MODIFIED MICROORGANISM AND METHODS OF USING SAME FOR PRODUCING BUTADIENE AND 1-PROPANOL AND/OR 1,2-PROPANEDIOL

BACKGROUND

Butadiene (1,3-butadiene, CH₂═CH—CH═CH₂, CAS 106-99-0) is a linear, 4-carbon molecule with 2 conjugated double bonds typically manufactured (along with other 4-carbon molecules) by steam cracking petroleum-based hydrocarbons. This process involves harsh conditions and high temperatures 850° C.). Other methods of butadiene production involve toxic and/or expensive catalysts, highly flammable and/or gaseous carbon sources, and high temperatures. Globally, several million tons of butadiene-containing polymers are produced annually. Butadiene can be polymerized to form polybutadiene, or reacted with hydrogen cyanide (prussic acid) in the presence of a nickel catalyst to form adiponitrile, a precursor to nylon. More commonly, however, butadiene is polymerized with other olefins to form copolymers such as acrylonitrile-butadiene-styrene (ABS), acrylonitrile-butadiene (ABR), or styrene-butadiene (SBR) copolymers.

1-propanol (n-propanol, CH₃CH₂CH₂OH, CAS 71-23-8) is a primary alcohol typically manufactured by catalytic hydrogenation of propionaldehyde, which is generally synthesized in large scale from ethylene in an energy-intensive multi-step industrial process. This process involves use of toxic chemicals such as carbon monoxide and hydrogen at high pressure (e.g., 10-100 ATM) and high temperature (up to 200° C.). 1-propanol can be used as an intermediate for further organic reactions or as a building block for polymers such as propylene. Propylene is a chemical compound that is widely used to synthesize a wide range of petrochemical products. For instance, this olefin is the raw material used for the production of polypropylene, its copolymers and other chemicals such as acrylonitrile, acrylic acid, epichloridrine and acetone. Propylene is typically obtained in large quantity scales as a byproduct of catalytical or thermal oil cracking, or as a co-product of ethylene production from natural gas. (Propylene, Jamie G. Lacson, CEH Marketing Research Report-2004, Chemical Economics Handbook-SRI International). Propylene is polymerized to produce thermoplastics resins for innumerous applications such as rigid or flexible packaging materials, blow molding and injection molding.

1,2-propanediol (propylene glycol, HO—CH₂—CHOH—CH₃, CAS 57-55-6) is an organic compound with formula C₃H₈0₂. Industrially, propylene glycol is produced from propylene oxide. Propylene glycol may be manufactured using either a non-catalytic high-temperature process at 200° C. (392° F.) to 220° C. (428F), or a catalytic method, which proceeds at 150° C. (302° F.) to 180° C. (356° F.) in the presencefdon exchange resin or a small amount of sulfuric acid or alkali. Propylene glycol can be used as a solvent, nontoxic antifreeze and to produce polyesteres compounds.

Given the world-wide demand for butadiene, 1-propanol, and 1,2-propanediol, there exits a need in the art for improved methods for their production that overcome their current production drawbacks including the use of toxic and/or expensive catalysts, and highly flammable and/or gaseous carbon sources.

SUMMARY

The present disclosure provides a non-naturally occurring microorganism comprising: one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA (i.e., acetyl-CoA is produced in the cytosol of the microorganism), wherein the microorganism has reduced levels of pyruvate decarboxylase enzymatic activity, and wherein the microorganism is capable of growing on a C6 sugar as a sole carbon source and under anaerobic conditions.

The present disclosure also provides a non-naturally occurring microorganism comprising: a disruption of one or more enzymes that decarboxylate pyruvate and/or a disruption of one or more transcription factors of one or more enzymes that decarboxylate pyruvate; a genetic modification that substantially decreases glucose import into the microorganism; one or more polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA; one or more polynucleotides encoding one or more enzymes in a pathway that catalyze a conversion of crotonyl alcohol, 5-hydroxy-3-ketovaleryl-CoA, 3-ketopent-4-enoyl-CoA, or 3,5-ketovaleryl-CoA to butadiene; and one or more polynucleotides encoding one or more enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to 1-propanol and/or 1,2-propanediol.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more enzymes that decarboxylate pyruvate is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more enzymes that decarboxylate pyruvate include pdc1, pdc 5, and/or pdc6, and wherein the one or more transcription factors of the one or more enzymes that decarboxylate pyruvate include pdc2.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises an exogenous polynucleotide that encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises a genetic modification in an endogenous polynucleotide that encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentioned embodiments, the genetic modification is a truncation of the MTH1 transcription factor.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces acetyl-CoA encode i.) pyruvate formate lyase and pyruvate formate lyase activating enzyme, ii) pyruvate dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoamide dehydrogenase, iii) pyruvate dehydrogenase, dihydrolipoyl transacetylase, dihydrolipoamide dehydrogenase, and pyruvate dehydrogenase complex protein X, or any combination thereof.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism is a eukaryote selected from the group consisting of: yeast, filamentous fungi, protozoa, and algae.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of crotonyl alcohol to butadiene include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonyl-alcohol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-alcohol to butadiene, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-alcohol to 2-butenyl-4-phosphate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-butenyl-4-phosphate to 2-butenyl-4-diphosphate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-butenyl-4-diphosphate to butadiene, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-alcohol to 2-butenyl-4-diphosphate; wherein the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 5-hydroxy-3-ketovaleryl-CoA to butadiene include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-ketovaleryl-CoA to R/S-3,5-dihydroxy-valeryl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S-3,5-dihydroxy-valeryl-CoA to (R/S)-3-hydroxy-4-pentenoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of (R/S)-3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene; wherein the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 3-ketopent-4-enoyl-CoA to butadiene include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-ketopent-4-enoyl-CoA to R/S-3-hydroxy-4-pentenoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S-3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene; or wherein the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 3,5-ketovaleryl-CoA to butadiene include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3,5-ketovaleryl-CoA to 5-hydroxy-3-ketovaleryl-CoA or R/S 5-keto-3-hydroxyvaleryl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-ketovaleryl-CoA or R/S 5-keto-3-hydroxyvaleryl-CoA to R/S-3,5-dihydroxy-valeryl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S-3,5-dihydroxy-valeryl-CoA to R/S-3-hydroxy-4-pentenoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S-3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanol and/or 1,2 propanediol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

The present disclsoure also provides a non-naturally occurring microorganism comprising: one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA; one or more polynucleotides encoding one or more enzymes in a pathway that catalyze a conversion of crotonyl alcohol, 5-hydroxy-3-ketovaleryl-CoA, 3-ketopent-4-enoyl-CoA, or 3,5-ketovaleryl-CoA to butadiene; and one or more polynucleotides encoding one or more enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to 1-propanol and/or 1,2-propanediol, wherein the microorganism has reduced levels of pyruvate decarboxylase enzymatic activity, and wherein the microorganism is capable of growing on a C6 sugar as a sole carbon source under anaerobic conditions.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has a disruption in one or more polynucleotides that code for one or more enzymes that decarboxylate pyruvate or a disruption in one or more polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more enzymes that decarboxylate pyruvate is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanol and/or 1,2-propanediol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

The present disclosure also provides methods for co-producing butadiene with 1-propanol and/or 1,2-propanediol from a fermentable carbon source under anaerobic conditions, the method comprising: providing a fermentable carbon source; contacting the fermentable carbon source with the non-naturally occurring microorganism as disclosed herein in a fermentation media, and expressing the polynucleotides in the microorganism for the co-production of butadiene with 1-propanol and/or 1,2-propanediol under substantially anaerobic conditions, wherein the microorganism co-produces butadiene with 1-propanol and/or 1,2-propanediol.

In some embodiments of each or any of the above or below mentioned embodiments, the fermentable carbon source is sugarcane juice, sugarcane molasses, hydrolyzed starch, hydrolyzed lignocellulosic materials, glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, or glycerol in any form or mixture thereof.

In some embodiments of each or any of the above or below mentioned embodiments, the fermentable carbon source is a monosaccharide, oligosaccharide, or polysaccharide.

In some embodiments of each or any of the above or below mentioned embodiments, the produced butadiene and 1-propanol and/or 1,2-propanediol are secreted by the microorganism into the fermentation media.

In some embodiments of each or any of the above or below mentioned embodiments, the methods further comprise recovering the produced butadiene and 1-propanol and/or 1,2-propanediol from the fermentation media.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has no detectable pyruvate decarboxylase enzymatic activity.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has a disruption in one or more polynucleotides that code for one or more enzymes that decarboxylate pyruvate (e.g., a pyruvate decarboxylase) or a disruption in one or more polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has a disruption in each of the one or more polynucleotides that code for enzymes that decarboxylate pyruvate or a disruption in each of the polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more polynucleotides is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides that code for enzymes that decarboxylate pyruvate code for pdc1, pdc2, and/or pdc6. In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate code for pdc2.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises an exogenous polynucleotide that encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentioned embodiments, the genetic modification is a truncation of the MTH1 transcription factor. In an embodiment, the MTH1 transcription factor may have the amino acid sequence as set forth in SEQ ID NO: 1 and the truncated MTH1 transcription factor may have the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments of each or any of the above or below mentioned embodiments, the truncated MTH1 transcription factor has a longer half-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA encode i.) pyruvate formate lyase and pyruvate formate lyase activating enzyme, ii) pyruvate dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoamide dehydrogenase, iii) pyruvate dehydrogenase, dihydrolipoyl transacetylase, dihydrolipoamide dehydrogenase, and pyruvate dehydrogenase complex protein X, or any combination thereof.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism is a eukaryote.

In some embodiments of each or any of the above or below mentioned embodiments, the eukaryote is a yeast, filamentous fungi, protozoa, or algae.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides coding for an acetoacetyl-CoA hydrolase.

In some embodiments of each or any of the above or below mentioned embodiments, the acetoacetyl-CoA hydrolase is produced by introducing a mutation into the polynucleotide that encodes acetoacetyl-CoA:acetate transferase. In some embodiments of each or any of the above or below mentioned embodiments, the mutation is a E51D Glu-Asp mutation corresponding to the numbering of SEQ ID NO: 3.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 1,2-propanediol to propanaldehyde.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme is a B12-independent dehydratase.

In some embodiments of each or any of the above or below mentioned embodiments, the B12-independent dehydratase is from Clostridium acetobutylicum, Clostridium glycolicum, Clostridium butyricum or Roseburia inulinivorans.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1,2-propanediol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to 1,2-propanediol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to 1-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises one or more exogenous polynucleotides encoding one or more enzymes in pathways for the co-production of 1-propanol and butadiene from a fermentable carbon source under anaerobic or micro-anaerobic conditions.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of one or more intermediates to butadiene, wherein the one or more intermediates in the pathway for the production of butadiene are selected from the group consisting of: crotonyl alcohol, 5-hydroxy-3-ketovaleryl-CoA, 3-ketopent-4-enoyl-CoA and 3,5-ketovaleryl-CoA.

The present disclosure also provides a non-naturally occurring microorganism comprising: a disruption of one or more enzymes that decarboxylate pyruvate and/or a transcription factor of an enzyme that decarboxylates pyruvate; a genetic modification that decreases glucose import into the microorganism; and one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in one or more enzymes that decarboxylate pyruvate and/or a transcription factor of an enzyme that decarboxylates pyruvate results in reduced levels of pyruvate decarboxylase enzymatic activity or no detectable pyruvate decarboxylase enzymatic activity.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more enzymes that decarboxylate pyruvate is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more enzymes that decarboxylate pyruvate include pdc 1, pdc 5, and/or pdc 6. In some embodiments of each or any of the above or below mentioned embodiments, the transcription factor of an enzyme that decarboxylates pyruvate includes pdc2.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises an exogenous polynucleotide that encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentioned embodiments, the truncated MTH1 transcription factor has a longer half-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism is a eukaryote.

In some embodiments of each or any of the above or below mentioned embodiments, the eukaryote is a yeast, filamentous fungi, protozoa, or algae.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides coding for an acetoacetyl-CoA hydrolase.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1,2-propanediol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to 1,2-propanediol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde ando/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentioned embodiments, the enzyme is a B12-independent dehydratase.

In some embodiments of each or any of the above or below mentioned embodiments, the B12-independent dehydratase is from Clostridium butyricum, or Roseburia inuvolurans.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to 1-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

The present disclosure also provides a non-naturally occurring microorganism comprising: one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA; one or more polynucleotides coding for enzymes that produce 1,2-propanediol, and wherein the microorganism has reduced levels of pyruvate decarboxylase enzymatic activity, and wherein the microorganism is capable of growing on a C6 sugar as a sole carbon source and under anaerobic conditions.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides encoding one or more enzymes in a pathway that produces acetate.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides encoding an acetyl-CoA hydrolase.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides encoding a phosphate acetyltransferase and acetyl-phosphate kinase.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides encoding a lactate CoA-transferase.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 1,2-propanediol to 1-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has no detectable pyruvate decarboxylase enzymatic activity.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has a disruption in one or more polynucleotides that code for one or more enzymes that decarboxylate pyruvate (e.g., a pyruvate decarboxylase) or a disruption in one or more polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more polynucleotides is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises an exogenous polynucleotide that encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentioned embodiments, the truncated MTH1 transcription factor has a longer half-life than an untruncated MTH1 transcription factor.

The present disclosure also provides a non-naturally occurring microorganism comprising: one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA; one or more polynucleotides coding for an acetyl-CoA acetyltransferase; one or more polynucleotides coding for enzymes that produce 1,2-propanediol, wherein the microorganism has reduced levels of pyruvate decarboxylase enzymatic activity, and wherein the microorganism is capable of growing on a C6 sugar as a sole carbon source and under anaerobic conditions.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides coding for an acetoacetyl-CoA hydrolase.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism further comprises one or more polynucleotides coding for a HMG-CoA synthase and HMG-CoA lyase.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has no detectable pyruvate decarboxylase enzymatic activity.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more polynucleotides is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides code for pyruvate decarboxylase 1, 2, 5, and/or 6.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises an exogenous polynucleotide that encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentioned embodiments, the truncated MTH1 transcription factor has a longer half-life than an untruncated MTH1 transcription factor.

C6 sugar as a sole carbon source The present disclosure also provides a non-naturally occurring microorganism comprising: one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA, one or more polynucleotides coding for an acetoacetyl-CoA hydrolase, one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihidroxyacetone phosphate to 1-propanol or one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1-propanol, and one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of one or more intermediates to butadiene, wherein the one or more intermediates in the pathway for the production of butadiene are selected from the group consisting of: crotonyl alcohol, 5-hydroxy-3-ketovaleryl-CoA, 3-ketopent-4-enoyl-CoA and 3,5-ketovaleryl-CoA, wherein the microorganism has reduced levels of pyruvate decarboxylase enzymatic activity, and wherein the microorganism is capable of growing on a C6 sugar as a sole carbon source and under anaerobic conditions.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has no detectable pyruvate decarboxylase enzymatic activity.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has a disruption in one or more polynucleotides that code for one or more enzymes that decarboxylate pyruvate (e.g., a pyruvate decarboxylase) or a disruption in one or more polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more polynucleotides is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises an exogenous polynucleotide that encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentioned embodiments, the truncated MTH1 transcription factor has a longer half-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1,2-propanodiol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1,2-propanediol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

In some embodiments of each or any of the above or below mentioned embodiments, the one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

The present disclosure also provides a non-naturally occurring microorganism comprising: a disruption of one or more enzymes that decarboxylate pyruvate; a genetic modification that permits growth of microorganism on a C6 sugar as a sole carbon source; one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA, one or more polynucleotides coding for an acetoacetyl-CoA hydrolase, one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 1,2-propanediol to 1-propanol, and one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of one or more intermediates to butadiene, wherein the one or more intermediates in the pathway for the production of butadiene are selected from the group consisting of: crotonyl alcohol, 5-hydroxy-3-ketovaleryl-CoA, 3-ketopent-4-enoyl-CoA and 3,5-ketovaleryl-CoA.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has no detectable pyruvate decarboxylase enzymatic activity.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism has a disruption in one or more polynucleotides that code for one or more enzymes that decarboxylate pyruvate (e.g., a pyruvate decarboxylase) or a disruption in one or more polynucleotides that code for a transcription factor of an enzyme that decarboxylates pyruvate.

In some embodiments of each or any of the above or below mentioned embodiments, the disruption in the one or more polynucleotides is a deletion or a mutation.

In some embodiments of each or any of the above or below mentioned embodiments, the microorganism comprises an exogenous polynucleotide that encodes a transcription factor involved in glucose import.

In some embodiments of each or any of the above or below mentioned embodiments, the truncated MTH1 transcription factor has a longer half-life than an untruncated MTH1 transcription factor.

In some embodiments of each or any of the above or below mentioned embodiments, polynucleotides coding for enzymes in a pathway that catalyzes a conversion of 1,2-propanediol to 1-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol.

The present disclosure also provides methods for co-producing 1-propanol and butadiene from a fermentable carbon source under anaerobic conditions, the method comprising: a.) providing a fermentable carbon source in substantially anaerobic culture media; and b.) contacting the fermentable carbon source with any of the non-naturally occurring microorganisms disclosed herein in a fermentation media, wherein the microorganism co-produces 1-propanol and butadiene from the fermentable carbon source.

The present disclosure also provides methods for co-producing 1-propanol and butadiene from a fermentable carbon source, the method comprising: a.) growing any of the non-naturally occurring microorganisms disclosed herein in a culture media under aerobic condtions, b) providing a fermentable carbon source to the culture media; and c.) co-producing 1-propanol and butadiene from the fermentable carbon source under anerobic conditions.

In some embodiments of each or any of the above or below mentioned embodiments, the fermentable carbon source is a monosaccharide, oligosaccharide, or polysaccharide.

The present disclosure also provides methods of making a non-naturally occurring microorganism that lacks pyruvate decarboxylase enzymatic activity, that is capable of growing on a C6 sugar as a sole carbon source, and that is capable of producing 1-propanol and butadiene from a fermentable carbon source under anaerobic conditions, the method comprising: introducing a disruption in one or more polynucleotides in the microorganism that encode enzymes that decarboxylate pyruvate; introducing a genetic modification in the microorganism that decreases import of glucose into the microorganism; introducing into the microorganism one or more exogenous polynucleotides encoding one or more enzymes in a pathway that produces cytosolic acetyl-CoA; introducing into the microorganism one or more polynucleotides coding for an acetoacetyl-CoA hydrolase or acetoacetyl-CoA transferase; introducing into the microorganism one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone phosphate or lactate to 1-propanol; and introducing into the microorganism one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of one or more intermediates are selected from the group consisting of: crotonyl alcohol, 5-hydroxy-3-ketovaleryl-CoA, 3-ketopent-4-enoyl-CoA and 3,5-ketovaleryl-CoA to butadiene.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the disclosure, one preferred embodiment is shown in the following figure. It should be understood, however, that the disclosure is not limited to the precise arrangements, examples and instrumentalities shown.

FIG. 1 depicts an exemplary pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol, where butadiene is produced via a crotonyl alcohol intermediate.

FIG. 2 depicts an exemplary pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol, where butadiene is produced via a 5-hydroxy-3-ketovaleryl-CoA intermediate.

FIG. 3 depicts an exemplary pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol, where butadiene is produced via a 3-ketopent-4-enoyl-CoA intermediate.

FIG. 4 depicts an exemplary pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol, where butadiene is produced via a 3,5-ketovaleryl-CoA intermediate.

DETAILED DESCRIPTION

The present disclosure generally relates to microorganisms (e.g., non-naturally occurring microorganisms) that comprise a genetically modified pathway and uses of the microorganisms for the conversion of a fermentable carbon source to butadiene and 1-propanol and/or 1,2-propanediol. Such microorganisms may comprise one or more polynucleotides coding for enzymes that catalyze a conversion of a fermentable carbon source to butadiene, one or more polynucleotides coding for enzymes that catalyze a conversion of a fermentable carbon source to 1,2-propanediol, one or more polynucleotides coding for enzymes that catalyse a conversion of 1,2-propanediol to 1-propanol.

This disclosure provides, in part, the discovery of novel anaerobic enzymatic pathways including, for example, novel combinations of enzymatic pathways, for the production of butadiene and 1-propanol and/or 1,2-propanediol from a carbon source (e.g., a fermentable carbon source).

The present disclosure provides microorganisms (e.g., S. cerevisiae) for the co-production of butadiene and 1-propanol and/or 1,2-propanediol. Microorganisms may be modified so that they may co-produce butadiene and 1-propanol and/or 1,2-propanediol. In an embodiment, a microorganism may have its native ethanol production reduced or elimiated (i.e., shut off). In an embodiment, to eliminate ethanol production in the microorganism the activity of pyruvate decarboxylase (i.e., the enzyme which decarboxylates pyruvate and in the process makes acetaldehyde and CO₂) may be disrupted including, for example, knocked-out. Pyruvate decarboxylase comes in three isoforms in yeast and its activity can be mostly knocked out by deleting the genes PDC1, PDC5, and PDC6. Without wishing to be bound by a theory of the invention, the elimination of the pyruvate decarboxylase activity in the cell's cytoplasm renders the yeast cell unable to grow under anaerobic conditions due to two factors: (1) the lack of an alternative route for cytoplasmic acetyl-CoA production, due to the lack of acetaldehyde that would be converted to acetate and acetyl-coA; and (2) a redox imbalance due to excess NADH because the NADH is no longer oxidized in the conversion of acetaldehyde to ethanol. Thus, it is necessary to also alter the ability of the microorgansim to import glucose by truncating a transcription factor of the glucose importer called MTH1. This truncation then restores the ability of the ΔPDC1,5,6 mutant microorganism to survive on C6 sugars. In an embodiment, one or more polynucleotides coding for a bacterial pyruvate formate lyase or cytosolic pyruvate dehydrogenase complex may be inserted into the microorganism to convert pyruvate into Acetyl CoA in the cytosol. In an embodiment, the microorganism may be modified to comprise one or more polynucleotides that code for enzymes in a pathway for the coproduction of butadiene and 1-propanol and/or 1,2-propanediol. In a further embodiment, the microorganism may be modified to comprise an acetoacetylCoA hydrolase. Such an acetoacetylCoA hydrolase may be engineered from an acetoacetylCoA:acetate transferase by making a single Glu-Asp mutation in the acetoacetylCoA:acetate transferase (e.g., a E51 D Glu-Asp mutation corresponding to the numbering of SEQ ID NO: 3). In an additional embodiment, a microorganism may be modified to comprise one or more polynucleotides coding for a B12-independent dehydratase from the organism Roseburia inulinivorans to convert 1,2-propanediol to propanaldehyde. Microorganims that comprise one or more of the modifications set forth above are termed a non-naturally occuring microroganism or a modified microorganism.

WO2004099425 discloses the overproduction of pyruvate in S. cerevisiae by knocking out pyruvate decarboxylase activity and a directed evolution process that allowed this triple mutant to grow on glucose due to a truncation of the MTH1 transcription factor. However, the scope stopped at the overproduction of pyruvate in aerobic fermentation systems. The use of oxygen, in this context, was essential as there is a huge buildup of NADH in the cell due to the fact that NADH is no longer oxidized in the conversion of acetaldehyde to ethanol.

The present disclosure further comprises a pyruvate overproducing cell able to produce cytosolic Acetyl-CoA inserting for example, bacterial pyruvate formate lyase or cytosolic pyruvate dehydrogenase complex to convert pyruvate into Acetyl-CoA in the cytosol of the eukaryote cell. The insertion of pyruvate formate lyase in to a PDC-negative yeast strain was disclosed by Waks and Silver in Engineering a Synthetic Dual-Organism System for Hydrogen Production (Applied and Environmental Microbiology, vol. 75, n. 7, 2009, p. 1867-1875) without success in anaerobic growth or metabolism. Furthermore, the present disclosure further comprises a pyruvate overproducing cell able to produce cytosolic Acetyl-CoA and to grow under anaerobic conditions by providing a temporary redox sink that allows reoxidation of NADH by introducing a gene coding for a bacterial soluble NAD(P)+transhydrogenase (Si-specific) (udhA gene from E. coli, E.C. number 1.6.1.1.) that catalyzes the interconversion of NADP++NADH=NADPH+NAD+. The concomitant expression of the PFL and udhA enzymes to restore anaerobic growth to the PDC-null yeast strain expressing the truncated MTH1 constitutes the first report of anaerobic growth of a PDC-null yeast strain and serves as a new eukaryotic chassis for the production of commodity chemicals.

Moreover, the present disclosure teaches how to make the 1,2-propanol or 1-propanol and butadiene pathways work in the new eukaryote chassis. Since the cell had the production of acetaldehyde knocked out, acetate is no longer formed and a new CoA receptor is necessary for the butadiene metabolic pathway to work. To solve this matter, the present disclosure proposes, for example, to engineer an acetoacetyl-CoA hydrolase from an acetoacetyl-CoA:acetate transferase (EC number 2.8.3.8.) by applying a mutation to it that was reported by Mack and Buckel in Conversion of glutaconate CoA-transferase from Acidaminococcus fermentans into an acyl-CoA hydrolase by site-directed mutagenesis (FEBS Letters, v. 405, n. 2, 1997, p. 209-212) but applied to another transferase. In that case, the “glucatonate CoA transferase” was transformed into a hydrolase by a single Glu-Asp mutation. The main advantage of this strategy is that the specificity of the enzyme for acetoacetyl-CoA is maintained since the transferase activity of a protein that already has high specificity for acetoacetyl-CoA is knocked out. The methods provided herein may also provide end-results similar to those of sterilization without the high capital expenditure and continuing higher management costs required to establish and maintain sterility throughout a production process. In this regard, most industrial-scale isoprene production processes are operated in the presence of measurable numbers of bacterial contaminants. Such drawbacks of prior methods are avoided by the presently disclosed methods as the toxic nature of the produced butadiene and/or 1-propanol reduces contaminants in the production process.

Additionally, the non-naturally occurring eukaryotic microorganism disclosed herein is capable of anaerobic growth and concomitant production of butadiene and 1-propanol and/or 1,2-propanediol. The supplementation of oxygen and nitrogen in a fermenter requires an additional investment for aerobic process. Additionally, aerobic fermentation processes for the production of butadiene and 1-propanol and/or 1,2-propanediol present several drawbacks at industrial scale (where it is technically challenging to maintain aseptic conditions) such as the fact that: (i) greater biomass is obtained reducing overall yields on carbon; (ii) the presence of oxygen favors the growth of contaminants (Weusthuis et al., 2011, Trends in Biotechnology, 2011, Vol. 29, No. 4, 153-158) and (iii) the mixture of oxygen and gaseous compounds poses serious risks of explosion, (iv) the oxygen can catalyze the unwanted reaction of polymerization of the olefinic compounds and, finally, (v) higher costs of fermentation and purification in aerobic conditions. Each of the drawbacks associated with aerobic fermentation including, for example, the risk of an explosion during the manufacture of butadiene and 1-propanol and/or 1,2-propanediol including dilution by oxygen and nitrogen are overcome by the anaerobic fermentation methods provided herein.

The present disclosure provides microorganisms comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and 1-propanol and/or 1,2-propanediol in a fermentation media, wherein 1,2-propanediol and 1-propanol are produced via a dihydroxyacetone phosphate intermediate or a pyruvate intermediate. In some embodiments, butadiene is produced via an acetyl-CoA intermediate.

The present disclosure also provides methods of co-producing 2-propanol and 1-propanol and/or 1,2-propanediol from a fermentable carbon source by providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of 2-propanol and 1-propanol and/or 1,2-propanediol, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to 2-propanol and 1-propanol and/or 1,2-propanediol in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of 2-propanol and 1-propanol and/or 1,2-propanediol and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to 2-propanol and 1-propanol and/or 1,2-propanediol in the microorganism to produce 12-propanol and 1-propanol and/or 1,2-propanediol, wherein 2-propanol and 1-propanol and/or 1,2-propanediol are produced via a dihydroxyacetone phosphate intermediate and/or a pyruvate intermediate, and wherein the co-production method is anaerobic.

It will be understood that the steps involved in any and all of the methods described herein may be performed in any order and are not to be limited or restricted to the order in which they are particularly recited. For example, the present disclosure provides methods of co-producing butadiene and 1-propanol and/or 1,2-propanediol from a fermentable carbon source, comprising: providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and 1-propanol and/or 1,2-propanediol in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and 1-propanol and/or 1,2-propanediol in the microorganism to produce butadiene and 1-propanol and/or 1,2-propanediol. As such, expression of the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and 1-propanol and/or 1,2-propanediol in the microorganism to produce 1 butadiene and 1-propanol and/or 1,2-propanediol may be preformed prior to or after contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the co-production of butadiene and 1-propanol and/or 1,2-propanediol, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and 1-propanol and/or 1,2-propanediol in a fermentation media.

Any of the intermediates produced in any of the enzymatic pathways disclosed herein may be an intermediate in the classical sense of the word in that they may be enzymatically converted to another intermediate or an end product. Alternatively, the intermediates themselves may be considered an end product.

As used herein, the term “biological activity” or “functional activity,” when referring to a protein, polypeptide or peptide, may mean that the protein, polypeptide or peptide exhibits a functionality or property that is useful as relating to some biological process, pathway or reaction. Biological or functional activity can refer to, for example, an ability to interact or associate with (e.g., bind to) another polypeptide or molecule, or it can refer to an ability to catalyze or regulate the interaction of other proteins or molecules (e.g., enzymatic reactions).

As used herein, “butadiene” is intended to mean 1,3-butadiene with a general formula CH₂CHCHCH₂(CAS number—106-99-0).

As used herein, the term “culturing” may refer to growing a population of cells, e.g., microbial cells, under suitable conditions for growth, in a liquid or on solid medium.

As used herein, the term “derived from” may encompass the terms originated from, obtained from, obtainable from, isolated from, and created from, and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to the another specified material.

As used herein, “exogenous polynucleotide” refers to any deoxyribonucleic acid that originates outside of the microorganism.

As used herein, the term “an expression vector” may refer to a DNA construct containing a polynucleotide or nucleic acid sequence encoding a polypeptide or protein, such as a DNA coding sequence (e.g. gene sequence) that is operably linked to one or more suitable control sequence(s) capable of affecting expression of the coding sequence in a host. Such control sequences include a promoter to affect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, cosmid, phage particle, bacterial artificial chromosome, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome (e.g., independent vector or plasmid), or may, in some instances, integrate into the genome itself (e.g., integrated vector). The plasmid is the most commonly used form of expression vector. However, the disclosure is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.

As used herein, the term “expression” may refer to the process by which a polypeptide is produced based on a nucleic acid sequence encoding the polypeptides (e.g., a gene). The process includes both transcription and translation.

As used herein, the term “gene” may refer to a DNA segment that is involved in producing a polypeptide or protein (e.g., fusion protein) and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).

As used herein, the term “heterologous,” with reference to a nucleic acid, polynucleotide, protein or peptide, may refer to a nucleic acid, polynucleotide, protein or peptide that does not naturally occur in a specified cell, e.g., a host cell. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes. In contrast, the term homologous, with reference to a nucleic acid, polynucleotide, protein or peptide, refers to a nucleic acid, polynucleotide, protein or peptide that occurs naturally in the cell.

As used herein, the term a “host cell” may refer to a cell or cell line, including a cell such as a microorganism which a recombinant expression vector may be transfected for expression of a polypeptide or protein (e.g., fusion protein). Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell may include cells transfected or transformed in vivo with an expression vector.

As used herein, the term “introduced,” in the context of inserting a nucleic acid sequence or a polynucleotide sequence into a cell, may include transfection, transformation, or transduction and refers to the incorporation of a nucleic acid sequence or polynucleotide sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence or polynucleotide sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

As used herein, the term “non-naturally occurring” when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Non-naturally occurring microbial organisms of the disclosure can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

As used herein, the term “operably linked” may refer to a juxtaposition or arrangement of specified elements that allows them to perform in concert to bring about an effect. For example, a promoter may be operably linked to a coding sequence if it controls the transcription of the coding sequence.

As used herein, “1,2-propanediol” is intended to mean propylene glycol with general formula CH₃CH(OH)CH₂OH (CAS number—57-55-6).

As used herein, “1-propanol” is intended to mean n-propanol with a general formula CH₃CH₂CH₂OH (CAS number—71-23-8).

As used herein, the term “a promoter” may refer to a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene. A promoter may be an inducible promoter or a constitutive promoter. An inducible promoter is a promoter that is active under environmental or developmental regulatory conditions.

As used herein, the term “a polynucleotide” or “nucleic acid sequence” may refer to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Such polynucleiotides or nucleic acid sequences may encode amino acids (e.g., polypeptides or proteins such as fusion proteins). Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present disclosure encompasses polynucleotides which encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2′-O-Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin. The term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring. The terms polynucleotide, nucleic acid, and oligonucleotide are used herein interchangeably. Polynucleotides may contain RNA, DNA, or both, and/or modified forms and/or analogs thereof. A sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR₂(amidate), P(O)R, P(O)OR′, COCH₂(formacetal), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions.

As used herein, the term a “protein” or “polypeptide” may refer to a composition comprised of amino acids and recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms protein and polypeptide are used interchangeably herein to refer to polymers of amino acids of any length, including those comprising linked (e.g., fused) peptides/polypeptides (e.g., fusion proteins). The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

As used herein, related proteins, polypeptides or peptides may encompass variant proteins, polypeptides or peptides. Variant proteins, polypeptides or peptides differ from a parent protein, polypeptide or peptide and/or from one another by a small number of amino acid residues. In some embodiments, the number of different amino acid residues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, variants differ by about 1 to about 10 amino acids. Alternatively or additionally, variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g., as determined using a sequence alignment tool, such as BLAST, ALIGN, and CLUSTAL (see, infra). For example, variant proteins or nucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with a reference sequence.

As used herein, the term “recovered,” “isolated,” “purified,” and “separated” may refer to a material (e.g., a protein, peptide, nucleic acid, polynucleotide or cell) that is removed from at least one component with which it is naturally associated. For example, these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.

As used herein, the term “recombinant” may refer to nucleic acid sequences or polynucleotides, polypeptides or proteins, and cells based thereon, that have been manipulated by man such that they are not the same as nucleic acids, polypeptides, and cells as found in nature. Recombinant may also refer to genetic material (e.g., nucleic acid sequences or polynucleotides, the polypeptides or proteins they encode, and vectors and cells comprising such nucleic acid sequences or polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another coding sequence or gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at decreased or elevated levels, expressing a gene conditionally or constitutively in manners different from its natural expression profile, and the like.

As used herein, the term “selective marker” or “selectable marker” may refer to a gene capable of expression in a host cell that allows for ease of selection of those hosts containing an introduced nucleic acid sequence, polynucleotide or vector. Examples of selectable markers include but are not limited to antimicrobial substances (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.

As used herein, the term “substantially anaerobic” means that growth of the modified micororganism takes place in culture media that comprises a dissolved oxygen concentration of less than 5 ppm.

As used herein, the term “substantially similar” and “substantially identical” in the context of at least two nucleic acids, polynucleotides, proteins or polypeptides may mean that a nucleic acid, polynucleotide, protein or polypeptide comprises a sequence that has at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference (e.g., wild-type) nucleic acid, polynucleotide, protein or polypeptide. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altshul et al. (1990) J. Mol. Biol. 215:403-410; Henikoff et al. (1989) Proc. Natl. Acad. Sci. 89:10915; Karin et al. (1993) Proc. Natl. Acad. Sci. 90:5873; and Higgins et al. (1988) Gene 73:237). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Person et al. (1988) Proc. Natl. Acad. Sci. 85:2444-2448.) In some embodiments, substantially identical polypeptides differ only by one or more conservative amino acid substitutions. In some embodiments, substantially identical polypeptides are immunologically cross-reactive. In some embodiments, substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).

As used herein, the term “transfection” or “transformation” may refer to the insertion of an exogenous nucleic acid or polynucleotide into a host cell. The exogenous nucleic acid or polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome. The term transfecting or transfection is intended to encompass all conventional techniques for introducing nucleic acid or polynucleotide into host cells. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, and microinjection.

As used herein, the term “transformed,” “stably transformed,” and “transgenic” may refer to a cell that has a non-native (e.g., heterologous) nucleic acid sequence or polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.

As used herein, the term “vector” may refer to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, single and double stranded cassettes and the like.

As used herein, the term “wild-type,” “native,” or “naturally-occurring” proteins may refer to those proteins found in nature. The terms wild-type sequence refers to an amino acid or nucleic acid sequence that is found in nature or naturally occurring. In some embodiments, a wild-type sequence is the starting point of a protein engineering project, for example, production of variant proteins.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, second ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure. Further, it will be understood that any of the substrates disclosed in any of the pathways herein may alternatively include the anion or the cation of the substrate.

Numeric ranges provided herein are inclusive of the numbers defining the range.

Unless otherwise indicated, nucleic acids sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

While the present disclosure is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the disclosure, and is not intended to limit the disclosure to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the disclosure in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values recited as well as any ranges that can be formed by such values. Also disclosed herein are any and all ratios (and ranges of any such ratios) that can be formed by dividing a disclosed numeric value into any other disclosed numeric value. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios can be unambiguously derived from the numerical values presented herein and in all instances such ratios, ranges, and ranges of ratios represent various embodiments of the present disclosure.

Modification of Microorganism

In some embodiments, the microorganism may comprise one or more exogenous polynucleotides encoding one or more enzymes in pathways for the co-production of butadiene and 1-propanol and/or 1,2-propanediol from a fermentable carbon source under anaerobic conditions.

In some embodiments, the non-naturally occurring microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1,2-propanediol including, for example: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol. Enzymes catalyzing any of these conversions may include, for example, those enzymes listed in Table 1.

In some embodiments, the non-naturally occurring microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1,2-propanediol including, for example, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol. Enzymes catalyzing any of these conversions may include, for example, those enzymes listed in Table 2.

In some embodiments, the non-naturally occurring microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone-phosphate to 1-propanol including, for example: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol. Enzymes catalyzing any of these conversions may include, for example, those enzymes listed in Table 3.

In some embodiments, the non-naturally occurring microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1-propanol including, for example, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to lactaldehyde, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactaldehyde to 1,2-propanediol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 1,2-propanediol to propionaldehyde, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propionaldehyde to 1-propanol. Enzymes catalyzing any of these conversions may include, for example, those enzymes listed in Table 4.

TABLE 1

Pathway B1 (1,2-propanediol from Dihydroxyacetone phosphate)

Enzyme

EC

No.
Enzyme name
Number
Reaction

F1.
methylglyoxal synthase
4.2.3.3
dihydroxyacetone phosphate →

methylglyoxal

F2.
methylglyoxal synthase, phosphate
4.2.3.3
dihydroxyacetone phosphate →

insensitive

methylglyoxal

G.
Methylglyoxal reductase, secondary
1.1.1.—
Methylglyoxal → lactaldehyde

alcohol dehydrogenase

H.
Methylglyoxal reductase, primary
1.1.1.78
methylglyoxal →

alcohol dehydrogenase

hydroxyacetone

I.
methylglyoxal reductase
1.1.1.—
Hydroxyacetone +NAD(P)H +H⁺ →

[multifunctional], secondary alcohol

1,2-propanediol +NAD(P)⁺

dehydrogenase

J.
methylglyoxal reductase
1.1.1.—
Lactaldehyde +NAD(P)H +H⁺

[multifunctional], primary alcohol

→1,2-propanediol +NAD(P)⁺

dehydrogenase

TABLE 2

Pathway C1 (1,2-propanediol from lactate)

Enzyme

EC

No.
Enzyme name
Number
Reaction

M1.
D-Lactate dehydrogenase
1.1.1.28
Pyruvate +NAD(P)H +H⁺→ D-

Lactate +NAD(P)⁺

M2.
L-Lactate dehydrogenase
1.1.1.27
Pyruvate +NAD(P)H +H⁺→ L-

Lactate +NAD(P)⁺

N.
propionate CoA-transferase*
2.8.3.1
Lactate + Acetyl-CoA →

Lactoyl-CoA + Acetic acid

O.
Lactoyl-CoA synthase
2.3.3.—
Lactate + CoA + ATP → lactoyl-

CoA + AMP

O2.
Carboxylic acid reductase
1.2.1.30
Lactate +ATP +NADPH →

lactaldehyde + AMP +NADP⁺

P.
aldehyde dehydrogenase, acid-CoA
1.2.1.—
Lactoyl-CoA +NAD(P)H +H⁺

reductase

→Lactaldehyde +NAD(P)⁺

Q.
Lactaldehyde reductase, 1,2-
1.1.1.77
L-Lactaldehyde +NAD(P)H +H⁺ →

propanediol oxidoreductase

L-1,2-propanediol +NAD(P)⁺

J.
methylglyoxal reductase
1.1.1.—
Lactaldehyde +NAD(P)H +H⁺ →

[multifunctional], primary alcohol

1,2-propanediol +NAD(P)⁺

dehydrogenase

*enzyme with homologous function but altered substrate specificity is required/preferred

TABLE 3

Pathway B2 (1-propanol from Dihydroxyacetone phosphate

Enzyme

EC

No.
Enzyme name
Number
Reaction

F1.
methylglyoxal synthase
4.2.3.3
dihydroxyacetone phosphate →

methylglyoxal

F2.
methylglyoxal synthase, phosphate
4.2.3.3
dihydroxyacetone phosphate →

insensitive

methylglyoxal

G.
Methylglyoxal reductase, secondary
1.1.1.—
Methylglyoxal → lactaldehyde

alcohol dehydrogenase

H.
Methylglyoxal reductase, primary
1.1.1.78
methylglyoxal →

alcohol dehydrogenase

hydroxyacetone

I.
methylglyoxal reductase
1.1.1.—
Hydroxyacetone +NAD(P)H +H⁺ →

[multifunctional], secondary alcohol

1,2-propanediol +NAD(P)⁺

dehydrogenase

J.
methylglyoxal reductase
1.1.1.—
Lactaldehyde +NAD(P)H +H⁺ →

[multifunctional], primary alcohol

1,2-propanediol +NAD(P)⁺

dehydrogenase

K.
1,2 propanediol dehydratase
4.2.1.30
R/S 1,2 propanediol → propanal

L.
1-propanol dehydrogenase
1.1.1.—
propanal + NADH → propanol +

NAD+

TABLE 4

Pathway C2 (1-propanol from lactate)

Enzyme

EC

No.
Enzyme name
Number
Reaction

M1.
D-Lactate dehydrogenase
1.1.1.28
Pyruvate +NAD(P)H +H⁺→ D-

Lactate +NAD(P)⁺

M2.
L-Lactate dehydrogenase
1.1.1.27
Pyruvate +NAD(P)H +H⁺→ L-

Lactate +NAD(P)⁺

N.
propionate CoA-transferase*
2.8.3.1
Lactate + Acetyl-CoA →

Lactoyl-CoA + Acetic acid

O.
Lactoyl-CoA synthase
2.3.3.—
Lactate + CoA + ATP → lactoyl-

CoA + AMP

O2.
Carboxylic acid reductase
1.2.1.30
Lactate +ATP +NADPH →

lactaldehyde + AMP +NADP⁺

P.
aldehyde dehydrogenase, acid-CoA
1.2.1.—
Lactoyl-CoA +NAD(P)H +H⁺ →

reductase

Lactaldehyde +NAD(P)⁺

Q.
Lactaldehyde reductase, 1,2-
1.1.1.77
L-Lactaldehyde +NAD(P)H +H⁺ →

propanediol oxidoreductase

L-1,2-propanediol +NAD(P)⁺

J.
methylglyoxal reductase
1.1.1.—
Lactaldehyde +NAD(P)H +H⁺ →

[multifunctional], primary alcohol

1,2-propanediol +NAD(P)⁺

dehydrogenase

K.
1,2 propanediol dehydratase
4.2.1.28
R/S 1,2 propanediol → propanal

L.
1-propanol dehydrogenase
1.1.1.—
propanal + NADH → propanol +

NAD+

*enzyme with homologous function but altered substrate specificity is required/preferred

In some embodiments, the non-naturally occurring microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of one or more intermediates to butadiene, wherein the one or more intermediates in the pathway for the production of butadiene are selected from the group consisting of: crotonyl alcohol, 5-hydroxy-3-ketovaleryl-CoA, 3-ketopent-4-enoyl-CoA and 3,5-ketovaleryl-CoA.

A microorganism may be modified (e.g., genetically engineered) by any method known in the art to comprise and/or express one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to one or more intermediates in a pathway for the co-production of butadiene and 1-propanol. Such enzymes may include any of those enzymes as are set forth in any one of FIGS. 1-4. For example, the microorganism may be modified to comprise one or more polynucleotides coding for enzymes that catalyze a conversion of crotonyl alcohol (Pathway D, Table 5), 5-hydroxy-3-ketovaleryl-CoA (Pathway E, Table 6), 3-ketopent-4-enoyl-CoA (Pathway F, Table 7), or 3,5-ketovaleryl-CoA (Pathway G, Table 8) to butadiene. Additionally, for example, the microorganism may be modified to comprise one or more polynucleotides coding for enzymes that catalyze a conversion of methylglyoxal and/or lactate to 1,2-propanediol or 1-propanol (Pathways B and C, Tables 1 to 4).

A modified microorganism as provided herein may comprise one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-alcohol to butadiene (Pathway D) and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal and/or lactate to 1,2-propanediol or 1-propanol (pathways B and C). In some embodiments, the one or more polynucleotides include:

- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glucose to dihydroxyacetone-phosphate,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal (e.g., methylglyoxal synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to R/S lactaldehyde (e.g., methylglyoxal reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone (e.g., methylglyoxal reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactaldehyde to R/S 1,2-propanediol (e.g., lactaldehyde reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to R/S 1,2-propanediol (e.g., 1,2-propanediol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 1,2-propanediol to propanal (e.g., 1,2-propanediol dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propanal to 1-propanol (e.g., 1-propanol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glucose to fructose,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of fructose to glyceraldehyde-3P (glyceraldehyde-3 phosphate),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glyceraldehyde-3P to pyruvate,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to R/S lactate (e.g., lactate dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactate to R/S lactaldehyde (e.g.,carboxylic acid reductase and phosphopantetheinyl transferase; lactoyl-CoA synthase or propionate CoA-transferase and lactoyl-CoA reductase or lactaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetaldehyde (e.g., pyruvate decarboxylase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetaldehyde to acetic acid (e.g., acetaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetic acid to acetyl-CoA (e.g., acetyl-CoA synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA (e.g., pyruvate dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetyl-CoA and acetyl-CoA to acetoacetyl-CoA (e.g., acetoacetyl-CoA thiolase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA (e.g., 3-hydroxybutyryl-CoA dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (e.g., crotonase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonyl alcohol (e.g., crotonyl-CoA reductase (bifuncional)),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonaldehyde (e.g., crotonaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonaldehyde to crotonyl alcohol (e.g., alcohol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl alcohol to butadiene (e.g., crotonyl alcohol dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl alcohol to 2-butenyl-4-phosphate (e.g., crotonyl alcohol kinase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-butenyl-4-phosphate to 2-butenyl-4-diphosphate (e.g., 2-butenyl-4-phosphate kinase), and/or
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2-butenyl-4-diphosphate to butadiene (e.g., butadiene kinase).

Exemplary enzymes which convert crotonyl-alcohol to butadiene (Pathway D) are presented in Table 5 below, as well as, the substrates that they act upon and product that they produce. The enzyme number represented in Table 5 correlates with the enzyme numbering used in FIG. 1 which schematically represents the enzymatic conversion of a fermentable carbon source to butadiene and 1-propanol through a crotonyl-alcohol intermediate, and methylglyoxal and lactate intermediates, respectively (Pathways B and C).

TABLE 5

Pathway D (butadiene via a crotonyl-alcohol intermediate)

Enzyme

E.C.

No.
Enzyme Name
number
Mediated Conversion

A1.
Pyruvate
1.2.4.1
Pyruvate + CoA + NAD⁺→ Acetyl-CoA +

dehydrogenase
2.3.1.12
CO₂+ NADH

1.8.1.4

A2.
Formate-C
2.3.1.54
Pyruvate + CoA → Acetyl-CoA +

acetyltransferase and
1.97.1.4
formate

Formate-C acetyl

transferase activating

enzyme

B.
acetoacetyl-CoA
2.3.1.9
Acetyl-CoA → Acetoacetyl-CoA

thiolase

R.
3-hydroxybutyryl-CoA
1.1.1.35;
Acetoacetyl-CoA → 3-hydroxybutyryl-

dehydrogenase
1.1.1.157
CoA

S.
Crotonase
4.2.1.17
3-hydroxybutyryl-CoA → Crotonyl-CoA

T.
Crotonyl-CoA
1.1.1
Crotonyl-CoA → crotonyl alcohol

reductase
1.1.1.34

(bifunctional)
1.1.1.88

U.
Crotonaldehyde
1.2.1.
Crotonyl-CoA → crotonaldehyde

dehydrogenase

V.
Alcohol
1.1.1
Crotonaldehyde → crotonyl alcohol

dehydrogenase

W.
Crotonyl alcohol
4.2.1
Crotonyl alcohol → butadiene

dehydratase
4.2.1.127

X.
crotonyl alcohol
2.7.1.36
Crotonyl alcohol →2-butenyl-4-

kinase

phosphate

Y.
2-butenyl-4-
2.7.4.2
2-butenyl-4-phosphate → 2-butenyl-4-

phosphate kinase

diphosphate

Z.
crotonyl alcohol
2.7.1.33
Crotonyl alcohol →2-butenyl-4-

diphosphokinase

diphosphate

AA.
butadiene synthase
4.2.3.27
2-butenyl-4-diphosphate → butadiene

A modified microorganism as provided herein may comprise one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-ketovaleryl-CoA to butadiene and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal and/or lactate to 1-propanol and/or 1,2-propanediol. In some embodiments, the one or more polynucleotides include:

- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of fructose to dihydroxyacetone-phosphate,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal (e.g., methylglyoxal synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to R/S lactaldehyde (e.g., methylglyoxal reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone (e.g., methylglyoxal oxidoreductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactaldehyde to R/S 1,2-propanediol (e.g., lactaldehyde reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to R/S 1,2-propanediol (e.g., 1,2-propanediol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 1,2-propanediol to propanal (e.g., 1,2-propanediol dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propanal to 1-propanol (e.g., 1-propanol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glucose to fructose, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of fructose to glyceraldehyde-3P,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glyceraldehyde-3P to pyruvate,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to R/S lactate (e.g., lactate dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactate to R/S lactaldehyde (e.g.,carboxylic acid reductase and phosphopantetheinyl transferase; lactoyl-CoA synthase or propionate CoA-transferase and lactoyl-CoA reductase or lactaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetaldehyde (e.g., pyruvate decarboxylase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetaldehyde to acetic acid (e.g., acetaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetic acid to acetyl-CoA (e.g., acetyl-CoA synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA (e.g., pyruvate dehydrogenase, pyruvate formate lyase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactate to lactoyl-CoA (e.g., lactoyl-CoA transferase, or synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA (e.g., lactoyl-CoA dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA to 3-hydroxypropionyl-CoA (e.g., acryloyl-CoA hydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetyl-CoA and 3-hydroxypropionyl-CoA to 5-hydroxy-3-ketovaleryl-CoA (e.g., a 5-hydroxy-3-ketovaleryl-CoA thiolase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-ketovaleryl-CoA to R/S 3,5-dihydroxy-valeryl-CoA (e.g., 5-hydroxy-3-ketovaleryl-CoA dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3,5-dihydroxy-valeryl-CoA to R/S 3-hydroxy-4-pentenoyl-CoA (e.g., 3,5-hydroxyvaleryl-CoA dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid (e.g., 3-hydroxy-4-pentenoyl-CoA hydrolase, transferase or synthase), and /or
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene (e.g., 3-hydroxy-4-pentenoic acid decarboxylase).

Exemplary enzymes which convert 5-hydroxy-3-ketovaleryl-CoA to butadiene (Pathway E) and methylglyoxal and lactate to 1-propanol are presented in Table 6 below, as well as, the substrates that they act upon and product that they produce. The enzyme number represented in Table 6 correlates with the enzyme numbering used in FIG. 2 which schematically represents the enzymatic conversion of a fermentable carbon source to butadiene and 1-propanol through a 5-hydroxy-3-ketovaleryl-CoA intermediate, and a methylglyoxal and lactate intermediates, respectively.

TABLE 6

Pathway E (butadiene via a 5-hydroxy-3-ketovaleryl-CoA intermediate)

Enzyme

E.C.

No.
Enzyme Name
number
Mediated Conversion

M.
Lactate
1.1.1.27
Pyruvate → Lactate

dehydrogenase

A1.
Pyruvate
1.2.4.1
Pyruvate + CoA + NAD⁺→ Acetyl-CoA + CO₂+

dehydrogenase
2.3.1.12
NADH

1.8.1.4

A2.
Formate-C
2.3.1.54
Pyruvate + CoA → Acetyl-CoA +

acetyltransferase
1.97.1.4
formate

and Formate-C

acetyl transferase

activating enzyme

N.
lactoyl-CoA
2.8.3.1
Lactate → Lactoyl-CoA

transferase
2.3.3

or synthase

AB.
lactoyl-CoA
4.2.1.54
Lactoyl-CoA → Acryloyl-CoA

dehydratase

AC.
acryloyl-CoA
4.2.1.17
Acryloyl-CoA →3-hydroxypropyonyl-

hydratase

CoA

AD.
5-hydroxy-3-
2.3.1.16
Acetyl-CoA + 3-hydroxypropionyl-CoA →

ketovaleryl-CoA

5-hydroxy-3-ketovaleryl-CoA

thiolase

AE.
5-hydroxy-3-
1.1.1.35
5-hydroxy-3-Ketovaleryl-CoA → R/S

Ketovaleryl-CoA
1.1.1.36
3,5-dihydroxy-valeryl-CoA

dehydrogenase

AF.
3,5-hydroxyvaleryl-
4.2.1
R/S 3,5-dihydroxy-valeryl-CoA → 3-

CoA dehydratase
4.2.1.17
hydroxy-4-pentenoyl-CoA

4.2.1.54

AG.
3-hydroxy-4-
3.1.2,
3-hydroxy-4-pentenoyl-CoA → 3-

pentenoyl-CoA
2.8.3 or
hydroxy-4-pentenoic acid

hydrolase,
2.3.3

transferase or

synthase

AH.
3-hydroxy-4-
4.1.1.33
3-hydroxy-4-pentenoic acid →

pentenoic acid

butadiene

decarboxylase

A modified microorganism as provided herein may comprise one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-keto-pent-4-enoyl-CoA to butadiene and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal and/or lactate to 1-propanol and/or 1,2-propanediol. In some embodiments, the one or more polynucleotides include:

- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of fructose to dihydroxyacetone-phosphate,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal (e.g., methylglyoxal synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to R/S lactaldehyde (e.g., methylglyoxal reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone (e.g., methylglyoxal oxidoreductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactaldehyde to R/S 1,2-propanediol (e.g., lactaldehyde reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to R/S 1,2-propanediol (e.g., 1,2-propanediol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 1,2-propanediol to propanal (e.g., 1,2-propanediol dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propanal to 1-propanol (e.g., 1-propanol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glucose to fructose, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of fructose to glyceraldehyde-3P,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glyceraldehyde-3P to pyruvate,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to R/S lactate (e.g., lactate dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactate to R/S lactaldehyde (e.g.,carboxylic acid reductase and phosphopantetheinyl transferase; lactoyl-CoA synthase or propionate CoA-transferase and lactoyl-CoA reductase or lactaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetaldehyde (e.g., pyruvate decarboxylase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetaldehyde to acetic acid (e.g., acetaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetic acid to acetyl-CoA (e.g., acetyl-CoA synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA (e.g., pyruvate dehydrogenase, pyruvate formate lyase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactate to lactoyl-CoA (e.g., lactoyl-CoA transferase, or synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA (e.g., lactoyl-CoA dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA and acetyl-CoA to 3-keto-4-pentenoyl-CoA (e.g., 3-keto-4-pentenoyl-CoA thiolase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-keto-4-pentenoyl-CoA to R/S 3-hydroxy-4-pentenoyl-CoA (e.g., 3-keto-4-pentenoyl-CoA dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid (e.g., hydroxy-4-pentenoyl-CoA transferase, hydrolase, or synthase), and/or
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene (e.g., 3-hydroxy-4-pentenoic acid decarboxylase).

Exemplary enzymes which convert 3-keto-4-pentenoyl-CoA to butadiene (Pathway F) are presented in Table 7 below, as well as, the substrates that they act upon and product that they produce. The enzyme number represented in Table 7 correlates with the enzyme numbering used in FIG. 3 which schematically represents the enzymatic conversion of a fermentable carbon source to butadiene through a 3-keto-4-pentenoyl-CoA intermediate, and 1-propanol through a methylglyoxal and lactate intermediate (Pathways B and C), respectively.

TABLE 7

Pathway F (butadiene via a 3-keto-4-pentenoyl-CoA intermediate)

Enzyme

E.C.

No.
Enzyme Name
number
Mediated Conversion

M.
Lactate dehydrogenase
1.1.1.27
Pyruvate → Lactate

A1.
Pyruvate dehydrogenase
1.2.4.1
Pyruvate + CoA + NAD⁺→ Acetyl-CoA +

2.3.1.12
CO₂+ NADH

1.8.1.4

A2.
Formate-C
2.3.1.54
Pyruvate + CoA → Acetyl-CoA +

acetyltransferase and
1.97.1.4
formate

Formate-C acetyl

transferase activating

enzyme

N.
lactoyl-CoA transferase
2.8.3.1
Lactate → Lactoyl-CoA

or synthase
2.3.3

AB.
lactoyl-CoA dehydratase
4.2.1.54
Lactoyl-CoA → Acryloyl-CoA

AI.
3-keto-4-pentenoyl-CoA
2.3.1.9
Acryloyl-CoA + Acetyl-CoA → 3-

thiolase
2.3.1.16
keto-4-pentenoyl-CoA

AJ.
3-keto-4-pentenoyl-CoA
1.1.1.35
3-keto-4-pentenoyl-CoA →

dehydrogenase
1.1.1.36
R/S 3-hydroxy-4-pentenoyl-CoA

AK.
3-hydroxy-4-pentenoyl-
2.8.3
R/S 3-hydroxy-4-pentenoyl-CoA →

CoA transferase
3.1.2
3-hydroxy-4-pentenoic acid

or hydrolase or synthase
6.2.1

AL.
3-hydroxy-4-pentenoic
4.1.1.33
3-hydroxy-4-pentenoic acid →

acid decarboxylase

butadiene

A modified microorganism as provided herein may comprise one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3,5-ketovaleryl-CoA to butadiene and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal and/or lactate to 1-propanol and/or 1,2-propanediol. In some embodiments, the one or more polynucleotides include:

- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of fructose to dihydroxyacetone-phosphate,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to methylglyoxal (e.g., methylglyoxal synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to R/S lactaldehyde (e.g., methylglyoxal reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of methylglyoxal to hydroxyacetone (e.g., methylglyoxal oxidoreductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactaldehyde to R/S 1,2-propanediol (e.g., lactaldehyde reductase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of hydroxyacetone to R/S 1,2-propanediol (e.g., 1,2-propanediol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 1,2-propanediol to propanal (e.g., 1,2-propanediol dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of propanal to 1-propanol (e.g., 1-propanol dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glucose to fructose,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of fructose to glyceraldehyde-3P,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glyceraldehyde-3P to pyruvate,
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to R/S lactate (e.g., lactate dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S lactate to R/S lactaldehyde (e.g.,carboxylic acid reductase and phosphopantetheinyl transferase; lactoyl-CoA synthase or propionate CoA-transferase and lactoyl-CoA reductase or lactaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetaldehyde (e.g., pyruvate decarboxylase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetaldehyde to acetic acid (e.g., acetaldehyde dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetic acid to acetyl-CoA (e.g., acetyl-CoA synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA (e.g., pyruvate dehydrogenase, pyruvate formate lyase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of CO₂to formate (e.g., a formate dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate and CoA to acetyl-CoA and formate (e.g., an Acetyl-CoA:formate C-acetyltran sf erase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of formate to formyl-CoA (e.g., a formyl-CoA transferase, or formyl-CoA synthase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA and formyl-CoA to 3,5-ketovaleryl-CoA (e.g., 3,5-ketovaleryl-CoA thiolase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3,5-ketovaleryl-CoA to 5-hydroxy-3-ketovaleryl-CoA (e.g., a 5-hydroxy-3-ketovaleryl-CoA dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3,5-ketovaleryl-CoA to 5-hydroxy-3-ketovaleryl-CoA (e.g., a 5-hydroxy-3-ketovaleryl-CoA dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-ketovaleryl-CoA or R/S 3-hydroxy-5-ketovaleryl-CoA to R/S 3,5-hydroxyvaleryl-CoA (e.g., a 3,5-hydroxyvaleryl-CoA dehydrogenase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3,5-hydroxyvaleryl-CoA to R/S 3-hydroxy-4-pentenoyl-CoA (e.g., a 3,5-hydroxyvaleryl-CoA dehydratase),
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid (e.g., a 3-hydroxy-4-pentenoyl-CoA hydrolase, transferase or synthase), and/or
- one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene (e.g., a 3-hydroxy-4-pentenoic acid decarboxylase).

Exemplary enzymes which convert 3,5-ketovaleryl-CoA to butadiene (Pathway G) are presented in Table 8 below, as well as, the substrates that they act upon and product that they produce. The enzyme number represented in Table 8 correlates with the enzyme numbering used in FIG. 4 which schematically represents the enzymatic conversion of a fermentable carbon source to butadiene and 1-propanol and/or 1,2-propanediol through a 3,5-ketovaleryl-CoA intermediate, and a methylglyoxal and lactate intermediate (Pathways B and C), respectively.

TABLE 8

Pathway G (butadiene via a 3,5-ketovaleryl-CoA intermediate)

Enzyme

E.C.

No.
Enzyme Name
number
Mediated Conversion

A1.
Pyruvate
1.2.4.1
Pyruvate + CoA + NAD⁺→ Acetyl-CoA +

dehydrogenase
2.3.1.12
CO₂+ NADH

1.8.1.4

A2.
Formate-C
2.3.1.54
Pyruvate + CoA → Acetyl-CoA + formate

acetyltransferase and
1.97.1.4

Formate-C acetyl

transferase activating

enzyme

B.
acetoacetyl-CoA
2.3.1.9
Acetyl-CoA → Acetoacetyl-CoA

thiolase

AM.
Formate
1.2.1.2
CO₂→ Formate

dehydrogenase

AN.
formyl-CoA
2.8.3.16
Formate → formyl-CoA

transferase,
6.2.1

formyl-CoA synthase

AO.
3,5-ketovaleryl-CoA
2.3.1.16
Acetoacetyl-CoA + formyl-CoA → 3,5-

thiolase

ketovaleryl-CoA

AE.
5-hydroxy-3-
1.1.1.35
3,5-ketovaleryl-CoA → 5-hydroxy-3-

Ketovaleryl-CoA
1.1.1.36
Ketovaleryl-CoA

dehydrogenase

AP.
3-hydroxy-5-
1.1.1.35
3,5-ketovaleryl-CoA → R/S 3-hydroxy-5-

Ketovaleryl-CoA
1.1.1.36
Ketovaleryl-CoA

dehydrogenase

AQ.
3,5-hydroxyvaleryl-
1.1.1.35
5-hydroxy-3-Ketovaleryl-CoA or R/S 3-

CoA dehydrogenase
1.1.1.36
hydroxy-5-Ketovaleryl-CoA → R/S 3,5-

hydroxyvaleryl-CoA

AF.
3,5-hydroxyvaleryl-
4.2.1.17
R/S 3,5-hydroxyvaleryl-CoA → R/S 3-hydroxy-

CoA dehydratase
4.2.1.54
4-pentenoyl-CoA

AK.
3-hydroxy-4-
3.1.2,
R/S 3-hydroxy-4-pentenoyl-CoA → 3-

pentenoyl-CoA
2.8.3 or
hydroxy-4-pentenoic acid

hydrolase,
2.3.3

transferase or

synthase

AL.
3-hydroxy-4-
4.1.1.33
3-hydroxy-4-pentenoic acid → butadiene

pentenoic acid

decarboxylase

The microorganism may be an archea, bacteria, or eukaryote. In some embodiments, the bacteria is a Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus including, for example, Pelobacter propionicus, Clostridium propionicum, Clostridium acetobutylicum, Lactobacillus, Propionibacterium acidipropionici or Propionibacterium freudenreichii. In some embodiments, the eukaryote is a yeast, filamentous fungi, protozoa, or algae. In some embodiments, the yeast is Saccharomyces cerevisiae, Kluyveromyces lactis or Pichia pastoris.

In some embodiments, the microorganism is additionally modified to comprise one or more tolerance mechanisms including, for example, tolerance to a produced molecule (i.e., methylglyoxal, 1,2-propanediol, 1-propanol, or butadiene), and/or organic solvents. A microorganism modified to comprise such a tolerance mechanism may provide a means to increase titers of fermentations and/or may control contamination in an industrial scale process.

In some embodiments, the disclosure contemplates the modification (e.g., engineering) of one or more of the enzymes provided herein. Such modification may be performed to redesign the substrate specificity of the enzyme and/or to modify (e.g., reduce) its activity against others substrates in order to increase its selectivity for a given substrate. Additionally or alternatively, one or more enzymes as provided herein may be engineered to alter (e.g., enhance including, for example, increase its catalytic activity or its substrate specificity) one or more of its properties, including acceptance of different co-factors such as NADH instead of NADPH.

In some embodiments, sequence alignment and comparative modeling of proteins may be used to alter one or more of the enzymes disclosed herein. Homology modeling or comparative modeling refers to building an atomic-resolution model of the desired protein from its primary amino acid sequence and an experimental three-dimensional structure of a similar protein. This model may allow for the enzyme substrate binding site to be defined, and the identification of specific amino acid positions that may be replaced to other natural amino acid in order to redesign its substrate specificity.

Variants or sequences having substantial identity or homology with the polynucleotides encoding enzymes as disclosed herein may be utilized in the practice of the disclosure. Such sequences can be referred to as variants or modified sequences. That is, a polynucleotide sequence may be modified yet still retain the ability to encode a polypeptide exhibiting the desired activity. Such variants or modified sequences are thus equivalents in the sense that they retain their intended function. Generally, the variant or modified sequence may comprise at least about 40%-60%, preferably about 60%-80%, more preferably about 80%-90%, and even more preferably about 90%-95% sequence identity with the native sequence.

One example of such a variant is described in SEQ ID NO: 3 wherein a E51D Glu-Asp mutation that renders the coded acetoacetyl-CoA transferase into a acetoacetyl-CoA hydrolase. Further modifications to SEQ ID NO: 3 through rational and/or random approaches may be further performed to improve hydrolase activity.

In some embodiments, a microorganism may be modified to express including, for example, overexpress, one or more enzymes as provided herein. The microorganism may be modified by genetic engineering techniques (i.e., recombinant technology), classical microbiological techniques, or a combination of such techniques and can also include naturally occurring genetic variants to produce a genetically modified microorganism. Some of such techniques are generally disclosed, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press; and Selifonova et al. (2001) Appl. Environ. Microbiol. 67(8):3645).

A genetically modified microorganism may include a microorganism in which a polynucleotide has been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect of expression (e.g., over-expression) of one or more enzymes as provided herein within the microorganism. Genetic modifications which result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene. Addition of cloned genes to increase gene expression can include maintaining the cloned gene(s) on replicating plasmids or integrating the cloned gene(s) into the genome of the production organism. Furthermore, increasing the expression of desired cloned genes can include operatively linking the cloned gene(s) to native or heterologous transcriptional control elements.

Where desired, the expression of one or more of the enzymes provided herein are under the control of a regulatory sequence that controls directly or indirectly the expression of the enzyme in a time-dependent fashion during a fermentation reaction.

In some embodiments, a microorganism is transformed or transfected with a genetic vehicle such as, an expression vector comprising an exogenous polynucleotide sequence coding for the enzymes provided herein.

Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may typically, but not always, comprise a replication system (i.e. vector) recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and may preferably, but not necessarily, also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (expression vectors) may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, mRNA stabilizing sequences, nucleotide sequences homologous to host chromosomal DNA, and/or a multiple cloning site. Signal peptides may also be included where appropriate, preferably from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.

The vectors can be constructed using standard methods (see, e.g., Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989; and Ausubel, et al., Current Protocols in Molecular Biology, Greene Publishing, Co. N.Y, 1995).

The manipulation of polynucleotides of the present disclosure including polynucleotides coding for one or more of the enzymes disclosed herein is typically carried out in recombinant vectors. Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes, episomal vectors and gene expression vectors, which can all be employed. A vector of use according to the disclosure may be selected to accommodate a protein coding sequence of a desired size. A suitable host cell is transformed with the vector after in vitro cloning manipulations. Host cells may be prokaryotic, such as any of a number of bacterial strains, or may be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells including, for example, rodent, simian or human cells. Each vector contains various functional components, which generally include a cloning site, an origin of replication and at least one selectable marker gene. If given vector is an expression vector, it additionally possesses one or more of the following: enhancer element, promoter, transcription termination and signal sequences, each positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a polypeptide repertoire member according to the disclosure.

Vectors, including cloning and expression vectors, may contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. For example, the sequence may be one that enables the vector to replicate independently of the host chromosomal DNA and may include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. For example, the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as COS cells.

A cloning or expression vector may contain a selection gene also referred to as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate, hygromycin, thiostrepton, apramycin or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.

The replication of vectors may be performed in E. coli (e.g., strain TB1 or TG1, DH5α, DH10β, JM110). An E. coli-selectable marker, for example, the β-lactamase gene that confers resistance to the antibiotic ampicillin, may be of use. These selectable markers can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19, or pUC119.

Expression vectors may contain a promoter that is recognized by the host organism. The promoter may be operably linked to a coding sequence of interest. Such a promoter may be inducible or constitutive. Polynucleotides are operably linked when the polynucleotides are in a relationship permitting them to function in their intended manner.

Promoters suitable for use with prokaryotic hosts may include, for example, the a-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, the erythromycin promoter, apramycin promoter, hygromycin promoter, methylenomycin promoter and hybrid promoters such as the tac promoter. Moreover, host constitutive or inducible promoters may be used. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno sequence operably linked to the coding sequence.

Viral promoters obtained from the genomes of viruses include promoters from polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2 or 5), herpes simplex virus (thymidine kinase promoter), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (e.g., MoMLV, or RSV LTR), Hepatiti B virus, Myeloproliferative sarcoma virus promoter (MPSV), VISNA, and Simian Virus 40 (SV40). Heterologous mammalian promoters include, e.g., the actin promoter, immunoglobulin promoter, heat-shock protein promoters.

The early and late promoters of the SV40 virus are conveniently obtained as a restriction fragment that also contains the SV40 viral origin of replication (see, e.g., Fiers et al., Nature, 273:113 (1978); Mulligan and Berg, Science, 209:1422-1427 (1980); and Pavlakis et al., Proc. Natl. Acad. Sci. USA, 78:7398-7402 (1981)). The immediate early promoter of the human cytomegalovirus (CMV) is conveniently obtained as a Hind III E restriction fragment (see, e.g., Greenaway et al., Gene, 18:355-360 (1982)). A broad host range promoter, such as the SV40 early promoter or the Rous sarcoma virus LTR, is suitable for use in the present expression vectors.

Generally, a strong promoter may be employed to provide for high level transcription and expression of the desired product. Among the eukaryotic promoters that have been identified as strong promoters for high-level expression are the SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus long terminal repeat, and human cytomegalovirus immediate early promoter (CMV or CMV IE). In an embodiment, the promoter is a SV40 or a CMV early promoter.

The promoters employed may be constitutive or regulatable, e.g., inducible. Exemplary inducible promoters include jun, fos and metallothionein and heat shock promoters. One or both promoters of the transcription units can be an inducible promoter. In an embodiment, the GFP is expressed from a constitutive promoter while an inducible promoter drives transcription of the gene coding for one or more enzymes as disclosed herein and/or the amplifiable selectable marker.

The transcriptional regulatory region in higher eukaryotes may comprise an enhancer sequence. Many enhancer sequences from mammalian genes are known e.g., from globin, elastase, albumin, α-fetoprotein and insulin genes. A suitable enhancer is an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the enhancer of the cytomegalovirus immediate early promoter (Boshart et al. Cell 41:521 (1985)), the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers (see also, e.g., Yaniv, Nature, 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters). The enhancer sequences may be introduced into the vector at a position 5′ or 3′ to the gene of interest, but is preferably located at a site 5′ to the promoter.

Yeast and mammalian expression vectors may contain prokaryotic sequences that facilitate the propagation of the vector in bacteria. Therefore, the vector may have other components such as an origin of replication (e.g., a nucleic acid sequence that enables the vector to replicate in one or more selected host cells), antibiotic resistance genes for selection in bacteria, and/or an amber stop codon which can permit translation to read through the codon. Additional eukaryotic selectable gene(s) may be incorporated. Generally, in cloning vectors the origin of replication is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known, e.g., the CoIE1 origin of replication in bacteria. Various viral origins (e.g., SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, a eukaryotic replicon is not needed for expression in mammalian cells unless extrachromosomal (episomal) replication is intended (e.g., the SV40 origin may typically be used only because it contains the early promoter).

To facilitate insertion and expression of different genes coding for the enzymes as disclosed herein from the constructs and expression vectors, the constructs may be designed with at least one cloning site for insertion of any gene coding for any enzyme disclosed herein. The cloning site may be a multiple cloning site, e.g., containing multiple restriction sites.

The plasmids may be propagated in bacterial host cells to prepare DNA stocks for subcloning steps or for introduction into eukaryotic host cells. Transfection of eukaryotic host cells can be any performed by any method well known in the art. Transfection methods include lipofection, electroporation, calcium phosphate co-precipitation, rubidium chloride or polycation mediated transfection, protoplast fusion and microinjection. Preferably, the transfection is a stable transfection. The transfection method that provides optimal transfection frequency and expression of the construct in the particular host cell line and type, is favored. Suitable methods can be determined by routine procedures. For stable transfectants, the constructs are integrated so as to be stably maintained within the host chromosome.

Vectors may be introduced to selected host cells by any of a number of suitable methods known to those skilled in the art. For example, vector constructs may be introduced to appropriate cells by any of a number of transformation methods for plasmid vectors. For example, standard calcium-chloride-mediated bacterial transformation is still commonly used to introduce naked DNA to bacteria (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), but electroporation and conjugation may also be used (see, e.g., Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).

For the introduction of vector constructs to yeast or other fungal cells, chemical transformation methods may be used (e.g., Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Transformed cells may be isolated on selective media appropriate to the selectable marker used. Alternatively, or in addition, plates or filters lifted from plates may be scanned for GFP fluorescence to identify transformed clones.

For the introduction of vectors comprising differentially expressed sequences to mammalian cells, the method used may depend upon the form of the vector. Plasmid vectors may be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation (see, e.g., Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).

Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™ (Stratagene) kits are available. Other companies offering reagents and methods for lipofection include Bio-Rad Laboratories, CLONTECH, Glen Research, InVitrogen, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

The host cell may be capable of expressing the construct encoding the desired protein, processing the protein and transporting a secreted protein to the cell surface for secretion. Processing includes co- and post-translational modification such as leader peptide cleavage, GPI attachment, glycosylation, ubiquitination, and disulfide bond formation. Immortalized host cell cultures amenable to transfection and in vitro cell culture and of the kind typically employed in genetic engineering are preferred. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (CO 7, ATCC CRL 1651); human embryonic kidney line (293 or 293 derivatives adapted for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977); baby hamster kidney cells (BHK, ATCC CCL 10); DHFR-Chinese hamster ovary cells (ATCC CRL-9096); dp12.CHO cells, a derivative of CHO/DHFR-(EP 307,247 published 15 Mar. 1989); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); PEER human acute lymphoblastic cell line (Ravid et al. Int. J. Cancer 25:705-710 (1980)); MRC 5 cells; FS4 cells; human hepatoma line (Hep G2), human HT1080 cells, KB cells, JW-2 cells, Detroit 6 cells, NIH-3T3 cells, hybridoma and myeloma cells. Embryonic cells used for generating transgenic animals are also suitable (e.g., zygotes and embryonic stem cells).

Suitable host cells for cloning or expressing polynucleotides (e.g., DNA) in vectors may include, for example, prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), E. coli JM110 (ATCC 47,013) and E. coli W3110 (ATCC 27,325) are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast may be suitable cloning or expression hosts for vectors comprising polynucleotides coding for one or more enzymes. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

When the enzyme is glycosylated, suitable host cells for expression may be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silk moth) have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, tobacco, lemna, and other plant cells can also be utilized as host cells.

Examples of useful mammalian host cells are Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (CO 7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36: 59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-described expression or cloning vectors for production of one or more enzymes as disclosed herein or with polynucleotides coding for one or more enzymes as disclosed herein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Host cells containing desired nucleic acid sequences coding for the disclosed enzymes may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44, (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adeNOSine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Polynucleotides and Encoded Enzymes

Any known polynucleotide (e.g., gene) that codes for an enzyme or variant thereof that is capable of catalyzing an enzymatic conversion including, for example, an enzyme as set forth in any one of Tables 1-8 or FIGS. 1-4, is contemplated for use by the present disclosure. Such polynucleotides may be modified (e.g., genetically engineered) to modulate (e.g., increase or decrease) the substrate specificity of an encoded enzyme, or the polynucleotides may be modified to change the substrate specificity of the encoded enzyme (e.g., a polynucleotide that codes for an enzyme with specificity for a substrate may be modified such that the enzyme has specificity for an alternative substrate). Preferred microorganisms may comprise polynucleotides coding for one or more of the enzymes as set forth in Tables 1-8 and FIGS. 1-4.

Enzymes for catalyzing the conversions set forth in the pathways of Tables 1-8 and FIGS. 1-4 are categorized in Table 9 and 10 below. Enzyme numbers presented in Tables 9 and 10 that are followed by a numeral, e.g., Al or A2, represent alternative enzymes that can catalyze a particular conversion and may be generally referred to throughout this disclosure and figures by the first letter that precedes the numeral, e.g., A.

TABLE 9

Exemplary Gene Identifier (GI) numbers

Enzyme

FIGS.
No.
EC No.
Enzyme candidate
GI No.

1, 2, 3, 4
A1
2.3.1.54/1.97.1.4
Formate-C acetyltransferase
48994873

1, 2, 3, 4
A1
2.3.1.54/1.97.1.4
Formate-C acetyltransferase
48994873

(activating enzyme)

1, 2, 3, 4
A1
2.3.1.54/1.97.1.4
Formate-C acetyltransferase
387233059

1, 2, 3, 4
A1
2.3.1.54/1.97.1.4
Formate-C acetyltransferase
41400040

(activating enzyme)

1, 2, 3, 4
A2
1.2.4.1/2.3.1.12/
Pyruvate dehydrogenase
298058

1.8.1.4
complex

1, 2, 3, 4
A2
1.2.4.1/2.3.1.12/
Pyruvate dehydrogenase
171428

1.8.1.4
complex

1, 2, 3, 4
A2
1.2.4.1/2.3.1.12/
Pyruvate dehydrogenase
170971

1.8.1.4
complex

1, 2, 3, 4
A2
1.2.4.1/2.3.1.12/
Pyruvate dehydrogenase
171847

1.8.1.4
complex

1, 2, 3, 4
A2
1.2.4.1/2.3.1.12/
Pyruvate dehydrogenase
172267

1.8.1.4
complex

1, 2, 3, 4
A2
1.2.4.1/2.3.1.12/
Pyruvate dehydrogenase
327533853

1.8.1.4
complex (E1 aplha)

1, 4
B
2.3.1.9
acetyl coenzyme A
3309200

acetyltransferase

1, 4
B
2.3.1.9
acetyl coenzyme A
3309200

acetyltransferase

1, 2, 3, 4
F1
4.2.3.3
methylglyoxal synthase
1146223

1, 2, 3, 4
F1
4.2.3.3
methylglyoxal synthase
1845160

1, 2, 3, 4
F2
4.2.3.3
methylglyoxal synthase
Engineered

1, 2, 3, 4
G
1.1.1.—
methylglyoxal reductase,
3907615

multifunctional

1, 2, 3, 4
H
1.1.1.78
methylglyoxal reductase
48994873

1, 2, 3, 4
I
1.1.1.304
methylglyoxal reductase,
3907615

multifunctional

1, 2, 3, 4
J1
1.1.1.77
lactaldehyde reductase
146044

1, 2, 3, 4
J2
1.1.1.—
methylglyoxal reductase
944901

[multifunctional]

1, 2, 3, 4
K1
4.2.1.30
glycerol dehydratase
384086948

1, 2, 3, 4
K1
4.2.1.30
glycerol dehydratase activator
384086948

1, 2, 3, 4
K2
4.2.1.30
diol dehydratase
83596364

1, 2, 3, 4
K2
4.2.1.30
diol dehydratase activator
83596364

1, 2, 3, 4
L
1.1.1.1
alcohol dehydrogenase
308066805

1, 2, 3, 4
M1
1.1.1.28
D-Lactate dehydrogenase
946315

1, 2, 3, 4
M2
1.1.1.27
L-Lactate dehydrogenase
1063343

1, 2, 3, 4
M2
1.1.1.27
L-lactate dehydrogenase
217591

1, 2, 3, 4
N
2.8.3.1
propionate CoA-transferase*
7242549

1, 2, 3, 4
O
2.3.3.—
Lactoyl-CoA Synthase
296142482

1, 2, 3, 4
P
1.2.1.—
CoA-dependent
1253572

propionaldehyde

dehydrogenase*

1, 2, 3, 4
Q
1.1.1.77
L-1,2-propanediol
947273

oxidoreductase

1
R
1.1.1.157
3-hydroxybutyryl-CoA
1118891

dehydrogenase

S.
1.2.1.
Crotonaldehyde
856044

dehydrogenase

1
T.
1.2.1.10
crotonaldehyde dehydrogenase
12932628

1
U.
4.2.1.127
crotonyl alcohol dehydratase
302064203

1
V.
2.7.1.36
crotonyl alcohol kinase
855248

1
X.
2.7.4.2
2-butenyl-4-phosphate kinase
855260

1
Z.
2.7.1.33
crotonyl alcohol
12934389

diphosphokinase

1
AA.
4.2.3.27
butadiene synthase
16079305

2,3
AB.
4.2.1.54
lactoyl-CoA dehydratase
343794933

2,3
AB.
4.2.1.54
lactoyl-CoA dehydratase
343794931

2,3
AB.
4.2.1.54
lactoyl-CoA dehydratase
343794935

2
AC
4.2.1.116
acryloyl-CoA hydratase
5103388

2
AC
4.2.1.116
acryloyl-CoA hydratase
M2W248

2
AC
4.2.1.116
acryloyl-CoA hydratase
10922910

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
12934018

thiolase

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
10441755

thiolase

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
11639550

thiolase

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
4490319

thiolase

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
4997503

thiolase

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
4383639

thiolase

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
428815

thiolase

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
10921806

thiolase

2
AD.
2.3.1
5-hydroxy-3-kethovaleryl-CoA
12421448

thiolase

2, 4
AE.
1.1.1.35
5-hydroxy-3-Ketovaleryl-CoA
12934454

dehydrogenase

2, 4
AE.
1.1.1.35
5-hydroxy-3-Ketovaleryl-CoA
12931539

dehydrogenase

2, 4
AE.
1.1.1.36
5-hydroxy-3-Ketovaleryl-CoA
10920675

dehydrogenase

2, 4
AE.
1.1.1.36
5-hydroxy-3-Ketovaleryl-CoA
9410631

dehydrogenase

2, 4
AF.,
4.2.1.80
3,5-hydroxyvaleryl-CoA
87081722

AL.

dehydratase

2, 4
AF., AL.
4.2.1.132
3,5-hydroxyvaleryl-CoA
1263188

dehydratase

2, 4
AF., AL.
4.2.1
3,5-hydroxyvaleryl-CoA
8178258

dehydratase

2, 4
AF., AL.
4.2.1.33/4.2.1.35
3,5-hydroxyvaleryl-CoA
2122345

dehydratase

2, 4
AF., AL.
4.2.1.85
3,5-hydroxyvaleryl-CoA
9884634

dehydratase

2, 4
AF., AL.
4.2.1.85
3,5-hydroxyvaleryl-CoA
9884633

dehydratase

2, 4
AF., AL.
4.2.1.55
3,5-hydroxyvaleryl-CoA
1118895

dehydratase

2, 4
AF., AL.
4.2.1.54
3,5-hydroxyvaleryl-CoA
343794933

dehydratase

2, 4
AF., AL.
4.2.1.54
3,5-hydroxyvaleryl-CoA
343794931

dehydratase

2, 4
AF., AL.
4.2.1.54
3,5-hydroxyvaleryl-CoA
343794935

dehydratase

2, 3, 4
AH., AL.
4.1.
3-hydroxy-4-pentenoic acid
145235771

decarboxylase

2, 3, 4
AH., AL.
4.1.
3-hydroxy-4-pentenoic acid
145235769

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
2845318

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
855779

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
162312575

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
257051090

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
8741675

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
9132821

decarboxylase

2, 3, 4
AH., AL.
4.1
3-hydroxy-4-pentenoic acid
1447408

decarboxylase

2, 3, 4
AH., AL.
4.1
3-hydroxy-4-pentenoic acid
12170895

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
11027973

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
8433456

decarboxylase

2, 3, 4
AH., AL.
4.1.1.33
3-hydroxy-4-pentenoic acid
12158799

decarboxylase

2, 3, 4
AH., AL.
4.1.1.1
3-hydroxy-4-pentenoic acid
851654

decarboxylase

2, 3, 4
AH., AL.
4.1.1.1
3-hydroxy-4-pentenoic acid
12759328

decarboxylase

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
12934018

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
10441755

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
11639550

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
4490319

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
4997503

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
4383639

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
428815

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
10921806

3
AI.
2.3.1
3-keto-4-pentenoyl-CoA thiolase
12421448

3
AJ.
1.1.1.35
3-keto-4-pentenoyl-CoA
12934454

dehydrogenase

3
AJ.
1.1.1.35
3-keto-4-pentenoyl-CoA
12931539

dehydrogenase

3
AJ.
1.1.1.36
3-keto-4-pentenoyl-CoA
10920675

dehydrogenase

3
AJ.
1.1.1.36
3-keto-4-pentenoyl-CoA
9410631

dehydrogenase

3, 4
AK.
3.1.2
3-hydroxy-4-pentenoyl-CoA
23664428

transferase or hydrolase or

synthase

3, 4
AK.
3.1.2
3-hydroxy-4-pentenoyl-CoA
46200680

transferase or hydrolase or

synthase

3, 4
AK.
3.1.2
3-hydroxy-4-pentenoyl-CoA
89052491

transferase or hydrolase or

synthase

3, 4
AK.
3.1.2
3-hydroxy-4-pentenoyl-CoA
126729407

transferase or hydrolase or

synthase

3, 4
AK.
3.1.2
3-hydroxy-4-pentenoyl-CoA
1786813

transferase or hydrolase or

synthase

3, 4
AK.
2.8.3
3-hydroxy-4-pentenoyl-CoA
2492994

transferase or hydrolase or

synthase

3, 4
AK.
2.8.3
3-hydroxy-4-pentenoyl-CoA
2492990

transferase or hydrolase or

synthase

3, 4
AK.
2.8.3
3-hydroxy-4-pentenoyl-CoA
62391407

transferase or hydrolase or

synthase

3, 4
AK.
2.8.3
3-hydroxy-4-pentenoyl-CoA
62289399

transferase or hydrolase or

synthase

3, 4
AK.
2.8.3
3-hydroxy-4-pentenoyl-CoA
15004866

transferase or hydrolase or

synthase

3, 4
AK.
2.8.3
3-hydroxy-4-pentenoyl-CoA
15004867

transferase or hydrolase or

synthase

3, 4
AK.
2.8.3
3-hydroxy-4-pentenoyl-CoA
150019354

transferase or hydrolase or

synthase

3, 4
AK.
2.8.3
3-hydroxy-4-pentenoyl-CoA
141776

transferase or hydrolase or

synthase

4
AM.
1.2.1.2
Formate dehydrogenase
12933956

4
AM.
1.2.1.2
Formate dehydrogenase
12933907

4
AM.
1.2.1.2
Formate dehydrogenase
854570

4
AM.
1.2.1.2
Formate dehydrogenase
9444316

4
AM.
1.2.1.2
Formate dehydrogenase
9444319

4
AN.
2.8.3.16
formyl-CoA transferase
12931869

4
AN.
2.8.3.16
formyl-CoA transferase
4209557

4
AN.
2.8.3.16
formyl-CoA transferase
1213305

4
AN.
2.8.3.16
formyl-CoA transferase
10846643

4
AN.
2.8.3.16
formyl-CoA transferase
2688995

4
AO.
2.3.1.16
3,5-ketovaleryl-CoA thiolase
10441755

4
AO.
2.3.1.16
3,5-ketovaleryl-CoA thiolase
11639550

4
AO.
2.3.1.16
3,5-ketovaleryl-CoA thiolase
4490319

4
AO.
2.3.1.16
3,5-ketovaleryl-CoA thiolase
4997503

4
AO.
2.3.1.16
3,5-ketovaleryl-CoA thiolase
4383639

4
AO.
2.3.1.16
3,5-ketovaleryl-CoA thiolase
428815

4
AO.
2.3.1.16
3,5-ketovaleryl-CoA thiolase
10921806

4
AO.
2.3.1.16
3,5-ketovaleryl-CoA thiolase
12421448

4
AP.
1.1.1.35
3-hydroxy-5-Ketovaleryl-CoA
12934454

dehydrogenase

4
AP.
1.1.1.35
3-hydroxy-5-Ketovaleryl-CoA
12931539

dehydrogenase

4
AP.
1.1.1.36
3-hydroxy-5-Ketovaleryl-CoA
10920675

dehydrogenase

4
AP.
1.1.1.36
3-hydroxy-5-Ketovaleryl-CoA
9410631

dehydrogenase

4
AQ.
4.2.1.80
3,5-hydroxyvaleryl-CoA
87081722

dehydratase

4
AQ.
4.2.1.132
3,5-hydroxyvaleryl-CoA
1263188

dehydratase

4
AQ.
4.2.1
3,5-hydroxyvaleryl-CoA
8178258

dehydratase

4
AQ.
4.2.1.85
3,5-hydroxyvaleryl-CoA
9884634

dehydratase

4
AQ.
4.2.1.85
3,5-hydroxyvaleryl-CoA
9884633

dehydratase

4
AQ.
4.2.1.55
3,5-hydroxyvaleryl-CoA
1118895

dehydratase

4
AQ.
4.2.1.54
3,5-hydroxyvaleryl-CoA
343794933

dehydratase

4
AQ.
4.2.1.54
3,5-hydroxyvaleryl-CoA
343794931

dehydratase

4
AQ.
4.2.1.54
3,5-hydroxyvaleryl-CoA
343794935

dehydratase

TABLE 10

Exemplary Gene Identifier (GI) numbers

Enzyme

Enzyme

EC
Source
GI
SEQ

No.
Gene
candidate
Reaction
number
Organism
(nt)
ID NO.

A1
pflB
Formate-C
Pyruvate + CoA →
2.3.1.54/

Escherichia

116182, 8116182
93

acetyltransferase
acetyl-CoA + formate
1.97.1.4

coli

A1
pflA
Formate-C
Pyruvate + CoA →
2.3.1.54/

Escherichia

387233060
94

acetyltransferase
acetyl-CoA + formate
1.97.1.4

coli

(activating enzyme)

A1
pflB
Formate-C
Pyruvate + CoA →
2.3.1.54/

Neocallimastix

41400040
95

acetyltransferase
acetyl-CoA + formate
1.97.1.4

frontalis

A1
pflA
Formate-C
Pyruvate + CoA →
2.3.1.54/

Neocallimastix

298058
96

acetyltransferase
acetyl-CoA + formate
1.97.1.4

frontalis

(activating enzyme)

A2
pda1
Pyruvate
Pyruvate + CoA + NAD+ →
1.2.4.1/

Saccharomyces

171428
97

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

cerevisiae

complex

1.8.1.4

A2
pdb1
Pyruvate
Pyruvate + CoA + NAD+ →
1.2.4.1/

Saccharomyces

170971
98

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

cerevisiae

complex

1.8.1.4

A2
lat1
Pyruvate
Pyruvate + CoA + NAD+ →
1.2.4.1/

Saccharomyces

171847
99

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

cerevisiae

complex

1.8.1.4

A2
lpd1
Pyruvate
Pyruvate + CoA + NAD+ →
1.2.4.1/

Saccharomyces

172267
100

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

cerevisiae

complex

1.8.1.4

A2
pdx1
Pyruvate
Pyruvate + CoA + NAD+ -->
1.2.4.1/

Saccharomyces

327533853
101

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

cerevisiae

complex

1.8.1.4

A2
pdhA
Pyruvate
Pyruvate + CoA + NAD+ →
1.2.4.1/

Enterococcus

327533853
102

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

faecalis

complex (E1 aplha)

1.8.1.4

A2
pdhB
Pyruvate
Pyruvate + CoA + NAD+ →
1.2.4.1/

Enterococcus

327533853
103

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

faecalis

complex (E2 beta)

1.8.1.4

A2
aceF
Pyruvate
Pyruvate + CoA + NAD+ →
1.2.4.1/

Enterococcus

327533853
104

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

faecalis

complex (E2)

1.8.1.4

A2
lpd
Pyruvate
Pyruvate + CoA + NAD+ →
1.2.4.1/

Enterococcus

3309200
105

dehydrogenase
acetyl-CoA + CO2 + NADH
2.3.1.12/

faecalis

complex (E3)

1.8.1.4

B
thlA
acetyl coenzyme A
2 acetyl-Coa →
2.3.1.9

Clostridium

3309200
106

acetyltransferase
acetoacetyl-CoA + CoA

acetobutylicum

B
Erg10
acetyl coenzyme A
2 acetyl-Coa →
2.3.1.9

Saccharomyces

48994873
107

acetyltransferase
acetoacetyl-CoA + CoA

cerevisiae

C1
atoA
Acetyl-
acetoacetyl-Coa + acetate →
2.8.3.8

Escherichia

48994873
108

CoA: acetoacetate-
acetoacetate + acetyl-CoA

coli

CoA transferase

subunit

C1
atoD
Acetyl-
acetoacetyl-Coa + acetate →
2.8.3.8

Escherichia

109

CoA:acetoacetate-
acetoacetate + acetyl-CoA

coli

CoA transferase

subunit

C2
atoA*
Acyl-CoA
acetoacetyl-CoA→
3.1.2.—

Escherichia

6466901
3

thioesterase 2
acetoacetate + CoA

coli,

engineered

D
adc
acetoacetate
acetoacetate →
4.1.1.4

Clostridium

149901357
110

decarboxylase
acetone + CO2

acetobutylicum

D
adc
acetoacetate
acetoacetate → acetone +
4.1.1.4

Clostridium

60592972
111

decarboxylase
CO2

beijerinckii

E
adh
secondary alcohol
acetone + NAD(P)H→
1.1.1.2

Clostridium

1146223
112

dehydrogenase
2-propanol + NAD(P)+

beijerinckii

F1
mgsA
methylglyoxal
dihydroxyacetone
4.2.3.3

Bacillus

1845160
113

synthase
phosphate →

subtilis

methylglyoxal

F1
mgsA
methylglyoxal
dihydroxyacetone
4.2.3.3

Escherichia

modified
114

synthase
phosphate →

coli

version

methylglyoxal

of 1845160

F2
mgsA*
methylglyoxal
dihydroxyacetone
4.2.3.3

Escherichia

3907615
115

synthase
phosphate →

coli

methylglyoxal

G
budC
methylglyoxal
methylglyoxal →
1.1.1.—

Klebsiella

48994873
116

reductase,
lactaldehyde

pneumoniae

multifunctional

H
ydjG
methylglyoxal
methylglyoxal →
1.1.1.78

Escherichia

3907615
117

reductase
hydroxyacetone

coli

I
budC
methylglyoxal
hydroxyacetone + NAD(P)H →
1.1.1.304

Klebsiella

146044
118

reductase,
1,2-propanediol + NAD(P)+/

pneumoniae

multifunctional
methylglyoxal + NAD(P)H→

lactaldehyde + NAD(P)+

J1
fucO
lactaldehyde
lactaldehyde + NAD(P)H→
1.1.1.77

Escherichia

GeneID: 944901
119

reductase
1,2-propanediol + NAD(P)+

coli

J2
yafB
methylglyoxal
Lactaldehyde +
1.1.1.—

Escherichia

384086948
120

reductase
NAD(P)H + H⁺ →1,2-

coli

[multifunctional]
propanediol + NAD(P)⁺

K1
dhaB1
glycerol
1,2-propanediol → propanal
4.2.1.30

Clostridium

384086948
121

dehydratase

butyricum

K1
dhaB2
glycerol
1,2-propanediol → propanal
4.2.1.30

Clostridium

83596364
122

dehydratase

butyricum

activator

K2
ORF18
diol dehydratase
1,2-propanediol → propanal
4.2.1.30

Roseburia

83596364
123

inulinivorans

K2
ORF19
diol dehydratase
1,2-propanediol → propanal
4.2.1.30

Roseburia

308066805
124

activator

inulinivorans

L
adh
alcohol
propanal + NADH →
1.1.1.1

Clostridium

946315
125

dehydrogenase
propanol + NAD+

carboxidivorans

M1
ldhA
D-Lactate
Pyruvate + NAD(P)H +
1.1.1.28

Escherichia

1063343
126

dehydrogenase
H⁺ →D-Lactate +

coli

NAD(P)⁺

M2
ldh2
L-Lactate
Pyruvate + NAD(P)H +
1.1.1.27

Lactobacillus

217591
127

dehydrogenase
H⁺ →L-Lactate +

plantarum

NAD(P)⁺

M2
ldh2
L-lactate
Pyruvate + NAD(P)H +
1.1.1.27

Bos taurus

7242549
128

dehydrogenase
H⁺ →L-Lactate +

NAD(P)⁺

N
pct
propionate CoA-
Lactate + Acetyl-CoA →
2.8.3.1

Clostridium

296142482
129

transferase*
Lactoyl-CoA + Acetic acid

propionicum

O
ACS1
Lactoyl-CoA
Lactate + CoA + ATP→
2.3.3.—

Saccharomyces

40796034
130

Synthase
Lactoyl-CoA + AMP

cerevisiae

O2
car
Carboxylic acid
Lactate + ATP + NADPH →
1.2.1.30

Nocardia

1253572
131

reductase
lactaldehyde + AMP + NADP+

iowensis

P
pduP
CoA-dependent
Lactoyl-CoA + NAD(P)H +
1.2.1.—

Salmonella

947273
132

propionaldehyde
H⁺ →Lactaldehyde +

enterica

dehydrogenase*
NAD(P)⁺

Q
fucO
L-1,2-propanediol
L-Lactaldehyde + NAD(P)H +
1.1.1.77

Escherichia

117623121, 4494334
133

oxidoreductase
H⁺ → L-1,2-propanediol +

coli

NAD(P)⁺

Enzyme

Enzyme

EC
Source
GI
SEQ

No.
Gene
Candidate
Reaction
No.
Organism
(nt)
ID NO.

R.
hbd

Acetoacetyl-CoA →
1.1.1.35

Clostridium

20162442
4

3-hydroxybutyryl-CoA

beijerinckii

R.
hbd
3-
Acetoacetyl-CoA →
1.1.1.157

Clostridium

1118891
5

hydroxybutyryl-
3-hydroxybutyryl-CoA

acetobutylicum

CoA

dehydrogenase

S.
Mcup_1680
3-
3-hydroxybutyryl-CoA →
4.2.1.17

Metallosphaera

10493869
6

hydroxybutyryl-
Crotonyl-CoA

cuprina

CoA

dehydrogenase

T.
adhE2
Crotonase
Crotonyl-CoA →
1.2.1.10/

Clostridium

12958625
7

crotonyl alcohol
1.1.1.1

acetobutylicum

U.
Ald6p
Crotonyl-CoA
Crotonyl-CoA →
1.2.1.

Saccharomyces

856044
8

reductase
crotonaldehyde

cerevisiae

(Bifuncional)

adhE
Crotonaldehyde

1.2.1.

Streptococcus

251817349
9

dehydrogenase

suis

V.
yqhD

Crotonaldehyde →
1.1.1

Escherichia

Gene ID: 12933386,
10

crotonyl alcohol

coli

GI: 388479012

ydjG
Alcohol
Crotonaldehyde →
1.1.1

Escherichia

Gene ID: 12930149,
11

dehydrogenase
crotonyl alcohol

coli

GI: 388477844

W.
ldi

Crotonyl alcohol →
4.2.1.127

Castellaniella

302064203
12

butadiene

defragrans

X.
Erg12
Crotonyl alcohol
Crotonyl alcohol →2-
2.7.1.36

Saccharomyces

855248
13

dehydratase
butenyl-4-phosphate

cerevisiae

Y.
SSO2988
crotonyl alcohol
2-butenyl-4-phosphate →
2.7.4.2

Sulfolobus

Gene ID: 1453012
14

kinase
2-butenyl-4-diphosphate

solfataricus P2

Z.
Erg12
2-butenyl-4-
Crotonyl alcohol →2-
2.7.1.33

Saccharomyces

855248
15

phosphate
butenyl-4-diphosphate

cerevisiae

kinase

ispS
crotonyl alcohol
2-butenyl-4-diphosphate →
4.2.3.27

Populus alba

13539551
16

diphosphokinase
butadiene

N.
pct
butadiene
lactate + acetyl-CoA →
2.8.3.1

Clostridium

7242549
17

synthase
lactoyl-CoA + acetic acid

propionicum

acs
lactoyl-CoA
lactate + acetyl-CoA →
6.2.1.1

Escherichia

16131895
18

transferase
lactoyl-CoA + acetic acid

coli

AB.
lcdA
lactoyl-CoA
Lactoyl-CoA →Acryloyl-CoA
4.2.1.54

Clostridium

343794933
19

synthase

propionicum

lcdB
lactoyl-CoA

4.2.1.54

Clostridium

343794931
20

dehydratase

propionicum

lcdC

4.2.1.54

Clostridium

343794935
21

propionicum

AC.
Mcup_1680

Acryloyl-CoA →3-
4.2.1.17

Metallosphaera

10493869
22

hydroxypropyonyl-CoA

cuprina

AD.
phaD
acryloyl-CoA
Acetyl-CoA +
2.3.1.16

Pseudomonas

10441755
23

hydratase
3-hydroxypropionyl-CoA →

putida

5-hydroxy-3-ketovaleryl-CoA

pcaF
5-hydroxy-3-

2.3.1.16

Pseudomonas

4383639
24

ketovaleryl-CoA

aeroginosa

thiolase

pcaF

2.3.1.16

Acinetobacter

11639550
25

calcoaceticus

fadA

2.3.1.16

Aeromonas

4490319
26

hydrophila

AE.
fadB

5-hydroxy-3-
1.1.1.36

Escherichia

12934454
27

Ketovaleryl-CoA →

coli

R/S 3,5-dihydroxy-

valeryl-CoA

yfcX
5-hydroxy-3-

1.1.1.36

Escherichia

12931539
28

Ketovaleryl-CoA

coli

dehydrogenase

phbB

1.1.1.36

Cupriavidus

10920675
29

necator

phaB

1.1.1.36

Rastonia

9410631
30

solanacearum

AF.
mhpD

R/S 3,5-dihydroxy-
4.2.1.80

Escherichia

87081722
31

valeryl-CoA →

coli

3-hydroxy-4-

pentenoyl-CoA

ctmF
3,5-

4.2.1.132

Pseudomonas

1263188
32

hydroxyvaleryl-CoA

putida

dehydratase

hpaH

4.2.1

Escherichia

8178258
33

coli

cnbE

4.2.1.33/

Methanocaldococcus

2122345
34

4.2.1.35

jannaschii

dmdA

4.2.1.85

Eubacterium

9884634
35

limosum

dmdB

4.2.1.85

Eubacterium

9884633
36

limosum

crt

4.2.1.55

Clostridium

1118895
37

acetobutylicum

lcdA

4.2.1.54

Clostridium

343794933
38

propionicum

lcdB

4.2.1.54

Clostridium

343794931
39

propionicum

lcdC

4.2.1.54

Clostridium

343794935
40

propionicum

AG.
menD

3-hydroxy-4-
2.3.3/

Escherichia

12665319
41

AK.

pentenoyl-CoA →
3.1.2/

coli

3-hydroxy-4-
2.8.3

pentenoic acid

leuA
3-hydroxy-4-

2.3.3

Escherichia

947465
42

pentenoyl-CoA

coli

hydrolase,

transferase

or synthase

AH.
mvd

3-hydroxy-4-
4.1.1.33

Picrophilus

2845318
43

pentenoic acid →

torridus

butadiene

mvd
3-hydroxy-4-

4.1.1.33

Saccharomyces

855779
44

pentenoic acid

cerevisiae

decarboxylase

mvaD

4.1.1.33

Streptococcus

11027973
45

pseudopneumoniae

AI.
paaJ

Acryloyl-CoA +
2.3.1

Escherichia

12934018
46

Acetyl-CoA →

coli

3-keto-4-pentenoyl-

CoA

phaD
3-keto-4-

2.3.1

Pseudomonas

10441755
47

pentenoyl-CoA

putida

thiolase

pcaF

2.3.1

Acinetobacter

11639550
48

calcoaceticus

AJ.
fadB

3-keto-4-
1.1.1.35

Escherichia

12934454
49

pentenoyl-CoA→

coli

R/S 3-hydroxy-4-

pentenoyl-CoA

yfcX
3-keto-4-

1.1.1.35

Escherichia

12931539
50

pentenoyl-CoA

coli

dehydrogenase

phbB

1.1.1.36

Cupriavidus

10920675
51

necator

phaB

1.1.1.36

Rastonia

9410631
52

solanacearum

AK.
Orf1

R/S 3-hydroxy-4-
3.1.2

Azoarcus

23664428
53

pentenoyl-CoA →

evansii

3-hydroxy-4-

pentenoic acid

COG0824
3-hydroxy-4-

3.1.2

Magnetospirillum

46200680
54

pentenoyl-CoA

magnetotacticum

transferase

or hydrolase

or synthase

atoA

2.8.3

Escherichia

2492994
55

coli

atoD

2.8.3

Escherichia

2492990
56

coli

actA

2.8.3

Corynebacterium

62391407
57

glutamicum

AL.
oleT_JE

3-hydroxy-4-
4.1.

Jeotgalicoccus

58

pentenoic acid →

sp; ATCC8456

butadiene

padA1
3-hydroxy-4-

4.1.

Aspergillus

145235771
59

pentenoic acid

niger

decarboxylase

mvd

4.1.1.33

Picrophilus

2845318
60

torridus

mvd

4.1.1.33

Saccharomyces

855779
61

cerevisiae

dvd

4.1

Halobacterium

1447408
62

salinarum

dfd

4.1

Aspergillus

12170895
63

clavatu

mvaD

4.1.1.33

Streptococcus

11027973
64

pseudopneumoniae

mvaD

4.1.1.33

Lactobacillus

8433456
65

rhamnosus

pdc2

4.1.1.1

Saccharomyces

851654
66

cerevisiae

pdc1

4.1.1.1

Escherichia

12759328
67

coli

AM.
fdhF

CO2 → Formate
1.2.1.2

Escherichia

12933956
68

coli

fdnH
Formate

1.2.2.1

Escherichia

12933907
69

dehydrogenase

coli

fdh1

1.2.1.2

Saccharomyces

854570
70

cerevisiae

CLJU_c06990

1.2.1.2

Clostridium

9444316
71

ljungdahlii

CLJU_c07020

1.2.1.2

Clostridium

9444319
72

ljungdahlii

AN.
frc

Formate → formyl-CoA
2.8.3.16

Escherichia

12931869
73

coli

frc
formyl-CoA

2.8.3.16

Shigella

4209557
74

transferase

flexneri

frc

2.8.3.16

Streptomyces

1213305
75

avermitilis

AN.
acs

Formate → formyl-CoA
6.2.1

Escherichia

16131895
76

coli

phaD
formyl-CoA
Acetoacetyl-CoA +
2.3.1.16

Pseudomonas

10441755
77

synthase
formyl-CoA →

putida

3,5-ketovaleryl-CoA

pcaF
3,5-

2.3.1.16

Acinetobacter

11639550
78

ketovaleryl-CoA

calcoaceticus

thiolase

fadA

2.3.1.16

Aeromonas

4490319
79

hydrophila

AP.
fadB

3,5-ketovaleryl-CoA →
1.1.1.35

Escherichia

12934454
80

R/S 3-hydroxy-5-

coli

Ketovaleryl-CoA

yfcX
3-hydroxy-5-

1.1.1.35

Escherichia

12931539
81

Ketovaleryl-CoA

coli

dehydrogenase

phbB

1.1.1.36

Cupriavidus

10920675
82

necator

phaB

1.1.1.36

Rastonia

9410631
83

solanacearum

AQ.
mhpD

R/S 3,5-
4.2.1.80

Escherichia

87081722
84

hydroxyvaleryl-CoA →

coli

R/S 3-hydroxy-4-

pentenoyl-CoA

ctmF
3,5-

4.2.1.132

Pseudomonas

1263188
85

hydroxyvaleryl-CoA

putida

dehydratase

hpaH

4.2.1

Escherichia

8178258
86

coli

dmdA

4.2.1.85

Eubacterium

9884634
87

limosum

dmdB

4.2.1.85

Eubacterium

9884633
88

limosum

crt

4.2.1.55

Clostridium

1118895
89

acetobutylicum

lcdA

4.2.1.54

Clostridium

343794933
90

propionicum

lcdB

4.2.1.54

Clostridium

343794931
91

propionicum

lcdC

Acetoacetyl-CoA →
4.2.1.54

Clostridium

343794935
92

3-hydroxybutyryl-CoA

propionicum

Methods for the Co-Production of Butadiene and 1-Propanol and/or 1,2-Propanediol

Butadiene and 1-propanol and/or 1,2-propanediol may be produced by contacting any of the genetically modified microorganisms provided herein with a fermentable carbon source. Such methods may preferably comprise contacting a fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to any of the intermediates provided in FIGS. 1-4 (Tables 1-4) and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates provided in FIGS. 1-4 (tables 1-4) to butadiene and 1-propanol and/or 1,2-propanediol in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to the one or more intermediates provided in FIGS. 1-4 (tables 1-4) and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates provided in FIGS. 1-4 (tables 1-4) to butadiene and 1-propanol and/or 1,2-propanediol.

The metabolic pathways that lead to the production of industrially important compounds involve oxidation-reduction (redox) reactions. For example, during fermentation, glucose is oxidized in a series of enzymatic reactions into smaller molecules with the concomitant release of energy. The electrons released are transferred from one reaction to another through universal electron carriers, such Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NAD(P)), which act as cofactors for oxidoreductase enzymes. In microbial catabolism, glucose is oxidized by enzymes using the oxidized form of the cofactors (NAD(P)+ and/or NAD+) as cofactor thus generating reducing equivalents in the form of the reduced cofactor (NAD(P)H and NADH). In order for fermentation to continue, redox-balanced metabolism is required, i.e., the cofactors must be regenerated by the reduction of microbial cell metabolic compounds.

Microorganism-catalyzed fermentation for the production of natural products is a widely known application of biocatalysis. Industrial microorganisms can affect multistep conversions of renewable feedstocks to high value chemical products in a single reactor. Products of microorganism-catalyzed fermentation processes range from chemicals such as ethanol, lactic acid, amino acids and vitamins, to high value small molecule pharmaceuticals, protein pharmaceuticals, and industrial enzymes. In many of these processes, the biocatalysts are whole-cell microorganisms, including microorganisms that have been genetically modified to express heterologous genes.

Some key parameters for efficient microorganism-catalyzed fermentation processes include the ability to grow microorganisms to a greater cell density, increased yield of desired products, increased amount of volumetric productivity, removal of unwanted co-metabolites, improved utilization of inexpensive carbon and nitrogen sources, adaptation to varying fermenter conditions, increased production of a primary metabolite, increased production of a secondary metabolite, increased tolerance to acidic conditions, increased tolerance to basic conditions, increased tolerance to organic solvents, increased tolerance to high salt conditions and increased tolerance to high or low temperatures. Inefficiencies in any of these parameters can result in high manufacturing costs, inability to capture or maintain market share, and/or failure to bring fermented end-products to market.

The methods and compositions of the present disclosure can be adapted to conventional fermentation bioreactors (e.g., batch, fed-batch, cell recycle, and continuous fermentation).

In some embodiments, a microorganism (e.g., a genetically modified microorganism) as provided herein is cultivated in liquid fermentation media (i.e., a submerged culture) which leads to excretion of the fermented product(s) into the fermentation media. In one embodiment, the fermented end product(s) can be isolated from the fermentation media using any suitable method known in the art.

In some embodiments, formation of the fermented product occurs during an initial, fast growth period of the microorganism. In one embodiment, formation of the fermented product occurs during a second period in which the culture is maintained in a slow-growing or non-growing state. In one embodiment, formation of the fermented product occurs during more than one growth period of the microorganism. In such embodiments, the amount of fermented product formed per unit of time is generally a function of the metabolic activity of the microorganism, the physiological culture conditions (e.g., pH, temperature, medium composition), and the amount of microorganisms present in the fermentation process.

In some embodiments, the fermentation product is recovered from the periplasm or culture medium as a secreted metabolite. In one embodiment, the fermentation product is extracted from the microorganism, for example when the microorganism lacks a secretory signal corresponding to the fermentation product. In one embodiment, the microorganisms are ruptured and the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions may then be separated if necessary. The fermentation product of interest may then be purified from the remaining supernatant solution or suspension by, for example, distillation, fractionation, chromatography, precipitation, filtration, and the like.

The methods of the present disclosure are preferably preformed under anaerobic conditions. Both the degree of reduction of a product as well as the ATP requirement of its synthesis determines whether a production process is able to proceed aerobically or anaerobically. To produce butadiene and 1-propanol and/or 1,2-propanediol via anaerobic microbial conversion, or at least by using a process with reduced oxygen consumption, redox imbalances should be avoided. Several types of metabolic conversion steps involve redox reactions including some of the conversions as set forth in FIG. 1. Such redox reactions involve electron transfer mediated by the participation of redox cofactors such as NADH, NADPH and ferredoxin. Since the amounts of redox cofactors in the cell are limited to permit the continuation of metabolic processes, the cofactors have to be regenerated. In order to avoid such redox imbalances, alternative ways of cofactor regeneration may be engineered, and in some cases additional sources of ATP generation may be provided. Alternatively, oxidation and reduction processes may be separated spatially in bioelectrochemical systems (Rabaey and. Rozendal, 2010, Nature reviews, Microbiology, vol 8: 706-716).

In some embodiment, redox imbalances may be avoided by using substrates (e.g., fermentable carbon sources) that are more oxidized or more reduced. for example, if the utilization of a substrate results in a deficit or surplus of electrons, a requirement for oxygen can be circumvented by using substrates that are more reduced or oxidized, respectively. For example, glycerol which is a major byproduct of biodiesel production is more reduced than sugars, and is therefore more suitable for the synthesis of compounds whose production from sugar results in cofactor oxidation, such as succinic acid. In some embodiments, if the conversion of a substrate to a product results in an electron deficit, co-substrates can be added that function as electron donors (Babel 2009, Eng. Life Sci. 9,285-290). An important criterion for the anaerobic use of co-substrates is that their redox potential is higher than that of NADH (Geertman et al., 2006, FEMS Yeast Res. 6, 1193-1203). If the conversion of substrate to produce results in an electron surplus, co-substrates can be added that function as electron acceptors.

Methods for the Production of Polypropylene

1-propanol produced via methods disclosed herein may be dehydrated to form propylene, which may then be polymerized to produce polypropylene in a cost-effective manner.

Propylene is a chemical compound that is widely used to synthesize a wide range of petrochemical products. For instance, this olefin is the raw material used for the production of polypropylene, its copolymers and other chemicals such as acrylonitrile, acrylic acid, epichloridrine and acetone. Propylene demand is growing faster than ethylene demand, mainly due to the growth of market demand for polypropylene. Propylene is polymerized to produce thermoplastics resins for innumerous applications such as rigid or flexible packaging materials, blow molding and injection molding.

Propylene is typically obtained in large quantity scales as a byproduct of catalytical or thermal oil cracking, or as a co-product of ethylene production from natural gas. (Propylene, Jamie G. Lacson, CEH Marketing Research Report-2004, Chemical Economics Handbook-SRI International). The use of alternative routes for the production of propylene has been continuously evaluated using a wide range of renewable raw materials (“Green Propylene”, Nexant, January 2009). These routes include, for example, dimerization of ethylene to yield butylene, followed by metathesis with additional ethylene to produce propylene. Another route is biobutanol production by sugar fermentation followed by dehydration and methatesis with ethylene. Some thermal routes are also being evaluated such as gasification of biomass to produce a syngas followed by synthesis of methanol, which may then produce green propylene via methanol-to-olefin technology.

Propylene production by 2-propanol dehydration has been well-described in document EP00498573B1, wherein all examples show propylene selectivity higher than 90% with high conversions. Dehydration of 1-propanol has also been studied in the following articles: “Mechanism and Kinetics of the Acid-Catalyzed Dehydration of 1- and iso-propanol in Hot Compressed Liquid Water” (Antal, M et al., Ind. Eng. Chem. Res. 1998, 37, 3820-3829) and “Fischer-Tropsch Aqueous Phase Refining by Catalytic Alcohol Dehydration” (Nel, R. et al., Ind. Eng. Chem. Res. 2007, 46, 3558-3565). The reported yield is higher than 90%.

Methods for the Production of Polybutadiene and Other Compounds from Butadiene

Butadiene is gaseous at room temperature or in fermentative conditions (20-45° C.), and their production from a fermentation process results in a gas that could accumulate in the headspace of a fermentation tank, and be siphoned and concentrated. Butadiene may be purified from fermentation of gases, including gaseous alcohol, CO₂and other compound by solvent extraction, cryogenic processes, distillation, fractionation, chromatography, precipitation, filtration, and the like.

Butadiene produced via any of the processes or methods disclosed herein may be converted to polybutadiene. Alternatively, butadiene produced via methods disclosed herein may be polymerized with other olefins to form copolymers such as acrylonitrile-butadiene-styrene (ABS), acrylonitrile-butadiene (ABR), or styrene-butadiene (SBR) copolymers, BR butyl rubber (RB), poly butadiene rubber (PBR), nitrile rubber and polychloroprene (Neoprene). Those synthetic rubbers or plastic elastomers applications include productions of tires, plastic materials, sole, shoe hills, technical goods, home appliance, neoprene, paper coatings, gloves, gaskets and seals.

Without further description, it is believed that one of ordinary skill in the art may, using the preceding description and the following illustrative examples, make and utilize the agents of the present disclosure and practice the claimed methods. The following working examples are provided to facilitate the practice of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES
Example 1
Modification of Microorganism for Production of Butadiene and 1-Propanol and/or 1,2-Propanediol

A microorganism such as a bacterium is genetically modified to produce butadiene and 1-propanol and/or 1,2-propanediol from a fermentable carbon source including, for example, glucose.

In an exemplary method, a microorganism may be genetically engineered by any methods known in the art to comprise: i.) one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the fermentable carbon source to dihydroxyacetone-phosphate or glyceraldehyde 3-phosphate and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate or glyceraldehyde 3-phosphate to butadiene and 1-propanol and/or 1,2-propanediol.

Alternatively, a microorganism that lacks one or more enzymes (e.g., one or more functional enzymes that are catalytically active) for the conversion of a fermentable carbon source to butadiene and 1-propanol and/or 1,2-propanediol may be genetically modified to comprise one or more polynucleotides coding for enzymes (e.g., functional enzymes including, for example any enzyme disclosed herein) in a pathway that the microorganism lacks to catalyze a conversion of the fermentable carbon source to butadiene and 1-propanol and/or 1,2-propanediol.

Example 2
Fermentation of Glucose by Genetically Modified Microorganism to Produce Butadiene and 1-Propanol and/or 1,2-Propanediol

A genetically modified microorganism, as produced in Example 1 above, may be used to ferment a carbon source to produce butadiene and 1-propanol and/or 1,2-propanediol.

In an exemplary method, a previously-sterilized culture medium comprising a fermentable carbon source (e.g., 9 g/L glucose, 1 g/L KH2PO4, 2 g/L (NH4)2HPO4, 5 mg/L FeSO4.7H2O, 10 mg/L MgSO4.7H2O, 2.5 mg/L MnSO4.H2O, 10 mg/L CaCl2.6H2O, 10 mg/L CoCl2.6H2O, and 10 g/L yeast extract) is charged in a bioreactor.

During fermentation, anaerobic conditions are maintained by, for example, sparging nitrogen through the culture medium. A suitable temperature for fermentation (e.g., about 30° C.) is maintained using any method known h the art. A near physiological pH (e.g., about 6.5) is maintained by, for example, automatic addition of sodium hydroxide. The bioreactor is agitated at, for example, about 50 rpm. Fermentation is allowed to run to completion.

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

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein can be further limited in the claims using consisting of or and consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure can be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

While the present disclosure has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the disclosure is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the disclosure being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.

MODIFIED MICROORGANISM AND METHODS OF USING SAME FOR PRODUCING BUTADIENE AND 1-PROPANOL AND/OR 1,2-PROPANEDIOL

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