the recombinant microorganism comprises (a) deletion or inactivation of aldA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar cotransporter operably linked to one or more constitutive promoters, wherein the recombinant microorganism expresses a pathway for MEG and / or acetone production.

[0174] In some embodiments, the microorganism further comprises a deletion or inactivation of glycolic acid dehydrogenase glcDEF. In some embodiments, the C5 co-transporter is controlled by the GAPDH promoter of the araFGH locus. In some embodiments, the one or more nucleic acid molecules encoding the GAPDH promoter comprise the nucleic acid sequence set forth in SEQ ID NO: 95. In some embodiments, the C5 sugar co-transporter is the xylose co-transporter XylE. In some embodiments, the one or more nucleic acid molecules encoding XylE comprise the nucleic acid sequence set forth in SEQ ID NO: 48. In some embodiments, the one or more amino acid sequences encoding XylE comprise the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the xylose co-transporter is endogenous to the microorganism. In some embodiments, the C5 sugar co-transporter is the arabinose co-transporter AraE. In some embodiments, the arabinose co-transporter is endogenous to the microorganism. In some embodiments, the one or more nucleic acid molecules encoding AraE comprise the nucleic acid sequence set forth in SEQ ID NO: 46. In some embodiments, the one or more amino acid sequences encoding AraE comprise the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, xylose is not sensitive to catabolite repression by other monosaccharides.

[0175] In some embodiments, the microorganism comprises a functional phosphotransferase system. In some embodiments, the microorganism comprises a native wild-type nucleic acid sequence encoding a cAMP receptor protein (CRP). In some embodiments, the one or more nucleic acid molecules encoding CRP comprise the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the one or more amino acid sequences encoding CRP comprise the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, constitutive overexpression of the xylose co-transporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, constitutive overexpression of the arabinose co-transporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, continuous xylose import occurs independently of the presence of other sugars in the feedstock.

[0176] Production of MEG and / or acetone involving the xylulose pathway In one aspect, the recombinant microorganism comprises (a) deletion or inactivation of aldA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, the recombinant microorganism comprises (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose isomerase and / or ketohexokinase and / or fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycoaldehyde reductase that catalyzes the conversion of glycoaldehyde to MEG; and (e) a pathway for MEG production involving one or more of deletion or inactivation of one or more xylulokinases from the genome of the parental microorganism, the recombinant microorganism comprises a pathway for MEG and / or acetone production.

[0177] In some embodiments, (c) and (d) are present in an operon controlled by the proD promoter. In some embodiments, one or more nucleic acid molecules encoding the proD promoter comprise the nucleic acid sequence set forth in SEQ ID NO: 53. In some embodiments, the xylose isomerase is XylA. In some embodiments, one or more nucleic acid molecules encoding XylA comprise the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, one or more amino acid sequences encoding XylA comprise the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the xylose isomerase is endogenous to the microorganism. In some embodiments, the ketohexokinase is Khk-C. In some embodiments, the ketohexokinase is of human origin. In some embodiments, the ketohexokinase is heterologous to the microorganism. In some embodiments, one or more nucleic acid molecules encoding Khk-C comprise the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, one or more amino acid sequences encoding Khk-C comprise the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the fructose-bisphosphate aldolase is aldoB. In some embodiments, the fructose-bisphosphate aldolase is of human origin. In some embodiments, the fructose-bisphosphate aldolase is heterologous to the microorganism. In some embodiments, one or more nucleic acid molecules encoding aldoB comprise the nucleic acid sequence set forth in SEQ ID NO: 50. In some embodiments, one or more amino acid sequences encoding aldoB comprise the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the glycolaldehyde reductase is endogenous to the microorganism. In some embodiments, the glycolaldehyde reductase is fucO. In some embodiments, one or more nucleic acid molecules encoding fucO comprise the nucleic acid sequence set forth in SEQ ID NO: 52. In some embodiments, one or more amino acid sequences encoding fucO comprise the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the xylulokinase is XylB. In some embodiments, one or more nucleic acid molecules encoding XylB comprise the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, one or more amino acid sequences encoding XylB comprise the amino acid sequence set forth in SEQ ID NO: 14.

[0178] Production of MEG and / or acetone, including the xylonate pathway In some embodiments, the recombinant microorganism comprises (a) deletion or inactivation of aldA, araFGH, and xylFGH from the genome of the parent microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, the recombinant microorganism comprises (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that catalyzes the conversion of glycolaldehyde to MEG; and (e) a pathway for MEG production involving one or more of deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases from the genome of the parent microorganism, the recombinant microorganism comprises a pathway for MEG and / or acetone production.

[0179] In some embodiments, the xylose dehydrogenase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylose dehydrogenase is heterologous to the microorganism. In some embodiments, one or more nucleic acid molecules encoding the Caulobacter crescentus xylose dehydrogenase comprise the nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, one or more amino acid sequences encoding the Caulobacter crescentus xylose dehydrogenase comprise the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, one or more nucleic acid molecules encoding the Burkholderia xenovorans xylose dehydrogenase comprise the nucleic acid sequence set forth in SEQ ID NO: 97. In some embodiments, one or more amino acid sequences encoding the Burkholderia xenovorans xylose dehydrogenase comprise the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, one or more nucleic acid molecules encoding the Haloferax volcanii xylose dehydrogenase comprise the nucleic acid sequence set forth in SEQ ID NO: 18. In some embodiments, one or more amino acid sequences encoding the Haloferax volcanii xylose dehydrogenase comprise the amino acid sequence set forth in SEQ ID NO: 19.

[0180] In some embodiments, the xylonolactonase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylonolactonase is heterologous to the microorganism. In some embodiments, the xylonolactonase is endogenous to the microorganism. In some embodiments, the one or more nucleic acid molecules encoding Caulobacter crescentus xylonolactonase comprise the nucleic acid sequence set forth in SEQ ID NO: 54. In some embodiments, the one or more amino acid sequences encoding Caulobacter crescentus xylonolactonase comprise the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the one or more nucleic acid molecules encoding Burkholderia xenovorans xylonolactonase comprise the nucleic acid sequence set forth in SEQ ID NO: 56. In some embodiments, the one or more amino acid sequences encoding Burkholderia xenovorans xylonolactonase comprise the amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the one or more nucleic acid molecules encoding Haloferax volcanii xylonolactonase comprise the nucleic acid sequence set forth in SEQ ID NO: 58. In some embodiments, the one or more amino acid sequences encoding Haloferax volcanii xylonolactonase comprise the amino acid sequence set forth in SEQ ID NO: 59.

[0181] In some embodiments, the xylose dehydratase is derived from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylose dehydratase is heterologous to the microorganism. In some embodiments, the xylose dehydratase is endogenous to the microorganism. In some embodiments, one or more nucleic acid molecules encoding the Caulobacter crescentus xylose dehydratase comprise the nucleic acid sequence set forth in SEQ ID NO: 60. In some embodiments, one or more amino acid sequences encoding the Caulobacter crescentus xylose dehydratase comprise the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, one or more nucleic acid molecules encoding the Burkholderia xenovorans xylose dehydratase comprise the nucleic acid sequence set forth in SEQ ID NO: 62. In some embodiments, one or more amino acid sequences encoding the Burkholderia xenovorans xylose dehydratase comprise the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, one or more nucleic acid molecules encoding the Haloferax volcanii xylose dehydratase comprise the nucleic acid sequence set forth in SEQ ID NO: 64. In some embodiments, one or more amino acid sequences encoding the Haloferax volcanii xylose dehydratase comprise the amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the glycolaldehyde reductase is endogenous to the microorganism. In some embodiments, the glycolaldehyde reductase is fucO. In some embodiments, the glycolaldehyde reductase is heterologous to the microorganism. In some embodiments, the xylose isomerase is XylA. In some embodiments, the xylulokinase is XylB.

[0182] Production of MEG and / or acetone comprising the xylulose pathway In one aspect, the recombinant microorganism comprises (a) deletion or inactivation of aldA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, the recombinant microorganism comprises (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose isomerase and / or ketohexokinase and / or fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that catalyzes the conversion of glycolaldehyde to MEG; and (e) a pathway for MEG production involving one or more of deletion or inactivation of one or more xylulokinases from the genome of the parental microorganism, the recombinant microorganism comprises (f) expression of at least one exogenous nucleic acid molecule encoding acetoacetyl-CoA thiolase; (g) expression of at least one exogenous nucleic acid molecule encoding acetate:acetoacetyl-CoA transferase; and (h) a pathway for acetone production involving one or more of expression of at least one exogenous nucleic acid molecule encoding acetoacetate decarboxylase that catalyzes the conversion of acetoacetate to acetone, the recombinant microorganism expresses a pathway for MEG and / or acetone production.

[0183] In some embodiments, (f), (g), and (h) are present in an operon controlled by the OXB11 promoter. In some embodiments, one or more nucleic acid molecules encoding OXB11 comprise the nucleic acid sequence set forth in SEQ ID NO: 78. In some embodiments, the acetoacetyl-CoA thiolase is Thl. In some embodiments, the thiolase is from Clostridium acetobutylicum or Clostridium beijerinckii. In some embodiments, one or more nucleic acid molecules encoding the Clostridium acetobutylicum thl thiolase comprise the nucleic acid sequence set forth in SEQ ID NO: 68. In some embodiments, one or more amino acid sequences encoding the Clostridium acetobutylicum thl thiolase comprise the amino acid sequence set forth in SEQ ID NO: 69. In some embodiments, one or more nucleic acid molecules encoding the Clostridium beijerinckii thl thiolase comprise the nucleic acid sequence set forth in SEQ ID NO: 66. In some embodiments, one or more amino acid sequences encoding the Clostridium beijerinckii thl thiolase comprise the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the acetate:acetoacetyl-CoA transferase is AtoDA. In some embodiments, the acetoacetate decarboxylase is Adc. In some embodiments, the decarboxylase is from Clostridium acetobutylicum or Clostridium beijerinckii. In some embodiments, one or more nucleic acid molecules encoding the Clostridium acetobutylicum Adc acetoacetate decarboxylase comprise the nucleic acid sequence set forth in SEQ ID NO: 74. In some embodiments, one or more amino acid sequences encoding the Clostridium acetobutylicum Adc acetoacetate decarboxylase comprise the amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, one or more nucleic acid molecules encoding the Clostridium beijerinckii Adc acetoacetate decarboxylase comprise the nucleic acid sequence set forth in SEQ ID NO: 76. In some embodiments, one or more amino acid sequences encoding the Clostridium beijerinckii Adc acetoacetate decarboxylase comprise the amino acid sequence set forth in SEQ ID NO: 77.

[0184] Production of MEG and / or acetone specific to the xylonate pathway In some embodiments, the recombinant microorganism comprises (a) deletion or inactivation of aldA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, and the recombinant microorganism comprises (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that catalyzes the conversion of glycolaldehyde to MEG; and (e) a pathway for MEG production involving one or more of deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases from the genome of the parental microorganism, and the recombinant microorganism comprises one or more of the following: (f) expression of at least one exogenous nucleic acid molecule encoding acetoacetyl-CoA thiolase; (g) expression of at least one exogenous nucleic acid molecule encoding acetate:acetoacetyl-CoA transferase; and (h) a pathway for acetone production involving one or more of expression of at least one exogenous nucleic acid molecule encoding acetoacetate decarboxylase that catalyzes the conversion of acetoacetate to acetone, and the recombinant microorganism expresses a pathway for MEG and / or acetone production.

[0185] In some embodiments, (f), (g), and (h) are present in an operon controlled by the OXB11 promoter. In some embodiments, the acetoacetyl-CoA thiolase is Thl. In some embodiments, the thiolase is from Clostridium acetobutylicum. In some embodiments, the acetate:acetoacetyl-CoA transferase is AtoDA. In some embodiments, one or more nucleic acid molecules encoding the AtoD subunit alpha of the acetate:acetoacetyl-CoA transferase comprise the nucleic acid sequence set forth in SEQ ID NO: 70. In some embodiments, one or more amino acid sequences encoding the AtoD subunit alpha of the acetate:acetoacetyl-CoA transferase comprise the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, one or more nucleic acid molecules encoding the AtoD subunit beta of the acetate:acetoacetyl-CoA transferase comprise the nucleic acid sequence set forth in SEQ ID NO: 71. In some embodiments, one or more amino acid sequences encoding the AtoD subunit beta of the acetate:acetoacetyl-CoA transferase comprise the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the acetoacetate decarboxylase is Adc. In some embodiments, the decarboxylase is from Clostridium acetobutylicum or Clostridium beijerinckii. In some embodiments, one or more nucleic acid molecules encoding the Clostridium acetobutylicum Adc acetoacetate decarboxylase comprise the nucleic acid sequence set forth in SEQ ID NO: 74. In some embodiments, one or more amino acid sequences encoding the Clostridium acetobutylicum Adc acetoacetate decarboxylase comprise the amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, one or more nucleic acid molecules encoding the Clostridium beijerinckii Adc acetoacetate decarboxylase comprise the nucleic acid sequence set forth in SEQ ID NO: 76. In some embodiments, one or more amino acid sequences encoding the Clostridium beijerinckii Adc acetoacetate decarboxylase comprise the amino acid sequence set forth in SEQ ID NO: 77.

[0186] Production of isopropanol In some embodiments, the recombinant microorganism is capable of producing isopropanol from any one or more raw materials capable of producing acetone. In some embodiments, a recombinant microorganism engineered to produce acetone is further engineered to express at least one exogenous nucleic acid molecule encoding an alcohol dehydrogenase that catalyzes the conversion of acetone to isopropanol. In some embodiments, (f), (g), and (h) of the acetone production disclosure above are further modified by (i) the expression of at least one exogenous nucleic acid molecule encoding an alcohol dehydrogenase that catalyzes the conversion of acetone to isopropanol. In some embodiments, the one or more nucleic acid molecules encoding alcohol dehydrogenase comprise the nucleic acid sequence set forth in SEQ ID NO: 93. In some embodiments, the one or more amino acid sequences encoding alcohol dehydrogenase comprise the amino acid sequence set forth in SEQ ID NO: 94.

[0187] Production of glycolic acid In some embodiments, the recombinant microorganism is capable of producing glycolic acid from a feedstock comprising xylose and glucose, and the recombinant microorganism uses xylose and glucose simultaneously and comprises one or more of the following: (a) deletion or inactivation of fucO, yqhD (SEQ ID NO: 1 or SEQ ID NO: 2), araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, and the recombinant microorganism further expresses one or more pathways for the production of glycolic acid.

[0188] In some embodiments, one or more nucleic acid molecules encoding the AraF periplasmic binding protein subunit of AraFGH comprise the nucleic acid sequence set forth in SEQ ID NO: 20. In some embodiments, one or more amino acid sequences encoding the AraF periplasmic binding protein subunit of AraFGH comprise the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, one or more nucleic acid molecules encoding the AraG ATP-binding subunit of AraFGH comprise the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, one or more amino acid sequences encoding the AraG ATP-binding subunit of AraFGH comprise the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, one or more nucleic acid molecules encoding the AraH membrane subunit of AraFGH comprise the nucleic acid sequence set forth in SEQ ID NO: 22. In some embodiments, one or more amino acid sequences encoding the AraH membrane subunit of AraFGH comprise the amino acid sequence set forth in SEQ ID NO: 25.

[0189] In some embodiments, one or more nucleic acid molecules encoding the xylF periplasmic binding protein subunit of xylFGH comprise the nucleic acid sequence set forth in SEQ ID NO: 26. In some embodiments, one or more amino acid sequences encoding the xylF periplasmic binding protein subunit of xylFGH comprise the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, one or more nucleic acid molecules encoding the xylG ATP-binding subunit of xylFGH comprise the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, one or more amino acid sequences encoding the xylG ATP-binding subunit of xylFGH comprise the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, one or more nucleic acid molecules encoding the xylH membrane subunit of xylFGH comprise the nucleic acid sequence set forth in SEQ ID NO: 28. In some embodiments, one or more amino acid sequences encoding the xylH membrane subunit of xylFGH comprise the amino acid sequence set forth in SEQ ID NO: 31.

[0190] In some embodiments, the microorganism further comprises a deletion or inactivation of glcDEF. In some embodiments, one or more nucleic acid molecules encoding the putative FAD-linked subunit GlcD comprise the nucleic acid sequence set forth in SEQ ID NO: 79. In some embodiments, one or more amino acid sequences encoding the putative FAD-linked subunit GlcD comprise the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, one or more nucleic acid molecules encoding the putative FAD-binding subunit GlcE comprise the nucleic acid sequence set forth in SEQ ID NO: 80. In some embodiments, one or more amino acid sequences encoding the putative FAD-binding subunit GlcE comprise the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, one or more nucleic acid molecules encoding the putative iron-sulfur subunit GlcF comprise the nucleic acid sequence set forth in SEQ ID NO: 81. In some embodiments, one or more amino acid sequences encoding the putative iron-sulfur subunit GlcF comprise the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the microorganism further comprises a deletion or inactivation of the aldehyde reductase dkgA. In some embodiments, one or more nucleic acid molecules encoding dkgA comprise the nucleic acid sequence set forth in SEQ ID NO: 85. In some embodiments, one or more amino acid sequences encoding dkgA comprise the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the microorganism further comprises a deletion or inactivation of the aldehyde reductase yahK. In some embodiments, one or more nucleic acid molecules encoding yahK comprise the nucleic acid sequence set forth in SEQ ID NO: 87. In some embodiments, one or more amino acid sequences encoding yahK comprise the amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the xylose cotransporter is controlled by the GAPDH promoter of the araFGH locus. In some embodiments, the C5 sugar cotransporter is the xylose cotransporter XylE. In some embodiments, one or more nucleic acid molecules encoding xylE comprise the nucleic acid sequence set forth in SEQ ID NO: 48. In some embodiments, one or more amino acid sequences encoding xylE comprise the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the xylose cotransporter is endogenous to the microorganism. In some embodiments, the C5 sugar cotransporter is the arabinose cotransporter AraE.In some embodiments, the one or more nucleic acid molecules encoding araE comprise the nucleic acid sequence set forth in SEQ ID NO: 46. In some embodiments, the one or more amino acid sequences encoding araE comprise the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the arabinose cotransporter is endogenous to the microorganism. In some embodiments, xylose uptake is not sensitive to catabolite repression by other monosaccharides. In some embodiments, the microorganism comprises a functional phosphotransferase system. In some embodiments, the microorganism comprises a native wild-type nucleic acid sequence encoding the cAMP receptor protein (CRP). In some embodiments, the one or more nucleic acid molecules encoding CRP comprise the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the one or more amino acid sequences encoding CRP comprise the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, constitutive overexpression of the xylose cotransporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, constitutive overexpression of the arabinose cotransporter enables continuous import of xylose from the feedstock into the microorganism. In some embodiments, continuous xylose import occurs independently of the presence of other sugars in the feedstock.

[0191] Production of glycolic acid, including the xylulose pathway In some embodiments, the recombinant microorganism has the ability to produce glycolic acid from a feedstock comprising xylose and glucose, and the recombinant microorganism simultaneously uses xylose and glucose, as follows: (a) deletion or inactivation of fucO, yqhD, yahK, dkgA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, wherein the recombinant microorganism further comprises: (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding a xylose isomerase and / or a ketohexokinase and / or a fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) expression of one or more pathways for the production of glycolic acid involving deletion or inactivation of one or more xylulokinases from the genome of the parental microorganism.

[0192] In some embodiments, (c) and (d) are present in an operon controlled by the proD promoter. In some embodiments, one or more nucleic acid molecules encoding the proD promoter comprise the nucleic acid sequence set forth in SEQ ID NO: 53. In some embodiments, the xylose isomerase is XylA. In some embodiments, one or more nucleic acid molecules encoding xylA comprise the nucleic acid sequence set forth in SEQ ID NO: 5. In some embodiments, one or more amino acid sequences encoding xylA comprise the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the xylose isomerase is endogenous to the microorganism. In some embodiments, the xylose isomerase is heterologous to the microorganism. In some embodiments, the ketohexokinase is Khk-C. In some embodiments, one or more nucleic acid molecules encoding khk-C comprise the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, one or more amino acid sequences encoding khk-C comprise the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the ketohexokinase is of human origin. In some embodiments, the fructose-bisphosphate aldolase is aldoB. In some embodiments, one or more nucleic acid molecules encoding alsoB comprise the nucleic acid sequence set forth in SEQ ID NO: 50. In some embodiments, one or more amino acid sequences encoding aldoB comprise the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the fructose-bisphosphate aldolase is of human origin. In some embodiments, the glycolaldehyde dehydrogenase is endogenous to the microorganism. In some embodiments, the glycolaldehyde dehydrogenase is heterologous to the microorganism. In some embodiments, the glycolaldehyde dehydrogenase is aldA. In some embodiments, one or more nucleic acid molecules encoding aldA comprise the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, one or more amino acid sequences encoding aldA comprise the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the xylulokinase is XylB. In some embodiments, one or more nucleic acid molecules encoding xylB comprise the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, one or more amino acid sequences encoding xylB comprise the amino acid sequence set forth in SEQ ID NO: 14.

[0193] Production of glycolic acid, including the xylonate pathway In some embodiments, the recombinant microorganism is capable of producing glycolic acid from a feedstock comprising xylose and glucose, the recombinant microorganism uses xylose and glucose simultaneously, and comprises one or more of the following: (a) deletion or inactivation of fucO, yqhD, yahK, dkgA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, the recombinant microorganism further expresses one or more pathways for the production of glycolic acid involving one or more of the following: (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases from the genome of the parental microorganism.

[0194] In some embodiments, (c) and (d) are controlled by the proD promoter. In some embodiments, the xylose isomerase is XylA. In some embodiments, the xylulokinase is XylB. In some embodiments, the xylose dehydrogenase is from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylose dehydrogenase is heterologous to the microorganism. In some embodiments, the xylonolactonase is from Caulobacter crescentus, Burkholderia xenovorans, or Haloferax volcanii. In some embodiments, the xylonolactonase is heterologous to the microorganism. In some embodiments, the xylonolactonase is endogenous to the microorganism. In some embodiments, the glycolaldehyde dehydrogenase is aldA. In some embodiments, the glycolaldehyde dehydrogenase is endogenous to the microorganism.

[0195] Production of glycolic acid (alternative pathway) involving the xylulose pathway In some embodiments, the recombinant microorganism has the ability to produce glycolic acid from a feedstock comprising xylose and glucose, and the recombinant microorganism uses xylose and glucose simultaneously and comprises one or more of the following: (a) deletion or inactivation of fucO, yqhD, yahK, dkgA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters. The recombinant microorganism further comprises one or more of the following: (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding a xylose isomerase and / or a ketohexokinase and / or a fructose-bisphosphate aldolase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) further expression of one or more pathways for the production of glycolic acid involving deletion or inactivation of one or more xylulokinases from the genome of the parental microorganism. The microorganism further comprises one or more of the following: (f) expression of at least one endogenous or exogenous nucleic acid molecule encoding isocitrate lyase; and / or (g) further expression of a pathway for the production of glycolic acid involving expression of at least one endogenous or exogenous nucleic acid molecule encoding glyoxylate reductase. In some embodiments, (f) and (g) are present in an operon controlled by the OXB20 promoter. In some embodiments, the one or more nucleic acid molecules encoding the OXB20 promoter comprise the nucleic acid sequence set forth in SEQ ID NO: 96. In some embodiments, the isocitrate lyase is AceA. In some embodiments, the glyoxylate reductase is YcdW. In some embodiments, the one or more nucleic acid molecules encoding ycdW comprise the nucleic acid sequence set forth in SEQ ID NO: 91. In some embodiments, the one or more amino acid sequences encoding ycdW comprise the amino acid sequence set forth in SEQ ID NO: 92.

[0196] Production of glycolic acid (alternative pathway) involving the xylonate pathway In some embodiments, the recombinant microorganism has the ability to produce glycolic acid from a feedstock comprising xylose and glucose, the recombinant microorganism uses xylose and glucose simultaneously, and comprises one or more of the following: (a) deletion or inactivation of fucO, yqhD, yahK, dkgA, araFGH, and xylFGH from the genome of the parental microorganism; and (b) expression of at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, the recombinant microorganism further expresses one or more pathways for the production of glycolic acid involving one or more of the following: (c) expression of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and / or xylonolactonase and / or xylose dehydratase operably linked to one or more constitutive promoters; (d) expression of at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde dehydrogenase that catalyzes the conversion of glycolaldehyde to glycolic acid; and (e) deletion or inactivation of one or more xylose isomerases and / or one or more xylulokinases from the genome of the parental microorganism, the microorganism further expresses one or more of the following: (f) expression of at least one endogenous or exogenous nucleic acid molecule encoding isocitrate lyase; and / or (g) expression of at least one endogenous or exogenous nucleic acid molecule encoding glyoxylate reductase for a pathway for the production of glycolic acid.

[0197] In some embodiments, (f) and (g) are present in an operon controlled by the OXB20 promoter. In some embodiments, the isocitrate lyase is AceA. In some embodiments, the glyoxylate reductase is YcdW.

[0198] Recombinant microorganisms comprising the sequences and modifications described herein In some embodiments, the present disclosure broadly relates to a recombinant microorganism of any one of the foregoing embodiments, the recombinant microorganism being obtained from a parent microorganism selected from the group consisting of Clostridium, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium ragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum, Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii, Alkalibaculum bacchi, Clostridium drakei, Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum, Blautia producta, Clostridium glycolicum, Clostridium magnum, Clostridium mayombei, Clostridium mesoxybovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacter phengii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusa termitida, Moorella glycerini, Eubacterium aggregans, Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus, Corynebacterium, Yarrowia lipolytica, Schizosaccharomyces stipitis, and Terrisporobacter glycolicus. In some embodiments, the parent microorganism is Escherichia coli.

[0199] In some embodiments, the enzymes, proteins, promoters, and nucleic acids of the present disclosure are summarized in Table 1.

[0200] (Table 1) Proteins and Nucleic Acids of the Present Disclosure TIFF0007715636000001.tif108158TIFF0007715636000002.tif214158TIFF0007715636000003.tif216158TIFF0007715636000004.tif214158TIFF0007715636000005.tif222158TIFF0007715636000006.tif235158

[0201] Method for detecting gene modification The present disclosure teaches primers, probes, and assays useful for detecting the microorganisms taught herein. In some aspects, the present disclosure provides methods for detecting a WT parental strain. In other aspects, the present disclosure provides methods for detecting engineered or modified microorganisms obtained from a parental or WT strain. In some aspects, the present disclosure provides methods for identifying genetic changes in microorganisms.

[0202] In some aspects, the genome engineering methods of the present disclosure result in the generation of "junction" sequences of unnatural nucleotides in the modified microorganisms. These non-naturally occurring nucleotide junctions can be used as a type of determination that indicates the presence of specific genetic changes in the microorganisms taught herein.

[0203] The present technology can detect these non-naturally occurring nucleotide junctions through the use of specialized quantitative PCR methods that include uniquely designed primers and probes. In some aspects, the probes of the present disclosure bind to non-naturally occurring nucleotide junction sequences. In some aspects, conventional PCR is used. In other aspects, real-time PCR is used. In some aspects, quantitative PCR (qPCR) is used. In some aspects, PCR methods are used to identify heterologous sequences inserted into the genomic DNA or extrachromosomal DNA of microorganisms.

[0204] Accordingly, the present disclosure can include the use of two general methods for detecting PCR products in real time: (1) non-specific fluorescent dyes that interact with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides labeled with fluorescent reporters that allow detection only after hybridization of a probe having its complementary sequence. In some embodiments, only non-naturally occurring nucleotide junctions are amplified via the taught primers and can thus be detected via either the use of non-specific dyes or specific hybridization probes. In other embodiments, the primers of the present disclosure are selected such that the primer flanks both sides of the junction sequence such that, when an amplification reaction occurs, the junction sequence is present.

[0205] Embodiments of the present disclosure involve nucleotide junction sequence molecules that are themselves non-naturally occurring, along with other nucleotide molecules that are capable of binding to such non-naturally occurring nucleotide junction sequences under mild to stringent hybridization conditions. In some embodiments, the nucleotide molecules that are capable of binding to such non-naturally occurring nucleotide sequences under mild to stringent hybridization conditions are referred to as "nucleotide probes."

[0206] In some embodiments, genomic DNA is extracted from a sample and used to quantify the presence of the microorganisms of the present disclosure using qPCR. The primers used in the qPCR reaction can be primers designed by Primer Blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to amplify unique regions of the wild-type genome or unique regions of engineered interspecies mutants. The qPCR reaction can be performed using only the forward amplification primer and the reverse amplification primer using the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher P / N 11762100) kit. Alternatively, the Kapa Probe Force kit (Kapa Biosystems P / N KK4301) can be used with the amplification primers and a TaqMan probe (Integrated DNA Technologies) containing a FAM dye label at the 5' end, an internal ZEN quencher, and a minor groove binder and a fluorescent quencher at the 3' end.

[0207] Quantitative polymerase chain reaction (qPCR) is a method for quantitatively amplifying one or more nucleic acid sequences in real time. Real-time quantification of a PCR assay enables determination of the amount of nucleic acid produced by the PCR amplification step by comparing the amplified nucleic acid of interest with an appropriate control nucleic acid sequence that can act as a calibration standard.

[0208] TaqMan probes are often used in qPCR assays that require improved specificity to quantify target nucleic acid sequences. A TaqMan probe comprises an oligonucleotide probe having a fluorophore attached to the 5' end and a quencher attached to the 3' end of the probe. When the TaqMan probe has the 5' and 3' ends of the probe in close contact with each other, the quencher prevents transmission of the fluorescence signal from the fluorophore. The TaqMan probe is designed to anneal within a nucleic acid region amplified by a specific primer set. When Taq polymerase extends the primer and synthesizes a nascent strand, the 5' to 3' exonuclease activity of Taq polymerase degrades the probe annealed to the template. This probe degradation releases the fluorophore, thus disrupting proximity to the quencher and enabling fluorescence of the fluorophore. The fluorescence detected in the qPCR assay is directly proportional to the amount of released fluorophore and the DNA template present in the reaction.

[0209] Due to the characteristics of qPCR, the labor-intensive post-amplification step of gel electrophoresis preparation, which is generally required for observation of the amplified product of a conventional PCR assay, can be eliminated. The advantages of qPCR over conventional PCR are significant and include increased speed, ease of use, reproducibility, and quantitative ability.

[0210] Microbial composition In some embodiments, the microorganisms of the present disclosure are combined in a microbial composition.

[0211] In some embodiments, the microbial composition of the present disclosure is solid. When a solid composition is used, it may be desirable to include one or more carrier materials including, but not limited to, silica, talc, kaolin, limestone, chalk, clay, dolomite, diatomaceous earth and other mineral earths, calcium sulfate, magnesium sulfate, magnesium oxide, zeolite, calcium carbonate, magnesium carbonate, trehalose, chitosan, shellac, albumin, starch, nonfat dry milk, sweet whey powder, maltodextrin, lactose, inulin, dextrose, and vegetable-derived products such as cereal meal, bark meal, wood flour, and nut shell meal.

[0212] In some embodiments, the microbial composition of the present disclosure is liquid. In further embodiments, the liquid comprises a solvent that may include water, alcohol, physiological saline, or a carbohydrate solution. In some embodiments, the microbial composition of the present disclosure includes a binder such as a polymer, carboxymethyl cellulose, starch, polyvinyl alcohol, and the like.

[0213] In some embodiments, the microbial composition of the present disclosure includes saccharides (e.g., monosaccharides, disaccharides, trisaccharides, polysaccharides, oligosaccharides, etc.), polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides, proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acid polymers, silica, inorganic salts, and combinations thereof. In further embodiments, the microbial composition includes polymers such as agar, agarose, gelrite, gellan gum, and the like. In some embodiments, the microbial composition includes plastic capsules, emulsions (e.g., water and oil), membranes, and artificial membranes. In some embodiments, an emulsion or a linked polymer solution may include the microbial composition of the present disclosure. See Harel and Bennett (U.S. Patent No. 8,460,726 B2).

[0214] In some embodiments, the microbial compositions of the present disclosure occur in solid form (e.g., lyophilized spores) or liquid form (microorganisms dispersed in a storage medium). In some embodiments, the microbial compositions of the present disclosure are added to a liquid in dry form immediately prior to use to form a suspension. In some embodiments, the microbial composition comprises vitrified microorganisms.

[0215] In some embodiments, the microbial compositions of the present disclosure have a water activity (aw) of less than 0.750, 0.700, 0.650, 0.600, 0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350, 0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0.150, 0.140, 0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080, 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, 0.010, or 0.005.

[0216] In some embodiments, the microbial compositions of the present disclosure have a water activity (aw) of less than about 0.750, about 0.700, about 0.650, about 0.600, about 0.550, about 0.500, about 0.475, about 0.450, about 0.425, about 0.400, about 0.375, about 0.350, about 0.325, about 0.300, about 0.275, about 0.250, about 0.225, about 0.200, about 0.190, about 0.180, about 0.170, about 0.160, about 0.150, about 0.140, about 0.130, about 0.120, about 0.110, about 0.100, about 0.095, about 0.090, about 0.085, about 0.080, about 0.075, about 0.070, about 0.065, about 0.060, about 0.055, about 0.050, about 0.045, about 0.040, about 0.035, about 0.030, about 0.025, about 0.020, about 0.015, about 0.010, or about 0.005.

[0217] The water activity value is determined by the method of a saturated aqueous solution (Multon, “Techniques d’Analyse E De Controle Dans Les Industries Agroalimentaires” APRIA (1981)), or by direct measurement using a viable Rotron BT hygrometer or other hygrometer or moisture detector.

[0218] raw material In some embodiments, the present disclosure relates to a method for producing, recovering / isolating one or more desired chemical substances. Recovery / collection / isolation can be performed by methods known in the art such as distillation, membrane-based separation gas stripping, solvent extraction, and expanded bed adsorption.

[0219] In some embodiments, the raw material contains a carbon source. In some embodiments, the carbon source may be selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins, carbon dioxide, and carbon monoxide. In one embodiment, the carbon source is a sugar. In one embodiment, the sugar is glucose or an oligomer of glucose. In one embodiment, the oligomer of glucose is selected from fructose, sucrose, starch, cellobiose, maltose, lactose, and cellulose. In one embodiment, the sugar is a pentose sugar. In one embodiment, the sugar is a hexose sugar. In some embodiments, the raw material contains one or more pentose sugars and / or one or more hexose sugars. In some embodiments, the raw material contains one or more of xylose, glucose, arabinose, galactose, maltose, fructose, mannose, sucrose, and / or combinations thereof. In some embodiments, the raw material contains one or more of xylose and / or glucose.In some embodiments, the raw material contains one or more of arabinose, galactose, maltose, fructose, mannose, sucrose, and / or combinations thereof. In some embodiments, the raw material contains xylose and glucose.

[0220] In some embodiments, the microorganism uses one or more pentoses and / or one or more hexoses. In some embodiments, the microorganism uses one or more of xylose and / or glucose. In some embodiments, the microorganism uses one or more of arabinose, galactose, maltose, fructose, mannose, sucrose, and / or combinations thereof. In some embodiments, the microorganism uses one or more of xylose, glucose, arabinose, galactose, maltose, fructose, mannose, sucrose, and / or combinations thereof.

[0221] In some embodiments, the hexose can be selected from D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-tagatose, D-sorbose, D-fructose, D-psicose, and other hexoses known in the art. In some embodiments, the pentose may be selected from D-xylose, D-ribose, D-arabinose, D-lyxose, D-xylulose, D-ribulose, and other pentoses known in the art. In some embodiments, the hexose and pentose can be selected from the left- or right-handed enantiomers of any of the hexoses and pentoses disclosed herein.

[0222] In some embodiments, the total amount of C5 and / or C6 carbohydrates supplied to the bioreactor / growth medium during the growth phase is at least 5 kg / m3 of carbohydrates, at least 10 kg / m3 of carbohydrates, at least 20 kg / m3 of carbohydrates, at least 30 kg / m3 of carbohydrates, at least 40 kg / m3 of carbohydrates, at least 50 kg / m3 of carbohydrates, at least 60 kg / m3 of carbohydrates, at least 70 kg / m3 of carbohydrates, at least 80 kg / m3 of carbohydrates, at least 90 kg / m3 of carbohydrates, at least 100 kg / m3 of carbohydrates, at least 150 kg / m3 of carbohydrates, at least 200 kg / m3 of carbohydrates, at least 250 kg / m3 of carbohydrates, at least 300 kg / m3 of carbohydrates, at least 400 kg / m3 of carbohydrates, at least 500 kg / m3 of carbohydrates, at least 600 kg / m3 of carbohydrates, at least 700 kg / m3 of carbohydrates, up to 800 kg / m3 of carbohydrates. In some embodiments, the total amount of C5 and / or C6 carbohydrates supplied to the bioreactor / growth medium during the growth phase is in the range of about 10 kg / m3 to 500 kg / m3 of carbohydrates.

[0223] In some embodiments, the time required for the growth phase varies between 1 and 200 hours. In a further embodiment, the time of the growth phase is 5 to 50 hours. The time depends on the supply amount of carbohydrates and / or the raw materials.

[0224] In some embodiments, the total amount of C5 and / or C6 carbohydrates supplied to the bioreactor / growth medium during the growth phase is at least 50 kg / m3 of carbohydrates, at least 60 kg / m3 of carbohydrates, at least 70 kg / m3 of carbohydrates, at least 80 kg / m3 of carbohydrates, at least 90 kg / m3 of carbohydrates, at least 100 kg / m3 of carbohydrates, at least 150 kg / m3 of carbohydrates, at least 200 kg / m3 of carbohydrates, at least 250 kg / m3 of carbohydrates, at least 300 kg / m3 of carbohydrates, at least 400 kg / m3 of carbohydrates, at least 500 kg / m3 of carbohydrates, at least 600 kg / m3 of carbohydrates, at least 700 kg / m3 of carbohydrates, at least 800 kg / m3 of carbohydrates, at least 900 kg / m3 of carbohydrates, up to 1000 kg / m3 of carbohydrates. In some embodiments, the total amount of C5 and / or C6 carbohydrates supplied to the bioreactor / growth medium during the production phase is in the range of about 100 kg / m3 to 800 kg / m3 of carbohydrates.

[0225] In some embodiments, the time required for the production phase varies between 5 and 500 hours. In further embodiments, the time of the production phase varies from 10 hours to 300 hours for batch and fed-batch operations. In other embodiments, the time of the production phase is up to 300 hours for continuous fermentation.

[0226] In some embodiments, the total amount of C5 and / or C6 carbohydrates supplied to the bioreactor / growth medium during a one-phase process is at least 50 kg / m3 of carbohydrates, at least 60 kg / m3 of carbohydrates, at least 70 kg / m3 of carbohydrates, at least 80 kg / m3 of carbohydrates, at least 90 kg / m3 of carbohydrates, at least 100 kg / m3 of carbohydrates, at least 150 kg / m3 of carbohydrates, at least 200 kg / m3 of carbohydrates, at least 250 kg / m3 of carbohydrates, at least 300 kg / m3 of carbohydrates, at least 400 kg / m3 of carbohydrates, at least 500 kg / m3 of carbohydrates, at least 600 kg / m3 of carbohydrates, at least 700 kg / m3 of carbohydrates, at least 800 kg / m3 of carbohydrates, at least 900 kg / m3 of carbohydrates, and up to 1000 kg / m3 of carbohydrates. In some embodiments, the total amount of C5 and / or C6 carbohydrates supplied to the bioreactor / growth medium during the production phase is in the range of about 100 kg / m3 of carbohydrates to 800 kg / m3 of carbohydrates.

[0227] In some embodiments, the time required for the production phase in a one-phase process varies between 5 and 500 hours. In a further embodiment, the time required for the production phase in a one-phase process varies between 5 and 300 hours.

[0228] In some embodiments, the production process for one or more phases takes about 5, about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500 hours.

[0229] In some embodiments, the production process for one or more phases takes 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 hours.

[0230] Trait improvement Using the methods of the present disclosure, one or more of various desired traits may be introduced or improved. Examples of traits that may be introduced or improved include MEG, glycolic acid, polyols, acetone, propene, increased rates and speeds of isopropanol, increased co-consumption of xylose and glucose, and decreased inhibitory effects of one or more sugars on sugar consumption and / or uptake.

[0231] In some embodiments, the microorganisms resulting from the methods described herein exhibit a trait difference that is at least about 1%, for example, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 9%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% higher than a reference under controlled conditions. In further examples, the microorganisms resulting from the methods described herein exhibit a trait difference that is at least about 5%, for example, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% or more than a reference control growth under similar conditions.

[0232] In one aspect, any one or more increases or decreases in the traits of the present disclosure are increases in traits that are an increase of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% compared to the unmodified microorganism.

[0233] In one aspect, any one or more increases or decreases in the traits of the present disclosure are at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0. compared to the unmodified microorganism.An increase of 9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

Example

[0234] Example 1: Co-production of monoethylene glycol (MEG) and acetone via the D-xylonate pathway in a strain capable of simultaneously consuming xylose and glucose - Path A in Figure 1 Escherichia coli K12 strain MG1655 was used as the host for deletion of three genes: aldA, xylA, and glcDEF, which can divert the carbon flux from the MEG + acetone pathway. The genes were successfully deleted, and the deletions were confirmed by PCR and sequencing. The next step was the integration of the MEG pathway. An operon expressing under the control of the proD promoter, containing the xdh gene (xylose dehydrogenase) and the fucO gene (glycoaldehyde reductase), which encode the first and final enzymes of the xylonate pathway, respectively, was integrated into the Escherichia coli genome, and an additional copy of the xdh gene was also placed under the control of the proD promoter and integrated at a different locus. The integration of the xdh gene enables the conversion of xylose to the intermediates D-xylonate and glycolaldehyde. The integration of the fucO gene reduces glycolaldehyde to MEG and is specific for MEG production. The next step was the integration of the acetone pathway. An operon expressing under the control of the OXB11 promoter, containing the thlA gene (acetoacetyl-CoA thiolase); the AtoDA gene (acetate:acetoacetyl-CoA transferase) and the adc gene (acetoacetate decarboxylase), was integrated into the Escherichia coli genome to obtain the basic strain. The basic strain was used as the host for modifications to promote the co-consumption of glucose and xylose. The first modification was the integration of an additional copy of xylE under the control of the GAPDH promoter at the araFGH locus, which thereby deleted araFGH. The second modification was the deletion of the xylFGH operon. All integrations and deletions were confirmed by PCR and sequencing.

[0235] Colonies from transformation were seeded into 5 mL of mineral medium containing either 12.85 g / L xylose and 2.15 g / L glucose (6:1 ratio) or 7.5 g / L xylose and 7.5 g / L glucose (1:1 ratio) for preculture. After 16 h of culture, 5% of the preculture was transferred to 100 mL of fresh medium. The flask was incubated at 37 °C and 250 rpm. The starting OD of the culture was 0.1.

[0236] For the 1:1 ratio culture, after 8 h of culture, the simultaneous use of glucose and xylose could be detected in the co - consumption strain, while in the parental strain, xylose began to be consumed only 18 h after glucose depletion (Figure 2). In the 36 - h culture, the co - consumption strain could consume 75% of the initial sugar mixture, while the parental strain consumed only 62%.

[0237] For the 6:1 ratio culture, both the parental strain and the co - consumption strain could completely consume the starting glucose and xylose with a similar profile of xylose consumption and biomass production (Figure 3). In the co - consumption strain, the total amount of MEG increased by 12% and the amount of acetone increased by 197% (Figure 4). The modification in xylose uptake led to an improvement in the co - production rate related to its parental strain.

[0238] Example 2: Co - production of monoethylene glycol (MEG) and acetone via the D - xylulose pathway in strains capable of simultaneously consuming xylose and glucose - Pathway B in Figure 1 Escherichia coli K12 strain MG1655 was used as a host with deletions of three genes: aldA, xylB, and glcDEF that can divert the carbon flux from the MEG+acetone pathway. The genes were successfully deleted, and the deletions were confirmed by PCR and sequencing. The next step was the integration of the MEG pathway. An operon expressed under the control of the proD promoter containing the khk-C gene (ketohexokinase), aldoB gene (fructose-1,6-bisphosphate aldolase), and fucO gene (glycoaldehyde reductase) was integrated into the Escherichia coli genome, and additional copies of the khk-C and aldoB genes under the control of the proD promoter were integrated at different loci. The integration of the khk-C and aldoB genes enables the conversion of xylose to the intermediate glycolaldehyde. The integration of the fucO gene reduces glycolaldehyde to MEG and is specific for MEG production. The next step was the integration of the acetone pathway. An operon expressed under the control of the OXB11 promoter containing the thlA gene (acetoacetyl-CoA thiolase); AtoDA gene (acetate:acetoacetyl-CoA transferase), and adc gene (acetoacetate decarboxylase) was integrated into the Escherichia coli genome to obtain a basic strain. The basic strain was used as a host for modifications to promote the co-consumption of glucose and xylose. The first modification was the integration of an additional copy of xylE under the control of the GAPDH promoter at the araFGH locus, which thereby deletes araFGH. The second modification was the deletion of the xylFGH operon and the replacement of the xylA promoter with the OXB15 promoter. The expression of xylA under a constitutive promoter enables the conversion of xylose to the intermediates D-xylonate and glycolaldehyde. All integrations and deletions were confirmed by PCR and sequencing.

[0239] Colonies from transformation were seeded into 5 mL of mineral medium containing either 12.85 g / L xylose and 2.15 g / L glucose (6:1 ratio) or 7.5 g / L xylose and 7.5 g / L glucose (1:1 ratio) for preculture. After 16 h of culture, 5% of the preculture was transferred to 100 mL of fresh medium. The flasks were incubated at 37 °C and 250 rpm. The starting OD of the culture was 0.1.

[0240] For the 1:1 ratio culture, simultaneous utilization of glucose and xylose could be detected in the co-consumer strain after 12 h of culture, but in the parental strain, xylose began to decrease only 18 h after glucose depletion (Figure 5). In the 36 h culture, the co-consumer strain could consume 61% of the initial sugar mixture, while the parental strain consumed only 52%.

[0241] For the 6:1 ratio culture, both the parental and co-consumer strains could completely consume the starting glucose and xylose with a similar profile of xylose consumption and biomass production (Figure 6). In the co-consumer strain, the total amount of MEG increased by 9% and the amount of acetone increased by 119% (Figure 7). The modification in xylose uptake led to an improvement in the co-production rate related to its parental strain.

[0242] Incorporation by reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, any reference to a reference, article, publication, patent, patent publication, and patent application cited herein is not an admission or any form of suggestion that it constitutes valid prior art or a part of common general knowledge in any country in the world, and should not be received as such. Further, the following references are incorporated herein by reference. TIFF0007715636000007.tif75161

Claims

1. A recombinant microorganism capable of producing a fermentation product from a raw material containing xylose and glucose, using said xylose and said glucose simultaneously, wherein there is (a) deletion or inactivation of a nucleic acid sequence encoding a xylose ABC transporter and an arabinose ABC transporter; (b) one or more endogenous or exogenous nucleic acid sequences encoding a C5 sugar co-transporter operably linked to one or more constitutive promoters, wherein said C5 sugar co-transporter comprises (1) a xylose co-transporter and / or (2) AraE of Escherichia coli (E. coli), said one or more endogenous or exogenous nucleic acid sequences; wherein there is (c) (1) a xylose isomerase operably linked to one or more constitutive promoters and deletion or inactivation of one or more nucleic acid sequences encoding xylulokinase and encodes, or wherein there is (2) a xylose dehydrogenase operably linked to one or more constitutive promoters and deletion or inactivation of one or more nucleic acid sequences encoding xylose isomerase and / or xylulokinase and encodes, one or more endogenous or exogenous nucleic acid sequences; wherein there is (d) deletion or inactivation of one or more nucleic acid sequences encoding glycolate dehydrogenase; and wherein there is (e) a native functional phosphotransferase system (PTS) and a native cAMP receptor protein (CRP) and which is Escherichia coli, said recombinant microorganism.

2. The recombinant microorganism according to claim 1, wherein said fermentation product comprises monoethylene glycol (MEG), acetone, isopropanol, glycolic acid, and / or propylene.

3. The recombinant microorganism according to claim 1, wherein said fermentation product comprises two or more molecules produced simultaneously.

4. A recombinant microorganism capable of producing monoethylene glycol (MEG), isopropanol, and / or acetone from a raw material containing xylose and glucose, using xylose and glucose simultaneously, wherein there is (a) deletion or inactivation of nucleic acid sequences encoding glycolaldehyde dehydrogenase, a xylose ABC transporter, and an arabinose ABC transporter; (b) At least one endogenous or exogenous nucleic acid molecule operably linked to one or more constitutive promoters and encoding a C5 sugar co-transporter, wherein the C5 sugar co-transporter comprises (1) a xylose co-transporter and / or (2) AraE of Escherichia coli, said at least one endogenous or exogenous nucleic acid molecule; (c) Deletion or inactivation of glycolic acid dehydrogenase and deletion or inactivation of either xylose isomerase or xylulokinase; and (d) A native functional phosphotransferase system (PTS) and a native cAMP receptor protein (CRP) comprising an enzyme pathway for MEG, isopropanol, and / or acetone production, and being Escherichia coli said recombinant microorganism. **Claim 5** The recombinant microorganism according to claim 4, wherein the glycolaldehyde dehydrogenase is aldA, the xylose ABC transporter is xylFGH, the arabinose ABC transporter is araFGH, the glycolic acid dehydrogenase is glcDEF, the xylose isomerase is xylA, and the xylulokinase is xylB. **Claim 6** The recombinant microorganism according to claim 4, further comprising one or more endogenous or exogenous nucleic acid sequences encoding a constitutive promoter, acetoacetyl-CoA thiolase, acetate:acetoacetyl-CoA transferase, and / or acetoacetate decarboxylase. **Claim 7** The nucleic acid sequence encoding the constitutive promoter comprises the sequence set forth in SEQ ID NO:

78. The nucleic acid sequence encoding the acetoacetyl-CoA thiolase comprises the sequence set forth in SEQ ID NO: 66 or 68. The nucleic acid sequence encoding the acetate:acetoacetyl-CoA transferase comprises the sequence set forth in SEQ ID NO: 70 or 71. The nucleic acid sequence encoding the acetoacetate decarboxylase comprises the sequence set forth in SEQ ID NO: 74 or 76. The amino acid sequence of the acetoacetyl-CoA thiolase has at least 95% sequence identity with SEQ ID NO: 67 or 69. The amino acid sequence of the acetate:acetoacetyl-CoA transferase has at least 95% sequence identity with SEQ ID NO: 72 or 73, and / or The amino acid sequence of the acetoacetate decarboxylase has at least 95% sequence identity with SEQ ID NO: 75 or 77, The recombinant microorganism according to claim 6.

8. The recombinant microorganism according to claim 4, wherein the C5 sugar co-transporter is a xylose co-transporter.

9. The nucleic acid sequence encoding the xylose co-transporter comprises the sequence set forth in SEQ ID NO: 48, or the amino acid sequence of the xylose co-transporter has at least 95% sequence identity with SEQ ID NO: 49, The recombinant microorganism according to claim 8.

10. The recombinant microorganism according to claim 4, further comprising one or more endogenous or exogenous nucleic acid sequences encoding a constitutive promoter, at least one xylose dehydrogenase, and glycolaldehyde reductase.

11. The nucleic acid sequence encoding the constitutive promoter comprises the sequence set forth in SEQ ID NO: 53, The nucleic acid sequence encoding the xylose dehydrogenase comprises the sequence set forth in SEQ ID NO: 15, 18, or 97, The nucleic acid sequence encoding the glycolaldehyde reductase comprises the sequence set forth in SEQ ID NO: 52, The amino acid sequence of the xylose dehydrogenase has at least 95% sequence identity with SEQ ID NO: 16, 17, or 19, and / or The amino acid sequence of the glycolaldehyde reductase has at least 95% sequence identity with SEQ ID NO: 98, The recombinant microorganism according to claim 10.

12. The recombinant microorganism according to claim 4, further comprising one or more endogenous or exogenous nucleic acid sequences encoding a constitutive promoter, at least one ketohexokinase, fructose-1,6-bisphosphate aldolase, and glycolaldehyde reductase.

13. The nucleic acid sequence encoding the constitutive promoter comprises the sequence set forth in SEQ ID NO: 53, The nucleic acid sequence encoding the ketohexokinase comprises the sequence set forth in SEQ ID NO: 11, The nucleic acid sequence encoding the fructose-1,6-bisphosphate aldolase comprises the sequence set forth in SEQ ID NO: 50, The nucleic acid sequence encoding the glycolaldehyde reductase comprises the sequence set forth in SEQ ID NO: 52, The amino acid sequence of the ketohexokinase has at least 95% sequence identity with SEQ ID NO: 12, The amino acid sequence of the fructose-1,6-bisphosphate aldolase has at least 95% sequence identity with SEQ ID NO: 51, and / or the amino acid sequence of the glycolaldehyde reductase has at least 95% sequence identity with SEQ ID NO: 98, The recombinant microorganism according to claim 12.

14. A recombinant microorganism capable of producing glycolic acid from a raw material containing xylose and glucose, using xylose and glucose simultaneously, (a) deletion or inactivation of nucleic acid sequences encoding one or more glycolaldehyde reductases, xylose ABC transporters, and arabinose ABC transporters; (b) at least one endogenous or exogenous nucleic acid molecule encoding a C5 sugar co-transporter, operably linked to one or more constitutive promoters, wherein the C5 sugar co-transporter comprises (1) a xylose co-transporter and / or (2) AraE of Escherichia coli, said at least one endogenous or exogenous nucleic acid molecule; (c) deletion or inactivation of xylose isomerase or xylulokinase; and (d) a native functional phosphotransferase system (PTS) and a native cAMP receptor protein (CRP) comprising, comprising an enzymatic pathway for glycolic acid production, being Escherichia coli, said recombinant microorganism.

15. The recombinant microorganism according to claim 14, wherein the glycolaldehyde reductase is fucO and yqhD, the xylose ABC transporter is xylFGH, the arabinose ABC transporter is araFGH, the C5 sugar co-transporter is xylE, the xylose isomerase is xylA, and the xylulokinase is xylB.

16. Deletion or inactivation of one or more endogenous or exogenous nucleic acid sequences encoding xylose dehydrogenase and glycolaldehyde reductase, operably linked to one or more constitutive promoters, and one or more nucleic acid sequences encoding xylose isomerase, in the recombinant microorganism according to claim 1.

17. The recombinant microorganism according to claim 1, wherein the fermentation product comprises monoethylene glycol (MEG) and / or acetone.

18. The recombinant microorganism according to claim 4, comprising deletion or inactivation of xylulokinase.

19. The recombinant microorganism according to claim 4, comprising a deletion or inactivation of xylose isomerase.

20. The recombinant microorganism according to claim 4, which has the ability to produce monoethylene glycol (MEG) and / or acetone and comprises an enzymatic pathway for MEG and / or acetone production.

Images