Hydrolase BioH knockout in E. coli enables efficient fatty acid methyl ester bioprocessing
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Fatty acid methyl esters (FAMEs) originating from plant oils are most interesting renewable feedstocks for biofuels and bio-based materials. FAMEs can also be produced and/or functionalized by engineered microbes to give access to, e.g., polymer building blocks. Yet, they are often subject to hydrolysis yielding free fatty acids, which typically are degraded by microbes. We identified BioH as the key enzyme responsible for the hydrolysis of medium-chain length FAME derivatives in different E. coli K-12 strains. E. coli ΔbioH strains showed up to 22-fold reduced FAME hydrolysis rates in comparison with respective wild-type strains. Knockout strains showed, beside the expected biotin auxotrophy, unchanged growth behavior and biocatalytic activity. Thus, high specific rates (~80 U g CDW −1 ) for terminal FAME oxyfunctionalization catalyzed by a recombinant alkane monooxygenase could be combined with reduced hydrolysis. Biotransformations in process-relevant two-liquid phase systems profited from reduced fatty acid accumulation and/or reduced substrate loss via free fatty acid metabolization. The BioH knockout strategy was beneficial in all tested strains, although its effect was found to differ according to specific strain properties, such as FAME hydrolysis and FFA degradation activities. BioH or functional analogs can be found in virtually all microorganisms, making bioH deletion a broadly applicable strategy for efficient microbial bioprocessing involving FAMEs.
KeywordsBioH Fatty acid methyl ester hydrolysis Whole-cell biocatalysis Industrial biotechnology Metabolic engineering
The results contribute to the programme topic Solar Fuels funded by the Helmholtz Research Programme, co-financed by the German Federal Ministry of Education and Research (BMBF, Grant Number 0316044A). The authors are grateful for financial support of the Centre for Biocatalysis (MiKat) at the Helmholtz Centre for Environmental Research by European Regional Development Funds (EFRE—Europe funds Saxony) and the Helmholtz Association. We thank Britta Dettweiler (former Laboratory of Chemical Biotechnology, TU Dortmund University) for experimental support. MK, AS, and BB are thankful for using infrastructure of TU Dortmund University at the former Laboratory of Chemical Biotechnology.
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