Efficient production of shinorine, a natural sunscreen material, from glucose and xylose by deleting HXK2 encoding hexokinase in Saccharomyces cerevisiae

Abstract Mycosporine-like amino acids (MAAs), microbial secondary metabolites with ultraviolet (UV) absorption properties, are promising natural sunscreen materials. Due to the low efficiency of extracting MAAs from natural producers, production in heterologous hosts has recently received attention. Shinorine is a well characterized MAA with strong UV-A absorption property. Previous, we developed Saccharomyces cerevisiae strain producing shinorine by introducing four shinorine biosynthetic genes from cyanobacterium Nostoc punctiforme. Shinorine is produced from sedoheptulose 7-phosphate (S7P), an intermediate in the pentose phosphate pathway. Shinorine production was greatly improved by using xylose as a co-substrate, which can increase the S7P pool. However, due to a limited xylose-utilizing capacity of the engineered strain, glucose was used as a co-substrate to support cell growth. In this study, we further improved shinorine production by attenuating glucose catabolism via glycolysis, which can redirect the carbon flux from glucose to the pentose phosphate pathway favoring shinorine production. Of the strategies we examined to reduce glycolytic flux, deletion of HXK2, encoding hexokinase, was most effective in increasing shinorine production. Furthermore, by additional expression Ava3858 from Anabaena variabilis, encoding a rate-limiting enzyme 2-demethyl 4-deoxygadusol synthase, 68.4 mg/L of shinorine was produced in an optimized medium containing 14 g/L glucose and 6 g/L xylose, achieving a 2.2-fold increase compared with the previous strain.

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Fine tuning the glycolytic flux ratio of EP-bifido pathway for mevalonate production by enhancing glucose-6-phosphate dehydrogenase (Zwf) and CRISPRi suppressing 6-phosphofructose kinase (PfkA) in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-021-01526-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Ying Li ◽

He Xian ◽

Ya Xu ◽

Yuan Zhu ◽

Zhijie Sun ◽

...

Keyword(s):

Pentose Phosphate Pathway ◽

Conversion Rate ◽

Metabolic Flux ◽

Flux Ratio ◽

Reducing Power ◽

Pentose Phosphate ◽

Fine Tuning ◽

High Yield ◽

Glycolytic Flux ◽

Acetyl Coa

Abstract Background Natural glycolysis encounters the decarboxylation of glucose partial oxidation product pyruvate into acetyl-CoA, where one-third of the carbon is lost at CO2. We previously constructed a carbon saving pathway, EP-bifido pathway by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and “bifid shunt”, to generate high yield acetyl-CoA from glucose. However, the carbon conversion rate and reducing power of this pathway was not optimal, the flux ratio of EMP pathway and pentose phosphate pathway (PPP) needs to be precisely and dynamically adjusted to improve the production of mevalonate (MVA). Result Here, we finely tuned the glycolytic flux ratio in two ways. First, we enhanced PPP flux for NADPH supply by replacing the promoter of zwf on the genome with a set of different strength promoters. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, pfkA was finely down-regulated by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system. The MVA yield of the regulated strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. Conclusion This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.

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Adaptation of central metabolite pools to variations in growth rate and cultivation conditions in Saccharomyces cerevisiae

Microbial Cell Factories ◽

10.1186/s12934-021-01557-8 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Kanhaiya Kumar ◽

Vishwesh Venkatraman ◽

Per Bruheim

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Rate ◽

Amino Acid ◽

Growth Rates ◽

Pentose Phosphate Pathway ◽

Pentose Phosphate ◽

Relative Reduction ◽

Central Metabolism ◽

Cultivation Conditions ◽

Biological Studies

Abstract Background Saccharomyces cerevisiae is a well-known popular model system for basic biological studies and serves as a host organism for the heterologous production of commercially interesting small molecules and proteins. The central metabolism is at the core to provide building blocks and energy to support growth and survival in normal situations as well as during exogenous stresses and forced heterologous protein production. Here, we present a comprehensive study of intracellular central metabolite pool profiling when growing S. cerevisiae on different carbon sources in batch cultivations and at different growth rates in nutrient-limited glucose chemostats. The latest versions of absolute quantitative mass spectrometry-based metabolite profiling methodology were applied to cover glycolytic and pentose phosphate pathway metabolites, tricarboxylic acid cycle (TCA), complete amino acid, and deoxy-/nucleoside phosphate pools. Results Glutamate, glutamine, alanine, and citrate were the four most abundant metabolites for most conditions tested. The amino acid is the dominant metabolite class even though a marked relative reduction compared to the other metabolite classes was observed for nitrogen and phosphate limited chemostats. Interestingly, glycolytic and pentose phosphate pathway (PPP) metabolites display the largest variation among the cultivation conditions while the nucleoside phosphate pools are more stable and vary within a closer concentration window. The overall trends for glucose and nitrogen-limited chemostats were increased metabolite pools with the increasing growth rate. Next, comparing the chosen chemostat reference growth rate (0.12 h−1, approximate one-fourth of maximal unlimited growth rate) illuminates an interesting pattern: almost all pools are lower in nitrogen and phosphate limited conditions compared to glucose limitation, except for the TCA metabolites citrate, isocitrate and α-ketoglutarate. Conclusions This study provides new knowledge-how the central metabolism is adapting to various cultivations conditions and growth rates which is essential for expanding our understanding of cellular metabolism and the development of improved phenotypes in metabolic engineering.

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Enhancing the flux of D-glucose to the pentose phosphate pathway in Saccharomyces cerevisiae for the production of D-ribose and ribitol

Applied Microbiology and Biotechnology ◽

10.1007/s00253-009-2184-4 ◽

2009 ◽

Vol 85 (3) ◽

pp. 731-739 ◽

Cited By ~ 19

Author(s):

Mervi H. Toivari ◽

Hannu Maaheimo ◽

Merja Penttilä ◽

Laura Ruohonen

Keyword(s):

Saccharomyces Cerevisiae ◽

Pentose Phosphate Pathway ◽

Pentose Phosphate

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Metabolic Changes Induced by Deletion of Transcriptional Regulator GCR2 in Xylose-Fermenting Saccharomyces cerevisiae

Microorganisms ◽

10.3390/microorganisms8101499 ◽

2020 ◽

Vol 8 (10) ◽

pp. 1499

Author(s):

Minhye Shin ◽

Soo Rin Kim

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Pentose Phosphate Pathway ◽

Carbon Metabolism ◽

Transcriptional Regulator ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Metabolic Changes ◽

Regulatory Systems ◽

Central Carbon

Glucose repression has been extensively studied in Saccharomyces cerevisiae, including the regulatory systems responsible for efficient catabolism of glucose, the preferred carbon source. However, how these regulatory systems would alter central metabolism if new foreign pathways are introduced is unknown, and the regulatory networks between glycolysis and the pentose phosphate pathway, the two major pathways in central carbon metabolism, have not been systematically investigated. Here we disrupted gcr2, a key transcriptional regulator, in S. cerevisiae strain SR7 engineered to heterologously express the xylose-assimilating pathway, activating genes involved in glycolysis, and evaluated the global metabolic changes. gcr2 deletion reduced cellular growth in glucose but significantly increased growth when xylose was the sole carbon source. Global metabolite profiling revealed differential regulation of yeast metabolism in SR7-gcr2Δ, especially carbohydrate and nucleotide metabolism, depending on the carbon source. In glucose, the SR7-gcr2Δ mutant showed overall decreased abundance of metabolites, such as pyruvate and sedoheptulose-7-phosphate, associated with central carbon metabolism including glycolysis and the pentose phosphate pathway. However, SR7-gcr2Δ showed an increase in metabolites abundance (ribulose-5-phosphate, sedoheptulose-7-phosphate, and erythrose-4-phosphate) notably from the pentose phosphate pathway, as well as alteration in global metabolism when compared to SR7. These results provide insights into how the regulatory system GCR2 coordinates the transcription of glycolytic genes and associated metabolic pathways.

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The synthetic cannabinoid JWH-018 modulates Saccharomyces cerevisiae energetic metabolism

FEMS Yeast Research ◽

10.1093/femsyr/foz042 ◽

2019 ◽

Vol 19 (5) ◽

Author(s):

Carla Ferreira ◽

Joana Couceiro ◽

Carlos Família ◽

Carolina Jardim ◽

Pedro Antas ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Mechanisms ◽

Synthetic Cannabinoids ◽

Cellular Level ◽

Pentose Phosphate ◽

Synthetic Cannabinoid ◽

Glycolytic Flux ◽

Vast Number ◽

2D Gel ◽

The Impact

ABSTRACT Synthetic cannabinoids are a group of novel psychoactive substances with similar properties to Δ9-THC. Among the vast number of synthetic cannabinoids, designed to be tested in clinical trials, JWH-018 was the first novel psychoactive substance found in the recreational drug marketplace. The consumption of JWH-018 shows typical effects of CB1 agonists including sedation, cognitive dysfunction, tachycardia, postural hypotension, dry mouth, ataxia and psychotropic effects, but appeared to be more potent than Δ9-THC. However, studies on human cells have shown that JWH-018 toxicity depends on the cellular line used. Despite these studies, the underlying molecular mechanisms to JWH-018 action has not been clarified yet. To understand the impact of JWH-018 at molecular and cellular level, we used Saccharomyces cerevisiae as a model. The results showed an increase in yeast growth rate in the presence of this synthetic cannabinoid due to an enhancement in the glycolytic flux at expense of a decrease in pentose phosphate pathway, judging by 2D-Gel proteomic analysis, qRT-PCR experiments and ATP measurements. Overall, our results provide insights into molecular mechanisms of JWH-018 action, also indicating that Saccharomyces cerevisiae is a good model to study synthetic cannabinoids.

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