Applications of Technology:
- High production volume of biosynthesis products such as nylons and fuels while consuming less sugar feedstock
Advantages:
- Dramatically increased efficiency in use of fuel due to no CO2 emission during glycolysis.
- 50% increase in maximum theoretical yield.
- Reduced gas buildup from biosynthesis tasks.
- This strain does not require acetate supplementation of the growth media.
- Grows on glucose and/or sucrose as the sole carbon source, which are widely available and cost effective media sources.
- Does not require plasmids for strain evolution, antibiotics, or inducer chemicals.
Background:
In traditional glycolysis, sugars are partially oxidized and split into pyruvate, which are then decarboxylated to produce acetyl-coenzyme A (CoA) for various biosynthetic purposes. This process, however, leads to one carbon equivalent lost as CO2 and therefore limits the theoretical yield to only two moles of C2 metabolites per mole of sugar.
Non-oxidative glycolysis (NOG), however, allows for complete carbon conservation in sugar catabolism to acetyl-CoA. Theoretically, this means 100% carbon yield converted to desirable fuels and chemicals. Current NOG pathways include inserting the fxpk gene from Bifidobacterium adolescentis into the E. coli genome. While this strategy has led to increased yield in comparison with the parent strain, significant efforts such as inserting additional promoters into the NOG gene of interest, and adding acetate to the growth media limit its applications. For example, addition of acetate may have damaging outcomes for industrial scale fermentation and product titers.
Therefore, there is a need for an E. coli strain that can efficiently and effectively use a non-oxidative glycolysis pathway without cofactor supplementation.
Technology Overview:
Researchers at Berkeley Lab and the Joint BioEnergy Institute have developed a strain of E. coli that achieves near theoretical yield of carbon conservation under non-oxidative glycolysis. During laboratory evolution of a previously developed E. coli strain, researchers at Berkeley Lab have characterized an enzyme which enables non-oxidative glycolysis with mostly genes native to E. coli. Metabolic labeling experiments were performed using cells grown anaerobically at an OD600 = 25 in the presence of 1% glucose-3,4-13C for 24 hours. Double labeled acetate formed in the NOG strain but not in the wild-type strain. The novel E. coli encodes an NOG pathway from a phosphoketolase (Xpk) enzyme in Bifidobacterium adolescentis, as well as additional genes that enable uptake and utilization of sucrose. Further, the researchers drove evolution of the strain to use media free of acetate.
Notably, the strain is able to bypass pyruvate dehydrogenase during glycolysis by converting a derivative of fructose (fructose 6-phosphate) to acetyl phosphate and subsequently acetyl coA, which resulted in no CO2 formation.
The strain has been validated in laboratory scale reactors and the researchers are looking for industry partners to validate the strain on a commercial scale. Next steps will include improving the strain’s resiliency to osmotic shock, and metabolic analysis and flux profiling.
Development Stage:
Proof of concept
Principal Investigators:
- Justin Panich
- Steven Singer
Status:
Patent Pending
Opportunities: Available for licensing or collaborative research.
SEE THESE OTHER BERKELEY LAB TECHNOLOGIES IN THIS FIELD:
Expression Platform for Soluble Hydrogenase Production via Escherichia coli 2022-146
Feedstocks With Reduced Acetylation for Higher Product Yields and Improved Properties EIB-2658