Metabolic engineering最新文献

{"title":"Precise biosynthesis of β-1,2-glucan from cellulosic materials by in vitro metabolic engineering","authors":"Yunjie Li ,&nbsp;Yujiao Lu ,&nbsp;Pingping Han ,&nbsp;Qingqing Guo ,&nbsp;Shuo Wang ,&nbsp;Zhongyi Jiang ,&nbsp;Yi-Heng P. Job Zhang","doi":"10.1016/j.ymben.2026.01.004","DOIUrl":"10.1016/j.ymben.2026.01.004","url":null,"abstract":"<div><div>The biosynthesis of polysaccharides with precisely defined structures, such as tunable degree of polymerization (DP) and low polydispersity index (PDI), remains a significant challenge in microbial cell factories due to their intricate endogenous metabolic networks. <em>In vitro</em> metabolic engineering (<em>iv</em>ME) has emerged as a promising alternative, offering simplified pathway design and easy process optimization. In this study, <em>iv</em>ME was utilized for the precise synthesis of β-1,2-glucans from β-1,4-linked cellobiose. A four-enzyme system comprising cellobiose phosphorylase, β-1,2-oligoglucan phosphorylase, glucose oxidase, and catalase operated under pH self-neutralized conditions, efficiently producing β-1,2-glucan at a concentration of 31.9 ± 0.4 g/L with a high molar yield (93.3 ± 1.3 %) and a rapid productivity of 4.0 ± 0.1 g/L/h. β-1,2-Glucans with tunable DPs (75–531) and narrow molecular weight distributions (PDI as low as 1.2) were synthesized by adjusting primer concentration, enzyme loadings, and reaction time. The low PDI values of β-1,2-glucans were attributed to the smart pathway design, the careful selection of β-1,2-oligoglucan phosphorylase, and the use of sophorose as the primer. The DP values were mainly influenced by the concentration and type of primers with sophorose outperforming glucose. This strategy of direct glycosidic bond rearrangement from β-1,4 to β-1,2 linkages without coenzymes (e.g., CoA, NAD, ATP, UTP) or external energy input provided a new route for lignocellulosic biomass utilization and significantly enhanced the capabilities of <em>iv</em>ME for the production of tailored polysaccharides.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 284-294"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145955932","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}

{"title":"Genome minimization of a Burkholderia bacterial host","authors":"Bruno S. Paulo,&nbsp;Sean B. Romanowski,&nbsp;Adjo E. Kadjo,&nbsp;Vitor B. Lourenzon,&nbsp;Alessandra S. Eustáquio","doi":"10.1016/j.ymben.2026.01.008","DOIUrl":"10.1016/j.ymben.2026.01.008","url":null,"abstract":"<div><div>Genome minimization, including the deletion of endogenous gene clusters that encode natural products, is a common strategy to improve the yield of heterologous products. We have been interested in developing <em>Burkholderia sp.</em> FERM BP-3421 as an alternative bacterial host. Instead of indiscriminately deleting gene clusters, which may have deleterious effects, we guided our efforts using transcriptomics data from production cultures. The genome of FERM BP-3421 is subdivided into two chromosomes and two plasmids. The top transcribed gene clusters were those encoding polyketide-nonribosomal peptide spliceostatins on plasmid p1 and nonribosomal peptide selethramide on chromosome 1. Deletion of the spliceostatin cluster had been shown to improve titers of the ribosomal peptide capistruin, whereas we showed that deletion of the selethramide cluster had no effect on capistruin titers. We next targeted the two endogenous plasmids using a CRISPR-Cas12a strategy, resulting in an 11 % reduction in genome size. The plasmid cured strains showed improved growth and 20–40 % increased production of capistruin depending on whether one or both plasmids were deleted. However, deletion of p2 alone negatively affected the heterologous production of two distinct polyketide-nonribosomal peptides. The p2⁻ strain produced only 5–23 % of the glidobactin A and megapolipeptin A titers compared to the wild type, respectively, whereas titers were restored to wild type levels in the p1⁻ p2⁻ strain. The observation that p2 appears to contain functions that support polyketide-nonribosomal peptide biosynthesis was unexpected and sets the stage for future studies aimed at identifying these functions and further enabling engineering efforts that may be widely applicable to other strains.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 305-314"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145995161","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}

{"title":"Novel routes for bioproduction of delta lactone aroma compounds","authors":"Sonali Srivastava ,&nbsp;Aakash Chandramouli ,&nbsp;Payal Gupta ,&nbsp;Abdur Rahman Manzer ,&nbsp;Rahul Choudhury ,&nbsp;D. Srinivasa Reddy ,&nbsp;Syed Shams Yazdani ,&nbsp;Siddhesh S. Kamat ,&nbsp;Debasisa Mohanty ,&nbsp;Vinay K. Nandicoori ,&nbsp;Rajesh S. Gokhale","doi":"10.1016/j.ymben.2026.01.005","DOIUrl":"10.1016/j.ymben.2026.01.005","url":null,"abstract":"<div><div>Delta lactones are fatty acid-derived aroma compounds that hold tremendous commercial value. As consumer demand for natural flavours continues to rise, the bioproduction of δ-lactones, including δ-decalactone and δ-dodecalactone, is attracting substantial interest. Our study brings forth a novel approach to the bioproduction of δ-lactones from glucose, deviating from existing methods that primarily rely on the biotransformation of fatty acids. The high cost of fatty acid raw material constrains the commercial viability of this traditional approach. We engineered surface-lipid producing type I polyketide synthase (PKS) from <em>Mycobacterium smegmatis</em> by incorporating macrolactone thioesterase (TE) domain. Two out of three fusion constructs produced an appropriately engineered PKS-TE fusion protein that facilitated the synthesis of δ-lactones. When grown on glucose as the sole carbon source, recombinant <em>E. coli</em> expressing the engineered PKS-TE fusion protein successfully made δ-lactones ranging from C8-C18 acyl chains. Our research further highlights the potential of <em>Mycobacterium smegmatis</em> as a cell factory for producing fatty acid-based δ-lactones. By genetically designing and engineering <em>Mycobacterium smegmatis</em> to express PKS-TE fusion protein, we achieved bioproduction of various δ-lactones. Batch fermentation of the engineered <em>E. coli</em> strain fed with 2 % glucose produced 786 mg/L of δ-dodecalactone and 444 mg/L of δ-decalactone at 120 h, underscoring the efficacy of our approach. Thus, this study is the first to demonstrate a methodology for redirecting primary metabolic intermediates towards δ-lactone biosynthesis in engineered bacteria, enabling the use of inexpensive and renewable feedstocks.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 295-304"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145962523","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}

{"title":"Engineered non-canonical reductive TCA pathway drives high-yield succinic acid biosynthesis in Yarrowia lipolytica","authors":"Huilin Tao,&nbsp;Aomei Hao,&nbsp;Xiaoyue Pan,&nbsp;Yutao Zhong,&nbsp;Zhiyong Cui,&nbsp;Qingsheng Qi","doi":"10.1016/j.ymben.2025.11.003","DOIUrl":"10.1016/j.ymben.2025.11.003","url":null,"abstract":"<div><div>The reductive tricarboxylic acid (rTCA) cycle is a crucial metabolic pathway employed in the microbial production of C4-dicarboxylic acids, especially succinic acid (SA). However, the inherent redox constraints associated with this cycle pose significant limitations on the yields of SA. Here, we address this critical bottleneck by engineering a non-canonical reductive TCA (Nc-rTCA) pathway in oleaginous yeast <em>Yarrowia lipolytica</em>. Our approach substitutes the NADH-dependent conversion of oxaloacetate to fumarate in the native rTCA cycle with an engineered cascade utilizing aspartate aminotransferase, aspartate ammonia-lyase, and glutamate dehydrogenase, effectively decoupling SA synthesis from NADH limitations. This NADPH-dependent module resulted in a remarkable 1.28-fold increase in fumarate titer. Further metabolic optimization in the engineered strain Ncr12 minimized malate byproduct formation, achieving an SA titer of 98.16 g/L with a high yield of 0.91 g/g glucose in 5-L bioreactors. Importantly, the Nc-rTCA pathway demonstrated potential for industrial application, yielding 74.34 g/L SA at 0.98 g/g glucose from lignocellulosic hydrolysate and 117.74 g/L SA at 0.74 g/g from glycerol. Our findings address the longstanding redox imbalance issues that have challenged rTCA-based engineering and establish a scalable platform for bio-based C4-dicarboxylic acid production.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 36-44"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145461946","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}

{"title":"Engineering Escherichia coli for the production of saturated archaeal lipids","authors":"Jiayi Jiang ,&nbsp;Mirthe Hoekzema ,&nbsp;Ruben Andringa ,&nbsp;Adriaan J. Minnaard ,&nbsp;Arnold J.M. Driessen","doi":"10.1016/j.ymben.2025.12.004","DOIUrl":"10.1016/j.ymben.2025.12.004","url":null,"abstract":"<div><div>Archaeal membrane phospholipids have a different chemical composition than the phospholipids found in bacteria and eukaryotes. Typically, in archaea, phospholipids consist of saturated isoprenoid chains that are ether-bonded to glycerol 1-phosphate whereas in bacteria and eukaryotes, the main phospholipids are fatty acyl chains ester-bonded to glycerol 3-phosphate. This distinct chemical structure of phospholipids is believed to play a crucial role in enabling archaea to survive extreme environments and energy-limited conditions. <em>Escherichia coli</em> has previously been engineered to synthesize archaeal phospholipids next to its endogenous bacterial phospholipids. Cells equipped with these mixed heterochiral membranes were found to be viable with some improvement in robustness. However, a complete biosynthetic pathway for the production of substantial amounts of saturated archaeal lipids has not yet been realized in <em>E. coli</em>. Here, we engineered <em>E. coli</em> for the production of saturated archaeal phospholipids by introducing next to the geranylgeranyl reductase (GGR) and ferredoxin (Fd) from <em>Methanosarcina acetivorans</em>, the pyruvate-ferredoxin oxidoreductase (PFOR) from <em>E. coli</em> to allow for an efficient reduction of Fd. This resulted in a strain where approximately 75 % of the produced archaeal lipids are partially or completely saturated. Importantly, <em>E. coli</em> cells containing this mixed heterochiral membrane showed improved resistance to both heat and cold shock as compared to native <em>E. coli</em> strain. This <em>E. coli</em> strain with saturated archaeal phospholipids can serve as a valuable model for further engineering to incorporate different types of more complex archaeal membrane lipids.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 213-222"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145777266","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}

{"title":"Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida","authors":"Ian S. Yunus ,&nbsp;David N. Carruthers ,&nbsp;Yan Chen ,&nbsp;Jennifer W. Gin ,&nbsp;Edward E.K. Baidoo ,&nbsp;Christopher J. Petzold ,&nbsp;Hector Garcia Martin ,&nbsp;Paul D. Adams ,&nbsp;Aindrila Mukhopadhyay ,&nbsp;Taek Soon Lee","doi":"10.1016/j.ymben.2025.11.007","DOIUrl":"10.1016/j.ymben.2025.11.007","url":null,"abstract":"<div><div>CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in <em>Pseudomonas putida</em> KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 67-76"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145447343","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}

{"title":"Pilot production of P(3HB-co-4HB) by engineered Halomonas bluephagenesis harboring an endogenous plasmid grown on glucose","authors":"Rou Wen ,&nbsp;Yiling Chen ,&nbsp;Jiale Wang ,&nbsp;Weinan Yang ,&nbsp;Fang Yang ,&nbsp;Qiong Wu ,&nbsp;Fuqing Wu ,&nbsp;Xu Yan ,&nbsp;Guo-Qiang Chen","doi":"10.1016/j.ymben.2025.11.017","DOIUrl":"10.1016/j.ymben.2025.11.017","url":null,"abstract":"<div><div>Poly (3-hydroxybutyrate<em>-co</em>-4-hydroxybutyrate) (P (3HB-<em>co</em>-4HB) or P34HB) is a promising biopolyester for applications in food packaging, medical sutures, drug delivery, and tissue engineering due to its tunable thermomechanical properties. However, industrial-scale production of P34HB from glucose remains challenging. In this study, scalable P34HB production by engineered <em>Halomonas bluephagenesis</em> was developed. A <em>de novo</em> 4HB synthesis pathway was introduced into an endogenous toxin-antitoxin plasmid, enabling stable expression in the absence of antibiotics. Promoter engineering and pathway optimization fine-tuned 4HB molar ratio in P34HB from 18 to 39 mol%. The engineered <em>H. bluephagenesis</em> WR3 demonstrated successful scale-up from 7-L to 100-L and 5000-L bioreactors, achieving a maximum cell dry weight (CDW) of 72 g/L and 84% P (3HB-<em>co</em>-30 mol% 4HB) from the cultures in 7-L bioreactor. In further scale-up studies, <em>H. bluephagenesis</em> WR3 maintained comparable 4HB ratios, producing 69 g/L and 71 g/L CDW containing 74% and 61% P34HB copolymer in 100-L and 5000-L scale bioreactors, respectively. The amorphous P (3HB-<em>co</em>-30 mol% 4HB) exhibited high ductility, with an elongation at break of over 800% and a Young's modulus of 164 MPa. Additionally, morphology engineering and a controllable cell lysis were applied to enhance downstream processing efficiency. The optimized <em>H. bluephagenesis</em> WR25 produced 97 g/L CDW containing 83% P (3HB-<em>co</em>-20 mol% 4HB) in 7-L bioreactor, and 83 g/L CDW with 80% P34HB in 100-L bioreactor, while maintaining a consistently high glucose to P34HB conversion efficiency of 37%. This study provides a robust and cost-effective platform for industrial P34HB production from glucose harboring the toxin-antitoxin stable plasmid encoded with the 4HB pathway.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 153-168"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145619676","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}

{"title":"Corrigendum to “Microbial production of propionic acid through a novel β-alanine route” [Metabol. Eng. (2026) 219–231 93]","authors":"Da-Hee Ahn ,&nbsp;Yoo-Sung Ko ,&nbsp;Cindy Pricilia Surya Prabowo ,&nbsp;Sang Yup Lee","doi":"10.1016/j.ymben.2025.11.008","DOIUrl":"10.1016/j.ymben.2025.11.008","url":null,"abstract":"","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Page 35"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145509381","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}

{"title":"PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification","authors":"Chao Wu ,&nbsp;Jeffrey N. Law ,&nbsp;Onyeka Onyenemezu ,&nbsp;Jetendra K. Roy ,&nbsp;Peter C. St. John ,&nbsp;Robert L. Jernigan ,&nbsp;Yannick J. Bomble ,&nbsp;Laura Jarboe","doi":"10.1016/j.ymben.2025.12.003","DOIUrl":"10.1016/j.ymben.2025.12.003","url":null,"abstract":"<div><div><em>Escherichia coli</em> employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of <em>E. coli</em>'s metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing <em>in silico</em> growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as <em>in silico</em> gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using <em>E. coli</em> as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization. This work also highlights the critical role of intracellular acidification in shaping enzyme performance and microbial adaptation. The PET-FBA framework is implemented as a Python package at <span><span>https://github.com/Chaowu88/etfba</span><svg><path></path></svg></span>, with detailed documentation provided at <span><span>https://etfba.readthedocs.io</span><svg><path></path></svg></span>.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 202-212"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145731099","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}

{"title":"Metabolic engineering of a plasmid-free, non-auxotrophic Escherichia coli for efficient glycolate production","authors":"Jinyu Cheng ,&nbsp;Xinyi Jiang ,&nbsp;Xiaomin Li ,&nbsp;Wanqing Wei ,&nbsp;Jia Liu ,&nbsp;Wei Song ,&nbsp;Guipeng Hu ,&nbsp;Cong Gao ,&nbsp;Liming Liu","doi":"10.1016/j.ymben.2025.12.006","DOIUrl":"10.1016/j.ymben.2025.12.006","url":null,"abstract":"<div><div>Glycolate, an α-hydroxycarboxylic acid, is widely used in industries such as bioplastics, food, and pharmaceuticals. However, current microbial production methods are limited by the use of plasmids and chemical inducers, hindering their industrial scalability. In this study, a stable and efficient <em>Escherichia coli</em> platform was developed for glycolate production. The glycolate biosynthetic pathway was reconstructed through the identification of a highly efficient glyoxylate reductase (GhrA) from <em>Acetobacter aceti</em>. Carbon flux toward glycolate synthesis was optimized through strategies including enhancing precursor supply, blocking competing pathways, and fine-tuning gene copy numbers. Cofactor engineering was employed by engineering GhrA cofactor preference from NADPH to NADH. Additionally, a non-auxotrophic strain (eliminating exogenous nutrient requirements) for glycolate production was engineered by implementing a growth-stage-dependent molecular switch to dynamically regulate the expression of isocitrate dehydrogenase. Through fermentation optimization, the engineered strain <em>E. coli</em> GA26 achieved a glycolate titer of 81.5 g/L, a yield of 0.49 g/g glucose, and a productivity of 1.9 g/L/h in a 5-L bioreactor, representing the highest reported glycolate titer from glucose to date. These results pave the way for sustainable and cost-effective industrial glycolate production.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"94 ","pages":"Pages 273-283"},"PeriodicalIF":6.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145924554","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}