Microorganisms and methods for reducing by-products

Abstract

The present disclosure provides microbial organisms whose undesirable byproducts (e.g., pyruvate-, CO 2 ‑, TCA‑derived byproducts; acetic acid; ethanol; and / or alanine) to enhance carbon flux through acetyl‑CoA, which can increase the production of acetyl‑CoA derived compounds (e.g., 1,3‑BDO, MMA, and (3R)‑hydroxybutyl (3R)‑hydroxybutyrate or any other acetyl‑CoA derived compounds) and products prepared from any of these compounds. Also provided are exogenous nucleic acids encoding one or more enzymes (e.g., aldehyde dehydrogenases, acetyl‑CoA synthases, D‑amino acid dehydrogenases, alanine racemases, and / or citrate synthases) that can reduce the production of unwanted byproducts, and / or one or more gene attenuations that occur in genes (e.g., acetolactate synthase) that cause reduced production of unwanted byproducts. The present disclosure also provides various combinations of exogenous nucleic acids and gene deletions. Also provided are methods of preparing and using the above-mentioned microbial organisms, exogenous genes, gene attenuations, and combinations, including methods of culturing cells and methods of producing various products.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 928,183, filed on October 30, 2019, the entire contents of which are incorporated herein by reference. 1. Technical Field

[0003] The present invention generally relates to organisms engineered to produce desired products, engineered enzymes that promote the production of desired products, and more specifically, to non-naturally occurring organisms that can reduce byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA and thereby increase one or more acetyl-CoA-derived products, including but not limited to 1,3-butanediol (1,3-BDO), methyl methacrylate (MMA), (3R)-hydroxybutyl (3R)-hydroxybutyrate, 3-hydroxybutyric acid (3-HB), hexamethylenediamine (HMDA), caprolactam, adipic acid, 6-aminocaproic acid (6-ACA), methacrylic acid (MAA), fatty acid methyl esters (FAME), and related products derived therefrom. 2. Background technology

[0004] Microbial organisms can be used to produce acetyl-CoA derivative compounds, such as 1,3-BDO, fatty acid methyl esters (e.g., (3R)-hydroxybutyl (3R)-hydroxybutyrate). The titer, rate, and yield of such production can be limited by the production of unwanted byproducts. In particular, the production of unwanted byproducts, such as pyruvate byproduct, acetate, and / or ethanol, can limit the amount of acetyl-CoA available for the production of desired acetyl-CoA derivative products. For example, the production of valine, a pyruvate byproduct, can limit the amount of pyruvate available for conversion to acetyl-CoA. Similarly, the production of unwanted ethanol and / or acetate from acetyl-CoA can limit the amount of acetyl-CoA available for conversion to desired acetyl-CoA byproducts. Therefore, reducing the production of unwanted byproducts, such as pyruvate byproduct, acetate, and / or ethanol, can help increase the titer, rate, and yield of acetyl-CoA derivative compounds, such as 1,3-BDO, MMA, and (3R)-hydroxybutyl (3R)-hydroxybutyrate.

[0005] 1,3-BDO is a four-carbon diol that is traditionally produced from acetylene by hydration. The resulting acetaldehyde is then converted to 3-hydroxybutyraldehyde, which is subsequently reduced to form 1,3-BDO. More recently, acetylene has been replaced by cheaper ethylene as a source of acetaldehyde. 1,3-BDO is commonly used as an organic solvent for food flavorings. It is also used as a comonomer for polyurethane and polyester resins and is widely used as a hypoglycemic agent. Optically active 1,3-BDO is a useful raw material for the synthesis of bioactive compounds and liquid crystals. Another use of 1,3-butanediol is that it is dehydrated to give 1,3-butadiene (Ichikawa et al. Journal of Molecular Catalysis A-Chemical 256:106-112 (2006); Ichikawa et al. Journal of Molecular Catalysis A-Chemical 231:181-189 (2005), which can be used to make synthetic rubber (e.g., tires), latex, and resins. The dependence of acetylene or ethylene on petroleum-based feedstocks warrants the development of routes based on renewable feedstocks for 1,3-butanediol and butadiene.

[0006] MMA is an organic compound with the molecular formula CH2=C(CH3)CO2CH3. This colorless liquid is methyl methacrylate (MAA), the monomer that produces the transparent plastic polymethyl methacrylate (PMMA). The main application of methyl methacrylate is in the production of polymethyl methacrylate acrylic plastics. In addition, methyl methacrylate is used to produce methyl methacrylate-butadiene-styrene copolymer (MBS), which is used as a PVC modifier. Methyl methacrylate polymers and copolymers are used in water-based coatings, such as latex paints. They are also used in adhesive formulations. Contemporary applications include flat panels used to keep light evenly distributed on liquid crystal display (LCD) computer and TV screens. Methyl methacrylate is also used to prepare corrosion models of anatomical organs, such as the coronary arteries of the heart.

[0007] It has been demonstrated that the intake of compounds and compositions containing (R)-3-hydroxybutyric acid derivatives, such as (3R)-hydroxybutyl (3R)-hydroxybutyrate, can increase the level of ketone bodies in the blood. Ketone bodies are compounds produced when fatty acids are metabolized by the body for energy, which in turn results in the ketone bodies themselves being used for energy. Ketone bodies have been shown to be suitable for reducing the level of free fatty acids circulating in an individual's plasma. Ingestion of ketone bodies can also bring various clinical benefits, including enhancing physical and cognitive abilities, treating cardiovascular disease, diabetes, and treating mitochondrial dysfunction disorders, as well as treating muscle fatigue and injury. However, direct administration of ketone bodies is impractical and dangerous. For example, direct administration of any (R)-3-hydroxybutyric acid can result in significant acidosis following rapid absorption from the gastrointestinal tract. Administration of the sodium salts of these compounds is also inappropriate due to the potentially harmful sodium overload that accompanies administration of therapeutically relevant amounts of these compounds. Administration of (R)-3-hydroxybutyric acid derivatives in oligomeric form has been used to circumvent this problem. To achieve the desired therapeutic and other benefits, ketone bodies generally need to be present in an individual's plasma at a threshold level, such as at least 1 mM (3R)-hydroxybutyl (3R)-hydroxybutyrate. However, production is hampered by low yields or the impracticality of large-scale production.

[0008] Therefore, there is a need to develop methods to reduce the production of unwanted byproducts such as pyruvate byproduct, acetate and / or ethanol) to improve efficiency and effectively produce commercial quantities of compounds such as 1,3-BDO, MMA and (3R)-hydroxybutyl (3R)-hydroxybutyrate. The present invention satisfies these needs and also provides related advantages. Other product molecules that can be produced by the teachings of the present invention include any acetyl-CoA derivative products, including but not limited to adipic acid, caprolactam, 6-aminocaproic acid (6-ACA), hexamethylenediamine (HMDA) or methacrylic acid (MAA). 3. Summary of the invention

[0009] In one aspect, provided herein are non-naturally occurring microbial organisms with reduced byproducts, including a microbial organism having a glycolytic pathway and enhanced carbon flux through acetyl-CoA, the microbial organism comprising one or more of: (a) an attenuated acetolactate synthase; (b) an acetaldehyde recycling loop.

[0010] In some embodiments, the attenuated acetolactate synthase comprises a lack of acetolactate synthase. In some embodiments, the attenuated acetolactate synthase comprises a non-functional acetolactate synthase. In some embodiments, the acetolactate synthase is ilvG. In some embodiments, the attenuated acetolactate synthase reduces the biosynthesis of valine.

[0011] In some embodiments, the acetaldehyde recycling loop comprises at least one exogenous nucleic acid encoding an acetaldehyde recycling loop enzyme selected from the group consisting of an aldehyde dehydrogenase and an acetyl-CoA synthase. In some embodiments, the aldehyde dehydrogenase is AldB. In some embodiments, the acetyl-CoA synthase is an acetyl-CoA synthase variant. In some embodiments, the at least one exogenous nucleic acid is a heterologous nucleic acid.

[0012] In certain embodiments, the non-naturally occurring microbial organisms of the present disclosure are reduced in acetate, ethanol, or a combination thereof.

[0013] In some embodiments, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

[0014] In some embodiments, the non-naturally occurring microbial organisms of the present disclosure include a 1,3-butanediol (1,3-BDO) pathway, a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway, a 3-hydroxybutyryl-CoA (3HB-CoA) pathway, a methyl methacrylate (MMA) pathway, an adipate pathway, a caprolactam pathway, a 6-aminocaproic acid (6-ACA) pathway, a hexamethylenediamine (HMDA) pathway, or a methacrylic acid (MAA) pathway.

[0015] In some embodiments, the microbial organism comprises a 1,3-BDO pathway. In specific embodiments, the 1,3-BDO pathway comprises a thiolase; an acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming); a 3-oxobutyraldehyde reductase (ketone reducing); a 3-hydroxybutyraldehyde reductase; an acetoacetyl-CoA reductase (CoA-dependent, alcohol forming); a 3-oxobutyraldehyde reductase (aldehyde reducing); a 4-hydroxy, 2-butanone reductase; an acetoacetyl-CoA reductase (ketone reducing); a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and a 3-hydroxybutyryl-CoA reductase (alcohol forming).

[0016] In some embodiments, the microbial organism comprises a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway. In specific embodiments, the (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway comprises a thiolase; a (3R)-hydroxybutyl (3R)-hydroxybutyrate forming enzyme; a (3R)-hydroxybutyryl-CoA:(R)-1,3-butanediol alcohol transferase; a (3R)-hydroxybutyl 3-oxobutyrate forming enzyme; an acetoacetyl-CoA:(R)-1,3-butanediol alcohol transferase; a (3R)-hydroxybutyl 3-oxobutyrate reductase; a (3R)-hydroxybutyryl-ACP:(R)-1,3-butanediol ester synthase and an acetoacetyl-ACP:(R)-1,3-butanediol ester synthase.

[0017] In some embodiments, the microbial organism comprises a 3HB-CoA pathway. In specific embodiments, the 3HB-CoA pathway comprises an acetyl-CoA thiolase and a 3-hydroxybutyryl-CoA dehydrogenase.

[0018] In some embodiments, the microbial organism comprises an MMA pathway. In specific embodiments, the MMA pathway comprises: (a) 4-hydroxybutyryl-CoA dehydratase, crotonase, 2-hydroxyisobutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase, and methacrylic acid (MAA)-CoA:methanol transferase; or (b) 4-hydroxybutyryl-CoA dehydratase, crotonase, 2-hydroxyisobutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA:methanol transferase, and 2-hydroxyisobutyrate methyl dehydratase. In some embodiments, the non-naturally occurring microbial organism having an MMA pathway also includes a second MMA pathway comprising: (c) methacrylic acid (MAA)-CoA:methanol transferase, 4-hydroxybutyryl-CoA mutase, and 3-hydroxyisobutyryl-CoA dehydratase; or (d) 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA:methanol transferase, and 3-hydroxyisobutyrate methyl dehydratase.

[0019] In some embodiments, the microbial organism comprises a 6-ACA pathway. In specific embodiments, the 6-ACA pathway comprises 2-amino-7-oxosuberic acid ketoacid decarboxylase, 2-amino-7-oxoheptanoic acid decarboxylase, 2-amino-7-oxoheptanoic acid oxidoreductase, 2-aminopimelate decarboxylase, 6-aminohexanal oxidoreductase, 2-amino-7-oxoheptanoic acid decarboxylase, or 2-amino-7-oxosuberic acid amino acid decarboxylase.

[0020] In some embodiments, the non-naturally occurring microbial organism comprises a caprolactam pathway. In specific embodiments, the caprolactam pathway comprises 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipoyl-CoA reductase (aldehyde forming), 6-aminohexanoate transaminase, 6-aminohexanoate dehydrogenase, 6-aminohexanoyl-CoA / acyl-CoA transferase, and 6-aminohexanoyl-CoA synthase.

[0021] In some embodiments, the microbial organism comprises an adipate pathway. In specific embodiments, the adipate pathway comprises 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA hydrolase, adipyl-CoA ligase, adipyl-CoA transferase, and phosphotransferase / adipate kinase.

[0022] In some embodiments, the microbial organism comprises a hexamethylenediamine (HMDA) pathway. In specific embodiments, the HMDA pathway comprises 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA reductase (aldehyde forming), 6-aminohexanoate transaminase, 6-aminohexanoate dehydrogenase, 6-aminohexanoyl-CoA / acyl-CoA transferase, 6-aminohexanoyl-CoA synthase, 6-aminohexanoyl-CoA reductase (aldehyde forming), HMDA transaminase, and HMDA dehydrogenase.

[0023] In some embodiments, the microbial organism comprises a MAA pathway. In specific embodiments, the MAA pathway comprises: (a) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA epimerase; (iv) a methylmalonyl-CoA reductase (aldehyde forming); (v) a methylmalonate semialdehyde reductase; (vi) a 3-hydroxyisobutyrate dehydratase; (b) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA reductase (aldehyde forming); (iv) a methylmalonate semialdehyde reductase; and (v) a 3-hydroxyisobutyrate dehydratase; or (c) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA reductase (alcohol forming); and (iv) a 3-hydroxyisobutyrate dehydratase.

[0024] In some embodiments, the non-naturally occurring microorganisms provided herein are bacteria, yeast, or fungi.

[0025] In another aspect, provided herein are methods for enhancing carbon flux through acetyl-CoA in a non-naturally occurring microbial organism to increase the yield of an acetyl-CoA-derived product, the method comprising culturing the non-naturally occurring microbial organism of the present disclosure under conditions and for a sufficient period of time to produce the acetyl-CoA-derived product.

[0026] In some embodiments, the acetyl-CoA derivative product is selected from the group consisting of 1,3-butanediol (1,3-BDO), methyl methacrylate (MMA), 3R-hydroxybutyric acid-3R-hydroxybutyric acid, 3-hydroxybutyric acid (3-HB), 4-hydroxy-2-butanone (4OH2B), hexamethylenediamine (HMDA), caprolactam, adipic acid, 6-aminocaproic acid (6-ACA) and methacrylic acid (MAA).

[0027] In some embodiments, the acetyl-CoA derivative product comprises 1,3-BDO. In other embodiments, the acetyl-CoA derivative product comprises MMA. In another embodiment, the acetyl-CoA derivative product comprises 3R-hydroxybutyrate-3R-hydroxybutyrate. 4. Description of the drawings

[0028] Figure 1A-Figure 1C The ilvGM deletion strains (L16410 and L16411) showed that the specific 1,3-BDO production ( Figure 1A ),rate( Figure 1B ) and yield [c-mol%] outperformed the control (L16375) ( Figure 1C ), and is independent of the oxygen transfer rate (OTR).

[0029] Figure 2A-2C showed an inverse relationship between valine production and 1,3-BDO yield among different strains ( Figure 2A For each culture volume, the ilvGM deletion strains (L16410 and L16411) had negligible valine levels ( Figure 2B ), and the level of 1,3-BDO increased ( Figure 2C ).

[0030] Figure 3 An exemplary 1,3-BDO production pathway is shown, involving the production of pyruvate by glycolysis, the conversion of pyruvate to acetyl-CoA, and the conversion of acetyl-CoA to 1,3-BDO. Unwanted byproducts ethanol (EtOH) and acetate can be generated from acetyl-CoA by ALD and ADH or by acetyl-CoA hydrolase / thioesterase.

[0031] Figure 4 Exemplary 1,3-BDO production pathways are shown that include an acetaldehyde recycling loop that converts acetaldehyde to acetate via aldB and / or acetate to acetyl-CoA via ACS, which can then be converted to 1,3-BDO.

[0032] Figure 5 A specific AldB enzyme was shown to have specific activity towards acetaldehyde but not 3-HB aldehyde.

[0033] Fig. 6A and Figure 6B The results show that expression of ACS* increased the production of 1,3-BDO in strains L16946 and L17787, respectively, relative to non-ACS* expressing strains L16768 and L17787 ( Fig. 6A ). In addition, ACS* expression significantly reduced acetate but not ethanol formation, indicating that acetate recycling effectively competes with CoA hydrolases ( Figure 6B).

[0034] Figure 7A-7D The overall product distribution demonstrated that the expression of the strain was significantly higher than that of the non-ACS* expression strain ( Fig. 7A and Figure 7B ), the expression of ACS* significantly reduced the byproducts ( Figure 7C ) and increased 1,3-BDO production ( Fig.7D ).

[0035] Figure 8 It was shown that co-expression of ACS* and AldB2886B significantly reduced the production of acetate and ethanol. In addition, the overall carbon-2 (i.e., ethanol and acetate) reduction was similar between the two strains.

[0036] Fig.9A and Fig. 9B It was shown that strains overexpressing ACS* and an alternative AldB candidate (AldB 1139A), with or without a NadK variant (NadK*), resulted in increased 1,3-BDO relative to their respective control strains without ACS* and AldB overexpression ( Fig.9A ) and reductions in ethanol and acetic acid ( Fig. 9B ).

[0037] Fig.10 An exemplary 1,3-BDO production pathway is shown, involving the production of pyruvate by glycolysis, and the conversion of pyruvate to acetyl-CoA or L-alanine. An alanine recycling loop involving dadX and dadA converts L-alanine to D-alanine and D-alanine to pyruvate, respectively, thereby reducing unwanted alanine production.

[0038] Figure 11A-11B The alanine recycle loop was shown to reduce alanine concentration levels across multiple aeration parameters. After fermentation, microorganisms expressing dadX and dadA ("recycled"; open bars) had lower levels of D-alanine, L-alanine, total alanine, and L-alanine / D-alanine ratios relative to control microorganisms without an alanine recycle loop (black bars). Fig.11A Measurements of alanine concentration (mmol / L) throughout the fermentation also showed that alanine levels were lower in the microorganism with an alanine recycling loop (solid grey line) relative to the control microorganism without an alanine recycling loop (dashed black line). Fig. 11B ).

[0039] Fig.12Exemplary 1,3-BDO production pathways involving the use of acetyl-CoA as a substrate for conversion to 1,3-BG or to citrate by citrate synthase (gltA) are shown. The citrate synthase variant (gltAR109L, "gltA*") has increased sensitivity to NADH relative to wild-type gltA.

[0040] Fig.13A and Fig. 13B Microorganisms expressing the citrate synthase variant gltA* (grey dashed line) show a rapid microaerobic shift relative to microorganisms expressing wild-type gltA (black solid line), as indicated by the arrows ( Fig.13A ). The 1,3-BDO titer of microorganisms expressing the citrate synthase variant gltA* (grey bars) was increased relative to that of microorganisms expressing wild-type gltA (black bars). Fig. 13B ). 5. Specific implementation methods

[0041] The present invention relates in part to reducing byproducts such as pyruvate byproducts (e.g., alanine and / or valine), tricarboxylic acid cycle (TCA) derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, thereby increasing engineered biosynthetic pathways for acetyl-CoA derived products. Exemplary product molecules include, but are not limited to, 1,3-butanediol (1,3-BDO), methyl methacrylate (MMA), (3R)-hydroxybutyl (3R)-hydroxybutyrate, 4-hydroxy-2-butanone (4OH2B), 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA, although in view of the teachings and guidance provided herein, it will be appreciated by those skilled in the art that any product molecule derived from acetyl-CoA can exhibit enhanced product production through reduction of byproducts. The present invention provides non-naturally occurring microbial organisms having one or more exogenous genes encoding enzymes and / or one or more attenuating enzymes that can reduce the production of byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA derived byproducts, acetate and / or ethanol. In some embodiments, these non-naturally occurring microbial organisms also have one or more exogenous genes encoding enzymes that can catalyze the production of a desired product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA.

[0042] In many engineering pathways, the achievement of maximum product yields based on carbohydrate feedstocks is hindered by the production of unwanted byproducts. According to some embodiments, the present invention increases the yield of acetyl-CoA products by (i) reducing the production of pyruvate byproducts such as valine to increase the conversion of pyruvate to acetyl-CoA, (ii) recycling unwanted pyruvate byproducts such as alanine back to pyruvate to increase the availability of pyruvate for conversion to acetyl-CoA, (iii) reducing the entry of acetyl-CoA into the TCA cycle, and / or (iv) recycling acetyl-CoA byproducts such as acetate and / or ethanol back to acetyl-CoA. Products that can be produced by non-naturally occurring organisms and methods described herein include, for example, but not limited to, 1,3-BDO, fatty acid methyl esters (e.g., MMA), (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA.

[0043] As used herein, the term "non-naturally occurring" when used to refer to the microbial organisms or microorganisms of the present invention is intended to mean that the microbial organism has at least one genetic alteration that is not normally present in naturally occurring strains of the species referred to (including wild-type strains of the species referred to). Genetic alterations include, for example, modifications that introduce expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions, and / or other functional disruptions of the genetic material of the microbial organism. Such modifications include, for example, coding regions of heterologous, homologous, or heterologous and homologous polypeptides of the species referred to, and functional fragments thereof. Other modifications include, for example, non-coding regulatory regions in which the modification alters the expression of a gene or operon. For example, exemplary metabolic polypeptides include enzymes or proteins that cause an increase or decrease in metabolic factors. An exemplary metabolic factor includes acetyl-CoA. Exemplary metabolic factors also include, for example, 1,3-butanediol (1,3-BDO), methyl methacrylate (MMA), and / or 3-hydroxybutyrate (3-HB). Other exemplary metabolic factors include, for example, acetolactate synthase, aldehyde dehydrogenase, and acetyl-CoA synthase.

[0044] As used herein, the terms "microbial," "microbial organism," or "microorganism" are intended to mean any organism that exists as a microscopic cell included within the domains Archaea, Bacteria, or Eukaryota. Thus, the term is intended to encompass prokaryotic or eukaryotic cells or organisms of microscopic size, and includes bacteria, archaea, and eubacteria of all species, as well as eukaryotic microorganisms, such as yeast and fungi. The term also includes cell cultures of any species that can be cultured to produce a biochemical.

[0045] As used herein, the term "byproduct" refers to an unwanted product produced during the production of a desired product. For example, as provided herein, the production of, for example, valine and / or alanine from pyruvate can be considered as a byproduct of the desire to convert pyruvate into acetyl-CoA. Similarly, exemplary byproducts include acetic acid from acetyl-CoA when it is desired to convert acetyl-CoA into 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product.

[0046] As used herein, the phrase "carbon flux enhancement" or "carbon flow enhancement" is intended to mean strengthening, increasing or further improving the degree or flow of metabolic carbon through or to a desired pathway, pathway product, intermediate or biologically derived compound. The intensity, increase or improvement can be relative to a predetermined baseline of a pathway product, intermediate or biologically derived compound. For example, an increase in acetyl-CoA yield can be achieved using an acetolactate synthase attenuated as described herein compared to a functional acetolactate synthase. Similarly, an increase in acetyl-CoA yield can be achieved by expressing one or more enzymes of an acetaldehyde recycling loop pathway compared to an enzyme in which an acetaldehyde recycling loop pathway is not present. It should be understood that, since an increase in acetyl-CoA yield can be achieved, any acetyl-CoA derivative compound of a higher yield, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA, can also be achieved.

[0047] As used herein, the term "recycling loop" refers to one or more reactions that convert a byproduct back into a substrate that can subsequently be metabolized and redirected into a desired product. For example, exemplary recycling loops provided herein include acetaldehyde recycling loops that, for example, convert byproduct acetaldehyde into acetyl-CoA to reduce acetaldehyde and increase the yield of a desired acetyl-CoA derivative product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product. Similarly, another exemplary recycling loop includes an alanine recycling loop that, for example, converts a byproduct alanine into pyruvate to reduce alanine levels and increase carbon flux from pyruvate to acetyl-CoA, thereby increasing the yield of a desired acetyl-CoA derivative product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product.

[0048] As used herein, the term "decay" or its grammatical equivalents are intended to represent the activity or amount of weakening, reducing or reducing enzymes or proteins. If the decay causes the enzyme or protein activity or amount to drop below the critical level required for a given pathway, reaction or series of reactions to play a role, the decay of the activity or amount can simulate complete destruction. However, the decay of the activity or amount of the enzyme or protein that simulates the complete destruction of a pathway, reaction or series of reactions is still enough to allow different pathways, reactions or series of reactions to continue to play a role. For example, the decay of endogenous enzymes or proteins may be enough to simulate the complete destruction of the same enzyme or protein that produces valine, but the remaining activity or amount of the enzyme or protein may still be enough to maintain other pathways, such as pathways that are vital to the survival, reproduction or growth of host microorganisms. The decay of enzymes or proteins can also be enough to increase the yield of the factor of the present invention (such as acetyl-CoA derivatives) but not necessarily simulate the amount of the complete destruction of enzymes or proteins, weaken, reduce or reduce the activity or amount of enzymes or proteins.

[0049] As used herein, "exogenous" means that the mentioned molecule or the mentioned activity is introduced into the host microorganism. For example, the molecule can be introduced by introducing the encoding nucleic acid into the host genetic material, for example, by integration into the host chromosome, or as a non-chromosomal genetic material, such as a plasmid. Therefore, when used to refer to the expression of the encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid in an expressible form into the microorganism. When used to refer to a biosynthetic activity, the term refers to the activity introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the mentioned activity after being introduced into the host microorganism. Therefore, the term "endogenous" refers to the mentioned molecule or activity present in the host. Similarly, when used to refer to the expression of the encoding nucleic acid, the term refers to the expression of the encoding nucleic acid contained in the microorganism. The term "heterologous" refers to a molecule or activity derived from a source other than the mentioned species, while "homologous" refers to a molecule or activity derived from the host microorganism. Therefore, the exogenous expression of the encoding nucleic acid of the present invention can utilize one or both of the heterologous or homologous encoding nucleic acids.

[0050] It should be understood that, as described above, when a microbial organism includes more than one exogenous nucleic acid, the more than one exogenous nucleic acid refers to the coding nucleic acid or biosynthetic activity mentioned. It should also be understood that, as disclosed herein, such more than one exogenous nucleic acid can be introduced into the host microbial organism on a separate nucleic acid molecule, on a polycistronic nucleic acid molecule, or a combination thereof, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein, a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired enzyme or protein required for a pathway, reaction, or series of reactions. In the case of introducing two exogenous nucleic acids encoding the desired activities into the host microbial organism, it should be understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on a separate plasmid, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it should be understood that more than two exogenous nucleic acids can be introduced into the host organism in any desired combination, for example, on a single plasmid, on a separate plasmid, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example, three exogenous nucleic acids. Thus, reference to the amount of exogenous nucleic acid or biosynthetic activity refers to the amount of encoding nucleic acid or the amount of biosynthetic activity, and not the amount of the individual nucleic acid introduced into the host organism.

[0051] As used herein, the term "variant" is intended to mean an enzyme form or pattern that is different from the wild-type enzyme. An exemplary variant is a mutant form of an enzyme, wherein the amino acid sequence of the variant enzyme is different from the amino acid sequence at one or more homologous amino acids. The variant may have a different function or activity relative to the wild-type enzyme. However, a variant need not be a mutant, and may include polymorphisms, paralogs, or orthologs.

[0052] As used herein, the term "substantially anaerobic" when used to refer to culture conditions or growth conditions means that the amount of oxygen is less than about 10% of the saturation amount of dissolved oxygen in a liquid medium, and is considered to be of this meaning. The term maintains an oxygen atmosphere of less than about 1%, and also includes sealed chambers of liquid or solid culture medium.

[0053] As used herein, the term "CoA" or "coenzyme A" means an organic cofactor or prosthetic group (non-protein portion of an enzyme) whose presence is required for many enzymes (apoenzymes) to form an active enzyme system. Coenzyme A plays a role in certain condensing enzymes, in acetyl or other acyl transfer, in fatty acid synthesis and oxidation, pyruvate oxidation, and in other acetylation.

[0054] As used herein, the term "1,3-butanediol" or "1,3-BDO" is intended to mean a diol having the chemical formula C4H 10One of the four stable isomers of butanediol with a molecular weight of 90.12 g / mol and 0.2. The compound 1,3-butanediol is known in the art as 1,3-butylene glycol (1,3-BG) and is also a chemical intermediate or precursor for a family of compounds generally referred to as BDO family compounds.

[0055] As used herein, "methyl methacrylate" or "MMA" having the chemical formula CH2=C(CH3)CO2CH3 and a molecular weight of 100.12 g / mol is the methyl ester of methacrylic acid (MAA). MMA is used as a monomer in the production of the transparent plastic polymethyl methacrylate (PMMA).

[0056] As used herein, the term "(3R)-hydroxybutyl (3R)-hydroxybutyrate" refers to a compound of formula (I):

[0057]

[0058] The term (3R)-hydroxybutyl (3R)-hydroxybutyrate is used interchangeably throughout with the terms (R)-(R)-3-hydroxybutyl 3-hydroxybutyrate, (3R)hydroxybutyl (3R)-hydroxybutyrate, and (R)-3-hydroxybutyl (R)-3-hydroxybutyrate.

[0059] As used herein, the term "gene disruption" or its grammatical equivalent is intended to represent a genetic alteration that renders the encoded gene product inactive. The genetic alteration can be, for example, the deletion of the entire gene, the deletion of regulatory sequences required for transcription or translation, the deletion of a gene portion resulting in a truncated gene product, or any of a variety of mutation strategies by which the encoded gene product is inactivated. A particularly useful gene disruption method is complete gene deletion because it reduces or eliminates the occurrence of gene back mutations in the non-naturally occurring microorganisms of the present invention.

[0060] The non-naturally occurring microbial organisms of the present invention can contain stable genetic alterations, which means that the microorganism can be cultured for more than five generations without losing the alteration. Generally, stable genetic alterations include modifications that persist for more than 10 generations, particularly stable modifications that persist for more than about 25 generations, and more particularly, stable genetic modifications that persist for greater than 50 generations, including indefinitely.

[0061] In the case of gene disruption, a particularly useful stable genetic alteration is gene deletion. The use of gene deletion to introduce a stable genetic alteration is particularly useful for reducing the likelihood of reversion to the phenotype prior to the genetic alteration. For example, stable growth-coupled production of biochemicals can be achieved by deleting genes encoding enzymes that catalyze one or more reactions in a set of metabolic modifications. The stability of growth-coupled production of biochemicals can be further enhanced by multiple deletions, thereby significantly reducing the likelihood of multiple compensatory reversals of each disrupted activity.

[0062] Metabolic modification refers to a biochemical reaction that is altered from a naturally occurring state. Thus, a non-naturally occurring microorganism can have a genetic modification to a nucleic acid encoding a metabolic polypeptide or a functional fragment thereof. Exemplary metabolic modifications are disclosed herein.

[0063] It will be appreciated by those skilled in the art that genetic alterations, including metabolic modifications illustrated herein, are described with reference to suitable host organisms such as Escherichia coli (E. coli) and their corresponding metabolic reactions or suitable source organisms for desired genetic material (e.g., genes for desired metabolic pathways, reactions, or series of reactions). However, in view of the high technical level of complete genome sequencing and genomics fields of a variety of organisms, those skilled in the art will be able to easily apply the teachings and guidance provided herein to substantially all other organisms. For example, the E. coli metabolic alterations illustrated herein can be easily applied to other species by incorporating the same or similar encoding nucleic acids from species different from the species mentioned. Such genetic alterations include, for example, common homologous gene replacements of species, and particularly orthologous, paralogous, or non-orthologous gene replacements.

[0064] Orthologous genes are one or more genes that are related by vertical descent and are responsible for essentially the same or identical function in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologous genes for the biological function of epoxide hydrolysis. For example, when the amount of sequence similarity shared by the genes is sufficient to indicate that they are homologous or related by evolution from a common ancestor, the genes are related by vertical descent. If the genes share a sufficient amount of three-dimensional structure, but not necessarily a common sequence similarity, indicating that they have evolved from a common ancestor to the extent that primary sequence similarity cannot be recognized, they can also be considered orthologous genes. Orthologous genes can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. If the three-dimensional structure of the gene encoding a protein with a common amino acid similarity of less than 25% also shows similarity, the gene can also be considered to be generated by vertical descent. Members of the serine protease enzyme family, including tissue plasminogen activator and elastase, are considered to be generated by vertical descent from a common ancestor.

[0065] Orthologous genes include genes or gene products encoded by them that differ in structure or overall activity, for example, by evolution. For example, when a species encodes a gene product that exhibits two functions, and such functions are separated into different genes in a second species, these three genes and their corresponding products are considered to be orthologous genes. For the production of biochemical products, those skilled in the art will appreciate that orthologous genes with metabolic activity to be introduced or destroyed are selected for the construction of non-naturally occurring microorganisms. Examples of orthologous genes that exhibit separable activity are those in which different activities have been separated into different gene products between two or more species or within a single species. A specific example is the separation of two types of serine protease activities, namely, elastase proteolysis and plasminogen proteolysis, into different molecules, as plasminogen activators and elastase. A second example is the separation of mycoplasma 5'-3' exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered as an orthologous gene of one or both of the exonuclease or polymerase from the second species, and vice versa.

[0066] In contrast, paralogous genes are homologs related to evolutionary divergence by, for example, duplication, and have similar or common but not identical functions. Paralogous genes can be derived from or derived from, for example, identical species or different species. For example, microsomal epoxide hydrolases (epoxide hydrolase I) and soluble epoxide hydrolases (epoxide hydrolase II) can be considered as paralogous genes, because they represent two kinds of co-evolution from common ancestors, catalyzing different reactions and having different enzymes with different functions in the same species. Paralogous homologs are proteins from the same species, with significant sequence similarity to each other, indicating that they are homologous, or are related by co-evolution from common ancestors. Paralogous protein family groups include HipA homologs, luciferase genes, peptidases, etc.

[0067] Non-orthologous gene replacement is a non-orthologous gene from a species that can replace the gene function mentioned in different species. Replacement includes, for example, being able to perform substantially the same or similar function in the source species compared to the function mentioned in different species. Although in general, non-orthologous gene replacement can be determined as structurally related to known genes of the function mentioned in the coding, structurally less related but functionally similar genes and their corresponding gene products will still fall within the meaning of the term used herein. Functional similarity requires, for example, that the active site or binding zone of the non-orthologous gene product has at least some structural similarities compared to the gene of the function that the coding seeks to be replaced. Therefore, non-orthologous genes include, for example, paralogous genes or unrelated genes.

[0068] Therefore, in identifying and constructing non-naturally occurring microbial organisms of the present invention with reduced byproducts such as pyruvic acid byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, it will be appreciated by those skilled in the art that identification of metabolic modifications can include identification and inclusion or inactivation of orthologous genes if the teachings and guidance provided herein are applied to specific species. Paralogous genes and / or non-orthologous gene replacements encoding enzymes that catalyze similar or substantially similar metabolic reactions in the microorganisms mentioned can also be utilized by those skilled in the art for these evolutionarily related genes. Similarly, for gene disruption, evolutionarily related genes can also be disrupted or missing in the host microorganism to reduce or eliminate the functional redundancy of the enzymatic activity targeted for disruption.

[0069] Orthologous genes, paralogous genes and non-orthologous gene displacement can be determined by methods well known to those skilled in the art.For example, checking the nucleic acid or amino acid sequence of two polypeptides will reveal the sequence identity and similarity between the comparison sequences.Based on such similarity, those skilled in the art can determine whether the similarity is high enough to indicate that the protein is related by evolving from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and other algorithms, compare and determine the similarity or identity of the original sequence, and also determine the existence or importance of the gap that can be assigned a weight or score in the sequence.Such algorithms are also known in the art, and are similarly applicable to determining nucleotide sequence similarity or identity.Parameters for determining the sufficient similarity of correlation are calculated based on the known methods for calculating statistical similarity or finding the chance of similar matching in random polypeptides and the importance of the determined matching.If necessary, those skilled in the art can also visually optimize the computer comparison of two or more sequences.It is expected that the related gene product or protein has a high degree of similarity, for example, 25% to 100% sequence identity. The identities of unrelated proteins may be as substantially identical as would be expected to occur by chance if a sufficiently large database (approximately 5%) were scanned. Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analyses to determine the significance of these matches given the size of the data set may be performed to determine the relatedness of these sequences.

[0070] For example, exemplary parameters for determining the relevance of two or more sequences using the BLAST algorithm can be as described below. In short, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (January 5, 1999) and the following parameters: Matrix: 0BLOSUM62; Gap opening: 11; Gap extension: 1; x_dropoff: 50; Expected: 10.0; Word length: 3; Filter: On. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (September 16, 1998) and the following parameters: Match: 1; Mismatch: -2; Gap: 5; Gap extension: 2; x_dropoff: 50; Expected: 10.0; Word length: 11; Filter: Off. Those skilled in the art will know which modifications can be made to the above parameters to increase or decrease the stringency of the comparison, for example, to determine the relevance of two or more sequences.

[0071] In certain embodiments, provided herein are non-naturally occurring microbial organisms with reduced byproducts, including microbial organisms with glycolytic pathways and carbon flux enhancement through acetyl-CoA. In some embodiments, the microbial organism includes one or more of the following: (a) attenuated acetolactate synthase; (b) acetaldehyde recycling loop; (c) alanine recycling loop; and (d) citrate synthase variants. In some embodiments, the microbial organism includes an attenuated acetolactate synthase. In some embodiments, the microbial organism includes an acetaldehyde recycling loop. In some embodiments, the microbial organism includes an alanine recycling loop. In some embodiments, the microbial organism includes a citrate synthase variant. In some embodiments, the microorganism includes under microaerobic conditions, such as by increasing the inhibition of NADH, increasing the Km of acetyl-CoA, expressing attenuation (e.g., by the absence, knocking down or expression reduction of citrate synthase (gltA)) or any combination thereof, any citrate synthase variant that reduces TCA activity.

[0072] In some embodiments, the microbial organism comprises two or more of the following: (a) attenuated acetolactate synthase; (b) acetaldehyde recycling loop; (c) alanine recycling loop; and (d) citrate synthase variants. In some embodiments, the microbial organism comprises an attenuated acetolactate synthase and an acetaldehyde recycling loop. In some embodiments, the microbial organism comprises an attenuated acetolactate synthase and an alanine recycling loop. In some embodiments, the microbial organism comprises an attenuated acetolactate synthase and a citrate synthase variant. In some embodiments, the microbial organism comprises an acetaldehyde recycling loop and an alanine recycling loop. In some embodiments, the microbial organism comprises an acetolactate synthase and a citrate synthase variant. In some embodiments, the microbial organism comprises an acetaldehyde recycling loop and an alanine recycling loop. In some embodiments, the microbial organism comprises an acetaldehyde recycling loop and a citrate synthase variant. In some embodiments, the microbial organism comprises an alanine recycling loop and a citrate synthase variant.

[0073] In some embodiments, the microbial organism comprises three or more of the following: (a) attenuated acetolactate synthase; (b) acetaldehyde recycling loop; (c) alanine recycling loop; and (d) citrate synthase variants. In some embodiments, the microbial organism comprises attenuated acetolactate synthase, acetaldehyde recycling loop, and alanine recycling loop. In some embodiments, the microbial organism comprises an acetaldehyde recycling loop, an alanine recycling loop, and a citrate synthase variant. In some embodiments, the microbial organism comprises attenuated acetolactate synthase, an alanine recycling loop, and a citrate synthase variant. In some embodiments, the microbial organism comprises attenuated acetolactate synthase, an acetaldehyde recycling loop, and a citrate synthase variant. In some embodiments, the microbial organism comprises attenuated acetolactate synthase, an acetaldehyde recycling loop, and a citrate synthase variant.

[0074] In some embodiments, the microbial organism includes each of: (a) an attenuated acetolactate synthase; (b) an acetaldehyde recycling loop; (c) an alanine recycling loop; and (d) a citrate synthase variant.

[0075] Acetolactate synthase (ALS) (Genbank Accession Nos. ACA79829.1 and ACA79830.1) (EC: 2.2.1.6), also known as acetohydroxyacid synthase (AHAS), catalyzes the first reaction in the branched-chain amino acid synthesis pathway. Acetolactate synthase is at a critical branch point because its reaction determines the extent of carbon flow through the branched-chain amino acids. The reaction involves irreversible decarboxylation of pyruvate and condensation of the acetaldehyde moiety with a second pyruvate molecule to produce 2-acetolactate or with a molecule of 2-ketobutyrate to produce 2-acetyl-2-hydroxybutyrate. Each product is then further transformed in three reactions catalyzed by ketol-acid reductoisomerase, dihydroxyacid dehydratase, and transaminase, respectively, to give valine and isoleucine. For leucine biosynthesis, four additional enzymes are required, using the valine precursor 2-ketoisovalerate as the starting point for synthesis. Thus, as provided herein, carbon flow can be directed away from the production of branched chain amino acids by attenuating acetolactate synthase activity, thereby increasing carbon flow from pyruvate to acetyl-CoA.

[0076] In some organisms, such as E. coli and Salmonella typhimurium, three different acetolactate synthase isozymes can be expressed: AHAS I (encoded by the ilvBN gene), AHAS II (encoded by the ilvGM gene), and AHAS III (encoded by the ilvIH gene). However, due to different chromosomal gene mutations, AHAS II from E. coli and AHAS III from Salmonella typhimurium can produce inactive proteins. In some strains of E. coli, such as Crooks (ATCC 8739), W (ATCC 9637), and B (REL606), ilvG is still intact and functional. Therefore, in some embodiments, the attenuation of acetolactate synthase involves the attenuation of one, two, three, or as many different isozymes expressed as possible. In some embodiments, the attenuation involves the attenuation of the active form of acetolactate synthase. In other embodiments, the attenuation of acetolactate synthase involves the attenuation of all forms of the enzyme.

[0077] In some embodiments, the microbial organism includes an attenuated acetolactate synthase, wherein the attenuated acetolactate synthase involves a reduction in the expression of acetolactate synthase. In some embodiments, the amount of acetolactate synthase expression reduction includes at least about 10% to about 90%. In some embodiments, the amount of acetolactate synthase expression reduction includes at least about 20% to about 80%. In some embodiments, the amount of acetolactate synthase expression reduction includes at least about 30% to about 70%. In some embodiments, the amount of acetolactate synthase expression reduction includes at least about 40% to about 60%. In some embodiments, the amount of acetolactate synthase expression reduction is about 50% reduction. In some embodiments, the amount of acetolactate synthase expression reduction is about 60% reduction. In some embodiments, the amount of acetolactate synthase expression reduction is about 70% reduction. In some embodiments, the amount of acetolactate synthase expression reduction is about 80% reduction. In some embodiments, the amount of acetolactate synthase expression reduction is about 90% reduction. In some embodiments, the amount of acetolactate synthase expression reduction is about 95% reduction. In some embodiments, the amount by which acetolactate synthase expression is reduced is about a 100% reduction.

[0078] In certain embodiments, the attenuated acetolactate synthase comprises the disappearance of acetolactate synthase. As mentioned above, in organisms expressing multiple enzyme forms, the attenuation can include the attenuation of one, two, three or as many different isozymes expressed as much as possible. Therefore, in some embodiments, the attenuation of acetolactate synthase comprises the disappearance of one, two, three or as many different isozymes expressed as much as possible. In some embodiments, the attenuation of acetolactate synthase comprises the disappearance of active form of acetolactate synthase. In other embodiments, the attenuation of acetolactate synthase relates to the disappearance of all forms of enzymes.

[0079] In some embodiments, the attenuated acetolactate synthase includes expression of a non-functional acetolactate synthase, such as a dominant negative form of the enzyme. As described above, AHAS II from Escherichia coli (encoded by the ilvGM gene) and AHAS III from Salmonella typhimurium (encoded by the ilvIH gene) can include an inactive form of the protein. Therefore, in some embodiments, the attenuated acetolactate synthase relates to the expression of a polynucleotide or polypeptide encoding an inactive form of acetolactate synthase.

[0080] In some embodiments, the attenuated acetolactate synthase comprises ilvGM. In some embodiments, the attenuated ilvGM involves reduced expression of the active form of ilvGM. In specific embodiments, the attenuated ilvGM involves a deletion of the active form of ilvGM. In some embodiments, the microorganism having attenuated ilvGM is an E. coli strain having intact ilvG.

[0081] In some embodiments, the attenuated acetolactate synthase reduces the biosynthesis of branched chain amino acids.

[0082] In some embodiments, the attenuated acetolactate synthase reduces the biosynthesis of valine, isoleucine and / or leucine. In some embodiments, the attenuated acetolactate synthase reduces the biosynthesis of valine. In some embodiments, the attenuated acetolactate synthase reduces the biosynthesis of isoleucine. In some embodiments, the attenuated acetolactate synthase reduces the biosynthesis of leucine.

[0083] In some embodiments, the non-naturally occurring microbial organisms with attenuated acetolactate synthases reduce the carbon flux into valine by 40 times. In some embodiments, the non-naturally occurring microbial organisms with attenuated acetolactate synthases reduce the carbon flux into valine by 30 times. In some embodiments, the non-naturally occurring microbial organisms with attenuated acetolactate synthases reduce the carbon flux into valine by 20 times. In some embodiments, the non-naturally occurring microbial organisms with attenuated acetolactate synthases reduce the carbon flux into valine by 15 times. In some embodiments, the non-naturally occurring microbial organisms with attenuated acetolactate synthases reduce the carbon flux into valine by 10 times. In some embodiments, the non-naturally occurring microbial organisms with attenuated acetolactate synthases reduce the carbon flux into valine by 5 times. In some embodiments, the non-naturally occurring microbial organisms with attenuated acetolactate synthases reduce the carbon flux into valine by 4 times. In some embodiments, the non-naturally occurring microbial organisms with attenuated acetolactate synthases reduce the carbon flux into valine by 3 times. In some embodiments, the non-naturally occurring microbial organism with attenuated acetolactate synthase reduces carbon flux into valine by 2-fold. In some embodiments, the non-naturally occurring microbial organism with attenuated acetolactate synthase reduces carbon flux into valine by 1.5-fold.

[0084] Pyruvate can also undergo transamination to form alanine. Alanine can be recycled back to form pyruvate through a two-step process. In the first step, L-alanine is converted to D-alanine by alanine racemase (EC 5.1.1.1). Alanine racemase can be constitutively active or inducible. For example, in Escherichia coli, dadX is responsible for most of the alanine racemase activity in the cell and can be induced by D-alanine or L-alanine (see, e.g., Wild J, et al., Mol Gen Genet. 1985; 198 (2): 315-322). In contrast, Alr is constitutively expressed, but shows a typical dependence on incubation temperature. In the second step, D-alanine is converted to pyruvate by D-amino acid dehydrogenase. For example, in E. coli and Salmonella typhimurium, dadA generates pyruvate from D-alanine (see, e.g., Wild and Klopotowski, Mol Gen Genet. 1981; 181(3):373-378).

[0085] Thus, in some embodiments, the disclosure also provides a non-naturally occurring microbial organism having an alanine recycling loop, the loop comprising at least one exogenous nucleic acid encoding an alanine recycling loop enzyme selected from the group consisting of a D-amino acid dehydrogenase and an alanine racemase. In some embodiments, the alanine recycling loop comprises at least one exogenous nucleic acid encoding a D-amino acid dehydrogenase. In some embodiments, the alanine recycling loop comprises at least one exogenous nucleic acid encoding an alanine racemase. In some embodiments, the alanine recycling loop comprises at least one exogenous nucleic acid encoding a D-amino acid dehydrogenase and an alanine racemase. In specific embodiments, the D-amino acid dehydrogenase is encoded by dadA. In some embodiments, the D-amino acid dehydrogenase is from Escherichia coli strain K-12 substr.MG1655 (NP_415707.1). In certain embodiments, the alanine racemase is encoded by dadX. In certain embodiments, the alanine racemase is from bacteria (NP_747370.1; WP_010955779.1).

[0086] As provided herein, non-naturally occurring microbial organisms with an alanine recycling loop are capable of redirecting the carbon converted from pyruvate to alanine back to pyruvate. Thus, in some embodiments, the alanine concentration of the non-naturally occurring microbial organisms with the alanine recycling loop is reduced compared to microbial organisms without the alanine recycling loop. In some embodiments, the alanine concentration reduction is a reduction in L-alanine concentration. In some embodiments, the ratio of L-alanine to D-alanine of the non-naturally occurring microbial organisms is reduced compared to microbial organisms without the alanine recycling loop.

[0087] In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 5% to 75% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 10% to 60% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 20% to 50% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 30% to 45% compared to a microbial organism without an alanine recycling loop.

[0088] In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 5% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 10% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 15% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 20% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 25% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 30% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 35% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 40% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 45% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 50% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 55% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of about 60% compared to a microbial organism without an alanine recycling loop. In some embodiments, the reduction in alanine concentration is a reduction in alanine concentration of greater than about 60% compared to a microbial organism without an alanine recycling loop.

[0089] As provided herein, reducing pyruvate byproducts, such as by reducing carbon flux to produce alanine and / or branched-chain amino acids, can increase carbon flux through acetyl-CoA and thus increase the production of acetyl-CoA derivatives, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivatives, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA. Thus, in some embodiments, branched-chain amino acids can be reduced in microorganisms attenuated with acetolactate, and the production of acetyl-CoA derivatives can be increased, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivatives, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA. In some embodiments, a microorganism having an alanine recycling loop can have reduced alanine and increased production of acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA.

[0090] In some embodiments, the reduction in the production of alanine and / or one or more branched-chain amino acids results in an increase in the yield of an acetyl-CoA-derived product by about 1 to about 2.5 times, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA. In some embodiments, the reduction in the production of alanine and / or one or more branched-chain amino acids results in an increase in the yield of an acetyl-CoA-derived product by more than 2.5 times, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA.

[0091] In addition, the microorganism that can produce acetyl-CoA derivative products may also have unwanted by-products, such as acetic acid and / or ethanol generated directly from acetyl-CoA. The generation of such by-products can limit the efficiency and / or amount of the carbon stream that can be converted into the required acetyl-CoA derivative products such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate or any other acetyl-CoA derivative products. However, as provided herein, the carbon stream can be redirected to the desired acetyl-CoA derivative product away from the unwanted by-product, and the disclosure provides that the unwanted by-product can be recycled back to acetyl-CoA to enhance. In some embodiments, the conversion of unwanted by-products such as acetic acid and / or ethanol back to acetyl-CoA can be realized by the acetaldehyde recycling loop.

[0092] The carbon flow from acetyl-CoA can be converted into unwanted byproducts, such as ethanol and / or acetic acid, by at least two exemplary reactions. In one exemplary reaction, acetyl-CoA is converted into acetaldehyde by CoA-dependent aldehyde dehydrogenase (ALDH; encoded by the ald gene), and then acetaldehyde is converted into ethanol by alcohol dehydrogenase (ADH). Alternatively, in another exemplary reaction, acetyl-CoA can be converted into acetic acid by CoA hydrolase / thioesterase.

[0093] As provided herein, the present disclosure provides an acetaldehyde recycling loop that can include at least one exogenous nucleic acid encoding an acetaldehyde recycling loop enzyme. In some embodiments, the acetaldehyde recycling loop enzyme is selected from the group consisting of an aldehyde dehydrogenase and an acetyl-CoA synthase.

[0094] Aldehyde dehydrogenase (ALDH) (Accession No. WP_000183980.1) (EC: 1.2.1.3) is a member of a diverse group of related enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. For example, in E. coli, more than a dozen aldehyde dehydrogenase genes have been identified, some of which have preferences for certain aldehyde substrates. For example, as provided herein, AldB can preferentially convert acetaldehyde to acetic acid and has little or no activity in converting 3HB-aldehyde to 3-hydroxybutyrate.

[0095] In some embodiments, the aldehyde dehydrogenase is AldB. In some embodiments, endogenous AldB is expressed at very low levels in microbial organisms. For example, after relative and absolute quantification (iTRAQ) global proteomics detection using LC / MS or using isobaric tags, endogenous AldB may be indistinguishable from background levels. In some embodiments, an exogenous nucleic acid encoding an acetaldehyde recycling loop enzyme includes AldB. In certain embodiments, the exogenous nucleic acid is a heterologous nucleic acid.

[0096] As provided herein, the acetic acid produced by the aldehyde dehydrogenase can be further converted into acetyl-CoA by acetyl-CoA synthase. Acetyl-CoA synthase (Accession No. WP_000078239.1) (EC: 6.2.1.1) catalyzes the connection of acetic acid to coenzyme A to produce acetyl-CoA. Therefore, in some embodiments, the acetaldehyde recycling loop includes at least one exogenous nucleic acid encoding acetyl-CoA synthase. In certain embodiments, acetyl-CoA synthase is an acetyl-CoA synthase variant. In some embodiments, acetyl-CoA synthase variant can be an acetyl-CoA synthase that is less sensitive to acetylation but retains acetyl-CoA synthase activity relative to wild-type acetyl-CoA synthase. An exemplary acetyl-CoA synthase variant is a mutant acetyl-CoA synthase that has replaced a leucyl residue (e.g., L641P) at position 641 with a prolyl residue. In certain embodiments, acetyl-CoA synthase variant is a Salmonella enterica acetyl-CoA synthase variant. In some embodiments, the acetaldehyde recycling loop includes one or more exogenous nucleic acids encoding both acetyl-CoA synthase and aldehyde dehydrogenase. In certain embodiments, at least one exogenous nucleic acid is a heterologous nucleic acid.

[0097] The present disclosure thus provides an acetaldehyde recycle loop that can reduce the production of unwanted byproducts such as acetic acid and / or ethanol. In some embodiments, the acetic acid, ethanol, or a combination thereof of a non-naturally occurring microbial organism is reduced. In some embodiments, the acetic acid of a non-naturally occurring microbial organism is reduced. In some embodiments, the ethanol of a non-naturally occurring microbial organism is reduced. In some embodiments, the acetic acid and ethanol of a non-naturally occurring microbial organism are reduced.

[0098] By reducing unwanted acetyl-CoA derived byproducts, such as acetate and / or ethanol, using, for example, an acetaldehyde recycle loop, the carbon flux of acetyl-CoA through a desired acetyl-CoA derived product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA, can be increased. Thus, in some embodiments, acetate and / or ethanol can be reduced for a microorganism having an acetaldehyde recycle loop, and the production of acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA, can be increased.

[0099] In some embodiments, the reduction in ethanol and / or acetate production results in an increase in the yield of an acetyl-CoA derived product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA, by about 1 to about 2.5 times. In some embodiments, the reduction in ethanol and / or acetate production results in an increase in the yield of an acetyl-CoA derived product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA, by more than 2.5 times.

[0100] Acetyl-CoA can also be converted into undesirable tricarboxylic acid (TCA) cycle (also known as citric acid cycle or Krebs cycle) derived byproducts. The TCA cycle begins with a reaction that combines acetyl-CoA with oxaloacetate to produce citric acid. Thus, when the desired product is an acetyl-CoA derived product that is not involved in the TCA cycle, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA, the TCA cycle can be responsible for the generation of the undesirable byproduct.

[0101] When acetyl-CoA combines with oxaloacetate to form citric acid, citrate synthase is responsible for the reaction rate in the first step of the cycle. Citrate synthase is inhibited by NADH, and at least one citrate synthase variant has increased sensitivity to NADH. Wild-type citrate synthase has arginine (R) at amino acid position 109, while variants contain leucine (L) instead of arginine (R109L) at amino acid position 109 (see Stokell, et al. J Biol Chem. 2003; 278 (37): 35435-35443). Due to increased sensitivity to NADH, the activity of citrate synthase variants is lower than that of wild-type citrate synthase, and therefore as much acetyl-CoA as possible will not be directed into the TCA cycle.

[0102] Thus, in some embodiments, the non-naturally occurring microbial organism comprises a citrate synthase variant. In some embodiments, the citrate synthase variant is a type II citrate synthase. In some embodiments, the citrate synthase variant binds NADH with greater affinity than wild-type citrate synthase. In a specific embodiment, the citrate synthase variant is encoded by gltAR109L.

[0103] As provided herein, compared with the microbial organism with wild-type citrate synthase, the tricarboxylic acid cycle (TCA) cycle derived byproducts of the non-naturally occurring microbial organism with citrate synthase variant are reduced. In some embodiments, compared with the microbial organism without citrate synthase variant, the reduction of TCA derived byproducts is to reduce about 5% to 75%. In some embodiments, compared with the microbial organism without citrate synthase variant, the reduction of TCA derived byproducts is to reduce about 10% to 60%. In some embodiments, compared with the microorganism without citrate synthase variant, the reduction of TCA derived byproducts is to reduce about 20% to 50%. In some embodiments, compared with the microbial organism without citrate synthase variant, the reduction of TCA derived byproducts is to reduce about 30% to 45%.

[0104] In some embodiments, the reduction of TCA-derived byproducts is a reduction of about 5% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction of TCA-derived byproducts is a reduction of about 10% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction of TCA-derived byproducts is a reduction of about 15% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction of TCA-derived byproducts is a reduction of about 20% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction of TCA-derived byproducts is a reduction of about 25% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction of TCA-derived byproducts is a reduction of about 30% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction of TCA-derived byproducts is a reduction of about 35% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction of TCA-derived byproducts is a reduction of about 40% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction in TCA-derived byproducts is a reduction of about 45% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction in TCA-derived byproducts is a reduction of about 50% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction in TCA-derived byproducts is a reduction of about 55% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction in TCA-derived byproducts is a reduction of about 60% compared to a microbial organism without a citrate synthase variant. In some embodiments, the reduction in TCA-derived byproducts is a reduction of greater than about 60% compared to a microbial organism without a citrate synthase variant.

[0105] In some embodiments, the reduction in the production of TCA-derived byproducts results in an increase in the yield of acetyl-CoA-derived products by about 1 to about 2.5 times, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA. In some embodiments, the reduction in the production of TCA-derived byproducts results in an increase in the yield of acetyl-CoA-derived products by more than 2.5 times, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA.

[0106] In some embodiments, the microbial organism comprises two or more of the following: (a) attenuated acetolactate synthase; (b) acetaldehyde recycling loop; (c) alanine recycling loop; and (d) citrate synthase variant. In some embodiments, the microbial organism comprises three or more of the following: (a) attenuated acetolactate synthase; (b) acetaldehyde recycling loop; (c) alanine recycling loop; and (d) citrate synthase variant. In some embodiments, the microbial organism comprises each of the following: (a) attenuated acetolactate synthase; (b) acetaldehyde recycling loop; (c) alanine recycling loop; and (d) citrate synthase variant. The combination of two or more mechanisms for reducing byproducts can be additive and allow even greater production of acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA. In some embodiments, the combination of two or more mechanisms for reducing byproducts can be synergistic and allow even greater production of acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA.

[0107] As provided herein, non-naturally occurring microbial organisms having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include a 1,3-butanediol (1,3-BDO) pathway, a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway, a 3-hydroxybutyryl-CoA (3HB-CoA) pathway, a methyl methacrylate (MMA) pathway, an adipate pathway, a caprolactam pathway, a 6-aminocaproic acid (6-ACA) pathway, a hexamethylenediamine (HMDA) pathway, or a methacrylic acid (MAA) pathway. In some embodiments, non-naturally occurring microbial organisms having reduced byproducts such as, for example, pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA include microbial organisms having a 1,3-butanediol (1,3-BDO) pathway, a methyl methacrylate (MMA) pathway, a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway, an amino acid biosynthetic pathway, a 3HB-CoA pathway, a methyl methacrylate (MMA) pathway, an adipate pathway, a caprolactam pathway, a 6-aminohexanoic acid (6-ACA) pathway, a hexamethylenediamine (HMDA) pathway, or a methacrylic acid (MAA) pathway.

[0108] In certain embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA further comprises a 1,3-butanediol (1,3-BDO) pathway. In some embodiments, the 1,3-BDO pathway comprises an enzyme selected from the group consisting of: 1) acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 2) 3-oxobutyraldehyde reductase (ketone reducing), 3) 3-hydroxybutyraldehyde reductase, 4) acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), 5) 3-oxobutyraldehyde reductase (aldehyde reducing), 6) 4-hydroxy-2-butanone reductase, 7) acetoacetyl-CoA reductase (ketone reducing), 8) 3-hydroxybutyryl-CoA reductase (aldehyde forming), and 9) 3-hydroxybutyryl-CoA reductase (alcohol forming). In some embodiments, the 1,3-BDO pathway comprises a nucleic acid encoding acetoacetyl-CoA reductase (phaB). In specific embodiments, the acetoacetyl-CoA reductase is a mutant acetoacetyl-CoA reductase. In some embodiments, the mutant acetoacetyl-CoA reductase uses NADH as a substrate. Any number of nucleic acids encoding these enzymes can be further introduced into the host microbial organism, including one, two, three, four, five, six, seven, eight, up to a total of nine nucleic acids encoding these enzymes. In the case of introducing one, two, three, four, five, six, seven, or eight exogenous nucleic acids, such nucleic acids can be any arrangement of the other nine nucleic acids.

[0109] In certain embodiments, the non-naturally occurring microbial organism having reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA further comprises a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway. In some embodiments, the (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of: 1) (3R)-hydroxybutyl (3R)-hydroxybutyrate forming enzyme, 2) (3R)-hydroxybutyryl-CoA:(R)-1,3-butanediol alcohol transferase, 3) (3R)-hydroxybutyl 3-oxobutyrate forming enzyme, 4) acetoacetyl-CoA:(R)-1,3-butanediol alcohol transferase, 5) (3R)-hydroxybutyl 3-oxobutyrate reductase, 6) (3R)-hydroxybutyryl-ACP:(R)-1,3-butanediol ester synthase, and 7) acetoacetyl-ACP:(R)-1,3-butanediol ester synthase. In some embodiments, the (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway comprises a nucleic acid encoding acetoacetyl-CoA reductase (phaB). In a specific embodiment, acetoacetyl-CoA reductase is the acetoacetyl-CoA reductase of mutation.In some embodiments, the acetoacetyl-CoA reductase of mutation uses NADH as substrate.The nucleic acid of these enzymes of coding of any number can be further introduced into the host microorganism organism, comprise one, two, three, four, five, six, seven, up to all eight nucleic acids of these enzymes of coding.When introducing one, two, three, four, five, six or seven kinds of exogenous nucleic acids, this type of nucleic acid can be any arrangement of other eight nucleic acids.

[0110] In certain embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include an MMA pathway. In specific embodiments, the MMA pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of: (a) 4-hydroxybutyryl-CoA dehydratase, crotonase, 2-hydroxyisobutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase, and methacrylic acid (MAA)-CoA:methanol transferase; or (b) 4-hydroxybutyryl-CoA dehydratase, crotonase, 2-hydroxyisobutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA:methanol transferase, and 2-hydroxyisobutyrate methyl ester dehydratase. In some embodiments, the MMA pathway further comprises at least one nucleic acid encoding an enzyme selected from the group consisting of: (c) methacrylic acid (MAA)-CoA:methanol transferase, 4-hydroxybutyryl-CoA mutase, and 3-hydroxyisobutyryl-CoA dehydratase; or (d) 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA:methanol transferase, and 3-hydroxyisobutyrate methyl dehydratase.

[0111] In other embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include a 3HB-CoA pathway. In specific embodiments, the 3HB-CoA pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of acetyl-CoA thiolase and 3-hydroxybutyryl-CoA dehydrogenase.

[0112] In some embodiments, the microbial organism with increased NADPH availability can also include an MMA approach. It is known in the art to use microorganisms to produce MMA, as illustrated in U.S. Patent Nos. 9,133,487 and 9,346,902, each of which is incorporated herein by reference in its entirety. In certain embodiments, the MMA approach comprises a MAA approach that is subsequently esterified with methanol to produce MMA. In a specific embodiment, the MMA approach comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of: (a) 4-hydroxybutyryl-CoA dehydratase, crotonase, 2-hydroxyisobutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase and methacrylic acid (MAA)-CoA: methanol transferase; or (b) 4-hydroxybutyryl-CoA dehydratase, crotonase, 2-hydroxyisobutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA: methanol transferase and 2-hydroxyisobutyrate methyl ester dehydratase. In some embodiments, the MMA pathway further comprises at least one exogenous nucleic acid encoding an enzyme selected from a second MMA pathway comprising: (c) methacrylic acid (MAA)-CoA:methanol transferase, 4-hydroxybutyryl-CoA mutase, and 3-hydroxyisobutyryl-CoA dehydratase; or (d) 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA:methanol transferase, and 3-hydroxyisobutyrate methyl dehydratase.

[0113] In certain embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include a 6-ACA pathway. In specific embodiments, the 6-ACA pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of 2-amino-7-oxosuberic acid ketoacid decarboxylase, 2-amino-7-oxoheptanoic acid decarboxylase, 2-amino-7-oxoheptanoic acid oxidoreductase, 2-aminopimelate decarboxylase, 6-aminohexanal oxidoreductase, 2-amino-7-oxoheptanoic acid decarboxylase, or 2-amino-7-oxosuberic acid amino acid decarboxylase. In other embodiments, the 6-ACA pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of: 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA reductase; and 6-aminohexanoyl-CoA / acyl-CoA transferase, 6-aminohexanoyl-CoA synthase, or 6-aminohexanoyl-CoA hydrolase.

[0114] In other embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include a caprolactam pathway. In specific embodiments, the caprolactam pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipoyl-CoA reductase (aldehyde forming), 6-aminohexanoate transaminase, 6-aminohexanoate dehydrogenase, 6-aminohexanoyl-CoA / acyl-CoA transferase, and 6-aminohexanoyl-CoA synthase.

[0115] In certain embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include an adipate pathway. In specific embodiments, the adipate pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA hydrolase, adipyl-CoA ligase, adipyl-CoA transferase, and phosphotransferase / adipate kinase.

[0116] In other embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include a hexamethylenediamine (HMDA) pathway. In specific embodiments, the HMDA pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA reductase (aldehyde forming), 6-aminohexanoate transaminase, 6-aminohexanoate dehydrogenase, 6-aminohexanoyl-CoA / acyl-CoA transferase, 6-aminohexanoyl-CoA synthase, 6-aminohexanoyl-CoA reductase (aldehyde forming), HMDA transaminase, and HMDA dehydrogenase. In other embodiments, the HMDA pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from: 6-aminohexanoyl-CoA / acyl-CoA transferase or 6-aminohexanoyl-CoA synthase; 6-aminohexanoyl-CoA reductase (aldehyde forming); and hexamethylenediamine transaminase or hexamethylenediamine dehydrogenase.

[0117] In some embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include a MAA pathway. In specific embodiments, the MAA pathway comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of: (1) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA epimerase; (iv) a methylmalonyl-CoA reductase (aldehyde forming); (v) a methylmalonate semialdehyde reductase; and (vi) a 3-hydroxyisobutyrate dehydratase; (2) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA epimerase; (iv) a methylmalonyl-CoA reductase (aldehyde forming); (v) a methylmalonate semialdehyde reductase; and (vi) a 3-hydroxyisobutyrate dehydratase; (ii) a methylmalonyl-CoA transferase, ligase or synthetase; (iii) a methylmalonyl-CoA mutase; (iv) a methylmalonate semialdehyde reductase; and (v) a 3-hydroxyisobutyrate dehydratase; or (3) (i) a succinyl-CoA transferase, ligase or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA reductase (aldehyde forming); and (iv) a 3-hydroxyisobutyrate dehydratase.

[0118] In some embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include a 1,3-butanediol pathway. In some embodiments, the 1,3-butanediol pathway includes at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of: glucose to pyruvate, pyruvate to acetyl-CoA, acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutyryl-CoA, 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde, and 3-hydroxybutyraldehyde to 1,3-BDO.

[0119] In some embodiments, the non-naturally occurring microbial organism having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway. In some embodiments, the (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway includes at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of: (R)-1,3-butanediol and (3R)-hydroxybutyrate to (3R)-hydroxybutyl (3R)-hydroxybutyrate, (R)-1,3-butanediol and (3R)-hydroxybutyryl-CoA to (3R)-hydroxybutyl (3R)-hydroxybutyrate, (R)-1,3-butanediol and (3R)-hydroxybutyl-ACP to (3R)-hydroxybutyl (3R)-hydroxybutyrate. Converted to (3R)-hydroxybutyl (3R)-hydroxybutyrate, (R)-1,3-butanediol and acetoacetate to (3R)-hydroxybutyl 3-oxobutyrate, (R)-1,3-butanediol and acetoacetyl-CoA to (3R)-hydroxybutyl 3-oxobutyrate, (R)-1,3-butanediol and acetoacetyl-ACP to (3R)-hydroxybutyl 3-oxobutyrate, (3R)-hydroxybutyl 3-oxobutyrate to (3R)-hydroxybutyl (3R)-hydroxybutyrate, acetyl-CoA to malonyl-C oA, malonyl-CoA is converted to acetoacetyl-CoA, acetyl-CoA is converted to acetoacetyl-CoA, acetoacetyl-CoA is converted to acetoacetate, acetoacetyl-CoA is converted to (3S)-hydroxybutyryl-CoA, (3S)-hydroxybutyryl-CoA is converted to (3R)-hydroxybutyryl-CoA, (3R)-hydroxybutyryl-CoA is converted to (3R)-hydroxybutyrate, (3R)-hydroxybutyryl-CoA is converted to (3R)-hydroxybutyraldehyde, (3R)-hydroxybutyrate is converted to (3R)-hydroxybutyraldehyde, (3R)-hydroxy Butyraldehyde is converted to (R)-1,3-butanediol, malonyl-ACP is converted to acetoacetyl-ACP, acetoacetyl-ACP is converted to acetoacetyl-CoA, acetoacetyl-ACP is converted to (3R)-hydroxybutyryl-ACP, (3R)-hydroxybutyryl-ACP is converted to (3R)-hydroxybutyryl-CoA, (3R)-hydroxybutyryl-ACP is converted to (3R)-hydroxybutyrate, (3R)-hydroxybutyryl-ACP is converted to (3R)-hydroxybutyric acid, (3R)-hydroxybutyryl-ACP is converted to (3R)-hydroxybutyraldehyde, and (3R)-hydroxybutyryl-ACP is converted to (R)-1,3-butanediol. It should be understood by those skilled in the art that these are merely exemplary, and any substrate-product pair disclosed herein that is suitable for producing the desired product and whose appropriate activity can be used to convert the substrate to the product can be easily determined by those skilled in the art based on the teachings herein.Thus, the invention provides non-naturally occurring microbial organisms containing at least one exogenous nucleic acid encoding an enzyme or protein that converts substrates and products of the (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway, such as those disclosed in U.S. Application No. 14 / 893,510, published as US2016-0108442A1, which is incorporated herein by reference in its entirety.

[0120] In some embodiments, non-naturally occurring microbial organisms with reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can also include an MMA pathway. In some embodiments, the MMA pathway includes at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate into a product, wherein the substrate conversion product is selected from the group consisting of: 4-HB-CoA to crotonyl-CoA and 3HIB-CoA, crotonyl-CoA to 3HB-CoA, 3HIB-CoA to MAA-CoA or methyl-3HIB, and MAA-CoA or methyl-3HIB to MMA. In another embodiment, the invention provides a non-naturally occurring microbial organism having an MMA pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of: conversion of 4HB-CoA to crotonyl-CoA, conversion of crotonyl-CoA to (3R)-HB-CoA or (3S)-HB-CoA, conversion of (3R)-HB-CoA or (3S)-HB-CoA to 2-HIB-CoA, conversion of 2-HIB-CoA to MAA-CoA or 2HIB-Me, and conversion of MAA-CoA or 2HIB-Me to MMA. In yet another embodiment, the invention provides a non-naturally occurring microbial organism having a 1,3-BDO pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of: conversion of 4HB-CoA to crotonyl-CoA, conversion of crotonyl-CoA to (3R)-HB-CoA or (3S)-HB-CoA, conversion of (3R)-HB-CoA or (3S)-HB-CoA to (3R)- or (3S)-1,3BDO.

[0121] In additional embodiments, the invention provides a non-naturally occurring microbial organism having a methacrylic acid pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of: acetyl-CoA and pyruvate to citramalate, citramalate to citraconic acid, and citraconate to methacrylic acid; acetyl-CoA and pyruvate to citramalyl-CoA, citramalyl-CoA to citramalate, citramalate to citraconic acid, and citraconate to methacrylic acid; aconitic acid to itaconic acid, itaconic acid to itaconyl-CoA, itaconyl-CoA to citramalyl-CoA, citramalyl-CoA to citramalate, citramalate to mesaconic acid, mesaconic acid to methacrylic acid, and the like, such as the reactions described herein. One skilled in the art will appreciate that these are exemplary only, and that any substrate-product pair disclosed herein that is suitable for producing a desired product and whose appropriate activity can be used to convert a substrate to a product can be readily determined by one skilled in the art based on the teachings herein. Thus, the invention provides non-naturally occurring microbial organisms containing at least one exogenous nucleic acid encoding an enzyme or protein that converts substrates and products of a methacrylic acid pathway, such as the pathway described herein. Additionally provided is a methacrylic acid pathway comprising an acetoacetyl-CoA thiolase, an acetoacetyl-CoA reductase, a crotonase, a 4-hydroxybutyryl-CoA dehydratase (or crotonyl-CoA hydratase, 4-hydroxy), a 4-hydroxybutyryl-CoA mutase, a 3-hydroxyisobutyryl-CoA synthetase or a 3-hydroxyisobutyryl-CoA hydrolase or a 3-hydroxyisobutyryl-CoA transferase, and a 3-hydroxyisobutyrate dehydratase. Also provided is a methacrylic acid pathway comprising acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, crotonase, 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA dehydratase and methacrylyl-CoA synthetase or methacrylyl-CoA hydrolase or methacrylyl-CoA transferase. The production of MAA is known in the art and can be found, for example, in U.S. Application No. 13 / 436,811, published as U.S. 2013-0065279A1, which is incorporated herein by reference in its entirety.

[0122] In additional embodiments, the invention provides a non-naturally occurring microbial organism having a caprolactam pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product selected from the group consisting of: adipoyl-CoA to adipic acid, adipoyl-CoA to adipic semialdehyde, adipic acid to adipic semialdehyde, adipic semialdehyde to 6-hydroxyhexanoic acid, 6-hydroxyhexanoic acid to 6-hydroxyhexanoyl-CoA, 6-hydroxyhexanoic acid to 6-hydroxyhexanoyl phosphate, 6-hydroxyhexanoic acid to caprolactone, 6-hydroxyhexanoyl-CoA to 6-hydroxyhexanoyl phosphate, 6-hydroxyhexanoic acid to caprolactone, 6-hydroxyhexanoyl-CoA to 6-hydroxyhexanoyl phosphate, 6-hydroxyhexanoyl phosphate to caprolactone, 6-hydroxyhexanoyl-CoA to caprolactone, 4-hydroxybutyryl-CoA to 3-oxo-6-hydroxyhexanoyl-CoA, 3-oxo- 6-hydroxyhexanoyl-CoA is converted to 3,6-dihydroxyhexanoyl-CoA, 3,6-dihydroxyhexanoyl-CoA is converted to 6-hydroxyhex-2-enoyl-CoA, 6-hydroxyhex-2-enoyl-CoA is converted to 6-hydroxyhexanoyl-CoA, 6-hydroxyhexanoyl-CoA is converted to 6-hydroxyhexanoic acid, cyclohexanone is converted to caprolactone, adipic acid semialdehyde is converted to cyclohexane-1,2-dione, cyclohexane-1,2-dione Ketone is converted to 2-hydroxycyclohexanone, 2-hydroxycyclohexanone is converted to cyclohexane-1,2-diol, cyclohexane-1,2-diol is converted to cyclohexanone, pimeloyl-CoA is converted to 2-ketocyclohexanone-1-carboxyl-CoA, 2-ketocyclohexanone-1-carboxyl-CoA is converted to 2-ketocyclohexane-1-carboxylic acid, 2-ketocyclohexane-1-carboxylic acid is converted to cyclohexanone, cyclohexanone is converted to cyclohexane oxime, and cyclohexane oxime is converted to caprolactam. Those skilled in the art will understand that these are merely exemplary, and any substrate-product pair disclosed herein that is suitable for producing a desired product and whose appropriate activity can be used to convert a substrate to a product can be easily determined by those skilled in the art based on the teachings herein. Therefore, the present invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, wherein the enzyme or protein converts substrates and products of the caprolactam pathway.

[0123] In some embodiments, the non-naturally occurring microbial organisms provided herein having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can further include an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; conversion of 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA; conversion of 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; conversion of 5-carboxy-2-pentenoyl-CoA to adipoyl-CoA; conversion of adipoyl-CoA to adipic acid (see, e.g., WO2012 / 177721, which is incorporated herein by reference in its entirety). In addition, the non-naturally occurring microbial organism can have an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; conversion of 3-oxoadipate-CoA to 3-oxoadipate; conversion of 3-oxoadipate to 3-hydroxyadipate; conversion of 3-hydroxyadipate to hexa-2-enedioic acid (also referred to herein as 5-carboxy-2-pentenoic acid); conversion of hexa-2-enedioic acid to adipic acid. In addition, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of adipyl-CoA to adipic acid semialdehyde; and conversion of adipic acid semialdehyde to 6-aminohexanoic acid. Additionally, the non-naturally occurring microbial organism can have a caprolactam pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: adipyl-CoA to adipic acid semialdehyde; adipate semialdehyde to 6-aminocaproic acid; and 6-aminocaproic acid to caprolactam. Additionally, the non-naturally occurring microbial organism can have an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: alpha-ketoadipate to alpha-ketoadipyl-CoA; alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipate-CoA to adipic acid.Additionally, the non-naturally occurring microbial organism can have an adipate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: alpha-ketoadipate to 2-hydroxyadipate; 2-hydroxyadipate to 2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipoyl-CoA; and adipoyl-CoA to adipic acid.

[0124] In addition, the non-naturally occurring microbial organism can have a 6-aminohexanoyl-CoA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: 4-aminobutyryl-CoA and acetyl-CoA to 3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to 3-hydroxy-6-aminohexanoyl-CoA; 3-hydroxy-6-aminohexanoyl-CoA to 6-aminohex-2-enoyl-CoA; 6-aminohex-2-enoyl-CoA to 6-aminohexanoyl-CoA. Other substrates and products of this pathway can include 6-aminohexanoyl-CoA to 6-aminohexanoic acid; 6-aminohexanoyl-CoA to caprolactam; or 6-aminohexanoyl-CoA to 6-aminohexanoic acid semialdehyde, and 6-aminohexanoic acid semialdehyde to hexamethylenediamine. The non-naturally occurring microbial organism can also have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: 4-aminobutyryl-CoA and acetyl-CoA to 3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to 3-oxo-6-aminohexanoic acid; 3-oxo-6-aminohexanoic acid to 3-hydroxy-6-aminohexanoic acid; 3-hydroxy-6-aminohexanoic acid to 6-aminohex-2-enoic acid; and 6-aminohex-2-enoic acid to 6-aminohexanoic acid. Other substrates and products of this pathway can include 6-aminohexanoic acid to caprolactam or 6-aminohexanoic acid to 6-aminohexanoyl-CoA, 6-aminohexanoyl-CoA to 6-aminohexanoic acid semialdehyde, and 6-aminohexanoic acid semialdehyde to hexamethylenediamine.

[0125] Additionally, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioic acid; conversion of 4-hydroxy-2-oxoheptane-1,7-dioic acid (HODH) to 2-oxohept-4-ene-1,7-dioic acid (OHED); conversion of 2-oxohept-4-ene-1,7-dioic acid (OHED) to 2-oxoheptane-1,7-dioic acid (2-OHD); conversion of 2-oxoheptane-1,7-dioic acid (2-OHD) to adipic semialdehyde; and conversion of adipic semialdehyde to 6-aminohexanoic acid. Alternatively, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioic acid; conversion of 4-hydroxy-2-oxoheptane-1,7-dioic acid (HODH) to 2-oxohept-4-ene-1,7-dioic acid (OHED); conversion of 2-oxohept-4-ene-1,7-dioic acid (OHED) to 6-oxohex-4-enoic acid (6-OHE); conversion of 6-oxohex-4-enoic acid (6-OHE) to adipic semialdehyde; and conversion of adipic semialdehyde to 6-aminohexanoic acid. Alternatively, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioic acid; conversion of 4-hydroxy-2-oxoheptane-1,7-dioic acid (HODH) to 2-oxohept-4-ene-1,7-dioic acid (OHED); conversion of 2-oxohept-4-ene-1,7-dioic acid (OHED) to 2-aminohept-4-ene-1,7-dioic acid (2-AHE); conversion of 2-aminohept-4-ene-1,7-dioic acid (2-AHE) to 2-aminoheptane-1,7-dioic acid (2-AHD); and conversion of 2-aminoheptane-1,7-dioic acid (2-AHD) to 6-aminohexanoic acid.Alternatively, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioic acid; conversion of 4-hydroxy-2-oxoheptane-1,7-dioic acid (HODH) to 2-oxohept-4-ene-1,7-dioic acid (OHED); conversion of 2-oxohept-4-ene-1,7-dioic acid (OHED) to 2-oxoheptane-1,7-dioic acid (2-OHD); conversion of 2-oxoheptane-1,7-dioic acid (2-OHD) to 2-aminoheptane-1,7-dioic acid (2-AHD); and conversion of 2-aminoheptane-1,7-dioic acid (2-AHD) to 6-aminohexanoic acid. Alternatively, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of pyruvate and succinate semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioic acid; conversion of 4-hydroxy-2-oxoheptane-1,7-dioic acid (HODH) to 3-hydroxyadipyl-CoA; conversion of 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA; conversion of 2,3-dehydroadipyl-CoA to adipoyl-CoA; conversion of adipoyl-CoA to adipic acid semialdehyde; and conversion of adipic acid semialdehyde to 6-aminohexanoic acid. Alternatively, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of pyruvate and succinate semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioic acid; conversion of 4-hydroxy-2-oxoheptane-1,7-dioic acid (HODH) to 2-oxohept-4-ene-1,7-dioic acid (OHED); conversion of 2-oxohept-4-ene-1,7-dioic acid (OHED) to 2,3-dehydroadipyl-CoA; conversion of 2,3-dehydroadipyl-CoA to adipoyl-CoA; conversion of adipoyl-CoA to adipic acid semialdehyde; and conversion of adipic acid semialdehyde to 6-aminohexanoic acid.Alternatively, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioic acid; conversion of 4-hydroxy-2-oxoheptane-1,7-dioic acid (HODH) to 2-oxohept-4-ene-1,7-dioic acid (OHED); conversion of 2-oxohept-4-ene-1,7-dioic acid (OHED) to 2-oxoheptane-1,7-dioic acid (2-OHD); conversion of 2-oxoheptane-1,7-dioic acid (2-OHD) to adipoyl-CoA; conversion of adipoyl-CoA to adipic acid semialdehyde; and conversion of adipic acid semialdehyde to 6-aminohexanoic acid.

[0126] Additionally, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: glutamate to glutamyl-CoA; glutamyl-CoA to 3-oxo-6-amino-pimeloyl-CoA; 3-oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-aminopimelate; and 2-aminopimelate to 6-aminohexanoic acid. Alternatively, a non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimeloic acid; 3-oxopimeloic acid to 3-aminopimeloic acid; 3-aminopimeloic acid to 2-aminopimeloic acid; and 2-aminopimeloic acid to 6-aminohexanoic acid. Alternatively, a non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: homolysine to 6-aminohexanamide; and 6-aminohexanamide to 6-aminohexanoic acid. Alternatively, the non-naturally occurring microbial organism can have a 6-aminocaproic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of adipic acid to adipic semialdehyde; conversion of adipic acid to adipoyl phosphate; and conversion of adipoyl phosphate to adipic semialdehyde.

[0127] Additionally, the non-naturally occurring microbial organism can have a 6-aminohexanoic acid pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: 2-amino-7-oxosuberic acid to 2-amino-7-oxoheptanoic acid; 2-amino-7-oxoheptanoic acid to 6-aminohexanal; 6-aminohexanal to 6-aminohexanoic acid; 2-amino-7-oxosuberic acid to 2-amino-7-oxoheptanoic acid; 2-amino-7-oxoheptanoic acid to 6-aminohexanal; 2-amino-7-oxoheptanoic acid to 2-aminopimelic acid; and 2-aminopimelic acid to 6-aminohexanoic acid. The non-naturally occurring microbial organism can also have a 2-amino-7-oxosuberate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of glutamate-5-semialdehyde to 2-amino-5-hydroxy-7-oxosuberate; conversion of 2-amino-5-hydroxy-7-oxosuberate to 2-amino-5-ene-7-oxosuberate; and conversion of 2-amino-5-ene-7-oxosuberate to 2-amino-7-oxosuberate.

[0128] Additionally, the non-naturally occurring microbial organism can have a hexamethylenediamine (HMDA) pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of 6-aminohexanoic acid to [(6-aminohexanoyl))oxy]phosphonic acid (6-AHOP); conversion of [(6-aminohexanoyl)oxy]phosphonic acid (6-AHOP) to 6-aminohexanoic acid semialdehyde; and conversion of 6-aminohexanoic acid semialdehyde to hexamethylenediamine. Alternatively, a non-naturally occurring microbial organism can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: 6-aminohexanoic acid to [(6-aminohexanoyl)oxy]phosphonic acid (6-AHOP); [(6-aminohexanoyl)oxy]phosphonic acid (6-AHOP) to 6-aminohexanoyl-CoA; 6-aminohexanoyl-CoA to 6-aminohexanoic acid semialdehyde; and 6-aminohexanoic acid semialdehyde to hexamethylenediamine. Alternatively, a non-naturally occurring microbial organism can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: 6-aminohexanoic acid to 6-aminohexanoyl-CoA; 6-aminohexanoyl acyl-CoA to 6-aminohexanoic acid semialdehyde; and 6-aminohexanoic acid semialdehyde to hexamethylenediamine. Alternatively, the non-naturally occurring microbial organism can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: 6-aminohexanoic acid to 6-acetylaminohexanoic acid; 6-acetylaminohexanoic acid to [(6-acetylaminohexanoyl)oxy]phosphonic acid (6-AAHOP); [(6-acetylaminohexanoyl)oxy]phosphonic acid (6-AAHOP) to 6-acetylaminohexanal; 6-acetylaminohexylaldehyde to 6-acetylaminohexylamine; and 6-acetylaminohexylamine to hexamethylenediamine. Alternatively, the non-naturally occurring microbial organism can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: 6-aminohexanoic acid to 6-acetylaminohexanoic acid; 6-acetylaminohexanoic acid to 6-acetylaminohexanoyl-CoA; 6-acetylaminohexanoyl-CoA to 6-acetylaminohexanal; 6-acetylaminohexanal to 6-acetylaminohexylamine; and 6-acetylaminohexylamine to hexamethylenediamine.Alternatively, the non-naturally occurring microbial organism can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: 6-aminohexanoic acid to 6-acetylaminohexanoic acid; 6-acetylaminohexanoic acid to [(6-acetylaminohexanoyl)oxy]phosphonic acid (6-AAHOP); [(6-acetylaminohexanoyl)oxy]phosphonic acid (6-AAHOP) to 6-acetylaminohexanoyl-CoA; 6-acetylaminohexanoyl-CoA to 6-acetylaminohexanal; 6-acetylaminohexylaldehyde to 6-acetylaminohexylamine; and 6-acetylaminohexylamine to hexamethylenediamine.

[0129] Additionally, the non-naturally occurring microbial organism can have a hexamethylenediamine (HMDA) pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: glutamate to glutamyl-CoA; glutamyl-CoA to 3-oxo-6-amino-pimeloyl-CoA; 3-oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoic acid; -amino-7-oxoheptanoic acid to homolysine; and homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-oxo-1-carboxyheptanal; conversion of 3-oxo-1-carboxyheptanal to 3-oxo-7-aminoheptanoic acid; conversion of 3-oxo-7-aminoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimeloic acid; conversion of 3-oxopimeloic acid to 5-oxopimeloylphosphonic acid; conversion of 5-oxopimeloylphosphonic acid to 3-oxo-1-carboxyheptanal; conversion of 3-oxo-1-carboxyheptanal to 3-oxo-7-aminoheptanoic acid; conversion of 3-oxo-7-aminoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine, and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimeloyl-CoA to 5-oxopimeloyl-CoA; conversion of 5-oxopimeloyl-CoA to 3-oxo-1-carboxyheptanal; conversion of 3-oxo-1-carboxyheptanal to 3-oxo-7-aminoheptanoic acid; conversion of 3-oxo-7-aminoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine; and conversion of homolysine to HMDA.Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-oxo-1-carboxyheptanal; conversion of 3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimeloyl-CoA to 5-oxopimeloyl-CoA; conversion of 5-oxopimeloyl-CoA to 3-oxo-1-carboxyheptanal; conversion of 3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimeloic acid; conversion of 3-oxopimeloic acid to 5-oxopimeloylphosphonic acid; conversion of 5-oxopimeloylphosphonic acid to 3-oxo-1-carboxyheptanal; conversion of 3-oxo-1-carboxyheptanal to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-aminopimelic acid; conversion of 3-aminopimelic acid to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 2-amino-7-oxoheptanoic acid; conversion of 2-amino-7-oxoheptanoic acid to homolysine; and conversion of homolysine to HMDA.Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-aminopimelic acid; conversion of 3-aminopimelic acid to 5-aminopimeloylphosphonic acid; conversion of 5-aminopimeloylphosphonic acid to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 2-amino-7-oxoheptanoic acid; conversion of 2-amino-7-oxoheptanoic acid to homolysine; and conversion of homolysine to acid HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimeloyl-CoA to 5-aminopimeloyl-CoA; conversion of 5-aminopimeloyl-CoA to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 2-amino-7-oxoheptanoic acid; conversion of 2-amino-7-oxoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-aminopimelic acid; conversion of 3-aminopimelic acid to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-aminopimelic acid; conversion of 3-aminopimelic acid to 5-aminopimeloyl-CoA; conversion of 5-aminopimeloyl-CoA to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine; and conversion of homolysine to HMDA.Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-aminopimelic acid; conversion of 3-aminopimelic acid to 5-aminopimeloylphosphonic acid; conversion of 5-aminopimeloylphosphonic acid to 3-amino-7-oxoheptanoic acid; conversion of 3-amino-7-oxoheptanoic acid to 3,7-diaminoheptanoic acid; conversion of 3,7-diaminoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-aminopimelic acid; conversion of 3-aminopimelic acid to 2-aminopimelic acid; conversion of 2-aminopimelic acid to 2-amino-7-oxoheptanoic acid; conversion of 2-amino-7-oxoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-aminopimelic acid; conversion of 3-aminopimelic acid to 2-aminopimelic acid; conversion of 2-aminopimelic acid to 6-aminopimeloylphosphonic acid; conversion of 6-aminopimeloylphosphonic acid to 2-amino-7-oxoheptanoic acid; conversion of 2-amino-7-oxoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: conversion of glutaryl-CoA to 3-oxopimeloyl-CoA; conversion of 3-oxopimeloyl-CoA to 3-oxopimelic acid; conversion of 3-oxopimelic acid to 3-aminopimelic acid; conversion of 3-aminopimelic acid to 2-aminopimelic acid; conversion of 2-aminopimelic acid to 6-aminopimeloyl-CoA; conversion of 6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoic acid; conversion of 2-amino-7-oxoheptanoic acid to homolysine; and conversion of homolysine to HMDA.Alternatively, the non-naturally occurring microbial organism can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of pyruvate and 4-aminobutyraldehyde to 2-oxo-4-hydroxy7-aminoheptanoic acid; conversion of 2-oxo-4-hydroxy7-aminoheptanoic acid to 2-oxo-7-aminohept-3-enoic acid; conversion of 2-oxo-7-aminohept-3-enoic acid to 2-oxo-7-aminoheptanoic acid; conversion of 2-oxo-7-aminoheptanoic acid to homolysine; and conversion of homolysine to HMDA. Alternatively, a non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: pyruvate and 4-aminobutyraldehyde into 2-oxo-4-hydroxy7-aminoheptanoic acid; 2-oxo-4-hydroxy7-aminoheptanoic acid into 2-oxo-7-aminohept-3-enoic acid; 2-oxo-7-aminohept-3-enoic acid into 2-oxo-7-aminoheptanoic acid; 2-oxo-7-aminoheptanoic acid into 6-aminohexanal; and 6-aminohexanal into HMDA. Alternatively, a non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: 6-aminohexanoic acid into 6-aminohexanoic acid semialdehyde; and 6-aminohexanoic acid semialdehyde into HMDA. Alternatively, the non-naturally occurring microbial organism can have a HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: 6-aminohexanoic acid to 6-acetylaminohexanoic acid; 6-acetylaminohexanoic acid to 6-acetylaminohexanal; 6-acetylaminohexanal to 6-acetylaminohexylamine; 6-acetylaminohexylamine to HMDA. Alternatively, the non-naturally occurring microbial organism can have an HMDA pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate to a product selected from the group consisting of: 2-amino-7-oxosuberate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal; 6-aminohexanal to HMDA; 2-amino-7-oxosuberate to 2-oxo-7-aminoheptanoate; 2-amino-7-oxoheptanoate to homolysine; homolysine to HMDA; 2-oxo-7-aminoheptanoate to homolysine; 2-oxo-7-aminoheptanoate to 6-aminohexanal; 2-amino-7-oxosuberate to 2,7-diaminosuberate; and 2,7-diaminosuberate to homolysine.The non-naturally occurring microbial organism can further have a 2-amino-7-oxosuberate pathway, wherein the microbial organism contains at least one exogenous nucleic acid encoding a polypeptide that converts a substrate into a product selected from the group consisting of: conversion of glutamate-5-semialdehyde to 2-amino-5-hydroxy-7-oxosuberate; conversion of 2-amino-5-hydroxy-7-oxosuberate to 2-amino-5-ene-7-oxosuberate; and conversion of 2-amino-5-ene-7-oxosuberate to 2-amino-7-oxosuberate.

[0130] Those skilled in the art will appreciate that these are merely exemplary and that any substrate-product pair disclosed herein that is suitable for producing a desired acetyl-CoA-derived product and whose appropriate activity can be used to convert the substrate to the product can be readily determined by those skilled in the art based on the teachings herein.

[0131] It is also understood that the expression of exogenous nucleic acid disclosed herein can be regulated by various promoters, such as endogenous promoters, constitutive promoters or inducible promoters. Given the required expression level and duration, those of ordinary skill in the art will understand which type of promoter to use. For example, if the required expression level is an endogenous level, those of ordinary skill will understand that endogenous promoters can be used to control the expression of exogenous nucleic acid. In addition, if it is desired that expression is, for example, temporary or at a specific point in the fermentation process, endogenous promoters can be used to control the expression of exogenous nucleic acid. However, if it is desired that expression is, for example, constant and robust, constitutive promoters can be used to control the expression of exogenous nucleic acid. Therefore, in some embodiments, the exogenous nucleic acid encoding enzyme is regulated by endogenous promoters, constitutive promoters or inducible promoters.

[0132] Although generally described herein as microbial organisms with reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, it is understood that the invention further provides non-naturally occurring microbial organisms comprising at least one exogenous nucleic acid encoding an enzyme expressed in sufficient amounts to increase the availability of a desired acetyl-CoA-derived product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA or MAA, and intermediates derived therefrom.

[0133] Thus, it is understood that the present invention, in some embodiments, further provides a non-naturally occurring microbial organism that includes an intermediate of a biosynthetic pathway for 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to a 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA biosynthetic pathway, wherein the pathway contains at least one enzyme that acts on an acetyl-CoA derivative product and the pathway enzymes are expressed in sufficient amounts to produce an intermediate of a biosynthetic pathway for 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to a 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA pathway. Thus, in addition to microbial organisms containing biosynthetic pathways that produce 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or an amino acid: 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to, 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA biosynthetic pathways, the present invention also provides non-naturally occurring microbial organisms, wherein the microbial organism produces an intermediate of an acetyl-CoA derived product, such as an intermediate of a biosynthetic pathway: 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to, 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA biosynthetic pathways.

[0134] It is understood that any of the pathways disclosed herein, which are described throughout and incorporated by reference in their entirety, can be utilized, if desired, by a non-naturally occurring microbial organism that reduces byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, thereby generating a non-naturally occurring microbial organism that further produces (as desired) any pathway intermediate or product derived from acetyl-CoA. As disclosed herein, such an intermediate-producing microbial organism can be used in combination with another microorganism expressing a downstream pathway enzyme to produce a desired product. However, it is understood that non-naturally occurring microbial organisms that produce intermediates of acetyl-CoA derived products, such as intermediates of the following biosynthetic pathways: 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA or MAA biosynthetic pathways, can be used to produce intermediates as desired products.

[0135] The invention is generally described herein for reducing byproducts of metabolic reactions, reactants or their products, or specifically for one or more nucleic acids or genes encoding enzymes or proteins, the enzymes being associated with the metabolic reactions, reactants or products mentioned or catalyzing the metabolic reactions, reactants or products mentioned, the proteins being associated with the metabolic reactions, reactants or products mentioned. Unless otherwise expressly stated herein, it will be understood by those skilled in the art that reference to a reaction also constitutes reference to the reactants and products of a reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also refers to a reaction, and reference to any of these metabolic components also refers to a gene or genes encoding an enzyme or protein, the enzyme catalyzing the reactions, reactants or products mentioned, the proteins being associated with the reactions, reactants or products mentioned. Similarly, in view of well-known fields such as metabolic biochemistry, enzymology and genomics, reference to a gene or encoding nucleic acid herein also constitutes reference to the corresponding encoded enzyme and the reaction catalyzed by it or the protein associated with the reaction and the reactants and reaction products of the reaction.

[0136] The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more enzymes or proteins of an acetaldehyde recycling loop and / or attenuating acetolactate synthase to reduce byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, and related products derived therefrom. In addition, in some embodiments, the non-naturally occurring microbial organisms of the invention can be produced by further introducing one or more enzymes or proteins involved in, for example, one or more of the following: 1,3-butanediol (1,3-BDO), methyl methacrylate (MMA), (3R)-hydroxybutyl (3R)-hydroxybutyrate biosynthesis or any other acetyl-CoA-derived product biosynthesis pathway.

[0137] Depending on the host microbial organism selected for biosynthesis, nucleic acids for some or all of a particular biosynthetic pathway for converting acetyl-CoA to a desired product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA, can be expressed. For example, if the selected host lacks one or more enzymes or proteins of the desired biosynthetic pathway, expressible nucleic acids for the deficient enzymes or proteins are introduced into the host for subsequent exogenous expression. Alternatively, if the selected host exhibits endogenous expression of certain pathway genes, but lacks other pathway genes, nucleic acids encoding the deficient enzymes or proteins are required to achieve biosynthesis of 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA. Thus, the non-naturally occurring microbial organisms of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway, or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produce a desired product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA.

[0138] The host microbial organism can be selected from and the non-naturally occurring microbial organism can be generated, for example, from bacteria, yeast, fungi, or any of a variety of other microorganisms suitable for use in fermentation processes. Exemplary bacteria include species selected from the group consisting of E. coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens. fluorescens and Pseudomonas putida. Exemplary yeasts or fungi include species selected from: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, etc. Escherichia coli is a particularly useful host organism because it is a well-characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast, such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolism and / or genetic modification to produce a desired product.

[0139] Depending on the biosynthetic pathway that utilizes acetyl-CoA to produce a desired product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA or MAA, and the components of the selected host microbial organism, the non-naturally occurring microbial organisms of the invention can include at least one exogenously expressed 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product pathway encoding nucleic acid, and up to all encoding nucleic acids for one or more 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product biosynthetic pathways. For example, 1,3-BDO, MMA or (3R)-hydroxybutyl (3R)-hydroxybutyrate biosynthesis can be established in a host lacking a pathway enzyme or protein by exogenous expression of the corresponding encoding nucleic acid. In a host lacking all enzymes or proteins of a pathway to 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, exogenous expression of all enzymes or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one pathway enzyme or protein. For example, exogenous expression of all enzymes or proteins in a pathway for producing 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or any other acetyl-CoA derived product can be included in a host expressing one or more enzymes or proteins of an acetaldehyde recycling loop and / or having an attenuated acetolactate synthase to reduce byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA derived byproducts, acetate, and / or ethanol.

[0140] In view of the teachings and guidance provided herein, it will be understood by those skilled in the art that the number of encoding nucleic acids introduced in an expressible form should at least parallel the pathway deficiencies of the selected host microbial organism for byproduct reduction. For example, the non-naturally occurring microbial organisms of the present invention can have one or two nucleic acids encoding enzymes or proteins that constitute the acetaldehyde recycling loop pathway disclosed herein for reducing byproducts, such as acetic acid and / or ethanol, to enhance carbon flux through acetyl-CoA. Enhancement of carbon flux through acetyl-CoA can thereby increase acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA. In some embodiments, the non-naturally occurring microbial organisms can also include other genetic modifications that promote or optimize the biosynthesis of acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, or that confer other useful functions to the host microbial organism. One such other function can include, for example, enhancing the synthesis of one or more of the following: acetyl-CoA derived product pathway precursors, such as 1,3-BDO precursors (e.g., 3HB-CoA), MMA precursors (e.g., MAA-CoA), (3R)-hydroxybutyl (3R)-hydroxybutyrate precursors (e.g., acetoacetyl-CoA), or any other acetyl-CoA derived product precursors.

[0141] Thus, the byproduct-reduced non-naturally occurring microbial organisms of the invention can have one or two nucleic acids encoding enzymes or proteins that constitute the acetaldehyde recycling pathways disclosed herein. In some embodiments, the byproduct-reduced non-naturally occurring microbial organisms of the invention can also have one, two, three, four, five, six, seven, or eight up to all nucleic acids encoding enzymes or proteins that constitute the biosynthetic pathways of the reactions disclosed herein. In some embodiments, the non-naturally occurring microbial organisms can also include other genetic modifications that promote or optimize the biosynthesis of acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived products, or that confer other useful functions to the host microbial organism. For example, one such other function can include, for example, enhancing the synthesis of one or more 1,3-BDO pathway precursors, such as 3HB-CoA.

[0142] Typically, the host microbial organism is selected so that it produces precursors for the acetyl-CoA dependent pathway, either as naturally occurring molecules or as engineered products that provide for the de novo production of the desired precursor or increase the production of a precursor naturally produced by the host microbial organism. For example, pyruvate is naturally produced in host organisms such as E. coli. As disclosed herein, the host organism can be engineered to increase the production of the precursor. In addition, microbial organisms engineered to produce the desired precursor can be used as host organisms and further engineered to express pathway enzymes or proteins for increasing acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA.

[0143] In some embodiments, the non-naturally occurring microbial organisms of the invention with reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol are generated by a host that contains enzymatic capabilities to synthesize acetyl-CoA-derived products (e.g., 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived products, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA. In this specific embodiment, it can be used to increase the synthesis or accumulation of acetyl-CoA-derived product pathway products, for example, to drive 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product pathway reactions toward the production of 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, respectively. Increased synthesis or accumulation can be achieved, for example, by overexpressing nucleic acids encoding enzymes or proteins involved in pathways for producing acetyl-CoA-derived products.

[0144] Overexpression of the enzymes or proteins disclosed herein (capable of reducing byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived products, including, but not limited to, 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA or MAA) can occur, for example, by exogenous expression of one or more endogenous genes or by exogenous expression of one or more heterologous genes. Thus, naturally occurring organisms can be readily generated as non-naturally occurring microbial organisms of the invention by overexpressing one, two, three, four, five, six, seven, or eight (depending on the number of enzymes in the pathway), i.e., up to all nucleic acids encoding enzymes or proteins disclosed herein that can reduce side products and increase, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product biosynthetic pathway enzymes or proteins that can reduce side products and increase, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, such as reducing side products such as pyruvate side products (e.g., alanine and / or valine), TCA-derived side products, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including, but not limited to, 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA. Additionally, non-naturally occurring organisms can be generated by mutagenesis of endogenous genes that result in increased enzyme activity disclosed herein, which can reduce byproducts and increase, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived products.

[0145] In particularly useful embodiments, exogenous expression of encoding nucleic acid is used. Exogenous expression confers the ability of customized expression elements and / or regulatory elements for the host and application, to achieve the desired expression level controlled by the user. However, endogenous expression can also be used for other embodiments, such as by removing negative regulatory effectors or inducing the promoter of a gene when connected with an inducible promoter or other regulatory elements. Therefore, an endogenous gene with a naturally occurring inducible promoter can be raised by providing a suitable inducing agent, or the regulatory region of the endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the increase of endogenous gene expression to be regulated at the desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

[0146] It is understood that in the methods of the invention, any one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention having a reduction in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA. Nucleic acids can be introduced to impart, for example, an attenuated acetolactate synthase and / or acetaldehyde recycling loop to the microbial organism. In some embodiments, the microbial organism with reduced byproducts can also include nucleic acids introduced to impart, for example, a biosynthetic pathway to the microbial organism for producing 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA. Alternatively, encoding nucleic acids can be introduced to generate an intermediate microbial organism with biosynthetic capabilities to catalyze some of the desired reactions to confer biosynthetic capabilities for 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product. For example, a non-naturally occurring microbial organism having reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA or MAA, can contain at least two exogenous nucleic acids encoding desired enzymes or proteins, such as a combination of: attenuated acetolactate synthase and acetyl-CoA synthase; and attenuated acetolactate synthase and aldehyde dehydrogenase; attenuated acetolactate synthase and aldehyde dehydrogenase; aldehyde dehydrogenase and acetyl-CoA synthase, etc. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in the non-naturally occurring microbial organism of the invention. Similarly, it is understood that all three enzymes or proteins of a biosynthetic pathway can be included in the non-naturally occurring microbial organisms of the invention, e.g., attenuated acetolactate synthase, acetyl-CoA synthase, and aldehyde dehydrogenase.

[0147] In addition to biosynthesis of, for example, 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or any other acetyl-CoA-derived product described herein, the non-naturally occurring microbial organisms of the invention having reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, and methods of the invention for reducing such byproducts, can also be used in various combinations with each other and with other microbial organisms and methods known in the art to achieve product biosynthesis through other pathways. For example, an alternative approach to producing, e.g., 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or any other acetyl-CoA-derived product in a non-naturally occurring microbial organism having reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, rather than using, e.g., 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or other acetyl-CoA-derived product producers, is by adding another microbial organism that is capable of converting, e.g., 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or other acetyl-CoA-derived product biosynthetic pathway intermediates to, e.g., 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or other acetyl-CoA-derived product, respectively.

[0148] One such procedure includes, for example, fermentation of a microbial organism that produces 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA or other acetyl-CoA derivative product biosynthetic pathway intermediates. The 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA or other acetyl-CoA derivative product biosynthetic pathway intermediates can then be used as substrates for a second non-naturally occurring microbial organism that converts the 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA or other acetyl-CoA derivative product biosynthetic pathway intermediates to 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA or other acetyl-CoA derivative products, respectively. The 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA or other acetyl-CoA derivative product biosynthetic pathway intermediate can be added directly to another culture of a second non-naturally occurring microbial organism, or an original culture of a 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA or other acetyl-CoA derivative product biosynthetic pathway intermediate producer can be depleted of these microbial organisms by, for example, cell separation, and utilizing subsequent addition of the second non-naturally occurring microbial organism to the fermentation broth to produce the final product without an intermediate purification step.

[0149] In other embodiments, the non-naturally occurring microbial organisms disclosed herein, which have reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, and the methods of the invention for reducing such byproducts, can be assembled in multiple sub-pathways to achieve the biosynthesis of, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA. In these embodiments, the biosynthetic pathways for the desired products of the invention can be separated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such biosynthetic schemes, the product of one microbial organism is a substrate for a second microbial organism until the final product is synthesized. For example, biosynthesis of 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or any other acetyl-CoA-derived product can be accomplished by constructing a microbial organism that contains a biosynthetic pathway that converts one pathway intermediate to another pathway intermediate or product, and one or more microbial organisms that convert one pathway intermediate to another pathway intermediate or product via an acetyl-CoA-derived pathway can be constructed to have reduced byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products. Alternatively, a desired acetyl-CoA derived product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA or MAA, 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA or an amino acid, can also be produced biosynthetically by one or more microbial organisms having enhanced carbon flux through acetyl-CoA by co-culturing or co-fermenting with two organisms in the same vessel, wherein the first microbial organism produces an intermediate such as a 1,3-BDO intermediate and the second microbial organism converts the intermediate to 1,3-BDO.

[0150] Given the teachings and guidance provided herein, those skilled in the art will appreciate that for the non-naturally occurring microbial organisms and methods of the invention, in combination with other microbial organisms, co-cultures of other microbial organisms having subpathways, and other chemical and / or biochemical procedures known in the art, there are various combinations and permutations to produce 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA.

[0151] Similarly, one skilled in the art will appreciate that a host organism can be selected based on desired characteristics for the introduction of one or more gene disruptions to increase production of 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA. Thus, it is understood that if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologous, orthologous, or paralogous genes that catalyze similar but not identical metabolic reactions can be similarly disrupted to ensure that the desired metabolic reaction is sufficiently disrupted. Because there are certain differences between the metabolic networks of different organisms, one skilled in the art will appreciate that the actual genes disrupted in a given organism may vary between organisms. However, given the teachings and guidance provided herein, those skilled in the art will also appreciate that the methods of the present invention can be applied to any suitable host microorganism to identify the homologous metabolic changes required to construct an organism in a species of interest that will increase the biosynthesis of 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product. In specific embodiments, increased production is coupled to the biosynthesis of, for example, 1,3-BDO, MMA, or (3R)-hydroxybutyl (3R)-hydroxybutyrate, and, if desired and as disclosed herein, production of, for example, 1,3-BDO, MMA, or (3R)-hydroxybutyl (3R)-hydroxybutyrate can be forced to be coupled to organism growth.

[0152] The source of the encoding nucleic acid of the pathway enzyme or protein of the acetaldehyde recycling loop or using acetyl-CoA as substrate can include, for example, any species whose encoded gene product can catalyze the reaction mentioned. Such species include prokaryotes and eukaryotes, including but not limited to bacteria, including archaea and true bacteria, and eukaryotes, including yeast, plants, insects, animals and mammals, including humans. Exemplary species of such sources include, for example, Escherichia coli, and other exemplary species disclosed herein or that can be used as corresponding gene source organisms. Exemplary species of such sources include, for example, Escherichia coli, Escherichia fergusonii, Methanocaldococcus jannaschii, Leptospira interrrogans, Geobacter sulfurreducens, Chloroflexus aurantiacus, Roseiflexus sp.) RS-1, Chloroflexus aggregans, Achromobacter xylosoxydans, Clostrdia species including Clostridium kluyveri, Clostridium symbiosum, Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium ljungdahlii, Trichomonas vaginalis G3, Trypanosoma brucei, Acidaminococcus fermentans, Fusobacterium species including Fusobacterium nucleatum, Fusobacterium mortiferum, Corynebacterium glutamicum glutamicum), Rattus norvegicus, Homo sapiens, Saccharomyces species including Saccharomyces cerevisiae, Apsergillus species including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger, Gibberella zeae, Pichia stipitis, Mycobacterium species including Mycobacterium smegmatis, Mycobacterium avium including Mycobacterium avium subsp. pratuberculosis, Salinispora arenicola), Pseudomonas species, including Pseudomonas sp.CF600), Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Ralstonia species including Ralstonia eutropha, Ralstonia eutropha JMP134, Ralstonia eutropha H16, Ralstonia pickettii, Lactobacillus plantarum, Klebsiella oxytoca, Bacillus species including Bacillus methanolicus, Bacillus subtilis, Bacillus pumilus, Bacillus megaterium, Pedicoccus pentosaceus pentosaceus), Chlorofexus species, including Chlorofexus aurantifolius and Chlorofexus agglomerans, Rhodobacter phaeroides, Methanococcus jannaschii, Leptospira interrogans, Candida maltosa, Salmonella species, including Salmonella enterica serovar Typhimurium, Shewanella species, including Shewanella oneidensis and Shewanella sp. MR-4, Alcaligenes faecalis, Geobacillus stearothermophilus, Serratia marcescens, Vibrio cholerae, cholerae), Eubacteriumbarkeri, Bacteroides capillosus, Archaeoglobusfulgidus, Archaeoglobusfulgidus, Haloarcula marismortui, Pyrobaculum aerophilum str.IM2.IM2), Rhizobium species, including Rhizobium leguminosarum, and other exemplary species disclosed herein or that can be used as source organisms for corresponding genes. However, now that complete genome sequences of more than 550 species (more than half of which are available in public databases such as NCBI) are available, including 395 microbial genomes and various yeast, fungal, plant and mammalian genomes, it is routine and well known in the art to identify genes encoding activities required to reduce byproducts such as pyruvate (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA, and to identify genes encoding 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA or other acetyl-CoA derivative biosynthetic activities for one or more genes in related or distantly related species, including, for example, homologous genes, orthologous genes, paralogous genes and non-orthologous gene replacements of known genes, and the exchange of genetic alterations between organisms. Thus, the metabolic alterations described herein with reference to a particular organism, such as E. coli, which allow for the reduction of byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived products, can be readily applied to other microorganisms, including prokaryotes and eukaryotes. Given the teachings and guidance provided herein, one skilled in the art will appreciate that metabolic alterations exemplified in one organism can be equally applied to other organisms. .

[0153] In some cases, for example, when an alternative biosynthetic pathway for producing 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA, exists in an unrelated species, the biosynthesis of 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA, can be conferred to the host species by, for example, exogenously expressing one or more orthologous genes from the unrelated species that catalyze similar but different metabolic reactions, in place of the mentioned reactions. Because certain differences between metabolic networks exist between different organisms, those skilled in the art will appreciate that actual gene usage may differ between different organisms. However, given the teachings and guidance provided herein, one skilled in the art will also appreciate that the teachings and methods of the present invention can be applied to all microbial organisms using homologous metabolic alterations of those exemplified herein to construct microbial organisms in a species of interest that will synthesize 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA.

[0154] Methods for constructing non-naturally occurring organisms and testing their expression levels can be performed by recombination and detection methods known in the art, wherein the organism has reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA. Such methods can be found in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[0155] Exogenous nucleic acid sequences involved in the reduction of byproduct pathways can be stably or transiently introduced into host cells using techniques well known in the art, including but not limited to conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasonic transformation, wherein the byproducts are, for example, pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, which are reduced to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals, such as N-terminal mitochondrial or other targeting signals, which can be removed before transformation into prokaryotic host cells if necessary. For example, removal of the mitochondrial leader sequence results in increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280: 4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, the gene can be expressed in the cytosol without the addition of a leader sequence, or can be targeted to mitochondria or other organelles or to secretion by adding a suitable targeting sequence, such as a mitochondrial targeting or secretion signal suitable for the host cell. Therefore, it is understood that appropriate modifications of the nucleic acid sequence to remove or include the targeting sequence can be incorporated into the exogenous nucleic acid sequence to impart the desired properties. In addition, the gene can be codon optimized using techniques known in the art to achieve optimized expression of the protein.

[0156] One or more expression vectors can be constructed to include one or more exogenous nucleic acids operably linked to expression control sequences that function in the host organism, each of which encodes an enzyme expressed in an amount sufficient to reduce byproducts to enhance carbon flux through acetyl-CoA, the byproduct being a pyruvate byproduct, acetate and / or ethanol, as exemplified herein. In some embodiments, one or more expression vectors can be further constructed to include one or more exogenous nucleic acids operably linked to expression control sequences that function in the host organism, each of which encodes an enzyme expressed in an amount sufficient to increase the production of acetyl-CoA derivatives, such as 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or other acetyl-CoA derivative biosynthetic pathway encoding nucleic acids, as exemplified herein. Expression vectors suitable for use in the microbial host organisms of the present invention include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, including operable vectors and selection sequences or markers that can be used for stable integration into host chromosomes. In addition, the expression vector can include one or more selective marker genes and appropriate expression control sequences. Selectable marker genes may also be included, for example, to provide resistance to antibiotics or toxins, to supplement nutritional deficiencies, or to provide key nutrients that are not in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., known in the art. When two or more exogenous coding nucleic acids are to be co-expressed, the two nucleic acids may be inserted into, for example, a single expression vector or a separate expression vector. For single vector expression, the encoding nucleic acid may be operably connected to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods known in the art may be used to confirm the conversion of exogenous nucleic acid sequences involved in metabolism or synthetic pathways. Such methods include, for example, nucleic acid analysis, such as RNA blotting or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of introduced nucleic acid sequences or their corresponding gene products. It is understood by those skilled in the art that exogenous nucleic acids are expressed in an amount sufficient to produce the desired product, and it is further understood that the expression level may be optimized using methods known in the art and disclosed herein to obtain sufficient expression.

[0157] Suitable purification and / or assays can be performed using known methods to test the production of byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, acetyl-CoA, or acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA. Suitable replicates, such as triplicate cultures, can be cultured for each engineered strain to be tested. For example, product and byproduct formation in an engineered production host can be monitored. Final products and intermediates and other organic compounds can be analyzed by methods such as HPLC (high performance liquid chromatography), GC-MS (gas chromatography-mass spectrometry), and LC-MS (liquid chromatography-mass spectrometry) or other suitable analytical methods, using conventional procedures known in the art. The release of products in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohol and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90: 775-779 (2005)), or other suitable assays and detection methods known in the art. Individual enzyme or protein activities from exogenous DNA sequences can also be measured using methods known in the art.

[0158] The production of acetyl-CoA derivative compounds, such as 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate, MMA, or any other acetyl-CoA derivative products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA, can be separated from other components in the culture using a variety of methods known in the art. Such separation methods include, for example, extraction procedures and methods including continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography and ultrafiltration. All of the above methods are well known in the art.

[0159] Any non-naturally occurring microbial organism described herein that has reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA can be cultured to produce and / or secrete the biosynthetic acetyl-CoA derivative products of the invention. For example, an acetyl-CoA derivative product producer can be cultured to biosynthetically produce a desired acetyl-CoA derivative product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA.

[0160] In order to produce the desired acetyl-CoA derivative product, the recombinant strain is cultured in a culture medium with a carbon source and other essential nutrients. It is sometimes necessary and may be very necessary to maintain anaerobic conditions in a fermentor tank to reduce the cost of the entire process. For example, such conditions can be obtained by first purging the culture medium with nitrogen, and then sealing the flask with a diaphragm and a crimp-cap. For strains that are not observed to grow under anaerobic conditions, limited ventilation can be performed by punching a small hole in the diaphragm, using microaerobic or basic anaerobic conditions. Exemplary anaerobic conditions have been previously described and are well known in the art. For example, exemplary aerobic and anaerobic conditions are described in the U.S. Publication 2009 / 0047719 submitted on August 10, 2007. As disclosed herein, fermentation can be carried out in batches, fed-batch or continuous modes.

[0161] The pH of the medium can be maintained at a desired pH, particularly a neutral pH, such as about pH 7, by the addition of a base (e.g., NaOH or other base) or an acid as needed to maintain the medium at the desired pH. The growth rate can be determined by measuring the optical density using a spectrophotometer (600 nm), while the glucose uptake rate can be determined by monitoring the consumption of the carbon source over time.

[0162] The growth medium can include any carbohydrate source that can provide carbon source to non-naturally occurring microorganisms. This type of source includes sugar, for example, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable raw materials and biomass. The exemplary types of biomass that can be used as raw materials in the inventive method include cellulose biomass, hemicellulose biomass and lignin raw materials or raw material parts. This type of biomass raw material contains, for example, carbohydrate substrates that can be used as carbon source, for example, glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will appreciate that renewable feedstocks and biomass other than those exemplified above can also be used to cultivate the microbial organisms of the present invention with reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, for producing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA.

[0163] In addition to renewable feedstocks such as those exemplified above, the microbial organisms of the invention can also be modified to grow on syngas as a carbon source, with reductions in byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including, but not limited to, 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA or MAA. In this specific embodiment, one or more proteins or enzymes are expressed in an organism that produces acetyl-CoA-derived products to provide a metabolic pathway for utilizing syngas or other gaseous carbon sources.

[0164] Synthesis gas, also known as syngas or producer gas, is the main product of the gasification of coal and carbonaceous materials (such as biomass materials, including crops and residues). Synthesis gas is mainly a mixture of H2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, kerosene, natural gas, biomass and waste organic matter. Gasification is usually carried out at a high fuel to oxygen ratio. Although mainly H2 and CO, syngas can also contain small amounts of CO2 and other gases. Therefore, syngas provides a cost-effective gaseous carbon source, such as CO, and additional CO2.

[0165] Wood-Ljungdahl pathway catalyzes the conversion of CO and H2 into acetyl-CoA and other products, such as acetic acid. Organisms that can utilize CO and syngas generally also have the ability to utilize CO2 and CO2 / H2 mixtures through the same basic enzyme group and conversions included in the Wood-Ljungdahl pathway. Long before it was revealed that CO could also be used by the same organisms and involved the same pathway, it was recognized that microorganisms converted CO2 into acetic acid in an H2-dependent manner. It has been demonstrated that many acetogenic bacteria grow and produce compounds such as acetic acid in the presence of CO2, as long as hydrogen is present to provide the necessary reducing equivalents (see, for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation:

[0166] 2CO2+4H2+n ADP+n Pi→CH3COOH+2H2O+n ATP

[0167] Therefore, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can also utilize a mixture of CO2 and H2 to produce acetyl-CoA and other desired products.

[0168] The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions that can be divided into two branches: (1) the methyl branch and (2) the carbonyl branch. The methyl branch converts syngas to methyltetrahydrofolate (methyl-THF), while the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in sequence by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methylenetetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase, and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in sequence by the following enzymes or proteins: methyltetrahydrofolate: corrinoid protein methyltransferase (e.g., AcsE), corrinoid iron-sulfur protein, nickel protein assembly protein (e.g., AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase, and nickel protein assembly protein (e.g., CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to reduce the production of unwanted byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA or MAA, one skilled in the art will understand that the same engineering design can also be performed for the introduction of nucleic acids encoding at least a Wood-Ljungdahl enzyme or protein not present in the host organism. Thus, the introduction of one or more encoding nucleic acids into the microbial organisms of the present invention such that the modified organism contains a complete Wood-Ljungdahl pathway will confer syngas utilization capability.

[0169] In addition, the reductive (reverse) tricarboxylic acid cycle is used for and / or hydrogenase activity can also be used to convert CO, CO2 and / or H2 into acetyl-CoA and other products, such as acetic acid. The organism that can fix carbon by the reductive TCA approach can utilize one or more of the following enzymes: ATP citrate lyase, citrate lyase, aconitase, isocitrate dehydrogenase, α-ketoglutarate: ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD (P) H: ferredoxin oxidoreductase, carbon monoxide dehydrogenase and hydrogenase. Specifically, the reduction equivalent extracted from CO and / or H2 by carbon monoxide dehydrogenase and hydrogenase is used for CO2 to be fixed as acetyl-CoA or acetic acid by the reductive TCA cycle. Acetic acid can be converted into acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase / phosphotransacetylase and acetyl-CoA synthetase. Acetyl-CoA can be converted to precursors of acetyl-CoA derivatives, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative, glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and enzymes of gluconeogenesis. Following the teachings and guidance provided herein, sufficient numbers of encoding nucleic acids are introduced to generate, for example, a 1,3-BDO pathway, a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway, an MMA pathway, or any other biosynthetic pathway that utilizes acetyl-CoA as a substrate in its pathway, and those skilled in the art will appreciate that the same engineering design can be performed for the introduction of nucleic acids encoding at least a reductive TCA pathway enzyme or protein that is not present in the host organism. Thus, the introduction of one or more encoding nucleic acids into the microbial organisms of the present invention such that the modified organism contains a complete reductive TCA pathway will confer syngas utilization capability.

[0170] Thus, in view of the teachings and guidance provided herein, those skilled in the art will appreciate that non-naturally occurring microbial organisms can be generated that secrete biosynthetic compounds of the invention when grown on a carbon source such as a carbohydrate, with reduced unwanted byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA. Such compounds include, for example, acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA, and any intermediate metabolites therein. All that is required is to engineer one or more desired enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate, including, for example, a biosynthetic pathway comprising some or all of the acetyl-CoA derived products. Thus, the invention provides non-naturally occurring microbial organisms that produce and / or secrete acetyl-CoA derivative products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative products when grown on carbohydrates or other carbon sources, and that produce and / or secrete any intermediate metabolites in the acetyl-CoA derivative product pathway when grown on carbohydrates or other carbon sources. The microbial organisms of the invention that produce acetyl-CoA derivative products can synthesize from intermediates.

[0171] In some embodiments, the non-naturally occurring microbial organisms of the invention are constructed using methods known in the art as exemplified herein to exogenously express at least one nucleic acid encoding an enzyme or protein in an amount sufficient to reduce byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA and thereby increase acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA. In other embodiments, the non-naturally occurring microbial organisms of the invention are constructed using methods known in the art as exemplified herein to attenuate the activity of an enzyme or protein in an amount sufficient to reduce byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol to enhance carbon flux through acetyl-CoA and thereby increase acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including but not limited to 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA.

[0172] It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, resulting in intracellular concentrations of about 0.1-200 mM or more. Typically, the intracellular concentration of acetyl-CoA derivative products such as 1,3-BDO, (3R)-hydroxybutyl (3R)-hydroxybutyrate or MMA is about 30-300 mM, particularly about 50-200 mM, more particularly about 70-150 mM, including about 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM or more. Intracellular concentrations between and above each of these exemplary ranges can also be achieved by the non-naturally occurring microbial organisms of the invention.

[0173] In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been previously described and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described in, for example, the U.S. Publication 2009 / 0047719 filed on August 10, 2007. Any of these conditions can be used together with non-naturally occurring microbial organisms and other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, acetyl-CoA derivative products such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA, can synthesize required acetyl-CoA derivative products at 5-10mM or higher intracellular concentrations and all other concentrations illustrated herein. It is understood that even though the above description refers to intracellular concentrations, a microbial organism that produces an acetyl-CoA-derived product can produce the acetyl-CoA-derived product, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, intracellularly and / or secrete the product into the culture medium.

[0174] In addition to the culture and fermentation conditions disclosed herein, growth conditions for achieving, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative biosynthesis, may include the addition of an osmotic protectant to the culture conditions. In certain embodiments, the non-naturally occurring microbial organisms of the present invention may be maintained, cultured, or fermented as described herein in the presence of an osmotic protectant. In short, an osmotic protectant refers to a compound that acts as an osmotic agent (osmolyte) and helps the microbial organisms described herein survive osmotic stress. Osmotic protectants include, but are not limited to, betaines, amino acids, and sugar trehalose. Non-limiting examples of this type are glycine betaine, praline betaine, dimethylthiotin, dimethylslfonioproprionate, 3-dimethylsulfono-2-methylpropionate, pipecolic acid, dimethylsulfonioacetate, choline, L-carnitine, and tetrahydropyrimidine. In one aspect, the osmotic protectant is glycine betaine. Those of ordinary skill in the art will appreciate that the amount and type of osmoprotectant suitable for protecting the microbial organisms described herein from osmotic stress will depend on the microbial organisms used. The amount of osmoprotectant in the culture conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about 10 mM, no more than about 50 mM, no more than about 100 mM or no more than about 500 mM.

[0175] Culture conditions can include, for example, liquid culture procedures as well as fermentation and other large-scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

[0176] As described herein, an exemplary growth condition for achieving biosynthesis of, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the present invention can be maintained, cultured, or fermented under anaerobic or substantially anaerobic conditions. In short, anaerobic conditions refer to an environment without oxygen. Substantially anaerobic conditions include, for example, culturing, batch fermentation, or continuous fermentation such that the dissolved oxygen concentration in the culture medium is maintained between 0% and 10% of saturation. Substantially anaerobic conditions also include growing or dormant cells on liquid culture medium or solid agar in a sealed chamber that maintains an atmosphere of less than 1% oxygen. For example, the percentage of oxygen can be maintained by purging the culture with a N2 / CO2 mixture or other suitable non-oxygen gas or gases.

[0177] The culture conditions described herein can be scaled up and grown continuously for the production of, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation, fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these methods are well known in the art. Fermentation procedures are particularly suitable for the biosynthetic production of commercial quantities of, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product. Generally, and as with discontinuous culture procedures, continuous and / or near-continuous production of, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, will include culturing the non-naturally occurring organisms of the invention that produce 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product in nutrients and culture medium sufficient to maintain and / or nearly maintain exponential phase growth. Continuous culturing under such conditions may include, for example, growth for 1, 2, 3, 4, 5, 6, or 7 days or longer. In addition, continuous culturing may include longer time periods of 1, 2, 3, 4, or 5 weeks or more, as well as months. Alternatively, the organisms of the invention may be cultured for several hours, if appropriate for a particular application. It should be understood that continuous and / or near-continuous culture conditions may also include all time intervals between these exemplary time periods. It should also be understood that the time for culturing the microbial organisms of the invention is a sufficient time period to produce sufficient amounts of the product for the desired purpose.

[0178] Fermentation procedures are well known in the art. Briefly, fermentation to biosynthetically produce, for example, 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derivative product, can be used, for example, in fed-batch fermentation and batch separation, fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

[0179] In addition to the above fermentation process using the 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate or any other acetyl-CoA derivative product producer of the present invention to continuously produce a large amount of 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate or any other acetyl-CoA derivative product, if desired, the 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate or any other acetyl-CoA derivative product producer can also be subjected to a chemical synthesis process, for example, simultaneously to convert the product into other compounds, or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product into other compounds.

[0180] In order to generate better producers, metabolic modeling can be used to optimize growth conditions. Modeling can also be used to design additional knockouts that optimize pathway utilization (see, e.g., U.S. Patent Publications US 2002 / 0012939, US 2003 / 0224363, US 2004 / 0029149, US 2004 / 0072723, US 2003 / 0059792, US 2002 / 0168654, and US2004 / 0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable prediction of the impact on cell growth of shifting metabolism to more efficiently produce 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyric acid, or any other acetyl-CoA derivative products.

[0181] A computational method for identifying and designing metabolic changes that are beneficial to the biosynthesis of desired products is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84: 647-657 (2003)). OptKnock is a metabolic modeling and simulation program that proposes a gene deletion or destruction strategy that causes genetically stable microorganisms to overproduce target products. Specifically, the framework examines the complete metabolism and / or biochemical network of microorganisms to propose genetic manipulations that force the desired biochemical substances to become essential byproducts of cell growth. Through strategically placed gene deletions or other functional gene disruptions, biochemical production is coupled to cell growth, and after a long time in a bioreactor, the growth selection pressure applied to the engineered strain leads to improved performance, which is the result of mandatory growth-coupled biochemical production. Finally, when constructing a gene deletion, the possibility of designing the strain to restore its wild-type state is negligible because the genes selected by OptKnock will be completely removed from the genome. Therefore, this computational approach can be used to identify alternative pathways leading to the biosynthesis of a desired product, or used in conjunction with non-naturally occurring microbial organisms to further optimize the biosynthesis of a desired product.

[0182] In short, OptKnock is a term used herein to refer to computational methods and systems for simulating cellular metabolism. The OptKnock program involves a model and method framework that incorporates specific constraints into a flux balance analysis (FBA) model. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and / or DNA microarray experimental data. OptKnock also calculates solutions to various metabolic problems, such as by tightening the flux boundaries derived by the flux balance model, and then probing the performance limitations of the metabolic network in the presence of gene additions or deletions. The OptKnock computational framework allows the construction of model formulas that allow effective query of metabolic network performance limitations, and provides methods for solving the resulting mixed integer linear programming problems. The metabolic modeling and simulation method referred to herein as OptKnock is described in, for example, U.S. Publication No. 2002 / 0168654, filed January 10, 2002, International Patent No. PCT / US02 / 00660, filed January 10, 2002, and U.S. Patent No. 2009 / 0047719, filed August 10, 2007, all of which are incorporated by reference in their entirety.

[0183] Another computational approach used to identify and design metabolic alterations that favor product biosynthesis is known as Metabolic modeling and simulation systems. Such computational methods and systems are described in, for example, U.S. Publication No. 2003 / 0233218, filed on June 14, 2002, and International Patent Application No. PCT / US03 / 18838, filed on June 13, 2003. It is a computational system that can be used to generate network models on a computer and simulate the flow of mass, energy, or charge through the chemical reactions of a biological system to define a solution space containing any and all possible functions of the chemical reactions in the system, thereby determining the range of activities allowed by the biological system. This approach is called constraint-based modeling because the solution space is defined by constraints, such as the known stoichiometry of the reactions included and the thermodynamic and capacity constraints of the reactions related to the maximum flux through the reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behaviors of the biological system or its biochemical components.

[0184] These computational approaches are consistent with biological reality, as biological systems are flexible and can reach the same outcome in many different ways. Biological systems are designed through evolutionary mechanisms that are subject to fundamental constraints that all living systems must face. Constraint-based modeling strategies therefore embrace these general realities. Furthermore, the ability to continually impose further restrictions on network models by tightening constraints leads to a reduction in the size of the solution space, thereby increasing the precision with which physiological performance or phenotypes can be predicted.

[0185] Given the teachings and guidance provided herein, one skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement the biosynthesis of desired compounds in host microbial organisms. Such metabolic modeling and simulation methods include, for example, and OptKnock. To illustrate the present invention, some methods are described herein with reference to the OptKnock computational framework for modeling and simulation. Those skilled in the art will know how to use OptKnock to apply the identification, design and implementation of metabolic alterations to any such other metabolic modeling and simulation computational frameworks and methods known in the art.

[0186] The above method will provide a set of metabolic reactions to be destroyed. Elimination of each reaction or metabolic modification within the set may result in the desired product as an essential product of the growth phase of the organism. Because the reactions are known, the solution to the two-level OptKnock problem will also provide one or more associated genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identifying a set of reactions and their corresponding genes encoding the enzymes involved in each reaction is usually an automated process and is accomplished by associating the reactions with a reaction database having relationships between enzymes and encoding genes.

[0187] Once identified, by making at least one gene encoding each metabolic reaction in the group be functionally destroyed, a group of reactions to be destroyed in order to achieve the desired product production is implemented in the target cell or organism. A particularly useful way to achieve the functional destruction of the reaction group is to make each coding gene missing. However, in some cases, by other genetic aberrations, including for example the disappearance of a sudden change, a regulatory region (for example a promoter or a cis-binding site of a regulatory factor), or by truncating the coding sequence at any position in a plurality of positions, it may be useful to destroy the reaction. These latter aberrations, resulting in less total deletion of the genome, may be useful, for example, when the coupling of the product needs to be rapidly assessed or when the gene back mutation is unlikely to occur.

[0188] In order to identify other productive schemes of the above-mentioned two-stage OptKnock problem, which scheme leads to other reaction groups that will be destroyed or metabolic modifications that can cause biosynthesis, the biosynthesis includes growth-coupled biosynthesis of the desired product, an optimization method called integer cut can be implemented. The method proceeds by iteratively solving the OptKnock problem illustrated above, and other constraints called integer cuts are added in each iteration. The integer cut constraint effectively prevents the solver from selecting the exact same reaction group identified in any previous iteration that forcibly couples product biosynthesis to growth. For example, if the metabolic modification of the previously identified even growth coupling specifies reactions 1, 2, and 3 for destruction, the following constraints prevent the same reaction from being considered simultaneously in subsequent schemes. Integer cut methods are well known in the art and can be found in, for example, Burgard et al., Biotechnol. Prog. 17: 791-797 (2001). As with all methods described herein, reference is made to their use in conjunction with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method for reducing redundancy in iterative computational analysis can also be applied with other computational frameworks known in the art, including, e.g.

[0189] The methods exemplified herein allow for the construction of cells and organisms that biosynthetically produce desired products, including the mandatory coupling of production of a target biochemical product to the growth of a cell or organism engineered to contain an identified genetic alteration. Thus, the computational methods described herein allow for the identification and implementation of a target biochemical product that is produced by a gene selected from the group consisting of OptKnock or The set of metabolic modifications can include, for example, the addition of one or more biosynthetic pathway enzymes and / or the functional disruption of one or more metabolic reactions, including, for example, disruptions caused by gene deletions.

[0190] As discussed above, the development of OptKnock methodology is to carry out under the premise that the mutant microbial network can evolve to the maximum growth phenotype predicted by its calculation when experiencing long-term growth selection. In other words, the method takes advantage of the ability of organisms to self-optimize under selection pressure. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force the coupling between biochemical production and cell growth based on network stoichiometry. The identification of the best gene / reaction knockout requires a solution for a two-stage optimization problem, which selects an active reaction group so that the optimal growth scheme of the resulting network overproduces the biochemical substance of interest (Burgard et al., Biotechnol. Bioeng. 84: 647-657 (2003)).

[0191] The computer stoichiometric model of Escherichia coli metabolism can be used to identify the essential genes of metabolic pathways, as previously illustrated and described in for example U.S. Patent Publications US 2002 / 0012939, US 2003 / 0224363, US 2004 / 0029149, US 2004 / 0072723, US 2003 / 0059792, US 2002 / 0168654 and US 2004 / 0009466, and U.S. Patent number 7,127,379. As disclosed herein, the OptKnock mathematical framework can be used to identify the gene deletion that causes the growth coupling production of the desired product. In addition, the scheme of the two-stage OptKnock problem only provides one group of deletions. In order to enumerate all meaningful schemes, i.e. all knockout groups that cause the growth coupling production to form, the optimization technique called integer cutting can be implemented. As discussed above, this requires iterative solution of the OptKnock problem, and in each iteration, other constraints called integer cutting are added.

[0192] As provided herein, the invention also provides non-naturally occurring microbial organisms with genetic alterations (e.g., gene disruptions) that reduce the production of byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA. In some embodiments, the non-naturally occurring microbial organisms of the invention include an acetolactate synthase deletion. In some embodiments, the acetolactate synthase deletion includes an ilvG deletion.

[0193] In view of the teachings and guidance provided herein, it will be understood by those skilled in the art that in order to introduce metabolic changes, such as the destruction of enzymatic reactions, it is necessary to destroy the catalytic activity of one or more enzymes involved in the reaction. Alternatively, metabolic changes may include the destruction of the expression of regulatory proteins or cofactors necessary for enzyme activity or maximum activity. Destruction may occur by a variety of methods, including, for example, deletion of coding genes or incorporation of genetic changes in one or more coding gene sequences. The targeted destroyed coding genes may be one, some or all genes encoding enzymes involved in catalytic activity. For example, when a single enzyme participates in targeted catalytic activity, destruction may be carried out by genetic changes that reduce or eliminate the catalytic activity of the coding gene product. Similarly, when a single enzyme is a polymer, including a heteropolymer, destruction may be carried out by genetic changes that reduce or destroy the function of one or all subunits of the coding gene product. The destruction of activity may be achieved by the following means: loss of the binding activity of one or more subunits required to form an active complex, destruction of the catalytic subunits of the multimeric complex, or both. Other functions of multimeric protein association and activity may also be targeted to destroy the metabolic reactions of the present invention. Such other functions are well known to those skilled in the art. Similarly, the target enzyme activity can be reduced or eliminated by destroying the expression of proteins or enzymes that modify and / or activate the target enzyme, for example, by converting the apoenzyme into the molecules required for the holoenzyme. In addition, some or all functions of a single polypeptide or multimeric complex can be destroyed according to the present invention to reduce or eliminate the catalytic activity of one or more enzymes involved in the reaction or metabolic modification of the present invention. Similarly, some or all enzymes involved in the reaction or metabolic modification of the present invention can be destroyed, as long as the targeted reaction is reduced or eliminated.

[0194] In view of the teaching and guidance provided herein, it will also be understood by those skilled in the art that enzymatic reactions can be destroyed by reducing or eliminating the reaction encoded by common genes and / or by one or more orthologous genes of the gene showing similar or substantially identical activity. The reduction of common genes and all orthologous genes can cause any catalytic activity of targeted reactions to be completely eliminated. However, the destruction of common genes or one or more orthologous genes can cause the reduction of targeted reaction catalytic activity to be enough to promote the coupling of growth and product biosynthesis. Common genes encoding multiple metabolically modified catalytic activities and their orthologous genes are illustrated herein. It will be understood by those skilled in the art that the destruction of genes of enzymes encoding some or all targeted metabolic reactions can be implemented in the method of the present invention, and incorporated into the non-naturally occurring microbial organisms of the present invention, to achieve the reduction of the production of byproducts such as pyruvic acid byproducts (e.g., alanine and / or valine), TCA derived byproducts, acetate and / or ethanol, to enhance the carbon flux by acetyl-CoA.

[0195] In some embodiments, microaerobic design can be used based on the growth coupling formation of desired product. In order to check this point, it is possible to first maximize and then minimize product output with feasible different biomass formation rates in the network, for each strategy to build production cone (production cone). If the rightmost boundary of all possible phenotypes of the mutant network is a point, this means that under the maximum biomass formation rate, a unique product optimal yield may exist in the network. In other cases, the rightmost boundary of feasible phenotype is a vertical line, indicating that at the maximum biomass point, the network can prepare the product of any amount in the calculation range, including the minimum amount at the bottom point of the vertical line. The priority of this type of design is lower.

[0196] In some embodiments, gene disruption can include complete gene deletion. In some embodiments, other methods of disrupting genes include, for example, frameshifting by omitting or adding oligonucleotides or by mutations that render the gene inoperable. However, those skilled in the art will recognize the advantages of gene deletion because it confers stability to non-naturally occurring organisms, preventing reversion to the parental phenotype in which gene disruption has not occurred. In particular, gene disruption is selected from the genome disclosed herein.

[0197] Once a gene set for disruption is computationally predicted to reduce byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, strains can be constructed, evolved, and tested. Gene disruption, including gene deletion, is introduced into the host organism by methods known in the art. As disclosed herein, a particularly useful gene disruption method is by homologous recombination.

[0198] Engineered strains can be characterized by measuring growth rate, substrate uptake rate, and / or product / byproduct secretion rate. Cultures can be grown and used as inoculum for fresh batch cultures measured during exponential growth. Growth rate can be determined by measuring optical density using a spectrophotometer (A600). The concentration of glucose and other organic acid byproducts in the culture supernatant, as disclosed herein, can be determined by known methods such as HPLC, GC-MS, or other well-known analytical methods suitable for analyzing the desired product, and used to calculate uptake rate and secretion rate.

[0199] Strains containing gene disruptions may show suboptimal growth rates until their metabolic networks have adapted to their missing functions. In order to help with this adaptation, strains can be subjected to adaptive evolution. By subjecting strains to adaptive evolution, cell growth rate becomes the main selection pressure, and mutant cells are forced to redistribute their metabolic fluxes to improve their growth rates. Recently, several E. coli mutants have been demonstrated to have been subjected to adaptive evolution on various substrates to achieve growth rates predicted a priori by computer models (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004)). The growth improvement brought by adaptive evolution is accompanied by the increased [INSERT PRODUCT (insertion product)] productivity. Adaptive evolution of strains is often performed in replicates run in parallel to account for differences in evolutionary patterns that may be exhibited by the host organism (Fong and Palsson, Nat. Genet. 36: 1056-1058 (2004); Fong et al., J. Bacteriol. 185: 6400-6408 (2003); Ibarra et al., Nature 420: 186-189 (2002)), which could potentially result in one strain having superior production quality over the other strains. Evolution can be run for a period of time, typically 2-6 weeks, depending on the rate of growth improvement achieved. In general, evolution is stopped once a stable phenotype is achieved.

[0200] The new strains are characterized again by measuring growth rate, substrate absorption rate and product / byproduct secretion rate after the adaptive evolution process. These results are compared with theoretical predictions by mapping the production envelope of actual growth and output and metabolic modeling together. The most successful design / evolution combination is selected to further explore and characterize in batch and continuous fermentation of laboratory scale. The growth-coupled biochemical production concept behind the method disclosed herein (such as OptKnock method) should also result in the generation of genetically stable overproducers. Therefore, the culture is maintained for a long time in a continuous mode, such as one month or longer, to assess long-term stability. Sampling can be performed regularly to ensure that productivity and productivity are maintained.

[0201] Adaptive evolution is a powerful technique that can be used to improve the growth rate of wild-type strains grown under mutation or engineering microbial strains or non-natural environmental conditions. It is particularly useful for strains designed by methods such as OptKnock, which can cause growth coupling products to form. Therefore, the evolution to the best growth strain will also indirectly optimize production. Unique strains (Fong and Palsson, Nat.Genet.36:1056-1058 (2004)) of Escherichia coli K-12MG1655 were created by gene knockout and adaptive evolution. In this work, all adaptive evolution cultures were maintained at long-term exponential growth by continuously passing batch cultures into fresh culture medium before reaching stationary phase, so that growth rate was used as the main selection pressure. Knockout strains were constructed and evolved on minimal medium supplemented with different carbon substrates (four carbon substrates for each knockout strain). Evolution cultures were carried out in duplicate or triplicate, and 50 evolution knockout strains were obtained in total. Evolution cultures were kept exponentially growing until stable growth rate was reached. The computational predictions were accurate (within 10%) when predicting the post-evolution growth rates of knockout strains in 38 of the 50 cases examined. In addition, the combination of the OptKnock design and adaptive evolution led to improvements in lactic acid production strains. (Fong et al., Biotechnol. Bioeng. 91: 643-648 (2005)). Similar methods can be applied to the strains disclosed herein and to various host strains.

[0202] There are many developed techniques that can be used to perform adaptive evolution. Exemplary methods are disclosed herein. In some embodiments, the optimization of non-naturally occurring organisms of the present invention with reduced byproducts such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, includes utilizing adaptive evolution techniques to enhance carbon flux through acetyl-CoA, and thereby increase acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived products, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA or MAA, to increase the yield and / or stability of the production strain.

[0203] Serial cultivation involves repeatedly transferring a small amount of growing culture to a much larger container containing fresh growth medium. When the cultured organism grows to saturation in the new container, the process is repeated. In the literature (Lenski and Travisano, Proc. Natl. Acad. Sci. USA 91: 6808-6814 (1994)), this method has been used to achieve the demonstration of the longest continuous cultivation in experiments, and the experiments clearly demonstrate the continuous improvement of the reproduction rate over several years. Usually, the transfer of the culture is usually carried out in the exponential phase, so the transfer amount is accurately calculated every day to maintain exponential growth in the next 24 hours. Manual serial dilution is low in cost and easy to parallelize.

[0204] In continuous culture, the growth of cells in the chemostat represents an extreme dilution situation, in which a very high proportion of cell groups are retained. Along with the growth and saturation of the culture, a small portion of the culture grown is replaced by fresh culture medium, so that the culture continues to grow when approaching its maximum population size. Chemostat has been used to demonstrate a short-term rapid improvement in reproduction rate (Dykhuizen, Methods Enzymol.613-631 (1993)). The potential usefulness of these devices has been recognized, but due to the unintentional selection of anti-dilution (static) variants, traditional chemostat cannot maintain the selection of the reproduction rate of improvement for a long time. These variants can resist dilution by adhering to the chemostat surface, and by doing so, outperform individuals with lower adhesion in competition, including those with higher reproduction rates, thereby excluding the intended purpose of the device (Chao and Ramsdell, J. Gen. Microbiol 20: 132-138 (1985)). One possible way to overcome this drawback is to implement an apparatus with two growth chambers which, as previously described (Marliere and Mutzel, US Patent No. 6,686,194), periodically undergo a transient sterilization phase.

[0205] EvolugatorTM is a continuous culture device developed by Evolugate, LLC (Gainesville, FL), which shows significant time and energy savings compared to traditional evolution techniques (de Crecy et al.,. Appl. Microbiol. Biotechnol. 77: 489-496 (2007)). Before reaching the stationary phase, cells are kept in long-term exponential growth by serially subculturing batch cultures into fresh culture medium. Through automated optical density measurement and liquid handling, EvolugatorTM can use large culture media to perform serial transfers at high rates, thereby approaching the efficiency of chemostat in cell fitness evolution. For example, an Acinetobacter ADP1 mutant that lacks translational elements and severely hinders growth evolved to 80% of the wild-type growth rate in 200 generations. However, compared to chemostat, which keeps cells in a single container, the machine operates by moving from one "reactor" to the next in a subdivided area of ​​a roll of tubes, thereby eliminating any selection for wall growth. The transfer volume is adjustable and is usually set to about 50%. The disadvantage of this device is that it is large and expensive, so it is impractical to run a large number of evolutions in parallel. In addition, gas addition is not well regulated and the current device configuration does not maintain strict anaerobic conditions. However, this is an alternative method for adaptive evolution of production strains.

[0206] As disclosed herein, nucleic acids encoding desired enzyme activities that reduce unwanted byproducts, such as pyruvate byproducts (e.g., alanine and / or valine), TCA-derived byproducts, acetate and / or ethanol, can be introduced into a host organism to enhance carbon flux through acetyl-CoA, thereby increasing acetyl-CoA-derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA-derived product, including, but not limited to, 4OH2B, 3HB, adipate, caprolactam, 6-ACA, HMDA, or MAA. In some cases, it may be desirable to modify the activity of an enzyme or protein that reduces unwanted byproducts or proteins to increase carbon flux through acetyl-CoA, thereby increasing acetyl-CoA derived products, such as 1,3-BDO, MMA, (3R)-hydroxybutyl (3R)-hydroxybutyrate, or any other acetyl-CoA derived product, including but not limited to 4OH2B, 3HB, adipic acid, caprolactam, 6-ACA, HMDA, or MAA. For example, known mutations that increase protein or enzyme activity can be introduced into the encoding nucleic acid molecule. In addition, optimization methods can be applied to increase the activity of an enzyme or protein and / or reduce inhibitory activity, for example, to reduce the activity of a negative regulator.

[0207] One such optimization method is directed evolution. Directed evolution is a powerful method that involves the introduction of mutations targeted to specific genes to improve and / or alter the properties of an enzyme. Improved and / or altered enzymes can be identified by developing and implementing sensitive high-throughput screening assays that allow for the automated screening of many enzyme variants (e.g., >10 4 ). Iterative rounds of mutagenesis and screening are usually performed to provide enzymes with optimized properties. Computational algorithms that help identify mutagenic gene regions have also been developed, and the algorithm can significantly reduce the number of enzyme variants that need to be generated and screened. Many directed evolution techniques that effectively create libraries of different variants have been developed (for review, see Hibbert et al., Biomol. Eng 22: 11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol. Eng 22: 1-9 (2005); and Sen et al., Appl Biochem. Biotechnol 143: 212-223 (2007)), and these methods have been successfully applied to improve the wide range of properties of many enzymes. Enzyme properties that have been improved and / or altered by directed evolution techniques include, for example: selectivity / specificity for conversion of non-natural substrates; temperature stability for robust high temperature processing; pH stability for bioprocessing at lower or higher pH conditions; substrate or product tolerance to achieve high product titers; binding (K m ), including expansion of substrate binding to include non-native substrates; inhibition (K i ), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increase enzymatic reaction rates and thus achieve desired flux; expression level, to increase protein yield and overall pathway flux; oxygen stability, for running air-sensitive enzymes under aerobic conditions; and anaerobic activity, for running aerobic enzymes in the absence of oxygen.

[0208] Many exemplary methods have been developed for the mutagenesis and diversification of genes to target the desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these methods can be used to change and / or optimize the activity of enzymes or proteins that reduce unwanted by-products, such as pyruvic acid by-products (such as alanine and / or valine), TCA derived by-products, acetate and / or ethanol, to enhance the carbon flux through acetyl-CoA. Such methods include, but are not limited to, EpPCR, which introduces random point mutations by reducing the fidelity of the DNA polymerase in the PCR reaction (Pritchard et al., J Theor. Biol. 234:497-509 (2005)); error-prone rolling circle amplification (epRCA), which is similar to epPCR, except that an intact circular plasmid is used as a template and the plasmid is amplified using random 6-mers with exonuclease-resistant phosphorothioate bonds on the last 2 nucleotides and then transformed into cells where the plasmid recircularizes as tandem repeats (Fujii et al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNA or family shuffling, which typically involves the use of a nuclease (e.g., DNase I or EndoV) to generate a pool of random fragments, which are reassembled by annealing and extension cycles in the presence of DNA polymerase to create a chimeric gene library (Stemmer, Proc Natl Acad Sci USA 91: 10747-10751 (1994); and Stemmer, Nature 370: 389-391 (1994)); Staggered Extension (StEP), which requires template priming followed by repeated cycles of 2-step PCR, denaturation and very short annealing / extension duration (as short as 5 seconds) (Zhao et al., Nat. Biotechnol. 16: 258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different fragments of the template (Shao et al., Nucleic Acids Res 26:681-683(1998)).

[0209] Other methods include heteroduplex recombination, in which linear plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); and Volkov et al, Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs DNase I fragmentation and size fractionation of single-stranded DNA (ssDNA) (Coco et al, Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated template (RETT), which requires template switching of a unidirectional growing chain from a primer in the presence of a unidirectional ssDNA fragment used as a template pool (Lee et al, J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules (Bergquist and Gibbs, Methods Mol. Biol 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzyme (ITCHY), which creates a combinatorial library in which the gene or gene fragment of interest is deleted by 1 base pair (Ostermeier et al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol.17:1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzyme (THIO-ITCHY), which is similar to ITCHY except that phosphorothioate dNTPs are used to generate the truncation (Lutz et al., Nucleic Acids Res 29:E16 (2001)); SCRATCHY, which combines two approaches to recombinant genes, ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations are generated by epPCR and then screened / selected for those that retain usable activity (Bergquist et al., al., Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), which is a random mutagenesis method that uses random incorporation and cleavage of phosphorothioate nucleotides to generate a pool of random-length fragments that are used as templates for extension in the presence of a "universal" base such as inosine, and replication of the inosine complement generates random base incorporation and, therefore, mutagenesis (Wong et al., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); synthetic shuffling, which uses overlapping oligonucleotides designed to encode "all the genetic diversity in the target" and allows for very high diversity in the shuffled progeny (Ness et al., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); al., Nat. Biotechnol. 20:1251-1255 (2002)); Nucleotide Exchange and Excision Technology (NexT), which utilizes a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine for endpoint DNA fragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)).

[0210] Further methods include sequence homology-independent protein recombination (SHIPREC), in which a linker is used to promote the fusion between two distantly related or unrelated genes and generate a series of chimeras between the two genes, thereby generating a single crossover hybrid library (Sieber et al., Nat. Biotechnol. 19:456-460 (2001)); gene site saturation mutagenesis TM (Gene SiteSaturation Mutagenesis TM )(GSSM TM ), in which the starting material includes a supercoiled double-stranded DNA (dsDNA) plasmid containing an insert and two primers that are degenerate at the desired mutation site (Kretz et al., Methods Enzymol. 388:3-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace a limited region with a large number of possible amino acid sequence changes (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is similar in nature to CCM and uses epPCR at a high mutation rate to identify hotspots and hotspot regions that are then extended by CMCM to cover defined regions of protein sequence space (Reetz et al., Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241:53-57 (1988)). al., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); mutant strain technology, in which a conditional ts mutant plasmid uses the mutD5 gene encoding a mutant subunit of DNA polymerase III, allowing a random and natural mutation frequency to increase by 20 to 4000-X during the selection process and preventing the accumulation of deleterious mutations when selection is not required (Selifonova et al., Appl. Environ. Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

[0211] Other exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method to evaluate and optimize combinatorial mutations of selected amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at once or to create large chimera (multiple mutation) libraries of a single gene (Tunable Gene Reassembly provided by Verenium Corporation); TM (TGR TM ) Technology); in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors a structurally well-defined protein backbone with a particular fold and searches the sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works best for proteins with known three-dimensional structures (Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using structure / function knowledge to select potential sites for improving the enzyme, performing saturation mutagenesis at the selected sites using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA), screening / selecting for desired properties, and using the improved clones, starting over at another site, and continuing to iterate until the desired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Nat. Protoc. 2:891-903 (2007); al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)). Any of the above mutagenesis methods can be used alone or in any combination. In addition, as described herein, any one or combination of directed evolution methods can be used in combination with adaptive evolution techniques.

[0212] 6. Examples

[0213] Embodiment 1

[0214] Attenuation of acetolactate synthase increases the titer, rate, and yield of 1,3-butanediol (1,3-BDO)

[0215] The following example demonstrates that attenuation of the valine biosynthetic enzyme acetolactate synthase (ilvG) increases the titer, rate, and yield of 1,3-butanediol (1,3-BDO). E. coli strain L16375 expressing nadK and pntAB and E. coli strains L16410 and L16411 expressing nadK and pntAB and having a deletion of the ilvGM operon were cultured and the production of 1,3-BDO was measured.

[0216] Three different bacterial strains (L16410, L16411 and L16375) were cultured in 2.5 mL, 2.0 mL or 1.5 mL of medium at a flow rate of 300 standard cubic centimeters per minute (sccm) under 100% air and a power of 48 WP using standard techniques. The cultures were incubated at 400 rpm for 24 hours with a starting OD of 0.4.

[0217] Table 1

[0218] strain Details L16375 ECh-10481(ECh-10437p108-pntAB-p115-nadK) L16410 ECh-10488(ECh-10452,ΔilvGM,p115-nadK-pntAB) L16411 ECh-10489(ECh-10481,ΔilvGM,p108-pntAB-p115-nadK)

[0219] The results showed that in the specific 1,3-BDO production ( Figure 1A ),rate( Figure 1B ) and yield [c-mol%]( Figure 1C ), the ilvGM deletion strains (L16410 and L16411) performed better than the control (L16375), regardless of the oxygen transfer rate (OTR). In addition, a comparison of valine levels with 1,3-BDO levels in multiple bacterial strains showed an inverse relationship between valine production and 1,3-BDO yield ( Figure 2A Consistent with these results, the ilvGM deletion strains (L16410 and L16411) had negligible levels of valine per culture volume ( Figure 2B ), and 1,3-BDO levels increased ( Figure 2C Specifically, the reduction in valine translated into an increase in 1,3-BDO yield of approximately 1.0-2.5 [c-mol %] ( Figure 2C ).

[0220] Collectively, these results suggest that attenuation of valine biosynthetic enzymes reduces valine levels, which translates into increased 1,3-BDO yield.

[0221] Example II

[0222] Use of aldehyde dehydrogenase with specific activity towards acetaldehyde but not R-3-hydroxybutyraldehyde in acetaldehyde recycling loop way

[0223] The following examples demonstrate that an aldehyde dehydrogenase specific for acetaldehyde can be used in an acetaldehyde recycling loop to reduce the levels of acetyl-CoA byproducts, acetic acid and / or ethanol. Figure 3 As shown, an exemplary pathway for producing 1,3-BDO involves a thiolase (e.g., THL), an acetoacetyl-CoA reductase (e.g., PhaB), a CoA-dependent aldehyde dehydrogenase (e.g., ALD), and an alcohol dehydrogenase (e.g., ADH). However, acetyl-CoA can also be converted to acetaldehyde by ALD, and acetaldehyde can be converted to ethanol by ADH, which limits the amount of acetyl-CoA available for the production of 1,3-BDO. In order to reduce ethanol and acetaldehyde byproducts, the inventors designed an exogenous recycling loop that can convert acetaldehyde to acetate by an aldehyde dehydrogenase (e.g., AldB) and convert acetate back to acetyl-CoA by an acetyl-CoA synthase (e.g., ACS). However, as Figure 4 As shown, some aldehyde dehydrogenases can act on 3HB-aldehyde as well as acetaldehyde.

[0224] To identify aldehyde dehydrogenases that are selective for acetaldehyde, the lysate activities of selected AldB candidates were measured using three different aldehyde substrates: acetaldehyde (AcAld) and R-3-hydroxybutyraldehyde (R-3HBuAld) to measure the selectivity for aldehydes.

[0225]

[0226]

[0227] The results showed that a specific AldB enzyme with specific activity towards acetaldehyde but not towards 3-HB aldehyde could be identified ( Figure 5 ).

[0228] Example III

[0229] Inclusion of acetyl-CoA synthetase variants in the acetaldehyde recycling loop increases 1,3-BDO production

[0230] The following examples demonstrate that expression of acetyl-CoA synthase variants in the acetaldehyde recycling loop increases 1,3-BDO production. The addition of acetyl-CoA synthase variants (ACS*) was analyzed in three different Ald expression strain backgrounds: L16768 (Ald only), L16933 (Ald+pntAB and NadK), and L17786 (Ald with NadK variant).

[0231] Table 2:

[0232] Ald ACS* NadK* L16768 714UC L16946 714UC X L16933 714AIX L17662 714AIX X L17786 714AIX X L17787 714AIX X X

[0233] Comparison of the carbon distribution between pathway products and carbon-3:carbon-2 nodes indicated that expression of ACS* improved 1,3-BDO production in strains L16946 and L17787 relative to strains L16768 and L17787, respectively ( Fig. 6A). In addition, ACS* expression significantly reduced acetate, but not ethanol, formation, suggesting that acetate recycling effectively competes with CoA hydrolases ( Figure 6B ). Consistent with the carbon distribution, the overall product distribution demonstrated that compared with the strain not expressing ACS* ( Fig. 7A )and( Figure 7B ), the expression of ACS* significantly reduced the byproducts ( Figure 7C ) and increased the production of 1,3-BDO ( Fig.7D ).

[0234] Taken together, these results demonstrate that exogenous expression of an acetyl-CoA synthase variant (ACS*) can reduce byproduct production and increase 1,3-BDO production by specifically reducing acetate levels.

[0235] Example IV

[0236] Inclusion of acetyl-CoA synthetase variants in the acetaldehyde recycling loop increases 1,3-BDO production

[0237] The following examples demonstrate that co-expression of acetyl-CoA synthase variants and aldehyde dehydrogenases as part of an acetaldehyde recycling loop can reduce acetate and ethanol byproduct levels and increase 1,3-BDO production in different strain backgrounds.

[0238] The effects of ACS* and AldB 2886B expression were tested in two different Ald-expressing strain backgrounds. Comparison of strains with or without co-expression of ACS* and AldB2886B demonstrated that ACS* and AldB expression significantly reduced the production of acetate and ethanol ( Figure 8 ). In addition, the overall carbon 2 (i.e., ethanol and acetate) reduction was similar between the two strains.

[0239] Table 3:

[0240] Ald ACS* AldB L16768 714UC L16948 714UC X p115-2886B (plasmid) L16933 714AIX L17663 714AIX X p108-2886B (plasmid)

[0241] Using a different AldB candidate, AldB 1139A ( Fig.9A and Fig. 9B ) obtained similar results. Comparison of strains overexpressing ACS* and an alternative AldB candidate (AldB 1139A), with or without a NadK variant (NadK*), resulted in an increase in 1,3-BDO relative to their respective control strains without ACS* and AldB overexpression ( Fig.9A ) and ethanol and acetic acid reduction ( Fig. 9B ).

[0242] Table 4:

[0243] Ald ACS* AldB NadK* L16933 X L17674 X X X L17786 X X L17892 X X X X

[0244] Example V

[0245] Includes an alanine recycling loop to reduce alanine

[0246] The following examples demonstrate that co-expression of a D-amino acid dehydrogenase (dadA) and an alanine racemase (dadX) as part of an alanine recycling loop reduces alanine byproduct levels and increases levels of an exemplary acetyl-CoA derived product, 1,3-BDO.

[0247] When carbon is diverted from pyruvate to alanine metabolism, carbon flux from pyruvate to acetyl-CoA is limited. To test whether the unwanted alanine byproduct can be recycled back to pyruvate, nucleic acids encoding D-amino acid dehydrogenase (dadA) and alanine racemase (dadX) were overexpressed in Escherichia coli to create an alanine recycling loop ( Fig.10 After fermentation, the alanine concentration was compared between the organisms with D-amino acid dehydrogenase (dadA) and alanine racemase (dadX) and the control organisms without D-amino acid dehydrogenase (dadA) and alanine racemase (dadX). The results showed that overexpression of D-amino acid dehydrogenase (dadA) and alanine racemase (dadX) significantly reduced the alanine concentration by about 43% ( Fig. 11B , Figure 11C and Table 5). Therefore, exogenous expression of D-amino acid dehydrogenase (dadA) and alanine racemase (dadX) can generate an alanine recycling loop that can reduce alanine levels in an organism.

[0248] Table 5:

[0249] D-Ala L-Ala Total Ala L-Ala / D-Ala ratio Comparison 2.34 6.09 8.43 2.60 dadA+dadX 1.91 3.76 5.67 1.97

[0250] Analysis of the L- and D-forms of alanine showed that the alanine recycling loop reduces the L-alanine fraction to a greater extent than the D-alanine fraction ( Fig. 11B and Table 5). Consistent with the reduced L-alanine fraction, the L-alanine / D-alanine ratio was also reduced in organisms expressing an alanine recycling loop ( Fig. 11B and Table 5).

[0251] Importantly, a decrease in alanine concentrations in organisms expressing an alanine recycling loop was detected across multiple aeration parameters without affecting other products, including pyruvate or 1,3BG ( Fig.11A ).

[0252] Taken together, these results demonstrate that an alanine recycling loop, involving co-expression of a D-amino acid dehydrogenase (dadA) and an alanine racemase (dadX), is able to reduce the unwanted byproduct alanine.

[0253] Example VI

[0254] Citrate synthase variants reduce TCA cycle-derived byproducts and increase 1,3-BDO

[0255] The following examples demonstrate that expression of citrate synthase variants reduces undesirable tricarboxylic acid (TCA) cycle derived byproducts and increases levels of an exemplary acetyl-CoA derived product, 1,3-BDO.

[0256] Citrate synthase is the enzyme that catalyzes the first step of TCA by converting acetyl-CoA and oxaloacetate to form citrate and is a key enzyme pulling carbon flux from acetyl-CoA to other products such as 1,3-BDO, fatty acid methyl esters, or other chain extension products ( Fig.12 ). As a result, citrate synthase produces unwanted TCA cycle-derived byproducts.

[0257] Citrate synthase can be strongly and specifically inhibited by NADH, and a citrate synthase variant (gltAR109L) has been reported to have increased sensitivity to NADH (see Stokell et al., J. Biol. Chem, 2003; 278(37): 35435-35443). Therefore, to test whether inhibition of citrate synthase can reduce unwanted tricarboxylic acid (TCA) cycle-derived byproducts and increase the production of 1,3-BDO, exogenous gltAR109L was expressed in E. coli.

[0258] Measurement of an exemplary acetyl-CoA-derived product 1,3-BDO after overexpression of a citrate synthase variant (gltA R109L) revealed that the citrate synthase variant increased the titer of 1,3-BDO ( Fig. 13B In addition, lower TCA cycle-derived byproducts were produced in microorganisms expressing the citrate synthase variant (gltA R109L). Notably, microorganisms expressing the citrate synthase variant (gltA R109L) also had a rapid microaerobic transition ( Fig.13A ).

[0259] Taken together, these results demonstrate that citrate synthase variants are able to reduce the production of unwanted TCA cycle-derived byproducts and increase the titer of acetyl-CoA-derived products such as 1,3-BDO.

[0260] In the present application, various publications are cited. The entire disclosure of these publications, including GenBank and GI numbers, is hereby incorporated by reference into the present application to more fully describe the prior art to which the present invention belongs. Although the present invention has been described with reference to the examples provided above, it should be appreciated that various modifications can be made without departing from the spirit of the present invention.

Claims

1. A non-naturally occurring microbial organism with reduced byproducts, comprising a microbial organism having a glycolytic pathway and increased carbon flux through acetyl-CoA, the microbial organism comprising one or more of: (a) Attenuated acetolactate synthase; (b) exogenous acetaldehyde recycling loop; (c) an exogenous alanine recycling loop; and (d) a citrate synthase variant comprising increased sensitivity to NADH compared to wild-type citrate synthase, wherein the exogenous acetaldehyde recycling loop comprises at least one exogenous nucleic acid encoding an aldehyde dehydrogenase and an acetyl-CoA synthase, wherein the exogenous alanine recycling loop comprises at least one exogenous nucleic acid encoding at least an alanine recycling loop enzyme of a D-amino acid dehydrogenase and an alanine racemase, and wherein said byproduct reduction is a result of said microbial organisms comprising said one or more of (a) to (d).

2. The non-naturally occurring microbial organism of claim 1, wherein the attenuated acetolactate synthase comprises reduced expression of acetolactate synthase.

3. The non-naturally occurring microbial organism of claim 1, wherein said attenuated acetolactate synthase comprises a deficiency of acetolactate synthase.

4. The non-naturally occurring microbial organism of claim 1, wherein said attenuated acetolactate synthase comprises a non-functional acetolactate synthase.

5. The non-naturally occurring microbial organism of any one of claims 1-4, wherein the acetolactate synthase is ilvG.

6. The non-naturally occurring microbial organism of claim 1, wherein the attenuated acetolactate synthase reduces valine biosynthesis compared to a microbial organism having a wild-type acetolactate synthase.

7. The non-naturally occurring microbial organism of claim 1, wherein said aldehyde dehydrogenase is encoded by aldB.

8. The non-naturally occurring microbial organism of claim 1, wherein the acetyl-CoA synthase is an acetyl-CoA synthase variant.

9. The non-naturally occurring microbial organism of claim 1, wherein the at least one exogenous nucleic acid encoding an aldehyde dehydrogenase or an acetyl-CoA synthase is a heterologous nucleic acid.

10. The non-naturally occurring microbial organism of claim 1, wherein said non-naturally occurring microbial organism has reduced acetic acid, ethanol, or a combination thereof.

11. The non-naturally occurring microbial organism of claim 1, wherein the at least one exogenous nucleic acid encoding a D-amino acid dehydrogenase or an alanine racemase is a heterologous nucleic acid.

12. The non-naturally occurring microbial organism of claim 1, wherein said D-amino acid dehydrogenase is encoded by dadA.

13. The non-naturally occurring microbial organism of claim 1, wherein said alanine racemase is encoded by dadX.

14. The non-naturally occurring microbial organism of claim 1, wherein said non-naturally occurring microbial organism has a reduced alanine concentration compared to a microbial organism without said exogenous alanine recycling loop.

15. The non-naturally occurring microbial organism of claim 14, wherein said reduced alanine concentration is a reduced L-alanine concentration.

16. The non-naturally occurring microbial organism of claim 1, wherein the non-naturally occurring microbial organism has a reduced ratio of L-alanine to D-alanine compared to a microbial organism without the exogenous alanine recycling loop.

17. The non-naturally occurring microbial organism of claim 1, wherein the citrate synthase variant is a type II citrate synthase.

18. The non-naturally occurring microbial organism of claim 17, wherein the citrate synthase variant binds NADH with a greater affinity than wild-type citrate synthase.

19. The non-naturally occurring microbial organism of claim 17 or 18, wherein said citrate synthase variant comprises at least one exogenous nucleic acid encoded by gltA R109L.

20. The non-naturally occurring microbial organism of claim 19, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

21. The non-naturally occurring microbial organism of claim 1, wherein the non-naturally occurring microbial organism has decreased tricarboxylic acid cycle (TCA) cycle derived byproducts and / or increased acetyl-CoA derived products compared to a microbial organism having a wild-type citrate synthase.

22. The non-naturally occurring microbial organism of claim 1, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

23. The non-naturally occurring microbial organism of claim 1, further comprising a 1,3-butanediol (1,3-BDO) pathway, a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway, a 3-hydroxybutyryl-coenzyme A (3HB-CoA) pathway, a methyl methacrylate (MMA) pathway, an adipate pathway, a caprolactam pathway, a 6-aminocaproic acid (6-ACA) pathway, a hexamethylenediamine (HMDA) pathway, or a methacrylic acid (MAA) pathway.

24. The non-naturally occurring microbial organism of claim 23, wherein said microorganism comprises a 1,3-BDO pathway.

25. The non-naturally occurring microbial organism of claim 24, wherein the 1,3-BDO pathway comprises a thiolase; a CoA-dependent, aldehyde-forming acetoacetyl-CoA reductase; a ketone-reducing 3-oxobutyraldehyde reductase; a 3-hydroxybutyraldehyde reductase; a CoA-dependent, alcohol-forming acetoacetyl-CoA reductase; an aldehyde-reducing 3-oxobutyraldehyde reductase; a 4-hydroxy, 2-butanone reductase; a ketone-reducing acetoacetyl-CoA reductase; an aldehyde-forming 3-hydroxybutyryl-CoA reductase; and an alcohol-forming 3-hydroxybutyryl-CoA reductase.

26. The non-naturally occurring microbial organism of claim 23, wherein said microbial organism comprises a (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway.

27. The non-naturally occurring microbial organism of claim 26, wherein the (3R)-hydroxybutyl (3R)-hydroxybutyrate pathway comprises a thiolase; a (3R)-hydroxybutyl (3R)-hydroxybutyrate forming enzyme; a (3R)-hydroxybutyryl-CoA:(R)-1,3-butanediol alcohol transferase; a (3R)-hydroxybutyl 3-oxobutyrate forming enzyme; an acetoacetyl-CoA:(R)-1,3-butanediol alcohol transferase; a (3R)-hydroxybutyl 3-oxobutyrate reductase; a (3R)-hydroxybutyryl-ACP:(R)-1,3-butanediol ester synthase and an acetoacetyl-ACP:(R)-1,3-butanediol ester synthase.

28. The non-naturally occurring microbial organism of claim 23, wherein said microbial organism comprises a 3HB-CoA pathway.

29. The non-naturally occurring microbial organism of claim 28, wherein said 3HB-CoA pathway comprises acetyl-CoA thiolase and 3-hydroxybutyryl-CoA dehydrogenase.

30. The non-naturally occurring microbial organism of claim 23, wherein said microbial organism comprises a MMA pathway.

31. The non-naturally occurring microbial organism of claim 30, wherein said MMA pathway comprises: (a) 4-hydroxybutyryl-CoA dehydratase, crotonase, 2-hydroxyisobutyryl-CoA mutase, 2-hydroxyisobutyryl-CoA dehydratase and methacrylic acid (MAA)-CoA:methanol transferase; or (b) 4-Hydroxybutyryl-CoA dehydratase, crotonase, 2-hydroxyisobutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA:methanol transferase and 2-hydroxyisobutyrate methyl dehydratase.

32. The non-naturally occurring microbial organism of claim 31, further comprising a second MMA pathway comprising: (c) methacrylic acid (MAA)-CoA:methanol transferase, 4-hydroxybutyryl-CoA mutase and 3-hydroxyisobutyryl-CoA dehydratase; or (d) 4-Hydroxybutyryl-CoA mutase, 3-hydroxyisobutyryl-CoA:methanol transferase, and 3-hydroxyisobutyrate methyl dehydratase.

33. The non-naturally occurring microbial organism of claim 23, wherein said microbial organism comprises a 6-ACA pathway.

34. The non-naturally occurring microbial organism of claim 33, wherein the 6-ACA pathway comprises 2-amino-7-oxosuberate keto-acid decarboxylase, 2-amino-7-oxoheptanoate decarboxylase, 2-amino-7-oxoheptanoate oxidoreductase, 2-aminopimelate decarboxylase, 6-aminohexanal oxidoreductase, 2-amino-7-oxoheptanoate decarboxylase, or 2-amino-7-oxosuberate amino acid decarboxylase.

35. The non-naturally occurring microbial organism of claim 23, wherein said microbial organism comprises a caprolactam pathway.

36. The non-naturally occurring microbial organism of claim 35, wherein said caprolactam pathway comprises 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, an aldehyde forming adipyl-CoA reductase, 6-aminohexanoate transaminase, 6-aminohexanoate dehydrogenase, 6-aminohexanoyl-CoA / acyl-CoA transferase, and 6-aminohexanoyl-CoA synthase.

37. The non-naturally occurring microbial organism of claim 23, wherein said microbial organism comprises an adipate pathway.

38. The non-naturally occurring microbial organism of claim 37, wherein said adipate pathway comprises 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA hydrolase, adipyl-CoA ligase, adipyl-CoA transferase, and phosphotransferase / adipate kinase.

39. The non-naturally occurring microbial organism of claim 23, wherein said microbial organism comprises a hexamethylenediamine (HMDA) pathway.

40. The non-naturally occurring microbial organism of claim 39, wherein said hexamethylenediamine (HMDA) pathway comprises 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, an aldehyde forming adipyl-CoA reductase, 6-aminohexanoate transaminase, 6-aminohexanoate dehydrogenase, 6-aminohexanoyl-CoA / acyl-CoA transferase, 6-aminohexanoyl-CoA synthase, aldehyde forming 6-aminohexanoyl-CoA reductase, HMDA transaminase, and HMDA dehydrogenase.

41. The non-naturally occurring microbial organism of claim 23, wherein said microbial organism comprises a MAA pathway.

42. The non-naturally occurring microbial organism of claim 41, wherein said MAA pathway comprises: (a) (i) a succinyl-CoA transferase, ligase or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA epimerase; (iv) an aldehyde-forming methylmalonyl-CoA reductase; (v) a methylmalonate semialdehyde reductase; and (vi) a 3-hydroxyisobutyrate dehydratase; (b) (i) a succinyl-CoA transferase, ligase or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) an aldehyde-forming methylmalonyl-CoA reductase; (iv) a methylmalonate semialdehyde reductase; and (v) a 3-hydroxyisobutyrate dehydratase; or (c) (i) a succinyl-CoA transferase, ligase or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) an alcohol-forming methylmalonyl-CoA reductase; and (iv) a 3-hydroxyisobutyrate dehydratase.

43. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism is a species of bacteria.

44. A method for enhancing carbon flux through acetyl-CoA in a non-naturally occurring microbial organism to increase the yield of an acetyl-CoA derived product, the method comprising culturing the non-naturally occurring microbial organism of any one of claims 1-43 under conditions and for a sufficient period of time to produce an acetyl-CoA derived product.

45. The method of claim 44, wherein the acetyl-CoA derivative product is selected from the group consisting of 1,3-butanediol (1,3-BDO), methyl methacrylate (MMA), 3R-hydroxybutyric acid-3R-hydroxybutyric acid, 3-hydroxybutyric acid (3-HB), 4-hydroxy-2-butanone (4OH2B), hexamethylenediamine (HMDA), caprolactam, adipic acid, 6-aminocaproic acid (6-ACA), and methacrylic acid (MAA).

46. ​​The method of claim 45, wherein the acetyl-CoA derivative product comprises 1,3-BDO.

47. The method of claim 45, wherein the acetyl-CoA derivative product comprises MMA.

48. The method of claim 45, wherein the acetyl-CoA derivative product comprises 3R-hydroxybutyrate-3R-hydroxybutyrate.

Images