Overcoming NADPH product inhibition improves D-sorbitol conversion to L-sorbose

Tae-Su Kim,Jung-Kul Lee,Hui Gao,In-Won Kim,Karthikeyan Muthusamy,Vipin C Kalia,Jinglin Li,Jae Kyung Sohng

doi:10.1038/s41598-018-37401-0

Tae-Su Kim, Jung-Kul Lee + Show 6 more

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https://doi.org/10.1038/s41598-018-37401-0

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Abstract

Gluconobacter oxydans sorbitol dehydrogenase (GoSLDH) exhibits a higher catalytic efficiency than other l-sorbose producing enzymes. During the reaction catalysed by GoSLDH, NADP+ is reduced to NADPH and d-sorbitol is oxidized to l-sorbose. However, GoSLDH activity is inhibited by the NADPH (Ki = 100 μM) formed during the enzymatic reaction. Therefore, Escherichia coligosldh-lrenox producing both GoSLDH for d-sorbitol oxidation and LreNOX (NAD(P)H oxidase from Lactobacillus reuteri) for NADP+ regeneration was generated and used for l-sorbose production. Whole cell biocatalysts with the LreNOX cofactor recycling system showed a high conversion rate (92%) of d-sorbitol to l-sorbose in the presence of low concentration of NADP+ (0.5 mM). By alleviating NADPH accumulation during the catalytic reactions, E. coligosldh-lrenox exhibited 23-fold higher conversion rate of d-sorbitol than E. coligosldh. l-Sorbose production by E. coligosldh-lrenox reached 4.1 g/L after 40 min, which was 20.5-fold higher than that of E. coligosldh. We also constructed G. oxydansgosldh and G. oxydansgosldh-lrenox strains, and they exhibited 1.2- and 2.9-fold higher conversion rates than the wild-type G. oxydans KCTC 1091. The results indicate that overcoming NADPH product inhibition using LreNOX improves chemical production in NADP+-dependent enzymatic reactions.

Highlights

Gluconobacter oxydans sorbitol dehydrogenase (GoSLDH) exhibits a higher catalytic efficiency than other l-sorbose producing enzymes
We developed a co-expression system in which GoSLDH encoded by the G. oxydans G624 gosldh was used as the l-sorbose producing enzyme and LreNOX was used as the cofactor-regenerating enzyme
During whole cell biocatalysis by E. coligosldh in the presence of 0.5 mM NADP+, the NADPH concentration reached 147 μM, showing a conversion rate of d-sorbitol to l-sorbose of only 2.6% (Fig. 1a,b)

Summary

Introduction

Gluconobacter oxydans sorbitol dehydrogenase (GoSLDH) exhibits a higher catalytic efficiency than other l-sorbose producing enzymes. During the reaction catalysed by GoSLDH, NADP+ is reduced to NADPH and d-sorbitol is oxidized to l-sorbose. Escherichia coligosldh-lrenox producing both GoSLDH for d-sorbitol oxidation and LreNOX (NAD(P)H oxidase from Lactobacillus reuteri) for NADP+ regeneration was generated and used for l-sorbose production. Whole cell biocatalysts with the LreNOX cofactor recycling system showed a high conversion rate (92%) of d-sorbitol to l-sorbose in the presence of low concentration of NADP+ (0.5 mM). By alleviating NADPH accumulation during the catalytic reactions, E. coligosldh-lrenox exhibited 23-fold higher conversion rate of d-sorbitol than E. coligosldh. NAD(P)H product inhibition must be overcome for rare sugar production using polyol dehydrogenases. During the reaction catalysed by GoSLDH, d-sorbitol is oxidized to l-sorbose, which involves the reduction of NADP+ to NADPH. Simultaneous overexpression of the target enzymes and cofactor regeneration biocatalyst has been implemented in many asymmetric reduction systems[22,23]

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