Production of 10‐Hydroxy‐2‐decenoic Acid from Decanoic Acid via Whole‐Cell Catalysis in Engineered Escherichia coli

Abstract

10‐Hydroxy‐2‐decenoic acid (10‐HDA) is a terminal hydroxylated medium‐chain α,β‐unsaturated carboxylic acid that performs various unique physiological activities and has a wide market value. Therefore, development of an environmentally friendly, safe, and high‐efficiency route to synthesize 10‐HDA is required. Here, the β‐oxidation pathway of Escherichia coli was modified and a P450 terminal hydroxylase (CYP153A33‐CPRBM3) was rationally designed to synthesize 10‐HDA using decanoic acid as a substrate via two‐step whole‐cell catalysis. Different homologues of FadDs, FadEs, and YdiIs were analyzed in the first step of the conversion of decanoic acid to trans‐ ‐2‐ decenoic acid. In the second step, CYP153A33 (M228L)‐CPRBM3 efficiently catalyzed the conversion of trans‐ ‐2‐ decenoic acid to 10‐HDA. Finally, 217 mg L−1 10‐HDA was obtained with 500 mg L−1 decanoic acid. This study provides a strategy for biosynthesis of 10‐HDA and other α, β‐unsaturated carboxylic acid derivatives from specific fatty acids.

Efficient Biosynthesis of 10-Hydroxy-2-decenoic Acid Using a NAD(P)H Regeneration P450 System and Whole-Cell Catalytic Biosynthesis

“…To identify the endogenous transporter that can accelerate trans -2-decenoic acid production, we constructed engineered strains of E. coli BL21(DE3) ( ΔfadBJR ), consisting of two different plasmids. The first plasmid, pCDFDuet-1 - Pp fadE - Ma MACS , was derived from our previous research, 9 and the second plasmid was used to coexpress the thioesterase gene Ct ydiI and the transporter gene ( Figure S1 ). Here, we investigated two types of transporters.…”

“…The plasmids pCDFDuet-1- Pp fadE - Ma MACS , pET-28a-sumo- Ct ydiI , and pET-21b- CYP153A33/M228L-CPR BM3 were all constructed as mentioned previously. 9 The first two plasmids were used for the synthesis of trans -2-decenoic acid. The third plasmid was used for 10-HDA from trans -2-decenoic acid.…”

“…The intracellular and extracellular production of fatty acids was analyzed using the fatty acid extraction and derivatization detection method of Li et al via gas chromatography-mass spectrometry (GC-MS, Agilent). 9 …”

“… 8 Recently, the biosynthesis of 10-HDA was reported using decanoic acid as a substrate via two-step whole-cell catalysis. 9 In the first step, decanoic acid was converted to trans -2-decenoic acid through the modified β-oxidation pathway in Escherichia coli . In the second step, CYP153A33/M228L-CPR BM3 efficiently catalyzed the conversion of trans -2-decenoic acid to 10-HDA.…”

“…We obtained a new type of P450 enzyme (CYP153A33/M228L-CPR BM3 ) through molecular rational design during the initial study, which has high catalytic activity on trans -2-decenoic acid. 9 CYP153A33/M228L-CPR BM3 is a self-sufficient P450 enzyme composed of CYP153A33/M228L and CPR BM3 , the latter being the reducing domain of CYP102A from Bacillus megaterium. The CYP102 family, CYP116 family, and CYP505 family are all self-sufficient P450 enzymes in nature, which encode their heme domain and reductase protein in a single polypeptide.…”