Metabolic engineering最新文献

{"title":"Rational design of a bacterial import system for new-to-nature molecules","authors":"","doi":"10.1016/j.ymben.2024.05.005","DOIUrl":"10.1016/j.ymben.2024.05.005","url":null,"abstract":"<div><p>Integration of novel compounds into biological processes holds significant potential for modifying or expanding existing cellular functions. However, the cellular uptake of these compounds is often hindered by selectively permeable membranes. We present a novel bacterial transport system that has been rationally designed to address this challenge. Our approach utilizes a highly promiscuous sulfonate membrane transporter, which allows the passage of cargo molecules attached as amides to a sulfobutanoate transport vector molecule into the cytoplasm of the cell. These cargoes can then be unloaded from the sulfobutanoyl amides using an engineered variant of the enzyme γ-glutamyl transferase, which hydrolyzes the amide bond and releases the cargo molecule within the cell. Here, we provide evidence for the broad substrate specificity of both components of the system by evaluating a panel of structurally diverse sulfobutanoyl amides. Furthermore, we successfully implement the synthetic uptake system <em>in vivo</em> and showcase its functionality by importing an impermeant non-canonical amino acid.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"85 ","pages":"Pages 26-34"},"PeriodicalIF":6.8,"publicationDate":"2024-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624000715/pdfft?md5=943ee8bd0b08d14fa53326f2db881676&pid=1-s2.0-S1096717624000715-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141158608","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":"Use of acetate as substrate for sustainable production of homoserine and threonine by Escherichia coli W3110: A modular metabolic engineering approach","authors":"Toan Minh Vo,&nbsp;Joon Young Park,&nbsp;Donghyuk Kim,&nbsp;Sunghoon Park","doi":"10.1016/j.ymben.2024.05.004","DOIUrl":"10.1016/j.ymben.2024.05.004","url":null,"abstract":"<div><p>Acetate, a promising yet underutilized carbon source for biological production, was explored for the efficient production of homoserine and threonine in <em>Escherichia coli</em> W. A modular metabolic engineering approach revealed the crucial roles of both acetate assimilation pathways (AckA/Pta and Acs), optimized TCA cycle flux and glyoxylate shunt activity, and enhanced CoA availability, mediated by increased pantothenate kinase activity, for efficient homoserine production. The engineered strain W–H22/pM2/pR1P exhibited a high acetate assimilation rate (5.47 mmol/g cell/h) and produced 44.1 g/L homoserine in 52 h with a 53% theoretical yield (0.18 mol/mol) in fed-batch fermentation. Similarly, strain W–H31/pM2/pR1P achieved 45.8 g/L threonine in 52 h with a 65% yield (0.22 mol/mol). These results represent the highest reported levels of amino acid production using acetate, highlighting its potential as a valuable and sustainable feedstock for biomanufacturing.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"84 ","pages":"Pages 13-22"},"PeriodicalIF":8.4,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141142614","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 Saccharomyces cerevisiae for enhanced metabolic robustness and L-lactic acid production from lignocellulosic biomass","authors":"Bohyun Choi ,&nbsp;Albert Tafur Rangel ,&nbsp;Eduard J. Kerkhoven ,&nbsp;Yvonne Nygård","doi":"10.1016/j.ymben.2024.05.003","DOIUrl":"10.1016/j.ymben.2024.05.003","url":null,"abstract":"<div><p>Metabolic engineering for high productivity and increased robustness is needed to enable sustainable biomanufacturing of lactic acid from lignocellulosic biomass. Lactic acid is an important commodity chemical used for instance as a monomer for production of polylactic acid, a biodegradable polymer. Here, rational and model-based optimization was used to engineer a diploid, xylose fermenting <em>Saccharomyces cerevisiae</em> strain to produce L-lactic acid. The metabolic flux was steered towards lactic acid through the introduction of multiple lactate dehydrogenase encoding genes while deleting <em>ERF2</em>, <em>GPD1</em>, and <em>CYB2</em>. A production of 93 g/L of lactic acid with a yield of 0.84 g/g was achieved using xylose as the carbon source. To increase xylose utilization and reduce acetic acid synthesis, <em>PHO13</em> and <em>ALD6</em> were also deleted from the strain. Finally, <em>CDC19</em> encoding a pyruvate kinase was overexpressed, resulting in a yield of 0.75 g lactic acid/g sugars consumed, when the substrate used was a synthetic lignocellulosic hydrolysate medium, containing hexoses, pentoses and inhibitors such as acetate and furfural. Notably, modeling also provided leads for understanding the influence of oxygen in lactic acid production. High lactic acid production from xylose, at oxygen-limitation could be explained by a reduced flux through the oxidative phosphorylation pathway. On the contrast, higher oxygen levels were beneficial for lactic acid production with the synthetic hydrolysate medium, likely as higher ATP concentrations are needed for tolerating the inhibitors therein. The work highlights the potential of <em>S. cerevisiae</em> for industrial production of lactic acid from lignocellulosic biomass.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"84 ","pages":"Pages 23-33"},"PeriodicalIF":8.4,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624000697/pdfft?md5=843ae8f101d942e166b7a1d8f4648a82&pid=1-s2.0-S1096717624000697-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141093523","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":"Engineering yeasts to Co-utilize methanol or formate coupled with CO2 fixation","authors":"Yuanke Guo,&nbsp;Rui Zhang,&nbsp;Jing Wang,&nbsp;Ruirui Qin,&nbsp;Jiao Feng,&nbsp;Kequan Chen,&nbsp;Xin Wang","doi":"10.1016/j.ymben.2024.05.002","DOIUrl":"10.1016/j.ymben.2024.05.002","url":null,"abstract":"<div><p>The development of synthetic microorganisms that could use one-carbon compounds, such as carbon dioxide, methanol, or formate, has received considerable interest. In this study, we engineered <em>Pichia pastoris</em> and <em>Saccharomyces cerevisiae</em> to both synthetic methylotrophy and formatotrophy, enabling them to co-utilize methanol or formate with CO₂ fixation through a synthetic C1-compound assimilation pathway (MFORG pathway). This pathway consisted of a methanol-formate oxidation module and the reductive glycine pathway. We first assembled the MFORG pathway in <em>P. pastoris</em> using endogenous enzymes, followed by blocking the native methanol assimilation pathway, modularly engineering genes of MFORG pathway, and compartmentalizing the methanol oxidation module. These modifications successfully enabled the methylotrophic yeast <em>P. pastoris</em> to utilize both methanol and formate. We then introduced the MFORG pathway from <em>P. pastoris</em> into the model yeast <em>S. cerevisiae</em>, establishing the synthetic methylotrophy and formatotrophy in this organism<em>.</em> The resulting strain could also successfully utilize both methanol and formate with consumption rates of 20 mg/L/h and 36.5 mg/L/h, respectively. The ability of the engineered <em>P. pastoris</em> and <em>S. cerevisiae</em> to co-assimilate CO₂ with methanol or formate through the MFORG pathway was also confirmed by ¹³C-tracer analysis. Finally, production of 5-aminolevulinic acid and lactic acid by co-assimilating methanol and CO₂ was demonstrated in the engineered <em>P. pastoris</em> and <em>S. cerevisiae</em>. This work indicates the potential of the MFORG pathway in developing different hosts to use various one-carbon compounds for chemical production.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"84 ","pages":"Pages 1-12"},"PeriodicalIF":8.4,"publicationDate":"2024-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140957015","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum to “Cyclo-diphenylalanine Production in Aspergillus nidulans through Stepwise Metabolic Engineering” [Metab. Eng. 82 (2024) 147–156]","authors":"Xiaolin Liu ,&nbsp;Kang Li ,&nbsp;Jing Yu ,&nbsp;Chuanteng Ma ,&nbsp;Qian Che ,&nbsp;Tianjiao Zhu ,&nbsp;Dehai Li ,&nbsp;Blaine A. Pfeifer ,&nbsp;Guojian Zhang","doi":"10.1016/j.ymben.2024.02.019","DOIUrl":"10.1016/j.ymben.2024.02.019","url":null,"abstract":"","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"83 ","pages":"Page 216"},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624000351/pdfft?md5=d5c332c5a8b6764f7d8e588b1f93dae6&pid=1-s2.0-S1096717624000351-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140094370","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 “Self-controlled in silico gene knockdown strategies to enhance the sustainable production of heterologous terpenoid by Saccharomyces cerevisiae” [Metab. Eng. 83 (2024) 172–182]","authors":"Na Zhang ,&nbsp;Xiaohan Li ,&nbsp;Qiang Zhou ,&nbsp;Ying Zhang ,&nbsp;Bo Lv ,&nbsp;Bing Hu ,&nbsp;Chun Li","doi":"10.1016/j.ymben.2024.04.006","DOIUrl":"10.1016/j.ymben.2024.04.006","url":null,"abstract":"","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"83 ","pages":"Page 217"},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717624000600/pdfft?md5=9a5d42bccde0e0414c09f6625165c309&pid=1-s2.0-S1096717624000600-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140909467","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":"Modularized Engineering of Shewanella oneidensis MR-1 for Efficient and Directional Synthesis of 5-Aminolevulinic Acid","authors":"Jie Wu ,&nbsp;Jing Wu ,&nbsp;Ru-Li He ,&nbsp;Lan Hu ,&nbsp;Dong-Feng Liu ,&nbsp;Wen-Wei Li","doi":"10.1016/j.ymben.2024.05.001","DOIUrl":"10.1016/j.ymben.2024.05.001","url":null,"abstract":"<div><p><em>Shewanella oneidensis</em> MR-1 has found widespread applications in pollutant transformation and bioenergy production, closely tied to its outstanding heme synthesis capabilities. However, this significant biosynthetic potential is still unexploited so far. Here, we turned this bacterium into a highly-efficient bio-factory for green synthesis of 5-Aminolevulinic Acid (5-ALA), an important chemical for broad applications in agriculture, medicine, and the food industries. The native C5 pathway genes of <em>S. oneidensis</em> was employed, together with the introduction of foreign anti-oxidation module, to establish the 5-ALA production module, resulting 87-fold higher 5-ALA yield and drastically enhanced tolerance than the wild type. Furthermore, the metabolic flux was regulated by using CRISPR interference and base editing techniques to suppress the competitive pathways to further improve the 5-ALA titer. The engineered strain exhibited 123-fold higher 5-ALA production capability than the wild type. This study not only provides an appealing new route for 5-ALA biosynthesis, but also presents a multi-dimensional modularized engineering strategy to broaden the application scope of <em>S. oneidensis</em>.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"83 ","pages":"Pages 206-215"},"PeriodicalIF":8.4,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140851997","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":"Self-controlled in silico gene knockdown strategies to enhance the sustainable production of heterologous terpenoid by Saccharomyces cerevisiae","authors":"Na Zhang ,&nbsp;Xiaohan Li ,&nbsp;Qiang Zhou ,&nbsp;Ying Zhang ,&nbsp;Bo Lv ,&nbsp;Bing Hu ,&nbsp;Chun Li","doi":"10.1016/j.ymben.2024.04.005","DOIUrl":"https://doi.org/10.1016/j.ymben.2024.04.005","url":null,"abstract":"<div><p>Microbial bioengineering is a growing field for producing plant natural products (PNPs) in recent decades, using heterologous metabolic pathways in host cells. Once heterologous metabolic pathways have been introduced into host cells, traditional metabolic engineering techniques are employed to enhance the productivity and yield of PNP biosynthetic routes, as well as to manage competing pathways. The advent of computational biology has marked the beginning of a novel epoch in strain design through <em>in silico</em> methods. These methods utilize genome-scale metabolic models (GEMs) and flux optimization algorithms to facilitate rational design across the entire cellular metabolic network. However, the implementation of <em>in silico</em> strategies can often result in an uneven distribution of metabolic fluxes due to the rigid knocking out of endogenous genes, which can impede cell growth and ultimately impact the accumulation of target products. In this study, we creatively utilized synthetic biology to refine <em>in silico</em> strain design for efficient PNPs production. OptKnock simulation was performed on the GEM of <em>Saccharomyces cerevisiae</em> OA07, an engineered strain for oleanolic acid (OA) bioproduction that has been reported previously. The simulation predicted that the single deletion of <em>fol1</em>, <em>fol2</em>, <em>fol3</em>, <em>abz1</em>, and <em>abz2</em>, or a combined knockout of <em>hfd1</em>, <em>ald2</em> and <em>ald3</em> could improve its OA production. Consequently, strains EK1∼EK7 were constructed and cultivated. EK3 (OA07△<em>fol3</em>), EK5 (OA07△<em>abz1</em>), and EK6 (OA07△<em>abz2</em>) had significantly higher OA titers in a batch cultivation compared to the original strain OA07. However, these increases were less pronounced in the fed-batch mode, indicating that gene deletion did not support sustainable OA production. To address this, we designed a negative feedback circuit regulated by malonyl-CoA, a growth-associated intermediate whose synthesis served as a bypass to OA synthesis, at <em>fol3, abz1</em>, <em>abz2</em>, and at acetyl-CoA carboxylase-encoding gene <em>acc1</em>, to dynamically and autonomously regulate the expression of these genes in OA07. The constructed strains R_3A, R_5A and R_6A had significantly higher OA titers than the initial strain and the responding gene-knockout mutants in either batch or fed-batch culture modes. Among them, strain R_3A stand out with the highest OA titer reported to date. Its OA titer doubled that of the initial strain in the flask-level fed-batch cultivation, and achieved at 1.23 ± 0.04 g L⁻¹ in 96 h in the fermenter-level fed-batch mode. This indicated that the integration of optimization algorithm and synthetic biology approaches was efficiently rational for PNP-producing strain design.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"83 ","pages":"Pages 172-182"},"PeriodicalIF":8.4,"publicationDate":"2024-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140639139","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":"Serial adaptive laboratory evolution enhances mixed carbon metabolic capacity of Escherichia coli","authors":"Kangsan Kim ,&nbsp;Donghui Choe ,&nbsp;Minjeong Kang ,&nbsp;Sang-Hyeok Cho ,&nbsp;Suhyung Cho ,&nbsp;Ki Jun Jeong ,&nbsp;Bernhard Palsson ,&nbsp;Byung-Kwan Cho","doi":"10.1016/j.ymben.2024.04.004","DOIUrl":"https://doi.org/10.1016/j.ymben.2024.04.004","url":null,"abstract":"<div><p>Microbes have inherent capacities for utilizing various carbon sources, however they often exhibit sub-par fitness due to low metabolic efficiency. To test whether a bacterial strain can optimally utilize multiple carbon sources, <em>Escherichia coli</em> was serially evolved in L-lactate and glycerol. This yielded two end-point strains that evolved first in L-lactate then in glycerol, and vice versa. The end-point strains displayed a universal growth advantage on single and a mixture of adaptive carbon sources, enabled by a concerted action of carbon source-specialists and generalist mutants. The combination of just four variants of <em>glpK</em>, <em>ppsA</em>, <em>ydcI</em>, and <em>rph-pyrE</em>, accounted for more than 80% of end-point strain fitness. In addition, machine learning analysis revealed a coordinated activity of transcriptional regulators imparting condition-specific regulation of gene expression. The effectiveness of the serial adaptive laboratory evolution (ALE) scheme in bioproduction applications was assessed under single and mixed-carbon culture conditions, in which serial ALE strain exhibited superior productivity of acetoin compared to ancestral strains. Together, systems-level analysis elucidated the molecular basis of serial evolution, which hold potential utility in bioproduction applications.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"83 ","pages":"Pages 160-171"},"PeriodicalIF":8.4,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140632558","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 a solar formic acid/pentose (SFAP) pathway in Escherichia coli for lactic acid production","authors":"Yajing Zhang ,&nbsp;Tao Sun ,&nbsp;Linqi Liu ,&nbsp;Xupeng Cao ,&nbsp;Weiwen Zhang ,&nbsp;Wangyin Wang ,&nbsp;Can Li","doi":"10.1016/j.ymben.2024.04.002","DOIUrl":"https://doi.org/10.1016/j.ymben.2024.04.002","url":null,"abstract":"<div><p>Microbial CO₂ fixation into lactic acid (LA) is an important approach for low-carbon biomanufacturing. Engineering microbes to utilize CO₂ and sugar as co-substrates can create efficient pathways through input of moderate reducing power to drive CO₂ fixation into product. However, to achieve complete conservation of organic carbon, how to engineer the CO₂-fixing modules compatible with native central metabolism and merge the processes for improving bioproduction of LA is a big challenge. In this study, we designed and constructed a solar formic acid/pentose (SFAP) pathway in <em>Escherichia coli</em>, which enabled CO₂ fixation merging into sugar catabolism to produce LA. In the SFAP pathway, adequate reducing equivalents from formate oxidation drive glucose metabolism shifting from glycolysis to the pentose phosphate pathway. The Rubisco-based CO₂ fixation and sequential reduction of C3 intermediates are conducted to produce LA stoichiometrically. CO₂ fixation theoretically can bring a 20% increase of LA production compared with sole glucose feedstock. This SFAP pathway in the integration of photoelectrochemical cell and an engineered <em>Escherichia coli</em> opens an efficient way for fixing CO₂ into value-added bioproducts.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"83 ","pages":"Pages 150-159"},"PeriodicalIF":8.4,"publicationDate":"2024-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140604816","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}