Engineered P450 BM3 and cpADH5 coupled cascade reaction for β-oxo fatty acid methyl ester production in whole cells
| Autor: | Anna Joëlle Ruff, Gustavo de Almeida Santos, Ulrich Schwaneberg, Yunus Ensari |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
0301 basic medicine Candida parapsilosis Ketone Bioengineering Hydroxylation Valerate 01 natural sciences Applied Microbiology and Biotechnology Biochemistry Substrate Specificity Catalysis 03 medical and health sciences chemistry.chemical_compound Cytochrome P-450 Enzyme System Cascade reaction 010608 biotechnology Operon Protein purification Escherichia coli Organic chemistry Caproates Fatty acid methyl ester chemistry.chemical_classification Escherichia coli Proteins Fatty Acids Alcohol Dehydrogenase Fatty acid Esters 030104 developmental biology chemistry Biocatalysis Bacillus megaterium Directed Molecular Evolution Bacterial Outer Membrane Proteins Biotechnology |
| Zdroj: | Enzyme and Microbial Technology. 138:109555 |
| ISSN: | 0141-0229 |
| DOI: | 10.1016/j.enzmictec.2020.109555 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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