Multi-enzyme cascade reaction catalyzed γ-butyrolactone to 1,4-butanediol
1,4-Butanediol is an important chemical intermediate included on the high valent industrial chemicals list(HVICL)of NICNAS.With the increasing demand for 1,4-butanediol in the global market,biosynthesis of 1,4-butanediol has attracted more attention in these years.The present study employed y-butyrolactone as a substrate to synthesize 1,4-butanediol based on a multi-enzyme cascade process.In this process,γ-butyrolactone is hydrolyzed into 4-hydroxybutyrate by a caprolactone hydrolase(ChnC);then 4-hydroxybutyrate is converted to 4-hydroxybutyraldehyde with a carboxylic acid reductase(Car);and subsequently,4-hydroxybutyraldehyde is reduced to 1,4-butanediol via alcohol dehydrogenase(YqhD).Meanwhile,a NADH regeneration system involving formate dehydrogenase(FDH)is introduced to enhance 1,4-butanediol production.Finally,the effects of substrate addition concentration,catalytic temperature,pH,and proportion of multi-enzyme dosage on 1,4-butanediol biosynthesis were observed.Based on the results of parameters optimization,the optimum reaction conditions are as follows:the ratio of the three enzyme dosages is 1∶3∶2,5 g/L y-butyrolactone,performed at 30 ℃,pH 7.0,the titer of 1,4-butanediol reached 2.41 g/L with a molar yield of 46.1％,which was improved by 375.46％and 374.79％,respectively,compared under the unoptimized conditions.Accordingly,the fed-batch reaction system showed a remarkable 1,4-butanediol-producing potency.The maximum1,4-butanediol reached 6.31 g/L with a molar yield of 67.3％.The results suggest that biosynthesis of 1,4-butanediol based on this multi-enzyme cascade process developed in this study is feasible.Meanwhile,the strategy is expected to provide an alternative for the enzyme-catalyzed synthesis of 1,4-butanediol and 1,4-butanediol-derived high-value-added products.
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