WO2014165763A1 - Microorganisms for the conversion of methane and methanol to higher value chemicals and fuels - Google Patents

Abstract

This invention relates to metabolically engineered microorganism strains in which there is an increased conversion efficiency of methane, methanol and/or formaldehyde to the intermediate acetyl-CoA. Utilization of acetyl-CoA is well known in the art as a starting metabolite for the production of various products.

Description

MICROORGANISMS FOR THE CONVERSION OF METHANE AND METHANOL TO HIGHER VALUE CHEMICALS AND FUELS

FIELD OF THE INVENTION

[0001] This invention relates to metabolically engineered microorganisms, such as bacterial and or fungal strains, in which there is an increased utilization of formaldehyde for the production of the intracellular intermediate acetyl-CoA. Acetyl-CoA can then be used as a substrate for the metabolic production of numerous products including but not limited to acetate, alcohols (ethanol, butanol, hexanol, and longer n-alcohols), fatty acids and there derivatives (fatty acid methyl esters (FAMEs), fatty aldehydes, alkenes, alkanes) and isoprenoids. Further downstream products can be made from such chemical products. Formaldehyde can be metabolically derived from methanol which in turn can be derived from natural gas or methane. Also, genetic modifications may be made to provide one or more chemical products, from methane, methanol and/or formaldehyde.

INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BACKGROUND OF THE INVENTION

[0003] With increasing acceptance that there are large reservoirs of natural gas, and the development of cost effective methods to make these gas reserves available, new technologies that can take advantage of low cost natural gas, particularly methane, and convert these feedstocks into higher value chemical and fuel products have a cost advantage. Interest has increased for developing and improving industrial microbial systems for production of chemicals and fuels using CI carbon feedstocks (1 carbon atom molecules), such as methane, methanol and formaldehyde. Such industrial microbial systems could completely or partially replace the use of petroleum hydrocarbons for production of certain chemicals and or fuels.

[0004] Commercial objectives for microbial fermentation of CI feedstocks include the increase of titer, production rate, and yield of a target chemical product. When the overall yield in a fermentation event is elevated, this may positively affect the production rate, titers and other economic factors, such as capital costs.

[0005] In spite of strong interest to improve microbial fermentation economics by improving yield and/or productivity for certain chemical products, there remains a need to increase net conversion, as may be quantified by yield over various periods of or an entire fermentation production run, in a fermentative microorganism cell to desired target chemical products employing commercially viable fermentation methods. Additionally, un-engineered natural microbial metabolic pathways to assimilate CI carbons are not capable of maximal theoretical conversion efficiencies. Among problems remaining to be solved are how to improve the conversion efficiencies of CI feedstocks into two carbon or larger carbon number products.

SUMMARY OF THE INVENTION

[0006] According to one embodiment, the invention is directed to a method for increasing the conversion efficiency of methane, methanol and/or formaldehyde into acetyl-CoA (a two carbon molecule). Specifically genetically modified micro-organisms are described that have an increased conversion efficiency of CI feedstocks including methane, methanol and formaldehyde into the intracellular intermediate acetyl-CoA. Additional genetic modifications may be added to a microorganism to enable the conversion of acetyl-CoA to other metabolic products, thereby increasing the yield of these products from CI feedstocks. Products that can be derived from acetyl-CoA include but are not limited to acetate, alcohols (ethanol, butanol, hexanol, and longer n-alcohols), fatty acids and there derivatives (fatty acid methyl esters (FAMEs), fatty aldehydes, alkenes, alkanes) and isoprenoids.

[0007] It is known in the art many natural organisms are capable assimilating formaldehyde via hexulose-6- phosphate in the ribulose -monophosphate pathway or uMP pathway via the actions of hexulse-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The RuMP pathway is depicted in Figure 1. It is also known in the art that many natural organisms are capable of converting fructose-6-phosphate to erythrose -4 -phosphate and acetyl -phosphate via part of the Bifidobacterium shunt, via the action of fructose-6-phosphate phosphoketolase. The Bifidobacterium shunt is depicted in Figure 2. In various embodiments of this invention, the increased production of acetyl-CoA from formaldehyde may occur via increased expression of an enzyme having fructose -6 -phosphate phosphoketolase activity in combination with expression of an enzymes having hexulse-6- phosphate synthase and 6-phospho-3-hexuloisomerase activity, combining enzyme activities of both the RuMP pathway and the Bifidobacterium shunt. This new hybrid pathway termed here the Bifi-RuMP pathway, depicted in Figure 3, can offer significantly higher yields of acetyl-CoA dependent products from methanol.

[0008] In various embodiments of this invention, the increased production of acetyl-phosphate from formaldehyde may occur via increased expression of an enzyme having xylulose -5 -phosphate phosphoketolase activity in combination with expression of an enzymes having hexulse-6-phosphate synthase and 6-phospho-3- hexuloisomerase activity, combining enzyme activities of both the RuMP pathway and the Bifidobacterium shunt. This new hybrid pathway termed here the "modified Bifi-RuMP pathway", depicted in Figure 4, can offer significantly higher yields of acetyl-CoA dependent products from methanol. It is appreciated that many enzymes having xylulose -5 -phosphate phosphoketolase will also have fructose-6-phosphate phosphoketolase activity and that as a consequence the Bifi-RuMP and modified Bifi-RuMP pathways are expected to coexist in many cases. A comparison of the natural RuMP pathway with the Bifi-RuMP and modified Bifi-RuMP pathways for acetyl-CoA producton is given in Table 1, and several non-limiting examples enzymes that may be used for these pathways are listed in Table 2.

[0009] In various embodiments of this invention, the increased production of acetyl-CoA from formaldehyde may occur via increased expression of an enzyme having phosphoacetyltransferase activity. Phosphoacetyltransferase can convert acetyl-phosphate generated from the Bifi-RuMP and modified Bifi-RuMP pathways into acetyl-CoA. Several non-limiting example enzymes having phosphoacetyltransferase activity are listed in Table 3.

[0010] In certain embodiments, the cell culture comprises an inhibitor of phosphofructokinase and/or fructose 1,6 bisphosphate aldolase enzymes or said microorganism is genetically modified for reduced or eliminated enzymatic activity in the organism's phosphofructokinase and/or fructose 1,6 bisphosphate aldolase enzymes.

[0011] In various embodiments of this invention, the increased production of acetyl-CoA from formaldehyde may occur via increased expression of an enzyme having phosphoacetyltransferase activity. Phosphoacetyltransferase can convert acetyl -phosphate generated from the Bifi- uMP and modified Bifi-RuMP pathways into acetyl-CoA. Several non-limiting example enzymes having phosphoacetyltransferase activity are listed in Table 3.

[0012] In various embodiments of this invention, the increased production of acetyl-CoA from formaldehyde may occur via formaldehyde that is generated from methanol. This may be accomplished increased expression of an enzyme having methanol dehydrogenase activity. Several non-limiting examples enzymes that have methanol dehydrogenase activity are listed in Table 4.

[0013] In various embodiments of this invention, the increased production of acetyl-CoA from formaldehyde may occur via formaldehyde that is generated from methanol that is turn generated from methane. . This may be accomplished increased expression of an enzyme having methane monooxygenase activity. Several non- limiting examples enzymes that have methane monooxygenase activity are listed in Table 5. Alternatively the enzymes needed for the Bifi-RuMP, modified Bifi-RuMP in addition to methanol dehydrogenase and phosphoacetyltransferase may be expressed in an mico-organism naturally expressing methane monoxygenase activity. These micro-organisms may include methylotrophes such as but not limited to Methylococcus capsulatus, Methylococcus thermphilus, and Methylosinus sporium.

[0014] In various embodiments of this invention, the increased production of acetyl-CoA from formaldehyde may occur via formaldehyde that is generated from methanol that is turn generated from methane. Methanol may be obtained from a chemical conversion of methane and oxygen by methods well known in the art.

[0015] The carbon source according to the invention may be predominantly CI feedstocks, including methane, methanol formaldehyde or other one carbon feedstocks.

[0016] In various embodiments, the increase in conversion efficiency of CI feedstocks to acetyl-CoA is at least 5 percent, at least 10 percent, at least 20 percent, at least 50 percent, at least 75 percent, at least 100 percent, or at least 150 percent above the conversion efficiency in a microorganism that does not comprise the genetic modifications and/or culture system features of the invention.

[0017] In various embodiments, the increased production of a product from acetyl-CoA may occur via increased expression of a production pathway comprising enzymes that are oxygen tolerant.

[0018] In various embodiments, the increased production of a product from acetyl-CoA may occur via increased expression of a production pathway comprising enzymes that utilize the cofactor NADH as a reductant.

[0019] In various embodiments, the increased production of a product from acetyl-CoA may occur via increased expression of a production pathway comprising enzymes that utilize the cofactor NADPH as a reductant.

[0020] In various embodiments, the increased production of a product from acetyl-CoA may occur via increased expression of a production pathway comprising enzymes that utilize both the cofactor NADPH and NADH as a reductant. Alternatively, the increased production of a product from acetyl-CoA may occur via increased expression of a production pathway comprising multiple pathway enzymes of which at least one utilizes NADH as a reductant and one of which uses NADPH as a reductant.

[0021] In various embodiments, the increased production of acetate from acetyl-phosphate may occur via the increased expression of an acetate kinase. A non-limiting example is the acetate kinase from E. coli encoded by the ackA gene. Increased expression of an acetate kinase may optionally be combined with genetic modifications that result decreased activity phosphoacetyltransferase such as that encoded by the pta gene of E. coli.

[0022] In various embodiments, the increased production of ethanol from acetyl-CoA may occur via the increased expression of an oxygen tolerant ethanol dehydrogenase, such as the enzyme from E. coli encoded by the adhE gene with a mutation Glu568Lys as taught by 1. (Dellomonaco et al, AEM. August 2010, Vol. 76, No. 15, p 5067.) and 2. (Holland-Staley et al. JBACs. November 2000, Vol. 182, No. 21, p6049.)

[0023] In various embodiments, the increased production of butyrate from acetyl-CoA may occur via the increased expression of butyrate pathway enzymes including an acetoacetyl-CoA thiolase, crotonase, crotonyl- CoA reductase, butyrate phospho-transferase and butyrate kinase as taught by 1. (Fischer et al, Appl Microbiol Biotechnol. 2010, September, Vol. 88, No. l, p. 265-275). Alternatively, increased butyrate may be accomplished via the increased expression of butyrate pathway enzymes including an acetoacetyl-CoA synthase, crotonase, crotonyl-CoA reductase and butyryl-CoA thioesterase as taught by 2. (PCT/US2012/030209).

[0024] In various embodiments, the increased production of n-butanol from acetyl-CoA may occur via the increased expression of n-butanol pathway enzymes including an acetoacetyl-CoA thiolase, crotonase, crotonyl- CoA reductase, butyryl-CoA reductase and butyraldehyde reductase as taught by: (Atsumi et al, Metabolic Engineering. 2008. November, Vol. 10, No.6, p. 305).

[0025] In various embodiments, the increased production of fatty acids of chain length greater than 4, from acetyl-CoA may occur via the increased expression of a fatty acid synthesis pathway enzymes including an ketoacetyl-CoA synthase, 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA reductase, and a acyl-CoA thioesterase as taught by PCT/US2012/030209.

[0026] In various embodiments, the increased production of fatty acid methyl esters from acetyl-CoA may occur via the increased expression of fatty acid methyl ester synthesis pathway enzymes including an ketoacetyl-CoA synthase, 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA reductase, and a acyl-CoA wax ester synthase as taught by: 1. (PCT/US2012/030209) and 2. (US 20110146142 Al).

[0027] In various embodiments, the increased production of n-hexanol from acetyl-CoA may occur via the increased expression of a fatty acid synthesis pathway enzymes including an ketoacetyl-CoA thiolases, 3- hydroxyacyl-CoA dehydratase, an enoyl-CoA reductase, and a acyl-CoA thioesterase as taught by : ( Dekishima et al. J Am Chem Soc. 2011. August. Vol.133, No. 30, p. 1139).

[0028] In various embodiments, the increased production of n-alcohols of chain length greater than 4, from acetyl-CoA may occur via the increased expression of a fatty acid synthesis pathway enzymes including an ketoacetyl-CoA synthase, 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA reductase, as taught by PCT/US2012/030209 and a fatty acyl-CoA reductase and fatty aldehyde reductase as taught by (Yan-Ning Zheng et al. Microbial Cell Factories. 2012. Vol. 11 :65). [0029] In various embodiments, the increased production of n-alkenes can be accomplished by first producing n-alcohols as described elsewhere followed by the chemical dehydration of the n-alcohol to an n-alkene by catalytic methods well known in the art.

[0030] In various embodiments, the increased production of n-alkanes can be accomplished by first producing fatty acids as described elsewhere followed by the chemical decarboxylation of the n-alcohol to an alkane by catalytic methods well known in the art.

[0031] In various embodiments, the increased production of isoprene from acetyl-CoA may occur via the increased expression of pathway enzymes including an acetoacetyl-CoA thiolase, hydro xymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonte diphosphate decarboxylase, isopentenyl -diphosphate isomerase and isoprene synthase as taught by 1. (US 20120276603 Al).

[0032] In various embodiments, the increased production of a product from acetyl -CoA may occur via both the increased expression of an acetyl-CoA carboxylase enzyme which can convert acetyl-CoA into malonyl-CoA and the increased expression of a production pathway comprising multiple pathway enzymes which can convert malonyl-CoA further to a product.

[0033] In various embodiments, the increased production of a product from malonyl-CoA may occur via both the increased activity of an acetyl -CoA carboxylase enzyme which can caused by mutation of one or more fatty acid synthesis enzymes such as is taught by 1. (PCT/US2012/030209), 2. (PCT/US2011/0222790) and 3. ( UK Patent GB2473755) and the increased expression of a production pathway comprising multiple pathway enzymes which can convert malonyl-CoA further to a product.

[0034] Within the scope of the invention are genetically modified microorganism, wherein the microorganism is capable of producing an acetyl -CoA derived product at a specific rate selected from the rates of greater than 0.05 g/gDCW-hr, 0.08g/gDCW-hr, greater than O. lg/gDCW-hr, greater than 0.13g/gDCW-hr, greater than 0.15g/gDCW-hr, greater than 0.175g/gDCW-hr, greater than 0.2g/gDCW-hr, greater than 0.25g/gDCW-hr, greater than 0.3g/gDCW-hr, greater than 0.35g/gDCW-hr, greater than 0.4g/gDCW-hr, greater than 0.45g/gDCW-hr, or greater than 0.5g/gDCW-hr.

[0035] In various embodiments, the invention includes a culture system comprising a carbon source in an aqueous medium and a genetically modified microorganism according to any one of claims herein, wherein said genetically modified organism is present in an amount selected from greater than 0.05 gDCW/L, 0.1 gDCW/L, greater than 1 gDCW/L, greater than 5 gDCW/L, greater than 10 gDCW/L, greater than 15 gDCW/L or greater than 20 gDCW/L, such as when the volume of the aqueous medium is selected from greater than 5 mL, greater than 100 mL, greater than 0.5L, greater than 1L, greater than 2 L, greater than 10 L, greater than 250 L, greater than 1000L, greater than 10,000L, greater than 50,000 L, greater than 100,000 L or greater than 200,000 L, and such as when the volume of the aqueous medium is greater than 250 L and contained within a steel vessel.

[0036] Variously, the carbon source for such culture systems is selected from methane, methanol and formaldehyde, and combinations thereof, the pH of the aqueous medium is less than 7.5, the culture system is aerated.

BRIEF DESCRIPTION OF THE DRAWINGS [0037] The novel features of the invention are set forth with particularity in the claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0038] FIG. 1 depicts the Ribulose monophosphate ( uMP) formaldehyde assimilation pathway.

[0039] FIG. 2 depicts the Bifidobacterium shunt pathway.

[0040] FIG. 3 depicts the Bifidobacterium shunt- Ribulose monophosphate (Bifi-RuMP) formaldehyde assimilation pathway.

[0041] FIG. 4 depicts the modified Bifidobacterium shunt- Ribulose monophosphate (modified Bifi-RuMP) formaldehyde assimilation pathway.

[0042] FIG. 5 depicts the Bifidobacterium shunt-Ribulose monophosphate (Bifi-RuMP) formaldehyde assimilation pathway lacking phosphofructokinase and fructose- 1,6-bisphosphate aldolase enzyme activities.

[0043] FIG. 6 depicts pathways for several products that can be produced from acetyl-CoA

[0044] Tables also are provided herein and are part of the specification.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention is related to various production methods and/or genetically modified microorganisms that have utility for fermentative production of various chemical products, to methods of making such chemical products that utilize populations of these microorganisms in vessels, and to systems for chemical production that employ these microorganisms and methods. Among the benefits of the present invention is the increased yield of these products from one carbon feedstocks including methane, methanol and formaldehyde.

[0046] Definitions

[0047] As used in the specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "microorganism" includes a single microorganism as well as a plurality of microorganisms; and the like.

[0048] As used herein, "reduced enzymatic activity," "reducing enzymatic activity," and the like is meant to indicate that a microorganism cell's, or an isolated enzyme, exhibits a lower level of activity than that measured in a comparable cell of the same species or its native enzyme. That is, enzymatic conversion of the indicated substrate(s) to indicated product(s) under known standard conditions for that enzyme is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent less than the enzymatic activity for the same biochemical conversion by a native (non-modified) enzyme under a standard specified condition. This term also can include elimination of that enzymatic activity. A cell having reduced enzymatic activity of an enzyme can be identified using any method known in the art. For example, enzyme activity assays can be used to identify cells having reduced enzyme activity. See, for example, Enzyme Nomenclature, Academic Press, Inc., New York 2007.

[0049] The term "heterologous DNA," "heterologous nucleic acid sequence," and the like as used herein refers to a nucleic acid sequence wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host microorganism; (b) the sequence may be naturally found in a given host microorganism, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a heterologous nucleic acid sequence that is recombinantly produced will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid, such as an nonnative promoter driving gene expression.

[0050] The term "heterologous" is intended to include the term "exogenous" as the latter term is generally used in the art. With reference to the host microorganism's genome prior to the introduction of a heterologous nucleic acid sequence, the nucleic acid sequence that codes for the enzyme is heterologous (whether or not the heterologous nucleic acid sequence is introduced into that genome).

[0051] As used herein, the term "gene disruption," or grammatical equivalents thereof (and including "to disrupt enzymatic function," "disruption of enzymatic function," and the like), is intended to mean a genetic modification to a microorganism that renders the encoded gene product as having a reduced polypeptide activity compared with polypeptide activity in or from a microorganism cell not so modified. The genetic modification can be, for example, deletion of the entire gene, deletion or other modification of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product (e.g., enzyme) or by any of various mutation strategies that reduces activity (including to no detectable activity level) the encoded gene product. A disruption may broadly include a deletion of all or part of the nucleic acid sequence encoding the enzyme, and also includes, but is not limited to other types of genetic modifications, e.g., introduction of stop codons, frame shift mutations, introduction or removal of portions of the gene, and introduction of a degradation signal, those genetic modifications affecting mRNA transcription levels and/or stability, and altering the promoter or repressor upstream of the gene encoding the enzyme.

[0052] Bio-production, as used herein, may be aerobic, microaerobic, or anaerobic.

[0053] When the genetic modification of a gene product, i.e., an enzyme, is referred to herein, including the claims, it is understood that the genetic modification is of a nucleic acid sequence, such as or including the gene, that normally encodes the stated gene product, i.e., the enzyme.

[0054] Species and other phylogenic identifications are according to the classification known to a person skilled in the art of microbiology.

[0055] Enzymes are listed here within, with reference to a uniprot identification number, which would be well known to one skilled in the art. The uniprot database can be accessed at http://www.uniprot.org/.

[0056] Where methods and steps described herein indicate certain events occurring in certain order, those of ordinary skill in the art will recognize that the ordering of certain steps may be modified and that such modifications are in accordance with the variations of the invention. Additionally, certain steps may be performed concurrently in a parallel process when possible, as well as performed sequentially.

[0057] Prophetic examples provided herein are meant to be broadly exemplary and not limiting in any way.

[0058] The meaning of abbreviations is as follows: "C" means Celsius or degrees Celsius, as is clear from its usage, DCW means dry cell weight, "s" means second(s), "min" means minute(s), "h," "hr," or "hrs" means hour(s), "psi" means pounds per square inch, "nm" means nanometers, "d" means day(s), "μί" or "uL" or "ul" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "mm" means millimeter(s), "nm" means nanometers, "mM" means millimolar, "μΜ" or "uM" means micromolar, "M" means molar, "mmol" means millimole(s), "μηιοΓ' or "uMol" means micromole(s)", "g" means gram(s), '^g" or "ug" means microgram(s) and "ng" means nanogram(s), "PC " means polymerase chain reaction, "OD" means optical density, "OD₆₀o" means the optical density measured at a photon wavelength of 600 nm, "kDa" means kilodaltons, "g" means the gravitation constant, "bp" means base pair(s), "kbp" means kilobase pair(s), "% w/v" means weight/volume percent, "% v/v" means volume/volume percent, "IPTG" means isopropyl-μ-D-thiogalactopyranoiside, "RBS" means ribosome binding site, "rpm" means revolutions per minute, "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography.

[0059] I. Carbon Sources

[0060] Bio-production media, which is used in the present invention with recombinant microorganisms having a biosynthetic pathway for a product derived from acetyl-CoA, must contain suitable carbon sources or substrates for the intended metabolic pathways. Suitable substrates may include, but are not limited to methane,methanol and formaldehyde. In addition to one and two carbon substrates methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, methanethiol and a variety of other substrates for metabolic activity.

[0061] Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention as a carbon source, common carbon substrates used as carbon sources are glucose, fructose, and sucrose, as well as mixtures of any of these sugars as well as glycerol.

[0062] II. Microorganisms

[0063] Features as described and claimed herein may be provided in a microorganism selected from the listing herein, or another suitable microorganism, that also comprises one or more natural, introduced, or enhanced product bio-production pathways. Thus, in some embodiments the microorganism comprise an endogenous product production pathway (which may, in some such embodiments, be enhanced), whereas in other embodiments the microorganism does not comprise an endogenous product production pathway.

[0064] The examples describe specific modifications and evaluations to certain bacterial and fungal microorganisms. The scope of the invention is not meant to be limited to such species, but to be generally applicable to a wide range of suitable microorganisms.

[0065] More particularly, based on the various criteria described herein, suitable microbial hosts for the bio- production of a chemical product generally may include, but are not limited to the organisms described in the Common Methods Section

[0066] III. Media and Culture Conditions

[0067] In addition to an appropriate carbon source, such as selected from one of the herein -disclosed types, bio- production media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for chemical product bio-production under the present invention.

[0068] Another aspect of the invention regards media and culture conditions that comprise genetically modified microorganisms of the invention and optionally supplements.

[0069] Typically cells are grown at a temperature in the range of about 25° C to about 40° C in an appropriate medium, as well as up to 70° C for thermophilic microorganisms. Suitable growth media are well characterized and known in the art. [0070] Suitable H ranges for the bio -production are between pH 3.0 to pH 10.0, where pH 6.0 to pH 8.0 is a typical pH range for the initial condition. However, the actual culture conditions for a particular embodiment are not meant to be limited by these pH ranges.

[0071] Bio-productions may be performed under aerobic, or microaerobic conditions with or without agitation. [0072] IV. Bio-production Reactors and Systems

[0073] Fermentation systems utilizing methods and/or compositions according to the invention are also within the scope of the invention.

[0074] Any of the recombinant microorganisms as described and/or referred to herein may be introduced into an industrial bio -production system where the microorganisms convert a carbon source into a product in a commercially viable operation. The bio -production system includes the introduction of such a recombinant microorganism into a bioreactor vessel, with a carbon source substrate and bio -production media suitable for growing the recombinant microorganism, and maintaining the bio-production system within a suitable temperature range (and dissolved oxygen concentration range if the reaction is aerobic or microaerobic) for a suitable time to obtain a desired conversion of a portion of the substrate molecules to a selected chemical product. Bio-productions may be performed under aerobic, microaerobic, or anaerobic conditions, with or without agitation. Industrial bio -production systems and their operation are well-known to those skilled in the arts of chemical engineering and bioprocess engineering.

[0075] The following published resources are incorporated by reference herein for their respective teachings to indicate the level of skill in these relevant arts, and as needed to support a disclosure that teaches how to make and use methods of industrial bio -production of chemical product(s) produced under the invention, from sugar sources, and also industrial systems that may be used to achieve such conversion with any of the recombinant microorganisms of the present invention (Biochemical Engineering Fundamentals, 2^nd Ed. J. E. Bailey and D. F. Ollis, McGraw Hill, New York, 1986, entire book for purposes indicated and Chapter 9, pages 533 -657 in particular for biological reactor design; Unit Operations of Chemical Engineering, 5^th Ed., W. L. McCabe et al, McGraw Hill, New York 1993, entire book for purposes indicated, and particularly for process and separation technologies analyses; Equilibrium Staged Separations, P. C. Wankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988, entire book for separation technologies teachings).

[0076] The amount of a product produced in a bio -production media generally can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC), gas chromatography (GC), or GC/Mass Spectroscopy (MS).

[0077] V. Genetic Modifications, Nucleotide Sequences, and Amino Acid Sequences

[0078] Embodiments of the present invention may result from introduction of an expression vector into a host microorganism, wherein the expression vector contains a nucleic acid sequence coding for an enzyme that is, or is not, normally found in a host microorganism.

[0079] The ability to genetically modify a host cell is essential for the production of any genetically modified (recombinant) microorganism. The mode of gene transfer technology may be by electroporation, conjugation, transduction, or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors are tailored to the host organisms based on the nature of antibiotic resistance markers that can function in that host. Also, as disclosed herein, a genetically modified (recombinant) microorganism may comprise modifications other than via plasmid introduction, including modifications to its genomic DNA.

[0080] More generally, nucleic acid constructs can be prepared comprising an isolated polynucleotide encoding a polypeptide having enzyme activity operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a microorganism, such as E. coli, under conditions compatible with the control sequences. The isolated polynucleotide may be manipulated to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well established in the art.

[0081] The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence may contain transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The techniques for modifying and utilizing recombinant DNA promoter sequences are well established in the art.

[0082] For various embodiments of the invention the genetic manipulations may be described to include various genetic manipulations, including those directed to change regulation of, and therefore ultimate activity of, an enzyme or enzymatic activity of an enzyme identified in any of the respective pathways. Such genetic modifications may be directed to transcriptional, translational, and post-translational modifications that result in a change of enzyme activity and/or selectivity under selected and/or identified culture conditions and/or to provision of additional nucleic acid sequences such as to increase copy number and/or mutants of an enzyme related to product production. Specific methodologies and approaches to achieve such genetic modification are well known to one skilled in the art.

[0083] In various embodiments, to function more efficiently, a microorganism may comprise one or more gene deletions. For example, in E. coli, the genes encoding the lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvate-formate lyase (pflB), methylglyoxal synthase (mgsA), and cetate kinase (ackA) may be disrupted, including deleted. Such gene disruptions, including deletions, are not meant to be limiting, and may be implemented in various combinations in various embodiments. Gene deletions may be accomplished by numerous strategies well known in the art, as are methods to incorporate foreign DNA into a host chromosome. .

[0084] For all nucleic acid and amino acid sequences provided herein, it is appreciated that conservatively modified variants of these sequences are included, and are within the scope of the invention in its various embodiments. Functionally equivalent nucleic acid and amino acid sequences (functional variants), which may include conservatively modified variants as well as more extensively varied sequences, which are well within the skill of the person of ordinary skill in the art, and microorganisms comprising these, also are within the scope of various embodiments of the invention, as are methods and systems comprising such sequences and/or microorganisms.

[0085] Accordingly, as described in various sections above, some compositions, methods and systems of the present invention comprise providing a genetically modified microorganism that comprises both a production pathway from acetyl -CoA or acetyl-phosphate to a product of interest and additional modifications that enable the Bifi- uMP pathway to function in that microorganism.

[0086] Aspects of the invention also regard provision of multiple genetic modifications to improve microorganism overall effectiveness in converting a selected carbon source into a selected product. Particular combinations are shown, such as in the Examples, to increase specific productivity, volumetric productivity, titer and yield substantially over more basic combinations of genetic modifications.

[0087] In addition to the above-described genetic modifications, in various embodiments genetic modifications also are provided to increase the pool and availability of the cofactor NADPH and/or NADH which may be consumed in the production of a product from acetyl-CoA.

[0088] More generally, and depending on the particular metabolic pathways of a microorganism selected for genetic modification, any subgroup of genetic modifications may be made to decrease cellular production of fermentation product(s) other than the desired fermentation product, selected from the group consisting of acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene, isobutylene, ethyl acetate, vinyl acetate, other acetates, 1,4-butanediol, 2,3-butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate, 2-OH-isobutryate, 3-OH-butyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate, caprolactam, adipic acid, propanol, isopropanol, fusel alcohols, and 1,2-propanediol, 1,3 -propanediol, formate, fumaric acid, propionic acid, succinic acid, valeric acid, maleic acid and poly-hydro xybutyrate. Gene deletions may be made as disclosed generally herein, and other approaches may also be used to achieve a desired decreased cellular production of selected fermentation products other than the desired products.

[0089] VI. The Bifi-RuMP and Modified Bifi-RuMP Pathways

[0090] In particular the invention describes the construction of a hybrid metabolic pathway for the assimilation of formaldehyde that enables higher yield production of the metabolites acetyl-phosphate and acetyl -CoA. In turn these key intermediates can be used to produce various products by pathways well known in the art. The hybrid pathway combines components of two natural metabolic pathways, first the Ribulose monophosphate pathway or RuMP pathway for formaldehyde assimilation, with the Bifidobacterium shunt for high yield acetyl- phosphate production from six carbon sugar units. This new pathway is termed the Bifidobacterium Shunt - Ribulose Monophosphate pathway or the Bifi-RuMP pathway. The RuMP pathway is capable of assimilating formaldehyde via hexulose-6-phosphate in the ribulose -monophosphate pathway or RuMP pathway via the actions of hexulse-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The RuMP pathway is depicted in Figure 1. The Bifidobacterium shunt is capable of converting fructose-6-phosphate to erythrose-4-phosphate and acetyl-phosphate at high yields via the action of fructose-6-phosphate phosphoketolase. The Bifidobacterium shunt is depicted in Figure 2.

[0091] The Bifi-RuMP pathway relies on the assimilation of formaldehyde into six carbon units via the one carbon molecules via the enzymes hexulse-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The fructose-6-phospate that results can be converted to erythrose -4 -phosphate and acetyl-phosphate by the actions of fructose-6-phosphate phosphoketolase from the Bifidobacterium shunt, allowing for high yield acetyl- phosphate production. While the natural RuMP cycle links the production of acetyl-CoA from formaldehyde with the concomitant production of NADH and carbon dioxide, the Bifi-RuMP Pathway allows for the production of acetyl-CoA from formaldehyde without the need for carbon dioxide and NADH production. See Table 1 for a comparison. This enables a higher yield of product formation using the Bifi-RuMP pathway, as carbon dioxide (yield loss) is not a required product.

[0092] In addition, it is noted that many natural fructose-6-phosphate phosphoketolase enzymes also possess xylulose -5 -phosphate phosphoketolase activity. The effect of this enzyme activity is shown in modified Bifi- RuMP pathway shown in Figure 4. The net effect of this pathway is shown in Table 1. The modified Bifi- RuMP pathway does allow for an increased yield of acetyl-CoA from formaldehyde than the RuMP pathway albeit lower than that achieved by the Bifi-RuMP Pathway.

[0093] The enzymatic steps necessary for the Bifi-RuMP and modified Bifi-RuMP pathways are illustrated in Figures 3 and 4.Nonlimiting candidate enzymes for these steps are given in Table 2.

Table 1: Pathway Summary

Table 2: Non-limiting Candidate Enzymes for use in the Bifi-RuMP and modified Bifi-RuMP pathways

[0094] Importantly the Bifi- uMP and modified Bifi-RuMP pathways generate acetyl-phosphate as an intermediate. Acetyl-phosphate can be used as a precursor for the production of acetate. Alternatively, a phosphoacetyltransferase enzyme can be used to convert the acetyl-phosphate to acetyl-CoA which is the precursor for a much larger number of products. Table 3 gives several non-limiting examples of phosphoacetyltransferase enzymes.

Table 3: Non-limiting Candidate phosphoacetyltransferase Enzymes.

[0095] Formaldehyde that enters the Bifi-RuMP or modified Bifi-RuMP pathways can be obtained directly, from methanol via the actions of a methanol dehydrogenase, several enzymes that can convert methanol to formaldehyde are disclosed in Table 4. In turn, methanol can be obtained from methane either by traditional chemical catalytic steps well known in the art or via enzymatic means such as by the actions of methane mono- oxygenase enzymes, several of which are disclosed in Table 5.

Table 4: Non-limiting Candidate Methanol Dehydrogenase Enzymes.

Table 5: Non-limiting Candidate Methanol Mono-oxygenase Enzymes.

[0096] A microorganism's intracellular acetyl-CoA product may be in turn on converted to a chemical product including acetate,ethanol, fatty acids, longer chain-alcohols , fatty aldehydes, fatty acid methylesters, alkanes and alkenes. One product may be a fatty acid of any chain length from 4 to greater than 18 carbons. This group of chemical products includes: butyrate or butyric acid, hexanoate or hexanoic acid, octanoate or octanoic acid, decanoate or decanoic acid, dodecanoate or dodecanoic acid, myristate or myristic acid, palmitate or palmitic acid, palmitoleate or plamitoleic acid, stearate or stearic acid, and oleate or oleic acid. These fatty acid products may be produced from a fatty acyl-CoA intermediate via the activity of a fatty acyl-CoA thioesterase. Alternatively, these fatty acids may be produced from a fatty acyl-CoA intermediate via concerted activities of a fatty acyl-CoA phosphotransferase first producing a fatty acyl-phosphate and then the action of a fatty acid kinase operating to produce a fatty acid from the fatty acyl-phosphate. [0097] Another chemical product may be a fatty aldehyde of any chain length from 4 to greater than 18 carbons.

This group of chemical products includes: butanal, hexanal, octanal, decanal, octanal, decanal, dodecanal, myristaldehyde, palmitaldehyde, palmitoleic aldehyde, stearaldehyde and oleic aldehyde. These aldehyde products may be produced from a fatty acyl-CoA intermediate via the activity of a fatty acyl-CoA reductase or acyl-CoA reductase. Production strains making fatty acids may also be used to produce fatty aldehydes.

[0098] Another chemical product may be a fatty alcohol of any chain length from 4 to greater than 18 carbons.

This group of chemical products includes: butanol, hexanol, octanol, decanol, dodecanol, C14 fatty alcohol,

C16 fatty alcohol or CI 8 fatty alcohol. These fatty acid products may be produced from a fatty aldehyde via the activity of an aldehyde reductase. Production strains making fatty acids may also be used to produce fatty alcohols by expressing genes encoding enzymes that convert fatty acyl-CoA or free fatty acids to fatty alcohols.

Examples of these enzymes include an alcohol-forming acyl-CoA reductase (EC 1.2.1.-), or a long-chain-fatty- acyl-CoA reductase (EC 1.2.1.50) plus an alcohol dehydrogenase (EC 1.1.1.1), or a combination of an aldehyde dehydrogenase (EC 1.2.1.-) and an alcohol dehydrogenase. A polypeptide with fatty acyl-CoA reductase activity is provided by the fabG gene of Acinetobacter SP. ADP1, accession number YP_047869. A polypeptide with fatty-acyl reductase activity is provided by the FAR-N_SDR_e gene of Bombyx mori, accession number

BAC79425. A polypeptide with aldehyde dehydrogenase is provided by the ALDH gene of GeobaciUus thermodenitrificans NG80-2, accession number YP_001125970. A polypeptide with alcohol dehydrogenase activity is provided by the yqhD gene of E. coli, accession number AP_003562.1. Additional sources of these activities are known to the art and can be combined to generate a production strain that produces fatty alcohols.

[0099] Another chemical product may be an alpha olefin of any chain length from 4 to greater than 18 carbons.

[00100] Another chemical product may be an alkane of any chain length from 4 to greater than 18 carbons.

[00101] Another chemical product may be a diacid of any chain length from 4 to greater than 18 carbons. These fatty acid products may be produced from a fatty acid via omega or terminal oxidation by enzymes known in the art.

[00102] For several of the products discussed, the impact of using the Bifi-RuMP pathway as an alternative to the RuMP pathway on the theoretical maximal yields from methane and methanol are shown in Figure 6. Importantly, it is contemplated that in various embodiments of this invention that the Bifi-RuMP pathway will operate in parallel with the RuMP pathway , which would result in a maximal theoretical yield of a product higher than that resulting from the RuMP pathway alone and lower than that resulting from the Bifi-RuMP pathway alone. The exact yield would depend on the balance of carbon flux between the two pathways.

Table 6: Example Maximal Theoretical Product Yields from CI Feedstocks

Fatty acids Methanol Bifi-RuMP 0.5

FAMEs Methanol RuMP 0.33

FAMEs Methanol Bifi-RuMP >0.5

FAMEs Methane RuMP 0.33

FAMEs Methane Bifi-RuMP >.34

[00103] Any of these may be described herein as a selected chemical product, or a product of interest. Also, any grouping, including any sub-group, of the above listing may be considered what is referred to by "selected product," "product of interest," and the like. For any of these products a microorganism may inherently comprise a biosynthesis pathway to such chemical product and/or may require addition of one or more heterologous nucleic acid sequences to provide or complete such a biosynthesis pathway, in order to achieve a desired production of such chemical product.

[00104] Other additional genetic modifications are disclosed herein for various embodiments. [00105] VII. Disclosed Embodiments Are Non-Limiting

[00106] While various embodiments of the present invention have been shown and described herein, it is emphasized that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may be made without departing from the invention herein in its various embodiments. Specifically, and for whatever reason, for any grouping of compounds, nucleic acid sequences, polypeptides including specific proteins including functional enzymes, metabolic pathway enzymes or intermediates, elements, or other compositions, or concentrations stated or otherwise presented herein in a list, table, or other grouping (such as metabolic pathway enzymes shown in a figure), unless clearly stated otherwise, it is intended that each such grouping provides the basis for and serves to identify various subset embodiments, the subset embodiments in their broadest scope comprising every subset of such grouping by exclusion of one or more members (or subsets) of the respective stated grouping. Moreover, when any range is described herein, unless clearly stated otherwise, that range includes all values therein and all sub-ranges therein.

[00107] Also, and more generally, in accordance with disclosures, discussions, examples and embodiments herein, there may be employed conventional molecular biology, cellular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. (See, e.g., Sambrook and Russell, "Molecular Cloning: A Laboratory Manual," Third Edition 2001 (volumes 1 - 3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed., 1986.) These published resources are incorporated by reference herein for their respective teachings of standard laboratory methods found therein. Such incorporation, at a minimum, is for the specific teaching and/or other purpose that may be noted when citing the reference herein. If a specific teaching and/or other purpose is not so noted, then the published resource is specifically incorporated for the teaching(s) indicated by one or more of the title, abstract, and/or summary of the reference. If no such specifically identified teaching and/or other purpose may be so relevant, then the published resource is incorporated in order to more fully describe the state of the art to which the present invention pertains, and/or to provide such teachings as are generally known to those skilled in the art, as may be applicable. However, it is specifically stated that a citation of a published resource herein shall not be construed as an admission that such is prior art to the present invention. Also, in the event that one or more of the incorporated published resources differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Subject matter in the Examples is incorporated into this section to the extent not already present.

EXAMPLES

[00108] The examples herein provide some examples, not meant to be limiting. All reagents, unless otherwise indicated, are obtained commercially. Species and other phylogenic identifications are according to the classification known to a person skilled in the art of microbiology.

[00109] The names and city addresses of major suppliers are provided herein.

[00110] Example 1 : General example of genetic modification to a host cell .

[00111] In addition to the above or below specific examples, this example is meant to describe a non-limiting approach to genetic modification of a selected microorganism to introduce a nucleic acid sequence of interest. Alternatives and variations are provided within this general example. The methods of this example are conducted to achieve a combination of desired genetic modifications in a selected microorganism species, such as a combination of genetic modifications as described in sections herein, and their functional equivalents, such as in other bacterial and other microorganism species.

[00112] A gene or other nucleic acid sequence segment of interest is identified in a particular species (such as E. coli as described herein) and a nucleic acid sequence comprising that gene or segment is obtained.

[00113] Based on the nucleic acid sequences at the ends of or adjacent the ends of the segment of interest, 5' and 3 ' nucleic acid primers are prepared. Each primer is designed to have a sufficient overlap section that hybridizes with such ends or adjacent regions. Such primers may include enzyme recognition sites for restriction digest of transposase insertion that could be used for subsequent vector incorporation or genomic insertion. These sites are typically designed to be outward of the hybridizing overlap sections. Numerous contract services are known that prepare primer sequences to order (e.g., Integrated DNA Technologies, Coralville, IA USA).

[00114] Once primers are designed and prepared, polymerase chain reaction (PC ) is conducted to specifically amplify the desired segment of interest. This method results in multiple copies of the region of interest separated from the microorganism's genome. The microorganism's DNA, the primers, and a thermophilic polymerase are combined in a buffer solution with potassium and divalent cations (e.g., Mg or Mn) and with sufficient quantities of deoxynucleoside triphosphate molecules. This mixture is exposed to a standard regimen of temperature increases and decreases. However, temperatures, components, concentrations, and cycle times may vary according to the reaction according to length of the sequence to be copied, annealing temperature approximations and other factors known or readily learned through routine experimentation by one skilled in the art.

[00115] In an alternative embodiment the segment of interest may be synthesized, such as by a commercial vendor, and prepared via PCR, rather than obtaining from a microorganism or other natural source of DNA.

[00116] The nucleic acid sequences then are purified and separated, such as on an agarose gel via electrophoresis. Optionally, once the region is purified it can be validated by standard DNA sequencing methodology and may be introduced into a vector. Any of a number of vectors may be used, which generally comprise markers known to those skilled in the art, and standard methodologies are routinely employed for such introduction. Commonly used vector systems are well known in the art. Similarly, the vector then is introduced into any of a number of host cells. Commonly used host cells are E. coli strains. Some of these vectors possess promoters, such as inducible promoters, adjacent the region into which the sequence of interest is inserted (such as into a multiple cloning site). The culturing of such plasmid-laden cells permits plasmid replication and thus replication of the segment of interest, which often corresponds to expression of the segment of interest.

[00117] Various vector systems comprise a selectable marker, such as an expressible gene encoding a protein needed for growth or survival under defined conditions. Common selectable markers contained on backbone vector sequences include genes that encode for one or more proteins required for antibiotic resistance as well as genes required to complement auxotrophic deficiencies or supply critical nutrients not present or available in a particular culture media. Vectors also comprise a replication system suitable for a host cell of interest.

[00118] The plasmids containing the segment of interest can then be isolated by routine methods and are available for introduction into other microorganism host cells of interest. Various methods of introduction are known in the art and can include vector introduction or genomic integration. In various alternative embodiments the DNA segment of interest may be separated from other plasmid DNA if the former will be introduced into a host cell of interest by means other than such plasmid.

[00119] While steps of the general prophetic example involve use of plasmids, other vectors known in the art may be used instead. These include cosmids, viruses (e.g., bacteriophage, animal viruses, plant viruses), and artificial chromosomes (e.g., yeast artificial chromosomes (YAC) and bacteria artificial chromosomes (BAC)).

[00120] Host cells into which the segment of interest is introduced may be evaluated for performance as to a particular enzymatic step, and/or tolerance or bio-production of a chemical compound of interest. Selections of better performing genetically modified host cells may be made, selecting for overall performance, tolerance, or production or accumulation of the chemical of interest.

[00121] It is noted that this procedure may incorporate a nucleic acid sequence for a single gene (or other nucleic acid sequence segment of interest), or multiple genes (under control of separate promoters or a single promoter), and the procedure may be repeated to create the desired heterologous nucleic acid sequences in expression vectors, which are then supplied to a selected microorganism so as to have, for example, a desired complement of enzymatic conversion step functionality for any of the herein-disclosed metabolic pathways. However, it is noted that although many approaches rely on expression via transcription of all or part of the sequence of interest, and then translation of the transcribed mRNA to yield a polypeptide such as an enzyme, certain sequences of interest may exert an effect by means other than such expression.

[00122] The specific laboratory methods used for these approaches are well-known in the art and may be found in various references known to those skilled in the art, such as Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition 2001 (volumes 1 -3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (hereinafter, Sambrook and Russell, 2001).

[00123] As an alternative to the above, other genetic modifications may also be practiced, such as a deletion of a nucleic acid sequence of the host cell's genome. One non-limiting method to achieve this is by use of Red/ET recombination, known to those of ordinary skill in the art and described in U.S. Patent Nos. 6,355,412 and 6,509,156, issued to Stewart et al. and incorporated by reference herein for its teachings of this method. Material and kits for such method are available from Gene Bridges (Gene Bridges GmbH, Dresden, Germany, «www.genebridges.com»), and the method may proceed by following the manufacturer's instructions. Targeted deletion of genomic DNA may be practiced to alter a host cell's metabolism so as to reduce or eliminate production of undesired metabolic products. This may be used in combination with other genetic modifications such as described herein in this general example.

[00124] Example 2: Methanol utilization in E. coli - 1

[00125] This example describes methanol utilization in E. coli by expression of enzymes in the uMP pathway in addition to a methanol dehydrogenase. Briefly, genetically modified E. coli can be constructed using methods well known in the art to have deletions in one or more of the following genes: IdhA, pflB, poxB, pta, and mgsA. A strain with any of these gene deletions, or no gene deletions, may be used for the heterologous expression of enzymes in the RuMP pathway including hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. Importantly, E. coli already expresses transaldolase and transketolase, ribose-5-phosphate isomerase and ribulose-5-phosphate-3-epimerase to some degree. In addition, any of the above strains may be further modified to express methanol dehydrogenase, which can convert methanol to formaldehyde. An example of genes that may be used for overexpression include: the hexulose-6-phosphate synthase gene hps and 6-phospho-3- hexuloisomerase gene rmpB from BrevibaciUus brevis, as well as the methanol dehydrogenase gene mdh and its activator protein (Uniprot # Q8KP10) from Bacillus methanolicus. In addition any of these strains may also express 6-phosphate phosphoketolase such as that encoded by the xfp gene from Bifidobacerium longum or Bifidobacerium animalis lactis. These genetically modified E. coli strains may then be grown aerobically in a minimal salts medium at 37 degrees Celsius with methanol as the sole carbon source.

[00126] Example 3: High Yield Acetate Production from Methanol in E. coli - 1

[00127] An of the above strains including those described in Example 2 may be further genetically modified for increased expression of the acetate kinase gene from E.coli, ackA. In addition, these strains may be further modified to delete genes encoding phosphofructokinase including pfkA and pfkB or fructose-l,6-bisphosphate including fbaA and fbaB. These genetically modified E. coli strains may then be grown aerobically in a minimal salts medium at 37 degrees Celsius with methanol as the sole carbon source for the production of acetate from methanol.

[00128] Example 4: High Yield Ethanol Production from Methanol in ii. coli - 1

[00129] An of the above strains including those described in Example 2 may be further genetically modified for to reduce the expression of the acetate kinase gene from E.coli, ackA. In addition, these strains may be further modified to delete genes encoding phosphofructokinase including pfkA and pfkB or fructose-l,6-bisphosphate including fbaA and fbaB. Any of these modified strains will be further modified to express an oxygen tolerant alcohol dehydrogenase such as that encoded by the mutant adhE gene from E. coli encoding the Glu568Lys mutation. These genetically modified E. coli strains may then be grown aerobically in a minimal salts medium at 37 degrees Celsius with methanol as the sole carbon source for the production of ethanol from methanol.

[00130] Example 5: Acetate Production from Methanol or Methane in Methylococcus capsulatus- 1

[00131] Methylococcus capsulatus strains may be genetically modified for increased expression of the acetate kinase gene from E.coli, ackA. In addition they may also be modified for the heterologous expression of enzymes in the Bifi-RuMP pathway including 6-phosphate phosphoketolase such as that encoded by the xfp gene from Bifidobacerium longum or Bifidobacerium animalis lactis. Importantly, Methylococcus strains already express enymes in the RuMP pathway including: hexulose-6-phosphate synthase, 6-phospho-3- hexuloisomerase, transaldolase and transketolase, ribose-5-phosphate isomerase and ribulose-5-phosphate-3- epimerase. In addition, these strains may be further modified to delete genes encoding phosphofructokinase (gene MCA1521) and fructose- 1,6-bisphosphate ( genes MCA3041 and MCA3047) genes. These genetically modified Methlococcus capsulatus strains may then be grown aerobically in a medium at 45 degrees Celsius with methanol or methane as the sole carbon source for the production of acetate.

[00132] Example 6: Ethanol Production from Methanol or Methane in Methylococcus capsulatus- 1

[00133] Methylococcus capsulatus strains may be genetically modified for increased expression of an oxygen tolerant alcohol dehydrogenase such as that encoded by the mutant adhE gene from E. coli encoding the Glu568Lys mutation. In addition they may also be modified for the heterologous expression of enzymes in the Bifi-RuMP pathway including 6-phosphate phosphoketolase such as that encoded by the xfp gene from Bifidobacerium longum or Bifidobacerium animalis lactis. Importantly, Methylococcus strains already express enymes in the RuMP pathway including: hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, transaldolase and transketolase, ribose-5-phosphate isomerase and ribulose-5-phosphate-3-epimerase. In addition, these strains may be further modified to delete genes encoding phosphofructokinase (gene MCA1521) and fructose- 1,6-bisphosphate ( genes MCA3041 and MCA3047) genes. These genetically modified Methlococcus capsulatus strains may then be grown aerobically in a medium at 45 degrees Celsius with methanol or methane as the sole carbon source for the production of ethanol.

[00134] Example 7: Bifi-RuMP pathway expression and product formation in heterologous host.

[00135] Numerous microbial strains, such as any of the strains listed in the Common Methods Section, may be genetically modified to express all of the enzymes of the Bifi-RuMP or modified Bifi-RuMP pathways by methods well known in the art. Any of the microbial strains may be genetically modified to heterologously express hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, fructose -6 -phosphate phosphoketolase, transaldolase and transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate-3-epimerase enzymatic activities. Any of these strains may be further genetically modified to have decreased phosphofructokinase or fructose-l,6-bisphosphate aldolase enzyme activity. In addition, any of these strains may be further genetically modified to express an enzymatic pathway to produce a product from acetyl -CoA, such as those well known in the art.

[00136] COMMON METHODS SECTION

[00137] All methods in this Section are provided for incorporation into the Examples where so referenced.

[00138] Subsection I. Microorganism Species and Strains, Cultures, and Growth Media

[00139] Microbial species, that may be utilized as needed, are as follows:

[00140] Acinetobacter calcoaceticus (DSMZ # 1139) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended A. calcoaceticus culture are made into BHI and are allowed to grow for aerobically for 48 hours at

37°C at 250 rpm until saturated.

[00141] Bacillus subtilis is a gift from the Gill lab (University of Colorado at Boulder) and is obtained as an actively growing culture. Serial dilutions of the actively growing B. subtilis culture are made into Luria Broth ( PI Corp, Mt. Prospect, IL, USA) and are allowed to grow for aerobically for 24 hours at 37°C at 250 rpm until saturated.

[00142] Chlorobium limicola (DSMZ# 245) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended using Pfennig's Medium I and II (#28 and 29) as described per DSMZ instructions. C. limicola is grown at 25°C under constant vortexing.

[00143] Citrobacter braakii (DSMZ # 30040) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion(BHI) Broth ( RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended C. braakii culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30°C at 250 rpm until saturated.

[00144] Clostridium acetobutylicum (DSMZ # 792) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Clostridium acetobutylicum medium (#411) as described per DSMZ instructions. C. acetobutylicum is grown anaerobically at 37°C at 250 rpm until saturated.

[00145] Clostridium aminobutyricum (DSMZ # 2634) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Clostridium aminobutyricum medium (#286) as described per DSMZ instructions. C. aminobutyricum is grown anaerobically at 37°C at 250 rpm until saturated.

[00146] Clostridium kluyveri (DSMZ #555) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as an actively growing culture. Serial dilutions of C. kluyveri culture are made into Clostridium kluyveri medium (#286) as described per DSMZ instructions. C. kluyveri is grown anaerobically at 37°C at 250 rpm until saturated.

[00147] Cupriavidus metallidurans ( DMSZ # 2839) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth ( RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended C. metallidurans culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30°C at 250 rpm until saturated.

[00148] Cupriavidus necator (DSMZ # 428) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended C. necator culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30°C at 250 rpm until saturated. As noted elsewhere, previous names for this species are Alcaligenes eutrophus and Ralstonia eutrophus.

[00149] Desulfovibrio fructosovorans (DSMZ # 3604) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Desulfovibrio fructosovorans medium (#63) as described per DSMZ instructions. D. fructosovorans is grown anaerobically at 37°C at 250 rpm until saturated.

[00150] Escherichia coli Crooks (DSMZ#1576) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, IL, USA). Serial dilutions of the resuspended E. coli Crooks culture are made into BHI and are allowed to grow for aerobically for 48 hours at 37°C at 250 rpm until saturated.

[00151] Escherichia coli K12 is a gift from the Gill lab (University of Colorado at Boulder) and is obtained as an actively growing culture. Serial dilutions of the actively growing E. coli K12 culture are made into Luria Broth (RPI Corp, Mt. Prospect, IL, USA) and are allowed to grow for aerobically for 24 hours at 37°C at 250 rpm until saturated.

[00152] Halobacterium salinarum (DSMZ# 1576) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Halobacterium medium (#97) as described per DSMZ instructions. H. salinarum is grown aerobically at 37°C at 250 rpm until saturated.

[00153] Lactobacillus delbrueckii (#4335) is obtained from WYEAST USA (Odell, OR, USA) as an actively growing culture. Serial dilutions of the actively growing L. delbrueckii culture are made into Brain Heart Infusion (BHI) broth (RPI Corp, Mt. Prospect, IL, USA) and are allowed to grow for aerobically for 24 hours at 30°C at 250 rpm until saturated.

[00154] Metallosphaera sedula (DSMZ #5348) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as an actively growing culture. Serial dilutions of M. sedula culture are made into Metallosphaera medium (#485) as described per DSMZ instructions. M. sedula is grown aerobically at 65°C at 250 rpm until saturated.

[00155] Methylococcus capsulatus Bath (ATCC # 33009) is obtained from the American Type Culture Collection (ATCC) (Manassas, VA 20108 USA ) as a vacuum dried culture. Cultures are then resuspended in ATCC^®_Medium 1306: Nitrate mineral salts medium (NMS) under a 50% air 50% methane atmosphere (ATCC, Manassas, VA 20108 USA ) and are allowed to grow at 45°C .

[00156] Methylococcus thermophilus IMV 2 Yu T is obtained. Cultures are then resuspended in ATCC^®_Medium 1306: Nitrate mineral salts medium (NMS) under a 50% air 50% methane atmosphere (ATCC, Manassas, VA 20108 USA ) and are allowed to grow at 50°C .

[00157] Methylosinus tsporium (ATCC # 35069) is obtained from the American Type Culture Collection (ATCC) (Manassas, VA 20108 USA ) as a vacuum dried culture. Cultures are then resuspended in ATCC^®_Medium 1306: Nitrate mineral salts medium (NMS) under a 50% air 50% methane atmosphere (ATCC, Manassas, VA 20108 USA ) and are allowed to grow at 30°C .

[00158] Pichia pastoris (Komagataella pastoris) (DSMZ# 70382) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in YPD-medium (#393) as described per DSMZ instructions. Pichia pastoris is grown aerobically at 30°C at 250 rpm until saturated.

[00159] Propionibacterium freudenreichii subsp. shermanii (DSMZ# 4902) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in PYG-medium (#104) as described per DSMZ instructions. P. freudenreichii subsp. shermanii is grown anaerobically at 30°C at 250 rpm until saturated.

[00160] Pseudomonas putida is a gift from the Gill lab (University of Colorado at Boulder) and is obtained as an actively growing culture. Serial dilutions of the actively growing P. putida culture are made into Luria Broth (RPI Corp, Mt. Prospect, IL, USA) and are allowed to grow for aerobically for 24 hours at 37°C at 250 rpm until saturated.

[00161] Streptococcus mutans (DSMZ# 6178) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Luria Broth (RPI Corp, Mt. Prospect, IL, USA). S. mutans is grown aerobically at 37°C at 250 rpm until saturated.

Claims

CLAIMS claimed is:

A genetically modified microorganism capable of converting the one carbon compounds: methane, methanol and formaldehyde to acetyl-CoA at molar yields greater than 0.35 moles acetyl-CoA per mole of a one carbon compound, greater than 0.40 moles of acetyl-CoA per mole of a one carbon compound, greater than 0.45 moles of acetyl-CoA per mole of a one carbon compound, or greater than 0.49 moles of acetyl-CoA per mole of a one carbon compound.

A genetically modified microorganism capable of converting the one carbon compounds: methane, methanol and formaldehyde to a two or more carbon product at molar yields greater than 0.35 moles a product per mole of a one carbon compound, greater than 0.40 moles of product per mole of a one carbon compound, greater than 0.45 moles of product per mole of a one carbon compound, or greater than 0.49 moles of product per mole of a one carbon compound.

A genetically modified microorganism of any of the above claims, wherein the micro-organism is genetically modified to have hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, fructose - 6-phosphate phosphoketolase, transaldolase and transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate-3-epimerase enzymatic activities.

A genetically modified microorganism of any of the above claims, wherein the micro-organism is further genetically modified to have increased expression of phosphoacetyltransferase activity.

A genetically modified microorganism of any of the above claims, wherein the micro-organism is further genetically modified to have decreased phosphofructokinase or fructose-l,6-bisphosphate aldolase enzyme activity.

A genetically modified microorganism of any of the above claims, wherein the micro-organism is additionally genetically modified to produce a product from acetyl-CoA, including: acetate, ethanol, ethylene, n-alcohols, alcohols, alkenes, fatty acids, fatty acid methyl-esters, alkanes, isoprene and others.

A method of making a product from methane, methanol or formaldehyde utilizing any microorganism of any of the above claims.