Metabolic engineering最新文献

{"title":"Cell factory design with advanced metabolic modelling empowered by artificial intelligence","authors":"Hongzhong Lu ,&nbsp;Luchi Xiao ,&nbsp;Wenbin Liao ,&nbsp;Xuefeng Yan ,&nbsp;Jens Nielsen","doi":"10.1016/j.ymben.2024.07.003","DOIUrl":"10.1016/j.ymben.2024.07.003","url":null,"abstract":"<div><p>Advances in synthetic biology and artificial intelligence (AI) have provided new opportunities for modern biotechnology. High-performance cell factories, the backbone of industrial biotechnology, are ultimately responsible for determining whether a bio-based product succeeds or fails in the fierce competition with petroleum-based products. To date, one of the greatest challenges in synthetic biology is the creation of high-performance cell factories in a consistent and efficient manner. As so-called white-box models, numerous metabolic network models have been developed and used in computational strain design. Moreover, great progress has been made in AI-powered strain engineering in recent years. Both approaches have advantages and disadvantages. Therefore, the deep integration of AI with metabolic models is crucial for the construction of superior cell factories with higher titres, yields and production rates. The detailed applications of the latest advanced metabolic models and AI in computational strain design are summarized in this review. Additionally, approaches for the deep integration of AI and metabolic models are discussed. It is anticipated that advanced mechanistic metabolic models powered by AI will pave the way for the efficient construction of powerful industrial chassis strains in the coming years.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"85 ","pages":"Pages 61-72"},"PeriodicalIF":6.8,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624000879/pdfft?md5=4fdbb7076fbe0228648e0c190857b323&pid=1-s2.0-S1096717624000879-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141748551","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":"Corrigendum to “Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440” (Metab. Eng. 67 (2021) 250–261)","authors":"Allison Z. Werner ,&nbsp;Rita Clare ,&nbsp;Thomas D. Mand ,&nbsp;Isabel Pardo ,&nbsp;Kelsey J. Ramirez ,&nbsp;Stefan J. Haugen ,&nbsp;Felicia Bratti ,&nbsp;Gara N. Dexter ,&nbsp;Joshua R. Elmore ,&nbsp;Jay D. Huenemann ,&nbsp;George L. Peabody ,&nbsp;Christopher W. Johnson ,&nbsp;Nicholas A. Rorrer ,&nbsp;Davinia Salvachúa ,&nbsp;Adam M. Guss ,&nbsp;Gregg T. Beckham","doi":"10.1016/j.ymben.2024.07.004","DOIUrl":"10.1016/j.ymben.2024.07.004","url":null,"abstract":"","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"85 ","pages":"Pages 131-132"},"PeriodicalIF":6.8,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624000946/pdfft?md5=8ab3e9ce82ae834b7115e1bf4caf1d12&pid=1-s2.0-S1096717624000946-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141727401","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":"Functional genomic screening in Komagataella phaffii enabled by high-activity CRISPR-Cas9 library","authors":"Aida Tafrishi ,&nbsp;Varun Trivedi ,&nbsp;Zenan Xing ,&nbsp;Mengwan Li ,&nbsp;Ritesh Mewalal ,&nbsp;Sean R. Cutler ,&nbsp;Ian Blaby ,&nbsp;Ian Wheeldon","doi":"10.1016/j.ymben.2024.07.006","DOIUrl":"10.1016/j.ymben.2024.07.006","url":null,"abstract":"<div><p>CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast <em>Komagataella phaffii</em> is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in <em>K. phaffii</em>, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guide's targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of <em>K. phaffii</em> essential genes, many of which relate to metabolic engineering targets, including protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in <em>K. phaffii</em>, applied here to gene essentiality classification, and promises to enable other genetic screens.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"85 ","pages":"Pages 73-83"},"PeriodicalIF":6.8,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S109671762400096X/pdfft?md5=a05347382c4764a1110a8983923a2bc4&pid=1-s2.0-S109671762400096X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141633918","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 of “last-line antibiotic” colistin in Paenibacillus polymyxa","authors":"Nanzhu Chen,&nbsp;Peiyan Cai,&nbsp;Dengwei Zhang,&nbsp;Junliang Zhang,&nbsp;Zheng Zhong,&nbsp;Yong-Xin Li","doi":"10.1016/j.ymben.2024.07.005","DOIUrl":"10.1016/j.ymben.2024.07.005","url":null,"abstract":"<div><p>Colistin, also known as polymyxin E, is a lipopeptide antibiotic used to treat infections caused by multidrug-resistant gram-negative bacteria. It is considered a “last-line antibiotic”, but its clinical development is hindered by low titer and impurities resulting from the presence of diverse homologs in microbial fermentation. To ensure consistent pharmaceutical activity and kinetics, it is crucial to have high-purity colistin active pharmaceutical ingredient (API) in the pharmaceutical industry. This study focused on the metabolic engineering of a natural colistin producer strain to produce colistin with a high titer and purity. Guided by genome mining, we identified <em>Paenibacillus polymyxa</em> ATCC 842 as a natural colistin producer capable of generating a high proportion of colistin A. By systematically inactivating seven non-essential biosynthetic gene clusters (BGCs) of peptide metabolites that might compete precursors with colistin or inhibit colistin production, we created an engineered strain, P14, which exhibited an 82% increase in colistin titer and effectively eliminated metabolite impurities such as tridecaptin, paenibacillin, and paenilan. Additionally, we engineered the L-2,4-diaminobutyric acid (L-2,4-DABA) pathway to further enhance colistin production, resulting in the engineered strain P19, which boosted a remarkable colistin titer of 649.3 mg/L – a 269% improvement compared to the original strain. By concurrently feeding L-isoleucine and L-leucine, we successfully produced high-purity colistin A, constituting 88% of the total colistin products. This study highlights the potential of metabolic engineering in improving the titer and purity of lipopeptide antibiotics in the non-model strain, making them more suitable for clinical use. These findings indicate that efficiently producing colistin API in high purity directly from fermentation can now be achieved in a straightforward manner.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"85 ","pages":"Pages 35-45"},"PeriodicalIF":6.8,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141633919","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":"Multidimensional engineering of Saccharomyces cerevisiae for the efficient production of heme by exploring the cytotoxicity and tolerance of heme","authors":"Qidi Guo,&nbsp;Jiacun Li,&nbsp;Ming-Rui Wang,&nbsp;Ming Zhao,&nbsp;Gege Zhang,&nbsp;Shuyan Tang,&nbsp;Liang-Bin Xiong,&nbsp;Bei Gao,&nbsp;Feng-Qing Wang,&nbsp;Dong-Zhi Wei","doi":"10.1016/j.ymben.2024.07.007","DOIUrl":"10.1016/j.ymben.2024.07.007","url":null,"abstract":"<div><p>Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood. In this study, the effect of heme toxicity on <em>Saccharomyces cerevisiae</em> was evaluated by enhancing its synthesis using metabolic engineering. The results showed that the accumulation of heme after the disruption of heme homeostasis caused serious impairments in cell growth and metabolism, as demonstrated by significantly poor growth, mitochondrial damage, cell deformations, and chapped cell surfaces, and these features which were further associated with substantially elevated reactive oxygen species (ROS) levels within the cell (mainly H₂O₂ and superoxide anion radicals). To improve cellular tolerance to heme, 5 rounds of laboratory evolution were performed, increasing heme production by 7.3-fold and 4.2-fold in terms of the titer (38.9 mg/L) and specific production capacity (1.4 mg/L/OD₆₀₀), respectively. Based on comparative transcriptomic analyses, 32 genes were identified as candidates that can be modified to enhance heme production by more than 20% in <em>S. cerevisiae</em>. The combined overexpression of 5 genes (<em>SPS22</em>, <em>REE1</em>, <em>PHO84</em>, <em>HEM4</em> and <em>CLB2</em>) was shown to be an optimal method to enhance heme production. Therefore, a strain with enhanced heme tolerance and ROS quenching ability (R5-M) was developed that could generate 380.5 mg/L heme with a productivity of 4.2 mg/L/h in fed-batch fermentation, with <em>S. cerevisiae</em> strains being the highest producers reported to date. These findings highlight the importance of improving heme tolerance for the microbial production of heme and provide a solution for efficient heme production by engineered yeasts.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"85 ","pages":"Pages 46-60"},"PeriodicalIF":6.8,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141633920","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":"Auto-inducible synthetic pathway in E. coli enhanced sustainable indigo production from glucose","authors":"","doi":"10.1016/j.ymben.2024.07.002","DOIUrl":"10.1016/j.ymben.2024.07.002","url":null,"abstract":"<div><p>Indigo is widely used in textile industries for denim garments dyeing and is mainly produced by chemical synthesis which, however, raises environmental sustainability issues. Bio-indigo may be produced by fermentation of metabolically engineering bacteria, but current methods are economically incompetent due to low titer and the need for an inducer. To address these problems, we first characterized several synthetic promoters in <em>E. coli</em> and demonstrated the feasibility of inducer-free indigo production from tryptophan using the inducer-free promoter. We next coupled the tryptophan-to-indigo and glucose-to-tryptophan pathways to generate a <em>de novo</em> glucose-to-indigo pathway. By rational design and combinatorial screening, we identified the optimal promoter-gene combinations, which underscored the importance of promoter choice and expression levels of pathway genes. We thus created a new <em>E. coli</em> strain that exploited an indole pathway to enhance the indigo titer to 123 mg/L. We further assessed a panel of heterologous tryptophan synthase homologs and identified a plant indole lyase (<em>Ta</em>IGL), which along with modified pathway design, improved the indigo titer to 235 mg/L while reducing the tryptophan byproduct accumulation. The optimal <em>E. coli</em> strain expressed 8 genes essential for rewiring carbon flux from glucose to indole and then to indigo: <em>mFMO</em>, <em>ppsA</em>, <em>tktA</em>, <em>trpD, trpC</em>, <em>TaIGL</em> and feedback-resistant <em>aroG</em> and <em>trpE</em>. Fed-batch fermentation in a 3-L bioreactor with glucose feeding further increased the indigo titer (≈965 mg/L) and total quantity (≈2183 mg) at 72 h. This new synthetic glucose-to-indigo pathway enables high-titer indigo production without the need of inducer and holds promise for bio-indigo production.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"85 ","pages":"Pages 14-25"},"PeriodicalIF":6.8,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545022","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 sub-organelles of a diploid Saccharomyces cerevisiae to enhance the production of 7-dehydrocholesterol","authors":"Ke Bi ,&nbsp;Wenguang Wang ,&nbsp;Dandan Tang ,&nbsp;Zhuwei Shi ,&nbsp;Shuyu Tian ,&nbsp;Lei Huang ,&nbsp;Jiazhang Lian ,&nbsp;Zhinan Xu","doi":"10.1016/j.ymben.2024.06.011","DOIUrl":"10.1016/j.ymben.2024.06.011","url":null,"abstract":"<div><p>7-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D₃. Despite significant improvements in the biosynthesis of 7-DHC, it remains insufficient to meet the industrial demands. In this study, we reported high-level production of 7-DHC in an industrial <em>Saccharomyces cerevisiae</em> leveraging subcellular organelles. Initially, the copy numbers of <em>DHCR24</em> were increased in combination with sterol transcriptional factor engineering and rebalanced the redox power of the strain. Subsequently, the effects of compartmentalizing the post-squalene pathway in peroxisomes were validated by assembling various pathway modules in this organelle. Furthermore, several peroxisomes engineering was conducted to enhance the production of 7-DHC. Utilizing the peroxisome as a vessel for partial post-squalene pathways, the potential of yeast for 7-dehydrocholesterol production was demonstrated by achieving a 26-fold increase over the initial production level. 7-DHC titer reached 640.77 mg/L in shake flasks and 4.28 g/L in a 10 L bench-top fermentor, the highest titer ever reported. The present work lays solid foundation for large-scale and cost-effective production of 7-DHC for practical applications.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"84 ","pages":"Pages 169-179"},"PeriodicalIF":6.8,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469295","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 CHO cells towards cysteine prototrophy and systems analysis of the ensuing phenotype","authors":"Laura Greenfield ,&nbsp;Mariah Brantley ,&nbsp;Pauline Geoffroy ,&nbsp;Jeffrey Mitchell ,&nbsp;Dylan DeWitt ,&nbsp;Fang Zhang ,&nbsp;Bhanu Chandra Mulukutla","doi":"10.1016/j.ymben.2024.06.003","DOIUrl":"10.1016/j.ymben.2024.06.003","url":null,"abstract":"<div><p>Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (βME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and βME levels, were able to proliferate in the HCY-free, βME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn't completely adapt to βME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, ‘omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"84 ","pages":"Pages 128-144"},"PeriodicalIF":6.8,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141440642","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":"Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering","authors":"Fuqiang Song ,&nbsp;Zhijie Qin ,&nbsp;Kun Qiu ,&nbsp;Zhongshi Huang ,&nbsp;Lian Wang ,&nbsp;Heng Zhang ,&nbsp;Xiaoyu Shan ,&nbsp;Hao Meng ,&nbsp;Xirong Liu ,&nbsp;Jingwen Zhou","doi":"10.1016/j.ymben.2024.06.006","DOIUrl":"10.1016/j.ymben.2024.06.006","url":null,"abstract":"<div><p>Vitamin B₅ [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (<em>R</em>)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel “PEACES” system by (1) expressing the NAD ⁺ kinase gene <em>ppnk</em> from <em>Clostridium glutamicum</em> and the NADP ⁺ -dependent <em>gapC</em>_cae from <em>Clostridium acetobutyricum</em> and (2) knocking-out the endogenous <em>sthA</em> gene, which interacts with <em>ilvC</em> and <em>panE</em> in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"84 ","pages":"Pages 158-168"},"PeriodicalIF":6.8,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469293","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":"Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440","authors":"Alissa C. Bleem ,&nbsp;Eugene Kuatsjah ,&nbsp;Josefin Johnsen ,&nbsp;Elsayed T. Mohamed ,&nbsp;William G. Alexander ,&nbsp;Zoe A. Kellermyer ,&nbsp;Austin L. Carroll ,&nbsp;Riccardo Rossi ,&nbsp;Ian B. Schlander ,&nbsp;George L. Peabody V ,&nbsp;Adam M. Guss ,&nbsp;Adam M. Feist ,&nbsp;Gregg T. Beckham","doi":"10.1016/j.ymben.2024.06.009","DOIUrl":"10.1016/j.ymben.2024.06.009","url":null,"abstract":"<div><p>Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and <em>O</em>-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic <em>O</em>-demethylation, but they are rarely compared <em>in vivo</em> to determine an optimal biocatalytic strategy. Here, two pathways for aromatic <em>O</em>-demethylation were compared in <em>Pseudomonas putida</em> KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in <em>P. putida</em>, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate <em>O-</em>demethylase, PP_3494, a global regulator of vanillate catabolism, and <em>fghA</em>, involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms<em>,</em> yielding a platform strain for efficient <em>O</em>-demethylation of lignin-related aromatic compounds to value-added products.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"84 ","pages":"Pages 145-157"},"PeriodicalIF":6.8,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469296","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}