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Fermentation of dihydroxyacetone by engineered Escherichia coli and Klebsiella variicola to products

Significance World-wide natural gas production in 2016 was 3.55 trillion cubic meters, and the natural gas flared is estimated to contribute about 350 million tons of CO2. The global warming potential of CH4 is several orders of magnitude higher than that of CO2. Upgrading CH4 to chemicals and liquid fuels converts low-cost natural gas to high-value products and traps it from release into atmosphere. Current chemical technology can produce dihydroxyacetone (DHA) from CH4 provided a microorganism can ferment this growth-inhibitory sugar. Here we report metabolically engineered microorganisms that ferment DHA to products. Combining the existing technology of chemical conversion of CH4 to DHA and the fermentation of this sugar is a strategy to transform inexpensive CH4 to chemicals and liquid fuels. Methane can be converted to triose dihydroxyacetone (DHA) by chemical processes with formaldehyde as an intermediate. Carbon dioxide, a by-product of various industries including ethanol/butanol biorefineries, can also be converted to formaldehyde and then to DHA. DHA, upon entry into a cell and phosphorylation to DHA-3-phosphate, enters the glycolytic pathway and can be fermented to any one of several products. However, DHA is inhibitory to microbes due to its chemical interaction with cellular components. Fermentation of DHA to d-lactate by Escherichia coli strain TG113 was inefficient, and growth was inhibited by 30 g⋅L−1 DHA. An ATP-dependent DHA kinase from Klebsiella oxytoca (pDC117d) permitted growth of strain TG113 in a medium with 30 g⋅L−1 DHA, and in a fed-batch fermentation the d-lactate titer of TG113(pDC117d) was 580 ± 21 mM at a yield of 0.92 g⋅g−1 DHA fermented. Klebsiella variicola strain LW225, with a higher glucose flux than E. coli, produced 811 ± 26 mM d-lactic acid at an average volumetric productivity of 2.0 g−1⋅L−1⋅h−1. Fermentation of DHA required a balance between transport of the triose and utilization by the microorganism. Using other engineered E. coli strains, we also fermented DHA to succinic acid and ethanol, demonstrating the potential of converting CH4 and CO2 to value-added chemicals and fuels by a combination of chemical/biological processes.