Metabolic engineering最新文献

{"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}

{"title":"Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440","authors":"Allison Z. Werner ,&nbsp;Young-Saeng C. Avina ,&nbsp;Josefin Johnsen ,&nbsp;Felicia Bratti ,&nbsp;Hannah M. Alt ,&nbsp;Elsayed T. Mohamed ,&nbsp;Rita Clare ,&nbsp;Thomas D. Mand ,&nbsp;Adam M. Guss ,&nbsp;Adam M. Feist ,&nbsp;Gregg T. Beckham","doi":"10.1016/j.ymben.2024.12.006","DOIUrl":"10.1016/j.ymben.2024.12.006","url":null,"abstract":"<div><div>Poly (ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, we expand on our previous efforts engineering and applying <em>Pseudomonas putida</em> KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three <em>P. putida</em> strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h⁻¹ on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators <em>gacS, gacA,</em> and <em>turA</em> along with large duplication and intergenic deletion events that impacted the heterologously-expressed <em>tphAB</em>_II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of <em>tphAB</em>_II and <em>tpaK</em> displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve <em>P. putida</em> growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 196-205"},"PeriodicalIF":6.8,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864064","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 of insect juvenile hormone III biosynthesis in the plant Nicotiana benthamiana","authors":"Angeliki Stathaki,&nbsp;Ryan M. Alam,&nbsp;Tobias G. Köllner,&nbsp;Sarah E. O'Connor","doi":"10.1016/j.ymben.2024.12.005","DOIUrl":"10.1016/j.ymben.2024.12.005","url":null,"abstract":"<div><div>Juvenile hormones (JHs) are farnesoic acid-derived sesquiterpenoids that play a crucial role in regulating various developmental processes in insects. Based on these reported biological activities, JHs and their synthetic analogs have been utilized as insecticides with significant commercial success over the past years. Here we describe the engineering of the JH pathway of the yellow fever mosquito (<em>Aedes aegypti</em>) by transient gene expression in the plant <em>Nicotiana benthamiana</em>. This approach led to the successful production of JH III in <em>N. benthamiana</em> leaves at a concentration of <em>ca</em>. 10 μg/g fresh weight. The co-expression of a feedback-insensitive version of 3-hydroxy-3-methylglutaryl coenzyme A reductase from <em>Arabidopsis thaliana</em> further increased the titer eight-fold from 10 to 80 μg/g fresh weight. Our efforts also revealed that the rich endogenous metabolic background of <em>N. benthamiana</em> can generate farnesoic acid, a key precursor to JH III, and thus, only 3 genes need to be expressed to provide high titers of this compound. Our study demonstrates the production of high titers of JH III in <em>N. benthamina</em> via heterologous expression of insect JH biosynthetic genes.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 77-84"},"PeriodicalIF":6.8,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864366","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":"Construction of a synthetic metabolic pathway for biosynthesis of threonine from ethylene glycol","authors":"Cláudio J.R. Frazão,&nbsp;Nils Wagner,&nbsp;T.A. Stefanie Nguyen,&nbsp;Thomas Walther","doi":"10.1016/j.ymben.2024.12.002","DOIUrl":"10.1016/j.ymben.2024.12.002","url":null,"abstract":"<div><div>Ethylene glycol is a promising substrate for bioprocesses which can be derived from widely abundant CO₂ or plastic waste. In this work, we describe the construction of an eight-step synthetic metabolic pathway enabling carbon-conserving biosynthesis of threonine from ethylene glycol. This route extends the previously disclosed synthetic threose-dependent glycolaldehyde assimilation (STEGA) pathway for the synthesis of 2-oxo-4-hydroxybutyrate with three additional reaction steps catalyzed by homoserine transaminase, homoserine kinase, and threonine synthase. We first validated the functionality of the new pathway in an <em>Escherichia coli</em> strain auxotrophic for threonine, which was also employed for discovering a better-performing D-threose dehydrogenase enzyme activity. Subsequently, we transferred the pathway to producer strains and used ¹³C-tracer experiments to improve threonine biosynthesis starting from glycolaldehyde. Finally, extending the pathway for ethylene glycol assimilation resulted in the production of up to 6.5 mM (or 0.8 g L⁻¹) threonine by optimized <em>E. coli</em> strains at a yield of 0.10 mol mol⁻¹ (corresponding to 20 % of the theoretical yield).</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 50-62"},"PeriodicalIF":6.8,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142822085","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":"Metabolic engineering improves transduction efficiency and downstream vector isolation by altering the lipid composition of extracellular vesicle-enclosed AAV","authors":"Paula Espinoza ,&nbsp;Ming Cheng ,&nbsp;Carrie Ng ,&nbsp;Demitri de la Cruz ,&nbsp;Elizabeth D. Wasson ,&nbsp;Deirdre M. McCarthy ,&nbsp;Pradeep G. Bhide ,&nbsp;Casey A. Maguire ,&nbsp;Miguel C. Santoscoy","doi":"10.1016/j.ymben.2024.12.003","DOIUrl":"10.1016/j.ymben.2024.12.003","url":null,"abstract":"<div><div>Adeno-associated viruses (AAV) are promising vectors for gene therapy due to their efficacy <em>in vivo</em>. However, there is room for improvement to address key limitations such as the pre-existing immunity to AAV in patients, high-dose toxicity, and relatively low efficiency for some cell types. This study introduces a metabolic engineering approach, using knockout of the enzyme phosphatidylserine synthase 1 (PTDSS1) to increase the abundance of extracellular vesicle-enclosed AAV (EV-AAV) relative to free AAV in the supernatant of producer cells, simplifying downstream purification processes. The lipid-engineered HEK293T-ΔPTDSS1 cell line achieved a 42.7-fold enrichment of EV-AAV9 compared to free AAV9 in the supernatant. The rational genetic strategy also led to a 300-fold decrease of free AAV in supernatant compared to wild-type HEK293T. The membrane-engineered EV-AAV9 (mEV-AAV9) showed unique envelope composition alterations, including cholesterol enrichment and improved transduction efficiency in human AC16 cardiomyocytes by 1.5-fold compared to conventional EV-AAV9 and by 11-fold compared to non-enveloped AAV9. Robust <em>in-vivo</em> transduction four weeks after intraparenchymal administration of mEV-AAV9 was observed in the murine brain. This study shows promise in the potential of lipid metabolic engineering strategies to improve the efficiency and process development of enveloped gene delivery vectors.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 40-49"},"PeriodicalIF":6.8,"publicationDate":"2024-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142801209","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":"Deciphering molecular drivers of lactate metabolic shift in mammalian cell cultures","authors":"Mauro Torres ,&nbsp;Ellie Hawke ,&nbsp;Robyn Hoare ,&nbsp;Rachel Scholey ,&nbsp;Leon P. Pybus ,&nbsp;Alison Young ,&nbsp;Andrew Hayes ,&nbsp;Alan J. Dickson","doi":"10.1016/j.ymben.2024.12.001","DOIUrl":"10.1016/j.ymben.2024.12.001","url":null,"abstract":"<div><div>Lactate metabolism plays a critical role in mammalian cell bioprocessing, influencing cellular performance and productivity. The transition from lactate production to consumption, known as lactate metabolic shift, is highly beneficial and has been shown to extend culture lifespan and enhance productivity, yet its molecular drivers remain poorly understood. Here, we have explored the mechanisms that underpin this metabolic shift through two case studies, illustrating environmental- and genetic-driven factors. We characterised these study cases at process, metabolic and transcriptomic levels. Our findings indicate that glutamine depletion coincided with the timing of the lactate metabolic shift, significantly affecting cell growth, productivity and overall metabolism. Transcriptome analysis revealed dynamic regulation the ATF4 pathway, involved in the amino acid (starvation) response, where glutamine depletion activates ATF4 gene and its targets. Manipulating ATF4 expression through overexpression and knockdown experiments showed significant changes in metabolism of glutamine and lactate, impacting cellular performance. Overexpression of ATF4 increased cell growth and glutamine consumption, promoting a lactate metabolic shift. In contrast, ATF4 downregulation decreased cell proliferation and glutamine uptake, leading to production of lactate without any signs of lactate shift. These findings underscore a critical role for ATF4 in regulation of glutamine and lactate metabolism, related to phasic patterns of growth during CHO cell culture. This study offers unique insight into metabolic reprogramming during the lactate metabolic shift and the molecular drivers that determine cell status during culture.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 25-39"},"PeriodicalIF":6.8,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142791593","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":"Model-assisted CRISPRi/a library screening reveals central carbon metabolic targets for enhanced recombinant protein production in yeast","authors":"Xin Chen ,&nbsp;Feiran Li ,&nbsp;Xiaowei Li ,&nbsp;Maximilian Otto ,&nbsp;Yu Chen ,&nbsp;Verena Siewers","doi":"10.1016/j.ymben.2024.11.010","DOIUrl":"10.1016/j.ymben.2024.11.010","url":null,"abstract":"<div><div>Production of recombinant proteins is regarded as an important breakthrough in the field of biomedicine and industrial biotechnology. Due to the complexity of the protein secretory pathway and its tight interaction with cellular metabolism, the application of traditional metabolic engineering tools to improve recombinant protein production faces major challenges. A systematic approach is required to generate novel design principles for superior protein secretion cell factories. Here, we applied a proteome-constrained genome-scale protein secretory model of the yeast <em>Saccharomyces cerevisiae</em> (pcSecYeast) to simulate α-amylase production under limited secretory capacity and predict gene targets for downregulation and upregulation to improve α-amylase production. The predicted targets were evaluated using high-throughput screening of specifically designed CRISPR interference/activation (CRISPRi/a) libraries and droplet microfluidics screening. From each library, 200 and 190 sorted clones, respectively, were manually verified. Out of them, 50% of predicted downregulation targets and 34.6% predicted upregulation targets were confirmed to improve α-amylase production. By simultaneously fine-tuning the expression of three genes in central carbon metabolism, i.e. <em>LPD1</em>, <em>MDH1</em>, and <em>ACS1</em>, we were able to increase the carbon flux in the fermentative pathway and α-amylase production. This study exemplifies how model-based predictions can be rapidly validated via a high-throughput screening approach. Our findings highlight novel engineering targets for cell factories and furthermore shed light on the connectivity between recombinant protein production and central carbon metabolism.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 1-13"},"PeriodicalIF":6.8,"publicationDate":"2024-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142770364","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}