WO2016048828A1

WO2016048828A1 - Genetically modified microbes for the biological conversion of carbonaceous materials to isobutanol and isoamyl alcohol

Info

Publication number: WO2016048828A1
Application number: PCT/US2015/050953
Authority: WO
Inventors: Marthah Jeanne Margaret DE LORME; Gianguido COFFA
Original assignee: Ciris Energy, Inc.
Priority date: 2014-09-24
Filing date: 2015-09-18
Publication date: 2016-03-31

Abstract

A method for synthesizing isoamyl alcohol and/or isobutanol from a low value carbon source, comprising the steps of oxidizing the low value carbon source with at least one oxidizing agent to produce a modified, partially solubilized low value carbon source; and contacting the modified, partially solubilized low value carbon source with a genetically engineered microorganism adapted for growth in the modified, partially solubilized low value carbon source, which is derived from a wild-type microorganism comprising an isobutanol and/or isoamyl alcohol biosynthesis pathway from pyruvate.

Description

GENETICALLY MODIFIED MICROBES FOR THE BIOLOGICAL CONVERSION OF CARBONACEOUS MATERIALS TO ISOBUTANOL AND ISOAMYL

ALCOHOL

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0001] The present invention relates to biosynthesis of isobutanol or isoamyl alcohol using a microorganism. In particular, the present invention is directed to a biosynthesis process using a genetically engineered microorganism to synthesize isobutanol or isoamyl alcohol from a low value carbon source.

2. Description of the Related Technology

[0002] Isobutanol is an important industrial chemical, useful as a fuel additive, a feedstock chemical in the plastics industry, and a food grade extractant in the food and flavor industry. Isobutanol is a better fuel additive than ethanol, though ethanol is widely used as an additive in gasoline today. Specifically, isobutanol is compatible with gasoline at any ratio and can also be used as a pure fuel in existing combustion engines without modifications to the engines. Unlike ethanol, isobutanol does not absorb water and can thus be stored and distributed using the existing petrochemical infrastructure. Isobutanol has a high energy content that is close to the energy content of gasoline and provides better fuel economy (miles per gallon) than ethanol. Also, isobutanol-gasoline blends have lower vapor pressure than ethanol-gasoline blends, which is important for reducing evaporative hydrocarbon emissions. This may be due to isobutanol' s higher octane number for having a branched carbon chain.

[0003] Isoamyl alcohol is a clear, colorless alcohol, which may be used as a flavoring agent in the food industry. Isoamyl alcohol is also the main ingredient of Kovac's reagent, used for the bacterial diagnostic indole test. Isoamyl alcohol is also used as an antifoaming agent (e.g., as the chloroform:isomyl Alcohol reagent).

[0004] Several industrial methods for chemical synthesis of isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6^th edition, 2003, Wiley- VCHVerlag GmbH and Co., Weinheim, Germany, vol. 5, pages 716-719) and Guerbet condensation of methanol with n-propanol (Carlini et al., "Guerbet Condensation of Methanol with N-Propanol to Isobutanol Alcohol Over Heterogeneous Copper Chromite/Mg-Al Mixed Oxides Catalysts," /. Mol. Catal. A: Chem. , vol. 220, pages 215-220, 2004). These methods use starting materials derived from petrochemicals and are generally expensive and not environmentally friendly.

[0005] Some genetically engineered microorganisms have been developed for producing isobutanol from renewable resources such as sugars or plant waste. U.S. Patent No. 8,017,375 discloses a microorganism with an isobutanol biosynthesis pathway containing at least one exogenous gene. The microorganism may be a yeast with a Crabtree-negative phenotype or a Crabtree-positive phenotype. The recombinant microorganism may also be engineered to disrupt, mutate, or delete an endogenous pyruvate decarboxylase gene such that the recombinant microorganism has reduced endogenous pyruvate decarboxylase activity. The recombinant microorganism produces isobutanol at a yield which is at least 10% of the theoretical yield of isobutanol from glucose. The carbon sources used by the recombinant microorganism include glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof.

[0006] U.S. Patent No. 8,530,226 discloses a yeast cell for producing isobutanol, where the yeast cell has an increased metabolic flow of material from pyruvate to acetolactate, 2,3- dihydroxy isovalerate, to 2-ketoisovalerate, to isobutyraldehyde and finally to isobutanol. The yeast cell has at least one gene that is over-expressed that encodes at least one enzyme involved in these conversion from pyruvate and acetolactate to isobutanol. The over- expressed genes are not heterologous to the yeast cell. The carbon source used by the yeast cell may be monosaccharides, oligosaccharides or polysaccharides. Examples include glucose, fructose, mannose, galactose, saccharose, maltose, xylose and arabinose.

[0007] US 2007/0092957 provides a recombinant microbial host cell comprising at least one DNA molecule that encodes an enzyme catalyzing a reaction in the isobutanol synthesis pathway. The at least one DNA molecule is heterologous to the microbial host cell. The microbial host cell is a member of a genus selected from Clostridium, Zymomonas,

Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. The fermentable carbon substrate used by the microbial host cell in the production of isobutanol is selected from monosaccharides, oligosaccharides, and polysaccharides. Examples include glucose, sucrose, and fructose.

[0008] US 2011/0281314 discloses a recombinant microorganism comprising at least one genetic modification to provide nucleic acids that encode enzymes for catalyzing one or more conversions from pyruvate to isobutanol. The pathway from pyruvate to isobutanol includes the conversion of isobutyryl-CoA to isobutanol, which requires at least an aldehyde dehydrogenase. The carbon source for supporting the growth of the recombinant

microorganism is selected from monosaccharides, oligosaccharides, and polysaccharides. Examples include glucose, sucrose, and fructose. The recombinant microorganism is a member selected from a genus selected from Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

[0009] US 2009/0305369 discloses an enteric production host for producing isobutanol, which comprises genetic modifications in endogenous enzyme activities of pyruvate formate lyase, fumarate reductase, alcohol dehydrogenase, and lactate dehydrogenase. Additionally, the production host is substantially free of the enzymatic activities of at least one of the following enzymes pyruvate formate lyase, fumarate reductase, alcohol dehydrogenase, and lactate dehydrogenase. The production host is selected from Escherichia, Salmonella, Erwinia, Shigella, Kelbsiella, Serratia. The carbon sources for the production host may be monosaccharides, such as glucose or fructose; disaccharides, such as lactose or sucrose; oligosaccharides; polysaccharides, such as starch or cellulose; one carbon substrates; and mixtures thereof.

[00010] Many of these genetically engineered microorganisms for the production of isobutanol rely on using expensive feedstocks, such as glucose, sucrose or starch, which makes the isobutanol biosynthesis process costly and commercially less viable. Thus, there remains a need for an efficient, cost effective isobutanol or isoamyl alcohol biosynthesis method using low value carbon sources by fermentation with a genetically engineered microorganism. The present invention provides one or more genetically engineered microorganisms and processes that produce isobutanol or isoamyl alcohol cost-effectively from a low cost carbon source such as coal, biomass or industrial waste.

SUMMARY OF THE INVENTION

[00011] In one aspect, the present invention provides a method for synthesizing isoamyl alcohol and/or isobutanol from a low value carbon source, comprising the steps of oxidizing the low value carbon source with at least one oxidizing agent to produce a modified, partially solubilized low value carbon source and contacting the modified, partially solubilized low value carbon source with a genetically engineered microorganism adapted for growth in the modified, partially solubilized low value carbon source, which is derived from a wild-type microorganism comprising an isobutanol and/or isoamyl alcohol biosynthesis pathway from pyruvate, wherein the genetically engineered microorganism comprises a genetic

modification selected from: increasing an enzymatic activity of at least one enzyme in the isobutanol and/or isoamyl alcohol biosynthesis pathway relative to the activity of the same enzyme in the wild-type microorganism, and decreasing an enzymatic activity of at least one enzyme in a pathway competitive to the isobutanol and/or isoamyl alcohol biosynthesis pathway relative to the activity of the same enzyme in the wild-type microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

[00012] Figure 1 is a flow chart showing a method for biosynthesizing isobutanol or isoamyl alcohol from a low value carbon source, according to an embodiment of the present invention.

[00013] Figure 2A shows an isobutanol and/or isoamyl alcohol biosynthesis pathway starting from pyruvate and comprising a step (d) that converts a-ketoisovalerate to a- ketoisocaproate, whereby isobutanol may also be produced by this biosynthesis pathway.

[00014] Figure 2B shows step (d) in Figure 2A, which comprises a series steps for producing a-ketoisocaproate and leucine.

[00015] Figures 3A-3H show examples of vectors that can be used to genetically engineer a microorganism.

[00016] Figure 4A is a schematic representation of a gene knock-out process where a gene in the microorganism host genome is deleted.

[00017] Figure 4B is a schematic representation of a gene knock-in process where a fragment in the microorganism host genome is replaced with an exogenous DNA sequence.

[00018] Figure 4C is a schematic representation of a gene knock-in process where an exogenous DNA sequence is inserted into the microorganism host genome.

[00019] Figure 5 represents three promoter/terminator systems that may be used in the present invention.

[00020] Figures 6A-6C represent the structure and sequence of the three

promoter/terminator systems depicted in Figure 5.

[00021] Figure 7 is a schematic representation of a pretreatment method for converting a carbonaceous raw material to a feedstock for microorganisms.

[00022] Figure 8 is a flow chart depicting a pretreatment method for black liquor to obtain a feedstock for microorganisms.

[00023] Figure 9 is a flow chart depicting a pretreatment method for black liquor according to another embodiment, wherein selected organic polymers are recovered from the raw black liquor and only selected components of the black liquor are used to generate one or more organic compounds comprising from about 2 to about 20 carbon atoms, which can be used as a feedstock for microorganisms.

[00024] Figure 10 is a flow chart depicting a pretreatment method for black liquor according to yet another embodiment, wherein only selected components of the black liquor are used in to generate one or more organic compounds comprising from about 2 to about 20 carbon atoms (used as feedstock for microorganisms.

[00025] Figure 11 is a flow chart depicting a pretreatment method for black liquor according to yet another embodiment, wherein selected organic polymers are recovered from the raw black liquor, and only selected components of black liquor are used to generate one or more organic compounds comprising from about 2 to about 20 carbon atoms (used as feedstock for microorganisms).

[00026] Figure 12 shows Rhodococcus bacterial strains grown on petri dishes that were transformed by plasmids pCE7 and pCE8, together with negative controls.

[00027] Figure 13 shows the results of gel electrophoresis for mutant microorganisms prepared in Example 5 using a gene knock-out process.

[00028] Figure 14 shows the results of gel electrophoresis for different microorganism colonies after a second recombination as described in Example 6.

[00029] Figure 15 shows the results of gel electrophoresis for different microorganism colonies after a first recombination as described in Example 7.

[00030] Figure 16 shows an expression vector pCE46 that was used to introduce exogenous genes into Rhodococcus bacterial cells in Example 10.

[00031] Figure 17 shows a representative chromatogram that is produced in Example 10.

DEFINITIONS

[00032] The term "algae concentrate" as used herein refers to an algae paste or an algae cake, which is a residue obtained by separating algae from the medium in which they grow. The medium in which the algae grows is typically water based. The algae concentrate may be able to be processed in a form containing a small amount of residual water. The algae concentrate may be separated from the medium in a variety of ways, for example, by filtration.

[00033] The term "biomass" as used herein refers to virtually any plant-derived organic matter containing cellulose and/or hemicellulose as its primary carbohydrates. Biomass can include, but is not limited to, agricultural residues such as corn stover, wheat straw, rice straw, sugar cane bagasse and the like. Biomass further includes, but is not limited to, woody energy crops, wood wastes and residues such as trees, including fruit trees, such as fruit- bearing trees, (e.g., apple trees, orange trees, and the like), softwood forest thinnings, barky wastes, sawdust, paper and pulp industry waste streams, wood fiber, and the like.

Additionally, perennial grass crops, such as various prairie grasses, including prairie cord grass, switchgrass, Miscanthus, big bluestem, little bluestem, side oats grama, and the like, have potential to be produced large-scale as additional plant biomass sources. For urban areas, potential biomass includes yard waste (e.g., grass clippings, leaves, tree clippings, brush, etc.) and vegetable processing waste. Biomass is known to be the most prevalent form of carbohydrate available in nature and corn stover is currently the largest source of readily available biomass in the United States.

[00034] The term "biosynthetic pathway," also referred to as "metabolic pathway," as used herein refers to is a set of anabolic or catabolic biochemical reactions for transmuting one chemical species into another. Anabolic reactions involve constructing a larger molecule from smaller molecules, a process requiring energy. Catabolic reactions involve breaking down of larger molecules, often releasing energy. Gene products belong to the same

"biosynthetic pathway" if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.

[00035] The term "carbon bearing material" as used herein refers any high carbon-content material that exists in a subterraneous formation. Examples of carbon bearing material include, but not limited to, oil shale, coal, coal seam, waste coal, coal derivatives, lignite, peat, oil formations, tar sands, hydrocarbon-contaminated soil, petroleum sludge, drill cuttings, and the like and may even include those conditions or even surroundings in addition to oil shale, coal, coal seam, waste coal, coal derivatives, lignite, peat, bitumen, oil formations, tar sands, hydrocarbon-contaminated soil, petroleum sludge, drill cuttings, and the like.

[00036] The term "carbon source" as used herein means any source of carbon containing molecules that has at least one carbon atom.

[00037] The term, "low value carbon source" as used herein includes biomass, industrial wastes (such as sewage sludge), carbon bearing materials, or derivatives thereof. Examples of low value carbon sources include coal, lignite, tar sands, tars, peat, pitch, resins, lignin, latex rubber, waxes, agricultural wastes, bark, wood, any type of renewable biomass and other products from trees, algae cake, and other recalcitrant organic matter, and may also include lower-valued by-products from petroleum refining and chemical manufacturing, such as crude oil atmospheric bottoms, crude oil vacuum residues, residua from fluid catalytic cracking, petroleum coke, coker and other thermal cracking gas oils and bottoms, raffinates, asphalts, polynuclear aromatics, and the like, as well as synthetic polymer wastes such as polyethylene, polypropylene, polystyrene, polyesters, polyacrylics, and the like.

[00038] The term "coal" as used herein refers to any of the series of carbonaceous fuels ranging from lignite to anthracite. The members of the series differ from each other in the relative amounts of moisture, volatile matter, and fixed carbon they contain. Coal is comprised mostly of carbon, hydrogen and entrained water, predominantly in the form of large molecules having numerous double carbon bonds. Low rank coal deposits are mostly comprised of coal and water. Energy can be derived from the combustion of carbonaceous molecules, such as coal, or carbonaceous molecules derived from the solubilization of coal molecules. Of the coals, those containing the largest amounts of fixed carbon and the smallest amounts of moisture and volatile matter are the most useful.

[00039] The term "endogenous" to an organism as used herein refers to molecules, and in particular enzymes and polynucleotides, that originate or are found in the organism in nature. It is understood that expression of endogenous enzymes or polynucleotides may be modified in genetically engineered microorganisms.

[00040] The term "enzyme" as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide. The term "enzymatic activity" refers to a function of the enzyme for catalyzing the chemical or biochemical reaction without destruction or modification at the end of the reaction. The enzymes involve in metabolism in a microorganism have the enzymatic activity of converting one metabolite to another.

[00041] The term "exogenous" to a organism as used herein refers molecules, and in particular enzymes and polynucleotides, that are introduced into the organism. The molecule can be introduced into an organism, for example, by integration of a nucleic acid into the host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microorganism.

[00042] The term "expression" of a gene sequence as used herein refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product (protein) encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein.

[00043] The term "fermentation" or "fermentation process" is defined as a process in which a microorganism is cultivated in a culture medium containing a carbon source, wherein the microorganism converts the carbon source into at least one desirable product.

[00044] The term "gene" as used herein refers to a nucleotide sequence of a nucleic acid molecule (chromosome, plasmid, etc.) related to a genetic function. A gene is a genetic unit of an organism including, for example, a polynucleotide sequence occupying a specific physical location ("genetic locus") in the genome of a microorganism. A gene may code for an expression product such as a polypeptide or a polynucleotide. Generally, a gene includes a coding sequence, such as a polypeptide coding sequence, and may also include non-coding sequences, such as a promoter sequence, a polyadenylated sequence, or a transcription control sequence (e.g., an enhancer sequence). Genes of various eukaryotic organisms have "exons" (coding sequences) between which an "intron" (non-coding sequence)" is interposed.

[00045] The terms "genetically engineered" or "genetically engineering" as used herein refers to altering the genetic material (DNA or RNA) existed in a natural microorganism, or introducing exogenous genetic material into a natural microorganism. In one aspect, when an exogenous gene encoding a protein is introduced into a natural microorganism, the gene can be codon-optimized for expression in the microorganism.

[00046] The term "genetically engineered microorganism" or "non-natural microorganism" as used herein refers to a microorganism that has at least one genetic alteration not normally found in a naturally occurring strain of the referenced microorganism species, including wild- type strains of the referenced species. The term "wild-type" refers to the common genotype or phenotype, or genotypes or phenotypes, of a microorganism as it is found in the nature for the given microorganism species. Genetic alterations include, for example, a gene deletion or some other functional disruption of the genetic material. Genetic alterations also include modifications that introduce expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the genetic material in the microorganism. Such modification include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Genetically engineered microorganisms are often derived from wild-type microorganisms by making one or more genetic

modifications to the wild-type microorganism.

[00047] The term, "genetically engineered microorganism" refers not only to the particular genetically engineered microorganism but also to the progenies of such a genetically engineered microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent microorganism, but are still included within the scope of the term as used herein.

[00048] The term "genus" as used herein is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity et al, 2007, TOB A Release 7.7, Michigan State University Board of Trustees).

[00049] The term "homologous recombination" as used herein refers to the process wherein two nucleic acid molecules associate with each other in regions of homology, leading to recombination between those nucleic acid molecules. For purposes of the present invention, homologous recombination is determined according to the procedures summarized by Paques and Haber, Microbiology and Molecular Biology Reviews, vol. 63, pages 349-404, 1999. Homologous recombination can cause reciprocal exchange of a region on the genome of the microorganism and a region on a vector. The term is intended to encompass the reciprocal exchange of any nucleic acid segment, including the reciprocal exchange of a short segment of a gene as well as the exchange of an entire gene.

[00050] The term "knock-in" as used herein refers to a genetic engineering technique that replaces a genetic sequence (e.g. a target gene, a promoter) on the host microorganism genome with a different genetic sequence. Knock-in technique can modify an endogenous gene sequence to create a loss-of-function or gain-of-function mutation.

[00051] The term "knockout" as used herein means a genetic engineering technique that renders deletion of a sequence segment from a microorganism genome. Knock-out often causes inactivation of the product of a target gene. Knock-out can be, for example, deletion of the entire gene, deletion of a regulatory sequence (e.g. promoter) required for transcription or translation, deletion of a portion of the gene with results in a truncated gene product or by any of various mutation strategies that inactivate the encoded gene product. One particularly useful method of gene knock-out is complete gene deletion because it reduces or eliminates the occurrence of genetic reversions in the genetically engineered microorganisms of the invention.

[00052] As used herein, the terms "microorganism," "microbial organism," "microbe," or "microbial" is intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya, including bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The microorganisms may include at least the following types of microorganisms: Thermoto gales, Cytophaga group, Azospirillum group, Paracoccus subgroup, Sphingomonas group,

Nitrosomonas group, Azoarcus group, Acidovorax subgroup, Oxalobacter group,

Rhodococcus group, Thiobacillus group, Xanthomonas group, Oceanospirillum group, Pseudomonas and relatives, Marinobacter hydrocarbonoclaticus group, Pseudoalteromonas group, Vibrio subgroup, Aeromonas group, Desulfovibrio group, Desulfuromonas group, Desulfobulbus assemblage, Campylobacter group, Acidimicrobium group, Frankia subgroup, Arthrobacter and relatives, Nocardiodes subgroup, Thermoanaerobacter and relatives, Bacillus megaterium group, Carnobacterium group, Clostridium and relatives, and archaea such as Archaeo glob ales, Methanobacteriales, Methanomicrobacteria and relatives, Methanopy rales, and Methanococcales.

[00053] Some species of microorganism are selected from Escherichia (and particularly E. coli), Corynebacterium, Clostridium, Zymonomas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klesiella, Paenibacillus, Arthrobacter, Brevibacterium, Pichia, Candida, Hansenula, Synechococcus, Synechocystis, Anabaena, Ralstonia, Lactococcus, or Saccharomyces.

[00054] The term "operably linked" as used herein indicates that nucleic acid sequence elements are arranged to permit the general functions of the elements. Therefore, a certain promoter operably linked to a coding sequence (e.g., a sequence coding for a polypeptide of interest) may enable expression of the coding sequence in the presence of a control protein and a suitable enzyme. In some cases, such a promoter is not necessarily adjacent to the coding sequence as long as the promoter can direct the expression of the coding sequence.

[00055] The term "operon" as used herein refers to two or more genes which are transcribed as a single transcriptional unit from a common promoter. In some embodiments, the genes comprising the operon are contiguous genes. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter. Alternatively, any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide. The modification can result in an increase in the activity of the encoded polypeptide. Further, the modification can impart new activities on the encoded polypeptide. Exemplary new activities include the use of alternative substrates and/or the ability to function in alternative environmental conditions.

[00056] The term "overexpressed," or "overexpression" as used herein refers to a level of expression of a gene in a genetically engineered microorganism which is greater than the level of the gene in the wild type microorganism under the same condition. Overexpression can be achieved by removing repressor of gene expression, or adding activators of gene expression, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like. Overexpression thus results in a greater than natural level of the gene production, or "overproduction" of the target gene product in the microorganism. The term "underexpressed" or "underexpression" as used herein refers to a level of expression of a gene in a genetically engineered microorganism that is lower than the level of the gene in the wild type microorganism under the same condition. Underexpression leads to production of lower level of the gene product.

[00057] A "polypeptide" as used herein is a single linear chain of amino acids bonded together by peptide bonds. A "polypeptide" also includes a polypeptide modified after translation. The terms "protein" herein can encompass a single polypeptide chain or complexes of two or more polypeptide chains. The term "protein" may also include a fragment, an analog, or a derivative of a natural protein.

[00058] The term "pretreatment" as used herein refers to any step, i.e., treatment intended to alter native carbon sources so they can be more efficiently and economically converted to reactive intermediate chemical compounds such as sugars, organic acids, etc., which can then be further processed to an end product such as 3-HP, ethanol, isobutanol, long chain alkanes, etc. Pretreatment can reduce the degree of crystallinity of a polymeric substrate, reduce the interference of lignin with biomass conversion by hydrolyzing some of the structural carbohydrates, thus increasing their enzymatic digestibility and accelerating the degradation of biomass to useful products. Pretreatment methods can utilize hydrothermal treatments including water, heat, steam or pressurized steam pre treatments, including, but not limited to, wet oxidization, hydro-thermolysis pretreatment and liquid hot water pretreatment, further including, for example, acid catalyzed steam explosion pretreatment. Pretreatment can take place in or be deployed in various types of containers, reactors, pipes, flow through cells and the like.

[00059] The term "promoter" as used herein refers to a regulatory DNA sequence which is necessary to effect the expression of coding sequences to which they are operably linked. Alternatively the term "promoter" refers to a region located upstream or downstream from the start of transcription and which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.

[00060] The term "stable transformation" or "stably transformed" as used herein means that a nucleic acid is introduced into a host cell of a microorganism and integrates into the genome of the cell. As such, the integrated nucleic acid is capable of being inherited by the progeny of the microorganism, more particularly, by the progeny of multiple successive generations. Stable transformation as used herein can also refer to introducing a nucleic acid that is maintained extrachromasomally in a microorganism host cell, for example, as a plasmid in a microorganism, yeast artificial chromosome, bacterial phage, all of which can replicate autonomously in the microorganism host cell .

[00061] The term "substrate" as used herein refers to any substance or compound that is converted or meant to be converted into another compound by action of an enzyme. The term includes not only a single compound, but also solutions, mixtures, and other materials, which contain at least one substrate, or derivative thereof. Substrates include suitable carbon sources ordinarily used by microorganisms. Further, the term "substrate" encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass or carbon bearing material, but also intermediate and end product metabolites used in a biosynthesis pathway of a genetically engineered microorganism as described herein.

[00062] As used herein, the term "thermochemical treating" or "thermochemical treatment" refers to a process in which the chemical nature of a low value carbon source comprising carbon-containing compounds is changed by the treatment conditions including application of elevated temperatures. More particularly, a thermochemical treatment can be selected from gasification, pyrolysis, reforming, and partial oxidation.

[00063] The terms "transformation" and "transfection" as used herein refer to the process by which an exogenous nucleic acid molecule is introduced into a microorganism host cell. A "transformed" or "transfected" cell is a host cell into which a nucleic acid molecule has been introduced by, for example, molecular biology techniques. As used herein, the term

"transformation" encompasses all techniques by which a nucleic acid molecule might be introduced into such a host cell including, without limitation, transfection with a viral vector, conjugation, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. When DNA is introduced into the host cell, the nucleic acid may be inserted into a chromosome or replicated as extrachromosomal material (e.g. a plasmid in bacteria).

[00064] The term "vector" as used herein refers to a nucleic acid, which once is introduced into a host cell, can either be recombined with the genome of a host cell or autonomously replicate within the host cell as an episome. Such a vector may be a linear nucleic acid, a plasmid, a cosmid, an RNA vector, or a viral vector.

[00065] The term "yield" is defined as the amount of product obtained per unit weight of carbon source and may be expressed as gram product per gram carbon source (g/g). Yield may also be expressed as a percentage of the theoretical yield. "Theoretical yield" is defined as the maximum amount of product that can be generated per a given amount of carbon source as dictated by the stoichiometry of the metabolic pathway used to make the product.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S

[00066] For illustrative purposes, the principles of the present invention are described by referencing various exemplary embodiments. Although certain embodiments of the invention are specifically described herein, one of ordinary skill in the art will readily recognize that the same principles are equally applicable to, and can be employed in other systems and methods. Before explaining the disclosed embodiments of the present invention in detail, it is to be understood that the invention is not limited in its application to the details of any particular embodiment shown. Additionally, the terminology used herein is for the purpose of description and not of limitation. Furthermore, although certain methods are described with reference to steps that are presented herein in a certain order, in many instances, these steps may be performed in any order as may be appreciated by one skilled in the art; the novel method is therefore not limited to the particular arrangement of steps disclosed herein.

[00067] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. The terms "comprising", "including", "having" and "constructed from" can also be used interchangeably.

[00068] The present invention provides a method of producing isobutanol or isoamyl alcohol using a genetically engineered microorganism from a low value carbon source such as biomass, carbon bearing materials and industrial wastes. Referring to Figure 1, the method comprises an oxidizing step 100 wherein one or more components of the low value carbon source are oxidized with at least one oxidizing agent to produce a modified, partially solubilized low value carbon source; and a contacting step wherein the modified, partially solubilized low value carbon source is contacted with a genetically engineered

microorganism adapted for growth in the modified, partially solubilized low value carbon source, which is derived from a wild-type microorganism comprising an isoamyl alcohol and/or isobutanol biosynthesis pathway from pyruvate.

[00069] The genetically engineered microorganism has one or more genetic modifications selected from: (a) an increased enzymatic activity of at least one enzyme in the isoamyl alcohol and/or isobutanol biosynthesis pathway relative to the activity of the same enzyme in the wild-type microorganism, and (b) a decreased enzymatic activity of at least one enzyme in a pathway that is competitive to the isoamyl alcohol and/or isobutanol biosynthesis pathway relative to the activity of the same enzyme in the wild-type microorganism.

The Isobutanol and/or isoamyl Alcohol Biosynthesis Pathway

[00070] Referring to Figure 2A, the isobutanol and/or isoamyl alcohol biosynthesis pathway starts from pyruvate, which is a central metabolite of carbohydrates, especially cellulose, hemicellulose, or other components of biomass. In step (a), 2-pyruvates are condensed to form 2-acetolactate, in a reaction catalyzed by acetolactate synthase, as shown in the following reaction scheme:

(a) 2-pyruvate→ 2-acetolactate + C0₂

[00071] In step (b), the 2-acetolactate is converted to 2,3-dihydroxy-3-methylbutanoate, in a reaction catalyzed by ketolacid reductoisomerase, as shown in the following reaction scheme:

(b) 2-acetolactate + NADPH + H⁺→ 2,3-dihydroxy-3-methylbutanoate + NADP⁺

[00072] In step (c), the 2,3-dihydroxy-3-methylbutanoate is converted to a-ketoisovalerate, in a reaction catalyzed by dihydroxy-acid dehydrogenase, as shown in the following scheme:

(c) 2,3-dihydroxy-3-methylbutanoate→ a-ketoisovalerate + H₂0

[00073] Step (d) comprises multiple steps that can convert α-ketoisovalerate to a- ketoisocaproate. These steps are represented as steps (dl) to (d5) in Figure 2B. In step (dl), α-ketoisovalerate is converted to 2-isopropylmalate, in a reaction catalyzed by 2- isopropylmalate synthase, as shown in the following reaction scheme:

(dl) a-ketoisovalerate + acetyl-CoA→ 2-isopropylmalate + CoA

[00074] In step (d2), 2-isopropylmalate is converted to 3-isopropylmalate, in a reaction catalyzed by 3-isopropylmalate dehydratase. In step (d3), the 3-isopropylmalate is converted to 2-isopropyl-3-oxosuccinate, in a reaction catalyzed by 3-isopropylmalate dehydrogenase. These two steps (d2-d3) are shown as the following reaction schemes:

(d2) 2-isopropylmalate→ 3-isopropylmalate

(d3) 3-isopropylmalate + NAD⁺→ 2-isopropyl-3-oxosuccinate + NADH + H⁺

[00075] Step (d4) is a spontaneous reaction where 2-isopropyl-3-oxosuccinate is converted to a-ketoisocaproate by decarboxylation, as shown in the following reaction scheme:

(d4) 2-isopropyl-3-oxosuccinate→ a-ketoisocaproate + C0₂

[00076] Step (d5) converts α-ketoisocaproate to leucine, which is a further step beyond the step of conversion of a-ketoisovalerate to a-ketoisocaproate.

[00077] Referring to Figure 2A, in step (e2), the α-ketoisocaproate is converted to isoamylaldehyde, in a reaction catalyzed by ketoisovalerate decarboxylase, as shown in the following reaction scheme:

(e2) a-ketoisocaproate + H⁺→ isoamylaldehyde + C0₂

[00078] In step (f2), the isoamylaldehyde is converted to isoamyl alcohol, in a reaction catalyzed by alcohol dehydrogenase, as shown in the following reaction scheme:

(f2) isoamylaldehyde + NAD(P)H + H⁺→ isoamyl alcohol + NAD(P)⁺

[00079] The isobutanol and/or isoamyl alcohol biosynthesis pathway has a branch shown as steps (el)-(fl), where the α-ketoisovalerate may be converted to isobutanol by the same two enzymes that catalyze the steps (e2) and (f2) described above. More specifically, in step (el), the a- ketoisovalerate may be converted to isobutyraldehyde, in a reaction catalyzed by ketoisovalerate decarboxylase, as shown in the following reaction scheme:

(el) a-ketoisovalerate + H⁺→ isobutyraldehyde + C0₂

[00080] In step (fl), the isobutyraldehyde is converted to isobutanol, in a reaction catalyzed by alcohol dehydrogenase, as shown in the following reaction scheme:

(fl) isobutyraldehyde + NAD(P)H + H⁺→ isobutanol + NAD(P)⁺

[00081] Therefore, the isobutanol and/or isoamyl alcohol biosynthesis pathway can produce both isoamyl alcohol and isobutanol, using the same enzymes.

Pathways Competitive to the Isobutanol and/or isoamyl Alcohol Biosynthesis Pathway A. Competitive pathways to produce amino acids

[00082] In one aspect, two intermediates of the isobutanol and/or isoamyl alcohol biosynthesis pathway, α-ketoisovalerate and α-ketoisocaproate, may be converted to the amino acids valine and leucine, respectively. The formation of valine is catalyzed by valine:2-oxoglutarate aminotransferase or valine -pyruvate aminotransferase as shown in the following reaction scheme: α-ketoisovalerate + glutamate→ valine + 2-oxoglutarate

[00083] The formation of leucine is catalyzed by leucine:2-oxoglutarate aminotransferase or branched-chain-amino-acid transaminase, step (d5), as shown in the following reaction scheme:

(d5) a-ketoisocaproate + glutamate→ leucine + 2-oxoglutarate

B. Competitive pathway to produce ethanol

[00084] Pyruvate may be converted to ethanol by a two-step process, where the pyruvate is first converted to acetaldehyde, in a reaction catalyzed by pyruvate decarboxylase, followed by conversion of acetaldehyde to ethanol, in a reaction catalyzed by alcohol dehydrogenase. These two steps are shown as the following reaction schemes:

pyruvate→ acetaldehyde + C0₂

acetaldehyde + NADH + H⁺→ ethanol + NAD⁺

C. Competitive pathway to produce fumarate

[00085] Pyruvate may be converted to fumarate with malate as an intermediate. Pyruvate is first converted to malate, in a reaction catalyzed by malate dehydrogenase, as shown in the following reaction scheme:

pyruvate + NADPH + H⁺ + C0₂CoA→ malate + NAD⁺

[00086] The pyruvate may also be converted to malate via a two-step process, in reactions catalyzed by pyruvate carboxylase and malate dehydrogenase. These two steps are shown as the following reaction schemes:

pyruvate + ATP + HCO₃ ^"→ oxoloacetate + ADP + Orthophosphate oxoloacetate + NADPH + H⁺→ malate + NADP⁺

[00087] The malate is then converted to fumarate, in a reaction catalyzed by fumarate hydratase, as shown in the following reaction scheme:

malate→ fumarate + H₂0

D. Competitive pathways to produce acetyl-CoA, lactate or formate

[00088] Pyruvate may be converted to acetyl-CoA, in a reaction catalyzed by pyruvate dehydrogenase, as shown in the following reaction scheme:

pyruvate + CoA + NAD+→ acetyl-CoA + NADH + H⁺ + C0₂

[00089] Pyruvate may also be converted to lactate, in a reaction catalyzed by lactate dehydrogenase, as shown in the following reaction scheme: pyruvate + 2 ferricytochrome c + 2 H → lactate + 2 ferricytochrome c

[00090] Pyruvate may also be converted to formate, in a reaction catalyzed by formate C- acetyltransferase, as shown in the following reaction scheme:

pyruvate + Co A→ formate + acetyl- Co A

Source of Pyruvate

[00091] The starting material for the isobutanol and/or isoamyl alcohol biosynthesis pathway is pyruvate, which is an important product of metabolism of carbohydrates. Many low value carbon sources are rich in carbohydrates and/or rich in other organic compounds that may be metabolized to produce carbohydrates. Using the low valued carbon sources as feedstock for microorganisms will allow production of pyruvate by the microorganisms. The pyruvate in turn can be used by the isobutanol and/or isoamyl alcohol biosynthesis pathway to produce isoamyl alcohol and isobutanol. Accordingly, the genetically engineered microorganism can utilize one or more components in a pretreated low value carbon source as nutrients to naturally generate pyruvate. The present invention avoids using an expensive sugar based feedstock, such as glucose, sucrose or starch for this purpose, as is the case in many prior art processes for the production of isobutanol.

[00092] The pretreated low value carbon sources can be used by the genetically engineered microorganisms as nutrients more efficiently, in comparison with a untreated low value carbon source. The present invention involves pretreatment of a low value carbon source by oxidation of one or more components of the low value carbon source to provide nutrients for the genetically engineered microorganism as shown in Figure 1. Oxidizing 100 components of the low value carbon source to produce a modified, partially solubilized low value carbon source typically involves breaking down large, insoluble molecules to produce smaller, soluble molecules that can be used as nutrients by microorganisms. Metabolism of these smaller molecules by the microorganisms, leads to generation of pyruvate. Examples of such small molecules that may be present in the modified, partially solubilized low value carbon source after oxidation of one or more components of the low value carbon source include fatty acids, hydrocarbons, carbohydrates, oxides, aldehydes, alcohols, aliphatic and aromatic compounds such as polycyclic aromatics and aromatic carboxylic acids.

[00093] Conventional techniques for oxidizing 100 a low value carbon source include hydroliquefaction or direct liquefaction, pyrolysis and gasification. In these processes, components of the low value carbon source are depolymerized to varying degrees to their organic constituents with or without oxygen, with or without water. For example, an indirect coal liquefaction (ICL) process consists of a gasification step at temperatures greater than about 700 °C in the presence of oxygen or air to make syngas (a mix of CO & H₂) followed by at least one catalytic step which converts syngas to liquid hydrocarbons.

[00094] A direct coal liquefaction process (DCL) converts one or more components of the low value carbon source directly into one or more liquids, without the intermediate step of gasification, by breaking down high molecular weight molecules with application of solvents and catalysts in a high pressure and high temperature environment using hydrogen. Since liquid hydrocarbons generally have a higher hydrogen to carbon molar ratio than a low value carbon source, either hydrogenation or carbon-rejection processes may be employed in both ICL and DCL technologies.

[00095] Generally, the gasification process consists of feeding a low value carbon source into a heated chamber (the "gasifier") along with a controlled and/or limited amount of oxygen and optionally steam. Gasification processes produce a raw gas composition comprising CO, H₂, H₂S, and N¾. After clean-up, the primary gasification products of interest are H₂ and CO. See Demirbas, "Recovery of Energy and Chemicals from

Carbonaceous Materials," Energy Sources, Part A, vol. 28, pages 1473-1482, 2006. During the gasification process, large carbonaceous molecules are oxidized into small carbon molecules, with many being soluble and capable of being used by microorganisms.

[00096] In some embodiments, the oxidizing step 100 uses mild conditions for efficient oxidative depolymerization large carbonaceous molecules in the low value carbon source. The oxidizing step 100 is performed in the presence of at least one oxidizing agent, and optionally at least one solubilizing agent. The oxidizing step 100 may comprise heating the low value carbon source to a temperature below 300 °C and at a pressure below 1230 psig. The oxidizing step 100 may comprise using a combination of steam and air in a solid- vapor (non-aqueous) environment for treatment of the low value carbon source. The conditions of the oxidizing step 100 in terms of the ratio of 0₂/low value carbon source, the ratio of steam/low value carbon source, vapor and solids residence times, and temperature can be varied to alter the final product distribution, gain selectivity and to yield specific chemical products. This oxidizing step 100 does not require pure oxygen, nor is pure oxygen desirable.

[00097] The mild conditions for the oxidizing step 100 allow the production of various product distributions based on varying operating conditions. The oxidizing step 100 can simultaneously oxidize, depolymerize, reform and/or solubilize high molecular weight large molecules in the low value carbon source to smaller low molecular weight hydrocarbons, oxo-chemicals and other chemicals. This conversion of large molecules to small molecules provides nutrients suitable for use by the genetically engineered microorganisms.

[00098] Under certain conditions, a mixture of water soluble oxo-chemicals may be produced. Here, oxo-chemicals are organic compounds that comprise at least one oxygen atom. For example, a mixture of aliphatic and aromatic carboxylic acids can be produced. At other conditions, a mixture of these oxo-chemicals and waxy hydrocarbons containing paraffins and olefins having chain lengths ranging from C₁₀ to C44 can be produced. The small molecules in the modified, partially solubilized low value carbon source resulting from the oxidizing step 100 are often water soluble and may be used by microorganisms as nutrients (biodegradable), thus producing pyruvate for the isoamyl alcohol and isobutanol biosynthesis process.

[00099] The oxidizing step 100 may comprise raising the temperature of the low value carbon source to a desired temperature and/or keeping the low value carbon source at a pressure at or above the steam saturation pressure. The product after the oxidizing step 100 may be used as a feedstock for the genetically engineered microorganisms. In some embodiments, the product may optionally be subjected to chemical and/or physical separation before being used as a feedstock. Chemical and/or physical separation may be employed for separation of various components in the product. For example, some high- valued minerals and chemicals may be retrieved from the product using conventional chemical and/or physical separation methods. Such chemicals include, for example, oxo-chemicals.

Applicable chemical and physical separation technologies that may be used include any of those known to one skilled in the art, including fractional distillation, liquid/liquid extraction, adsorption, ion exchange, membrane filtering, and hybrid systems.

[000100] In some embodiments, the low value carbon source may be too impermeable, e.g. due to limited porosity, to be efficiently treated by the oxidizing step 100. In such a case, the low value carbon source may be preprocessed (e.g. comminuted) to increase its permeability or available surface area, thus increasing the susceptibility of the large carbonaceous molecules in the low value carbon source to the oxidizing agent of the oxidizing step 100. Any method known to a skilled person in the art that is suitable for reducing the particle size and/or increasing surface area of the low value carbon sources may be used. For example, physical (e.g., grinding, milling, fracture and the like) and chemical approaches (e.g., treating with surfactants, acids, bases, oxidants, such as but not limited to acetic acid, sodium hydroxide, percarbonate, peroxide and the like) can be applied to reduce the size and/or increase the surface area of the carbonaceous materials in the low value carbon source. [000101] In some embodiments, preprocessing may be used to break down coal, oil shale, lignite, coal derivatives and similar structures to release more organic matter, or to make them more susceptible to degradation into smaller organic compounds. In one embodiment, coal and water at about a 1 :2 weight ratio are loaded into a mill with steel media. The duration of milling may be in the range from about 60 to about 90 minutes. After milling, the coal slurry may be used as an input to the oxidizing step 100.

[000102] The solubilizing agent used in the oxidizing step 100 may be selected from mineral acids or mineral bases. Preferred bases include Group I (alkali metals) and Group II (alkaline earth) oxides, hydroxides, carbonates, borates, or halogenates. In particular, sodium, potassium, calcium, and magnesium compounds are preferred. Examples of the solubilizing agents include sodium hydroxide, potassium hydroxide, sodium carbonate and potassium carbonate. Naturally occurring minerals of some of these materials are also appropriate for use in this process. These include, but are not limited to Nahcolite, Trona, Thermonatrite, Gaylussite, Hydromagnesite, Lansfordite, Ikaite, Hydrocalcite, Dolomite, Huntite, Aragonite, Natrite, Magnesite, Calcite, Kalcinite, Gregoryite, and others.

[000103] The low value carbon source, oxidizing agent, and optionally a solubilizing agent, and a solvent such as water are mixed and fed to the oxidizing step 100. Mineral bases generally comprise no more than about 15 wt of the mixture provided to the oxidizing step 100, and preferably comprise below 10 about wt and most preferably at or below about 6 wt of the mixture provided to the oxidizing step 100. In some embodiments, the mineral base comprises at least about 1 wt or at least about 3 wt or at least about 5 wt of the mixture provided to the oxidizing step 100.

[000104] In some embodiments, the solubilizing agent may be a mineral acid, such as phosphoric acid, nitric acid, boric acid, hydrochloric acid, and sulfuric acid.

[000105] The low value carbon source may be mixed with the solubilizing agent provided in an aqueous solution to provide the mixture fed to the oxidizing step 100. In some alternative embodiments, the low value carbon source may be combined with steam or water vapor containing solubilizing agent. In these embodiments, the vapor or steam may be blown onto the low value carbon source.

[000106] In some embodiments, the low value carbon source is dispersed in an aqueous solution of the solubilizing agent to make the mixture. The amount of low value carbon source dispersed in the aqueous solution is limited by the average size of the monomer molecules that may be oxidatively reformed from the low value carbon source and their solubility in water based on their functional groups, their degree of ionization in water, and physical and chemical attributes of the aqueous system, such as temperature, pH, pressure, activity coefficient, and other considerations. Solution viscosity also increases with higher low value carbon source loading in the slurry-like mixture and is a limitation that may reduce mass transfer and mixing between the solid and liquid. In some embodiments, the low value carbon source content in the mixture may be less than about 40% by weight. The low value carbon source content of the mixture may be at or below about 30% by weight, or at or below about 25% by weight.

[000107] The low value carbon source may be heated in a reaction vessel in the presence of at least one oxidizing agent. The oxidizing step 100 may comprise raising the temperature of the low value carbon source to a desired temperature by any suitable means and/or subjecting the mixture to a pressure at or above the steam saturation pressure. Multiple reactions may occur during the oxidizing step 100, including oxidization, depolymerization, reforming and solubilization. In a reforming process, the molecular structure of high molecular weight carbonaceous molecules such as hydrocarbons is rearranged to produce smaller, lower molecular weight molecules that may be used effectively as nutrients for the microorganisms.

[000108] The oxidizing agent may be selected from air, oxygen enriched air, ozone, sulfuric acid, permanganates, carbon dioxide, nitrous oxide, nitric acid, chromates, perchlorates, persulfates, superoxides, chlorates, peroxides, hypochlorites, Fenton's reagent and nitrates in which the cations may comprise metal cations, hydrogen ions and/or ammonium ions.

[000109] Oxidizing agents may be ranked by their strength. See Holleman et al. "Inorganic Chemistry," Academic Press, 2001, page 208. A skilled person will appreciate that, to prevent over-oxidation of the low value carbon source, the conditions in the oxidizing step may be adjusted according to the strength of the oxidizing agent used. For example, when a strong oxidizing agent is used, one or more of temperature, pressure, and duration of the oxidizing step 100 may be reduced to prevent over-oxidation and/or ensure that the desired degree of conversion is not exceeded. On the other hand, when a weak oxidizing agent is used, one or more of temperature, pressure, and duration of the oxidizing step 100 may be increased to ensure that the desired degree of oxidation and/or conversion is achieved. When the oxidizing agent is gaseous, the pressure in the reaction vessel for the oxidizing step 100 is important for ensuring the desired degree of oxidation and/or conversion.

[000110] In some embodiments, oxygen is used as the oxidizing agent. In one embodiment, oxygen can be delivered to the reaction vessel as air. In some other embodiments, depending on the susceptibility of the low value carbon source to oxidation, oxygen-enriched air can be used. Suitable enrichment percentages can be from an oxygen concentration slightly above that of atmospheric air to substantially pure oxygen.

[000111] In alternative embodiments, depending on the types of small organic molecules sought, instead of using a base, a mineral acid may be used to provide more acidic conditions for carrying out the oxidation reaction. Examples of suitable mineral acids include phosphoric acid, nitric acid, boric acid, hydrochloric acid, and sulfuric acid.

[000112] In some embodiments, at least one catalyst may optionally be added to the mixture fed to the oxidizing step 100. The catalyst may catalyze the oxidation reaction by, for example, causing or enhancing formation of peroxides and superoxides, which may enhance the rate of oxygen insertion into the large carbonaceous molecules, leading to a more complete breakdown of the large carbonaceous molecules in the low value carbon source relative to oxidation of the same low value carbon source in the absence of such catalysts. The catalyst may be selected from water insoluble metals, transition metals, and precious metals. Examples of these metals include nickel, cobalt, platinum, palladium, rhenium, copper, vanadium and ruthenium. The catalyst may be unsupported or may be supported on an inert or active matrix material such as clay, alumina, silica, silica alumina, zeolites, activated carbon, diatomaceous earth, titania, zirconia, molybdena, ceramics, and the like. Such catalysts can enhance rates of oxygen insertion and reforming of high molecular weight carbonaceous molecules as well as being able to enhance the degree of relative oxidation. Examples of the catalysts include metal oxides, mixed metal oxides, hydroxides, and carbonates, of ceria, lanthanum, mixed rare earths, brucite, hydrotalcite, iron, clays, copper, tin, and vanadium.

[000113] The reaction vessel in which the oxidizing step 100 is conducted is not limited to any particular reactor design, but may be any sealable reaction vessel that can tolerate the temperature and pressure required for the present invention. In some embodiments, the mixture is fed to a reaction vessel, which has been pre-heated to the desired temperature. Then, air or oxygen enriched air is slowly added to the reaction vessel until the desired pressure is reached. The temperature and pressure in the reaction vessel may be monitored to ensure a desired level of oxidization of the low value carbon sources during the filling of air or oxygen enriched air, as well as during the oxidizing step 100.

[000114] For the oxidizing step 100, the mixture in the reaction vessel, including the low value carbon source, an oxidizing agent, and optionally a solubilizing agent, a catalyst, and solvent such as water, is heated to a temperature below about 300 °C (572 °F), or below about 220 °C (428 °F), or below about 150 °C (302 °F). A positive pressure in the reaction vessel is maintained at saturated steam pressure or slightly higher, for example below about 1230 psig, or below about 322 psig, or below about 54 psig respectively. A minimum temperature is approximately 130 °C and a respective minimum pressure is approximately 24 psig.

[000115] In some embodiments, the mixture in the reaction vessel has at least two phases: a liquid phase (water/solubilizing agent/oxidizing agent) and a solid phase (low value carbon source). In some embodiments, there are three phases in the reaction vessel: gas (oxygen/air and/or steam), liquid (water/solubilizing agent) and solid (low value carbon source). To ensure efficient heat and mass transfer among these phases, the mixture may be subjected to mechanical agitation. The reaction vessel may include structural features to facilitate interactions among the phases. For example, an unstirred reaction vessel with gas dispersion features, a reaction vessel with mechanical agitation devices as well as reaction vessels with gas entrainment devices or combinations thereof may be used. Exemplary reaction vessels include a co-current flow tubular reactor with gas dispersion, a counter-current flow tubular reactor with gas dispersion, and a flowing tubular reactor with static mixers.

[000116] The duration of the oxidizing step 100 may be determined, for example, by the oxidative stress induced in the mixture and the desired product. As a general rule, a higher oxidative stress requires a shorter duration oxidizing step 100. In addition, if the desired products are generated by more complete oxidization of the low value carbon source, e.g. via a series of sequential reaction steps, a longer duration for the oxidizing step 100 may be required.

[000117] In some embodiments, the duration of the oxidizing step 100 can vary from a few seconds to several hours, depending on the degree of conversion required, the reduction in molecular weight desired, the reactivity of the low value carbon source, process economics, the amount of carbon dioxide, carbon monoxide, and hydrogen generated, and other constraints. In one embodiment, the low value carbon source is coal and the reaction time is in the range from about 0.02 to about 4 hours, or about 0.2 to about 4 hours, or about 0.5 to about 4 hours, or about 1 to about 3 hours, or about 1.5 to about 2.5 hours.

[000118] In some embodiments, the reaction conditions including temperature, pressure and reaction time may also depend on molecular and elemental characteristics of the particular low value carbon source. Examples of the characteristics of the low value carbon source which may be taken into consideration are the degree of aromaticity, the hydrogen to carbon ratio, oxygen to carbon ratio, nitrogen to carbon ratio, sulfur to carbon ratio, mineral or ash content, and other factors. Thus, in some embodiments, a blend of low value carbon sources of different characteristics may enhance the efficiency of the pretreatment method by adjusting one or more of these characteristics. For example, blending a highly aromatic, more difficult to react, carbon source, such as coal, with a more acyclic carbonaceous material, such as agricultural waste or synthetic polymer waste, may result in an oxidized product stream that is more biodegradable and will support greater microbial population densities, as well as increasing the rate and depth of conversion of the less reactive large molecules in these low value carbon sources.

[000119] The extent of conversion can be controlled by using different reaction conditions to yield different types and amounts of, for example, partial oxidation products. The reaction conditions may also be adjusted to convert substantially all of the solids of the low value carbon source, other than inorganics concentrated in an ash stream, without significant loss of carbonaceous compounds to C0₂ production.

[000120] In some embodiments, a portion of the gaseous phase in the reaction vessel may optionally be continuously or periodically withdrawn and replaced. Carbon dioxide formed during the reaction has several roles, including acting as an excess base neutralizer and forming a carbonate buffering system in the water. A carbonate buffered system is a desirable feature for subsequent conversion of the treatment product by the genetically engineered microorganisms. In many cases, microorganisms prefer a system at or around pH 7. The C0₂ produced in the pretreatment reacts with excess base and reduces or eliminates the need to adjust the pH of the product stream resulting from depolymerization by the addition of acid, thereby lowering costs. Any excess carbon dioxide formed during the reaction is preferably removed from the reaction vessel. In one embodiment, gas is withdrawn from the reaction vessel, the carbon dioxide content of the withdrawn gas is reduced and the gas with the reduced carbon dioxide content is optionally resupplied back to the reaction vessel, with or without being enriched with oxygen. This embodiment may also be used for maintaining a desired partial pressure of oxygen in the reaction vessel during the pretreatment method.

[000121] In some embodiments, samples of the gas phase in the reaction vessel may be taken periodically in order to monitor the progress of pretreatment. The gas sample may be analyzed by, for example, a gas chromatograph to identify the content of one or more components to provide an indication of the progress of the pretreatment. Once the desired degree of conversion is reached, the oxidizing step 100 may be terminated. Carbon dioxide may be withdrawn or oxygen may be periodically or continuously added to the reaction vessel to maintain the desired level of oxidant. [000122] The oxidizing step 100 can be conducted in batch mode, semi-batch mode, or continuously. In one aspect, at least a portion of the carbonaceous material in the low value carbon source may be oxidized by the oxidizing step 100. The carbonaceous material may be oxidized to organic acids, such as oxalic acid, mellitic acid, benzoic acid and acetic acid. In addition, high molecular weight carbonaceous molecules may be depolymerized/reformed to lower molecular weight carbonaceous molecules.

[000123] In some embodiments, mineral bases are used to increase the pH of the mixture to a caustic alkaline pH of greater than about 7, greater than about 9 or greater than about 10. In such mixtures, the formed organic acids will be present in salt form due to the presence of the mineral base. Such salts may be recovered from the products of the oxidizing step 100 by filtering off the solid material and extracting the oxalic acid therefrom with dilute hydrochloric or sulfuric acid. The salts of mellitic acid and like acids can be isolated from the filtrate by acidifying, warming, and filtering the warm liquid, while acetic acid can be recovered from the residual liquid by, for example, steam distillation.

[000124] The products of the oxidizing step 100 may include minerals, chemicals and low- molecular weight carbonaceous compounds. After optionally extracting the minerals and high- value chemicals, the remainder of the product may be used as a feedstock for the genetically engineered microorganisms. This portion of the product includes solubilized carbonaceous compounds, and possibly some solid high molecular weight carbonaceous molecules. The product may be used for fermentation by the genetically engineered microorganisms to produce isoamyl alcohol and isobutanol, where the carbonaceous molecules, especially the low-molecular weight carbonaceous molecules produced by oxidation and depolymerization, are used as feedstock for the genetically engineered microorganisms to produce pyruvate and eventually isoamyl alcohol and isobutanol.

[000125] Thus, in one aspect of the invention, the conditions of the oxidizing step 100 are selected on the basis of producing modified, partially solubilized low value carbon source that may include larger quantities of biodegradable materials and/or may exhibit an enhanced rate of biodegradation or an enhanced tendency to biodegrade.

[000126] In some embodiments, the oxidizing step 100 is part of pretreatment process schematically represented in Figure 7. The low value carbon source (carbonaceous raw material) is mixed with reagents, water and air or oxygen-enriched air in a pretreatment process. The reagents include at least one oxidizing agent, and optionally at least one solubilizing agent or a catalyst. The oxidizing step 100 comprises heating the mixture to a suitable temperature and at a suitable pressure. In some embodiments, two or more sequential heating stages may be conducted under different conditions using the product of a prior heating stage as the feed to the following heating stage. The reaction conditions at each heating stage are adjusted to favor different reactions, rates of reaction, degrees of conversion, etc. For example, one heating stage may have reaction conditions selected for the production of valuable oxo-chemicals and another heating stage may have its reaction conditions selected for enhancing biodegradability of the modified, partially solubilized low value carbon source.

[000127] In some embodiments, the oxidizing step 100 is used to treat black liquor in a reactor, which is the liquor resulting from the cooking of pulpwood in an alkaline solution in a soda or sulfate, such as a Kraft, paper making process by removing lignin, hemicelluloses, tall oil, and other extractives from the wood to free the cellulose fibers. One embodiment of the pretreatment of black liquor is exemplified in Figure 8. The black liquor and an oxidizing agent are fed into a reactor, along with optional additional reagents, and heated under pressure. The reaction within the reactor creates a reaction mixture, which can then be treated and/or separated by chemical, physical or microbial means, to yield organic compounds. These organic compounds include organic compounds comprising from about 2 to about 20 carbon atoms that are suitable as nutrients for the genetically engineered microorganisms.

[000128] In alternative embodiments, the black liquor is separated, or fractionated, into various components prior to the oxidizing step 100 in a reactor. One possible embodiment is exemplified in Figure 9. The black liquor is separated by a chemical, a physical or a microbial process, selected organic polymers are recovered as an economically valuable commodity, and the balance of the black liquor is a black liquor component reactor feedstock. This black liquor component reactor feedstock is fed, along with an oxidizing agent and optional additional reagents, into the reactor, and is heated under pressure. The reaction within the reactor creates a reaction mixture, which can then be treated and/or separated by chemical, physical or microbial means, to yield organic compounds. These organic compounds include organic compounds comprising from about 2 to about 20 carbon atoms that are suitable as nutrients for the genetically engineered microorganisms.

[000129] In another alternative embodiment the black liquor is separated, or fractionated, into various components prior to the oxidizing step 100 in a reactor, as exemplified in Figure 10. The black liquor from the pulp line ("raw black liquor") is separated by a chemical, physical or microbial process, to obtain the black liquor component reactor feedstock, and a residue. The black liquor component reactor feedstock is fed, along with an oxidizing agent and optional additional reagents, into the reactor, and is heated under pressure. The reaction within the reactor creates a reaction mixture, which can then be treated and/or separated by chemical, physical or microbial means, to yield organic compounds. These organic compounds include organic compounds comprising from about 2 to about 20 carbon atoms that are suitable as nutrients for the genetically engineered microorganisms. The residue from the separation of raw black liquor is further dewatered, and burned in a recovery boiler to produce energy.

[000130] In still another alternative embodiment the black liquor is separated, or

fractionated, into various components prior to the oxidizing step 100 in a reactor, as exemplified in Figure 11. The black liquor is separated by a chemical, physical or microbial process, to obtain the selected organic polymers, the black liquor component reactor feedstock, and the residue. The black liquor component reactor feedstock is fed, along with an oxidizing agent and optional additional reagents, into the reactor, and is heated under pressure. The reaction within the reactor creates a reaction mixture, which can then be treated and/or separated by chemical, physical or microbial means, to yield organic compounds. These organic compounds include organic compounds comprising from about 2 to about 20 carbon atoms that are suitable as nutrients for the genetically engineered microorganisms. The residue from the separation of raw black liquor is further dewatered, and burned in a recovery boiler to produce energy.

[000131] Such separation of components from the raw black liquor may decrease the concentration of some components, and thus increase the relative concentration of other components. For example, removal of soaps and/or tall oils will increase the concentration of the lignin. In one embodiment the concentration of lignin is increased from about 35 to about 45 wt with respect to the total organics to at least about 55 wt . Under another embodiment, the concentration is increased to at least about 65 wt . Under another embodiment, the concentration is increased to at least about 75 wt .

[000132] In one embodiment, the black liquor may be heated in a reaction vessel in the presence of at least one oxidizing agent. The oxidizing step 100 may comprise raising the temperature of the mixture to a desired temperature by any suitable means and/or subjecting the mixture to a pressure at or above the steam saturation pressure. Multiple reactions may occur during the pretreatment step, including oxidization, depolymerization, reforming and solubilization. In a reforming process, the molecular structure of a hydrocarbon is rearranged. Without being bound by theory, it is believe that the pretreatment step of the present invention may oxidatively crack wood polymers to provide small organic compounds. [000133] The black liquor stream as generated by the pulping process is typically very caustic. Such a caustic environment is typically sufficient to allow oxidative breaking down of wood polymers to generate one or more small organic compounds comprising from about 2 to about 20 carbon atoms that are suitable as nutrients for the genetically engineered microorganisms. However, in some cases, the black liquor stream may have a lower pH that does not readily allow for acceptable oxidative breaking down of wood polymers. Under such circumstances, a mineral base may be added to the black liquor. Exemplary bases that may be used include Group I (alkali metal) and Group II (alkaline earth) oxides, hydroxides, carbonates, borates, and halogenates. In particular, sodium, potassium, calcium, and magnesium compounds are preferred. Examples of suitable bases include sodium hydroxide and potassium hydroxide.

[000134] The oxidizing 100 of black liquor occurs at a temperature sufficient to oxidize components of the black liquor to generate one or more organic compounds comprising from about 2 to about 20 carbon atoms that are suitable as nutrients for the genetically engineered microorganisms. This temperature has been found to be up to about 300 °C, or between about 150 °C and about 250 °C. In another embodiment, the treatment of the black liquor occurs at a temperature between about 150 °C and about 220 °C. In yet another embodiment, the pretreatment of the black liquor occurs at a temperature below about 150 °C.

[000135] The oxidizing of black liquor occurs at a pressure sufficient to oxidize components of the black liquor to generate one or more organic compounds comprising from about 2 to about 20 carbon atoms that are suitable as nutrients for the genetically engineered microorganisms. This pressure has been found to be below about 1230 psig or about 322 psig. In another embodiment, this pressure has been found to be below about 54 psig. In certain embodiments, this pressure ranges from atmospheric pressure to about 1230 psig, or about 322 psig or about 54 psig.

[000136] In some embodiments, the oxidizing conditions including temperature, pressure and reaction time may also depend on the molecular and elemental characteristics of the particular black liquor feedstock. Different species of wood may result in differing compositions of the black liquor. The characteristics of the black liquor used in the pulping process that may affect pretreatment include the degree of aromaticity, the hydrogen to carbon ratio, the oxygen to carbon ratio, the nitrogen to carbon ratio, the sulfur to carbon ratio, and the mineral or ash content, as well as other factors.

[000137] Referring to Figure 1, the product of the oxidizing step 100 is a modified, partially solubilized low value carbon source that may be cooled to a temperature suitable for the growth of the wild-type microorganisms and genetically engineered microorganisms.

Temperatures between about 25 °C and about 40 °C are suitable for growth of most microorganisms. For example, a suitable growth temperature for yeast is about 28 °C and many bacteria grow at a temperature of about 37 °C. A suitable growth temperature for the wild-type microorganism and genetically engineered microorganism may be established by a person skilled in the art following routine procedures.

[000138] In some embodiments, before contacting the modified, partially solubilized low value carbon source with the genetically engineered microorganism, the product of oxidizing step 100 may be subjected to one or more additional optional steps to prepare the modified, partially solubilized low value carbon source to be used by the genetically engineered microorganism more efficiently to produce isoamyl alcohol and isobutanol.

[000139] One optional step involves adding one or more microorganisms to the modified, partially solubilized low value carbon source to convert one or more components in the modified, partially solubilized low value carbon source to intermediate products that can be more efficiently metabolized by the genetically engineered microorganisms to produce pyruvate, and/or isoamyl alcohol and isobutanol. Another optional step involves adding at least one nutrient to the modified, partially solubilized low value carbon source in order to enhance growth of the microorganism involved in the biosynthesis of isoamyl alcohol and isobutanol. The nutrient may be selected from substances upon which the genetically engineered microorganisms are dependent or substances that can be converted in situ to another product upon which the genetically engineered microorganisms are dependent.

Suitable nutrients may include ammonium, ascorbic acid, biotin, calcium, calcium

pantothenate, chlorine, cobalt, copper, folic acid, iron, K₂HPO₄, KNO₃, magnesium, manganese, molybdenum, Na₂HP0₄, NaNC>3, NH₄C1, NH4NO₃, nickel, nicotinic acid, p- aminobenzoic acid, biotin, lipoic acid, mercaptoethanesulfonic acid, nicotinic acid, phosphorus, potassium, pyridoxine HCl, riboflavin, selenium, sodium, thiamine, thioctic acid, tungsten, vitamin B6, vitamin B2, vitamin Bl, vitamin B12, vitamin K, yeast extract, zinc and mixtures of one or more of these nutrients.

[000140] Yet another optional step involves adding at least one enzyme to the modified, partially solubilized low value carbon source to increase the yield of, and/or selectivity for, isoamyl alcohol and isobutanol biosynthesis, for example, to enhance utilization of the modified, partially solubilized low value carbon source by the genetically engineered microorganism. For example, an enzyme may be used to assist in converting one or more components of the low value carbon source to intermediate(s) that may be more effectively used by the genetically engineered microorganism. In some exemplary embodiments, enzymes may be used to further to enhance the yield and/or selectivity of the biosynthesis of isoamyl alcohol and isobutanol.

[000141] Enzymes suitable for the present invention may include Acetyl xylan esterase, Alcohol oxidases, Allophanate hydrolase, Alpha amylase, Alpha mannosidase, Alpha-L- arabinofuranosidase, Alpha-L-rhamnosidases, Ammoniamonooxygenase, Amylases, Amylo- alpha-l,6-lucosidase, Arylesterase, Bacterial alpha-L-rhamnosidase, Bacterial pullanases, Beta-galactosidase, Beta-glucosidase, Carboxylases, Carboxylesterase,

Carboxymuconolactone decarboxylase, Catalases, Catechol dioxygenase, Cellulases, Chitobiase/beta-hexo-aminidase, CO dehydrogenase, CoA ligase, Dexarboxylases,

Dienelactone hydrolase, Dioxygenases, Dismutases, Dopa 4,5-dioxygenase, Esterases, Family 4 glycosylhydrolases, Glucanaeses, Glucodextranases, Glucosidases, Glutathione S- transferase, Glycosyl hydrolases, Hyaluronidases, Hydratases/decarboxylases, Hydrogenases, Hydrolases, Isoamylases, Laccases, Levansucrases/Invertases, Mandelate racemases, Mannosyl oligosaccharide glucosidases, Melibiases, Methanomicrobialesopterin S- me thy transferases, Methenyl tetrahydro-methanopterin cyclohydrolases, Methyl-coenzyme M reductase, Methylmuconolactone methyl-isomerase, Monooxygenases, Muconolactone delta-isomerase, Nitrogenases, O-methy transferases, Oxidases, Oxidoreductases,

Oxygenases, Pectinesterases, Periplasmic pectate lyase, Peroxidases, Phenol hydroxylase, Phenol oxidases, Phenolic acid decarboxylase, Phytanoyl-CoA dioxygenase, Polysaccharide deacetylase, Pullanases, Reductases, Tetrahydromethan- opterin S-methyltransferase, Thermotoga glucanotransferase and Tryptophan 2,3-dioxygenase.

[000142] The modified, partially solubilized low value carbon source comprises nutrients that may be used by the genetically engineered microorganism to produce pyruvate, which may then be used to produce isoamyl alcohol and isobutanol. Oxygenated organic compounds that may be present in the modified, partially solubilized low value carbon source include oxygenated hydrocarbons, carboxylic acids, and oxygenated compounds comprising additional heteroatoms. Oxygenated organic compounds are organic compounds that comprise at least one oxygen atom. Heteroatom means any atom besides hydrogen or carbon. Examples of heteroatoms other than oxygen include nitrogen, phosphorus, sulfur, fluorine, and chlorine.

[000143] Examples of oxygenated hydrocarbons that may be present in the modified carbon source include alcohols, aldehydes, carboxylic acids, salts of carboxylic acids, esters, ethers, anhydrides, and like. Oxygenated compounds may be monofunctional, difunctional, trifunctional, or polyfunctional. Included in the definition of oxygenated hydrocarbons are also compounds with more than one functional group, such as polyols, dicarboxylic acids, triacids, polyesters, polyethers, aldehydic acids, and the like. Also included in the definition of oxygenated hydrocarbons are compounds in which there is more than one type of functional group.

[000144] Examples of carboxylic acids include compounds of the formula R-COOH, wherein R is an alkyl group. Particular examples include formic acid, methanoic acid, acetic acid, ethanoic acid, propionic acid, butyric acid, butanoic acid, valeric acid, pentanoic acid, caproic acid, hexanoic acid, enanthic acid, heptanoic acid, caprylic acid, octanoic acid, pelargonic acid, nonanoic acid, capric acid, decanoic acid, undecylic acid, undecanoic acid, lauric acid, dodecanoic acid, tridecylic acid, tridecanoic acid, myristic acid, tetradecanoic acid, pentadecanoic acid, palmitic acid, hexadecanoic acid, margaric acid, heptadecanoic acid, stearic acid, octadecanoic acid, arachidic acid, and icosanoic acid.

[000145] Dicarboxylic acids of the present invention are organic compounds that contain two carboxylic acid groups. Such dicarboxylic acids may comprise additional heteroatoms, such as oxygen, nitrogen, or sulfur. Dicarboxylic acids may be aliphatic or aromatic. Aside from the two -COOH groups, dicarboxylic acids may be saturated or unsaturated. The dicarboxylic acids may be represented by the formula HOOC-R-COOH, wherein R is a difunctional organic group, such as alkylene, alkenylene, alkynylene, arylene, and any of the preceding modified by a one or more heteroatoms.

[000146] Examples of dicarboxylic acids include compounds such as alkylene dicarboxylic acids, having the general formula HOOC-(CH₂)„-COOH wherein n is 0 to about 12; mono- unsaturated forms thereof; di-unsaturated forms thereof; tri-unsaturated forms thereof; and polyunsaturated forms thereof. Examples of dicarboxylic acids also include oxalic acid, ethanedioic acid, malonic acid, propanedioic acid, succinic acid, butanedioic acid, glutaric acid, pentanedioic acid, adipic acid, hexanedioic acid, pimelic acid, heptanedioic acid, suberic acid, octanedioic acid, azelaic acid, nonanedioic acid, sebacic acid, decanedioic acid, undecanedioic acid, and dodecanedioic acid.

[000147] Examples of aromatic dicarboxylic acids include phthalic acid, benzene- 1 ,2- dicarboxylic acid, o-phthalic acid, isophthalic acid, benzene-l,3-dicarboxylic acid, m-phthalic acid, terephthalic acid, benzene- 1,4-dicarboxylic acid, and p-phthalic acid.

[000148] Examples of monounsaturated acids include maleic acid, (Z)-butenedioic acid, fumaric acid, (E)-butenedioic acid, glutaconic acid, pent-2-enedioic acid, traumatic acid, and dodec-2-enedioic acid. Examples of di-unsaturated acids include the three isomeric forms of muconic acid, and (2E,4E)-hexa-2,4-dienedioic acid.

[000149] The identity and amounts of the small organic compounds in the modified, partially solubilized low value carbon source depend on the heating parameters, such as the reaction conditions including the pressure, and temperature, the type of oxidant used, and the weight ratio of the oxidant to the low value carbon source. In one embodiment, the modified, partially solubilized low value carbon source comprises primarily alcohols and ethers. In another embodiment, involving more complete oxidation, the modified, partially solubilized low value carbon source comprises greater relative amounts of aldehydes. By increasing the degree of oxidation further, the modified, partially solubilized low value carbon source may comprise greater relative amounts of carboxylic acids and esters. The alcohols, ethers, aldehydes, esters, and carboxylic acids may be monofunctional, or polyfunctional.

[000150] The modified, partially solubilized low value carbon source is used as the feedstock for the genetically engineered microorganisms of the present invention. The composition of the modified, partially solubilized low value carbon source obtained from the oxidizing step 100 may affect one or both of the yield and selectivity of isoamyl alcohol and isobutanol biosynthesis. Thus, in one aspect of the invention, the conditions of the oxidizing step 100 are selected on the basis of producing a modified, partially solubilized low value carbon source that may include larger quantities of biodegradable materials and/or may exhibit an enhanced yield and/or selectivity in the process for biosynthesis of isoamyl alcohol and/or isobutanol.

Genetically Engineered Microorganisms

[000151] Referring to Figure 1, in the contacting step 200 the modified, partially solubilized low value carbon source is placed in contact with the genetically engineered microorganism. The genetically engineered microorganism of the present invention may be derived from a wild-type microorganism that comprises the isobutanol and/or isoamyl alcohol biosynthesis pathway as shown in Figure 2A. Such a wild-type microorganism is capable of producing isoamyl alcohol and/or isobutanol, though typically at a low level that is not viable for commercial production of isoamyl alcohol and/or isobutanol. Genetic engineering of the wild-type microorganism can significantly enhance the yield and/or selectivity of a process for producing isoamyl alcohol and/or isobutanol using the genetically engineered

microorganism. [000152] in some embodiments, the wild-type microorganism is also selected for strains that, are tolerant of isoamyl alcohol and/or isobutanoi. Alcohols such as isoamyl alcohol and isobutanoi are toxic to many microorganisms. The toxicity is caused primarily by alcohol- induced leakage of the plasma membrane through fluidizing the membrane, resulting in uncontrolled transport of solutes that, can decrease the proton flux across the membrane and cause leakage of important cof actors. Alcohols can also inactivate membrane and cytosolic enzymes, for example, ATPase and glycolytic enzymes, causing a decrease in cell growth. The alcohol tolerance of different microorganisms appears to result in large part from adaptive and evolutionary changes in cell membrane composition. Wild-type microorganisms sensiti ve to isoamyl alcohol and/or isobutanoi may be less desirable for use in the present invention. The present invention preferably selects a wild-type microorganism that is tolerant, of isoamyl alcohol and/or isobutanoi. Suitable wild-type microorganisms with a tolerance for isoamyl alcohol and/or isobutanoi may be identified by screening assays that are based on an intrinsic tolerance of the microorganism.

[000153] A microorganism's intrinsic tolerance to isoamyl alcohol and/or isobutanoi may be measured by determining the concentration of isoamyl alcohol and/or isobutanoi that can cause 50% inhibition of the growth rate (IC5₀) of the microorganism when grown in a minimal medium. The IC5.3 values may be determined using methods known in the art. For example, a wild-type microorganism of interest may be grown in the presence of various amounts of isoamyl alcohol and/or isobutanoi and the growth rate monitored by measuring the optical density at 600 nanometers. The doubling time may be calculated from the logarithmic part of the growth curve and used as a measure of the growth rate. The concentration of isoamyl alcohol and/or isobutanoi that produces 50% inhibition of growth may be determined from a graph of the percent inhibition of growth versus the isoamyl alcohol and/or isobutanoi concentration. In some embodiments, suitable wild-type microorganisms may have an TC₅o for an isoamyl alcohol and/or isobutanoi concentration of greater than about 5 wt.%, or an IC₅₀ for an isoamyl alcohol and/or isobutanoi concentration of greater than about 10 wt.%, or an ICso for an isoamyl alcohol and/or isobutanoi concentration of greater than about 15 wt.%, or an IC₅₀ for an isoamyl alcohol and/or isobutanoi concentration of greater than about 20 wt.%, or an ICso for an isoamyl alcohol and/or isobutanoi concentration of greater than about 30 wt.%.

[000154] In some embodiments, the wild-type microorganism is capable of growing on a particular class of modified, partially solubilized low value carbon sources described herein. Different types of low value carbon sources can yield different modified, partially solubilized low value carbon sources that may support a different spectrum of microorganisms. In addition, the same low value carbon sources oxidized under different conditions or using different procedures may yield different modified, partially solubilized low value carbon sources that support different microorganisms. Thus, the present invention may involve selection of a suitable wild-type microorganism depending on the characteristics of the modified, partially solubilized low value carbon source that is obtained from the oxidizing step 100 that is to be used as a feedstock for the production of isoamyl alcohol and/or isobutanol.

[000155] A simple test may be employed to select a suitable wild-type microorganism for growing in a particular modified, partially solubilized low value carbon source. One option involves measuring the growth potential of the wild-type microorganism in the modified, partially solubilized low value carbon source. Wild-type microorganisms showing good growth potential in the modified, partially solubilized low value carbon source may be selected for further genetic engineering.

[000156] A suitable wild-type microorganism may also need to have the ability to be transformed by exogenous nucleic acids (vectors) to generate a genetically engineered microorganism. Gene transformation may be accomplished by techniques known in the art, such as electroporation, conjugation, transduction, or natural transformation.

[000157] In some embodiments, the wild-type microorganisms may be selected from various groups including Thermotogales group, Cytophaga group, Azospirillum group, Paracoccus subgroup, Sphingomonas group, Nitrosomonas group, Azoarcus group, Acidovorax subgroup, Oxalobacter group, Rhodococcus group, Thiobacillus group, Xanthomonas group,

Oceanospirillum group, Pseudomonas and relatives, Marinobacter hydrocarbonoclaticus group, Pseudoalteromonas group, Vibrio subgroup, Aeromonas group, Desulfovibrio group, Desulfuromonas group, Desulfobulbus assemblage, Campylobacter group, Acidimicrobium group, Frankia subgroup, Arthrobacter and relatives, Nocardiodes subgroup,

Thermo anaerobacter and relatives, Bacillus megaterium group, Carnobacterium group, Clostridium and relatives, and archaea such as Archaeoglobales, Methanobacteriales, Methanomicrobacteria and relatives, Methanopy rales, and Methanococcales.

[000158] Suitable microorganisms may preferably be selected from a genus such as

Escherichia (and particularly E. coli), Corynebacterium, Clostridium, Zymonomas,

Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klesiella, Paenibacillus, Arthrobacter, Brevibacterium, Pichia, Candida, Hansenula, Synechococcus, Synechocystis, Anabaena, Ralstonia, Lactococcus, or Saccharomyces. In one embodiment, the wild-type microorganism is selected from the Rhodococcus genus.

[000159] In some embodiments, the wild-type microorganisms may be a strain selected from strains of Pseudomonas oleovornas subspecies lubricantis DSM 21016, Rhodococcus opacus DSM 43251, Rhodococcus ruber DSM 44190, Rhodococcus ruber DSM 45332,

Rhodococcus ruber DSM 43338, Rhodococcus ruber DSM 7512, Rhodococcus ruber DSM 6264, Rhodococcus erythropolis DSM 8424, Rhodococcus erythropolis DSM 312,

Rhodococcus erythropolis DSM 43066 and Rhodococcus rhodochrous DSM 43269.

[000160] The genetically engineered microorganism of the present invention may comprise at least one genetic modification that increases the enzymatic activity of at least one enzyme in the isobutanol and/or isoamyl alcohol biosynthesis pathway in comparison with the wild- type microorganism. Throughout the present application, unless otherwise noted, the increase/decrease of enzymatic activity or expression level in the genetically engineered microorganism is in comparison with the corresponding enzymatic activity of the wild-type microorganism from which the genetically engineered microorganism is derived. The increased enzymatic activity of at least one enzyme in the isobutanol and/or isoamyl alcohol biosynthesis pathway can enhance the yield and/or selectivity of isoamyl alcohol and/or isobutanol biosynthesis process. These enzymes functioning in the isobutanol and/or isoamyl alcohol biosynthesis pathway include acetolactate synthase, ketolacid reductoisomerase, dihydroxy-acid dehydrogenase, 2-isopropylmalate synthase, 3-isopropylmalate dehydratase, 3-isopropylmalate dehydrogenase, ketoisovalerate decarboxylase, and alcohol

dehydrogenase.

[000161] The increase or decrease in the enzymatic activity of a particular enzyme may be measured by incubating the enzyme with a substrate for the enzyme. The product of the enzyme produced in a unit of time under a set of specific conditions or in a specific environment, preferably a set of conditions or an environment suitable for growing the genetically engineered microorganism, is a measurement of the enzyme activity of the enzyme. The enzymatic activity of the enzyme in the genetically engineered microorganism and the wild-type microorganism measured under the same conditions or in the same environment can be compared to determine the increase or decrease in the enzymatic activity of the particularly enzyme, as described in the present application.

[000162] In one embodiment, the genetically engineered microorganism is Rhodococcus erythropolis transformed with plasmid pCE46 which contains the a-ketoisovalerate decarboxylase gene from Lactococcus lactis (SEQ ID NO: l) and the alcohol dehydrogenase from Synechocystis Sp. PCC6803 (SEQ ID NO:2). The two genes are under the control of a trc promoter in the plasmid pCE46, which provide increased enzymatic activities of a- ketoisovalerate decarboxylase and alcohol dehydrogenase to the genetically engineered microorganism for enhancing yield and/or selectivity of isoamyl alcohol and/or isobutanol biosynthesis.

[000163] In some embodiments, the wild-type microorganism may be genetically engineered to completely or partially inactivate a pathway that is competitive with the isobutanol and or isoamyl alcohol biosynthesis pathway. The competitive pathway may be inactivated by- reducing or eliminating an enzymatic activity in the competitive pathway. The isobutanol and/or isoamyl alcohol biosynthesis pathway in some microorganisms may have several competitive pathways as discussed herein. The genetically engineered microorganism may have at least one enzyme with reduced enzymatic activity where the at least one enzyme is selected from valine:2-oxoglutarate aminotransferase, valine -pyruvate aminotransferase, leucine:2-oxoglutarate aminotransferase, branched-chain-amino-acid transaminase, pyruvate decarboxylase, alcohol dehydrogenase, malate dehydrogenase, pyruvate carboxylase, malate dehydrogenase, fumarate hydratase, pyruvate dehydrogenase, lactate dehydrogenase, and formate C-acetyltransferase.

[000164] The microorganism is genetically engineered to have reduced or eliminated enzymatic activity for one of these enzymes in the competitive pathways, which can partially or completely inactivate one of the competitive pathways. When at least one of the competitive pathways is blocked/deactivated or partially blocked/deactivated, the pyruvate is mostly directed via carbon flux to produce isoamyl alcohol and/or isobutanol, thus leading to an improvement of the yield and/or selectivity of isoamyl alcohol and/or isobutanol biosynthesis.

[000165] In some other embodiments, the genetically engineered microorganism may further comprise an increased enzymatic activity for at least one enzyme that contributes to improved NAD(P)H regeneration activity. As discussed above, there are multiple steps in the isobutanol and/or isoamyl alcohol biosynthesis pathway that use NAD(P)H as a co-factor.

[000166] The improved NAD(P)H regeneration activity may lead to a higher NAD(P)H concentration in the cytoplasm of genetically engineered microorganism, and thus a higher yield and/or selectivity of isoamyl alcohol and/or isobutanol biosynthesis. The enzymatic activity contributing to regeneration of NAD(P)H may be a pyridine nucleotide

transhydrogenase activity, for example, pntAB from E. coli and/or a glyceraldehyde-3- phosphate dehydrogenase activity (for example, gapN from S. mutans). Therefore, the genetically engineered microorganism may further have an increased pyridine nucleotide transhydrogenase enzymatic activity and/or a glyceraldehyde- 3 -phosphate dehydrogenase enzymatic activity. The NAD(P)H concentration may thus be increased in the cells of the genetically engineered microorganism to achieve a higher yield of and/or selectivity for isoamyl alcohol and/or isobutanol biosynthesis.

[000167] In some embodiments, the genetically engineered microorganism comprises at least one enzyme with increased enzymatic activity that enhances the production of pyruvate, which is the starting material for the isobutanol and/or isoamyl alcohol biosynthesis pathway as shown in Figure 2A. In some embodiments, the microorganism grows on a modified, partially solubilized low value carbon source containing aromatic compounds and small carbohydrate molecules. The enzymes that enhance production of pyruvate in such a medium include enzymes in the glycolysis pathway, which breaks down sugar to pyruvate. Some examples of these enzymes include hexokinase, phosphoglucoisomerase,

phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde- 3 -phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase. The genetically engineered microorganism may comprise enhanced enzymatic activity for at least one of these enzymes in order to enhance the production of pyruvate.

[000168] To increase the amount of aromatic compounds (aromaticity) in the modified, partially solubilized low value carbon source, the temperature and/or pressure in the oxidizing step 100 may be increased. In some embodiments, the low value carbon source may be selected such that it can yield a modified, partially solubilized low value carbon source with an increased aromaticity. For example, older coals generally yield a modified, partially solubilized low value carbon source with a higher aromaticity, since as coal ages, cyclization of carbon compounds occurs after deoxygenation. Thus, selection of a low-rank coal with higher aromaticity will yield a modified, partially solubilized low value carbon source with a higher aromaticity. Alternatively or additionally, less severe conditions during the oxidizing step in terms of pressure, temperature, and a less powerful oxidizing agent will favor preservation of aromatics and lead to a higher aromatic content in the modified, partially solubilized low value carbon source.

Genetic Engineering Technologies

[000169] The genetically engineered microorganism is derived from a wild-type

microorganism that comprises an isobutanol and/or isoamyl alcohol biosynthetic pathway from pyruvate to isoamyl alcohol and/or isobutanol. The technologies for deriving the genetically engineered microorganism from the wild-type microorganism are described in detail in this section. It is understand that one or a combination of the techniques described in this section may be used to genetically modify any enzymes in the wild-type microorganism that may be involved in the isobutanol and/or isoamyl alcohol biosynthesis pathway and/or its competitive pathways, and thereby derive the genetically engineered microorganism.

A. Increase or decrease an enzymatic activity

[000170] The target gene, which is an enzyme that directly or indirectly affects the yield or selectivity of the isobutanol and/or isoamyl alcohol biosynthesis pathway, may have its enzymatic activity increased or decreased, depending on whether the enzyme contributes to or hinders the biosynthesis of isoamyl alcohol and/or isobutanol, as discussed herein. When the target gene encodes an enzyme that contributes to the biosynthesis of isoamyl alcohol and/or isobutanol, the present invention uses the genetic engineering technologies to increase the enzymatic activity of the enzyme. The increase of an enzymatic activity of a target gene (encoding the enzyme) in a genetically engineered microorganism may be achieved in one of three ways: by either replacing the native enzyme with an exogenous enzyme which is more active than the native enzyme encoded by the target gene, or enhancing the expression level of the target gene, or both. The exogenous enzyme may be an ortholog, a homolog, or an up- mutant of the target gene. When the target gene is overexpressed in the genetically engineered microorganism, the enzyme encoded by the target gene is produced in larger quantity. Thus the genetically engineered microorganism will have increased enzymatic activity of the target gene. The up-mutant can have point mutations, deletions or insertions.

[000171] On the other hand, when the target encodes an enzyme that hinders the

biosynthesis of isoamyl alcohol and/or isobutanol, the present invention uses the genetic engineering technologies to decrease the enzymatic activity of the enzyme. The decrease of an enzymatic activity of a target gene (encoding the enzyme) in a genetically engineered microorganism may also be achieved in one of three ways: by either replacing the native enzyme with an exogenous enzyme which is less active than the native enzyme, deleting part or all of the target gene, suppressing the expression level of the target gene. The exogenous enzyme may be an ortholog, a homolog, or a down-mutant of the target gene. When the target gene is underexpressed in the genetically engineered microorganism, the enzyme encoded by the target gene is produced in smaller quantity. Thus the genetically engineered

microorganism will have decreased enzymatic activity of the target gene in comparison with the wild-type microorganism. The down-mutant can have point mutations, deletions or insertions. Partial or complete deletion of the target gene often causes complete loss of the enzymatic activity in the genetically engineered microorganism.

[000172] In some embodiments, enzymatic activities may be increased/decreased by either random mutagenesis or by DNA shuffling of the coding sequence of the target gene. The increase/decrease in enzymatic activity can be monitored in an enzyme specific fashion, depending on the characteristics of the enzyme. Enzymatic activity often can be measured by known methods such as a colorimetric/fluorometric/spectrophotometric assay with a proper substrate or product which produces measurable signals.

B. Vectors

[000173] The present invention uses vectors to introduce genetic materials (e.g. a gene, a promoter) into a wild-type microorganism to genetically engineer the microorganism to improve the yield and/or selectivity of isoamyl alcohol and/or isobutanol biosynthesis. In some embodiments, the vectors can autonomously replicate and operate irrespective of the host genome in the genetically engineered microorganism to create a stably transformed microorganism. Such vectors include plasmids, cosmids, and bacteriophages. In some other embodiments, the vectors are suicide vectors designed to cause homologous recombination between the microorganism modifications to the microorganism genome (e.g. inserting a gene, deleting a gene partially or completely, replacing a gene, replacing a promoter). The suicide vectors are not capable of replicating in the genetically engineered microorganism. Plasmids are often used as suicide vectors to cause homologous recombination with the host microorganism genome.

[000174] A typical plasmid vector that can replicate autonomously comprises (a) an origin of replication functioning in the wild-type microorganism so that the vector can effectively replicate and result in several hundred copies of the plasmid vector in each microorganism host cell, (b) an antibiotic -resistance gene so that a microorganism host cell transformed with the plasmid vector can be identified, and (c) a sequence comprising at least one restriction enzyme site where an exogenous DNA fragment can be inserted into the vector. In some embodiments, even in the absence of a suitable restriction enzyme site, a vector and the exogenous DNA fragment can easily be ligated by using a synthetic oligonucleotide adaptor or a linker according to conventional methods known in the art.

[000175] When a vector is designed to replicate autonomously in the genetically engineered microorganism, the vector can express the gene in the vector thus to produce the protein (en enzyme in the present invention) encoded by the gene. This will result in increased enzymatic activity for the target gene. This type of vectors, commonly referred to as expression vectors, have an exogenous gene operably linked to expression control sequences (e.g. promoters) for control of transcription of the gene in the genetically engineered microorganism. The expression vector is used for enhancing enzymatic activity for an enzyme in the genetically engineered microorganism. For example, the promoter and the exogenous gene are included in an expression vector together with a selection marker and a replication origin. The exogenous genes that may be inserted into an expression vector include the enzymes in the isobutanol and/or isoamyl alcohol biosynthesis pathway, the enzymes contributing to regeneration of NAD(P)H and enzymes enhancing production of pyruvate.

[000176] Some vectors that may be used in the present invention are presented in Figures 3A-3H.

[000177] It is well understood that not all vectors function equally in expressing an exogenous gene in a particular microorganism. Likewise, not all microorganisms are equally suitable for hosting an identical expression vector. However, those skilled in the art are able to make a suitable selection from various vectors that is suitable for a microorganism host without undue experimentation. For example, a vector may be selected taking into consideration the microorganism host cell since the vector should be replicated in the host cell. In addition, the copy number of a vector, the ability to control the copy number, expression of the enzyme encoded by the gene in the vector, and an antibiotic marker should all be considered in selecting a suitable vector for the microorganism. Also, various vectors/microorganism host combinations suitable for use in the present invention may be selected by those skilled in the art taking these factors into consideration.

C. Overexpression or underexpression of a target gene

[000178] As described here, the target gene may be overexpressed or underexpressed in the genetically engineered microorganism. The present invention provides two methods for overexpressing a target gene in the genetically engineered microorganism: (a) introducing a strengthened promoter to replace the native promoter of the target gene, and (b) introducing an extra copy of the target gene. The strengthened promoter is more active than the native promoter, thus can drive a higher expression level for the target gene in comparison with the wild-type microorganism. In addition, an extra copy of the target gene in the genetically engineered microorganism means that the enzyme encoded by the target gene may be produced from both the native target gene and the introduced extra copy of the target gene, thus a significantly higher expression level for the target gene in the genetically engineered microorganism may be achieved, in comparison with the wild-type microorganism.

[000179] In some embodiments, overexpression of the target gene may involve introducing a homolog of the target gene that encodes a protein with the same enzymatic activity. The homolog will produce an enzyme to enhance the enzymatic activity of the target gene.

[000180] The methods for underexpressing a target gene in the genetically engineered microorganism may be (c) introducing a weakened promoter to replace the native promoter of the target gene, and (d) knock-out the target gene. The weakened promoter is less active than the native promoter, thus producing a lower expression level for the target gene compared with the wild-type microorganism. Further, knock out of the target gene in the microorganism will completely diminish the expression of the target gene. Thus no functional product of the target gene will be produced in the genetically engineered microorganism.

[000181] The introduction of a strengthened promoter or a weakened promoter to replace the native promoter of the target gene, as well as introducing an extra copy of the target gene or its homolog may be accomplished by the knock-in technique. Alternatively, the introduction of an extra copy of the target gene or its homolog may also be achieved by transforming the microorganism with a plasmid containing the target gene so long as the plasmid can independently and autonomously replicate in the transformed microorganism.

[000182] The introduced promoter, strengthened or weakened, may affect the expression of all the genes in an operon to which the target gene belongs. The genes in an operon are evolutionarily selected to carry out the same or related function. For example, genes in the same metabolic pathway are often organized in the same operon. Thus, replacing the promoter of an operon provides the potential of affecting multiple genes that enhance biosynthesis of isoamyl alcohol and/or isobutanol in a coordinated fashion.

D. Knock-out and knock-in techniques

[000183] Figure 4A represents an embodiment of knock-out technique where the target gene is completely deleted from the wild-type microorganism genome. Figure 4B shows an embodiment of knock-in technique where a native sequence (e.g. promoter, target gene) is replaced with an exogenous sequence. Figure 4C represents an embodiment of knock-in technique where an exogenous sequence (target gene or its homolog) is inserted into the genome of microorganism. The vectors used in the knock-out and knock-in techniques are preferably suicide vectors, which cannot replicate in the wild-type microorganism. The suicide vectors thus do not have a functional origin of replication in the microorganism and have the sole purpose of causing homologous recombination with the host genome.

[000184] In one embodiment, a suicide vector for knock-out/knock-in in Rhodococcus is built using the backbone of a pUC57-Kan vector from E. coli that contains an origin of replication not functioning in the Rhodococcus species. The sacB (levansucrase) gene from Bacillus subtilis is synthesized and inserted into the pUC57-Kan backbone. The suicide vector further comprises an antibiotic resistance cassette. For deleting a target gene in Rhodococcus, the suicide vector may contain a region that fuses the two regions (two arms) surrounding the target gene (Figure 4A). For example, a 1.5kb DNA fragment is used for each "arm". The two 1.5 kb DNA "arms" are fused and inserted into the suicide vector (pCE20, SEQ ID NO:5) by Gibson assembly (https://www.neb.com/applications/cloning- and-synthetic-biology/gibson-assembly-cloning). The suicide vector may then be

electroporated to the wild-type microorganism. Mutants containing integrated plasmid, caused by a single crossover are selected based on their antibiotic resistance and sucrose sensitivity, since the first round of recombination generates a chromosomal mutant which is sensitive to sucrose and resistant to antibiotics (Figure 4A). A 2^nd recombination event is selected for lack of antibiotic resistance and no sucrose sensitivity, since the second round of recombination generates two possible mutants (Figure 4A). Of these two products, the intended one, with a genomic deletion of the target gene, is selected based on its resistance to sucrose and antibiotic sensitivity. A similar approach is used to insert a sequence

(strengthened or weakened promoter, homolog of the target gene) in the microorganism genome (Figures 4B-4C).

[000185] In some embodiments, the electroporation process may be carried out as follows. Rhodococcus strains (either wild-type or containing a plasmid) are inoculated in LB (Luria- Bertani) broth (with appropriate antibiotic if containing plasmid) in a shaker at 28 °C until they reach mid- log phase. Cultures are cooled to 4 °C and cells are harvested by

centrifugation at 3000 g for 10 minutes. Bacterial cells are washed twice with ice-cold water and centrifuged again at 3000 g for 10 minutes. In the final step, bacterial cells are resuspended in water containing 10% aliquoted glycerol solution. The treated bacterial cells can be used directly for electroporation or stored in a -80 °C freezer for later electroporation.

[000186] For electroporation, competent cells are thawed on ice if retrieved from the freezer. Plasmid DNA is added to the competent cells and incubated on ice for 30 minutes. Cells are then transferred to a pre-chilled 2 mm electroporation cuvette and electroporated with a single pulse at 2.5 kV followed by addition of 1 ml of super optimal broth with catabolite repression (SOC) medium and 3 hours recovery in a shaker at 28 °C. Cells are then plated in LB-Agar plates under the appropriate selection and grown for 2-3 days at 28 °C.

[000187] In other embodiments, a strategy for gene knock-out/knock-in uses the phage λ- Red Recombinase system. λ-Red Recombinase genes are amplified from pKD46 (Datsenko et al., "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products," Proc. Nat. Acad. Sci., vol. 97, pages 6640-5, 2000, incorporated herein by reference) and assembled to an expression vector (e.g. pCE17) capable of replicating in Rhodococcus which contains an inducible promoter (e.g. pTrc, SEQ ID:62). The sacB counter- selectable marker is also inserted to cure from plasmid after recombination.

[000188] A wild-type Rhodococcus strain is first made electrocompetent by transforming with a λ-Red Recombinase plasmid (e.g. pCE58). Successful transformation is verified by PCR amplification of the λ-Red Recombinase cassette from bacterial colonies. Colonies positive for λ-Red Recombinase are then grown overnight and after dilution with fresh media, induced for λ-Red Recombinase expression (e.g. by addition of IPTG). The Rhodococcus cells are thus made electrocompetent.

[000189] Competent Rhodococcus cells expressing the λ-Red Recombinase genes are transformed with PCR products obtained by amplification of an antibiotic cassette flanked by short DNA sequences homologous to the arms outside the region to be knocked-out. In some cases antibiotic cassettes are also flanked by recombination sites (e.g. flippase recognition target (FRT) or Cre-LOX) to allow for removal of antibiotic marker if desired. Transformants are selected based on their capability to grow under antibiotic selection and successful gene deletion is confirmed by colony PCR with primers designed to amplify the region of DNA knocked out or a combination of a primer that either amplifies the inserted antibiotic cassette or one of the flanking "arms".

E. Promoters

[000190] The expression level of a target gene is mostly controlled by the promoter of the target gene. A strong promoter leads to a higher expression level for the target gene, while a weak promoter leads to a lower expression level for the target gene. The present invention provides strengthened promoters and weakened promoters to increase or decrease the expression level of a target gene. Particularly, an exogenous promoter (strengthened or weakened promoter) may be introduced into the microorganism genome to replace the native promoter of a target gene using the knock-in technique disclosed herein, thus the expression level of the target gene may be enhanced or suppressed. Note here that the strengthened promoter or weakened promoter is determined by comparison with the native promoter. If the exogenous promoter has a higher transcription initiation activity than the native promoter, the exogenous promoter is a strengthened promoter that may be used to replace the native promoter to drive overexpression of the target gene. Conversely, the weakened promoter has a lower transcription initiation activity than the native promoter, and thus may be used to replace the native promoter to drive underexpression of the target gene.

[000191] There are many promoters that have strong transcription initiation activities.

Examples include an SV40 promoter or the early and late promoters of adenovirus, the promoter of lac operon, the promoter of trp operon, the promoter of TAC or TRC system, T3 and T7 promoters, the major operator and promoter domain of phage lambda, the promoter of bacteriophage fd coat protein, the promoter of 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of a phosphatase (e.g. Pho5), and the promoter of the yeast alpha- mating system.

[000192] There are numerous other promoters can be used in the present invention, depending on the level of expression desired for the target gene in the genetically engineered microorganism. A skilled person can easily assess the strength of a promoter by making a construct with a reporter gene where the expression of the reporter gene is driven by the promoter. For example, a reporter gene may be green fluorescent protein gene or luciferase. The product of green fluorescent protein may be visible under blue light and the amount of luciferase expressed may be measured by simple enzymatic reaction. Thus, the strength of a promoter can be determined in this manner.

[000193] In some embodiments, three promoter/terminator systems (Figure 5) may be used. Two of them are based on the pTrc promoter made inducible or constitutive by the presence (inducible) or absence (constitutive) of a lacl gene. The third promoter is based on a Pnit promoter. To modify the strength of a promoter, a ribosomal binding site (RBS) may be inserted into a promoter to enhance the binding of ribosomes, thus increasing expression of the downstream target gene. Transcriptional terminators used are (rrnB Tl and T2, and Thca terminator as shown in Figure 5. The three promoter/terminators systems are represented by SEQ ID NOS: 62-64. The three promoter/terminator systems are also shown in Figures 6A- 6C.

[000194] Genes in microorganisms, especially in bacteria, are often organized in operons where transcription of the genes in the same operon is controlled by a common promoter. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying a common promoter. Alternatively, any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide (up-mutants or down-mutants). These modification can result in an increase or decrease in the activity of the encoded enzyme.

[000195] The weakened and strengthened promoters may be built by these skilled in the art with known techniques. For example, Baliga ("Promoter analysis by saturation mutagenesis," Biol. Proced. , vol. 3, pages 64-69, 2001) describes techniques changing a promoters for strengthening or weakening it. Kagawa et al. ("Identification of a methanol-inducible promoter from Rhodococcus erythropolis PR4 and its use as an expression vector," /. Biosci. Bioeng., vol. 113, pages 596-603, 2012) describes strengthening a promoter by making it constitutive after removal one DNA fragment on its 5 ' end.

F. General Methods

[000196] Standard procedures for cloning a recombinant DNA and transformation of microorganisms used in the present invention are known in the art. Techniques suitable for use in the following examples may be found in Sambrook et al., (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989), Silhavy et al., (Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1984), and Ausubel et al., (Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, 1987).

[000197] Materials and methods suitable for maintenance and growth of a bacterial culture are known in the art. Suitable techniques can be seen in the following: Phillipp et al., eds., Manual of Methods for General Bacteriology, American Society for Microbiology,

Washington, D.C., 1994 and Thomas D. Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989).

Further Processes

A. Fermentation

[000198] The modified, partially solubilized low value carbon source may be at least partially solubilized after the oxidizing step 100. The modified, partially solubilized low value carbon source is placed in contact with the genetically engineered microorganism, whereby the microorganism may utilize the solubilized carbonaceous materials as nutrients to first produce pyruvate, which is then converted into isoamyl alcohol and/or isobutanol through the isobutanol and/or isoamyl alcohol biosynthesis pathway. This process is called fermentation where the genetically engineered microorganisms are cultured under suitable conditions in a medium comprising the modified, partially solubilized low value carbon source in order to produce isoamyl alcohol and/or isobutanol.

[000199] The fermentation medium may comprise, in addition to the modified, partially solubilized low value carbon source, a suitable mineral, salt, enzymatic cofactor, buffer, and other components known to those skilled in the art which are suitable for stimulating the disclosed isobutanol and/or isoamyl alcohol biosynthesis pathway and/or for growth of the genetically engineered microorganism in the culture.

[000200] Typically, microorganism cells are grown at a temperature in the range of about 25 °C to about 40 °C. A suitable pH for the fermentation is between about pH 5.0 to about pH 9.0. More specifically, the initial pH for culture and fermentation may be about pH 6.0 to about pH 8.0. Fermentation may be performed under aerobic or anaerobic conditions. In one embodiment, fermentation is performed under an anaerobic or microaerobic condition.

[000201] The fermentation process may be a batch fermentation process. A classical batch fermentation is a closed system in which the composition of the medium is established at the beginning of the fermentation and not subjected to artificial alterations during the fermentation. Therefore, at the beginning of the fermentation, the medium is inoculated with the desired organism, and the fermentation is permitted to occur without anything being added to or retrieved from the medium during the fermentation. However, the batch fermentation is often not completely closed with respect to the addition of the modified, partially solubilized low value carbon source, as well as altering controlling factors such as pH and oxygen concentration during the fermentation.

[000202] In the batch fermentation system, the metabolite and carbon source composition of the system change constantly up to the time the fermentation is stopped. Within batch cultures, cell growth proceeds through a static lag phase to an exponential growth (log) phase and finally to a stationary phase where the growth rate is decreased or stopped. The microorganism cells in the stationary phase will eventually die. Generally, microorganism cells in the log phase are involved in bulk-formation of the final product and/or any intermediates.

[000203] A modified version of the standard batch fermentation process is a called fed-batch fermentation process. A fed-batch fermentation process is identical to a typical batch fermentation process except that a growth-limiting substrate is incrementally added as the fermentation progresses. A fed-batch process is useful in various circumstances, such as when catabolite repression is apt to inhibit metabolic action in the cells or where it is desirable to have limited amounts of substrate in the medium, for example to control the reaction rate. In a fed-batch system, the actual substrate concentration is difficult to measure, and is therefore calculated based on the change in measurable factors such as pH, dissolved oxygen, or the partial pressure of a waste gas, such as C0₂. Batch and fed-batch

fermentations are common and well known in the art (Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Desphande and Mukund, Appl. Biochem. Biotechnol., vol. 36, pages 227-234, 1992).

[000204] The fermentation process may also be a continuous fermentation process. A continuous fermentation is carried out in an open system in which fresh fermentation medium or at least one of its components is added continuously to the reaction vessel and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation permits microorganism cells to be maintained at a constant high density where the cells are mainly in log-phase growth.

[000205] Continuous fermentation processes allow for modulation of one or more factors that affect cell growth and/or yield/selectivity of isoamyl alcohol and/or isobutanol biosynthesis. For example, continuous fermentation allows maintaining a limiting nutrient such as the carbon source or nitrogen level at a fixed concentration while letting all other parameters moderate. In another example, a number of factors affecting cell growth can be altered continuously while the cell concentration, measured by medium turbidity, is kept constant. Continuous fermentation systems strive to maintain steady-state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate during the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the rate of cell duplication, are well known in the art of industrial microbiology and a variety of methods are detailed in Brock's textbook (Thomas D. Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989)).

B. Cell immobilization

[000206] The fermentation process may present a challenge when recovering the genetically engineered microorganism cells from the medium is desirable. For instance, the continuous fermentation process allows constant withdrawal of medium from the fermentation system whereby the microorganism cells are lost from the reaction vessel. In addition, at the end of fermentation process, the microorganism cells in the medium may all be lost if not recovered from the medium. Though some old medium with the microorganism cells may be added to fresh medium for the next batch of fermentation, this may be undesirable when the old medium contains some substances that could hinder or slow the start of the next fermentation. Thus, a technique making the microorganism cells in the fermentation medium easily recoverable may present significant advantages in some embodiments.

[000207] In some embodiments, the genetically engineered microorganism cells may be immobilized on a supporting structure, which is then placed in the reaction vessel in contact with the modified, partially solubilized low value carbon source. Therefore the withdrawal of medium from the reaction vessel causes no or minimal loss to the microorganism cells in the reaction vessel. In some embodiments, the supporting structure is an integral portion of the reaction vessel such that the microorganism cannot be easily washed out of the reaction vessel even if the fermentation medium is repeatedly retrieved from the reaction vessel. Immobilization of the genetically engineered microorganism cells can improve the yield, selectivity and/or reduce the cost of isoamyl alcohol and/or isobutanol production.

[000208] There are a variety of microorganism immobilization technologies known in the art, such as adsorption immobilization, self-immobilization, embedding immobilization and crosslinking immobilization, etc. Cell immobilization technologies are described in detail by Martins et al., "Immobilization of microbial cells: A promising tool for treatment of toxic pollutants in industrial wastewater," Afr. J. Biotechnol., vol. 12, pages 4412-4418, 2013, incorporated herein by reference.

[000209] A traditional method of cell immobilization is the piercing method, in which a film-forming material containing microbial cells is immobilized by dripping into a liquid fixative. For example, sodium alginate is immobilized in CaCl₂ solution; polyvinyl alcohol is immobilized in boric acid solution. The process is complex, with long immobilizing and washing times and low preparation efficiency, and difficult for large-scale production. [000210] Microencapsulation is an immobilization method using natural or synthetic polymer materials to embed living microorganism cells, even with some liquid substances to form micro-particles with a semipermeable or sealed membrane. The polymer materials may be agar, alginate, carrageenan, cellulose and its derivatives, collagen, gelatin, epoxy resin, photo cross- linkable resins, polyacrylamide, polyester, polystyrene and polyurethane. The microencapsulation method encapsulates microorganism cells within a rigid polymeric network to prevent the cells from diffusing into surrounding medium while still allowing penetration of nutrients and substrates (Ramakrishna et al., "Microbial fermentations with immobilized cells," Current Science, vol. 77, pages 87-100, 1999).

[000211] Immobilization of the genetically engineered microorganism cells eliminates most of the constraints faced with free-cell fermentation systems. A remarkable advantage of cell immobilization is the freedom to determine cell density prior to fermentation. It also facilitates operation of microbial fermentation on continuous mode without cell washout, where the solubilized carbon source moves continuously through the reaction vessel, while the genetically engineered microorganism cells are almost completely retained in the reaction vessel during the continuous fermentation process.

[000212] The following examples are illustrative, but not limiting, of the methods of the present disclosure. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in the field, and which are obvious to those skilled in the art, are within the scope of this disclosure.

EXAMPLES

Example 1

[000213] Selected strains of Rhodococcus were transformed with an expression vector, plasmid pCE46 as shown in Figure 16. The plasmid contains two genes under the control of a trc promoter, where the two genes are the a-ketoisovalerate decarboxylase gene from

Lactococcus lactis and the alcohol dehydrogenase from Synechocystis Sp. PCC6803. The transformed cells were cultured in LB media which were induced for 24 hours with ImM isopropyl β-D- 1 -thiogalactopyranoside (IPTG). The medium samples were analyzed for isoamyl alcohol and isobutanol production by headspace Gas Chromatography - Flame Ionization Detector (GC-FID). A representative chromatogram of the medium is shown in Figure 17, where both peaks for isoamyl alcohol and isobutanol were detected. Interestingly, isoamyl alcohol was produced in higher amounts compared to isobutanol as shown in Figure 17. In this example, the competitive pathway for ethanol biosynthesis was not inactivated, thus a significant amount of ethanol was produced.

Example 2

[000214] The following wild-type bacterial strains were tested for their ability to grow on a medium comprising a modified, partially solubilized low value carbon source derived from pretreatment of low-rank coal. Two types of medium containing dissolved organic carbon (DOC) from pretreated coal were used (>15,000 DOC and 3,000 DOC). The ability of these bacterial strains to grow on the medium is presented in Table 1 below. The results demonstrate that the ten Rhodococcus strains have a reasonable ability to grow in a medium comprising a modified, partially solubilized low value carbon source obtained by

pretreatment of coal, and thus are suitable to be used as wild-type microorganisms for the genetic engineering process of the present invention.

[000215]

Table 1. Growth of bacterial strain in carbonaceous materials

[000216] The ability of the Rhodococcus strains to be transformed by vectors was further tested. Two shuttle vectors, pCE7 and pCE8, were built using the backbone of the E. coli plasmid pACYC177 (containing the E. coli origin of replication and antibiotic resistance cassettes) and origins of replication of plasmid pRC4 from Rhodococcus rhodochrous DSM 43269 and plasmid pRE8424 from Rhodococcus erythropolis DSM 8424 respectively. Fragments were amplified by PCR and assembled by using the Gibson assembly reaction in E. coli (https://www.neb.com/applications/cloning-and-synthetic-biology/gibson-assembly- cloning).

[000217] The ten Rhodococcus strains were made competent by standard molecular biology techniques, followed by transformation by the pCE7 and pCE8 plasmids using electroporation of the plasmids into the Rhodococcus cells. Strains were plated under kanamycin selective conditions. Of the ten Rhodococcus strains tested, five strains produced transformed colonies when transformed with pCE7 and three strains produced transformed colonies when transformed with pCE8 as shown in Table 2 and Figure 12.

Table 2. Ability of Rhodococcus strains being transformed by a plasmid

[000218] To verify the success of transformation and replication of intact pCE7 and pCE8, plasmid DNA was extracted from colonies, retransformed in E. coli, digested and compared with original pCE7 and pCE8 plasmids. Plasmid DNA extracted from transformed

Rhodococcus colonies was able to replicate in E. coli, indicating the presence of a functional origin of replication for these organisms. The extracted plasmid DNA was indistinguishable from the original pCE7 and pCE8 plasmids as analyzed by restriction enzyme digestion. These results indicate successful transformation of Rhodococcus strains by the pCE7 and pCE8 plasmids. These two plasmids are potent vectors for genetic engineering of the Rhodococcus strains. Example 3

[000219] A suicide vector (pCE20, SEQ ID NO:5) was built using the backbone of a pUC57-Kan plasmid from E. coli that contains an origin of replication not functional in Rhodococcus species. The sacB (levansucrase) gene from Bacillus subtilis was synthesized and inserted into the pUC57-Kan backbone (the Gene synthesis was done by Genscript®). The suicide vector pCE20, containing the SacB gene and an antibiotic resistance cassette, may be used as a backbone to build a vector for targeting a specific gene. For example, a knock-out vector based on pCE20 can be built by assembly of the two regions ("arms") surrounding a target gene to be deleted. Generally a 1.5kb DNA fragment is used for each "arm." The two 1.5 kb DNA "arms" may be assembled to the pCE20 plasmid by Gibson assembly. The knock-out plasmid may then be electroporated to Rhodococcus host cells. Transformed cells containing integrated plasmid, caused by a single crossover may be selected based on their antibiotic resistance and sucrose sensitivity. A 2^nd recombination event is selected for lack of antibiotic resistance and no sucrose sensitivity, as shown in Figure 4A.

Example 4

[000220] A vector pCE58 (SEQ ID NO:61) based on the phage λ-Red Recombinase system was built for transformation of Rhodococcus strains. The λ-Red Recombinase genes were amplified from pKD46 (Datsenko et al., "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products," Proc. Nat. Acad. Sci., vol. 97, pages 6640-6645, 2000) and assembled to an expression vector pCE17 that is capable of replicating in

Rhodococcus cells. The pCE58 vector contains an inducible promoter (e.g. pTrc, SEQ ID NO: 62). The sacB counter-selectable marker was also inserted into the pCE58 vector to cure from plasmid after recombination.

[000221] The pCE58 vector can be used to make a Rhodococcus strain electrocompetent by transforming the bacteria with the vector. Successful transformation may be verified by PCR amplification of the λ-Red Recombinase cassette from bacterial colonies. Transformed cells may then grow overnight. After dilution with fresh media, the bacterial cells can be induced for λ-Red Recombinase expression (e.g. by addition of isopropyl β-D-l- thiogalactopyranoside (IPTG)) thus making the Rhodococcus cells electrocompetent.

[000222] Electrocompetent Rhodococcus cells expressing the λ-Red Recombinase cassette can be transformed with PCR products obtained by amplification of an antibiotic cassette flanked by short DNA sequences homologous to the "arms" outside the region to be knocked- out. In some cases the antibiotic cassette is also flanked by recombination sites (e.g. FRT or Cre-LOX) to allow for removal of antibiotic cassette if desired. Transformed cells can be selected based on their capability to grow under antibiotic selection and successful gene deletion may be confirmed by colony PCR with primers designed to amplify the region of DNA knocked-out or a combination of a primer amplifying the inserted antibiotic cassette and another primer amplifying one of the flanking "arms".

Example 5

[000223] In this example, a two-step process was used to knock-out a dihydroperoate synthase gene in a Rhodococcus strain CEBl {Rhodococcus erythropolis DSM-43066). First, a knock-out plasmid is assembled by Gibson assembly from the following PCR fragments:

• pCE20 backbone (from pCE20 template, SEQ ID NO:5, with primers pCE20-F, SEQ ID NO:6 and pCE20-R, SEQ ID NO:7);

• Left arm Dihydroperoate synthase (SEQ ID NO: 3) of CEBl (from CEBl template with primers Up_RER_41670 Up (SEQ ID NO:8) and Up_RER_41670 Do (SEQ ID NO:9);

• Right arm Dihydroperoate synthase (SEQ ID NO:4) of CEBl (from CEBl template with primers Do_RER_41670 Up (SEQ ID NO: 10) and Do_RER_41670 Do (SEQ ID NO: 11).

[000224] These DNA fragments were generated by PCR amplification using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs) following the manufacturer' s instructions. DNA fragments were purified using a DNA Clean & Concentrator™- 5 kit from Zymo Research and assembled using the Gibson Assembly® Master Mix (New England Biolabs) according to the manufacturer's instructions. Assembled mix was used to transform New England Biolabs Turbo Competent E. coli. Transformation mixture was plated on LB- Kanamycin (50ug/ml) agarose plates. Successful assembly of the DNA fragments was confirmed by colony PCR using primers RER_41670 KO Check Up (SEQ ID NO: 13) and RER_41670 KO Check Do (SEQ ID NO: 14) and the Q5® Hot Start High-Fidelity 2X Master Mix. Colonies containing the correct plasmid (pCE41, SEQ ID NO: 12) were grown overnight in LB-Kanamycin (50ug/ml) liquid media and plasmid DNA was purified using a GeneJET Plasmid Miniprep Kit (Thermo Scientific).

[000225] Purified pCE41 DNA was used to transform CEBl as described in Example 2. After 2-3 days of growth, colony PCR was performed using primers RER_41670 KO Check Up (SEQ ID NO:13) and RER_41670 KO Check Do (SEQ ID NO: 14) to identify successful first single crossover, characterized by amplification of two bands. For the second crossover, positive colonies were streaked on LB -Sucrose 20% agarose plates and checked by colony PCR to distinguish between reversion to the wild-type and generation of the knock-out mutant. Gel electrophoresis shows analysis of the knock-out mutant and comparison to the wild- type microorganism, as shown in Figure 13.

Example 6

[000226] In this example, two plasmids were constructed. One plasmid is for knocking-out of the muconate cycloisomerase (catB) gene in Rhodococcus strain CEB2 (Rhodococcus erythropolis DSM-312), assembled by Gibson assembly. The second plasmid is for knocking-in of an antibiotic resistance cassette to replace the catB gene in CEB2, flanked by recombination sites for later removal if desired, in place of the catB gene. This approach can be applied for insertion of gene(s) and/or genetic elements to replace a gene or simply insert in any other location in the bacterial genome.

[000227] The knock-out plasmid pCE50 for deletion of catB was assembled using the following DNA fragments:

• catb left arm CEB002 (From CEB2 template with primers catb left arm CEB002 Up, SEQ ID NO: 15 and catb left arm CEB002 Do, SEQ ID NO: 16);

• catb right arm CEB002 (From CEB2 template with primer catb right arm CEB002 Up, SEQ ID NO:17 and catb right arm CEB002 Do, SEQ ID NO:18).

[000228] The knock-in plasmid pCE57 for the substitution of catB with a kanamycin resistance cassette was assembled using the following DNA fragments:

• pCE20 extraction (sacB-Ori no Kan), SEQ ID NO:31 (From pCE20 template, SEQ ID NO:5, with primers pCE20_extraction_(sacBOri_no_Kan) Rev, SEQ ID NO:25 and pCE20_extraction_(sacBOri_no_Kan) Fwd, SEQ ID NO:26);

• catb left arm CEB002, SEQ ID NO:29 (From CEB2 template with primers catB left arm CEB2 FRT FWD, SEQ ID NO: 19 and catB left arm CEB2 FRT Rev, SEQ ID NO:20);

• pKD4 Kan-FRT extraction, SEQ ID NO:32 (From pKD4 (Gene Bank accession No: AY048743) template with primers pKD4 Kan-FRT catB fwd, SEQ ID NO:23 and pKD4 Kan-FRT catB Rev, SEQ ID NO:24);

• catb right arm CEB002, SEQ ID NO:30 (From CEB2 template with primers catB right arm CEB2 FRT Bis Fwd, SEQ ID NO:21 and catB right arm CEB2 FRT Rev, SEQ ID NO:22).

[000229] The DNA fragments were generated by PCR amplification using Q5® Hot Start High- Fidelity 2X Master Mix (New England Biolabs) following the manufacturer' s instructions. DNA fragments were purified using a DNA Clean & Concentrator™- 5 kit from Zymo Research and assembled using the Gibson Assembly® Master Mix (New England Biolabs) according to the manufacturer's instructions. The assembled mix was used to transform NEB Turbo Competent E. coli (High Efficiency). The transformation mixture was plated on LB-Kanamycin (50ug/ml) agarose plates. Successful assembly of the DNA fragments was confirmed by using a colony PCR using primers catB CEB002 KO Check Do, SEQ ID NO:27 and catB CEB002 KO Check Up, SEQ ID NO:28 and the Q5® Hot Start High-Fidelity 2X Master Mix.

[000230] Colonies containing correct plasmid (pCE50, SEQ ID NO:33 or pCE57, SEQ ID NO: 34) were grown in LB-Kanamycin (50ug/ml) liquid media overnight and the plasmid DNA was purified using a GeneJET Plasmid Miniprep Kit (Thermo Scientific). Purified pCE57 DNA was used to transform CEB-2 as described in Example 2. After growing 2-3 days, the colony PCR was performed using primers catB CEB002 KO Check Do, SEQ ID NO:27 and catB CEB002 KO Check Up, SEQ ID NO:28 to identify successful first single crossover, characterized by the amplification of two bands. For the second crossover, positive colonies were streaked on LB -Sucrose 20% agarose plates and checked by using the colony PCR to distinguish between reversion to the wild-type microorganism and generation of the knock-out mutant. Gel electrophoresis, shown in Figure 14, shows analysis of different colonies of microorganisms after second recombination. The presence of two bands indicates that second recombination did not take place. The presence of a single upper band indicates a successful second recombination.

Example 7

[000231] In this example, two plasmids were constructed. One plasmid was for knocking-out of protocatechuate 3,4-dioxygenase (pcaGH) in a Rhodococcus strain CEB2 (Rhodococcus erythropolis DSM-312), assembled by Gibson assembly. The second plasmid is for knocking-in of an antibiotic resistance cassette to replace the pcaGH gene in CEB2, flanked by recombination sites for later removal if desired, in place of the deleted gene. [000232] The knock-out plasmid pCE51 , SEQ ID NO:49 for deletion of pcaGH was assembled using the following DNA fragments:

• pcaG-H left arm of CEB002, SEQ ID NO:47 (From CEB2 template with primers pcaG-H left arm CEB002 Up, SEQ ID NO:35 and pcaG-H left arm CEB002 Do, SEQ ID NO:36);

• pcaG-H right arm of CEB002, SEQ ID NO:48 (From CEB2 template with primers pcaG-H right arm CEB002 Up, SEQ ID NO: 37 and pcaG-H right arm CEB002 Do, SEQ ID NO:38).

[000233] The knock-in plasmid pCE55, SEQ ID NO:50 for the substitution of pcaG-H with a kanamycin resistance cassette was assembled using the following DNA fragments:

• pcaG-H left arm of CEB002, SEQ ID NO:47 (From CEB2 template with primers pcaG-H left arm CEB2 FRT FWD, SEQ ID NO:39 and pcaG-H left arm CEB2 FRT Rev, SEQ ID NO:40);

• pKD4 Kan-FRT extraction (From pKD4 (Gene Bank accession No:AY048743) template with primers pKD4 Kan-FRT pcaG-H fwd, SEQ ID NO:43 and pKD4 Kan-FRT pcaG-H Rev, SEQ ID NO:44);

• pcaG-H right arm CEB002, SEQ ID NO:48 (From CEB2 template with primers pcaG-H right arm CEB2 FRT FWD, SEQ ID NO:41 and pcaG-H right arm CEB2 FRT Rev, SEQ ID NO:42).

[000234] The DAN fragments were generated by PCR amplification using Q5® Hot Start High- Fidelity 2X Master Mix (New England Biolabs) following the manufacturer' s instructions and the primers listed below. DNA fragments were purified using a DNA Clean & Concentrator™-5 kit from Zymo Research and assembled using the Gibson Assembly® Master Mix (New England Biolabs) according to the manufacturer's instructions. The assembled mix was used to transform NEB Turbo Competent E. coli (High Efficiency). The transformation mixture was plated on LB-Kanamycin (50ug/ml) agarose plates. Successful assembly of the DNA fragments was confirmed by colony PCR using primers pcaG-H KO check Up CEB002, SEQ ID NO:45 and pcaG-H KO check Do CEB002, SEQ ID NO:46 and the Q5® Hot Start High-Fidelity 2X Master Mix.

[000235] Colonies containing correct plasmid (pCE50 or pCE57) were grown overnight in LB-Kanamycin (50ug/ml) liquid media overnight and plasmid DNA was purified using a GeneJET Plasmid Miniprep Kit (Thermo Scientific). Purified pCE55 DNA was used to transform CEB-2 as described in Example 2. After growing 2-3 days, colony PCR was performed using primers pCE20-F, SEQ ID NO:6 and pCE20-R, SEQ ID NO:7 to confirm integration of the plasmid into the chromosome of CEB2. Gel electrophoresis, shown in Figure 15, shows analysis of different colonies after the first recombination. The presence of a band of the size expected for pCE20 backbone indicates that the plasmid was successfully recombined. No band indicates either a failed PCR or no integration of the plasmid.

Example 8

[000236] In this example, a curable plasmid for adaptation of the λ-red recombinase system to Rhodococcus was constructed. An antibiotic cassette and a counterselectable marker were inserted into the inducible expression plasmid pCE17, SEQ ID NO:57, which is capable of replicating in both E. coli and Rhodococcus. The following DNA fragments were used:

• pCE17 backbone (From pCE17 template, SEQ ID NO:57 with primers pTrcHis2A Fwd, SEQ ID NO:51 and pTrcffis2A Rev, SEQ ID NO:52);

• lambda red extraction pKD46 (From pKD46 (Gene Bank accession No.

AY048746) template with primers lambda_red Up, SEQ ID NO:53 and lambda_red Do, SEQ ID NO:54);

• sacB_neg marker from pCE20, SEQ ID NO: 59 (From pCE20 template with

primers sacB Up, SEQ ID NO:55 and sacB Do, SEQ ID NO:56).

[000237] The DNA fragments were generated by PCR amplification using Q5® Hot Start High-Fidelity 2X Master Mix (New England Biolabs) following the manufacturer' s instructions and primers listed below. DNA fragments were purified using a DNA Clean & Concentrator™-5 kit from Zymo Research and assembled using the Gibson Assembly® Master Mix (New England Biolabs) according to the manufacturer's instructions. The assembled mix was used to transform NEB Turbo Competent E. coli (High Efficiency). The transformation mixture was plated on LB-Kanamycin (50ug/ml) agarose plates. Successful assembly of DNA fragments was confirmed by a colony PCR using primers lambda_red Up, SEQ ID NO:53 and lambda_red Do, SEQ ID NO:54 and the Q5® Hot Start High-Fidelity 2X Master Mix. Colonies containing correct plasmid (pCE58) were grown overnight in LB- Kanamycin (50ug/ml) liquid media and plasmid DNA was purified using a GeneJET Plasmid Miniprep Kit (Thermo Scientific). Purified pCE58 DNA was used to transform CEB-2 as described in Example 2. After growing 2-3 days, a colony PCR was performed using primers lambda_red Up, SEQ ID NO:53 and lambda_red Do, SEQ ID NO:54 to identify successful transformation.

Example 9

[000238] Coal or another other low value carbon source was wet milled to provide an aqueous slurry with a median particle size of about 20 μιη. The slurry was then fed to a continuous stirred-tank reactor (CSTR), operated in a batch or continuous mode. An alkali base such as NaOH was added to the aqueous slurry. 0₂ was introduced to the CSTR via pressurization of the headspace with compressed air or 0₂-enriched air in a batch mode, or via a continuous flow of air for continuous mode. The solid content, alkali base

concentration, temperature, pressure and stirring rate were adjusted to achieve various degrees of oxidative depolymerization of the low value carbon source.

Example 10

[000239] A bacterial strain Rhodococcus erythropoUs DSM-312 is transformed with plasmid pCE43 which contains an exogenous, ketolacid reductoisomerase gene. The exogenous ketolacid reductoisomerase gene is derived from E. coli (GenBank Accession Number: NC_..000913.3). The ketolacid reductoisomerase may be codon optimized for Rhodococcus erythropoUs. The genetically engineered Rhodococcus erythropoUs DSM-312 will have increased enzymatic activity for ketolacid reductoisomerase, which will lead to an increased yield/selectivity of isoamyl alcohol and/or isobutanol production.

Example 11

[000240] A bacterial strain Rhodococcus erythropoUs PR4 is genetically engineered to have an increased enzymatic activity of ketolacid reductoisomerase. The expression of the gene is enhanced (overexpression) by introducing a strong promoter: SV40 promoter for the gene. The position of the gene in the Rhodococcus erythropoUs PR4 genome may be found from GenBank Accession Number NC_012490.1. The genetically engineered Rhodococcus erythropoUs PR4 bacteria have overexpressed ketolacid reductoisomerase, which lead to increased enzymatic activity for ketolacid reductoisomerase. The increased enzymatic activity will lead to an increased yield/selectivity of isoamyl alcohol and/or isobutanol production.

Example 12

[000241] A bacterial strain Rhodococcus jostii RHA1 is genetically engineered to have a decreased enzymatic activity of pyruvate carboxylase. The pyruvate carboxylase gene in the Rhodococcus jostii RHA.1 may be found from GenBank Accession Number NC_008268.1. The gene in Rhodococcus jostii RHA1 is knocked out using a suicide vector pUC57-Kan from E. coli. The genetically engineered Rhodococcus jostii RHA1 bacteria have a decreased enzymatic activity for pyruvate carboxylase. The increased enzymatic activity will lead to an increased yield/selectivity of isoamyl alcohol and/or isobutanol production.

[000242] It is to be understood, however, that even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and function of the invention, the disclosure is illustrative only, and changes may be made in detail, especially in matters of shape, size and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meanings of the terms in which the appended claims are expressed.

Claims

WHAT IS CLAIMED IS:

1. A method for synthesizing isoamyl alcohol and/or isobutanol from a low value carbon source, comprising steps of:

oxidizing the low value carbon source with at least one oxidizing agent to produce a modified, partially solubilized low value carbon source; and

contacting the modified, partially solubilized low value carbon source with a genetically engineered microorganism adapted for growth in the modified, partially solubilized low value carbon source, which is derived from a wild-type microorganism comprising an isobutanol and/or isoamyl alcohol biosynthesis pathway from pyruvate,

wherein the genetically engineered microorganism comprises a genetic modification selected from:

increasing an enzymatic activity of at least one enzyme in the isobutanol and/or isoamyl alcohol biosynthesis pathway relative to the activity of the same enzyme in the wild- type microorganism, and

decreasing an enzymatic activity of at least one enzyme in a pathway competitive to the isobutanol and/or isoamyl alcohol biosynthesis pathway relative to the activity of the same enzyme in the wild-type microorganism.

2. The method of claim 1 , further comprising a step of adding at least one other microorganism to the modified, partially solubilized low value carbon source to convert one or more components in the modified, partially solubilized low value carbon source to intermediates that are metabolizable by the genetically engineered microorganisms

3. The method of any one of claims 1-2, further comprising a step of adding at least one nutrient to the modified, partially solubilized low value carbon source wherein the at least one nutrient is metabolizable by the genetically engineered microorganism.

4. The method of any one of claims 1-3, further comprising a step of adding at least one other enzyme to the modified, partially solubilized low value carbon source to convert one or more components in the modified, partially solubilized low value carbon source to one or more intermediates capable of being metabolized by the genetically engineered

microorganism.

5. The method of any one of claims 1-4, wherein the low value carbon source is selected from coal, lignite, tar sands, tars, crude oils, peat, pitch, resins, lignin, latex rubber, waxes, agricultural wastes, bark, wood, lack liquor, band algae cake.

6. The method of any one of claims 1-5, further comprising a step of selecting the microorganism by testing a growth rate of the microorganism in the modified, partially solubilized low valued carbon source.

7. The method of any one of claims 1-6, wherein the microorganism is selected from a genus of Escherichia, Cory neb acterium, Clostridium, Zymonomas, Salmonella,

Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klesiella, Paenibacillus, Arthrobacter, Br evib acterium, Pichia, Candida, Hansenula, Synechococcus, Synechocystis, Anabaena, Ralstonia, Lactococcus, or Saccharomyces.

8. The method of any one of claims 1-7, wherein the wild-type microorganism has an IC₅₀ concentration of isoamyl alcohol or isobutanol at greater than about 3 wt.%, or greater than about 5 wt.%, or greater than about 7 wt.%, or greater than about 9 wt.%, or greater than about 10 wt.%.

9. The method of any one of claims 1-8, wherein the wild-type microorganism is selected from a genus Rhodococcus.

10. The method of any one of claims 1-9, wherein the at least one enzyme in the isobutanol and/or isoamyl alcohol biosynthesis pathway is selected from acetolactate synthase, ketolacid reductoisomerase, dihydroxy-acid dehydrogenase, 2-isopropylmalate synthase, 3-isopropylmalate dehydratase, 3-isopropylmalate dehydrogenase, ketoisovalerate decarboxylase, and alcohol dehydrogenase.

11. The method of any one of claims 1-10, wherein the at least one enzyme in the competitive pathway is selected from valine:2-oxoglutarate aminotransferase, valine-pyruvate aminotransferase, leucine:2-oxoglutarate aminotransferase, branched-chain-amino-acid transaminase, pyruvate decarboxylase, malate dehydrogenase, pyruvate carboxylase, malate dehydrogenase, fumarate hydratase, pyruvate dehydrogenase, lactate dehydrogenase and formate C-acetyltransferase.

12. The method of claim 11, wherein the at least one enzyme in the competitive pathway is inactivated in the genetically engineered microorganism.

13. The method of any one of claims 1-12, wherein the genetically engineered microorganism further comprises an increased enzymatic activity of at least one enzyme for generation of NAD(P)H relative to the activity of the same enzyme in the wild-type microorganism.

14. The method of claim 13, wherein the at least one enzyme for generation of NAD(P)H is selected from pyridine nucleotide transhydrogenase and glyceraldehyde-3-phosphate dehydrogenase.

15. The method of any one of claims 1-14, wherein the genetically engineered microorganism further comprises an increased enzymatic activity of at least one enzyme for generation of pyruvate relative to the activity of the same enzyme in the wild-type microorganism.

16. The method of claim 15, wherein the at least one enzyme for generation of pyruvate is selected from enzymes in a glycolysis pathway.

17. The method of claim 15, wherein the at least one enzyme for generation of pyruvate is selected from hexokinase, phosphoglucoisomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde- 3 -phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase.

18. The method of any one of claims 1-17, wherein the contacting step comprises a fermentation process selected from a batch fermentation process and a continuous fermentation process.

19. The method of any one of claims 1-18, further comprises a step of immobilizing the genetically engineered microorganism on a support structure.

20. The method of claim 19, wherein the genetically engineered microorganism is immobilized using a method selected from adsorption immobilization, self-immobilization, embedding immobilization, microencapsulation and crosslinking immobilization.

21. The method of claim 20, wherein the microencapsulation uses a polymer selected from agar, alginate, carrageenan, cellulose and its derivatives, collagen, gelatin, epoxy resin, photo cross-linkable resin, polyacrylamide, polyester, polystyrene and polyurethane.