Development of a whole-cell biocatalyst co-expressing P450 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane

Abstract

Oxygenases-based Escherichia coli whole-cell biocatalyst can be applied for catalysis of various commercially interesting reactions that are difficult to achieve with traditional chemical catalysts. However, substrates and products of interest are often toxic to E. coli, causing a disruption of cell membrane. Therefore, organic solvent-tolerant bacteria became an important tool for heterologous expression of such oxygenases. In this study, the organic solvent-tolerant Bacillus subtilis 3C5N was developed as a whole-cell biocatalyst for epoxidation of a toxic terminal alkene, 1-hexene. Comparing to other hosts tested, high level of tolerance towards 1-hexene and a moderately hydrophobic cell surface of B. subtilis 3C5N were suggested to contribute to its higher 1,2-epoxyhexane production. A systematic optimization of reaction conditions such as biocatalyst and substrate concentration resulted in a 3.3-fold increase in the specific rate. Co-expression of glucose dehydrogenase could partly restored NADPH-regenerating ability of the biocatalyst (up to 38 % of the wild type), resulting in approximately 53 % increase in specific rate representing approximately 22-fold increase in product concentration comparing to that obtained prior to an optimization.

Microbial Production of Aliphatic ( S )-Epoxyalkanes by Using Rhodococcus sp. Strain ST-10 Styrene Monooxygenase Expressed in Organic-Solvent-Tolerant Kocuria rhizophila DC2201

“…No product was observed for Rhodococcus (pK-ST10styAB) and Pseudomonas (pNUK-ST10styAB) biocatalysts, despite the StyA and StyB activities in these transformant cells. Siriphongphaew et al reported that Bacillus subtilis 3C5N expressing P450 BM3 showed higher production of 1,2-epoxyhexane from 1-hexene than the E. coli biocatalyst because of its superior tolerance to toxic compounds (28). These facts strongly suggest that epoxyalkane production depends on factors other than SMO activity in the cells to accelerate the reaction.…”

“…In our previous study, the E. coli cell biocatalyst was inactivated by the decrease in SMO activity during the reaction because of the organic solvent and toxic substrates and products (25). Using host microorganisms that tolerate organic solvents is a good strategy to overcome this problem (27)(28)(29). We constructed five different biocatalysts using organic solvent-tolerant host microorganisms and evaluated their RhSMO activity for producing epoxides.…”

“…As a result, further improvement of this process was difficult, especially for straightchain alkene substrates that require a long reaction time. To overcome these problems, several organic solvent-tolerant microorganisms have been studied recently as heterologous gene expression hosts for producing enantiopure epoxides (27)(28)(29). In particular, Pseudomonas taiwanensis VLB120, from which SMOcoding genes were isolated, is well known as a solvent-tolerant microorganism.…”

“…However, the E. coli biocatalyst was easily inactivated by organic solvents and toxic substrates and was not suitable for prolonged reaction times. Using organic solvent-tolerant microorganisms as heterologous expression hosts is an effective strategy for overcoming this problem, and it has been studied for producing several epoxides (27)(28)(29).In this report, we describe the development of a biocatalytic system for the production of enantiopure aliphatic (S)-epoxyalkanes using RhSMO, which was overproduced in Kocuria rhizophila DC2201 (NBRC 103217). K. rhizophila DC2201 is a coccoid, Gram-positive bacterium isolated from the rhizosphere.…”

“…However, the E. coli biocatalyst was easily inactivated by organic solvents and toxic substrates and was not suitable for prolonged reaction times. Using organic solvent-tolerant microorganisms as heterologous expression hosts is an effective strategy for overcoming this problem, and it has been studied for producing several epoxides (27)(28)(29).…”