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Development of an industrial yeast strain for efficient production of 2,3-butanediol.

ABSTRACT: As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol (2,3-BDO) has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high concentration of glycerol caused by the excess NADH produced in the pathway from glucose to 2,3-BDO was eliminated by overexpression of NoxE and also in a novel way by combined overexpression of NDE1, encoding mitochondrial external NADH dehydrogenase, and AOX1, encoding a heterologous alternative oxidase expressed inside the mitochondria. This was combined with strong downregulation of GPD1 and deletion of GPD2, to minimize glycerol production while maintaining osmotolerance. The HGS50 strain produced a 2,3-BDO titer of 121.04 g/L from 250 g/L glucose, the highest ever reported in batch fermentation, with a productivity of 1.57 g/L.h (0.08 g/L.h per gCDW) and a yield of 0.48 g/g glucose or with 96% the closest to the maximum theoretical yield ever reported. Expression of Lactococcus lactis NoxE, encoding a water-forming NADH oxidase, combined with similar genetic modifications, as well as expression of Candida albicans STL1, also minimized glycerol production while maintaining high osmotolerance. The HGS37 strain produced 130.64 g/L 2,3-BDO from 280 g/L glucose, with productivity of 1.58 g/L.h (0.11 g/L.h per gCDW). Both strains reach combined performance criteria adequate for industrial implementation.

SUBMITTER: Huo G

PROVIDER: S-EPMC9520875 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Development of an industrial yeast strain for efficient production of 2,3-butanediol.

Huo Guangxin G Foulquié-Moreno María R MR Thevelein Johan M JM

Microbial cell factories 20220929 1

As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol (2,3-BDO) has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high c ...[more]

PMID: 36175998

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Project description:Background2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol.ResultsEndogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C2-compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae. A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h.ConclusionsThe microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.

Project description:Background2,3-Butanediol (2,3-BD), a valuable compound used for chemicals, cosmetics, pesticides and pharmaceuticals, has been produced by various microbes. However, no high-temperature fermentation of the compound at high productivity has been reported.MethodsThermotolerant xylose-utilizing microbes were isolated from 6 different districts in Laos and screened for a low accumulation of xylitol in a xylose medium at 37 ˚C. One isolate was found to produce 2,3-BD and identified by 16S rDNA sequencing. The 2,3-BD fermentation capacity was investigated at different temperatures using xylose and glucose as carbon sources, and the fermentation parameters were determined by a high-performance liquid chromatography system.ResultsBy screening for a low accumulation of xylitol in a xylose medium, one isolate that accumulated almost no xylitol was obtained. Further analyses revealed that the isolate is Cronobacter sakazakii and that it has the ability to produce 2,3-BD at high temperatures. When xylose and glucose were used, this strain, named C. sakazakii OX-25, accumulated 2,3-BD in a short period before the complete consumption of these sugars and then appeared to convert 2,3-BD to acetoin. The optimum temperature of the 2,3-BD fermentation was 42 ˚C to 45 ˚C, and the maximum yield of 2,3-BD was 0.3 g/g at 12 h in 20 g/l xylose medium and 0.4 g/g at 6 h in 20 g/l glucose medium at 42 ˚C. The 2,3-BD productivity of the strain was higher than the 2,3-BD productivities of other non-genetically engineered microorganisms reported previously, and the highest productivity was 0.6 g/l·h and 1.2 g/l·h for xylose and glucose, respectively.ConclusionsAmong thermotolerant microbes isolated in Laos, we discovered a strain, C. sakazakii OX-25, that can convert xylose and glucose to 2,3-BD with high efficiency and high productivity at high temperatures, suggesting that C. sakazakii OX-25 has the potential for industrial application to produce 2,3-BD as an important platform chemical.

Dataset Information

Development of an industrial yeast strain for efficient production of 2,3-butanediol.

Publications

Development of an industrial yeast strain for efficient production of 2,3-butanediol.

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