Homoethanol production from Glycerol and Gluconate using recombinant Klebsiella Oxytoca strains

Authors: TAO, WEIYI - University Of California; WANG, YI - University Of California; WALTERS, ERIC - University Of California; LIN, HUI - University Of California; LI, SHUANG - University Of California; HE, HUANG - University Of California; Kasuga, Takao; ZHILIANG, FAN - University Of California

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/30/2018
Publication Date: 2/20/2019
Citation: Tao, W., Wang, Y., Walters, E., Lin, H., Li, S., He, H., Kasuga, T., Zhiliang, F. 2019. Homoethanol production from glycerol and gluconate using recombinant Klebsiella oxytoca strains. Applied and Environmental Microbiology. 85:e02122-18. https://doi.org/10.1128/AEM.02122-18.
DOI: https://doi.org/10.1128/AEM.02122-18

Interpretive Summary: Lignocellulosic biomass is a renewable, low cost, and abundant source for the production of biofuels. Fungi associate with plants such as pathogens are capable of decomposing lignocellulosic materials to sugar, therefore have industrial potential for bioenergy production. We have been developing fungal mutants to produce gluconic acid (sugar acid) from cellulosic material, but cannot consume gluconic acid. On the other hand, plant endophytic bacterium such as Klebsiella oxytoca is capable of fermenting diverse plant metabolites including gluconic acid. Taking advantage of the genome sequence information we genetically engineered K. oxytoca to produce ethanol from gluconic acid.

Technical Abstract: Gluconate is a more oxidized substrate than glucose, while glycerol is a more reduced substrate than glucose. The homo-production of ethanol from glucose can be achieved, the conversion of the gluconate to ethanol is accompanied by the production of oxidized byproduct such as acetate, and the conversion of glycerol to ethanol is accompanied by the production of the reduced byproducts such as 1,3-propanediol. In this study, we seek to achieve homo-ethanol production by co-utilizing of gluconate and glycerol as substrates using mutant Klebsiella oxytoca mutant strains in batch and fed batch fermentation. Ethanol yield from gluconate and glycerol was about 62% when they are used as the carbon source alone. Co-utilization of both gluconate and glycerol and improve the yield of ethanol to about 78.66%. Byproducts such as acetate and 1,3-propanediol produced in the co-fermentation was substantially lower than when these two substrates were used alone. Decreasing of byproduct formation by deleting competing pathways including pta, frd, ldh, pflA and pduC improved the yield of ethanol to 89.29% in batch fermentation of glycerol and gluconate mixture, resulting the strain BW26. However, the glycerol utilization rate was substantially slower than that of gluconate in batch fermentation. Besides, substantial amount of glycerol remain un-utilized after gluconate was depleted. Continuous fed-batch fermentation was used to solve the utilization rate mis-match problem for gluconate and glycerol. Ethanol yield of 97.5% was achieved in the fed batch fermentation of these two substrates and glycerol was completely used at the end of the fermentation. Co-utilization of gluconate and glycerol can lead to higher ethanol yield and lower byproduct production. The deletion of competing pathways can direct more carbon flow toward ethanol production. The fed-batch fermentation strategy can successfully match the rate of glycerol utilization with that of gluconate and lead to high ethanol yield.