Engineered P450 BM3 and cpADH5 coupled cascade reaction for β-oxo fatty acid methyl ester production in whole cells

• Published in: Enzyme and microbial technology 2020-08, Vol.138, p.109555-109555, Article 109555
• Main Authors: Ensari, Yunus, de Almeida Santos, Gustavo, Ruff, Anna Joëlle, Schwaneberg, Ulrich
• Format: Article
• Language: English
• Subjects: Alcohol dehydrogenase; Alcohol Dehydrogenase - genetics; Alcohol Dehydrogenase - metabolism; Bacillus megaterium - enzymology; Bacillus megaterium - genetics; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Biocatalysis; Candida parapsilosis - enzymology; Candida parapsilosis - genetics; Caproates - metabolism; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; directed evolution; Directed Molecular Evolution; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Esters - chemistry; Esters - metabolism; fatty acid methyl ester; Fatty Acids - biosynthesis; Fatty Acids - chemistry; Hydroxylation; Operon; P450 BM3; Substrate Specificity; whole cell
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2020.109555

Generated whole cell E.coli catalyst co-expressing P450 BM3 variant YE_M1_2 with previously engineered cpADH5 W286A and FhuA Δ1-160 variant, able to synthesize hydroxy- and keto-fatty acid methyl esters from fatty acid methyl esters as substrate. Substrate bioconversion is conducted through P450 BM3 and cpADH5 coupled cascade reaction while, substrate uptake into whole cell catalyst is boosted by passive diffusion channel protein FhuA Δ1-160. [Display omitted] •Directed evolution of P450 BM3 for improved hydroxylation of methyl hexanoate.•Whole cell catalyst development for hydroxy- and keto-FAMEs production.•Solving the whole cell catalyst substrate uptake limitation and unbalanced co-factor regeneration. Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.