Metabolic engineering最新文献

{"title":"CIFR (Clone–Integrate–Flip-out–Repeat): A toolset for iterative genome and pathway engineering of Gram-negative bacteria","authors":"Filippo Federici ,&nbsp;Francesco Luppino ,&nbsp;Clara Aguilar-Vilar,&nbsp;Maria Eleni Mazaraki,&nbsp;Lars Boje Petersen,&nbsp;Linda Ahonen,&nbsp;Pablo I. Nikel","doi":"10.1016/j.ymben.2025.01.001","DOIUrl":"10.1016/j.ymben.2025.01.001","url":null,"abstract":"<div><div>Advanced genome engineering enables precise and customizable modifications of bacterial species, and toolsets that exhibit broad-host compatibility are particularly valued owing to their portability. Tn<em>5</em> transposon vectors have been widely used to establish random integrations of desired DNA sequences into bacterial genomes. However, the iteration of the procedure remains challenging because of the limited availability and reusability of selection markers. We addressed this challenge with CIFR, a mini-Tn<em>5</em> integration system tailored for iterative genome engineering. The pCIFR vectors incorporate <em>attP</em> and <em>attB</em> sites flanking an antibiotic resistance marker used to select for the insertion. Subsequent removal of antibiotic determinants is facilitated by the Bxb1 integrase paired to a user-friendly counter-selection marker, both encoded in auxiliary plasmids. CIFR delivers engineered strains harboring stable DNA insertions and free of any antibiotic resistance cassette, allowing for the reusability of the tool. The system was validated in <em>Pseudomonas putida, Escherichia coli</em>, and <em>Cupriavidus necator</em>, underscoring its portability across diverse industrially relevant hosts. The CIFR toolbox was calibrated through combinatorial integrations of chromoprotein genes in <em>P. putida</em>, generating strains displaying a diverse color palette. We also introduced a carotenoid biosynthesis pathway in <em>P. putida</em> in a two-step engineering process, showcasing the potential of the tool for pathway balancing. The broad utility of the CIFR toolbox expands the toolkit for metabolic engineering, allowing for the construction of complex phenotypes while opening new possibilities in bacterial genetic manipulations.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 180-195"},"PeriodicalIF":6.8,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142951312","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}

{"title":"Cross-feeding of amino acid pathway intermediates is common in co-cultures of auxotrophic Escherichia coli","authors":"Yu-Jun Hong,&nbsp;Yijing Cai,&nbsp;Maciek R. Antoniewicz","doi":"10.1016/j.ymben.2025.01.003","DOIUrl":"10.1016/j.ymben.2025.01.003","url":null,"abstract":"<div><div>Amino acid auxotrophy refers to an organism's inability to synthesize one or more amino acids that are required for cell growth. In microbiome research, co-cultures of amino acid auxotrophs are often used to investigate metabolite cross-feeding interactions and model community dynamics. Thus far, it has been implicitly assumed that amino acids are mainly cross-fed between these auxotrophs. However, this assumption has not been fully verified. For example, it could be that intermediates of amino acid biosynthesis pathways are exchanged instead, or in addition to amino acids. If true, this would significantly increase the complexity of metabolic interactions that needs to be considered. Here, we show that metabolic pathway intermediates are indeed exchanged in many co-cultures of amino acid auxotrophs. To demonstrate this, we selected 25 <em>E. coli</em> single gene knockouts that are auxotrophic for five different amino acids: arginine, histidine, isoleucine, proline, and tryptophan. In co-culture experiments, we paired strains that shared the same amino acid auxotrophy and monitored cell growth. We observed growth in 23 out of 55 strain pairings, indicating that pathway intermediates were exchanged between the strains. To provide further support for cross-feeding of pathway intermediates, auxotrophic <em>E. coli</em> strains were cultured in media supplemented with commercially available metabolic pathway intermediates at different concentrations. Supplementing media with these metabolites recovered cell growth as was predicted from the co-culture experiments. Most of these metabolites supported high growth rates, even when present at low concentrations (10 μM), suggesting the presence of high affinity transporters for these metabolites. In total, we identified eight metabolic pathway intermediates that were likely exchanged between the auxotrophic <em>E. coli</em> strains and verified six of these, including histidinol, <em>N</em>-acetyl-L-ornithine, L-ornithine, L-citrulline, keto-isoleucine and anthranilate. Taken together, this work demonstrates that exchange of metabolic pathway intermediates is more common than has been assumed so far. In future, these exchanges must be explicitly considered when constructing models of metabolite cross-feeding interactions in microbial communities and when interpreting results from microbiome studies involving auxotrophic strains.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 172-179"},"PeriodicalIF":6.8,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142951314","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":"Deep learning for NAD/NADP cofactor prediction and engineering using transformer attention analysis in enzymes","authors":"Jaehyung Kim ,&nbsp;Jihoon Woo ,&nbsp;Joon Young Park ,&nbsp;Kyung-Jin Kim ,&nbsp;Donghyuk Kim","doi":"10.1016/j.ymben.2024.11.007","DOIUrl":"10.1016/j.ymben.2024.11.007","url":null,"abstract":"<div><div>Understanding and manipulating the cofactor preferences of NAD(P)-dependent oxidoreductases, the most widely distributed enzyme group in nature, is increasingly crucial in bioengineering. However, large-scale identification of the cofactor preferences and the design of mutants to switch cofactor specificity remain as complex tasks. Here, we introduce DISCODE (Deep learning-based Iterative pipeline to analyze Specificity of COfactors and to Design Enzyme), a novel transformer-based deep learning model to predict NAD(P) cofactor preferences. For model training, a total of 7,132 NAD(P)-dependent enzyme sequences were collected. Leveraging whole-length sequence information, DISCODE classifies the cofactor preferences of NAD(P)-dependent oxidoreductase protein sequences without structural or taxonomic limitation. The model showed 97.4% and 97.3% of accuracy and F1 score, respectively. A notable feature of DISCODE is the interpretability of its transformer layers. Analysis of attention layers in the model enables identification of several residues that showed significantly higher attention weights. They were well aligned with structurally important residues that closely interact with NAD(P), facilitating the identification of key residues for determining cofactor specificities. These key residues showed high consistency with verified cofactor switching mutants. Integrated into an enzyme design pipeline, DISCODE coupled with attention analysis, enables a fully automated approach to redesign cofactor specificity.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"87 ","pages":"Pages 86-94"},"PeriodicalIF":6.8,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142687367","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":"High-level sustainable production of complex phenylethanoid glycosides from glucose through engineered yeast cell factories","authors":"Penggang Bai ,&nbsp;Yihan Yang ,&nbsp;Jun Tang ,&nbsp;Daoyi Xi ,&nbsp;Yongya Hao ,&nbsp;Lili Jiang ,&nbsp;Hua Yin ,&nbsp;Tao Liu","doi":"10.1016/j.ymben.2024.11.012","DOIUrl":"10.1016/j.ymben.2024.11.012","url":null,"abstract":"<div><div>Complex phenylethanoid glycosides (PhGs), such as verbascoside and echinacoside, comprise a vital family of natural products with renowned nutraceutical and pharmaceutical significance. Despite the high demand for these compounds across various industries, traditional plant extraction methods yield insufficient quantities, highlighting the need for alternative production methods. Therefore, this paper reports the successful engineering of <em>Saccharomyces cerevisiae</em> cell factories for the efficient production of complex PhGs from glucose. First, key pathway enzymes with enhanced catalytic activities in yeast were primarily screened from various verbascoside-producing plants. Second, intermediate osmanthuside B was produced with a titer of 21.5 ± 1.5 mg/L from glucose by overexpressing several enzymes, including glucosyltransferase RrUGT33 from <em>Rhdiola rosea</em>, acyltransferase SiAT, and 1,3-rhamnosyltransferase SiRT from <em>Sesamum indicum</em>, UDP-L-rhamnose synthase AtRHM2, and 4-coumarate: coenzyme A ligase At4CL1 from <em>Arabidopsis thaliana</em> in a <em>p</em>-coumaric acid-overproducing <em>S. cerevisiae</em> strain. Third, the production of osmanthuside B was further enhanced by increasing the copy number of <em>SiAT</em> and <em>AtRHM2</em> in genome and diverting L-tyrosine into tyrosol biosynthesis by introducing an aromatic aldehyde synthase PcAAS from <em>Petroselinum crispum</em> with a titer of 320.6 ± 59.3 mg/L. Fourth, the biosynthesis of verbascoside was accomplished by integrating genes <em>CYP98A20</em> and <em>AtCPR1</em> into the chromosomes of the osmanthuside B-producing strain, the titer reached 184.7 ± 5.7 mg/L. Furthermore, the overexpression of the glucose-6-phosphate dehydrogenase (ZWF1) led to significantly enhanced verbascoside production to 230.6 ± 11.8 mg/L. The strains were further engineered to produce echinacoside with a titer of 184.2 ± 11.2 mg/L. Finally, the fed-batch fermentation in a 5-L bioreactor yielded 4497.9 ± 285.2 mg/L of verbascoside or 3617.4 ± 117.4 mg/L of echinacoside. This work provides a crucial foundation for the green, industrial, and sustainable production of verbascoside and echinacoside and sets an initial point for the microbial production of other complex PhG derivatives.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"87 ","pages":"Pages 95-108"},"PeriodicalIF":6.8,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142739901","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":"Synthetic translational coupling system for accurate and predictable polycistronic gene expression control in bacteria","authors":"Yong Hee Han ,&nbsp;Hyeon Jin Kim ,&nbsp;Keonwoo Kim ,&nbsp;Jina Yang ,&nbsp;Sang Woo Seo","doi":"10.1016/j.ymben.2024.12.011","DOIUrl":"10.1016/j.ymben.2024.12.011","url":null,"abstract":"<div><div>Precise and predictable genetic elements are required to address various issues, such as suboptimal metabolic flux or imbalanced protein assembly caused by the inadequate control of polycistronic gene expression in bacteria. Here, we devised a synthetic biopart based on the translational coupling to control polycistronic gene expression. This module links the translation of genes within a polycistronic mRNA, maintaining their expression ratios regardless of coding sequences, transcription rate, and upstream gene translation rate. By engineering the Shine-Dalgarno sequences within these synthetic bioparts, we adjusted the expression ratios of polycistronic genes. We created 41 bioparts with varied relative expression ratios, ranging from 0.03 to 0.92, enabling precise control of pathway enzyme gene expression in a polycistronic manner. This led to up to a 7.6-fold increase in the production of valuable biochemicals such as 3-hydroxypropionic acid, poly(3-hydroxybutyrate), and lycopene. Our work provides genetic regulatory modules for precise and predictable polycistronic gene expression, facilitating efficient protein assembly, biosynthetic gene cluster expression, and pathway optimization.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 148-159"},"PeriodicalIF":6.8,"publicationDate":"2024-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142915230","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":"Protein engineering of an oxidative cleavage-free pathway for crocetin-dialdehyde production in Escherichia coli","authors":"Jun Ho Lee ,&nbsp;Jeong-Yang Park ,&nbsp;Min-Duk Seo ,&nbsp;Pyung Cheon Lee","doi":"10.1016/j.ymben.2024.12.009","DOIUrl":"10.1016/j.ymben.2024.12.009","url":null,"abstract":"<div><div>The growing depletion of petroleum resources and the increasing demand for sustainable alternatives have spurred advancements in microorganism-based biofactories. Among high-value compounds, carotenoids are widely sought after in pharmaceuticals, cosmetics, and nutrition, making them prime candidates for microbial production. In this study, we engineered an efficient biosynthetic pathway in <em>Escherichia coli</em> for the production of the C₂₀-carotenoid crocetin-dialdehyde. By bypassing traditional oxidative cleavage reactions mediated by carotenoid cleavage dioxygenases (CCDs), our approach reduces the enzymatic complexity of the pathway. Using the crystal structure of a CrtMLIKE enzyme identified in this study, we developed a mutant enzyme capable of condensing two C₁₀-geranyl pyrophosphate molecules to form C₂₀-phytoene. This intermediate was then desaturated and oxidized by CrtN and CrtP to produce crocetin-dialdehyde, achieving a yield of 1.13 mg/L. By reducing enzyme requirements from six to three and eliminating the need for CCDs, this pathway alleviates metabolic stress on the host and enhances the scalability of production for industrial applications. Overall, our research presents a streamlined and innovative approach to carotenoid biosynthesis, advancing sustainable production methods for short-chain carotenoids.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 137-147"},"PeriodicalIF":6.8,"publicationDate":"2024-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142915277","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":"Developing a Bacillus licheniformis platform for de novo production of γ-aminobutyric acid and other glutamate-derived chemicals","authors":"Shiyi Wang ,&nbsp;Jiang Zhu ,&nbsp;Yiwen Zhao ,&nbsp;Shufen Mao ,&nbsp;Yihui He ,&nbsp;Feixiang Wang ,&nbsp;Tianli Jia ,&nbsp;Dongbo Cai ,&nbsp;Junyong Chen ,&nbsp;Dong Wang ,&nbsp;Shouwen Chen","doi":"10.1016/j.ymben.2024.12.010","DOIUrl":"10.1016/j.ymben.2024.12.010","url":null,"abstract":"<div><div>Microbial cell factories (MCFs) have emerged as a sustainable tool for the production of value-added biochemicals. However, developing high-performance MCFs remains a major challenge to fulfill the burgeoning demands of global markets. This study aimed to establish the B. licheniformis cell factory for the cost-effective production of glutamate-derived chemicals by modular metabolic engineering. Initially, the glutamate decarboxylase from E. coli was introduced into B. licheniformis DW2 to construct the artificial γ-aminobutyric acid (GABA) pathway. By systematically optimizing the central metabolic pathway, boosting the L-Glu synthesis pathway and improving the cofactor NADPH supply, the strain G35/pHY-P_r5u12-gadB^E89Q/H465A achieved a remarkable yield of 62.9 g/L of GABA in a 5-L bioreactor, representing the highest yield of 0.5 g/g glucose with a significant 49.3-fold increase. Remarkably, bioinformatics analyses and function verification identified the putative glyoxylate to glycolic acid synthesis pathway and KipR, an inhibitor of the glyoxylate cycle, as the rate-limiting steps in GABA production. Additionally, a versatile and robust platform using engineered B. licheniformis for efficient production of diverse glutamate-derived chemicals was established and the titer of 5-aminolevulinic acid, heme and indigoidine was improved by 5.3-, 4.7- and 1.9-fold, respectively. This study not only facilitates extensive application of B. licheniformis for chemical production, but also sheds light on research to improve the performance of other MCFs.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 124-136"},"PeriodicalIF":6.8,"publicationDate":"2024-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142906870","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":"Nucleotide distribution analysis of 5′UTRs in genome-scale directs their redesign and expression regulation in yeast","authors":"Chaoying Yao ,&nbsp;Yu Yin ,&nbsp;Qingyang Li ,&nbsp;Hanqi Zhang ,&nbsp;Yilun Zhang ,&nbsp;Qianqian Shao ,&nbsp;Qi Liu ,&nbsp;Yanna Ren ,&nbsp;Menghao Cai","doi":"10.1016/j.ymben.2024.12.008","DOIUrl":"10.1016/j.ymben.2024.12.008","url":null,"abstract":"<div><div>Non-conventional yeasts have emerged as important sources of valuable products in bioindustries. However, tools for the control of expression are limited in these hosts. In this study, we aimed to excavate the tools for the regulation of translation that are often overlooked. 5′UTR analysis of genome-scale annotated genes of four yeast species revealed a distinct decreasing ‘G’ frequency in −100 ∼ −1 region from 5040 5′UTRs in <em>Komagataella phaffii</em>. New 5′UTRs were regenerated by base substitutions in defined regions, and replacement of ‘G’ by ‘A’ or ‘T’ in the −50 ∼ −1 region highly facilitated gene expression. Preference analysis of all nucleotide triplets in 5′UTRs revealed a KZ₃ (−3 ∼ −1) that dominantly affected gene expression. A total of 128 KZ₃ variants were constructed to work with promoters of methanol-inducible <em>P</em>_{<em>AOX1</em>} and constitutive P_<em>GAP</em>, of which 58 KZ₃ variants increased gene expression and maximum difference in strength was 15-fold among all variants. Polysome profiling analysis clarified that 5′UTR-KZ₃ enhanced gene expression at translational but not transcriptional levels. Finally, improved production of three industrial proteins and one platform compound were achieved by ready-made 5′UTR-KZ₃ or <em>in situ</em> modification of the 5′UTR. This study provides new references and tools for the fine-tuning of translational regulation in yeast and other fungi.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 113-123"},"PeriodicalIF":6.8,"publicationDate":"2024-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142903378","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 metabolic flux for the microbial synthesis of aromatic compounds","authors":"Zhendong Li ,&nbsp;Xianghe Wang ,&nbsp;Guipeng Hu ,&nbsp;Xiaomin Li ,&nbsp;Wei Song ,&nbsp;Wanqing Wei ,&nbsp;Liming Liu ,&nbsp;Cong Gao","doi":"10.1016/j.ymben.2024.12.007","DOIUrl":"10.1016/j.ymben.2024.12.007","url":null,"abstract":"<div><div>Microbial cell factories have emerged as a sustainable alternative to traditional chemical synthesis and plant extraction methods for producing aromatic compounds. However, achieving economically viable production of these compounds in microbial systems remains a significant challenge. This review summarizes the latest advancements in metabolic flux regulation during the microbial production of aromatic compounds, providing an overview of its applications and practical outcomes. Various strategies aimed at improving the utilization of extracellular substrates, enhancing the efficiency of synthetic pathways for target products, and rewiring intracellular metabolic networks to boost the titer, yield, and productivity of aromatic compounds are discussed. Additionally, the persistent challenges in this field and potential solutions are highlighted.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 94-112"},"PeriodicalIF":6.8,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142887943","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":"De novo 2′-fucosyllactose biosynthesis using glucose as the sole carbon source by multiple engineered Bacillus subtilis","authors":"Quanwei Zhang ,&nbsp;Xianhao Xu ,&nbsp;Wei Zhang ,&nbsp;Ziyang Huang ,&nbsp;Yaokang Wu ,&nbsp;Yanfeng Liu ,&nbsp;Jianghua Li ,&nbsp;Guocheng Du ,&nbsp;Xueqin Lv ,&nbsp;Long Liu","doi":"10.1016/j.ymben.2024.12.004","DOIUrl":"10.1016/j.ymben.2024.12.004","url":null,"abstract":"<div><div>2′-Fucosyllactose (2′-FL) is the most abundant human milk oligosaccharide and plays significant roles in gut microbiome balance, neural development, and immunoregulation. However, current fermentation schemes using multiple carbon sources increase production cost and metabolism burden. This study reported the development of an engineered <em>Bacillus subtilis</em> strain that produces 2′-FL using glucose as the sole carbon source. First, a lactose biosynthesis module was constructed by expressing β-1,4-galactosyltransferase gene from <em>Neisseria meningitidis</em>. A 2′-FL titer of 2.53 ± 0.07 g/L was subsequently achieved using glucose as the sole carbon source by the combination of lactose and GDP-L-fucose (GDP-Fuc) biosynthesis modules. Introducing an exogenous nonphosphorylated transport system enhanced the supply of intracellular nonphosphorylated glucose, and the 2′-FL titer increased to 4.94 ± 0.35 g/L. Next, a transcription factor screening platform was designed. Based on this platform, the ligand of the transcription factor LacI was changed from isopropyl β-D-thiogalactoside to lactose. A lactose-responsive genetic circuit was then constructed and used for the dynamic regulation of metabolic fluxes between lactose and GDP-Fuc biosynthesis modules. Ultimately, the 2′-FL titer of the dynamically regulated strain improved by 107% to 9.67 ± 0.65 g/L in shake-flask, and the titer and yield in a 3-L bioreactor reached 30.1 g/L and 0.15 g/g using glucose as the sole carbon source. By using multidimensional engineering strategies, this study constructed a <em>B. subtilis</em> strain capable of efficiently producing 2′-FL with glucose as the sole carbon source, paving the way for the industrial production of 2′-FL with low cost in the future.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 85-93"},"PeriodicalIF":6.8,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854688","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}