PROTEINIC BIOMASS PREPARATION COMPRISING A NON-NATIVE ORGANISM OF THE CLOSTRIDIA CLASS

Abstract

A proteinic biomass preparation comprising a non-native organism of the Clostridia class, which organism expresses (i) a modified aspartate kinase; (ii) a modified homoserine dehydrogenase; (iii) a modified homoserine kinase; (iv) a modified anthranilate synthase; (v) a functional lycopene pathway and the genes crtY, crtW, and crtZ; and/or (vi) a functional oleic acid pathway and the four gene operon (pfaABCD). Methods of producing proteinic biomass preparations are also described.

Claims

1. A proteinic biomass preparation comprising a non-native organism of the Clostridia class, which organism expresses (i) a modified aspartate kinase characterized by reduced lysine inhibition, reduced threonine inhibition, and/or reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (ii) a modified homoserine dehydrogenase characterized by reduced threonine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (iii) a modified homoserine kinase characterized by reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (iv) a modified anthranilate synthase characterized by reduced tryptophan inhibition compared with the unmodified enzyme in native organism of the same genus and species; (v) a functional lycopene pathway and the genes crtY, crtW, and crtZ and/or (vi) a functional oleic acid pathway and the four gene operon (pfaABCD).

2-15. (canceled)

16. A preparation according to claim 1, wherein said non-native organism is an acetogen.

17-18. (canceled)

19. A preparation according to claim 1, comprising, on a dry basis, at least 55% wt protein.

20. A preparation according to claim 1, comprising at least one selected from the group consisting of: on total protein content, at least 6% wt lysine; (ii) on total protein content, at least 3% wt threonine; (iii) on total protein content, at least 1.5% wt methionine; (iv) on total protein content, at least 0.5% wt tryptophan; (v) on a dry basis, at least 0.01% wt astaxanthin; (vi) on a dry basis, at least 0.1% wt eicosapentaenoic acid; and (vii) on a dry basis, at least 0.1% wt docosahexaenoic acid.

21-43. (canceled)

44. A proteinic biomass preparation comprising an organism of the Clostridia class, wherein said preparation comprises, (i) on dry basis at least 55% wt protein; (ii) on total protein content, at least 6% wt lysine; (iii) on total protein content, at least 3% wt threonine; (iv) on total protein content, at least 1.5% wt methionine; (v) on total on total protein content, at least 0.5% wt tryptophan; (vi) on a dry basis, at least 0.01% wt astaxanthin; (vii) on a dry basis, at least 0.1wt eicosapentaenoic acid, and/or (viii) on a dry basis, at least 0.1% wt docosahexaenoic acid.

45. (canceled)

46. A preparation according to claim 44, comprising (i) and at least one of (ii) to (v).

47-50. (canceled)

51. A preparation according to claim 44, wherein said organism expresses (i) a modified aspartate kinase characterized by reduced lysine inhibition, reduced threonine inhibition, and/or reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (ii) a modified homoserine dehydrogenase characterized by reduced threonine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (iii) a modified homoserine kinase characterized by reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (iv) a modified anthranilate synthase characterized by reduced tryptophan inhibition compared with the unmodified enzyme in native organism of the same genus and species; (v) a functional lycopene pathway and the genes crtY, crtW, and crtZ and/or (vi) a functional oleic acid pathway and the four gene operon (pfaABCD)

52-55. (canceled)

56. A biomass according to claim 44, wherein said organism further expresses the gene pfaE.

57. A preparation according to claim 44, wherein said organism is selected from Butyribacterium methylotrophicum, Eubacterium limosum, and Clostridium kluyveri.

58. A preparation according to claim 44, wherein said organism is an acetogen.

59-61. (canceled)

62. A preparation according to claim 44, further comprising digestibility-enhancing enzymes selected from the group consisting of phytases, cellulases, lipases, amylases, arabinases, pectinases, mannases, keratinases, proteases, tannases, galactosidases, glucosidases, invertases and combinations thereof.

63. (canceled)

64. A preparation according to claim 51, wherein said non-native organism further expresses a diphosphate-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90).

65. A preparation according to claim 44, wherein phosphofructokinase 1 (EC 2.7.1.11, pfkA, BUME_09340) has been deleted from the genome of said non-native organism.

66. A preparation according to claim 44, wherein acetyl-CoA acetyltransferase gene (th1A, EC 2.3.1.9, BUME_07140) been deleted from the genome of said non-native organism.

67. Animal feed comprising a preparation according to claim 44.

68. (canceled)

69. A method for producing of a biomass comprising culturing an organism according to claim 44 in a fermentation medium comprising a carbon source and a nitrogen source, whereby biomass is generated in a fermentation broth.

70. A method according to claim 69, wherein said culturing is anaerobic.

71-73. (canceled)

74. A method according to claim 69, wherein said non-native organism fixes CO.sub.2.

75. (canceled)

76. A method according to claim 69, wherein biomass generation yield is greater than 35 gram biomass per 100 gram of carbon source consumed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 depicts an exemplary co-location integrated method for producing ethanol.

[0033] FIG. 2 shows exemplary results of B. methylotrophicum fermentation on glucose.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The particulars shown herein are by way of example and for purposes of illustrative discussion of the various embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0035] The present invention will now be described by reference to more detailed embodiments. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms a, an, and the are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0037] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0038] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0039] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0040] As used herein the term proteinic biomass refers to biomass comprising at least 50% protein.

[0041] As used herein the term comprising an amino acid refers to either comprising the amino acid in its free form or comprising peptides or proteins, the hydrolysate of which comprises that amino acid.

[0042] As used herein, the term genetically modified organisms refers to organism comprising specific modifications to the genome. These can include chromosomal deletions or insertions and expression of exogenous genes on a replicating plasmid. As used herein, the term genetic modifications does not refer to single point mutations or mutations arising from adaptation experiments or induced mutatgenesis experiments. As used herein, the term non-genetically modified organisms includes organisms comprising single point mutations or mutations arising from adaptation experiments or induced mutatgenesis experiments.

[0043] According to one aspect, provided is a proteinic biomass preparation comprising a non-native organism of the Clostridia class, which organism expresses (i) a modified aspartate kinase characterized by reduced lysine inhibition, reduced threonine inhibition, and/or reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (ii) a modified homoserine dehydrogenase characterized by reduced threonine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (iii) a modified homoserine kinase characterized by reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (iv) a modified anthranilate synthase characterized by reduced tryptophan inhibition compared with the unmodified enzyme in native organism of the same genus and species; (v) a functional lycopene pathway and the genes crtY, crtW, and crtZ and/or (vi) a functional oleic acid pathway and the four gene operon (pfaABCD).

[0044] According to an embodiment, said organism expresses a modified aspartate kinase characterized by reduced lysine inhibition, reduced threonine inhibition, and/or reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species.

[0045] According to an embodiment, native organisms of the Clostridia class can express multiple aspartase kinases, some of which can be inhibited by lysine alone, some by threonine alone, some by methionine and some by their combination. According to various embodiments, said preparation non-native organism expresses a modified aspartate kinase characterized by reduced lysine inhibition compared with native aspartate kinase in native organism; a modified aspartate kinase characterized by reduced threonine inhibition compared with native aspartate kinase in native organism; a modified aspartate kinase characterized by reduced methionine inhibition compared with native aspartate kinase in native organism or a modified aspartate kinase characterized by reduced inhibition by multiple of said amino acids compared with native aspartate kinase in native organism.

[0046] According to an embodiment, said modified aspartate kinase is derived from the Butyribacterium methylotrophicum aspartate kinase and is characterized by reduced lysine inhibition, reduced threonine inhibition, and/or reduced methionine inhibition compared with the unmodified Butyribacterium methylotrophicum aspartate kinase. According to an embodiment, the gene of said aspartate kinase is selected from the group consisting of lysC1 (BUME_01940), lysC2 (BUME_01950), and lysC3 (BUME_08600).

[0047] According to an embodiment, said organism expresses a modified homoserine dehydrogenase characterized by reduced threonine inhibition compared with the unmodified enzyme in native organism of the same genus and species.

[0048] According to an embodiment, said modified homoserine dehydrogenase is derived from the Butyribacterium methylotrophicum homoserine dehydrogenase and is characterized by reduced threonine inhibition compared with the unmodified Butyribacterium methylotrophicum homoserine dehydrogenase. According to an embodiment, the gene of said homoserine dehydrogenase is hom (BUME_08590).

[0049] According to an embodiment, said organism expresses a modified homoserine kinase characterized by reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species.

[0050] According to an embodiment, said modified homoserine kinase is derived from the Butyribacterium methylotrophicum homoserine kinase and is characterized by reduced methionine inhibition compared with the unmodified Butyribacterium methylotrophicum homoserine kinase. According to an embodiment, the gene of said homoserine kinase is selected from the group consisting of thrB1 (BUME_06990) and thrB2 (BUME_08570).

[0051] According to an embodiment, said organism expresses a modified anthranilate synthase characterized by reduced tryptophan inhibition compared with the unmodified enzyme in native organism of the same genus and species.

[0052] According to an embodiment, said modified anthranilate synthase is derived from the Butyribacterium methylotrophicum anthranilate synthase and is characterized by reduced tryptophan inhibition compared with the unmodified Butyribacterium methylotrophicum anthranilate synthase. According to an embodiment, the gene of said anthranilate synthase is trpEG (BUME_17910-BUME_17900).

[0053] According to an embodiment, at least one of said modified enzymes comprises a spontaneous mutation, a random mutation, site-specific mutation, or a combination thereof. According to an embodiment, at least one of said modified enzymes comprises mutation to the regulatory domain of the enzymes. According to an embodiment, at least one of said modified enzymes comprises mutation to the binding site of lysine, threonine, methionine, and/or tryptophan.

[0054] According to an embodiment, said non-native organism expresses a functional lycopene pathway. According to an embodiment, said non-native organism is modified to express a functional lycopene pathway. According to an embodiment, said non-native organism expresses a functional lycopene pathway and the genes crtY, crtW, and crtZ.

[0055] According to an embodiment, said organism expresses a functional oleic acid pathway and the four gene operon (pfaABCD). According to an embodiment, said organism further expresses the gene pfaE.

[0056] According to an embodiment, said non-native organism further expresses a diphosphate-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90). According to an embodiment, phosphofructokinase 1 (EC 2.7.1.11, pfkA, BUME_09340) has been deleted from the genome of said non-native organism.

[0057] According to an embodiment, acetyl-CoA acetyltransferase gene (th1A, EC 2.3.1.9, BUME_07140) been deleted from the genome of said non-native organism.

[0058] According to an embodiment, said preparation confers a probiotic benefit. According to an embodiment, said preparation can help disrupt the propagation of pathogenic gut bacteria, thus conferring a probiotic benefit. According to an embodiment, said preparation can induce a positive host response within the gut, thus conferring a probiotic benefit.

[0059] According to an embodiment, said preparation comprises enzymes capable of assisting the digestibility of feed ingredients. According to an embodiment, said preparation comprises digestibility-enhancing enzymes selected from the group consisting of phytases, cellulases, lipases, amylases, arabinases, pectinases, mannases, keratinases, proteases, tannases, galactosidases, glucosidases, invertases and combinations thereof. According to an embodiment, said digestibility-enhancing enzymes are generated at least partially endogenously by said non-native organism.

[0060] According to an embodiment, amino acid transport occurs at a lower rate in the non-native organism than in a native organism of the same genus and species, e.g. at less than 50% the rate in the native organism, less than 30%, less than 20%, less than 10% or less than 5%. According to an embodiment, said lower rate transport is out of the cell, into the cell or both. According to an embodiment, said lower rate is for transport of intracellular amino acids into the extracellular environment. According to an embodiment, said lower rate is a result of a modification to a Basic Amino Acid Antiporter (ArcD)-family protein. According to an embodiment, lysine transport occurs at a lower rate in the non-native organism. According to an embodiment, threonine transport occurs at a lower rate in the non-native organism. According to an embodiment, tryptophan transport occurs at a lower rate in the non-native organism. According to an embodiment, methionine transport occurs at a lower rate in the non-native organism. According to an embodiment, transport of multiple amino acids occurs at a lower rate in the non-native organism.

[0061] According to an embodiment, said non-native organism is not genetically modified.

[0062] According to an alternative embodiment, said non-native organism is genetically modified.

[0063] According to an embodiment, said non-native organism is selected from Butyribacterium methylotrophicum, Eubacterium limosum, Clostridium kluyveri, Selenomonas bovis, Selenomonas ruminantium subsp. Lactilytica, Selenomonas ruminantium subsp. Ruminantium, Prevotella albensis, Prevotella bryantii, Prevotella brevi, and Megasphaera elsdenii. According to an embodiment, said non-native organism is an acetogen.

[0064] According to an embodiment, said preparation consists of more than one bacterial species. According to an embodiment, said preparation consists of an acetogenic species and a non-acetogenic species.

[0065] According to an embodiment, said preparation comprises, on a dry basis, at least 55% wt protein, at least 58% wt, at least 60% wt, at least 62% wt, at least 64% wt, at least 66% wt, at least 68% wt, at least 70% wt, at least 72% wt, or at least 74% wt.

[0066] According to an embodiment, said preparation comprises, on total protein content, at least 6% wt lysine, at least 7% wt, at least 8% wt, at least 9% wt, at least 10% wt, at least 11% wt, at least 12% wt, at least 13% wt, at least 14% wt, or at least 15% wt.

[0067] According to an embodiment, said preparation comprises, on total protein content, at least 3% wt threonine, at least 3.5% wt, at least 4% wt, at least 4.5% wt, at least 5% wt, at least 5.5% wt or at least 6% wt.

[0068] According to an embodiment, said preparation comprises, on total protein content, at least 1.5% wt methionine, at least 1.7% wt, at least 1.8% wt, at least 1.9% wt, at least 2% wt, at least 2.1% wt, at least 2.2% wt, at least 2.3% wt, at least 2.4% wt or at least 2.5% wt.

[0069] According to an embodiment, said preparation comprises, on total protein content, at least o.5% wt tryptophan, at least 0.7% wt, at least 0.8% wt, at least 0.9% wt, at least 1% wt, at least 1.1% wt, at least 1.2% wt, at least 1.3% wt, at least 1.4% wt or at least 1.5% wt.

[0070] According to an embodiment, said preparation comprises, on a dry basis, at least 0.01% wt astaxanthin, at least 0.02% wt, at least 0.03% wt, at least 0.04% wt, at least 0.05% wt, at least 0.06% wt, at least 0.07% wt, at least 0.08% wt, at least 0.09% wt, least 0.1% wt, at least 0.11% wt, at least 0.12% wt, least 0.13% wt, at least 0.14% wt or least 0.15% wt.

[0071] According to an embodiment, said preparation comprises, on a dry basis, at least 0.1% wt eicosapentaenoic acid, at least 0.2% wt, at least 0.3% wt, at least 0.4% wt, at least 0.5% wt, at least 0.6% wt, at least 0.7% wt, at least 0.8% wt, at least 0.9% wt, least 1.0% wt, at least 1.1% wt, at least 1.2% wt, least 1.3% wt, at least 1.4% wt or least 1.5% wt.

[0072] According to an embodiment, said preparation comprises, on a dry basis, at least 0.1% wt docosahexaenoic acid, at least 0.2% wt, at least 0.3% wt, at least 0.4% wt, at least 0.5% wt, at least 0.6% wt, at least 0.7% wt, at least 0.8% wt, at least 0.9% wt, least 1.0% wt, at least 1.1% wt, at least 1.2% wt, least 1.3% wt, at least 1.4% wt or least 1.5% wt.

[0073] According to an embodiment, further provided is an animal feed comprising said proteinic biomass preparation. According to an embodiment, further provided is fish feed comprising said proteinic biomass preparation.

[0074] Also provided is a method for producing a proteinic preparation, which method comprises culturing said non-native Clostridia class organism in a fermentation medium comprising a carbon source and a nitrogen source, whereby said proteinic biomass is generated in a fermentation broth. According to an embodiment, said method further comprises separating said generated biomass from the fermentation medium. According to an embodiment, said separating comprises at least one of filtering and centrifugation and optionally washing said separated cells in order to wash off water-soluble compounds, such as ashes and carboxylic acid salts. According to an embodiment, said method further comprises at least one of lysing said biomass and drying it.

[0075] According to an embodiment, said fermentation broth further comprises a coproduct selected from the group consisting of acetic acid, butyric acid, lactic acid, ethanol, n-butanol, 1,3-propanediol, 2,3-butanediol, acetoin and combinations thereof. According to an embodiment, said method further comprises separating said coproduct from said fermentation broth. According to an embodiment, said separating comprises adjusting the pH of said broth to pH<4.

[0076] According to an embodiment, said culturing is anaerobic.

[0077] According to an embodiment, said fermentation medium comprises stillage. According to various embodiments, said stillage in whole stillage, thin stillage, combinations thereof or products thereof. According to an embodiment, said fermentation medium comprises glycerol.

[0078] According to an embodiment, said fermentation medium further comprises CO.sub.2 or a precursor thereof. According to an embodiment, said method comprises sparging CO.sub.2 through said medium and/or adding there a carbonate or a bicarbonate (e.g. sodium carbonate or sodium bicarbonate). According to an embodiment, said cultured organism fixes CO.sub.2.

[0079] According to an embodiment, said fermentation medium further comprises a non-sugar reductant.

[0080] According to an embodiment, in said method biomass generation yield is greater than 35 g biomass per 100 g of carbon source consumed greater than 40 g, greater than 45 g, greater than 50 g, or greater than 55 g. According to an embodiment, cell density in said fermentation broth is at least 15 gram cell mass per Liter (15 g/L), at least 20 g/L, at least 25 g/L, at least 30 g/L, at least 35 g/L or at least 40 g/L. According to an embodiment, cell culturing productivity in said fermentation broth is at least 0.5 gram/Liter/hour (g/L/hr), at least 0.6 g/L/hr, at least 0.7 g/L/hr, at least 0.8 g/L/hr, at least 0.9 g/L/hr, at least 1.0 g/L/hr, at least 1.1 g/L/hr, at least 1.2 g/L/hr or at least 1.3 g/L/hr.

[0081] According to an embodiment, said method further comprises combining said biomass, optionally lysed and/or dried, with other feed ingredients, such as fishmeal, fishoil, other animal proteins, other vegetable proteins, vitamins and/or minerals. According to an embodiment, said method further comprises pelletizing.

[0082] According to another aspect, provided is a proteinic biomass preparation comprising a non-native organism of the Clostridia class modified for expression of peptides and/or proteins, which peptides and/or proteins comprise, on a total protein content: (i), at least 6% wt lysine, at least 7% wt, at least 8% wt, at least 9% wt, at least 10% wt, at least 11% wt, at least 12% wt, at least 13% wt, at least 14% wt, or at least 15% wt; (ii) at least 3% wt threonine, at least 3.5% wt, at least 4% wt, at least 4.5% wt, at least 5% wt, at least 5.5% wt or at least 6% wt; (iii) at least 1.5% wt methionine, at least 1.7% wt, at least 1.8% wt, at least 1.9% wt, at least 2% wt, at least 2.1% wt, at least 2.2% wt, at least 2.3% wt, at least 2.4% wt or at least 2.5% wt; and/or (iv) at least 0.5% wt tryptophan at least 0.7% wt, at least 0.8% wt, at least 0.9% wt, at least 1% wt, at least 1.1% wt, at least 1.2% wt, at least 1.3% wt, at least 1.4% wt or at least 1.5% wt.

[0083] According to an embodiment, protein content and amino acid profile is modified by expression of a peptide sequence. This sequence can be a native peptide, an exogenous peptide, or a synthetic peptide sequence. The resulting peptide can be water insoluble.

[0084] According to another aspect, provided is proteinic biomass comprising an organism of the Clostridia class, wherein said preparation comprises, (i) on dry basis at least 55% wt protein, at least 58% wt, at least 60% wt, at least 62% wt, at least 64% wt, at least 66% wt, at least 68% wt, at least 70% wt, at least 72% wt, or at least 74% wt; (ii) on total protein content, at least 6% wt lysine at least 7% wt, at least 8% wt, at least 9% wt, at least 10% wt, at least 11% wt, at least 12% wt, at least 13% wt, at least 14% wt, or at least 15% wt; (iii) on total protein content, at least 3% wt threonine, at least 3.5% wt, at least 4% wt, at least 4.5% wt, at least 5% wt, at least 5.5% wt or at least 6% wt; (iv) on total protein content, at least 1.5% wt methionine, at least 1.7% wt, at least 1.8% wt, at least 1.9% wt, at least 2% wt, at least 2.1% wt, at least 2.2% wt, at least 2.3% wt, at least 2.4% wt or at least 2.5% wt; (v) on total on total protein content, at least 0.5% wt tryptophan, at least 0.7% wt, at least 0.8% wt, at least 0.9% wt, at least 1% wt, at least 1.1% wt, at least 1.2% wt, at least 1.3% wt, at least 1.4% wt or at least 1.5% wt; (vi) on a dry basis, at least 0.01% wt astaxanthin, at least 0.02% wt, at least 0.03% wt, at least 0.04% wt, at least 0.05% wt, at least 0.06% wt, at least 0.07% wt, at least 0.08% wt, at least 0.09% wt, least 0.1% wt, at least 0.11% wt, at least 0.12% wt, least 0.13% wt, at least 0.14% wt or least 0.15% wt; (vii) on a dry basis, at least 0.1wt eicosapentaenoic acid, at least 0.2% wt, at least 0.3% wt, at least 0.4% wt, at least 0.5% wt, at least 0.6% wt, at least 0.7% wt, at least 0.8% wt, at least 0.9% wt, least 1.0% wt, at least 1.1% wt, at least 1.2% wt, least 1.3% wt, at least 1.4% wt or least 1.5% wt; and/or (viii) on a dry basis, at least 0.1% wt docosahexaenoic acid, at least 0.2% wt, at least 0.3% wt, at least 0.4% wt, at least 0.5% wt, at least 0.6% wt, at least 0.7% wt, at least 0.8% wt, at least 0.9% wt, least 1.0% wt, at least 1.1% wt, at least 1.2% wt, least 1.3% wt, at least 1.4% wt or at least 1.5% wt.

[0085] According to various embodiment, said proteinic biomass comprises at least two of (i) to (viii), at least three, at least four, at least five, at least six, at least seven or all eight.

[0086] According to various embodiment, said proteinic biomass comprises (i) and at one of (ii) to (v), at least two, at least three or all four.

[0087] According to various embodiment, said proteinic biomass comprises (vi) and at one of (i) to (v), at least two, at least three, at least four or all five.

[0088] According to various embodiment, said proteinic biomass comprises at least one of (vii) and (viii) and at one of (i) to (v), at least two, at least three, at least four or all five.

[0089] According to various embodiment, said proteinic biomass comprises (vi); at least one of (vii) and (viii) and at one of (i) to (v), at least two, at least three, at least four or all five.

[0090] According to an embodiment, said organism is not genetically modified. According to an alternative embodiment, said organism is genetically modified.

[0091] According to an embodiment, said organism expresses (i) a modified aspartate kinase characterized by reduced lysine inhibition, reduced threonine inhibition, and/or reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (ii) a modified homoserine dehydrogenase characterized by reduced threonine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (iii) a modified homoserine kinase characterized by reduced methionine inhibition compared with the unmodified enzyme in native organism of the same genus and species; (iv) a modified anthranilate synthase characterized by reduced tryptophan inhibition compared with the unmodified enzyme in native organism of the same genus and species; (v) a functional lycopene pathway and the genes crtY, crtW, and crtZ and/or (vi) a functional oleic acid pathway and the four gene operon (pfaABCD).

[0092] According to an embodiment, said modified aspartate kinase is derived from the Butyribacterium methylotrophicum aspartate kinase and is characterized by reduced lysine inhibition, reduced threonine inhibition, and/or reduced methionine inhibition compared with the unmodified Butyribacterium methylotrophicum aspartate kinase. According to an embodiment, the gene of said aspartate kinase is selected from the group consisting of lysC1 (BUME_01940), lysC2 (BUME_01950), and lysC3 (BUME_08600).

[0093] According to an embodiment, said modified homoserine dehydrogenase is derived from the Butyribacterium methylotrophicum homoserine dehydrogenase and is characterized by reduced threonine inhibition compared with the unmodified Butyribacterium methylotrophicum homoserine dehydrogenase. According to an embodiment, the gene of said homoserine dehydrogenase is hom (BUME_08590).

[0094] According to an embodiment, said modified homoserine kinase is derived from the Butyribacterium methylotrophicum homoserine kinase and is characterized by reduced methionine inhibition compared with the unmodified Butyribacterium methylotrophicum homoserine kinase. According to an embodiment, the gene of said homoserine kinase is selected from the group consisting of thrB1 (BUME_06990) and thrB2 (BUME_08570).

[0095] According to an embodiment, said modified anthranilate synthase is derived from the Butyribacterium methylotrophicum anthranilate synthase and is characterized by reduced tryptophan inhibition compared with the unmodified Butyribacterium methylotrophicum anthranilate synthase. According to an embodiment, the gene of said anthranilate synthase is trpEG (BUME_17910-BUME_17900).

[0096] According to an embodiment, at least one of said modified enzymes comprises a spontaneous mutation, a random mutation, site-specific mutation, or a combination thereof. According to an embodiment, at least one of said modified enzymes comprises mutation to the regulatory domain of the enzymes. According to an embodiment, at least one of said modified enzymes comprises mutation to the binding site of lysine, threonine, methionine, and/or tryptophan.

[0097] According to an embodiment, amino acid transport occurs at a lower rate in the non-native organism than in a native organism of the same genus and species, e.g. at less than 50% the rate in the native organism, less than 30%, less than 20%, less than 10% or less than 5%. According to an embodiment, said lower rate transport is out of the cell, into the cell or both. According to an embodiment, said lower rate is for transport of intracellular amino acids into the extracellular environment. According to an embodiment, said lower rate is a result of a modification to a Basic Amino Acid Antiporter (ArcD)-family protein.

[0098] According to an embodiment, said non-native organism is selected from Butyribacterium methylotrophicum, Eubacterium limosum, Clostridium kluyveri, Selenomonas bovis, Selenomonas ruminantium subsp. Lactilytica, Selenomonas ruminantium subsp. Ruminantium, Prevotella albensis, Prevotella bryantii, Prevotella brevi, and Megasphaera elsdenii. According to an embodiment, said non-native organism is an acetogen.

[0099] According to an embodiment, further provided is an animal feed comprising said proteinic biomass preparation. According to an embodiment, further provided is fish feed comprising said proteinic biomass preparation.

[0100] According to an embodiment, further provided is a method for producing a proteinic biomass preparation comprising culturing said non-native Clostridia class organism in a fermentation medium comprising a carbon source and a nitrogen source, whereby said proteinic biomass is generated in fermentation broth. According to an embodiment, said culturing is anaerobic.

[0101] According to an embodiment, said fermentation medium comprises stillage. According to an embodiment, said fermentation medium further comprises a non-sugar reductant.

[0102] According to an embodiment, in said method biomass generation yield is greater than 35 g biomass per 100 g of carbon source consumed greater than 40 g, greater than 45 g, greater than 50 g, or greater than 55 g. According to an embodiment, cell density in said fermentation broth is at least 15 g cell mass per Liter (15 g/L), at least 20 g/L, at least 25 g/L, at least 30 g/L, at least 35 g/L or at least 40 g/L. According to an embodiment, cell culturing productivity in said fermentation broth is at least 0.5 gram/Liter/hour (g/L/hr), at least 0.6 g/L/hr, at least 0.7 g/L/hr, at least 0.8 g/L/hr, at least 0.9 g/L/hr, at least 1.0 g/L/hr, at least 1.1 g/L/hr, at least 1.2 g/L/hr or at least 1.3 g/L/hr.

[0103] Some embodiments herein, provide methods for producing the proteinic biomass preparation, comprising culturing said non-native Clostridia class organism in a fermentation medium comprising a carbon source and a nitrogen source, whereby said proteinic biomass is generated in a fermentation broth. According to an embodiment, said provided fermentation medium comprises stillage of ethanol production. According to an embodiment, ethanol production includes fermentation of carbohydrates-containing feedstock to form a fermentation broth comprising ethanol, biomass and non-fermented components of the feedstock, e.g. carbon sources and proteins. According to an embodiment, ethanol is distilled out of said broth to form distilled ethanol and a residue comprising said biomass and non-fermented components of the feedstock. This residue is referred to as whole stillage. According to an embodiment, the provided fermentation medium comprises said whole stillage. Alternatively, the whole stillage is filtered or centrifuged to generate wet solids and a solids-depleted liquid referred to as thin stillage. According to an embodiment, the provided fermentation medium comprises said thin stillage. A typical thin stillage contains glycerol at about 36 g/L, glucose, DP2, DP3 and DP4+ at 0.7 g/L, 17 g/L, 5 g/L and 28 g/L, respectively and lactic acid at 2.5 g/L.

[0104] Several of the above embodiment, provided methods for producing the proteinic biomass preparation, comprising culturing said non-native Clostridia class organism in a fermentation medium comprising a carbon source and a nitrogen source, whereby said proteinic biomass is generated in a fermentation broth. According to an embodiment said method is conducted at co-location with ethanol production. As used herein, the term co-location refers to location within 10 Km from each other, within 5 Km, within 2 Km or within 1 Km.

[0105] An exemplary co-location integrated method for producing ethanol is depicted in FIG. 1. It comprises, a primary ethanol fermentation [110] generating a primary ethanol stream [154] and stillage [156] and a secondary mixotrophic ethanol fermentation [120], wherein said stillage forms a fraction of the fermentation medium and wherein a secondary ethanol stream [194] is generated. According to an embodiment, the method further comprises milling [130] and liquefying [140] incoming corn grains [105] to form the feedstock [145] of the primary fermentation. According to an embodiment, the method further comprises fractionating the corn grains, e.g. for pre-removal of fiber and/or corn oil (not shown in the figure). The liquefied-corn-containing primary fermentation medium is metabolized by an ethanol producing organism, e.g. a yeast, in [110]. A primary fermentation broth is formed [114] containing ethanol. In distillation columns [150], ethanol is distilled out, forming a primary ethanol stream [154], which is optionally further dried on molecular sieves (not shown in the figure). The residue [156] is the whole stillage comprising the yeast, corn protein, optionally also fiber and oil, and soluble matter including glycerol and oligosaccharides. The whole stillage is centrifuged [160] to form wet distillers solids [166] and thin stillage [164].

[0106] The exemplary method further comprises gasification of corn stover [116] in a gasifier [170] to form a mixture of hydrogen, CO and CO2 [175] to be used as non-sugar reductant. Said non-sugar reductant is combined with said thin stillage (the carbon source) to form the feedstock for the fermentation [120] medium, wherein said non-native Clostridia class organism is cultured and whereby proteinic biomass is generated in a fermentation broth [121]. Optionally, said biomass is separated, dried and lysed (not shown in the figure).

EXAMPLES

Example 1Fermentation of Butyribacterium Methylotrophicum

[0107] A 3-L batch fermentation was conducted with Butyribacterium methylotrophicum grown on glucose. The fermenter was inoculated with a 10% (v/v) inoculum of an actively growing culture. The medium in the fermenter consisted of 0.2 g/L of K.sub.2HPO.sub.4.3H.sub.2O, 0.3 g/L of KH.sub.2PO.sub.4, 0.3 g/L of (NH.sub.4).sub.2SO.sub.4, 0.6 g/L of NaCl, 0.12 g/L of MgSO.sub.4.7H.sub.2O, 0.1 g/L of CaCl.sub.2.2H.sub.2O, 0.5 g/L of cysteine HCl, 1 g/L yeast extract, 3 g/L sodium acetate, 30 g/L of glucose, 10 mL/L Wolfe's Mineral Solution, and 10 mL/L Wolfe's Vitamin Solution. The fermenter was sparged with N.sub.2 until just after inoculation, at which time the sparging was turned off. The pH was bottom controlled at 6.5 with 6M NH.sub.4OH. Temperature was maintained at 37 C. with agitation of 100 rpm. At 19.5 hours after inoculation, the culture was fed another 14 g/L of glucose, as the culture was exhausted of glucose (FIG. 2).

[0108] The cell density reached over 20 g/L in the first 24 hours of fermentation with over 40 g/L of glucose consumed and about 14.5 g/L of acetate being produced. The cell mass yield was consistently over 0.5 g/g after 12 hours of growth.

Example 2Reduced Inhibition of Aspartate Kinase

[0109] Butyribacterium methylotrophicum has three annotated aspartate kinase genes: lysC1 (BUME_01940), lysC2 (BUME_01950), and lysC3 (BUME_08600).

TABLE-US-00001 BUME_01940 (amino acid sequence) MTTTYMESHSVDGLTVDHKNLMISLKKVPVNSIILTRCLSELSDADVNV DIITQTAPVKNAFDVSFIVLERDLEKVKDIVNALGEEYPEIKITINKDI TRLSVSGIGMRTQSGVAAKFFQVLADNDVQILMITTSEIRISCIIKIED TEKAVAATKEAFDLED BUME_01950 (amino acid sequence) MSIIVQKYGGTSMGTIDRIKNVARRIIKKREDGNQMVVVVSAMGKSTDE LVKMAYSISDAPPRRELDMLLATGEQVSISMLSMALNAMGYDAISFTGP QVGVHTMGHHGKSRIMDIETRKIEDALNDGKIVIIAGFQGVNENDDITT LGRGGSDTSAVALSCVLECPCEIYTDVDGIYGVDPRLYPPAKKLDTVSF DEMLEMASLGAGVMHARAIELGSKYNAEIYVASSIHDVPGTLIKEGDSN MSPMEQQAITGLAIDNDELMVSLKNVPFDMNITAQFFSDLAKKSINIDM ISQTAPVYGAINISFSAPIEDLSELRKILYDFMEKYPQVEMDINKEISK LSVVGIGMRSQSGVAAKFFQLLADNNIPMLMITTSEIRISCVIPSELRD TAVMATADAFDL BUME_08600 (amino acid sequence) MEDLIVQKYGGTSVGTVEKIKRVARRIVETKNAGNKVVVVVSAMGKTTD ELVDLALAINPNPPSREMDVLLATGEQVSISLLAMAIQTIGHDVVSLTG AQCGIQTSDVHKRARISGIDTARIERELADEKIVIVAGFQGVDENRDIT TLGRGGSDTSAVAIAAALEAKCCEIYTDVDGVYNADPRVVPTATKMDEV SYQEVLEMASLGAGVLHPRSVELAEKFKMPLIVRSSYNNNEGTIIKEDV KMEKVLVRGIALDENIAKISIFEVPDQPGIAFKLFSMLASANIHVDMIV QNVNRTAVNDISFTVDADELQEAVEVSQKFAFEVEAQKVAFDKGVAKLS VVGTGIVANAEIASKFFESLFELGINIQTISTSEIKISCLIDKERAKEA MIHIHKKFDM

[0110] One or more of these genes are subjected to mutagenesis, such as chemically-induced random mutagenesis or error-prone PCR amplification, and then screened for reduced inhibition by lyseine, threonine, and/or methionine.

Example 3Reduced Inhibition of Homoserine Dehydrogenase

[0111] Butyribacterium methylotrophicum has one annotated homoserine dehydrogenase gene: hom (BUME_08590).

TABLE-US-00002 BUME_08590 (amino acid sequence) MNIGLLGFGTIGTGVYELINLNKGRFAKNLDEKVVITKILDKDPNKKVA EEDKVARVVTNPDDIMDDPEIEIVIALLGGMDFEYGLIKRALQSGKHVV TANKAVISEYFEELLTIAAENNVILRYEASVGGGIPIIGSLKEELKINR VNEIKGILNGTTNFILSKMTEEGADFADTLKLAQSIGFAEADPTADIEG YDVSRKLAILSSLAYGGIIKDEDVRKRGLSDVRAVDIEMAGDYGYIIKY LGHSVLKEGNQVYTTVEPVMFKEASIMSNVNSEFNIISIVGDIIGELQF YGKGAGKDATANAVVGDALYIINCIKDNNFPKPLVFRKQLDKKGVGAFK GKYYLRVDIDSHETFEHALNAVDEVCARKNIIVSDNRVFFMTEPVEADV FDAMVAKIKEKQSECFYARIYE

[0112] This gene is subjected to mutagenesis, such as chemically-induced random mutagenesis or error-prone PCR amplification, and then screened for reduced inhibition by threonine.

Example 4Reduced Inhibition of Homoserine Kinase

[0113] Butyribacterium methylotrophicum has two annotated homoserine kinase genes: thrB1 (BUME_06990) or thrB2 (BUME_08570).

TABLE-US-00003 BUME_06990 (amino acid sequence) MVDGKFIEMQTEIMKNYPPANAQALLNIFNAASGMQEYLDDVLAKTRES YLYTQLRDLVENYYDIGTLLDVYQIFGGYINTSFGIYTEKNGEKQTWFV RKYKNGKELESLLFEHSMLKYARENGFSYGAVPIPAKDGKTYHEVTETT TEGETKSYFAVFNFVGGKAHYDWIPNWANAGVADLTVTSAAKSLAEFHN STRGFDPEGRHGDNIMDNEDITVNEIIRKFPKTLKEYRKSYAEAGFENV YTEYYDANYDYFAKMCERSVIPDADYNTMVSNVCHCDFHPGNFKYLDNG EVCGSFDYDMAKIDSRLFELGLAIHYCFSSWLSDTNGIINLGRASLFVK TYDEELHKAGGIEPLTAIEKKYLYEVTIQGALYDLGWCSSACVYDSTLD PYEYLFYTQHFVACLKWLEANEEAFRKAFK BUME_08570 (amino acid sequence) MIKVRVPATTANIGPGFDAFGMAFQLYNIFSFEERDNGKLTIRGVERRY QGKSNLVYKAMLKVFNRVHYRPKGIYIYTDVNIPVSRGLGSSAACIVGG LVGANTLCGAPLTGKELFDMAVEMEGHPDNVAPAMFGGLVVSLGLKEEN HYIKKEVSQCFEFYGLIPDFTLSTMEARKALPKKVFHKDAVFNVSRATM TYLALTEGRPDILKVSVEDKLHQPYREGLIAHYDEVSQKARELGALNTC ISGAGPTLLAITTRDNDQFYAEMGKYLKEKLPGWTLLKLEPDNTGVCTD QHS

[0114] One or more of these genes are subjected to mutagenesis, such as chemically-induced random mutagenesis or error-prone PCR amplification, and then screened for reduced inhibition by methionine.

Example 5Reduced Inhibition of Anthranilate Synthase

[0115] Butyribacterium methylotrophicum has one annotated anthranilate synthase consisting of two components: trpEG (BUME_17910-BUME_17900).

TABLE-US-00004 BUME_17910 (amino acid sequence) MKIPSLETVQRQSEGFAAFPVSCEIFADIKTPIQVLKILKSISSRCYLLE SVEGVEKWGRYSFLGFDPVVEVKCKDGLMEIKNGTAVRLETDDPGQEIRR ILSEYRSPKVAELPPFTGGFVGYFSYDYLKYSEPSLRFDGDDSAGFNDLD LMLFEKVIAFDQLRQKIVIMVTIKTDHLAVNYNRAVRELDYLVGLIHSDV PASGQLPRLLSDFKPHFDREAYCEIVKKTKGYIREGDIFQAVPSNRLTAE MEGSLFNTYRVLRTINPSPYMYYIACDDLEIAGASPETLVKLQDGELSTF PIAGTSPRGRTSEEDAELEERLLKDPKELAEHNMLVDLGRNDLGRVSRYG SVKVQEYLKVQKYSHVMHITSVVTGQLRENMDQLDAVTAVLPAGTLSGAP KIRACEIINELEGIRRGIYGGAIGYIDFTGNMDVCIAIRTAVKKDGRVYV QSGGGVVADSDPEKEYQESINKAMAVVEAVKQSVEVMD BUME_17900 (amino acid sequence) MILIIDNYDSFSYNLVQQVGRVNPDLKVIRNDALSVDEIEALHPSHIILS PGPGRPADAGVCEAVVRRFSGKLPVLGVCLGHQAICEAFGAEVTYASELI HGKRSSIHIANGSPLFKGLPPIMDAARYHSLAVSRSSLPDELLIIAEDNA DEVMGVKHRDFDVYGLQFHPESILTPQGNIIIENFLALGGKIQ

[0116] One or more of these genes are subjected to mutagenesis, such as chemically-induced random mutagenesis or error-prone PCR amplification, and then screened for reduced inhibition by tryptophan

Example 6Expression of Exogenous Peptide Sequences

[0117] In order to modify the protein content or amino acid profile of Butyribacterium methylotrophicum exogenous peptide sequences are expressed to change the composition of the prepared biomass. Examples of exogenous peptide sequences are:

Glb1

[0118]

TABLE-US-00005 Glb1 MVSARIVVLLAVLLCAAAAVASSWEDDNHHHHGGHKSGRCVRRCEDRPWH QRPRCLEQCREEEREKRQERSRHEADDRSGEGSSEDEREREQEKEEKQKD RRPYVFDRRSFRRVVRSEQGSLRVLRPFDEVSRLLRGIRDYRVAVLEANP RSFVVPSHTDAHCIGYVAEGEGVVTTIENGERRSYTIKQGHVFVAPAGAV TYLANTDGRKKLVITKILHTISVPGEFQFFFGPGGRNPESFLSSFSKSIQ RAAYKTSSDRLERLFGRHGQDKGIIVRATEEQTRELRRHASEGGHGPHWP LPPFGESRGPYSLLDQRPSIANQHGQLYEADARSFHDLAEHDVSVSFANI TAGSMSAPLYNTRSFKIAYVPNGKGYAEIVCPHRQSQGGESERERGKGRR SEEEEESSEEQEEVGQGYHTIRARLSPGTAFVVPAGHPFVAVASRDSNLQ IVCFEVHADRNEKVFLAGADNVLQKLDRVAKALSFASKAEEVDEVLGSRR EKGFLPGPKESGGHEEREQEEEEREERHGGRGERERHGREEREKEEEERE GRHGRGRREEVAETLLRMVTARM Glb3 MAKIAAAAAAALCFAALVAVAVCQGEVERQRLRDLQCWQEVQESPLDACR QVLDRQLTGGGGGGGVGPFRWGTGLRMRCCQQLQDVSRECRCAAIRSMVR GYEEAMPPLEKGWWPWGRQQQPPPQGGGGGQGGYYYPCSRPGEGYGYGQG GQRQMYPPCRPGTTGGGPRIGRVRLTKAREYAAGLPMMCRLSEPQECSIF SGGDQY Oat seed globulin MATTRFPSLLFYSCIFLLCNGSMAQLFGQSFTPWQSSRQGGLRGCKFDRL QAFEPLRQVRSQAGITEYFDEQNEQFRCAGVSVIRRVIEPQGLLLPQYHN APGLVYILQGRGFTGLTFPGCPATFQQQFQQFDQARFAQGQSKSQNLKDE HQRVHHIKQGDVVALPAGIVHWCYNDGDAPIVAVYVFDVNNNANQLEPRQ KEFLLAGNNKREQQFGQNIFSGFSVQLLSEALGISQQAAQKIQSQNDQRG EIIRVSQGLQFLKPFVSQQGPVEHQAYQPIQSQQEQSTQYQVGQSPQYQE GQSTQYQSGQSWDQSFNGLEENFCSLEARQNIENPKRADTYNPRAGRITH LNSKNFPTLNLVQMSATRVNLYQNAILSPYWNINAHSVMHMIQGRARVQV VNNHGQTVFNDILRRGQLLIIPQHYVVLKKAEREGCQYISFKTTPNSMVS YIAGKTSILRALPVDVLANAYRISRQESQNLKNNRGEEFGAFTPKFAQTG SQSYQDEGESSSTEKASE Sesame 11S globulin MVAFKFLLALSLSLLVSAAIAQTREPRLTQGQQCRFQRISGAQPSLRIQS EGGTTELWDERQEQFQCAGIVAMRSTIRPNGLSLPNYHPSPRLVYIERGQ GLISIMVPGCAETYQVHRSQRTMERTEASEQQDRGSVRDLHQKVHRLRQG DIVAIPSGAAHWCYNDGSEDLVAVSINDVNHLSNQLDQKFRAFYLAGGVP RSGEQEQQARQTFHNIFRAFDAELLSEAFNVPQETIRRMQSEEEERGLIV MARERMTFVRPDEEEGEQEHRGRQLDNGLEETFCTMKFRTNVESRREADI FSRQAGRVHVVDRNKLPILKYMDLSAEKGNLYSNALVSPDWSMTGHTIVY VTRGDAQVQVVDHNGQALMNDRVNQGEMFVVPQYYTSTARAGNNGFEWVA FKTTGSPMRSPLAGYTSVIRAMPLQVITNSYQISPNQAQALKMNRGSQSF LLSPGGRRS

Example 7Expression of Astaxanthin in Butyribacterium Methylotrophicum

[0119] Butyribacterium methylotrophicum do not natively produce astaxanthin but can produce lycopene, a key intermediate to astaxanthin. In order to enable B. methylotrophicum to produce astaxanthin from lycopene, three genes are needed: crtY, crtW, and crtZ.

[0120] A synthetic operon of these three genes is constructed with a constitutively active transcriptional promoter and then integrated into the chromosome of B. methylotrophicum or expressed from a replicating plasmid. Expression of these three genes allows astaxanthin to be produced.

[0121] Examples of the crtY, crtW, and crtZ genes are given.

crtY

TABLE-US-00006 crtY ATGAACGGGCGCAGGGCAGATCTGGCGATTGTTGGCGGCGGCCTGTCGGG CGGCCTGATCGCGCTGGCGCTGAGGAACGCGCGGCCCGAGCTCGACGTCA GGCTGATAGAAGCGGGCGAGAGGCTGGGCGGCAATCACCGCTGGAGCTGG TTCGAGAACGATCTCGGCAAAAGCGGCAACGAACTCATGCAGCCGTTTCG CAAGACCGAATGGCAGGGCTACGATGTTTATTTCCCGAAATATTCCCGCC GTCTGAAATCTCGCTACTATTCTCTGGCATCGCCCGATTTCGACGCCGGA TTGCGGCGGGAATTGGCGCAAGACACTGTTCATACCGGCAGGAAAGTGGT GGAATGCACGCCTGACAGTGTCACGCTGGAAGGGGGAGACGCCATACCGG CCCGTGCCGTGGTGGATTGCCGAGGGTTCGAGCCGAGCGAGCTCGTGCGT GGCGGCTGGCAGGTCTTCATGGGCCGCCACCTGCGCACCCATGCCCCGCA CGGCATTACCCGACCGGTCATCATGGACGTGACCGTAGAACAGCTCGACG GCTACCGCTTCGTTTACGTCCTCCCGCTCGGCGCAAGCGACATCTTCATC GAGGACACCTATTACAATTCCAACCCCGAGCTGGATCGCGGCGCGCTGTC GAGCCGGCTCGACCGATATTGCCGCCAGAACGGGTGGGAAGGCGACATCG TCGGCAGCGAGACCGGGGTCCTGCCGGTCATTACCGGCGGCGATTTCGCT GCCTATCGCCGCGAGCGCGAGCTCGACCGGATGCCGCAGGCGGGTGCCCG CGCGCTGCTGGCCCACCCGCTGACCAGCTACACCCTGCCGCAGGCGGTCG AAACCGCGCGCCTGATCGCGCGCAACGCGGACCTGCCTGGCGACCAGCTG GCCGCGCTGCTCGCCGCCCATGCCCAGCGGCACTGGAATCGCACGAGCTA TTACCGCCTATTGGGCAGGATACTTTTCGAAGCCCCGCAGCCTAAGGAAC GCTATCGAATCTTCGAGCGTTTCTACACACTCGACGAGGACTTGATCGAG CGGTTCTATGCCGCGCGTTCGCGCCCGCAGGAAAAGGCCCGCGTTTTGTG GGGCAATCCGCCGGTCGCGATCCACCGCGCCATCGCGGCGATCTTCAGCA AGAGTGCGCCGCTGGTGGTGCCGGACAGAACGAAAGGTCGCGTGTCGACA TGA crtW ATGTCTGACGGTCCTGCCTTTTCGTTGCCTGCGCGCTCGCGTCAGCAGGC GATCGGGTTGACGCTCGCCGTGCTGATCGCTGGCGCGTGGCTCGGCATCC ACGCCTATGCGATGTTCGTTTTCGAGCTGAGTTGGCAGAACCTGCCCTTC GCACTTCTGCTAGCAACGGTGCAAACGTGGCTGTCGGTTGGCGTCTTCAT CGTTAGTCATGACGCAATGCACGGCTCGCTGGCACCCGGCAGGCCGACGC TGAATTCGGCGATCGGCGCGTTTCTCTTGGCCCTTTACGCAGGCTTCGGC TGGCGACGAATGCGTGACGCGCATTTTACCCACCACAAGCTGGCGGGCCA TGCGGGAGACCCGGATTTCGACGAGCACAATCCGCGCAGTTTCTGGCGCT GGTACGGAACCTTCTTCCGGCGCTATTTCAGCTGGCAATCGATCCTGTTC GTGCATGTCGTTGTCGGCATCTACTGGCTAGTCCTCGACATTCCGATGGT GCAGATCGTCCTGCTCTACGGCGCACCGGCACTGCTTTCATCGCTGCAGC TTTTCTACTTCGGGACCTTTCGTCCACATCGGCGTTCCGAAGAGGGTTTT GCCGACCGGCACAACGCGCGCAGCGATGGCTTCGGCACGTTGGCCAGCCT CGCCACGTGCTTCCATTTCGGCTATCATCTCGAACATCATCGGCGTCCCG ACGTGCCGTGGTGGGCCTTGCCCGGCGCGCGAGAAGCGGGGATCGGAATG GAAGCGCAAAACGCATGA crtZ ATGAGCTGGTGGGCAATCGCCCTGATCGTATTCGGTGCCGTGGTCGGCAT GGAGTTTTTTGCCTGGTTTGCCCACAAATACATCATGCACGGCTGGGGCT GGTCCTGGCATCGCGACCATCATGAGCCGCACGACAACACACTGGAAAAG AACGATCTTTTCGCGGTCGTGTTCGGATCCGTAGCGGCGCTGCTTTTTGT TATAGGCGCCCTGTGGTCCGATCCTTTGTGGTGGGCTGCGGTCGGGATCA CGCTTTACGGCGTGATTTATACGCTGGTGCACGACGGGCTGGTCCATCAG CGCTACTGGCGCTGGACGCCCAAGCGCGGTTACGCGAAGCGGCTGGTCCA GGCCCACCGGCTGCATCATGCCACGGTAGGCAAGGAGGGCGGGGTGAGTT TCGGCTTCGTTTTCGCGCGCGATCCGGCAAAACTGAAGGCTGAACTCAAG CAGCAGCGCGAACAGGGTCTCGCGGTCGTGCGCGACAGCATGGGCGCCTG A

Example 8Expression of Omega-3 Fatty Acids in Butyribacterium Methylotrophicum

[0122] Butyribacterium methylotrophicum do not natively produce omega-3 fatty acids but can produce oleic acid from its native fatty acid biosynthesis. Eicosapentaenoic acid (EPA), an important omega-3 fatty acid, can be produced from oleic acid with expression of four genes pfaABCD, and then docosahexaenoic acid (DHA), another important omega-3 fatty acid, can be produced from EPA with an additional gene pfaE. In order to enable B. methylotrophicum to produce omega-3 fatty acids, these five genes are needed.

[0123] A synthetic operon of these five genes is constructed with a constitutively active transcriptional promoter and then integrated into the chromosome of B. methylotrophicum or expressed from a replicating plasmid. Expression of these five genes allows EPA and DHA to be produced.

Examples of the pfaA, pfaB, pfaC, pfaD, and pfaE genes are given.

TABLE-US-00007 pfaA ATGAGCCAGACCTCTAAACCTACAAACTCAGCAACTGAGCAAGCACAAGA CTCACAAGCTGACTCTCGTTTAAATAAACGACTAAAAGATATGCCAATTG CTATTGTTGGCATGGCGAGTATTTTTGCAAACTCTCGCTATTTGAATAAG TTTTGGGACTTAATCAGCGAAAAAATTGATGCGATTACTGAATTACCATC AACTCACTGGCAGCCTGAAGAATATTACGACGCAGATAAAACCGCAGCAG ACAAAAGCTACTGTAAACGTGGTGGCTTTTTGCCAGATGTAGACTTCAAC CCAATGGAGTTTGGCCTGCCGCCAAACATTTTGGAACTGACCGATTCATC GCAACTATTATCACTCATCGTTGCTAAAGAAGTGTTGGCTGATGCTAACT TACCTGAGAATTACGACCGCGATAAAATTGGTATCACCTTAGGTGTCGGC GGTGGTCAAAAAATTAGCCACAGCCTAACAGCGCGTCTGCAATACCCAGT ATTGAAGAAAGTATTCGCCAATAGCGGCATTAGTGACACCGACAGCGAAA TGCTTATCAAGAAATTCCAAGACCAATATGTACACTGGGAAGAAAACTCG TTCCCAGGTTCACTTGGTAACGTTATTGCGGGCCGTATCGCCAACCGCTT CGATTTTGGCGGCATGAACTGTGTGGTTGATGCTGCCTGTGCTGGATCAC TTGCTGCTATGCGTATGGCGCTAACAGAGCTAACTGAAGGTCGCTCTGAA ATGATGATCACCGGTGGTGTGTGTACTGATAACTCACCCTCTATGTATAT GAGCTTTTCAAAAACGCCCGCCTTTACCACTAACGAAACCATTCAGCCAT TTGATATCGACTCAAAAGGCATGATGATTGGTGAAGGTATTGGCATGGTG GCGCTAAAGCGTCTTGAAGATGCAGAGCGCGATGGCGACCGCATTTACTC TGTAATTAAAGGTGTGGGTGCATCATCTGACGGTAAGTTTAAATCAATCT ATGCCCCTCGCCCATCAGGCCAAGCTAAAGCACTTAACCGTGCCTATGAT GACGCAGGTTTTGCGCCGCATACCTTAGGTCTAATTGAAGCTCACGGAAC AGGTACTGCAGCAGGTGACGCGGCAGAGTTTGCCGGCCTTTGCTCAGTAT TTGCTGAAGGCAACGATACCAAGCAACACATTGCGCTAGGTTCAGTTAAA TCACAAATTGGTCATACTAAATCAACTGCAGGTACAGCAGGTTTAATTAA AGCTGCTCTTGCTTTGCATCACAAGGTACTGCCGCCGACCATTAACGTTA GTCAGCCAAGCCCTAAACTTGATATCGAAAACTCACCGTTTTATCTAAAC ACTGAGACTCGTCCATGGTTACCACGTGTTGATGGTACGCCGCGCCGCGC GGGTATTAGCTCATTTGGTTTTGGTGGCACTAACTTCCATTTTGTACTAG AAGAGTACAACCAAGAACACAGCCGTACTGATAGCGAAAAAGCTAAGTAT CGTCAACGCCAAGTGGCGCAAAGCTTCCTTGTTAGCGCAAGCGATAAAGC ATCGCTAATTAACGAGTTAAACGTACTAGCAGCATCTGCAAGCCAAGCTG AGTTTATCCTCAAAGATGCAGCAGCAAACTATGGCGTACGTGAGCTTGAT AAAAATGCACCACGGATCGGTTTAGTTGCAAACACAGCTGAAGAGTTAGC AGGCCTAATTAAGCAAGCACTTGCCAAACTAGCAGCTAGCGATGATAACG CATGGCAGCTACCTGGTGGCACTAGCTACCGCGCCGCTGCAGTAGAAGGT AAAGTTGCCGCACTGTTTGCTGGCCAAGGTTCACAATATCTCAATATGGG CCGTGACCTTACTTGTTATTACCCAGAGATGCGTCAGCAATTTGTAACTG CAGATAAAGTATTTGCCGCAAATGATAAAACGCCGTTATCGCAAACTCTG TATCCAAAGCCTGTATTTAATAAAGATGAATTAAAGGCTCAAGAAGCCAT TTTGACCAATACCGCCAATGCCCAAAGCGCAATTGGTGCGATTTCAATGG GTCAATACGATTTGTTTACTGCGGCTGGCTTTAATGCCGACATGGTTGCA GGCCATAGCTTTGGTGAGCTAAGTGCACTGTGTGCTGCAGGTGTTATTTC AGCTGATGACTACTACAAGCTGGCTTTTGCTCGTGGTGAGGCTATGGCAA CAAAAGCACCGGCTAAAGACGGCGTTGAAGCAGATGCAGGAGCAATGTTT GCAATCATAACCAAGAGTGCTGCAGACCTTGAAACCGTTGAAGCCACCAT CGCTAAATTTGATGGGGTGAAAGTCGCTAACTATAACGCGCCAACGCAAT CAGTAATTGCAGGCCCAACAGCAACTACCGCTGATGCGGCTAAAGCGCTA ACTGAGCTTGGTTACAAAGCGATTAACCTGCCAGTATCAGGTGCATTCCA CACTGAACTTGTTGGTCACGCTCAAGCGCCATTTGCTAAAGCGATTGACG CAGCCAAATTTACTAAAACAAGCCGAGCACTTTACTCAAATGCAACTGGC GGACTTTATGAAAGCACTGCTGCAAAGATTAAAGCCTCGTTTAAGAAACA TATGCTTCAATCAGTGCGCTTTACTAGCCAGCTAGAAGCCATGTACAACG ACGGCGCCCGTGTATTTGTTGAATTTGGTCCAAAGAACATCTTACAAAAA TTAGTTCAAGGCACGCTTGTCAACACTGAAAATGAAGTTTGCACTATCTC TATCAACCCTAATCCTAAAGTTGATAGTGATCTGCAGCTTAAGCAAGCAG CAATGCAGCTAGCGGTTACTGGTGTGGTACTCAGTGAAATTGACCCATAC CAAGCCGATATTGCCGCACCAGCGAAAAAGTCGCCAATGAGCATTTCGCT TAATGCTGCTAACCATATCAGCAAAGCAACTCGCGCTAAGATGGCCAAGT CTTTAGAGACAGGTATCGTCACCTCGCAAATAGAACATGTTATTGAAGAA AAAATCGTTGAAGTTGAGAAACTGGTTGAAGTCGAAAAGATCGTCGAAAA AGTGGTTGAAGTAGAGAAAGTTGTTGAGGTTGAAGCTCCTGTTAATTCAG TGCAAGCCAATGCAATTCAAACCCGTTCAGTTGTCGCTCCAGTAATAGAG AACCAAGTCGTGTCTAAAAACAGTAAGCCAGCAGTCCAGAGCATTAGTGG TGATGCACTCAGCAACTTTTTTGCTGCACAGCAGCAAACCGCACAGTTGC ATCAGCAGTTCTTAGCTATTCCGCAGCAATATGGTGAGACGTTCACTACG CTGATGACCGAGCAAGCTAAACTGGCAAGTTCTGGTGTTGCAATTCCAGA GAGTCTGCAACGCTCAATGGAGCAATTCCACCAACTACAAGCGCAAACAC TACAAAGCCACACCCAGTTCCTTGAGATGCAAGCGGGTAGCAACATTGCA GCGTTAAACCTACTCAATAGCAGCCAAGCAACTTACGCTCCAGCCATTCA CAATGAAGCGATTCAAAGCCAAGTGGTTCAAAGCCAAACTGCAGTCCAGC CAGTAATTTCAACACAAGTTAACCATGTGTCAGAGCAGCCAACTCAAGCT CCAGCTCCAAAAGCGCAGCCAGCACCTGTGACAACTGCAGTTCAAACTGC TCCGGCACAAGTTGTTCGTCAAGCCGCACCAGTTCAAGCCGCTATTGAAC CGATTAATACAAGTGTTGCGACTACAACGCCTTCAGCCTTCAGCGCCGAA ACAGCCCTGAGCGCAACAAAAGTCCAAGCCACTATGCTTGAAGTGGTTGC TGAGAAAACCGGTTACCCAACTGAAATGCTAGAGCTTGAAATGGATATGG AAGCCGATTTAGGCATCGATTCTATCAAGCGTGTAGAAATTCTTGGCACA GTACAAGATGAGCTACCGGGTCTACCTGAGCTTAGCCCTGAAGATCTAGC TGAGTGTCGAACGCTAGGCGAAATCGTTGACTATATGGGCAGTAAACTGC CGGCTGAAGGCTCTATGAATTCTCAGCTGTCTACAGGTTCCGCAGCTGCG ACTCCTGCAGCGAATGGTCTTTCTGCGGAGAAAGTTCAAGCGACTATGAT GTCTGTGGTTGCCGAAAAGACTGGCTACCCAACTGAAATGCTAGAGCTTG AAATGGATATGGAAGCCGATTTAGGCATAGATTCTATCAAGCGCGTTGAA ATTCTTGGCACAGTACAAGATGAGCTACCGGGTCTACCTGAGCTTAGCCC TGAAGATCTAGCTGAGTGTCGTACTCTAGGCGAAATCGTTGACTATATGA ACTCTAAACTCGCTGACGGCTCTAAGCTGCCGGCTGAAGGCTCTATGAAT TCTCAGCTGTCTACAAGTGCCGCAGCTGCGACTCCTGCAGCGAATGGTCT CTCTGCGGAGAAAGTTCAAGCGACTATGATGTCTGTGGTTGCCGAAAAGA CTGGCTACCCAACTGAAATGCTAGAACTTGAAATGGATATGGAAGCTGAC CTTGGCATCGATTCAATCAAGCGCGTTGAAATTCTTGGCACAGTACAAGA TGAGCTACCGGGTTTACCTGAGCTAAATCCAGAAGATTTGGCAGAGTGTC GTACTCTTGGCGAAATCGTGACTTATATGAACTCTAAACTCGCTGACGGC TCTAAGCTGCCAGCTGAAGGCTCTATGCACTATCAGCTGTCTACAAGTAC CGCTGCTGCGACTCCTGTAGCGAATGGTCTCTCTGCAGAAAAAGTTCAAG CGACCATGATGTCTGTAGTTGCAGATAAAACTGGCTACCCAACTGAAATG CTTGAACTTGAAATGGATATGGAAGCCGATTTAGGTATCGATTCTATCAA GCGCGTTGAAATTCTTGGCACAGTACAAGATGAGCTACCGGGTTTACCTG AGCTAAATCCAGAAGATCTAGCAGAGTGTCGCACCCTAGGCGAAATCGTT GACTATATGGGCAGTAAACTGCCGGCTGAAGGCTCTGCTAATACAAGTGC CGCTGCGTCTCTTAATGTTAGTGCCGTTGCGGCGCCTCAAGCTGCTGCGA CTCCTGTATCGAACGGTCTCTCTGCAGAGAAAGTGCAAAGCACTATGATG TCAGTAGTTGCAGAAAAGACCGGCTACCCAACTGAAATGCTAGAACTTGG CATGGATATGGAAGCCGATTTAGGTATCGACTCAATTAAACGCGTTGAGA TTCTTGGCACAGTACAAGATGAGCTACCGGGTCTACCAGAGCTTAATCCT GAAGATTTAGCTGAGTGCCGTACGCTGGGCGAAATCGTTGACTATATGAA CTCTAAGCTGGCTGACGGCTCTAAGCTTCCAGCTGAAGGCTCTGCTAATA CAAGTGCCACTGCTGCGACTCCTGCAGTGAATGGTCTTTCTGCTGACAAG GTACAGGCGACTATGATGTCTGTAGTTGCTGAAAAGACCGGCTACCCAAC TGAAATGCTAGAACTTGGCATGGATATGGAAGCAGACCTTGGTATTGATT CTATTAAGCGCGTTGAAATTCTTGGCACAGTACAAGATGAGCTCCCAGGT TTACCTGAGCTTAATCCTGAAGATCTCGCTGAGTGCCGCACGCTTGGCGA AATCGTTAGCTATATGAACTCTCAACTGGCTGATGGCTCTAAACTTTCTA CAAGTGCGGCTGAAGGCTCTGCTGATACAAGTGCTGCAAATGCTGCAAAG CCGGCAGCAATTTCGGCAGAACCAAGTGTTGAGCTTCCTCCTCATAGCGA GGTAGCGCTAAAAAAGCTTAATGCGGCGAACAAGCTAGAAAATTGTTTCG CCGCAGACGCAAGTGTTGTGATTAACGATGATGGTCACAACGCAGGCGTT TTAGCTGAGAAACTTATTAAACAAGGCCTAAAAGTAGCCGTTGTGCGTTT ACCGAAAGGTCAGCCTCAATCGCCACTTTCAAGCGATGTTGCTAGCTTTG AGCTTGCCTCAAGCCAAGAATCTGAGCTTGAAGCCAGTATCACTGCAGTT ATCGCGCAGATTGAAACTCAGGTTGGCGCTATTGGTGGCTTTATTCACTT GCAACCAGAAGCGAATACAGAAGAGCAAACGGCAGTAAACCTAGATGCGC AAAGTTTTACTCACGTTAGCAATGCGTTCTTGTGGGCCAAATTATTGCAA CCAAAGCTCGTTGCTGGAGCAGATGCGCGTCGCTGTTTTGTAACAGTAAG CCGTATCGACGGTGGCTTTGGTTACCTAAATACTGACGCCCTAAAAGATG CTGAGCTAAACCAAGCAGCATTAGCTGGTTTAACTAAAACCTTAAGCCAT GAATGGCCACAAGTGTTCTGTCGCGCGCTAGATATTGCAACAGATGTTGA TGCAACCCATCTTGCTGATGCAATCACCAGTGAACTATTTGATAGCCAAG CTCAGCTACCTGAAGTGGGCTTAAGCTTAATTGATGGCAAAGTTAACCGC GTAACTCTAGTTGCTGCTGAAGCTGCAGATAAAACAGCAAAAGCAGAGCT TAACAGCACAGATAAAATCTTAGTGACTGGTGGGGCAAAAGGGGTGACAT TTGAATGTGCACTGGCATTAGCATCTCGCAGCCAGTCTCACTTTATCTTA GCTGGGCGCAGTGAATTACAAGCTTTACCAAGCTGGGCTGAGGGTAAGCA AACTAGCGAGCTAAAATCAGCTGCAATCGCACATATTATTTCTACTGGTC AAAAGCCAACGCCTAAGCAAGTTGAAGCCGCTGTGTGGCCAGTGCAAAGC AGCATTGAAATTAATGCCGCCCTAGCCGCCTTTAACAAAGTTGGCGCCTC AGCTGAATACGTCAGCATGGATGTTACCGATAGCGCCGCAATCACAGCAG CACTTAATGGTCGCTCAAATGAGATCACCGGTCTTATTCATGGCGCAGGT GTACTAGCCGACAAGCATATTCAAGACAAGACTCTTGCTGAACTTGCTAA AGTTTATGGCACTAAAGTCAACGGCCTAAAAGCGCTGCTCGCGGCACTTG AGCCAAGCAAAATTAAATTACTTGCTATGTTCTCATCTGCAGCAGGTTTT TACGGTAATATCGGCCAAAGCGATTACGCGATGTCGAACGATATTCTTAA CAAGGCAGCGCTGCAGTTCACCGCTCGCAACCCACAAGCTAAAGTCATGA GCTTTAACTGGGGTCCTTGGGATGGCGGCATGGTTAACCCAGCGCTTAAA AAGATGTTTACCGAGCGTGGTGTGTACGTTATTCCACTAAAAGCAGGTGC AGAGCTATTTGCCACTCAGCTATTGGCTGAAACTGGCGTGCAGTTGCTCA TTGGTACGTCAATGCAAGGTGGCAGCGACACTAAAGCAACTGAGACTGCT TCTGTAAAAAAGCTTAATGCGGGTGAGGTGCTAAGTGCATCGCATCCGCG TGCTGGTGCACAAAAAACACCACTACAAGCTGTCACTGCAACGCGTCTGT TAACCCCAAGTGCCATGGTCTTCATTGAAGATCACCGCATTGGCGGTAAC AGTGTGTTGCCAACGGTATGCGCCATCGACTGGATGCGTGAAGCGGCAAG CGACATGCTTGGCGCTCAAGTTAAGGTACTTGATTACAAGCTATTAAAAG GCATTGTATTTGAGACTGATGAGCCGCAAGAGTTAACACTTGAGCTAACG CCAGACGATTCAGACGAAGCTACGCTACAAGCATTAATCAGCTGTAATGG GCGTCCGCAATACAAGGCGACGCTTATCAGTGATAATGCCGATATTAAGC AACTTAACAAGCAGTTTGATTTAAGCGCTAAGGCGATTACCACAGCAAAA GAGCTTTATAGCAACGGCACCTTGTTCCACGGTCCGCGTCTACAAGGGAT CCAATCTGTAGTGCAGTTCGATGATCAAGGCTTAATTGCTAAAGTCGCTC TGCCTAAGGTTGAACTTAGCGATTGTGGTGAGTTCTTGCCGCAAACCCAC ATGGGTGGCAGTCAACCTTTTGCTGAGGACTTGCTATTACAAGCTATGCT GGTTTGGGCTCGCCTTAAAACTGGCTCGGCAAGTTTGCCATCAAGCATTG GTGAGTTTACCTCATACCAACCAATGGCCTTTGGTGAAACTGGTACCATA GAGCTTGAAGTGATTAAGCACAACAAACGCTCACTTGAAGCGAATGTTGC GCTATATCGTGACAACGGCGAGTTAAGTGCCATGTTTAAGTCAGCTAAAA TCACCATTAGCAAAAGCTTAAATTCAGCATTTTTACCTGCTGTCTTAGCA AACGACAGTGAGGCGAATTAG pfaB GTGGAACAAACGCCTAAAGCTAGTGCGATGCCGCTGCGCATCGCACTTAT CTTACTGCCAACACCGCAGTTTGAAGTTAACTCTGTCGACCAGTCAGTAT TAGCCAGCTATCAAACACTGCAGCCTGAGCTAAATGCCCTGCTTAATAGT GCGCCGACACCTGAAATGCTCAGCATCACTATCTCAGATGATAGCGATGC AAACAGCTTTGAGTCGCAGCTAAATGCTGCGACCAACGCAATTAACAATG GCTATATCGTCAAGCTTGCTACGGCAACTCACGCTTTGTTAATGCTGCCT GCATTAAAAGCGGCGCAAATGCGGATCCATCCTCATGCGCAGCTTGCCGC TATGCAGCAAGCTAAATCGACGCCAATGAGTCAAGTATCTGGTGAGCTAA AGCTTGGCGCTAATGCGCTAAGCCTAGCTCAGACTAATGCGCTGTCTCAT GCTTTAAGCCAAGCCAAGCGTAACTTAACTGATGTCAGCGTGAATGAGTG TTTTGAGAACCTCAAAAGTGAACAGCAGTTCACAGAGGTTTATTCGCTTA TTCAGCAACTTGCTAGCCGCACCCATGTGAGAAAAGAGGTTAATCAAGGT GTGGAACTTGGCCCTAAACAAGCCAAAAGCCACTATTGGTTTAGCGAATT TCACCAAAACCGTGTTGCTGCCATCAACTTTATTAATGGCCAACAAGCAA CCAGCTATGTGCTTACTCAAGGTTCAGGATTGTTAGCTGCGAAATCAATG CTAAACCAGCAAAGATTAATGTTTATCTTGCCGGGTAACAGTCAGCAACA AATAACCGCATCAATAACTCAGTTAATGCAGCAATTAGAGCGTTTGCAGG TAACTGAGGTTAATGAGCTTTCTCTAGAATGCCAACTAGAGCTGCTCAGC ATAATGTATGACAACTTAGTCAACGCAGACAAACTCACTACTCGCGATAG TAAGCCCGCTTATCAGGCTGTGATTCAAGCAAGCTCTGTTAGCGCTGCAA AGCAAGAGTTAAGCGCGCTTAACGATGCACTCACAGCGCTGTTTGCTGAG CAAACAAACGCCACATCAACGAATAAAGGCTTAATCCAATACAAAACACC GGCGGGCAGTTACTTAACCCTAACACCGCTTGGCAGCAACAATGACAACG CCCAAGCGGGTCTTGCTTTTGTCTATCCGGGTGTGGGAACGGTTTACGCC GATATGCTTAATGAGCTGCATCAGTACTTCCCTGCGCTTTACGCCAAACT TGAGCGTGAAGGCGATTTAAAGGCGATGCTACAAGCAGAAGATATCTATC ATCTTGACCCTAAACATGCTGCCCAAATGAGCTTAGGTGACTTAGCCATT GCTGGCGTGGGGAGCAGCTACCTGTTAACTCAGCTGCTCACCGATGAGTT TAATATTAAGCCTAATTTTGCATTAGGTTACTCAATGGGTGAAGCATCAA TGTGGGCAAGCTTAGGCGTATGGCAAAACCCGCATGCGCTGATCAGCAAA ACCCAAACCGACCCGCTATTTACTTCTGCTATTTCCGGCAAATTGACCGC GGTTAGACAAGCTTGGCAGCTTGATGATACCGCAGCGGAAATCCAGTGGA ATAGCTTTGTGGTTAGAAGTGAAGCAGCGCCGATTGAAGCCTTGCTAAAA GATTACCCACACGCTTACCTCGCGATTATTCAAGGGGATACCTGCGTAAT CGCTGGCTGTGAAATCCAATGTAAAGCGCTACTTGCAGCACTGGGTAAAC GCGGTATTGCAGCTAATCGTGTAACGGCGATGCATACGCAGCCTGCGATG CAAGAGCATCAAAATGTGATGGATTTTTATCTGCAACCGTTAAAAGCAGA GCTTCCTAGTGAAATAAGCTTTATCAGCGCCGCTGATTTAACTGCCAAGC AAACGGTGAGTGAGCAAGCACTTAGCAGCCAAGTCGTTGCTCAGTCTATT GCCGACACCTTCTGCCAAACCTTGGACTTTACCGCGCTAGTACATCACGC CCAACATCAAGGCGCTAAGCTGTTTGTTGAAATTGGCGCGGATAGACAAA ACTGCACCTTGATAGACAAGATTGTTAAACAAGATGGTGCCAGCAGTGTA CAACATCAACCTTGTTGCACAGTGCCTATGAACGCAAAAGGTAGCCAAGA TATTACCAGCGTGATTAAAGCGCTTGGCCAATTAATTAGCCATCAGGTGC CATTATCGGTGCAACCATTTATTGATGGACTCAAGCGCGAGCTAACACTT TGCCAATTGACCAGCCAACAGCTGGCAGCACATGCAAATGTTGACAGCAA GTTTGAGTCTAACCAAGACCATTTACTTCAAGGGGAAGTCTAA pfaC ATGTCATTACCAGACAATGCTTCTAACCACCTTTCTGCCAACCAGAAAGG CGCATCTCAGGCAAGTAAAACCAGTAAGCAAAGCAAAATCGCCATTGTCG GTTTAGCCACTCTGTATCCAGACGCTAAAACCCCGCAAGAATTTTGGCAG AATTTGCTGGATAAACGCGACTCTCGCAGCACCTTAACTAACGAAAAACT CGGCGCTAACAGCCAAGATTATCAAGGTGTGCAAGGCCAATCTGACCGTT TTTATTGTAATAAAGGCGGCTACATTGAGAACTTCAGCTTTAATGCTGCA GGCTACAAATTGCCGGAGCAAAGCTTAAATGGCTTGGACGACAGCTTCCT TTGGGCGCTCGATACTAGCCGTAACGCACTAATTGATGCTGGTATTGATA TCAACGGCGCTGATTTAAGCCGCGCAGGTGTAGTCATGGGCGCGCTGTCG TTCCCAACTACCCGCTCAAACGATCTGTTTTTGCCAATTTATCACAGCGC CGTTGAAAAAGCCCTGCAAGATAAACTAGGCGTAAAGGCATTTAAGCTAA GCCCAACTAATGCTCATACCGCTCGCGCGGCAAATGAGAGCAGCCTAAAT GCAGCCAATGGTGCCATTGCCCATAACAGCTCAAAAGTGGTGGCCGATGC ACTTGGCCTTGGCGGCGCACAACTAAGCCTAGATGCTGCCTGTGCTAGTT CGGTTTACTCATTAAAGCTTGCCTGCGATTACCTAAGCACTGGCAAAGCC GATATCATGCTAGCAGGCGCAGTATCTGGCGCGGATCCTTTCTTTATTAA TATGGGATTCTCAATCTTCCACGCCTACCCAGACCATGGTATCTCAGTAC CGTTTGATGCCAGCAGTAAAGGTTTGTTTGCTGGCGAAGGCGCTGGCGTA TTAGTGCTTAAACGTCTTGAAGATGCCGAGCGCGACAATGACAAAATCTA TGCGGTTGTTAGCGGCGTAGGTCTATCAAACGACGGTAAAGGCCAGTTTG TATTAAGCCCTAATCCAAAAGGTCAGGTGAAGGCCTTTGAACGTGCTTAT GCTGCCAGTGACATTGAGCCAAAAGACATTGAAGTGATTGAGTGCCACGC AACAGGCACACCGCTTGGCGATAAAATTGAGCTCACTTCAATGGAAACCT TCTTTGAAGACAAGCTGCAAGGCACCGATGCACCGTTAATTGGCTCAGCT AAGTCTAACTTAGGCCACCTATTAACTGCAGCGCATGCGGGGATCATGAA GATGATCTTCGCCATGAAAGAAGGTTACCTGCCGCCAAGTATCAATATTA GTGATGCTATCGCTTCGCCGAAAAAACTCTTCGGTAAACCAACCCTGCCT AGCATGGTTCAAGGCTGGCCAGATAAGCCATCGAATAATCATTTTGGTGT AAGAACCCGTCACGCAGGCGTATCGGTATTTGGCTTTGGTGGCTGTAACG CCCATCTGTTGCTTGAGTCATACAACGGCAAAGGAACAGTAAAGGCAGAA GCCACTCAAGTACCGCGTCAAGCTGAGCCGCTAAAAGTGGTTGGCCTTGC CTCGCACTTTGGGCCTCTTAGCAGCATTAATGCACTCAACAATGCTGTGA CCCAAGATGGGAATGGCTTTATCGAACTGCCGAAAAAGCGCTGGAAAGGC CTTGAAAAGCACAGTGAACTGTTAGCTGAATTTGGCTTAGCATCTGCGCC AAAAGGTGCTTATGTTGATAACTTCGAGCTGGACTTTTTACGCTTTAAAC TGCCGCCAAACGAAGATGACCGTTTGATCTCACAGCAGCTAATGCTAATG CGAGTAACAGACGAAGCCATTCGTGATGCCAAGCTTGAGCCGGGGCAAAA AGTAGCTGTATTAGTGGCAATGGAAACTGAGCTTGAACTGCATCAGTTCC GCGGCCGGGTTAACTTGCATACTCAATTAGCGCAAAGTCTTGCCGCCATG GGCGTGAGTTTATCAACGGATGAATACCAAGCGCTTGAAGCCATCGCCAT GGACAGCGTGCTTGATGCTGCCAAGCTCAATCAGTACACCAGCTTTATTG GTAATATTATGGCGTCACGCGTGGCGTCACTATGGGACTTTAATGGCCCA GCCTTCACTATTTCAGCAGCAGAGCAATCTGTGAGCCGCTGTATCGATGT GGCGCAAAACCTCATCATGGAGGATAACCTAGATGCGGTGGTGATTGCAG CGGTCGATCTCTCTGGTAGCTTTGAGCAAGTCATTCTTAAAAATGCCATT GCACCTGTAGCCATTGAGCCAAACCTCGAAGCAAGCCTTAATCCAACATC AGCAAGCTGGAATGTCGGTGAAGGTGCTGGCGCGGTCGTGCTTGTTAAAA ATGAAGCTACATCGGGCTGCTCATACGGCCAAATTGATGCACTTGGCTTT GCTAAAACTGCCGAAACAGCGTTGGCTACCGACAAGCTACTGAGCCAAAC TGCCACAGACTTTAATAAGGTTAAAGTGATTGAAACTATGGCAGCGCCTG CTAGCCAAATTCAATTAGCGCCAATAGTTAGCTCTCAAGTGACTCACACT GCTGCAGAGCAGCGTGTTGGTCACTGCTTTGCTGCAGCGGGTATGGCAAG CCTATTACACGGCTTACTTAACTTAAATACTGTAGCCCAAACCAATAAAG CCAATTGCGCGCTTATCAACAATATCAGTGAAAACCAATTATCACAGCTG TTGATTAGCCAAACAGCGAGCGAACAACAAGCATTAACCGCGCGTTTAAG CAATGAGCTTAAATCCGATGCTAAACACCAACTGGTTAAGCAAGTCACCT TAGGTGGCCGTGATATCTACCAGCATATTGTTGATACACCGCTTGCAAGC CTTGAAAGCATTACTCAGAAATTGGCGCAAGCGACAGCATCGACAGTGGT CAACCAAGTTAAACCTATTAAGGCCGCTGGCTCAGTCGAAATGGCTAACT CATTCGAAACGGAAAGCTCAGCAGAGCCACAAATAACAATTGCAGCACAA CAGACTGCAAACATTGGCGTCACCGCTCAGGCAACCAAACGTGAATTAGG TACCCCACCAATGACAACAAATACCATTGCTAATACAGCAAATAATTTAG ACAAGACTCTTGAGACTGTTGCTGGCAATACTGTTGCTAGCAAGGTTGGC TCTGGCGACATAGTCAATTTTCAACAGAACCAACAATTGGCTCAACAAGC TCACCTCGCCTTTCTTGAAAGCCGCAGTGCGGGTATGAAGGTGGCTGATG CTTTATTGAAGCAACAGCTAGCTCAAGTAACAGGCCAAACTATCGATAAT CAGGCCCTCGATACTCAAGCCGTCGATACTCAAACAAGCGAGAATGTAGC GATTGCCGCAGAATCACCAGTTCAAGTTACAACACCTGTTCAAGTTACAA CACCTGTTCAAATCAGTGTTGTGGAGTTAAAACCAGATCACGCTAATGTG CCACCATACACGCCGCCAGTGCCTGCATTAAAGCCGTGTATCTGGAACTA TGCCGATTTAGTTGAGTACGCAGAAGGCGATATCGCCAAGGTATTTGGCA GTGATTATGCCATTATCGACAGCTACTCGCGCCGCGTACGTCTACCGACC ACTGACTACCTGTTGGTATCGCGCGTGACCAAACTTGATGCGACCATCAA TCAATTTAAGCCATGCTCAATGACCACTGAGTACGACATCCCTGTTGATG CGCCGTACTTAGTAGACGGACAAATCCCTTGGGCGGTAGCAGTAGAATCA GGCCAATGTGACTTGATGCTTATTAGCTATCTCGGTATCGACTTTGAGAA CAAAGGCGAGCGGGTTTATCGACTACTCGATTGTACCCTCACCTTCCTAG GCGACTTGCCACGTGGCGGAGATACCCTACGTTACGACATTAAGATCAAT AACTATGCTCGCAACGGCGACACCCTGCTGTTCTTCTTCTCGTATGAGTG TTTTGTTGGCGACAAGATGATCCTCAAGATGGATGGCGGCTGCGCTGGCT TCTTCACTGATGAAGAGCTTGCCGACGGTAAAGGCGTGATTCGCACAGAA GAAGAGATTAAAGCTCGCAGCCTAGTGCAAAAGCAACGCTTTAATCCGTT ACTAGATTGTCCTAAAACCCAATTTAGTTATGGTGATATTCATAAGCTAT TAACTGCTGATATTGAGGGTTGTTTTGGCCCAAGCCACAGTGGCGTCCAC CAGCCGTCACTTTGTTTCGCATCTGAAAAATTCTTGATGATTGAACAAGT CAGCAAGGTTGATCGCACTGGCGGTACTTGGGGACTTGGCTTAATTGAGG GTCATAAGCAGCTTGAAGCAGACCACTGGTACTTCCCATGTCATTTCAAG GGCGACCAAGTGATGGCTGGCTCGCTAATGGCTGAAGGTTGTGGCCAGTT ATTGCAGTTCTATATGCTGCACCTTGGTATGCATACCCAAACTAAAAATG GTCGTTTCCAACCTCTTGAAAACGCCTCACAGCAAGTACGCTGTCGCGGT CAAGTGCTGCCACAATCAGGCGTGCTAACTTACCGTATGGAAGTGACTGA AATCGGTTTCAGTCCACGCCCATATGCTAAAGCTAACATCGATATCTTGC TTAATGGCAAAGCGGTAGTGGATTTCCAAAACCTAGGGGTGATGATAAAA GAGGAAGATGAGTGTACTCGTTATCCACTTTTGACTGAATCAACAACGGC TAGCACTGCACAAGTAAACGCTCAAACAAGTGCGAAAAAGGTATACAAGC CAGCATCAGTCAATGCGCCATTAATGGCACAAATTCCTGATCTGACTAAA GAGCCAAACAAGGGCGTTATTCCGATTTCCCATGTTGAAGCACCAATTAC GCCAGACTACCCGAACCGTGTACCTGATACAGTGCCATTCACGCCGTATC ACATGTTTGAGTTTGCTACAGGCAATATCGAAAACTGTTTCGGGCCAGAG TTCTCAATCTATCGCGGCATGATCCCACCACGTACACCATGCGGTGACTT ACAAGTGACCACACGTGTGATTGAAGTTAACGGTAAGCGTGGCGACTTTA AAAAGCCATCATCGTGTATCGCTGAATATGAAGTGCCTGCAGATGCGTGG TATTTCGATAAAAACAGCCACGGCGCAGTGATGCCATATTCAATTTTAAT GGAGATCTCACTGCAACCTAACGGCTTTATCTCAGGTTACATGGGCACAA CCCTAGGCTTCCCTGGCCTTGAGCTGTTCTTCCGTAACTTAGACGGTAGC GGTGAGTTACTACGTGAAGTAGATTTACGTGGTAAAACCATCCGTAACGA CTCACGTTTATTATCAACAGTGATGGCCGGCACTAACATCATCCAAAGCT TTAGCTTCGAGCTAAGCACTGACGGTGAGCCTTTCTATCGCGGCACTGCG GTATTTGGCTATTTTAAAGGTGACGCACTTAAAGATCAGCTAGGCCTAGA TAACGGTAAAGTCACTCAGCCATGGCATGTAGCTAACGGCGTTGCTGCAA GCACTAAGGTGAACCTGCTTGATAAGAGCTGCCGTCACTTTAATGCGCCA GCTAACCAGCCACACTATCGTCTAGCCGGTGGTCAGCTGAACTTTATCGA CAGTGTTGAAATTGTTGATAATGGCGGCACCGAAGGTTTAGGTTACTTGT ATGCCGAGCGCACCATTGACCCAAGTGATTGGTTCTTCCAGTTCCACTTC CACCAAGATCCGGTTATGCCAGGCTCCTTAGGTGTTGAAGCAATTATTGA AACCATGCAAGCTTACGCTATTAGTAAAGACTTGGGCGCAGATTTCAAAA ATCCTAAGTTTGGTCAGATTTTATCGAACATCAAGTGGAAGTATCGCGGT CAAATCAATCCGCTGAACAAGCAGATGTCTATGGATGTCAGCATTACTTC AATCAAAGATGAAGACGGTAAGAAAGTCATCACAGGTAATGCCAGCTTGA GTAAAGATGGTCTGCGCATATACGAGGTCTTCGATATAGCTATCAGCATC GAAGAATCTGTATAA pfaD ATGAATCCTACAGCAACTAACGAAATGCTTTCTCCGTGGCCATGGGCTGT GACAGAGTCAAATATCAGTTTTGACGTGCAAGTGATGGAACAACAACTTA AAGATTTTAGCCGGGCATGTTACGTGGTCAATCATGCCGACCACGGCTTT GGTATTGCGCAAACTGCCGATATCGTGACTGAACAAGCGGCAAACAGCAC AGATTTACCTGTTAGTGCTTTTACTCCTGCATTAGGTACCGAAAGCCTAG GCGACAATAATTTCCGCCGCGTTCACGGCGTTAAATACGCTTATTACGCA GGCGCTATGGCAAACGGTATTTCATCTGAAGAGCTAGTGATTGCCCTAGG TCAAGCTGGCATTTTGTGTGGTTCGTTTGGAGCAGCCGGTCTTATTCCAA GTCGCGTTGAAGCGGCAATTAACCGTATTCAAGCAGCGCTGCCAAATGGC CCTTATATGTTTAACCTTATCCATAGTCCTAGCGAGCCAGCATTAGAGCG TGGCAGCGTAGAGCTATTTTTAAAGCATAAGGTACGCACCGTTGAAGCAT CAGCTTTCTTAGGTCTAACACCACAAATCGTCTATTACCGTGCAGCAGGA TTGAGCCGAGACGCACAAGGTAAAGTTGTGGTTGGTAACAAGGTTATCGC TAAAGTAAGTCGCACCGAAGTGGCTGAAAAGTTTATGATGCCAGCGCCCG CAAAAATGCTACAAAAACTAGTTGATGACGGTTCAATTACCGCTGAGCAA ATGGAGCTGGCGCAACTTGTACCTATGGCTGACGACATCACTGCAGAGGC CGATTCAGGTGGCCATACTGATAACCGTCCATTAGTAACATTGCTGCCAA CCATTTTAGCGCTGAAAGAAGAAATTCAAGCTAAATACCAATACGACACT CCTATTCGTGTCGGTTGTGGTGGCGGTGTGGGTACGCCTGATGCAGCGCT GGCAACGTTTAACATGGGCGCGGCGTATATTGTTACCGGCTCTATCAACC AAGCTTGTGTTGAAGCGGGCGCAAGTGATCACACTCGTAAATTACTTGCC ACCACTGAAATGGCCGATGTGACTATGGCACCAGCTGCAGATATGTTCGA GATGGGCGTAAAACTGCAGGTGGTTAAGCGCGGCACGCTATTCCCAATGC GCGCTAACAAGCTATATGAGATCTACACCCGTTACGATTCAATCGAAGCG ATCCCATTAGACGAGCGTGAAAAGCTTGAGAAACAAGTATTCCGCTCAAG CCTAGATGAAATATGGGCAGGTACAGTGGCGCACTTTAACGAGCGCGACC CTAAGCAAATCGAACGCGCAGAGGGTAACCCTAAGCGTAAAATGGCATTG ATTTTCCGTTGGTACTTAGGTCTTTCTAGTCGCTGGTCAAACTCAGGCGA AGTGGGTCGTGAAATGGATTATCAAATTTGGGCTGGCCCTGCTCTCGGTG CATTTAACCAATGGGCAAAAGGCAGTTACTTAGATAACTATCAAGACCGA AATGCCGTCGATTTGGCAAAGCACTTAATGTACGGCGCGGCTTACTTAAA TCGTATTAACTCGCTAACGGCTCAAGGCGTTAAAGTGCCAGCACAGTTAC TTCGCTGGAAGCCAAACCAAAGAATGGCCTAA pfaE ATGGTAAGAGGCTATTTGCGCGCTTTATTGTCACAACATAGTGAAATACG CCCCAATGAATGGCGCTTTGAATATGGCGACAAAGGTAAGCCTAGATTGA GTGATGCGCAATTTGCTCAAACCGGGGTCCACTTTAATGTGAGTCATAGT GGAGATTGGCTATTAGTAGGCATTTGCACTGCTGATAATAAAGGCGCCAG TCAGGCAAGCAAGGAGGAAACTGACTCTGCTAGTATTGAGTTTGGCGTCG ACATTGAGCGTTGCCGTAACAGCACCAATATCCACTCTATTCTTAGTCAT TATTTCTCTGAATCAGAAAAGCGAGCCTTGTTAGCGTTACCAGAGGCCTT GCAGCGAGACCGCTTTTTTGATTTGTGGGCGCTCAAGGAGTCTTACATTA AAGCGAAAGGACTTGGGCTGGCATTATCGCTAAAATCTTTTGCGTTTGAC TTCTCTGCACTGAGCGAAACTTTTCTTGGAGTTAATGCACCTAAAAGCTT GAGCCATTGTGTTGATATTTCCGATGCTATTGCGGATCACAAGGTTGAGC ATCAACTTAATCAGCGACAGGTTTTGTTAAAACAAGATATTGGTCTTGCT TTACTAGAGTCGAGTTCTAATAAGCCTAACGCTGAGCCACAAAAGTCTGG TTTAGGTTTGATTGAGGCTAAAGAACAGCAAATGAACGCTGCTGATAATT GGCATTGTTTACTGGGCCATCTTGATGATAGTTATCGTTTTGCACTGAGT ATTGGTCAGTGTCAGCAAATAAGTATTGCAGCAGAAGAAGTGAATTTTAA AGCTGTTGTTCGAGCTTCAGCTAAGACTAGCTAG