Metabolic engineering最新文献

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Enabling genetic manipulation and robustness of Bacillus methanolicus for methanol-based bio-manufacturing

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-26 DOI: 10.1016/j.ymben.2025.02.013

Bixiao Li , Zhiheng Yang , Zilong Li , Yuanyuan Zhang , Lixin Zhang , Weishan Wang

Methanol-based biomanufacturing holds great promise for sustainability but is currently limited by the slow growth and low efficiency of natural or synthetic methylotrophic strains. In contrast, the thermophilic methylotroph Bacillus methanolicus exhibits rapid growth, high-temperatures tolerance, and efficient methanol utilization in defined mineral medium, making it a promising candidate for industrial applications. However, its potential is constrained by reluctant genetic modification and suboptimal robustness under fluctuating methanol concentrations. To address these limitations, we developed a comprehensive genetic manipulation system that includes an improved transformation approach, a homologous recombination-based knock-out/knock-in method, a constitutive promoter library spanning a 600-fold range of strengths, and an stringent xylose-inducible promoter with a wide dynamic range. Using these enabling tools, we enhanced the robustness of B. methanolicus under varying methanol concentrations by introducing a xylose pathway, which buffered intracellular formaldehyde accumulation. Co-utilization of methanol and xylose achieved a molar consumption ratio exceeding 4:1, indicating methanol served as the primary carbon source while xylose was auxiliary to enhance robustness. Subsequently, we developed a riboflavin cell factory by systemic engineering of B. methanolicus, achieving 2579 mg/L production in a 5-L bioreactor—the highest riboflavin titer reported for methanol-based production. This study establishes B. methanolicus as a versatile and accessible platform for sustainable methanol-based bio-manufacturing.

{"title":"Enabling genetic manipulation and robustness of Bacillus methanolicus for methanol-based bio-manufacturing","authors":"Bixiao Li , Zhiheng Yang , Zilong Li , Yuanyuan Zhang , Lixin Zhang , Weishan Wang","doi":"10.1016/j.ymben.2025.02.013","DOIUrl":"10.1016/j.ymben.2025.02.013","url":null,"abstract":"<div><div>Methanol-based biomanufacturing holds great promise for sustainability but is currently limited by the slow growth and low efficiency of natural or synthetic methylotrophic strains. In contrast, the thermophilic methylotroph <em>Bacillus methanolicus</em> exhibits rapid growth, high-temperatures tolerance, and efficient methanol utilization in defined mineral medium, making it a promising candidate for industrial applications. However, its potential is constrained by reluctant genetic modification and suboptimal robustness under fluctuating methanol concentrations. To address these limitations, we developed a comprehensive genetic manipulation system that includes an improved transformation approach, a homologous recombination-based knock-out/knock-in method, a constitutive promoter library spanning a 600-fold range of strengths, and an stringent xylose-inducible promoter with a wide dynamic range. Using these enabling tools, we enhanced the robustness of <em>B. methanolicus</em> under varying methanol concentrations by introducing a xylose pathway, which buffered intracellular formaldehyde accumulation. Co-utilization of methanol and xylose achieved a molar consumption ratio exceeding 4:1, indicating methanol served as the primary carbon source while xylose was auxiliary to enhance robustness. Subsequently, we developed a riboflavin cell factory by systemic engineering of <em>B. methanolicus</em>, achieving 2579 mg/L production in a 5-L bioreactor—the highest riboflavin titer reported for methanol-based production. This study establishes <em>B. methanolicus</em> as a versatile and accessible platform for sustainable methanol-based bio-manufacturing.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"89 ","pages":"Pages 121-134"},"PeriodicalIF":6.8,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143527456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Recent advances on engineering Escherichia coli and Corynebacterium glutamicum for efficient production of L-threonine and its derivatives

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-26 DOI: 10.1016/j.ymben.2025.02.012

Guihong Zhao , Dezhi Zhang , Yaqun Tang , Xiaoqing Hu , Xiaoyuan Wang

L-threonine, one of the three major amino acids, plays a vital role in various industries such as food, feed, pharmaceuticals, and cosmetics. Currently, the fermentation-based production of L-threonine has evolved into an efficient, cost-effective, and environmentally friendly industrial process. Escherichia coli and Corynebacterium glutamicum, as the industrial workhorses of amino acids production, have long been widely studied due to their well-established genetic backgrounds and powerful molecular tools. This review focuses on recent advances in the microbial production of L-threonine by metabolic engineering. From three key modules, including L-threonine synthesis module, central metabolism module and global regulation module, we provide a comprehensive analysis on the entire metabolic pathway of L-threonine and the global regulation of the production process. Furthermore, we systematically summarize biotransformation methods for producing high-value derivatives of L-threonine, thereby broadening the application scope and market potential of L-threonine. Overall, this review shows many effective strategies for the biosynthesis of L-threonine, and offers guidance for the microbial production of L-aspartate family amino acids and their derivatives.

{"title":"Recent advances on engineering Escherichia coli and Corynebacterium glutamicum for efficient production of L-threonine and its derivatives","authors":"Guihong Zhao , Dezhi Zhang , Yaqun Tang , Xiaoqing Hu , Xiaoyuan Wang","doi":"10.1016/j.ymben.2025.02.012","DOIUrl":"10.1016/j.ymben.2025.02.012","url":null,"abstract":"<div><div>L-threonine, one of the three major amino acids, plays a vital role in various industries such as food, feed, pharmaceuticals, and cosmetics. Currently, the fermentation-based production of L-threonine has evolved into an efficient, cost-effective, and environmentally friendly industrial process. <em>Escherichia coli</em> and <em>Corynebacterium glutamicum</em>, as the industrial workhorses of amino acids production, have long been widely studied due to their well-established genetic backgrounds and powerful molecular tools. This review focuses on recent advances in the microbial production of L-threonine by metabolic engineering. From three key modules, including L-threonine synthesis module, central metabolism module and global regulation module, we provide a comprehensive analysis on the entire metabolic pathway of L-threonine and the global regulation of the production process. Furthermore, we systematically summarize biotransformation methods for producing high-value derivatives of L-threonine, thereby broadening the application scope and market potential of L-threonine. Overall, this review shows many effective strategies for the biosynthesis of L-threonine, and offers guidance for the microbial production of L-aspartate family amino acids and their derivatives.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"90 ","pages":"Pages 1-15"},"PeriodicalIF":6.8,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143526806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biosynthesis of 12-aminododecanoic acid from biomass sugars

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-23 DOI: 10.1016/j.ymben.2025.02.010

Haixin Gao , Qiang Fang , Yanfen Bai, Chunyue Hu, Howard H. Chou

Biosynthesis of 12-aminododecanoic acid (ADDA) directly from biomass-derived sugars would enable a more sustainable process for manufacturing the engineering polymer Nylon 12. ADDA biosynthesis is currently hindered by the cytotoxicity of dodecanoic acid (DDA) to growing cells, and the accumulation of the overoxidized byproduct dodecanedioic acid (DDDA). In this study, these challenges were addressed by engineering an autoinducible system to better control in vivo DDA synthesis without impacting growth, and deleting aldehyde dehydrogenases and oxidases to reduce DDDA accumulation. As a result, a one-step fermentation process was established to synthesize ADDA from glucose and cellobiose. Finally, batch fermentation achieved 1035 mg/L ADDA and 5% yield, which is the highest titer and yield accomplished directly from sugar to date. This research contributes to the mechanistic understanding of microbial DDA, ADDA, and DDDA synthesis, as well as the goal of developing more sustainable processes for nylon production.

引用次数: 0

Direct mRNA-to-sgRNA conversion generates design-free ultra-dense CRISPRi libraries for systematic phenotypic screening

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-22 DOI: 10.1016/j.ymben.2025.02.011

Jiseon Lee , Ha Hyeon Jeon , Euijin Seo , Sehyeon Park , Donghui Choe , Byung-Kwan Cho , Jeong Wook Lee

CRISPR interference (CRISPRi) is a versatile tool for high-throughput phenotypic screening. However, rational design and synthesis of the single-guide RNA (sgRNA) library required for each genome-wide CRISPRi application is time-consuming, expensive, and unfeasible if the target organisms lack comprehensive sequencing and characterization. We developed an ultra-dense random sgRNA library generation method applicable to any organism, including those that are not well-characterized. Our method converts transcriptome-wide mRNA into 20 nt of sgRNA spacer sequences through enzymatic reactions. The generated sgRNA library selectively binds to the non-template strand of the coding sequence, leading to more efficient repression compared to binding the template strand. We then generated a genome-scale library for Escherichia coli by applying this method and identified essential and auxotrophic genes through phenotypic screening. Furthermore, we tuned the production levels of lycopene and violacein and identified new repression targets for violacein production. Our results demonstrated that a genome-scale sgRNA library can be generated without rational design and can be utilized simultaneously in a range of phenotypic screenings.

{"title":"Direct mRNA-to-sgRNA conversion generates design-free ultra-dense CRISPRi libraries for systematic phenotypic screening","authors":"Jiseon Lee , Ha Hyeon Jeon , Euijin Seo , Sehyeon Park , Donghui Choe , Byung-Kwan Cho , Jeong Wook Lee","doi":"10.1016/j.ymben.2025.02.011","DOIUrl":"10.1016/j.ymben.2025.02.011","url":null,"abstract":"<div><div>CRISPR interference (CRISPRi) is a versatile tool for high-throughput phenotypic screening. However, rational design and synthesis of the single-guide RNA (sgRNA) library required for each genome-wide CRISPRi application is time-consuming, expensive, and unfeasible if the target organisms lack comprehensive sequencing and characterization. We developed an ultra-dense random sgRNA library generation method applicable to any organism, including those that are not well-characterized. Our method converts transcriptome-wide mRNA into 20 nt of sgRNA spacer sequences through enzymatic reactions. The generated sgRNA library selectively binds to the non-template strand of the coding sequence, leading to more efficient repression compared to binding the template strand. We then generated a genome-scale library for <em>Escherichia coli</em> by applying this method and identified essential and auxotrophic genes through phenotypic screening. Furthermore, we tuned the production levels of lycopene and violacein and identified new repression targets for violacein production. Our results demonstrated that a genome-scale sgRNA library can be generated without rational design and can be utilized simultaneously in a range of phenotypic screenings.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"89 ","pages":"Pages 108-120"},"PeriodicalIF":6.8,"publicationDate":"2025-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Switching the yeast metabolism via manipulation of sugar phosphorylation

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-21 DOI: 10.1016/j.ymben.2025.02.008

Cong Fan , Jian Fan , Haofeng Chen , Shujin Lin , Danli Zhang , Jingya Song , Junyi Wang , Yan Wang , Xiao Han , Jifeng Yuan

Saccharomyces cerevisiae predominantly ferments sugar to ethanol, irrespective of the presence of oxygen, which is known as the Crabtree-effect. Traditional methods rely on static controls of glycolytic flux to make S. cerevisiae Crabtree-negative, which are not favorable for future biomanufacturing applications. Considering native metabolic pathways typically harness dynamic regulatory networks, we therefore aim to develop an alternative strategy using dynamic regulation of the yeast central metabolism to generate Crabtree-negative S. cerevisiae. We report that manipulating a single step at sugar phosphorylation can alter the mode of yeast metabolism with an attenuated Crabtree-effect. By implementing catabolite-regulated sugar phosphorylation, the diauxic shift in budding yeast was effectively reduced. The Crabtree-attenuated metabolism in the engineered yeast was confirmed by multidimensional characterizations such as cell morphology, the measurements of sugar utilization rate and ethanol production, and transcriptomics. In addition, we demonstrated that the Crabtree-attenuated metabolism could substantially improve the mitochondrial synthesis of short branched-chain fatty acids from amino acid catabolism, and allow the synthesis and accumulation of retinaldehyde. Taken together, we present for the first time that manipulation of sugar phosphorylation can alter the mode of yeast metabolism, and the synthetic Crabtree-attenuated yeast factory established here might serve as a non-fermentative biomanufacturing chassis.

{"title":"Switching the yeast metabolism via manipulation of sugar phosphorylation","authors":"Cong Fan , Jian Fan , Haofeng Chen , Shujin Lin , Danli Zhang , Jingya Song , Junyi Wang , Yan Wang , Xiao Han , Jifeng Yuan","doi":"10.1016/j.ymben.2025.02.008","DOIUrl":"10.1016/j.ymben.2025.02.008","url":null,"abstract":"<div><div><em>Saccharomyces cerevisiae</em> predominantly ferments sugar to ethanol, irrespective of the presence of oxygen, which is known as the Crabtree-effect. Traditional methods rely on static controls of glycolytic flux to make <em>S. cerevisiae</em> Crabtree-negative, which are not favorable for future biomanufacturing applications. Considering native metabolic pathways typically harness dynamic regulatory networks, we therefore aim to develop an alternative strategy using dynamic regulation of the yeast central metabolism to generate Crabtree-negative <em>S. cerevisiae</em>. We report that manipulating a single step at sugar phosphorylation can alter the mode of yeast metabolism with an attenuated Crabtree-effect. By implementing catabolite-regulated sugar phosphorylation, the diauxic shift in budding yeast was effectively reduced. The Crabtree-attenuated metabolism in the engineered yeast was confirmed by multidimensional characterizations such as cell morphology, the measurements of sugar utilization rate and ethanol production, and transcriptomics. In addition, we demonstrated that the Crabtree-attenuated metabolism could substantially improve the mitochondrial synthesis of short branched-chain fatty acids from amino acid catabolism, and allow the synthesis and accumulation of retinaldehyde. Taken together, we present for the first time that manipulation of sugar phosphorylation can alter the mode of yeast metabolism, and the synthetic Crabtree-attenuated yeast factory established here might serve as a non-fermentative biomanufacturing chassis.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"89 ","pages":"Pages 76-85"},"PeriodicalIF":6.8,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143480544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Production and characterization of copolymers consisting of 3-hydroxybutyrate and increased 3-hydroxyvalerate by β-oxidation weakened Halomonas

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-21 DOI: 10.1016/j.ymben.2025.02.009

Huan Wang , Yunyun Ouyang , Weinan Yang , Hongtao He , Jiangnan Chen , Yiping Yuan , Helen Park , Fuqing Wu , Fang Yang , Guo-Qiang Chen

Polyhydroxyalkanoates (PHA) with high 3-hydroxyvalerate (3HV) monomer ratios lead to their accelerated biodegradation and improved thermal and mechanical properties. In this study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a broad range of 3HV ratios were produced and characterized using the next generation industrial biotechnology (NGIB) chassis Halomonas bluephagenesis (H. bluephagenesis). Wild type H. bluephagenesis was found to produce P(3HB-co-66.31mol% 3HV) when cultured in the presence of valerate. Deletion on the functional enoyl-CoA hydratase (fadB₁) increased to 93.11 mol% 3HV in the PHBV copolymers. Through tuning the glucose and valerate co-feeding, PHBV with controllable 3HV ratios were adjusted to range from 0-to-93.6 mol% in shake-flask studies. Metabolic weakening of the β-oxidation pathway paired with flux limitation to the native 3HB synthesis pathway were used to reach the highest reported 98.3 mol% 3HV by H. bluephagenesis strain G34B grown in shake flasks. H. bluephagenesis strain G34B was grown to 71.42 g/L cell dry weight (CDW) containing 74.12 wt% P(3HB-co-17.97 mol% 3HV) in 7 L fermentors. Mechanical properties of PHBV with 0, 22.81, 42.76, 73.49 and 92.17 mol% 3HV were characterized to find not linearly related to increased 3HV ratios. Engineered H. bluephagenesis has demonstrated as a platform for producing PHBV of various properties.

{"title":"Production and characterization of copolymers consisting of 3-hydroxybutyrate and increased 3-hydroxyvalerate by β-oxidation weakened Halomonas","authors":"Huan Wang , Yunyun Ouyang , Weinan Yang , Hongtao He , Jiangnan Chen , Yiping Yuan , Helen Park , Fuqing Wu , Fang Yang , Guo-Qiang Chen","doi":"10.1016/j.ymben.2025.02.009","DOIUrl":"10.1016/j.ymben.2025.02.009","url":null,"abstract":"<div><div>Polyhydroxyalkanoates (PHA) with high 3-hydroxyvalerate (3HV) monomer ratios lead to their accelerated biodegradation and improved thermal and mechanical properties. In this study, poly(3-hydroxybutyrate-<em>co</em>-3-hydroxyvalerate) (PHBV) with a broad range of 3HV ratios were produced and characterized using the next generation industrial biotechnology (NGIB) chassis <em>Halomonas bluephagenesis</em> (<em>H. bluephagenesis</em>). Wild type <em>H. bluephagenesis</em> was found to produce P(3HB-<em>co</em>-66.31mol% 3HV) when cultured in the presence of valerate. Deletion on the functional enoyl-CoA hydratase (<em>fadB</em><sub><em>1</em></sub>) increased to 93.11 mol% 3HV in the PHBV copolymers. Through tuning the glucose and valerate co-feeding, PHBV with controllable 3HV ratios were adjusted to range from 0-to-93.6 mol% in shake-flask studies. Metabolic weakening of the β-oxidation pathway paired with flux limitation to the native 3HB synthesis pathway were used to reach the highest reported 98.3 mol% 3HV by <em>H. bluephagenesis</em> strain G34B grown in shake flasks. <em>H. bluephagenesis</em> strain G34B was grown to 71.42 g/L cell dry weight (CDW) containing 74.12 wt% P(3HB-<em>co</em>-17.97 mol% 3HV) in 7 L fermentors. Mechanical properties of PHBV with 0, 22.81, 42.76, 73.49 and 92.17 mol% 3HV were characterized to find not linearly related to increased 3HV ratios. Engineered <em>H. bluephagenesis</em> has demonstrated as a platform for producing PHBV of various properties.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"89 ","pages":"Pages 97-107"},"PeriodicalIF":6.8,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483684","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Big data and machine learning: Metabolic engineering special issue

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-20 DOI: 10.1016/j.ymben.2025.02.007

Hal S. Alper

引用次数: 0

Engineering Komagataella phaffii for citric acid production through carbon-conserving supply of acetyl-CoA

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-17 DOI: 10.1016/j.ymben.2025.02.005

Evelyn Vásquez Castro , Özge Ata , Matthias G. Steiger , Tim Causon , Diethard Mattanovich

The oxidative formation of AcCoA limits the glycolytic pathway yield (Y^P_GLY) for citric acid due to the NADH overflow and carbon loss as CO₂. An interesting approach to enhance product yields is the incorporation of carbon-conserving pathways. This study assesses the potential of a carbon-conserving AcCoA pathway, the glycolysis alternative high carbon yield cycle (GATHCYC), to improve citric acid production, utilizing the non-native citric acid producer Komagataella phaffii as an orthogonal test system. The combination of different metabolic engineering strategies enabled K. phaffii to acquire the ability to produce extracellular citric acid. By constructing the GATHCYC in the cytosol and peroxisomes, the intracellular concentration of AcCoA increased. Overexpression of the genes encoding pyruvate carboxylase (PYC2), citrate synthase (CIT2) and citrate exporter protein (cexA) in the peroxisomal AcCoA strains boosted the citric acid production. Thus, the best producer strain reached a citric acid titer of 51.3 ± 0.9 g L⁻¹ and a yield of 0.59 ± 0.01 g g⁻¹ after 76 h of glucose-limited fed-batch cultivation. Our results highlight the potential of using GATHCYC to provide an efficient supply of acetyl-CoA to enhance citric acid production. This approach could be exploited for the production of other AcCoA-derived compounds of industrial relevance in different cell factories.

{"title":"Engineering Komagataella phaffii for citric acid production through carbon-conserving supply of acetyl-CoA","authors":"Evelyn Vásquez Castro , Özge Ata , Matthias G. Steiger , Tim Causon , Diethard Mattanovich","doi":"10.1016/j.ymben.2025.02.005","DOIUrl":"10.1016/j.ymben.2025.02.005","url":null,"abstract":"<div><div>The oxidative formation of AcCoA limits the glycolytic pathway yield (Y<sup>P</sup><sub>GLY</sub>) for citric acid due to the NADH overflow and carbon loss as CO<sub>2</sub>. An interesting approach to enhance product yields is the incorporation of carbon-conserving pathways. This study assesses the potential of a carbon-conserving AcCoA pathway, the glycolysis alternative high carbon yield cycle (GATHCYC), to improve citric acid production, utilizing the non-native citric acid producer <em>Komagataella phaffii</em> as an orthogonal test system. The combination of different metabolic engineering strategies enabled <em>K. phaffii</em> to acquire the ability to produce extracellular citric acid. By constructing the GATHCYC in the cytosol and peroxisomes, the intracellular concentration of AcCoA increased. Overexpression of the genes encoding pyruvate carboxylase (<em>PYC2</em>), citrate synthase (<em>CIT2</em>) and citrate exporter protein (<em>cexA</em>) in the peroxisomal AcCoA strains boosted the citric acid production. Thus, the best producer strain reached a citric acid titer of 51.3 ± 0.9 g L<sup>−1</sup> and a yield of 0.59 ± 0.01 g g<sup>−1</sup> after 76 h of glucose-limited fed-batch cultivation. Our results highlight the potential of using GATHCYC to provide an efficient supply of acetyl-CoA to enhance citric acid production. This approach could be exploited for the production of other AcCoA-derived compounds of industrial relevance in different cell factories.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"89 ","pages":"Pages 47-59"},"PeriodicalIF":6.8,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143458603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Dynamic regulation combined with systematic metabolic engineering for high-level palmitoleic acid accumulation in oleaginous yeast

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-17 DOI: 10.1016/j.ymben.2025.02.006

Yufan Zhou , Mei-Li Sun , Lu Lin , Rodrigo Ledesma-Amaro , Kaifeng Wang , Xiao-Jun Ji , He Huang

Palmitoleic acid (POA, C16:1Δ⁹) is widely recognized for its preventive and therapeutic effects in various chronic and cardiovascular diseases, but the current production practices based on plant extraction are both economically and ecologically unsustainable. Although Yarrowia lipolytica is capable of producing POA, it only accumulates to a small percentage of total fatty acids. The present study aimed to enhance the accumulation of POA by employing a two-layer engineering strategy, encompassing the modulation of the fatty acid profile and the promotion of the accumulation of POA-rich lipids. The fatty acid profile was subject to modulation through the engineering of the fatty acid metabolism by expressing heterologous specific fatty acid desaturases CeFat5 and implementing dynamic regulation based on a copper-responsive promoter. Then, the mechanism underlying this improvement of POA production capacity was elucidated. Finally, the POA-rich lipid accumulation ability was enhanced through engineering of the lipid metabolism by overexpressing the heterologous POA-specific triacylglycerol forming acyltransferase, introducing the artificial designed non-carboxylative malonyl-CoA production pathway, and preventing lipid degradation. The resulting optimized yeast strain achieved an impressive POA accumulation accounting for 50.62% of total fatty acids, marking a 37.7-fold improvement over the initial strain. Moreover, a record POA titer of 25.6 g/L was achieved in the bioreactor. Overall, this study introduces a framework for establishing efficient yeast platforms for the accumulation of valuable fatty acids.

{"title":"Dynamic regulation combined with systematic metabolic engineering for high-level palmitoleic acid accumulation in oleaginous yeast","authors":"Yufan Zhou , Mei-Li Sun , Lu Lin , Rodrigo Ledesma-Amaro , Kaifeng Wang , Xiao-Jun Ji , He Huang","doi":"10.1016/j.ymben.2025.02.006","DOIUrl":"10.1016/j.ymben.2025.02.006","url":null,"abstract":"<div><div>Palmitoleic acid (POA, C16:1Δ<sup>9</sup>) is widely recognized for its preventive and therapeutic effects in various chronic and cardiovascular diseases, but the current production practices based on plant extraction are both economically and ecologically unsustainable. Although <em>Yarrowia lipolytica</em> is capable of producing POA, it only accumulates to a small percentage of total fatty acids. The present study aimed to enhance the accumulation of POA by employing a two-layer engineering strategy, encompassing the modulation of the fatty acid profile and the promotion of the accumulation of POA-rich lipids. The fatty acid profile was subject to modulation through the engineering of the fatty acid metabolism by expressing heterologous specific fatty acid desaturases <em>Ce</em>Fat5 and implementing dynamic regulation based on a copper-responsive promoter. Then, the mechanism underlying this improvement of POA production capacity was elucidated. Finally, the POA-rich lipid accumulation ability was enhanced through engineering of the lipid metabolism by overexpressing the heterologous POA-specific triacylglycerol forming acyltransferase, introducing the artificial designed non-carboxylative malonyl-CoA production pathway, and preventing lipid degradation. The resulting optimized yeast strain achieved an impressive POA accumulation accounting for 50.62% of total fatty acids, marking a 37.7-fold improvement over the initial strain. Moreover, a record POA titer of 25.6 g/L was achieved in the bioreactor. Overall, this study introduces a framework for establishing efficient yeast platforms for the accumulation of valuable fatty acids.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"89 ","pages":"Pages 33-46"},"PeriodicalIF":6.8,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143453828","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Discovery, characterization, and application of chromosomal integration sites for stable heterologous gene expression in Rhodotorula toruloides

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-02-14 DOI: 10.1016/j.ymben.2025.02.004

Hao Xu , Longyuan Shi , Aashutosh Girish Boob , Wooyoung Park , Shih-I Tan , Vinh Gia Tran , John Carl Schultz , Huimin Zhao

Rhodotorula toruloides is a non-model, oleaginous yeast uniquely suited to produce acetyl-CoA-derived chemicals. However, the lack of well-characterized genomic integration sites has impeded the metabolic engineering of this organism. Here we report a set of computationally predicted and experimentally validated chromosomal integration sites in R. toruloides. We first implemented an in silico platform by integrating essential gene information and transcriptomic data to identify candidate sites that meet stringent criteria. We then conducted a full experimental characterization of these sites, assessing integration efficiency, gene expression levels, impact on cell growth, and long-term expression stability. Among the identified sites, 12 exhibited integration efficiencies of 50% or higher, making them sufficient for most metabolic engineering applications. Using selected high-efficiency sites, we achieved simultaneous double and triple integrations and efficiently integrated long functional pathways (up to 14.7 kb). Additionally, we developed a new inducible marker recycling system that allows multiple rounds of integration at our characterized sites. We validated this system by performing five sequential rounds of GFP integration and three sequential rounds of MaFAR integration for fatty alcohol production, demonstrating, for the first time, precise gene copy number tuning in R. toruloides. These characterized integration sites should significantly advance metabolic engineering efforts and future genetic tool development in R. toruloides.

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