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Title: Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening, more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wildmore » type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less
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  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Publication Date:
Report Number(s):
Journal ID: ISSN 2211-9264; PII: S2211926416302715
Grant/Contract Number:
AC04-94AL85000; ACO4-94AL85000; 26336
Accepted Manuscript
Journal Name:
Algal Research
Additional Journal Information:
Journal Volume: 19; Journal Issue: C; Journal ID: ISSN 2211-9264
Research Org:
Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; directed evolution; isobutanol, 2/3-methyl butanol; algae protein bioconversion; cofactor engineering; algal biofuel
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1402509