Metabolic engineering最新文献

Psilocybin biosynthesis enhancement through gene source optimization

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-04-16 DOI: 10.1016/j.ymben.2025.04.003

Madeleine R. Keller , Madeline G. McKinney , Abhishek K. Sen , Felicia G. Guagliardo , Elle B. Hellwarth , Khondokar Nowshin Islam , Nicholas A. Kaplan , William J. Gibbons Jr. , Grace E. Kemmerly , Chance Meers , Xin Wang , J. Andrew Jones

Psilocybin, the prodrug to the psychoactive compound in ‘magic’ mushrooms, is currently being studied in clinical trials as a treatment for severe mental health conditions, such as depression and anxiety. Previous reports of psilocybin biosynthesis as reconstituted in E. coli reported maximum titers of 1.16 g/L, exclusively using genes from the most common recreationally used mushroom, Psilocybe cubensis. This study explores the effect of gene species variation on psilocybin and baeocystin production using various exogenous genes sourced from psilocybin-producing mushrooms Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cyanescens, and Gymnopilus dilepis. The psiD and psiK genes sourced from P. cubensis demonstrated unequivocally superior performance, while psiM showed varied production levels of psilocybin and the pathway intermediate baeocystin with changes in gene source. Strains containing a psiM gene sourced from Psilocybe cyanescens demonstrated a higher degree of baeocystin selectivity as compared to other psiM genes, demonstrating a key difference between species. Most notably, the strain Gymdi30, containing psiM sourced from G. dilepis, achieved a psilocybin titer of 1.46 ± 0.13 g/L, the highest reported to date. Comparative proteomic analysis of Gymdi30 during periods of high and low productivity was also performed to investigate bottlenecks in cellular metabolism, which could be limiting strain performance. This work represents a significant improvement in psilocybin biosynthesis, a key step towards the development of a biosynthetic manufacturing route for psilocybin.

{"title":"Psilocybin biosynthesis enhancement through gene source optimization","authors":"Madeleine R. Keller , Madeline G. McKinney , Abhishek K. Sen , Felicia G. Guagliardo , Elle B. Hellwarth , Khondokar Nowshin Islam , Nicholas A. Kaplan , William J. Gibbons Jr. , Grace E. Kemmerly , Chance Meers , Xin Wang , J. Andrew Jones","doi":"10.1016/j.ymben.2025.04.003","DOIUrl":"10.1016/j.ymben.2025.04.003","url":null,"abstract":"<div><div>Psilocybin, the prodrug to the psychoactive compound in ‘magic’ mushrooms, is currently being studied in clinical trials as a treatment for severe mental health conditions, such as depression and anxiety. Previous reports of psilocybin biosynthesis as reconstituted in <em>E. coli</em> reported maximum titers of 1.16 g/L, exclusively using genes from the most common recreationally used mushroom, <em>Psilocybe cubensis</em>. This study explores the effect of gene species variation on psilocybin and baeocystin production using various exogenous genes sourced from psilocybin-producing mushrooms <em>Psilocybe cubensis</em>, <em>Psilocybe cyanescens</em>, <em>Panaeolus cyanescens</em>, and <em>Gymnopilus dilepis</em>. The <em>psiD</em> and <em>psiK</em> genes sourced from <em>P. cubensis</em> demonstrated unequivocally superior performance, while <em>psiM</em> showed varied production levels of psilocybin and the pathway intermediate baeocystin with changes in gene source. Strains containing a <em>psiM</em> gene sourced from <em>Psilocybe cyanescens</em> demonstrated a higher degree of baeocystin selectivity as compared to other <em>psiM</em> genes, demonstrating a key difference between species. Most notably, the strain <em>Gymdi30</em>, containing <em>psiM</em> sourced from <em>G. dilepis</em>, achieved a psilocybin titer of 1.46 ± 0.13 g/L, the highest reported to date. Comparative proteomic analysis of Gymdi30 during periods of high and low productivity was also performed to investigate bottlenecks in cellular metabolism, which could be limiting strain performance. This work represents a significant improvement in psilocybin biosynthesis, a key step towards the development of a biosynthetic manufacturing route for psilocybin.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 119-129"},"PeriodicalIF":6.8,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143850804","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

Multi-omics driven genome-scale metabolic modeling improves viral vector yield in HEK293

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-04-11 DOI: 10.1016/j.ymben.2025.03.011

L. Zehetner , D. Széliová , B. Kraus , J.A. Hernandez Bort , J. Zanghellini

HEK293 cells are a versatile cell line extensively used in the production of recombinant proteins and viral vectors, notably Adeno-associated virus (AAV) (Bulcha et al., 2021). Despite their high transfection efficiency and adaptability to various culture conditions, challenges remain in achieving sufficient yields of active viral particles. This study presents a comprehensive multi-omics analysis of two HEK293 strains under good manufacturing practice conditions, focusing on the metabolic and cellular responses during AAV production. The investigation included lipidomic, exometabolomic, and transcriptomic profiling across different conditions and time points. Genome-scale metabolic models (GSMMs) were reconstructed for these strains to elucidate metabolic shifts and identify potential bottlenecks in AAV production. Notably, the study revealed significant differences between a High-producing (HP) and a Low-producing (LP) HEK293 strains, highlighting pseudohypoxia in the LP strain. Key findings include the identification of hypoxia-inducible factor 1-alpha (HIF-1

α

) as a critical regulator in the LP strain, linking pseudohypoxia to poor AAV productivity. Inhibition of HIF-1

α

resulted in immediate cessation of cell growth and a 2.5-fold increase in viral capsid production, albeit with a decreased number of viral genomes, impacting the full-to-empty particle ratio. This trade-off is significant because it highlights a key challenge in AAV production: achieving a balance between capsid assembly and genome packaging to optimize the yield of functional viral vectors. Overall this suggests that while HIF-1

α

inhibition enhances capsid assembly, it simultaneously hampers nucleotide synthesis via the pentose phosphate pathway (PPP), necessary for nucleotide synthesis, and therefore for AAV genome replication.

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引用次数: 0

Carbon-conserving bioproduction of malate in an E. coli-based cell-free system

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-04-08 DOI: 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":"2025-04-08","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}

引用次数: 0

Enhancing oil feedstock utilization for high-yield low-carbon polyhydroxyalkanoates industrial bioproduction 提高石油原料利用率，促进高产低碳聚羟基烷酸酯工业生物生产

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-04-04 DOI: 10.1016/j.ymben.2025.04.001

Tianyu Jiang , Tingting Tan , Zhiyuan Zong , Dingding Fan , Jianxin Wang , Yanci Qiu , Xin Teng , Haoqian M. Zhang , Chitong Rao

Polyhydroxyalkanoates (PHAs) are biodegradable and environmentally sustainable alternatives to conventional plastics, yet their adoption has been hindered by the high production costs and scalability challenges. This study employed unbiased genomics approaches to engineer Cupriavidus necator H16, an industrial strain with intrinsic capabilities for PHA biosynthesis, for enhanced utilization of oil-based feedstocks, including food-grade palm oil and crude waste cooking oil. The engineered strain demonstrated significant improvements in PHA production, achieving a 264 g/L yield (25.4 % increase) and a 100 g/g conversion rate of palm oil (12 % increase) in 60-h fed-batch fermentation at 150 m³ production scale, the highest yield and conversion rate using food-grade palm oil as carbon source reported to the best of our knowledge. Notably, the carbon footprint of PHA production was reduced by 29.7 % using the engineered strain, and could be further reduced by adopting waste cooking oil. Mechanistic studies revealed that the H16_A3043/H16_A3044 two-component system plays a central role in regulating stress response and biogenesis, the deletion of which unlocked the regulatory constraint and enhanced oil feedstock consumption. This mutation, supplemented with the necessary lipase engineering as revealed during the scale-up troubleshooting, confered higher PHA production in a robust fermentation process scalable through 0.5 L, 200 L, 15 m³ and 150 m³. Additionally, the engineered strain demonstrated efficient utilization of waste cooking oil for PHA production. This study bridges laboratory-scale advancements and industrial feasibility, demonstrating a scalable, sustainable, and economically viable pathway for biopolymer production, contributing to the global shift toward a circular bioeconomy.

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引用次数: 0

Encapsulation of select violacein pathway enzymes in the 1,2-propanediol utilization bacterial microcompartment to divert pathway flux

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-04-03 DOI: 10.1016/j.ymben.2025.03.017

Brett Jeffrey Palmero , Emily Gamero , Niall M. Mangan , Danielle Tullman-Ercek

A continual goal in metabolic engineering is directing pathway flux to desired products and avoiding loss of pathway intermediates to competing pathways. Encapsulation of the pathway is a possible solution, as it creates a diffusion barrier between pathway intermediates and competing enzymes. It is hypothesized that bacteria use organelles known as bacterial microcompartments - proteinaceous shells encapsulating a metabolic pathway - for this purpose. We aim to determine to what degree this hypothesized benefit is conferred to encapsulated pathways. To this end, we used bacterial microcompartments to encapsulate select enzymes from the violacein pathway, which is composed of five enzymes that produce violacein as the main product and deoxyviolacein as a side product. Importantly, we studied the pathway in a cell-free context, allowing us to hold constant the concentration of unencapsulated and encapsulated enzymes and increase our control over reaction conditions. The VioE enzyme is a branch point in that it makes the precursor for both violacein and deoxyviolacein, the VioC enzyme is required for production of deoxyviolacein, and the VioD enzyme is required for violacein production. When we encapsulated VioE and VioC and left VioD unencapsulated, the product profile shifted toward deoxyviolacein and away from violacein compared to when VioC and VioD were both unencapsulated. This work provides the first fully quantitative evidence that microcompartment-based encapsulation can be used to divert pathway flux to the encapsulated pathway. It provides insight into why certain pathways are encapsulated natively and could be leveraged for metabolic engineering applications.

{"title":"Encapsulation of select violacein pathway enzymes in the 1,2-propanediol utilization bacterial microcompartment to divert pathway flux","authors":"Brett Jeffrey Palmero , Emily Gamero , Niall M. Mangan , Danielle Tullman-Ercek","doi":"10.1016/j.ymben.2025.03.017","DOIUrl":"10.1016/j.ymben.2025.03.017","url":null,"abstract":"<div><div>A continual goal in metabolic engineering is directing pathway flux to desired products and avoiding loss of pathway intermediates to competing pathways. Encapsulation of the pathway is a possible solution, as it creates a diffusion barrier between pathway intermediates and competing enzymes. It is hypothesized that bacteria use organelles known as bacterial microcompartments - proteinaceous shells encapsulating a metabolic pathway - for this purpose. We aim to determine to what degree this hypothesized benefit is conferred to encapsulated pathways. To this end, we used bacterial microcompartments to encapsulate select enzymes from the violacein pathway, which is composed of five enzymes that produce violacein as the main product and deoxyviolacein as a side product. Importantly, we studied the pathway in a cell-free context, allowing us to hold constant the concentration of unencapsulated and encapsulated enzymes and increase our control over reaction conditions. The VioE enzyme is a branch point in that it makes the precursor for both violacein and deoxyviolacein, the VioC enzyme is required for production of deoxyviolacein, and the VioD enzyme is required for violacein production. When we encapsulated VioE and VioC and left VioD unencapsulated, the product profile shifted toward deoxyviolacein and away from violacein compared to when VioC and VioD were both unencapsulated. This work provides the first fully quantitative evidence that microcompartment-based encapsulation can be used to divert pathway flux to the encapsulated pathway. It provides insight into why certain pathways are encapsulated natively and could be leveraged for metabolic engineering applications.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 91-102"},"PeriodicalIF":6.8,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143788669","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

Plasmid-based electroporation for efficient genetic engineering in immortalized T lymphocytes 基于质粒的电穿孔技术用于永生 T 淋巴细胞的高效基因工程。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-04-02 DOI: 10.1016/j.ymben.2025.03.019

Yu-Qing Xie , Martin Fussenegger

The recent clinical success of genetically modified T-cell therapies underscores the urgent need to accelerate fundamental studies and functional screening methods in T lymphocytes. However, a facile and cost-effective method for efficient genetic engineering of T-cells remains elusive. Current approaches often rely on viral transduction, which is labor-intensive and requires stringent biosafety measures. Plasmid-based electroporation presents an affordable alternative, but remains underexplored in T-cells. Moreover, the availability of prototypical T-cell lines is limited. Here, we address these challenges by focusing on two immortalized murine T-cell lines, HT-2 and CTLL-2, which recapitulate key characteristics of primary T-cells, including cytotoxicity and cytokine-dependent proliferation. Alongside the widely used Jurkat T-cell line, HT-2 and CTLL-2 were successfully transfected with single or multiple genes with high efficiencies by means of optimized electroporation in a cuvette-based system. Notably, optimization of plasmid constructs enabled the delivery of large gene-of-interest (GOI) cargos of up to 6.5 kilobase pairs, as well as stable integration of a GOI into the genome via the Sleeping Beauty transposon system. We also developed advanced methodologies for CRISPR/Cas9-mediated gene editing in immortalized T lymphocytes, achieving knockout efficiencies of up to 97 % and homology-directed repair (HDR)-based targeted knock-in efficiencies of up to 70 %. We believe this optimized plasmid-based electroporation approach will contribute to advances in basic research on lymphocyte biology, as well as providing a practical, cost-effective tool for preclinical studies of T-cell therapies.

{"title":"Plasmid-based electroporation for efficient genetic engineering in immortalized T lymphocytes","authors":"Yu-Qing Xie , Martin Fussenegger","doi":"10.1016/j.ymben.2025.03.019","DOIUrl":"10.1016/j.ymben.2025.03.019","url":null,"abstract":"<div><div>The recent clinical success of genetically modified T-cell therapies underscores the urgent need to accelerate fundamental studies and functional screening methods in T lymphocytes. However, a facile and cost-effective method for efficient genetic engineering of T-cells remains elusive. Current approaches often rely on viral transduction, which is labor-intensive and requires stringent biosafety measures. Plasmid-based electroporation presents an affordable alternative, but remains underexplored in T-cells. Moreover, the availability of prototypical T-cell lines is limited. Here, we address these challenges by focusing on two immortalized murine T-cell lines, HT-2 and CTLL-2, which recapitulate key characteristics of primary T-cells, including cytotoxicity and cytokine-dependent proliferation. Alongside the widely used Jurkat T-cell line, HT-2 and CTLL-2 were successfully transfected with single or multiple genes with high efficiencies by means of optimized electroporation in a cuvette-based system. Notably, optimization of plasmid constructs enabled the delivery of large gene-of-interest (GOI) cargos of up to 6.5 kilobase pairs, as well as stable integration of a GOI into the genome via the Sleeping Beauty transposon system. We also developed advanced methodologies for CRISPR/Cas9-mediated gene editing in immortalized T lymphocytes, achieving knockout efficiencies of up to 97 % and homology-directed repair (HDR)-based targeted knock-in efficiencies of up to 70 %. We believe this optimized plasmid-based electroporation approach will contribute to advances in basic research on lymphocyte biology, as well as providing a practical, cost-effective tool for preclinical studies of T-cell therapies.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"91 ","pages":"Pages 77-90"},"PeriodicalIF":6.8,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143788672","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

Insights into the methanol utilization capacity of Y. lipolytica and improvements through metabolic engineering

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-03-28 DOI: 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":"2025-03-28","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}

引用次数: 0

BGC heteroexpression strategy for production of novel microbial secondary metabolites

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-03-28 DOI: 10.1016/j.ymben.2025.03.018

Yuanyuan Liu, Yuqi Tang, Zhiyang Fu, Wangjie Zhu, Hong Wang, Huawei Zhang

Biosynthetic gene clusters (BGCs) in microbial genomes play a crucial role in the biosynthesis of diverse secondary metabolites (SMs) with pharmaceutical potential. However, most BGCs remain silent under conventional conditions, resulting in the frequently repeated discovery of known SMs. Fortunately, in the past two decades, the heterologous expression of BGCs in genetically tractable hosts has emerged as a powerful strategy to awaken microbial metabolic pathways for making novel microbial SMs. In this review, we comprehensively delineated the development and application of this strategy, highlighting various BGC cloning and assembly techniques and their technical characteristics. We also summarized 519 novel SMs from BGC hetero-expression-derived strains and described their occurrence, bioactivity, mode of action, and biosynthetic logic. Lastly, current challenges and future perspectives for developing more efficient BGC hetero-expression strategies were discussed in this review.

引用次数: 0

Elucidation of the plant progesterone biosynthetic pathway and its application in a yeast cell factory

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-03-27 DOI: 10.1016/j.ymben.2025.03.016

Rongsheng Li , Shuang Guo , Dong Wang , Tingting Yang , Wenhao Li , Jie Wang , Luqi Huang , Xueli Zhang , Zhubo Dai

Progesterone and its steroidal derivatives, including androgens, estrogens, glucocorticoids and mineralocorticoids are extensively utilized in pharmacotherapy, serving as predominant agents in anti-inflammatory, contraceptive, and anticancer treatments. From the 1990s to the present, scientists attempted to biosynthesize steroids such as progesterone and hydrocortisone from sugars in engineered microbial strains expressing pathway enzymes derived from animal sources. However, the low activity of the cytochrome P450 sterol side-chain cleavage (P450scc) system and their mitochondrial compartmentalization have limited the development of this route. Therefore, discovering an efficient P450scc system and developing innovative strategies will be necessary to overcome this bottleneck. Here, we elucidated the complete biosynthetic pathway of progesterone in Marsdenia tenacissima, a medicinal plant rich in steroids. The pathway comprises four enzymes, the two P450scc enzymes MtCYP108 and MtCYP150, as well as the two 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerases (HSDs) MtHSD5 and MtHSD6. Unlike their animal counterparts, the plant-derived P450scc enzymes were found to be localized to the endoplasmic reticulum in yeast and plants, and using the plant-type cytochrome P450 reductase (CPR) as electron transfer chain. The plant-derived HSDs are cytoplasmic in yeast and plants, whereas animal-derived HSDs localize to the endoplasmic reticulum. Based on this discovery, we engineered a yeast-based cell factory capable of synthesizing 1.06 g/L of progesterone from a simple carbon source. This discovery lays the groundwork for the sustainable synthesis of steroid hormone drugs through the use of plant-based systems or microbial host cells.

{"title":"Elucidation of the plant progesterone biosynthetic pathway and its application in a yeast cell factory","authors":"Rongsheng Li , Shuang Guo , Dong Wang , Tingting Yang , Wenhao Li , Jie Wang , Luqi Huang , Xueli Zhang , Zhubo Dai","doi":"10.1016/j.ymben.2025.03.016","DOIUrl":"10.1016/j.ymben.2025.03.016","url":null,"abstract":"<div><div>Progesterone and its steroidal derivatives, including androgens, estrogens, glucocorticoids and mineralocorticoids are extensively utilized in pharmacotherapy, serving as predominant agents in anti-inflammatory, contraceptive, and anticancer treatments. From the 1990s to the present, scientists attempted to biosynthesize steroids such as progesterone and hydrocortisone from sugars in engineered microbial strains expressing pathway enzymes derived from animal sources. However, the low activity of the cytochrome P450 sterol side-chain cleavage (P450scc) system and their mitochondrial compartmentalization have limited the development of this route. Therefore, discovering an efficient P450scc system and developing innovative strategies will be necessary to overcome this bottleneck. Here, we elucidated the complete biosynthetic pathway of progesterone in <em>Marsdenia tenacissima</em>, a medicinal plant rich in steroids. The pathway comprises four enzymes, the two P450scc enzymes MtCYP108 and MtCYP150, as well as the two 3β-hydroxysteroid dehydrogenase/Δ<sup>5</sup>-Δ<sup>4</sup> isomerases (HSDs) MtHSD5 and MtHSD6. Unlike their animal counterparts, the plant-derived P450scc enzymes were found to be localized to the endoplasmic reticulum in yeast and plants, and using the plant-type cytochrome P450 reductase (CPR) as electron transfer chain. The plant-derived HSDs are cytoplasmic in yeast and plants, whereas animal-derived HSDs localize to the endoplasmic reticulum. Based on this discovery, we engineered a yeast-based cell factory capable of synthesizing 1.06 g/L of progesterone from a simple carbon source. This discovery lays the groundwork for the sustainable synthesis of steroid hormone drugs through the use of plant-based systems or microbial host cells.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"90 ","pages":"Pages 197-208"},"PeriodicalIF":6.8,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143743649","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

Rhodotorula sp. as a promising host for microbial cell factories

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-03-24 DOI: 10.1016/j.ymben.2025.03.015

Baisong Tong, Yi Yu, Shuobo Shi

Rhodotorula sp. is a red yeast that has emerged as a promising host for microbial cell factories. Under specific conditions, Rhodotorula sp. can accumulate lipids that constitute over 70% of its dry cell weight, underscoring its potential in lipid compound production. Additionally, it can utilize a variety of carbon sources, including glucose, xylose, and volatile fatty acids, and exhibits high tolerance to low-cost carbon sources and industrial by-products, showcasing its excellent performance in industrial processes. Furthermore, the native mevalonate pathway of Rhodotorula sp. enables its efficient synthesis of antioxidant carotenoids and other terpenoids, which are widely applied in the food, pharmaceutical, and cosmetic industries. Due to its excellent accumulation ability of lipophilic compounds, metabolic diversity, and environmental adaptability, this review summarizes recent advances in genetic elements and metabolic engineering technologies for Rhodotorula sp., emphasizing its potential as a chassis cell factory for the production of lipids, carotenoids, and other chemicals. It also highlights key factors influencing commercial fermentation processes and concludes with challenges and solutions for further developing Rhodotorula sp. as microbial chassis.

{"title":"Rhodotorula sp. as a promising host for microbial cell factories","authors":"Baisong Tong, Yi Yu, Shuobo Shi","doi":"10.1016/j.ymben.2025.03.015","DOIUrl":"10.1016/j.ymben.2025.03.015","url":null,"abstract":"<div><div><em>Rhodotorula</em> sp. is a red yeast that has emerged as a promising host for microbial cell factories. Under specific conditions, <em>Rhodotorula</em> sp. can accumulate lipids that constitute over 70% of its dry cell weight, underscoring its potential in lipid compound production. Additionally, it can utilize a variety of carbon sources, including glucose, xylose, and volatile fatty acids, and exhibits high tolerance to low-cost carbon sources and industrial by-products, showcasing its excellent performance in industrial processes. Furthermore, the native mevalonate pathway of <em>Rhodotorula</em> sp. enables its efficient synthesis of antioxidant carotenoids and other terpenoids, which are widely applied in the food, pharmaceutical, and cosmetic industries. Due to its excellent accumulation ability of lipophilic compounds, metabolic diversity, and environmental adaptability, this review summarizes recent advances in genetic elements and metabolic engineering technologies for <em>Rhodotorula</em> sp., emphasizing its potential as a chassis cell factory for the production of lipids, carotenoids, and other chemicals. It also highlights key factors influencing commercial fermentation processes and concludes with challenges and solutions for further developing <em>Rhodotorula</em> sp. as microbial chassis.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"90 ","pages":"Pages 178-196"},"PeriodicalIF":6.8,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730738","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