High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate

Bae, Sang‐Jeong; Kim, Sujin; Park, Hyun June; Kim, Joonwon; Jin, Hyunbin; Kim, Byung-gee; Hahn, Jinyong

doi:10.1016/j.ymben.2021.04.001

Cited by 20 publications

(9 citation statements)

References 40 publications

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“…We next deleted the ORA1 gene, of which the product reduces (S)-α-acetolactate to 2,3-dimethylglycerate and thus competes with AlsD-mediated (R)-acetoin production [ 47 ]. This strongly reduced with 80% the 2,3-dimethylglycerate production of the resulting strain HGS48 in medium with 100 g/L glucose (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We have used 2 alternative approaches to address the redox imbalance: expression of NoxE encoding NADH oxidase as previously performed [ 35 , 37 , 38 , 47 , 51 ], and a new approach consisting of the combined overexpression of NDE1 , encoding mitochondrial external NADH dehydrogenase [ 42 ], and AOX1 , encoding a heterologous alternative oxidase [ 43 , 52 , 53 ]. The NoxE expression in HGS37 resulted in a similar 2,3-BDO productivity (1.58 g/L.h) compared to expression in HGS50 of the combination pathway with the alternative oxidase (1.57 g/L.h), but not when expressed per gCDW: 0.11 g/L.h per gCDW for the former versus 0.08 g/L.h per gCDW for the latter.…”

Section: Discussionmentioning

confidence: 99%

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

2022

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

2022

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“…[10,23]. Currently, researchers have obtained high optical pure AC producing strains by strain screening [16], recombinant strain construction [17,18], and gene knockout [19] strategies, and the purity of optical isomers in the fermentation broth reaches more than 98%. However, compared with research on the metabolic mechanisms of bacterial strains and the optimization of fermentation processes for AC production, the downstream separation of AC from fermentation broth is the critical step in obtaining AC products.…”

Section: Discussionmentioning

confidence: 99%

“…In general, bacteria contain more than one type of BDH [14,15], thereby producing both AC enantiomers. To obtain optically pure AC (i.e., only one AC enantiomer), researchers have used various approaches, including the screening of natural bacterial strains for the synthesis of AC with high optical purity [16], engineering strains that possess BDH with strict enantioselectivity [17,18], and deletion of bdhA gene in AC-producing bacteria, to block the conversion between AC and BD [19]. Several studies have demonstrated the effectiveness of these strategies and the optical purity of AC in the product can be increased to more than 98% [16,19].…”

Section: Introductionmentioning

confidence: 99%

Conversion of Enantiomers during the Separation of Acetoin from Fermentation Broth

Zhang

Yao

et al. 2022

Fermentation

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Acetoin (AC) is an important platform compound with two enantiomers (R)-AC and (S)-AC. Due to its unique spatial structure, optically pure AC has particularly high application in asymmetric synthesis. Highly optically pure AC could be produced from glucose using biofermentation technology. In this paper, we have observed that the recovered AC product from the fermentation broth containing (R)-AC was a racemic mixture. The changes of the enantiomeric excess (e.e.) of (R)-AC enantiomers in the feed solution during the recovery process were then investigated, confirming that the racemization occurs during solvent distillation. Further studies showed that high temperature is the main factor affecting the conversion of the two enantiomers, while low temperature significantly prevents this conversion reaction. Therefore, we optimized the solvent recovery process and used vacuum distillation to reduce the distillation process temperature, which effectively prevented the racemization: obtains AC products with more than 98% purity and successfully maintained the proportion of (R)-AC above 96%. To our knowledge, this is the first report on the factors affecting the enantiomeric purity in the downstream extraction process of AC production by fermentation.

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“…The compound acetoin (3-hydroxy-2-butanone, AC) is an indispensable flavor enhancer widely applied in foods, cosmetics and chemical synthesis ( Li et al, 2018 ; Xiao et al, 2018 ) and also plays a pivotal role in TMP synthesis as a precursor ( Demain et al, 1967 ; Xiao et al, 2014 ; Peng et al, 2020a ; Zhong et al, 2020a ). To improve the AC accumulation in microbial fermentation, various strategies have been employed, including screening of efficient natural producers of acetoin ( Xiao et al, 2006 ; Zhu et al, 2010 ; Zhang et al, 2011 ; Yin et al, 2018 ; Zhong et al, 2020b ), metabolic engineering ( Vivijs et al, 2014 ; Xu et al, 2015 ; Jang et al, 2017 ; Li et al, 2018 ; Bae et al, 2021 ) and fermentation optimization ( Xiao et al, 2007 ; Zhu and Xu 2010a ; Zhang et al, 2012 ; Yuan et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

A Green Route for High-Yield Production of Tetramethylpyrazine From Non-Food Raw Materials

et al. 2022

Front. Bioeng. Biotechnol.

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2,3,5,6-Tetramethylpyrazine (TMP) is an active pharmaceutical ingredient originally isolated from Ligusticum wallichii for curing cardiovascular and cerebrovascular diseases and is widely used as a popular flavoring additive in the food industry. Hence, there is a great interest in developing new strategies to produce this high-value compound in an ecological and economical way. Herein, a cost-competitive combinational approach was proposed to accomplish green and high-efficiency production of TMP. First, microbial cell factories were constructed to produce acetoin (3-hydroxy-2-butanone, AC), an endogenous precursor of TMP, by introducing a biosynthesis pathway coupled with an intracellular NAD+ regeneration system to the wild-type Escherichia coli. To further improve the production of (R)-AC, the metabolic pathways of by-products were impaired or blocked stepwise by gene manipulation, resulting in 40.84 g/L (R)-AC with a high optical purity of 99.42% in shake flasks. Thereafter, an optimal strain designated GXASR11 was used to convert the hydrolysates of inexpensive feedstocks into (R)-AC and achieved a titer of 86.04 g/L within 48 h in a 5-L fermenter under optimized fermentation conditions. To the best of our knowledge, this is the highest (R)-AC production with high optical purity (≥98%) produced from non-food raw materials using recombinant E. coli. The supernatant of fermentation broth was mixed with diammonium phosphate (DAP) to make a total volume of 20 ml and transferred to a high-pressure microreactor. Finally, 56.72 g/L TMP was obtained in 3 h via the condensation reaction with a high conversion rate (85.30%) under optimal reaction conditions. These results demonstrated a green and sustainable approach to efficiently produce high-valued TMP, which realized value addition of low-cost renewables.

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High-yield production of (R)-acetoin in Saccharomyces cerevisiae by deleting genes for NAD(P)H-dependent ketone reductases producing meso-2,3-butanediol and 2,3-dimethylglycerate

Cited by 20 publications

References 40 publications

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

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

Conversion of Enantiomers during the Separation of Acetoin from Fermentation Broth

A Green Route for High-Yield Production of Tetramethylpyrazine From Non-Food Raw Materials

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