Production host selection for asymmetric styrene epoxidation: Escherichia coli vs. solvent-tolerant Pseudomonas

Abstract

Selection of the ideal microbe is crucial for whole-cell biotransformations, especially if the target reaction intensively interacts with host cell functions. Asymmetric styrene epoxidation is an example of a reaction which is strongly dependent on the host cell owing to its requirement for efficient cofactor regeneration and stable expression of the styrene monooxygenase genes styAB. On the other hand, styrene epoxidation affects the whole-cell biocatalyst, because it involves toxic substrate and products besides the burden of additional (recombinant) enzyme synthesis. With the aim to compare two fundamentally different strain engineering strategies, asymmetric styrene epoxidation by StyAB was investigated using the engineered wild-type strain Pseudomonas sp. strain VLB120ΔC, a styrene oxide isomerase (StyC) knockout strain able to accumulate (S)-styrene oxide, and recombinant E. coli JM101 carrying styAB on the plasmid pSPZ10. Their performance was analyzed during fed-batch cultivation in two-liquid phase biotransformations with respect to specific activity, volumetric productivity, product titer, tolerance of toxic substrate and products, by-product formation, and product yield on glucose. Thereby, Pseudomonas sp. strain VLB120ΔC proved its great potential by tolerating high styrene oxide concentrations and by the absence of by-product formation. The E. coli-based catalyst, however, showed higher specific activities and better yields on glucose. The results not only show the importance but also the complexity of host cell selection and engineering. Finding the optimal strain engineering strategy requires profound understanding of bioprocess and biocatalyst operation. In this respect, a possible negative influence of solvent tolerance on yield and activity is discussed.

Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

“…The high biocatalytic potential of P. taiwanensis VLB120⌬C for the specific epoxidation of styrene to (S)-styrene oxide has been shown both on a small scale with resting cells (42) and in two-liquid-phase biotransformations with growing cells (65). The fact that styrene serves on the one hand as the inducer for styAB expression and on the other hand as the substrate for the epoxidation reaction complicates the differentiation between induction-and biotransformation-related effects.…”

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

“…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).…”