Metabolic engineering最新文献

{"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}

{"title":"Elucidation and engineering of Sphingolipid biosynthesis pathway in Yarrowia lipolytica for enhanced production of human-type sphingoid bases and glucosylceramides","authors":"Seo Hyeon Shin ,&nbsp;Hye Yun Moon ,&nbsp;Hae Eun Park ,&nbsp;Gi Jeong Nam ,&nbsp;Ju Hye Baek ,&nbsp;Che Ok Jeon ,&nbsp;Hyunwook Jung ,&nbsp;Myeong Seok Cha ,&nbsp;Sol Choi ,&nbsp;Jeong Jun Han ,&nbsp;Chen Yuan Hou ,&nbsp;Chang Seo Park ,&nbsp;Hyun Ah Kang","doi":"10.1016/j.ymben.2024.11.013","DOIUrl":"10.1016/j.ymben.2024.11.013","url":null,"abstract":"<div><div>Sphingolipids are vital membrane components in in mammalian cells, plants, and various microbes. We aimed to explore and exploit the sphingolipid biosynthesis pathways in an oleaginous and dimorphic yeast <em>Yarrowia lipolytica</em> by constructing and characterizing mutant strains with specific gene deletions and integrating exogenous genes to enhance the production of long-chain bases (LCBs) and glucosylceramides (GlcCers). To block the fungal/plant-specific phytosphingosine (PHS) pathway, we deleted the <em>SUR2</em> gene encoding a sphinganine C4-hydroxylase, resulting in a remarkably elevated secretory production of dihydrosphingosine (DHS) and sphingosine (So) without acetylation. The <em>Y. lipolytica SUR2</em> deletion (<em>Ylsur2</em>Δ) strain displayed retarded growth, increased pseudohyphal formation and stress sensitivity, along with the altered profiles of inositolphosphate-containing ceramides, GlcCers, and sterols. The subsequent disruption of the <em>SLD1</em> gene, encoding a fungal/plant-specific Δ8 sphingolipid desaturase, restored filamentous growth in the <em>Ylsur2</em>Δ strain to a yeast-type form and further increased the production of human-type GlcCers. Additional introduction of mouse alkaline ceramidase 1 (<em>maCER1</em>) into the <em>Ylsur2</em>Δ<em>sld1</em>Δ double mutants considerably increased DHS and So production while decreasing GlcCers. The production yields of LCBs from the <em>Ylsur2</em>Δ<em>sld1</em>Δ/<em>maCER1</em> strain increased in proportion to the C/N ratio in the N-source optimized medium, leading to production of 1.4 g/L non-acetylated DHS at the 5 L fed-batch fermentation with glucose feeding. This study highlights the feasibility of using the engineered <em>Y. lipolytica</em> strains as a cell factory for valuable sphingolipid derivatives for pharmaceuticals, cosmeceuticals, and nutraceuticals.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"87 ","pages":"Pages 68-85"},"PeriodicalIF":6.8,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142739900","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 Escherichia coli for N-methylserotonin biosynthesis","authors":"Qingchen Li ,&nbsp;Chenxi Li ,&nbsp;Jie Zhong ,&nbsp;Yukun Wang ,&nbsp;Qinghua Yang ,&nbsp;Bingmei Wang ,&nbsp;Wenjin He ,&nbsp;Jianzhong Huang ,&nbsp;Shengyuan Lin ,&nbsp;Feng Qi","doi":"10.1016/j.ymben.2024.11.011","DOIUrl":"10.1016/j.ymben.2024.11.011","url":null,"abstract":"<div><div>N-methylserotonin (NMS) is a valuable indole alkaloid with therapeutic potential for psychiatric and neurological disorders, and it is used in health foods, cosmetics, and weight loss supplements. However, environmental challenges and low reaction efficiencies significantly hinder cost-effective, large-scale production of NMS in plants or through chemical synthesis. Herein, we have successfully engineered <em>Escherichia coli</em> strains to enhance NMS production from L-tryptophan using whole-cell catalysis. We developed multiple biosynthesis pathways incorporating modules for serotonin (5-hydroxytryptamine, 5-HT), tetrahydromonapterin (MH₄), and S-adenosylmethionine (SAM) synthesis. To enhance MH₄ availability, we employed a high-activity <em>Bacillus subtilis</em> FolE and minimized carbon flux loss through targeted gene knockouts in competitive metabolic pathways, improving 5-HT production. Additionally, we constructed a comprehensive SAM biosynthesis module to facilitate transmethylation by a selected N-methyltransferase fused with ProS2. These engineered modules were coexpressed in two plasmids within the optimized strain NMS-19, producing 128.6 mg/L of NMS in a 5-L bioreactor using fed-batch cultivation—a 92-fold increase over the original strain. This study introduces a viable strategy for NMS production and provides insights into the biosynthesis of SAM-dependent methylated tryptamine derivatives.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"87 ","pages":"Pages 49-59"},"PeriodicalIF":6.8,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142739903","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":"Optimized production of concanamycins using a rational metabolic engineering strategy","authors":"Filipa Pereira ,&nbsp;Morgan McCauley ,&nbsp;Katherine Lev ,&nbsp;Linnea Verhey-Henke ,&nbsp;Alanna R. Condren ,&nbsp;Ralph J. Harte ,&nbsp;Jesus Galvez ,&nbsp;David H. Sherman","doi":"10.1016/j.ymben.2024.11.008","DOIUrl":"10.1016/j.ymben.2024.11.008","url":null,"abstract":"<div><div>Plecomacrolides, such as concanamycins and bafilomycins, are potent and specific inhibitors of vacuolar-type ATPase. Concanamycins are 18-membered macrolides with promising therapeutic potential against multiple diseases, including viral infection, osteoporosis, and cancer. Due to the complexity of their total synthesis, the production of concanamycins is only achieved through microbial fermentation. However, the low titers of concanamycin A and its analogs in the native producing strains are a significant bottleneck for scale-up, robust structure-activity relationship studies, and drug development. To address this challenge, we designed a library of engineered <em>Streptomyces</em> strains for the overproduction of concanamycin A-C by combining the overexpression of target regulatory genes with the optimization of fermentation media. Integration of two endogenous regulators from the concanamycin biosynthetic gene cluster (<em>cms</em>) and one heterologous regulatory gene from the bafilomycin biosynthetic gene cluster significantly increased production of concanamycin A and its less abundant analog concanamycin B in <em>Streptomyces eitanensis</em>. The highest titers reported to date were observed in the engineered <em>S. eitanensis</em> DHS10676, which produced over 900 mg/L of concanamycin A and 300 mg/L of concanamycin B. Heterologous overexpression of the identified target regulatory genes across a panel of <em>Streptomyces</em> spp. harboring a putative concanamycin biosynthetic gene cluster confirmed its identity, and significantly improved concanamycin A production in all tested strains. Strain engineering, optimization of fermentation, and extraction purification protocols enabled swift access to these structurally complex plecomacrolides for semi-synthetic medicinal chemistry-based approaches. Together, this work established a platform for robust overproduction of concanamycin analogs across species.</div></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"88 ","pages":"Pages 63-76"},"PeriodicalIF":6.8,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142710583","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}