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Increasing glycolysis by deletion of kcs1 and arg82 improved S-adenosyl-L-methionine production in Saccharomyces cerevisiae.

ABSTRACT: Reprogramming glycolysis for directing glycolytic metabolites to a specific metabolic pathway is expected to be useful for increasing microbial production of certain metabolites, such as amino acids, lipids or considerable secondary metabolites. In this report, a strategy of increasing glycolysis by altering the metabolism of inositol pyrophosphates (IPs) for improving the production of S-adenosyl-L-methionine (SAM) for diverse pharmaceutical applications in yeast is presented. The genes associated with the metabolism of IPs, arg82, ipk1 and kcs1, were deleted, respectively, in the yeast strain Saccharomyces cerevisiae CGMCC 2842. It was observed that the deletions of kcs1 and arg82 increased SAM by 83.3?% and 31.8?%, respectively, compared to that of the control. In addition to the improved transcription levels of various glycolytic genes and activities of the relative enzymes, the levels of glycolytic intermediates and ATP were also enhanced. To further confirm the feasibility, the kcs1 was deleted in the high SAM-producing strain Ymls1?GAPmK which was deleted malate synthase gene mls1 and co-expressed the Acetyl-CoA synthase gene acs2 and the SAM synthase gene metK1 from Leishmania infantum, to obtain the recombinant strain Ymls1?kcs1?GAPmK. The level of SAM in Ymls1?kcs1?GAPmK reached 2.89 g L^-1 in a 250-mL flask and 8.86 g L^-1 in a 10-L fermentation tank, increasing 30.2?% and 46.2?%, respectively, compared to those levels in Ymls1?GAPmK. The strategy of increasing glycolysis by deletion of kcs1 and arg82 improved SAM production in yeast.

SUBMITTER: Chen H

PROVIDER: S-EPMC7815874 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Increasing glycolysis by deletion of kcs1 and arg82 improved S-adenosyl-L-methionine production in Saccharomyces cerevisiae.

Chen Hailong H Zhu Nianqing N Wang Yan Y Gao Xinxin X Song Yuhe Y Zheng Jia J Peng Jiaping J Zhang Xin X

AMB Express 20210119 1

Reprogramming glycolysis for directing glycolytic metabolites to a specific metabolic pathway is expected to be useful for increasing microbial production of certain metabolites, such as amino acids, lipids or considerable secondary metabolites. In this report, a strategy of increasing glycolysis by altering the metabolism of inositol pyrophosphates (IPs) for improving the production of S-adenosyl-L-methionine (SAM) for diverse pharmaceutical applications in yeast is presented. The genes associa ...[more]

PMID: 33464427

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Project description:Protein folding is often considered the flux controlling process in protein synthesis and secretion. Here, two previously isolated Saccharomyces cerevisiae strains with increased α-amylase productivity were analyzed in chemostat cultures at different dilution rates using multi-omics data. Based on the analysis, we identified different routes of the protein folding pathway to improve protein production. In the first strain, the increased abundance of proteins working on the folding process, coordinated with upregulated glycogen metabolism and trehalose metabolism, helped increase α-amylase productivity 1.95-fold compared to the level in the original strain in chemostat culture at a dilution rate of 0.2/h. The second strain further strengthened the folding precision to improve protein production. More precise folding helps the cell improve protein production efficiency and reduce the expenditure of energy on the handling of misfolded proteins. As calculated using an enzyme-constrained genome-scale metabolic model, the second strain had an increased productivity of 2.36-fold with lower energy expenditure than that of the original under the same condition. Further study revealed that the regulation of N-glycans played an important role in the folding precision control and that overexpression of the glucosidase Cwh41p can significantly improve protein production, especially for the strains with improved folding capacity but lower folding precision. Our findings elucidated in detail the mechanisms in two strains having improved protein productivity and thereby provided novel insights for industrial recombinant protein production as well as demonstrating how multi-omics analysis can be used for identification of novel strain-engineering targets.IMPORTANCE Protein folding plays an important role in protein maturation and secretion. In recombinant protein production, many studies have focused on the folding pathway to improve productivity. Here, we identified two different routes for improving protein production by yeast. We found that improving folding precision is a better strategy. Dysfunction of this process is also associated with several aberrant protein-associated human diseases. Here, our findings about the role of glucosidase Cwh41p in the precision control system and the characterization of the strain with a more precise folding process could contribute to the development of novel therapeutic strategies.

Dataset Information

Increasing glycolysis by deletion of kcs1 and arg82 improved S-adenosyl-L-methionine production in Saccharomyces cerevisiae.

Publications

Increasing glycolysis by deletion of kcs1 and arg82 improved S-adenosyl-L-methionine production in Saccharomyces cerevisiae.

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