Biosynthetic Pathway for the Cyanide-Free Production of Phenylacetonitrile in Escherichia coli by Utilizing Plant Cytochrome P450 79A2 and Bacterial Aldoxime Dehydratase

Abstract

The biosynthetic pathway for the production of phenylacetonitrile (PAN), which has a wide variety of uses in chemical and pharmaceutical industries, was constructed in Escherichia coli utilizing enzymes from the plant glucosinolate-biosynthetic and bacterial aldoxime-nitrile pathways. First, the single-step reaction to produce E,Z-phenylacetaldoxime (PAOx) from L-Phe was constructed in E. coli by introducing the genes encoding cytochrome P450 (CYP) 79A2 and CYP reductase from Arabidopsis thaliana, yielding the E,Z-PAOx-producing transformant. Second, this step was expanded to the production of PAN by further introducing the aldoxime dehydratase (Oxd) gene from Bacillus sp. strain OxB-1, yielding the PAN-producing transformant. The E,Z-PAOx-producing transformant also produced phenethyl alcohol and PAN as by-products, which were suggested to be the metabolites of E,Z-PAOx produced by E. coli enzymes, while the PAN-producing transformant accumulated only PAN in the culture broth, which suggested that the CYP79A2 reaction (the conversion of L-Phe to E,Z-PAOx) was a potential bottleneck in the PAN production pathway. Expression of active CYP79A2 and concentration of biomass were improved by the combination of the autoinduction method, coexpression of groE, encoding the heat shock protein GroEL/GroES, N-terminal truncation of CYP79A2, and optimization of the culture conditions, yielding a >60-fold concentration of E,Z-PAOx (up to 2.9 mM). The concentration of PAN was 4.9 mM under the optimized conditions. These achievements show the potential of this bioprocess to produce nitriles and nitrile derivatives in the absence of toxic chemicals.

VOC Profiles of Saliva in Assessment of Halitosis and Submandibular Abscesses Using HS-SPME-GC/MS Technique

“…N -furfurylpyrrole is found in various foods, such as coffee, chocolate, popcorn, and roasted chicken [29]. Benzyl nitrile (also known as phenylacetonitrile) can be biochemically synthesized by bacterial aldoxime-nitrile pathway, reported in Escherichia coli [30] species. Increased number of hydrocarbons can be due to microbial production since bacteria are the main cause of abscesses in mouth.…”

Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli

“…Generally, P450 expression is difficult in E. coli . Because some P450 enzymes are successfully expressed in E. coli only after the deletion of their N-terminus 23 24 25 26 , N-terminal-deleted STORR (STORRNcut) was constructed in addition to full-length STORR. These STORRs were expressed with ATR2 and cultured in medium containing ( S )-reticuline, which was biosynthesized from glycerol as described previously 15 .…”

Modification of N-Terminal Amino Acids of Fungal Benzoate Hydroxylase (CYP53A15) for the Production of p-Hydroxybenzoate and Optimization of Bioproduction Conditions in Escherichia coli

“…3), suggesting that the optimum conditions for our CYP53A15 expression system are different from those of systems reported previously. In the case of bioconversion from L-phenylalanine to phenylacetonitrile in E. coli cells coexpressing plant P450 CYP79A2 and aldoxime dehydratase, culture at low temperature (16°C and 20°C) results in higher phenylacetonitrile production than that at high temperature (26°C, 30°C, and 37°C) [17]. Heterologous production of the P450 enzyme (P450SMO) from Rhodococcus sp.…”