Production of glycolate from ethylene glycol and related microbial engineering

Abstract

Processes, systems and microorganisms are described herein for producing glycolate from ethylene glycol. The processes generally comprise supplying a fermentation broth into a fermentation vessel, wherein the fermentation broth comprises ethylene glycol and a microorganism having a functional metabolic pathway for utilizing ethylene glycol as a carbon source. In a growth phase, an oxygen-containing gas is injected into the fermentation broth to provide oxygen bio-availability conditions to promote cell growth of the microorganism and limit accumulation of glycolate in the fermentation broth. In a production phase, an oxygen-containing gas is injected into the fermentation broth to provide oxygen bio-availability conditions to promote production of glycolate from ethylene glycol by the microorganism and accumulation of the glycolate in the fermentation broth, to produce a glycolate enriched broth.

1 - 124 . (canceled) 
     
     
         125 . A microorganism engineered to overexpress, relative to a corresponding wild-type microorganism, a polynucleotide encoding a zinc-dependent alcohol dehydrogenase and a polynucleotide encoding a lactaldehyde dehydrogenase, wherein overexpression of the alcohol dehydrogenase and the lactaldehyde dehydrogenase confer on the microorganism an increased ability to produce glycolate from ethylene glycol, relative to a corresponding microorganism that overexpresses none or only one of the alcohol dehydrogenase and the lactaldehyde dehydrogenase. 
     
     
         126 . The microorganism of  claim 125 , wherein at least one of the alcohol dehydrogenase and the lactaldehyde dehydrogenase is heterologous with respect to the microorganism. 
     
     
         127 . The microorganism of  claim 125 , wherein both the alcohol dehydrogenase and the lactaldehyde dehydrogenase are heterologous with respect to the microorganism. 
     
     
         128 . The microorganism of  claim 125 , wherein the alcohol dehydrogenase and the lactaldehyde dehydrogenase are expressed from exogenous polynucleotides introduced into the microorganism. 
     
     
         129 . The microorganism of  claim 128 , wherein at least one of the exogenous polynucleotides is integrated into the genome of the microorganism. 
     
     
         130 . The microorganism of  claim 125 , wherein the overexpression of at least one of the alcohol dehydrogenase and the lactaldehyde dehydrogenase is under control of an exogenous or heterologous regulatory element enabling control of expression in response to oxygen levels, pH, nutrient concentration, or the presence of an inducer. 
     
     
         131 . The microorganism of  claim 125 , wherein the overexpression of both of the alcohol dehydrogenase and the lactaldehyde dehydrogenase is under control of an exogenous or heterologous regulatory element enabling control of expression in response to oxygen levels, pH, nutrient concentration, or the presence of an inducer. 
     
     
         132 . The microorganism of  claim 125 , wherein the microorganism expresses a functional glycolate oxidase. 
     
     
         133 . The microorganism of  claim 125 , wherein the zinc-dependent alcohol dehydrogenase is a variant of a  Gluconobacter oxydans  alcohol dehydrogenase, wherein the  Gluconobacter oxydans  alcohol dehydrogenase is encoded by the  Gluconobacter oxydans  621H GOX0313 gene. 
     
     
         134 . The microorganism of  claim 125 , wherein the lactaldehyde dehydrogenase is a variant of an  Escherichia coli  lactaldehyde dehydrogenase, wherein the Escherichia coli lactaldehyde dehydrogenase is encoded by the  Escherichia coli  aldA gene. 
     
     
         135 . The microorganism of  claim 125 , which is comprised in a fermentation broth comprising ethylene glycol as a carbon source for the microorganism. 
     
     
         136 . The microorganism of  claim 125 , which is comprised in a fermentation broth comprising glycolate as a fermentative product. 
     
     
         137 . The microorganism of  claim 136 , wherein the concentration of glycolate in the fermentation broth is greater than 1 g/L. 
     
     
         138 . The microorganism of  claim 136 , wherein the concentration of glycolate in the fermentation broth is greater than 5 g/L. 
     
     
         139 . The microorganism of  claim 136  wherein the concentration of glycolate in the fermentation broth is greater than 10 g/L. 
     
     
         140 . A microorganism comprising a first polynucleotide encoding: a variant of Gluconobacter oxydans alcohol dehydrogenase, wherein the  Gluconobacter oxydans  alcohol dehydrogenase is encoded by the  Gluconobacter oxydans  621H GOX0313 gene; and a variant of an  Escherichia coli  lactaldehyde dehydrogenase, wherein the  Escherichia coli  lactaldehyde dehydrogenase is encoded by the  Escherichia coli  aldA gene. 
     
     
         141 . The microorganism of  claim 140 , wherein the overexpression of at least one of the alcohol dehydrogenase and the lactaldehyde dehydrogenase is under control of an exogenous or heterologous regulatory element enabling control of expression in response to oxygen levels, pH, nutrient concentration, or the presence of an inducer. 
     
     
         142 . The microorganism of  claim 125 , wherein the microorganism expresses a functional glycolate oxidase. 
     
     
         143 . A method for improving a microorganism's tolerance to oxygen in the presence of ethylene glycol as a carbon source, the method comprising overexpressing, relative to a corresponding wild-type microorganism, a polynucleotide encoding an alcohol dehydrogenase that utilizes zinc as a cofactor; and a polynucleotide encoding a lactaldehyde dehydrogenase, wherein overexpression of the alcohol dehydrogenase and the lactaldehyde dehydrogenase confer on the microorganism an increased ability to convert ethylene glycol to glycolate, relative to a corresponding microorganism that overexpresses none or only one of the alcohol dehydrogenase and the lactaldehyde dehydrogenase. 
     
     
         144 . The method of  claim 143 , wherein the microorganism expresses a functional glycolate oxidase.