MODIFIED MICROORGANISM AND METHODS OF USING SAME FOR PRODUCING 2-PROPANOL AND1-PROPANOL AND/OR 1,2-PROPANEDIOL

BACKGROUND

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.

2-propanol (isopropyl alcohol, CH₃CH₃CHOH, CAS 67-63-0) is a secondary alcohol and is a structural isomer of 1-propanol. 2-propanol is typically produced by the weak acid process in which propene gas is absorbed in, and reacted with, 60% sulfuric acid and the resulting sulfates hydrolyzed in a single step process. Another major current manufacturing process is catalytic hydration of propylene with water. Hydration can be gas phase with a phosphoric acid catalyst, mixed phase with a cation-exchange resin catalyst or liquid phase using a tungsten catalyst. 2-propanol is used as an industrial solvent, a component of industrial and consumer products and as a disinfectant. Most 2-propanol goes into the solvent market either directly or via conversion to acetone or one of acetone's derivatives—methyl isobutyl ketone, methyl isobutyl carbinol, diacetone alcohol, or isophorone. 2-propanol's major solvent uses include inks, coatings, cosmetics and pharmaceuticals.

1,2-propanediol (propylene glycol, HO—CH₂—CHOH—CH₃, CAS 57-55-6) is an organic compound with formula C₃H₈O₂. 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. (428 F), 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 2-propanol, 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 covers the co-production of 1,2-propanol or 1-propanol and 2-propanol in a eukaryote cell, such as a yeast, with reduced levels of pyruvate decarboxylase enzymatic activity, wherein the microorganism has its native ethanol production shut-off, and wherein the microorganism is capable of growing on a C6 sugar as a sole carbon source under anaerobic or microaerobic conditions.

In order to eliminate the ethanol production in yeast it is necessary to knock out the activity of pyruvate decarboxylase, the enzyme that decarboxylates pyruvate making acetaldehyde and carbon dioxide. In yeast, this enzyme comes in three isoforms, and its activity can be completely knocked out by deleting the genes PDC1, PDC5 and PDC6. As a consequence, the microorganism can not grow on C6 sugars as a sole carbon source such as glucose and consequently it is necessary to alter the ability of the microorganism to import glucose, for example, by truncating a transcription factor of the glucose importer MTH1. Also, the elimination of the pyruvate decarboxylase activity in the cell's cytoplasm renders the microorganism 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. Accordingly, the present disclosure discloses a series of complex deletions/truncations and gene integrations that enables a new acetil-CoA overproducing yeast chassis for the co-production of 1,2-propanol or 1-propanol and 2-propanol.

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 cytosolic acetyl-CoA to 2-propanol; 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 acetyl-CoA to 2-propanol include: one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA to acetoacetate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetate to acetone, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetone to 2-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 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 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 catalyze a conversion of cytosolic acetyl-CoA to 2-propanol; and one or more polynucleotides coding for 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 include and/or 1,2-propanediol: 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 methods for co-producing 2-propanol 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 under substantially anaerobic conditions, and expressing the polynucleotides in the microorganism for the co-production of 2-propanol with 1-propanol and/or 1,2-propanediol, wherein the microorganism co-produces 2-propanol 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 2-propanol with 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 2-propanol with 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, pdc5, 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 one or more enzymes 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 comprises one or more exogenous polynucleotides encoding one or more enzymes in pathways for the co-production of 1,2-propanediol and 2-propanol from a fermentable carbon source 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 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 microorganism comprises one or more exogenous polynucleotides encoding one or more enzymes in pathways for the co-production of 2-propanol, 1, propanol and/or 1,2-propanediol from a fermentable carbon source under 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 acetyl-CoA to 2-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 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.

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 microorganism comprises one or more exogenous polynucleotides encoding one or more enzymes in pathways for the co-production of 1,2-propanediol or 1-propanol and 2-propanol from a fermentable carbon source under anaerobic conditions.

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 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-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.

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 (see, e.g., WO2014076232).

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, 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.

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 dihydroxyacetone phosphate to 1,2-propanediol or 1-propanol or one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1,2-propanediol or 1-propanol, and one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetyl-CoA to 2-propanol, 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 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 1,2-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-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.

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.

C6 sugar as a sole carbon source

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 the microorganism on a C6 molecule 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 dihydroxyacetone phosphate to 1,2-propanediol or 1-propanol, and one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetyl-CoA to 2-propanol, and optionally one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1,2-propanediol or 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.

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.

The present disclosure also provides methods for co-producing 1,2-propanediol or 1-propanol and 2-propanol 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 1,2-propanediol or 1-propanol and 2-propanol from the fermentable carbon source.

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 growth on a C6 molecule as a sole carbon source, and that is capable of producing 1,2-propanediol or 1-propanol and 2-propanol 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 pyruvate to 1,2-propanediol or 1-propanol, and introducing into the microorganism one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetyl-CoA to 2-propanol, and optionally introducing into the microorganism one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to 1,2-propanediol or 1-propanol.

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 2-propanol and 1,2-propanediol, where 1,2-propanediol is produced via a dihydroxyacetone-phosphate intermediate.

FIG. 2 depicts an exemplary pathway for the co-production of 1,2-propanediol and 2-propanol, where 1,2-propanediol is produced via a glyceraldehyde 3-phosphate intermediate.

FIG. 3 depicts an exemplary pathway for the co-production of 2-propanol and 1-propanol, where 1-propanol is produced via a dihydroxyacetone-phosphate intermediate.

FIG. 4 depicts an exemplary pathway for the co-production of 2-propanol and 1-propanol, where 1-propanol is produced via a glyceraldehyde-3-phosphate intermediate.

FIG. 5 depicts a schematic representation of the anaerobic flask system used in Example 1.

FIG. 6A-C summarizes the results of Example 1 showing restored anaerobic growth of the BRKY-272 strain compared to the control (BRKY-397), carbon source consumption and metabolite profile. FIG. 6A shows increased growth of the BRKY-272 strain compared to the control (BRKY-397) in the anaerobic flask system shown in FIG. 5. FIGS. 6B and FIG. 6C show the kynetics of glucose consumption and metabolite production in strains BRKY-397 (control) and BRKY-272, respectively.

FIG. 7 summarizes the results of Example 3 showing co-production of 2-propanol and 1,2-propanediol in an ethanol-null strain BRKY-399.

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 2-propanol 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 2-propanol, 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 2-propanol 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 2-propanol and 1-propanol and/or 1,2-propanediol. Microorganisms may be modified so that they may co-produce 2-propanol 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 2-propanol 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 inuvolurans 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 2-propanol 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 2-propanol 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 2-propanol and/or 1-propanol reduce contaminants in the production process.

Additionally, the non-naturally occurring eukaryotic microorganism disclosed herein is capable of anaerobic growth and concomitant production of 2-propanol 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 2-propanol 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 2-propanol 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 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, wherein 1,2-propanediol and 1-propanol are produced via a dihydroxyacetone phosphate intermediate or a pyruvate intermediate. In some embodiments, 2-propanol 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 2-propanol 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 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 2-propanol 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 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 2-propanol 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 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.

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, 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, “2-propanol” is intended to mean isopropyl alcohol with a general formula CH₃CH₃CHOH (CAS number—67-63-0).

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

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 2-propanol and 1-propanol and/or 1,2-propanediol. Such enzymes may include any of those enzymes as set forth in FIGS. 1-4. For example, the microorganism may be modified to comprise one or more polynucleotides coding for enzymes that catalyze a conversion of dihydroxyacetone phosphate and/or pyruvate to 1,2-propanodiol or 1-propanol. Modified microorganisms may be referred to herein as non-naturally occuring microorganisms.

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

In some embodiments, the non-naturally microorganism may comprise one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to 2-propanol including, for example, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetyl-CoA to acetoacetyl-CoA, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA to acetoacetate, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetate to acetone, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetone to 2-propanol. 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 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 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 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 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 3.

A modified microorganism as provided herein may comprise:

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to cytosolic acetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetoacetyl-CoA to AcAcetate,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of AcAcetate to acetone,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetone to 2-propanol,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of dihydroxyacetone phosphate to methylglyoxal,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of methylglyoxal to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of methylglyoxal to hydroxyacetone,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of hydroxyacetone to 1,2-propanediol, and/or

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactaldehyde to 1,2-propanediol.

In some embodiments, the modified microorganism has a disruption in the one or more polynucleotides that code for enzymes that decarboxylate pyruvate and associated transcription factor (e.g., pyruvate decarboxylase 1, 2, 5, and 6). In some embodiments, the modified microorganism has a disruption in each polynucleotide that codes for enzymes that decarboxylate pyruvate and associated transcription factor (e.g., pyruvate decarboxylase 1, 2, 5, and 6). In some embodiments, the modified microorganism is capable of growth on a C6 sugar as a sole carbon source under anaerobic conditions. In some embodiments, the modified microorganism has a disruption in the one or more polynucleotides that code for enzymes that decarboxylate pyruvate and associated transcription factor (e.g., pyruvate decarboxylase 1, 2, 5, and 6) and is capable of growth on a C6 sugar as a sole carbon source under anaerobic conditions. In some embodiments, the modified microorganism has a disruption in each polynucleotide that codes for enzymes that decarboxylate pyruvate and associated transcription factor (e.g., pyruvate decarboxylase 1, 2, 5, and 6) and is capable of growth on a C6 sugar as a sole carbon source under anaerobic conditions.

A modified microorganism as provided herein may comprise:

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to lactate,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactate to lactoyl-CoA,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactoyl-CoA to lactaldehyde,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of lactaldehyde to 1,2-propanediol,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of pyruvate to acetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetyl-CoA to acetoacetyl-CoA,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetoacetyl-CoA to AcAcetate,

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of AcAcetate to acetone, and/or

one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of acetone to 2-propanol.

In some embodiments, the modified microorganism has a disruption in each of the one or more polynucleotides that code for enzymes that decarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5, and 6). In some embodiments, the modified microorganism is capable of growth on a C6 sugar as a sole carbon source under anaerobic conditions. In some embodiments, the modified microorganism has a disruption in each of the one or more polynucleotides that code for enzymes that decarboxylate pyruvate (e.g., pyruvate decarboxylase 1, 5, and 6) and is capable of growth on a C6 sugar as a sole carbon source under anaerobic conditions.