Abstract
An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.
Highlights
As promising bulk chemicals, 2,3-butanediol and acetoin (Figure 1) are proposed to have extensive applications in a variety of fields such as the fuel, chemical and food industries [1]
A two-step genetic switching circuit was constructed that consists of two plasmids: plasmid pSWNB (Figure 2A) that expresses T7 RNA polymerase that amplifies the transcriptional strength of promoter acetoactate decarboxylase (slaA), and plasmid pET-X that encodes the proteins of interest
The results showed that S. marcescens MG1ABC could convert diacetyl to 2,3-butanediol
Summary
2,3-butanediol and acetoin (Figure 1) are proposed to have extensive applications in a variety of fields such as the fuel, chemical and food industries [1]. These chemicals can be produced by chemical synthetic, fermentative, enzymatic or biocatalytic technologies [2,3]. Because of difficulties in chemical synthesis, the large-scale production of 2,3-butanediol and acetoin is limited using this technology. In most cases, 2,3butanediol and acetoin are co-produced, necessitating a downstream purification step. Enzymatic or biocatalytic technologies have been proposed as appropriate alternative production methods. As the conversion between 2,3-butanediol and acetoin is reversible and coupled with NADH/NAD+ transformation, the production of 2,3-butanediol or acetoin could be improved by regulating the level of intracellular cofactors [4,5]
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