Pub Date : 2025-09-01Epub Date: 2025-03-28DOI: 10.1016/j.ymben.2025.03.014
Wei Jiang , William Newell , Jingjing Liu , Lucas Coppens , Khushboo Borah Slater , Huadong Peng , David Bell , Long Liu , Victoria Haritos , Rodrigo Ledesma-Amaro
Methanol is a promising sustainable alternative feedstock for green biomanufacturing. The yeast Yarrowia lipolytica offers a versatile platform for producing a wide range of products but it cannot use methanol efficiently. In this study, we engineered Y. lipolytica to utilize methanol by overexpressing a methanol dehydrogenase, followed by the incorporation of methanol assimilation pathways from methylotrophic yeasts and bacteria. We also overexpressed the ribulose monophosphate (RuMP) and xylulose monophosphate (XuMP) pathways, which led to significant improvements in growth with methanol, reaching a consumption rate of 2.35 g/L in 24 h and a 2.68-fold increase in biomass formation. Metabolomics and Metabolite Flux Analysis confirmed methanol assimilation and revealed an increase in reducing power. The strains were further engineered to produce the valuable heterologous product resveratrol from methanol as a co-substrate. Unlike traditional methanol utilization processes, which are often resource-intensive and environmentally damaging, our findings represent a significant advance in green chemistry by demonstrating the potential of Y. lipolytica for efficient use of methanol as a co-substrate for energy, biomass, and product formation. This work not only contributes to our understanding of methanol metabolism in non-methylotrophic organisms but also paves the way for achieving efficient synthetic methylotrophy towards green biomanufacturing.
{"title":"Insights into the methanol utilization capacity of Y. lipolytica and improvements through metabolic engineering","authors":"Wei Jiang , William Newell , Jingjing Liu , Lucas Coppens , Khushboo Borah Slater , Huadong Peng , David Bell , Long Liu , Victoria Haritos , Rodrigo Ledesma-Amaro","doi":"10.1016/j.ymben.2025.03.014","DOIUrl":"10.1016/j.ymben.2025.03.014","url":null,"abstract":"<div><div>Methanol is a promising sustainable alternative feedstock for green biomanufacturing. The yeast <em>Yarrowia lipolytica</em> offers a versatile platform for producing a wide range of products but it cannot use methanol efficiently. In this study, we engineered <em>Y. lipolytica</em> to utilize methanol by overexpressing a methanol dehydrogenase, followed by the incorporation of methanol assimilation pathways from methylotrophic yeasts and bacteria. We also overexpressed the ribulose monophosphate (RuMP) and xylulose monophosphate (XuMP) pathways, which led to significant improvements in growth with methanol, reaching a consumption rate of 2.35 g/L in 24 h and a 2.68-fold increase in biomass formation. Metabolomics and Metabolite Flux Analysis confirmed methanol assimilation and revealed an increase in reducing power. The strains were further engineered to produce the valuable heterologous product resveratrol from methanol as a co-substrate. Unlike traditional methanol utilization processes, which are often resource-intensive and environmentally damaging, our findings represent a significant advance in green chemistry by demonstrating the potential of <em>Y. lipolytica</em> for efficient use of methanol as a co-substrate for energy, biomass, and product formation. This work not only contributes to our understanding of methanol metabolism in non-methylotrophic organisms but also paves the way for achieving efficient synthetic methylotrophy towards green biomanufacturing.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 30-43"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143753377","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}
Pub Date : 2025-09-01Epub Date: 2025-04-08DOI: 10.1016/j.ymben.2025.03.020
Ryan A.L. Cardiff , Shaafique Chowdhury , Widianti Sugianto , Benjamin I. Tickman , Diego Alba Burbano , Pimphan A. Meyer , Margaret Cook , Brianne King , David Garenne , Alexander S. Beliaev , Vincent Noireaux , Peralta-Yahya Pamela , James M. Carothers
Formate, a biologically accessible form of CO2, has attracted interest as a renewable feedstock for bioproduction. However, approaches are needed to investigate efficient routes for biological formate assimilation due to its toxicity and limited utilization by microorganisms. Cell-free systems hold promise due to their potential for efficient use of carbon and energy sources and compatibility with diverse feedstocks. However, bioproduction using purified cell-free systems is limited by costly enzyme purification, whereas lysate-based systems must overcome loss of flux to background reactions in the cell extract. Here, we engineer an E. coli-based system for an eight-enzyme pathway from DNA and incorporate strategies to regenerate cofactors and minimize loss of flux through background reactions. We produce the industrial di-acid malate from glycine, bicarbonate, and formate by engineering the carbon-conserving reductive TCA and formate assimilation pathways. We show that in situ regeneration of NADH drives metabolic flux towards malate, improving titer by 15-fold. Background reactions can also be reduced 6-fold by diluting the lysate following expression and introducing chemical inhibitors of competing reactions. Together, these results establish a carbon-conserving, lysate-based cell-free platform for malate production, producing 64 μM malate after 8 h. This system conserves 43 % of carbon otherwise lost as CO2 through the TCA cycle and incorporates 0.13 mol CO2 equivalents/mol glycine fed. Finally, techno-economic analysis of cell-free malate production from formate revealed that the high cost of lysate is a key challenge to the economic feasibility of the process, even assuming efficient cofactor recycling. This work demonstrates the capabilities of cell-free expression systems for both the prototyping of carbon-conserving pathways and the sustainable bioproduction of platform chemicals.
{"title":"Carbon-conserving bioproduction of malate in an E. coli-based cell-free system","authors":"Ryan A.L. Cardiff , Shaafique Chowdhury , Widianti Sugianto , Benjamin I. Tickman , Diego Alba Burbano , Pimphan A. Meyer , Margaret Cook , Brianne King , David Garenne , Alexander S. Beliaev , Vincent Noireaux , Peralta-Yahya Pamela , James M. Carothers","doi":"10.1016/j.ymben.2025.03.020","DOIUrl":"10.1016/j.ymben.2025.03.020","url":null,"abstract":"<div><div>Formate, a biologically accessible form of CO<sub>2</sub>, has attracted interest as a renewable feedstock for bioproduction. However, approaches are needed to investigate efficient routes for biological formate assimilation due to its toxicity and limited utilization by microorganisms. Cell-free systems hold promise due to their potential for efficient use of carbon and energy sources and compatibility with diverse feedstocks. However, bioproduction using purified cell-free systems is limited by costly enzyme purification, whereas lysate-based systems must overcome loss of flux to background reactions in the cell extract. Here, we engineer an <em>E. coli</em>-based system for an eight-enzyme pathway from DNA and incorporate strategies to regenerate cofactors and minimize loss of flux through background reactions. We produce the industrial di-acid malate from glycine, bicarbonate, and formate by engineering the carbon-conserving reductive TCA and formate assimilation pathways. We show that <em>in situ</em> regeneration of NADH drives metabolic flux towards malate, improving titer by 15-fold. Background reactions can also be reduced 6-fold by diluting the lysate following expression and introducing chemical inhibitors of competing reactions. Together, these results establish a carbon-conserving, lysate-based cell-free platform for malate production, producing 64 μM malate after 8 h. This system conserves 43 % of carbon otherwise lost as CO<sub>2</sub> through the TCA cycle and incorporates 0.13 mol CO<sub>2</sub> equivalents/mol glycine fed. Finally, techno-economic analysis of cell-free malate production from formate revealed that the high cost of lysate is a key challenge to the economic feasibility of the process, even assuming efficient cofactor recycling. This work demonstrates the capabilities of cell-free expression systems for both the prototyping of carbon-conserving pathways and the sustainable bioproduction of platform chemicals.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 59-76"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143825983","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}
Pub Date : 2025-09-01Epub Date: 2025-06-10DOI: 10.1016/j.ymben.2025.06.005
Daniel J. Willard , Robert M. Kelly
A genome-scale metabolic model was developed to explore metabolic engineering strategies for thermoacidophilic archaea, with a focus on the genetically tractable Sulfolobus acidocaldarius (Topt 75 °C, pHopt 2.5). S. acidocaldarius is natively neither an autotroph nor a sulfur oxidizer, although its genome suggests that this might have been the case at some evolutionary point. Comparative genomics provided insights into key genes and pathways missing from S. acidocaldarius necessary for chemolithotrophy. Growth data for the chemolithotrophic sulfur oxidizer, Sulfurisphaera ohwakuensis (Topt 85 °C, pHopt 2.0), provided metabolic data to inform model development. Previous metabolic engineering efforts enabled sulfur oxidation by S. acidocaldarius, albeit at levels below native sulfur oxidizers. Model analysis pointed to active sulfur transport as a key missing complement to passive diffusion. Modelling results predicted that sulfur oxidation could drive production of a bio-based chemical, acetone, in engineered strains of S. acidocaldarius with concomitant fixation of CO2 into product via the 3-Hydroxybutyrate/4-Hydroxybutyrate cycle. The findings here provide new insights into the basis for thermoacidophile chemolithotrophy and motivate further efforts to develop S. acidocaldarius into a valuable metabolic engineering platform.
{"title":"Metabolic engineering in Hot Acid: Strategies enabling chemolithotrophy in thermoacidophilic archaea","authors":"Daniel J. Willard , Robert M. Kelly","doi":"10.1016/j.ymben.2025.06.005","DOIUrl":"10.1016/j.ymben.2025.06.005","url":null,"abstract":"<div><div>A genome-scale metabolic model was developed to explore metabolic engineering strategies for thermoacidophilic archaea, with a focus on the genetically tractable <em>Sulfolobus acidocaldarius</em> (T<sub>opt</sub> 75 °C, pH<sub>opt</sub> 2.5). <em>S. acidocaldarius</em> is natively neither an autotroph nor a sulfur oxidizer, although its genome suggests that this might have been the case at some evolutionary point. Comparative genomics provided insights into key genes and pathways missing from <em>S. acidocaldarius</em> necessary for chemolithotrophy. Growth data for the chemolithotrophic sulfur oxidizer, <em>Sulfurisphaera ohwakuensis</em> (T<sub>opt</sub> 85 °C, pH<sub>opt</sub> 2.0), provided metabolic data to inform model development. Previous metabolic engineering efforts enabled sulfur oxidation by <em>S. acidocaldarius</em>, albeit at levels below native sulfur oxidizers. Model analysis pointed to active sulfur transport as a key missing complement to passive diffusion. Modelling results predicted that sulfur oxidation could drive production of a bio-based chemical, acetone, in engineered strains of <em>S. acidocaldarius</em> with concomitant fixation of CO<sub>2</sub> into product via the 3-Hydroxybutyrate/4-Hydroxybutyrate cycle. The findings here provide new insights into the basis for thermoacidophile chemolithotrophy and motivate further efforts to develop <em>S. acidocaldarius</em> into a valuable metabolic engineering platform.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 366-378"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144263771","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}
Pub Date : 2025-09-01Epub Date: 2025-04-25DOI: 10.1016/j.ymben.2025.04.005
Bradley Priem , Xiangchen Cai , Yu-Jun Hong , Karl Gilmore , Zijun Deng , Sabrina Chen , Harnish Mukesh Naik , Michael J. Betenbaugh , Maciek R. Antoniewicz
Chinese Hamster Ovary (CHO) cells are widely used in the pharmaceutical industry to produce therapeutic proteins. Increasing the productivity of CHO cells through media development and genetic engineering is a significant industry objective. Past research demonstrated the benefits of modulating fatty acid composition of CHO cells through genetic engineering. In this study, we describe an alternative approach to modulate fatty acid composition by directly feeding high levels of fatty acids in CHO cell culture. To accomplish this, we developed and optimized a pharmaceutically relevant feeding strategy using methyl-β-cyclodextrin (MBCD) to solubilize fatty acids. To quantify fatty acid composition of CHO cells, a new GC-MS protocol was developed and validated. In fed batch cultures, we found that the degree of saturation of fatty acids in CHO cell mass, i.e. the relative abundances of saturated, monounsaturated and polyunsaturated fatty acids, can be controlled by the choice of fatty acid supplement and feeding strategy. Feeding unsaturated fatty acids such as palmitoleic acid, oleic acid, and linoleic acid had the greatest impact the fatty acid composition of CHO cells, increasing their respective abundances in cell mass by upwards of 25x, 1.5x, and 50x, respectively. 13C-Tracing further revealed that the supplemented fatty acids were involved in a range of elongation, desaturation, and β-oxidation reactions to yield both common and uncommon fatty acids such as vaccenic acid and hypogeic acid. Finally, we show that CHO-K1 and CHO-GS cells take up fatty acids solubilized with MBCD at rates comparable to delivery using bovine serum albumin. Taken together, this work paves the way for new feed media formulations containing fatty acids to optimize CHO cell physiology in industrial cell cultures.
{"title":"Modulating fatty acid metabolism and composition of CHO cells by feeding high levels of fatty acids complexed using methyl-β-cyclodextrin","authors":"Bradley Priem , Xiangchen Cai , Yu-Jun Hong , Karl Gilmore , Zijun Deng , Sabrina Chen , Harnish Mukesh Naik , Michael J. Betenbaugh , Maciek R. Antoniewicz","doi":"10.1016/j.ymben.2025.04.005","DOIUrl":"10.1016/j.ymben.2025.04.005","url":null,"abstract":"<div><div>Chinese Hamster Ovary (CHO) cells are widely used in the pharmaceutical industry to produce therapeutic proteins. Increasing the productivity of CHO cells through media development and genetic engineering is a significant industry objective. Past research demonstrated the benefits of modulating fatty acid composition of CHO cells through genetic engineering. In this study, we describe an alternative approach to modulate fatty acid composition by directly feeding high levels of fatty acids in CHO cell culture. To accomplish this, we developed and optimized a pharmaceutically relevant feeding strategy using methyl-β-cyclodextrin (MBCD) to solubilize fatty acids. To quantify fatty acid composition of CHO cells, a new GC-MS protocol was developed and validated. In fed batch cultures, we found that the degree of saturation of fatty acids in CHO cell mass, i.e. the relative abundances of saturated, monounsaturated and polyunsaturated fatty acids, can be controlled by the choice of fatty acid supplement and feeding strategy. Feeding unsaturated fatty acids such as palmitoleic acid, oleic acid, and linoleic acid had the greatest impact the fatty acid composition of CHO cells, increasing their respective abundances in cell mass by upwards of 25x, 1.5x, and 50x, respectively. <sup>13</sup>C-Tracing further revealed that the supplemented fatty acids were involved in a range of elongation, desaturation, and β-oxidation reactions to yield both common and uncommon fatty acids such as vaccenic acid and hypogeic acid. Finally, we show that CHO-K1 and CHO-GS cells take up fatty acids solubilized with MBCD at rates comparable to delivery using bovine serum albumin. Taken together, this work paves the way for new feed media formulations containing fatty acids to optimize CHO cell physiology in industrial cell cultures.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 158-169"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143881601","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}
Pub Date : 2025-09-01Epub Date: 2025-05-02DOI: 10.1016/j.ymben.2025.04.004
Wei Jiang , William Newell , Jingjing Liu , Lucas Coppens , Khushboo Borah Slater , Huadong Peng , David Bell , Long Liu , Victoria Haritos , Rodrigo Ledesma-Amaro
{"title":"Corrigendum to “Insights into the methanol utilization capacity of Y. lipolytica and improvements through metabolic engineering” [Metabol. Eng. (2025) 91 30–43]","authors":"Wei Jiang , William Newell , Jingjing Liu , Lucas Coppens , Khushboo Borah Slater , Huadong Peng , David Bell , Long Liu , Victoria Haritos , Rodrigo Ledesma-Amaro","doi":"10.1016/j.ymben.2025.04.004","DOIUrl":"10.1016/j.ymben.2025.04.004","url":null,"abstract":"","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Page 170"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143899999","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}
Pub Date : 2025-09-01Epub Date: 2025-06-30DOI: 10.1016/j.ymben.2025.06.010
Yuanyuan Chen , Lianggang Huang , Tao Yu , Mingming Zhao , Junping Zhou , Lijuan Wang , Zhiqiang Liu , Yuguo Zheng
O-acetyl-L-homoserine (OAH) is a key precursor for the biosynthesis of L-methionine and various C4 compounds, with significant industrial potential. However, efficient microbial production of OAH remains challenging due to complex metabolic regulation and precursor limitations. In this study, we rationally developed a plasmid-free, non-auxotrophic Escherichia coli strain to produce OAH. We modularized the OAH synthetic pathway into L-homoserine and acetyl-CoA modules, enhanced each module individually, and identified a highly efficient L-homoserine O-acetyltransferase (MetX) from Cyclobacterium marinum. Using small RNA screening, we pinpointed critical metabolic nodes and fine-tuned the pathway flux through promoter engineering and regulatory elements. Notably, we balanced the acetyl-CoA and L-homoserine synthesis with moderate expression of pyruvate carboxylase, weakened the TCA cycle by modulating citrate synthase and the branched-chain amino acid pathway by attenuating BCAA aminotransferase, thereby redirecting carbon flux towards OAH production. Additionally, we optimized the threonine attenuator for dynamic regulation of the threonine pathway and enhanced intracellular ATP turnover. Under a two-stage pH control fermentation strategy, the final plasmid-free and non-auxotrophic strain OAH37 achieved a titer of 94.1 g/L OAH, with a yield of 0.42 g/g glucose and a productivity of 1.37 g/L/h. Our work demonstrates the potential of metabolic engineering strategies for efficient microbial synthesis of OAH, providing a foundation for industrial-scale production of this important precursor.
o -乙酰- l-高丝氨酸（OAH）是生物合成l-蛋氨酸和各种C4化合物的关键前体，具有重要的工业潜力。然而，由于复杂的代谢调节和前体的限制，高效的微生物生产OAH仍然具有挑战性。在这项研究中，我们合理地开发了一种无质粒、非营养大肠杆菌菌株来生产OAH。我们将OAH合成途径模块化为l-高丝氨酸和乙酰辅酶a模块，并分别对每个模块进行增强，从海洋环杆菌中鉴定出高效的l-高丝氨酸o -乙酰转移酶（MetX）。通过小RNA筛选，我们确定了关键的代谢节点，并通过启动子工程和调控元件微调了途径通量。值得注意的是，我们通过适度表达丙酮酸羧化酶来平衡乙酰辅酶a和l-高丝氨酸的合成，通过减弱BCAA转氨酶来调节柠檬酸合成酶和支链氨基酸途径，从而减弱TCA循环，从而将碳通量重定向到OAH的产生。此外，我们优化了苏氨酸衰减器，以动态调节苏氨酸途径并增强细胞内ATP的周转。在两阶段pH控制发酵策略下，最终无质粒、非营养不良菌株OAH37的OAH滴度为94.1 g/L，产率为0.42 g/g葡萄糖，产率为1.37 g/L/h。我们的工作证明了代谢工程策略在高效微生物合成OAH方面的潜力，为这种重要前体的工业规模生产提供了基础。
{"title":"Tailoring Escherichia coli for high-yield production of O-acetyl-L-homoserine through multi-node metabolic regulation","authors":"Yuanyuan Chen , Lianggang Huang , Tao Yu , Mingming Zhao , Junping Zhou , Lijuan Wang , Zhiqiang Liu , Yuguo Zheng","doi":"10.1016/j.ymben.2025.06.010","DOIUrl":"10.1016/j.ymben.2025.06.010","url":null,"abstract":"<div><div>O-acetyl-L-homoserine (OAH) is a key precursor for the biosynthesis of L-methionine and various C4 compounds, with significant industrial potential. However, efficient microbial production of OAH remains challenging due to complex metabolic regulation and precursor limitations. In this study, we rationally developed a plasmid-free, non-auxotrophic <em>Escherichia coli</em> strain to produce OAH. We modularized the OAH synthetic pathway into L-homoserine and acetyl-CoA modules, enhanced each module individually, and identified a highly efficient L-homoserine O-acetyltransferase (MetX) from <em>Cyclobacterium marinum</em>. Using small RNA screening, we pinpointed critical metabolic nodes and fine-tuned the pathway flux through promoter engineering and regulatory elements. Notably, we balanced the acetyl-CoA and L-homoserine synthesis with moderate expression of pyruvate carboxylase, weakened the TCA cycle by modulating citrate synthase and the branched-chain amino acid pathway by attenuating BCAA aminotransferase, thereby redirecting carbon flux towards OAH production. Additionally, we optimized the threonine attenuator for dynamic regulation of the threonine pathway and enhanced intracellular ATP turnover. Under a two-stage pH control fermentation strategy, the final plasmid-free and non-auxotrophic strain OAH37 achieved a titer of 94.1 g/L OAH, with a yield of 0.42 g/g glucose and a productivity of 1.37 g/L/h. Our work demonstrates the potential of metabolic engineering strategies for efficient microbial synthesis of OAH, providing a foundation for industrial-scale production of this important precursor.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 455-465"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144534257","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}
Pub Date : 2025-09-01Epub Date: 2025-05-02DOI: 10.1016/j.ymben.2025.04.008
Cody Kamoku , Pranav Bhavaraju , Collin Travis , Luis Taquillo , David R. Nielsen
Cyanobacteria represent promising biocatalysts for producing carbohydrates, including sorbitol, a naturally-occurring, fermentable sugar alcohol with conventional uses as a sweetener, pharmaceutical additive, and biodegradable plasticizer. Previously, Synechocystis sp. PCC 6803 was engineered to produce sorbitol, reaching a final titer of 2.3 g/L after 18 days. To improve upon this performance, sorbitol production was herein engineered in the faster growing strain Synechococcus sp. PCC 7002. Upon introducing the sorbitol biosynthetic pathway, up to 500 mg/L sorbitol was initially produced after seven days. However, due to the initial use of two highly promiscuous sugar phosphatase variants, this also resulted in the unwanted co-production of ribose and growth inhibition due to depletion of ribose-5-phosphate from the Calvin cycle. This off-target effect was ultimately mitigated via the discovery that mannitol-1-phosphate phosphatase from Eimeria tenella also dephosphorylates sorbitol-6-phosphate to sorbitol with greater specificity, leading to improved growth and sorbitol production. Next, two bottleneck enzymes in the Calvin cycle, namely fructose-bisphosphate aldolase (FBA) and bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (BiBPase), were overexpressed both individually and in combination, resulting in sorbitol production up to 1.3 g/L. Finally, upon optimizing the culture media to address nutrient limitation, the final strain produced up to 3.6 g/L sorbitol in nine days, respectively representing 1.5- and 3-fold increases in titer and productivity relative to previously-engineered Synechocystis sp. PCC 6803.
{"title":"Photosynthetic sorbitol production in Synechococcus sp. PCC 7002 is enhanced by addressing phosphatase promiscuity, nutrient availability and Calvin cycle bottlenecks","authors":"Cody Kamoku , Pranav Bhavaraju , Collin Travis , Luis Taquillo , David R. Nielsen","doi":"10.1016/j.ymben.2025.04.008","DOIUrl":"10.1016/j.ymben.2025.04.008","url":null,"abstract":"<div><div>Cyanobacteria represent promising biocatalysts for producing carbohydrates, including sorbitol, a naturally-occurring, fermentable sugar alcohol with conventional uses as a sweetener, pharmaceutical additive, and biodegradable plasticizer. Previously, <em>Synechocystis</em> sp. PCC 6803 was engineered to produce sorbitol, reaching a final titer of 2.3 g/L after 18 days. To improve upon this performance, sorbitol production was herein engineered in the faster growing strain <em>Synechococcus</em> sp. PCC 7002. Upon introducing the sorbitol biosynthetic pathway, up to 500 mg/L sorbitol was initially produced after seven days. However, due to the initial use of two highly promiscuous sugar phosphatase variants, this also resulted in the unwanted co-production of ribose and growth inhibition due to depletion of ribose-5-phosphate from the Calvin cycle. This off-target effect was ultimately mitigated via the discovery that mannitol-1-phosphate phosphatase from <em>Eimeria tenella</em> also dephosphorylates sorbitol-6-phosphate to sorbitol with greater specificity, leading to improved growth and sorbitol production. Next, two bottleneck enzymes in the Calvin cycle, namely fructose-bisphosphate aldolase (FBA) and bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (BiBPase), were overexpressed both individually and in combination, resulting in sorbitol production up to 1.3 g/L. Finally, upon optimizing the culture media to address nutrient limitation, the final strain produced up to 3.6 g/L sorbitol in nine days, respectively representing 1.5- and 3-fold increases in titer and productivity relative to previously-engineered <em>Synechocystis</em> sp. PCC 6803.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 181-191"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143911542","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}
Pub Date : 2025-09-01Epub Date: 2025-06-02DOI: 10.1016/j.ymben.2025.06.001
Maofang Teng , Juan Zhang , Jingwen Zhou , Jianghua Li , Guocheng Du , Jian Chen , Guoqiang Zhang
The protein-glutaminase (PG, EC 3.5.1.44) specifically targets glutamine residues in proteins and peptides, and has significant potential for enhancing the functional characteristics and processing efficiency of plant proteins. However, natural PG production faces challenges such as low enzymatic yield and difficult genetic manipulation. To address these challenges, a novel self-activating PG expression system was developed in Bacillus subtilis. First, pro-PG (PPG)-activated proteases were identified in B. subtilis by constructing a series of engineered strains. Second, the co-expression of PPG and PPG-activated protease in B. subtilis WB800 for mature PG (mPG) production was analyzed, and it was found that the supply and activation of PPG during fermentation was insufficient. Therefore, the gene expression components of PPG and protease, including the promoter and RBS, were further optimized. In addition, the key genes of the maltose metabolic pathway were knocked out, and the engineered strain W8ΔM2-AE-Pmal380 showed the highest capacity for PG production. Finally, a 53.0 U/mL mPG yield was achieved in a 5-L bioreactor within 64 h. This study establishes an efficient platform for industrial PG production and provides a reference for the expression and activation of other proenzymes.
{"title":"Regulation of proenzyme activation and metabolic engineering for protein-glutaminase production in Bacillus subtilis","authors":"Maofang Teng , Juan Zhang , Jingwen Zhou , Jianghua Li , Guocheng Du , Jian Chen , Guoqiang Zhang","doi":"10.1016/j.ymben.2025.06.001","DOIUrl":"10.1016/j.ymben.2025.06.001","url":null,"abstract":"<div><div>The protein-glutaminase (PG, EC 3.5.1.44) specifically targets glutamine residues in proteins and peptides, and has significant potential for enhancing the functional characteristics and processing efficiency of plant proteins. However, natural PG production faces challenges such as low enzymatic yield and difficult genetic manipulation. To address these challenges, a novel self-activating PG expression system was developed in <em>Bacillus subtilis</em>. First, pro-PG (PPG)-activated proteases were identified in <em>B. subtilis</em> by constructing a series of engineered strains. Second, the co-expression of PPG and PPG-activated protease in <em>B. subtilis</em> WB800 for mature PG (mPG) production was analyzed, and it was found that the supply and activation of PPG during fermentation was insufficient. Therefore, the gene expression components of PPG and protease, including the promoter and RBS, were further optimized. In addition, the key genes of the maltose metabolic pathway were knocked out, and the engineered strain W8ΔM2-AE-Pmal380 showed the highest capacity for PG production. Finally, a 53.0 U/mL mPG yield was achieved in a 5-L bioreactor within 64 h. This study establishes an efficient platform for industrial PG production and provides a reference for the expression and activation of other proenzymes.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 336-346"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144225885","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}
Pub Date : 2025-09-01Epub Date: 2025-06-16DOI: 10.1016/j.ymben.2025.06.007
Yusong Zou, Xinyu Gong, Jianli Zhang, Qi Gan, Yajun Yan
Genetic regulation tools have been examined for their ability to enable sophisticated dynamic control of biosynthesis in microbial cell factories, enhancing the production performance of valuable compounds. However, most genetic tools are pathway- or intermediate-specific, hindering their broad applicability in synthetic biology. Moreover, their potential to balance metabolic fluxes in central metabolism between cell growth and product formation remains under-explored, raising the question of whether they can facilitate efficient biosynthesis. To answer this, we established the PdhR biosensor system that responds to pyruvate to dynamically regulate metabolic flux distribution in central metabolism. In this study, we first characterized the dose response of PdhR biosensor system by screening multiple PdhR homologs derived from various microorganisms. Computational analysis further guided the identification of key factors contributing to their functional differences, enabling the optimization of biosensor properties through site-directed mutagenesis. As proof of concept, we employed our biosensor system to improve the biosynthesis of trehalose and 4-hydroxycoumarin (4HC), respectively. Specifically, trehalose titer increased to 3.72 g/L, which is 2.33-fold higher than the control group. In addition, we improved the 4HC titer to 491.5 mg/L, which possessed a 1.63-fold increase over the static strategy. In summary, the established central metabolism-responsive biosensor system underlined the necessity of metabolic flux distribution and validated its broad applicability in the biosynthesis of central metabolism-derived compounds.
{"title":"Engineering genetic circuits for dynamic control of central metabolism","authors":"Yusong Zou, Xinyu Gong, Jianli Zhang, Qi Gan, Yajun Yan","doi":"10.1016/j.ymben.2025.06.007","DOIUrl":"10.1016/j.ymben.2025.06.007","url":null,"abstract":"<div><div>Genetic regulation tools have been examined for their ability to enable sophisticated dynamic control of biosynthesis in microbial cell factories, enhancing the production performance of valuable compounds. However, most genetic tools are pathway- or intermediate-specific, hindering their broad applicability in synthetic biology. Moreover, their potential to balance metabolic fluxes in central metabolism between cell growth and product formation remains under-explored, raising the question of whether they can facilitate efficient biosynthesis. To answer this, we established the PdhR biosensor system that responds to pyruvate to dynamically regulate metabolic flux distribution in central metabolism. In this study, we first characterized the dose response of PdhR biosensor system by screening multiple PdhR homologs derived from various microorganisms. Computational analysis further guided the identification of key factors contributing to their functional differences, enabling the optimization of biosensor properties through site-directed mutagenesis. As proof of concept, we employed our biosensor system to improve the biosynthesis of trehalose and 4-hydroxycoumarin (4HC), respectively. Specifically, trehalose titer increased to 3.72 g/L, which is 2.33-fold higher than the control group. In addition, we improved the 4HC titer to 491.5 mg/L, which possessed a 1.63-fold increase over the static strategy. In summary, the established central metabolism-responsive biosensor system underlined the necessity of metabolic flux distribution and validated its broad applicability in the biosynthesis of central metabolism-derived compounds.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 405-414"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144307991","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}
Pub Date : 2025-09-01Epub Date: 2025-05-13DOI: 10.1016/j.ymben.2025.05.004
Dongpil Lee , Hyemin Park , Jae-Eung Kim , Yeonsoo Kim , Joo Hyun Park , Hyesoo Lee , Byoung Hoon Yoon , Boyoung Han , Joon Young Jung , Seungwoo Cha , Peter Lee , Ji-Sook Hahn
Climate change is reducing crop yields and increasing price volatility for commodities like cocoa and palm oil, thereby driving the need for sustainable alternatives such as microbial lipid production. The oleaginous yeast Yarrowia lipolytica is a promising platform for lipid synthesis. However, its lipid accumulation has traditionally relied on nitrogen limitation, posing challenges for achieving high yields under nutrient-rich conditions. In this study, we engineered Y. lipolytica to enhance lipid accumulation and productivity in nutrient-rich environments. Key modifications included deleting MHY1 to prevent filamentous growth, overexpressing triacylglycerol (TAG) biosynthetic genes, disrupting fatty acid degradation, and redirecting phosphatidic acid flux toward TAG biosynthesis by reducing phospholipid production through OPI3 deletion and CDS1 mutation. Furthermore, deletion of CEX1 to block citrate excretion significantly enhanced lipid accumulation. The resulting strain, CJ0415, achieved a lipid production of 54.6 g/L with a lipid content of 45.8 % and a record lipid productivity of 2.06 g/L/h under nutrient-rich conditions in a 5-L fermenter, representing a 2.6-fold increase compared to nitrogen-limited conditions. These findings underscore the potential of Y. lipolytica as a robust platform for scalable, industrial lipid production under nutrient-rich conditions.
{"title":"Engineering Yarrowia lipolytica for enhanced lipid productivity in nutrient-rich conditions: A scalable approach to microbial lipid production","authors":"Dongpil Lee , Hyemin Park , Jae-Eung Kim , Yeonsoo Kim , Joo Hyun Park , Hyesoo Lee , Byoung Hoon Yoon , Boyoung Han , Joon Young Jung , Seungwoo Cha , Peter Lee , Ji-Sook Hahn","doi":"10.1016/j.ymben.2025.05.004","DOIUrl":"10.1016/j.ymben.2025.05.004","url":null,"abstract":"<div><div>Climate change is reducing crop yields and increasing price volatility for commodities like cocoa and palm oil, thereby driving the need for sustainable alternatives such as microbial lipid production. The oleaginous yeast <em>Yarrowia lipolytica</em> is a promising platform for lipid synthesis. However, its lipid accumulation has traditionally relied on nitrogen limitation, posing challenges for achieving high yields under nutrient-rich conditions. In this study, we engineered <em>Y. lipolytica</em> to enhance lipid accumulation and productivity in nutrient-rich environments. Key modifications included deleting <em>MHY1</em> to prevent filamentous growth, overexpressing triacylglycerol (TAG) biosynthetic genes, disrupting fatty acid degradation, and redirecting phosphatidic acid flux toward TAG biosynthesis by reducing phospholipid production through <em>OPI3</em> deletion and <em>CDS1</em> mutation. Furthermore, deletion of <em>CEX1</em> to block citrate excretion significantly enhanced lipid accumulation. The resulting strain, CJ0415, achieved a lipid production of 54.6 g/L with a lipid content of 45.8 % and a record lipid productivity of 2.06 g/L/h under nutrient-rich conditions in a 5-L fermenter, representing a 2.6-fold increase compared to nitrogen-limited conditions. These findings underscore the potential of <em>Y. lipolytica</em> as a robust platform for scalable, industrial lipid production under nutrient-rich conditions.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 302-312"},"PeriodicalIF":6.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144065812","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}
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