Semi-rational evolution of pyruvate carboxylase from Rhizopus oryzae for elevated fumaric acid synthesis in Saccharomyces cerevisiae

Xu, Guoqiang; Shi, Xiangliu; Gao, Yuhao; Wang, Jiyue; Cheng, Hui; Liu, Yang; Chen, Yuanyuan; Li, Jiayu; Xu, Xiaopeng; Zha, Jian; Xia, Ke; Linhardt, Robert J.; Zhang, Xiaomei; Shi, Jin‐Song; Koffas, Mattheos; Xu, Zhenghong

doi:10.1016/j.bej.2021.108238

Cited by 4 publications

(4 citation statements)

References 32 publications

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“…[ 34 ] Xu conducted a heterologous expression of pyruvate carboxylase ( PYC ) encoding gene from R. oryzae resulted in an increase in fumaric acid titer in S. cerevisiae . [ 35 ] When overexpressing PYC , excess oxaloacetic acid will be transported to mitochondria for glucose synthesis, providing a substrate supply for SAM synthesis. As an agonist of PYC , AcCoA will increase when PYC is overexpressed, so that more NADH will be produced from the tricarboxylic acid cycle.…”

Section: Discussionmentioning

confidence: 99%

Enhanced synthesis of S‐adenosyl‐L‐methionine through combinatorial metabolic engineering and Bayesian optimization in Saccharomyces cerevisiae

Xiao,

Shi,

Huang

et al. 2024

Biotechnology Journal

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S‐Adenosyl‐L‐methionine (SAM) is a substrate for many enzyme‐catalyzed reactions and provides methyl groups in numerous biological methylations, and thus has vast applications in the agriculture and medical field. Saccharomyces cerevisiae has been engineered as a platform with significant potential for producing SAM, but the current production has room for improvement. Thus, a method that consists of a series of metabolic engineering strategies was established in this study. These strategies included enhancing SAM synthesis, increasing ATP supply, down‐regulating SAM metabolism, and down‐regulating competing pathway. After combinatorial metabolic engineering, Bayesian optimization was conducted on the obtained strain C262P6S to optimize the fermentation medium. A final yield of 2972.8 mg·L−1 at 36 h with 29.7% of the L‐Met conversion rate in the shake flask was achieved, which was 26.3 times higher than that of its parent strain and the highest reported production in the shake flask to date. This paper establishes a feasible foundation for the construction of SAM‐producing strains using metabolic engineering strategies and demonstrates the effectiveness of Bayesian optimization in optimizing fermentation medium to enhance the generation of SAM.

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Section: Discussionmentioning

confidence: 99%

Enhanced synthesis of S‐adenosyl‐L‐methionine through combinatorial metabolic engineering and Bayesian optimization in Saccharomyces cerevisiae

Xiao,

Shi,

Huang

et al. 2024

Biotechnology Journal

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“…Besides, it links the high-capacity glycolytic pathway in S. cerevisiae to the synthetic pathway of the desired product (Xu et al, 2017). Xu conducted a heterologous expression of pyruvate carboxylase (PYC) encoding gene from Rhizopus oryzae resulted in an increase in fumaric acid titer to 226.0 mg/L from 194.0 mg/L in S. cerevisiae (Xu et al, 2022). When overexpressing PYC, excess oxaloacetic acid will be transported to mitochondria for glucose synthesis, providing a substrate supply for SAM synthesis.…”

Section: Optimization Of Mediummentioning

confidence: 99%

Enhanced synthesis of S-adenosyl-L-methionine through Combinatorial metabolic engineering and Bayesian optimization in Saccharomyces cerevisiae

Xiao

Shi

et al. 2023

Preprint

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S-adenosyl-L-methionine (SAM) is a substrate for many enzyme-catalyzed reactions and provides methyl groups in numerous biological methylations, and thus has vast applications in the medical field. Saccharomyces cerevisiae has been engineered as a platform with significant potential for producing SAM, although the current production has room for improvement. To surpass the restriction, a series of metabolic engineering strategies were employed to enhance the synthesis of SAM in this study. These strategies included enhancing SAM synthesis by overexpression of SAM2, met6, and str2, increasing ATP supply by integration of adkI and PYC, and down-regulating SAM metabolism by disrupting erg4 and erg6 and replacing the original promoter of CYS4 with a weaker promoter. After combinatorial metabolic engineering, Bayesian optimization was conducted on the obtained strain C262P6 to optimize the fermentation medium. A final yield of 2972.8 mg/L at 36 h with 29.7% of the L-Met conversion rate in the shake flask was achieved, which was 26.3 times higher than that of its parent strain and the highest reported production in the shake flask to date. This paper establishes a feasible foundation for the construction of SAM-produced strains using metabolic engineering strategies and demonstrates the effectiveness of Bayesian optimization in optimizing fermentation medium to enhance the generation of SAM.

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“…Various four- and six-carbon dicarboxylic acids, including succinic and fumaric acid, have been included in the ‘Top Value-Added Chemicals from Biomass’ list compiled in the early 2000s (Werpy and Petersen 2004 ). Their applications in various important industries and global market values explain the substantial interest in the microbial production of these dicarboxylic acids from relatively cheap substrates (Yin et al 2015 , Njokweni et al 2021 , Xu et al 2022 ). Fumaric acid is traditionally produced via three routes (Xu et al 2012 ).…”

Section: Introductionmentioning

confidence: 99%

“…Potential substrates include renewable biomass and agricultural waste that could provide for a mixture of high- and low-value products, such as fumaric acid and ethanol. The yeast Saccharomyces cerevisiae is an ideal candidate for the bio-fermentative production of dicarboxylic acids, including fumaric acid (Xu et al 2012 , 2022 ). It is a well-established industrial microorganism in the food and beverage industry and is tolerant towards inhibitors, acidic conditions, and high sugar and ethanol concentrations (Nevoigt 2008 ).…”

Section: Introductionmentioning

confidence: 99%

Constructing recombinantSaccharomyces cerevisiaestrains for malic-to-fumaric acid conversion

Steyn

Viljoen-Bloom

Zyl

2023

FEMS Microbiology Letters

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Saccharomyces cerevisiae with its robustness and good acid tolerance, is an attractive candidate for use in various industries, including waste-based biorefineries where a high-value organic acid is produced, such as fumaric acid could be beneficial. However, this yeast is not a natural producer of dicarboxylic acids and genetic engineering of S. cerevisiae strains is required to achieve this outcome. Disruption of the natural FUM1 gene and the recombinant expression of fumarase and malate transporter genes improved the malic acid-to-fumaric acid conversion by engineered S. cerevisiae strains. The efficacy of the strains was significantly influenced by the source of the fumarase gene (yeast versus bacterial), the presence of the XYNSEC signal secretion signal and the available oxygen in synthetic media cultivations. The ΔFUM1Ckr_fum + mae1 and ΔFUM1(ss)Ckr_fum + mae1 strains converted extracellular malic acid to 0.98 g/L and 1.11 g/L fumaric acid under aerobic conditions.

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Semi-rational evolution of pyruvate carboxylase from Rhizopus oryzae for elevated fumaric acid synthesis in Saccharomyces cerevisiae

Cited by 4 publications

References 32 publications

Enhanced synthesis of S‐adenosyl‐L‐methionine through combinatorial metabolic engineering and Bayesian optimization in Saccharomyces cerevisiae

Enhanced synthesis of S‐adenosyl‐L‐methionine through combinatorial metabolic engineering and Bayesian optimization in Saccharomyces cerevisiae

Enhanced synthesis of S-adenosyl-L-methionine through Combinatorial metabolic engineering and Bayesian optimization in Saccharomyces cerevisiae

Constructing recombinantSaccharomyces cerevisiaestrains for malic-to-fumaric acid conversion

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