Pub Date : 2024-05-01DOI: 10.1016/j.ymben.2024.02.019
Xiaolin Liu , Kang Li , Jing Yu , Chuanteng Ma , Qian Che , Tianjiao Zhu , Dehai Li , Blaine A. Pfeifer , Guojian Zhang
{"title":"Corrigendum to “Cyclo-diphenylalanine Production in Aspergillus nidulans through Stepwise Metabolic Engineering” [Metab. Eng. 82 (2024) 147–156]","authors":"Xiaolin Liu , Kang Li , Jing Yu , Chuanteng Ma , Qian Che , Tianjiao Zhu , Dehai Li , Blaine A. Pfeifer , 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":null,"pages":null},"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}
Pub Date : 2024-05-01DOI: 10.1016/j.ymben.2024.04.006
Na Zhang , Xiaohan Li , Qiang Zhou , Ying Zhang , Bo Lv , Bing Hu , Chun Li
{"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 , Xiaohan Li , Qiang Zhou , Ying Zhang , Bo Lv , Bing Hu , 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":null,"pages":null},"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}
Pub Date : 2024-05-01DOI: 10.1016/j.ymben.2024.05.001
Jie Wu , Jing Wu , Ru-Li He , Lan Hu , Dong-Feng Liu , Wen-Wei Li
Shewanella oneidensis 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 S. oneidensis 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 S. oneidensis.
{"title":"Modularized Engineering of Shewanella oneidensis MR-1 for Efficient and Directional Synthesis of 5-Aminolevulinic Acid","authors":"Jie Wu , Jing Wu , Ru-Li He , Lan Hu , Dong-Feng Liu , 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":null,"pages":null},"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}
Pub Date : 2024-04-20DOI: 10.1016/j.ymben.2024.04.005
Na Zhang , Xiaohan Li , Qiang Zhou , Ying Zhang , Bo Lv , Bing Hu , Chun Li
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 in silico 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 in silico 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 in silico strain design for efficient PNPs production. OptKnock simulation was performed on the GEM of Saccharomyces cerevisiae OA07, an engineered strain for oleanolic acid (OA) bioproduction that has been reported previously. The simulation predicted that the single deletion of fol1, fol2, fol3, abz1, and abz2, or a combined knockout of hfd1, ald2 and ald3 could improve its OA production. Consequently, strains EK1∼EK7 were constructed and cultivated. EK3 (OA07△fol3), EK5 (OA07△abz1), and EK6 (OA07△abz2) 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 fol3, abz1, abz2, and at acetyl-CoA carboxylase-encoding gene acc1, 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.
近几十年来,利用宿主细胞中的异源代谢途径生产植物天然产物(PNPs)的微生物生物工程领域不断发展。异源代谢途径被引入宿主细胞后,传统的代谢工程技术被用来提高 PNP 生物合成途径的生产率和产量,以及管理竞争途径。计算生物学的出现标志着通过硅学方法进行菌株设计的新纪元的开始。这些方法利用基因组尺度代谢模型(GEM)和通量优化算法来促进整个细胞代谢网络的合理设计。然而,由于内源基因被硬性敲除,硅学策略的实施往往会导致代谢通量分布不均,从而阻碍细胞生长并最终影响目标产物的积累。在本研究中,我们创造性地利用合成生物学来完善高效生产 PNPs 的硅学菌株设计。OptKnock 模拟是在毕赤酵母(Saccharomyces cerevisiae)OA07 的 GEM 上进行的,OA07 是一种用于齐墩果酸(Oleanolic acid,OA)生物生产的工程菌株。模拟预测,单个删除 fol1、fol2、fol3、abz1 和 abz2,或联合敲除 hfd1、ald2 和 ald3 可提高其 OA 产量。因此,构建并培养了EK1∼EK7菌株。与原始菌株OA07相比,EK3(OA07△fol3)、EK5(OA07△abz1)和EK6(OA07△abz2)在批量培养中的OA滴度显著提高。然而,在批量喂养模式下,这些提高并不明显,这表明基因缺失并不支持可持续的 OA 生产。为了解决这个问题,我们在 fol3、abz1、abz2 和乙酰-CoA 羧化酶编码基因 acc1 上设计了一个由丙二酰-CoA(一种与生长相关的中间产物,其合成是 OA 合成的旁路)调控的负反馈回路,以动态、自主地调控 OA07 中这些基因的表达。构建的菌株 R_3A、R_5A 和 R_6A 在批次或喂养批次培养模式下的 OA 滴度均明显高于初始菌株和响应基因敲除突变体。其中,菌株 R_3A 的 OA 滴度最高。在烧瓶分批进行喂养培养时,其 OA 滴度是初始菌株的两倍;在发酵罐分批进行喂养培养时,其 OA 滴度在 96 小时内达到 1.23 ± 0.04 g L-1。这表明,优化算法与合成生物学方法的整合在设计生产 PNP 的菌株方面是有效合理的。
{"title":"Self-controlled in silico gene knockdown strategies to enhance the sustainable production of heterologous terpenoid by Saccharomyces cerevisiae","authors":"Na Zhang , Xiaohan Li , Qiang Zhou , Ying Zhang , Bo Lv , Bing Hu , 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<sup>−1</sup> 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":null,"pages":null},"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}
Pub Date : 2024-04-16DOI: 10.1016/j.ymben.2024.04.004
Kangsan Kim , Donghui Choe , Minjeong Kang , Sang-Hyeok Cho , Suhyung Cho , Ki Jun Jeong , Bernhard Palsson , Byung-Kwan Cho
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, Escherichia coli 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 glpK, ppsA, ydcI, and rph-pyrE, 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.
{"title":"Serial adaptive laboratory evolution enhances mixed carbon metabolic capacity of Escherichia coli","authors":"Kangsan Kim , Donghui Choe , Minjeong Kang , Sang-Hyeok Cho , Suhyung Cho , Ki Jun Jeong , Bernhard Palsson , 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":null,"pages":null},"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}
Pub Date : 2024-04-15DOI: 10.1016/j.ymben.2024.04.002
Yajing Zhang , Tao Sun , Linqi Liu , Xupeng Cao , Weiwen Zhang , Wangyin Wang , Can Li
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 Escherichia coli, 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 Escherichia coli opens an efficient way for fixing CO2 into value-added bioproducts.
微生物将二氧化碳固定为乳酸(LA)是低碳生物制造的重要方法。利用微生物工程技术将二氧化碳和糖作为共底物,可以通过输入适度的还原力来驱动二氧化碳固定到产品中,从而创建高效的途径。然而,要实现对有机碳的完全保护,如何设计出与原生中央代谢兼容的二氧化碳固定模块,并将这些过程合并以提高 LA 的生物生产是一个巨大的挑战。在这项研究中,我们在大肠杆菌中设计并构建了太阳能甲酸/戊糖(SFAP)途径,使二氧化碳固定与糖分解代谢相结合,生产 LA。在 SFAP 途径中,甲酸氧化产生的足够还原当量推动葡萄糖代谢从糖酵解转向磷酸戊糖途径。以 Rubisco 为基础的 CO2 固定和 C3 中间产物的顺序还原按比例产生 LA。与单纯的葡萄糖原料相比,二氧化碳固定理论上可使 LA 的产量提高 20%。这种将光电化学电池和工程大肠杆菌整合在一起的 SFAP 途径为将 CO2 固定为高附加值生物产品开辟了一条有效途径。
{"title":"Engineering a solar formic acid/pentose (SFAP) pathway in Escherichia coli for lactic acid production","authors":"Yajing Zhang , Tao Sun , Linqi Liu , Xupeng Cao , Weiwen Zhang , Wangyin Wang , 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 CO<sub>2</sub> fixation into lactic acid (LA) is an important approach for low-carbon biomanufacturing. Engineering microbes to utilize CO<sub>2</sub> and sugar as co-substrates can create efficient pathways through input of moderate reducing power to drive CO<sub>2</sub> fixation into product. However, to achieve complete conservation of organic carbon, how to engineer the CO<sub>2</sub>-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 CO<sub>2</sub> 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 CO<sub>2</sub> fixation and sequential reduction of C3 intermediates are conducted to produce LA stoichiometrically. CO<sub>2</sub> 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 CO<sub>2</sub> into value-added bioproducts.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"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}
Pub Date : 2024-04-15DOI: 10.1016/j.ymben.2024.03.008
Katherine J. Chou , Trevor Croft , Skyler D. Hebdon , Lauren R. Magnusson , Wei Xiong , Luis H. Reyes , Xiaowen Chen , Emily J. Miller , Danielle M. Riley , Sunnyjoy Dupuis , Kathrin A. Laramore , Lisa M. Keller , Dirk Winkelman , Pin-Ching Maness
Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different β-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the β-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.
{"title":"Engineering the cellulolytic bacterium, Clostridium thermocellum, to co-utilize hemicellulose","authors":"Katherine J. Chou , Trevor Croft , Skyler D. Hebdon , Lauren R. Magnusson , Wei Xiong , Luis H. Reyes , Xiaowen Chen , Emily J. Miller , Danielle M. Riley , Sunnyjoy Dupuis , Kathrin A. Laramore , Lisa M. Keller , Dirk Winkelman , Pin-Ching Maness","doi":"10.1016/j.ymben.2024.03.008","DOIUrl":"10.1016/j.ymben.2024.03.008","url":null,"abstract":"<div><p>Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and <em>Clostridium thermocellum</em> is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by <em>C. thermocellum</em> is approached with co-cultures, because <em>C. thermocellum</em> does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered <em>C. thermocellum</em> to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different β-xylosidase enzymes originating from <em>Thermoanaerobacterium saccharolyticum</em> into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the β-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported <em>C. thermocellum</em> strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140786785","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}
Pub Date : 2024-04-15DOI: 10.1016/j.ymben.2024.04.003
Armand Bernard , Seungwoo Cha , Hyesoo Shin , Daeyeol Lee , Ji-Sook Hahn
Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20K197G (Erg20G) and Erg20F96W, N127W (Erg20WW), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20WWG mutant with over four times higher GPP accumulating capability than Erg20WW, as observed through geraniol production levels. The Erg20WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20WWG-fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20WWG-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.
{"title":"Efficient production of (S)-limonene and geraniol in Saccharomyces cerevisiae through the utilization of an Erg20 mutant with enhanced GPP accumulation capability","authors":"Armand Bernard , Seungwoo Cha , Hyesoo Shin , Daeyeol Lee , Ji-Sook Hahn","doi":"10.1016/j.ymben.2024.04.003","DOIUrl":"https://doi.org/10.1016/j.ymben.2024.04.003","url":null,"abstract":"<div><p>Monoterpenes and monoterpenoids such as (<em>S</em>)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. <em>Saccharomyces cerevisiae</em> has proven to be an effective host to produce various terpenes and terpenoids. (<em>S</em>)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20<sup>K197G</sup> (Erg20<sup>G</sup>) and Erg20<sup>F96W, N127W</sup> (Erg20<sup>WW</sup>), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20<sup>WWG</sup> mutant with over four times higher GPP accumulating capability than Erg20<sup>WW</sup>, as observed through geraniol production levels. The Erg20<sup>WWG</sup> mutant was fused to the LS from <em>Mentha spicata</em> or the GES from <em>Catharanthus roseus</em> for efficient conversion of GPP to (<em>S</em>)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20<sup>WWG</sup>-fused enzymes in peroxisomes, while simultaneously downregulating the essential <em>ERG20</em> gene using the glucose-sensing <em>HXT1</em> promoter. In the case of (<em>S</em>)-limonene production, additional Erg20<sup>WWG</sup>-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (<em>S</em>)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20<sup>WWG</sup> mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140649963","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}
Pub Date : 2024-04-04DOI: 10.1016/j.ymben.2024.03.005
Axel Theorell , Johann F. Jadebeck , Wolfgang Wiechert , Johnjoe McFadden , Katharina Nöh
Metabolic reaction rates (fluxes) play a crucial role in comprehending cellular phenotypes and are essential in areas such as metabolic engineering, biotechnology, and biomedical research. The state-of-the-art technique for estimating fluxes is metabolic flux analysis using isotopic labelling (13C-MFA), which uses a dataset-model combination to determine the fluxes. Bayesian statistical methods are gaining popularity in the field of life sciences, but the use of 13C-MFA is still dominated by conventional best-fit approaches. The slow take-up of Bayesian approaches is, at least partly, due to the unfamiliarity of Bayesian methods to metabolic engineering researchers. To address this unfamiliarity, we here outline similarities and differences between the two approaches and highlight particular advantages of the Bayesian way of flux analysis. With a real-life example, re-analysing a moderately informative labelling dataset of E. coli, we identify situations in which Bayesian methods are advantageous and more informative, pointing to potential pitfalls of current 13C-MFA evaluation approaches. We propose the use of Bayesian model averaging (BMA) for flux inference as a means of overcoming the problem of model uncertainty through its tendency to assign low probabilities to both, models that are unsupported by data, and models that are overly complex. In this capacity, BMA resembles a tempered Ockham's razor. With the tempered razor as a guide, BMA-based 13C-MFA alleviates the problem of model selection uncertainty and is thereby capable of becoming a game changer for metabolic engineering by uncovering new insights and inspiring novel approaches.
{"title":"Rethinking 13C-metabolic flux analysis – The Bayesian way of flux inference","authors":"Axel Theorell , Johann F. Jadebeck , Wolfgang Wiechert , Johnjoe McFadden , Katharina Nöh","doi":"10.1016/j.ymben.2024.03.005","DOIUrl":"https://doi.org/10.1016/j.ymben.2024.03.005","url":null,"abstract":"<div><p>Metabolic reaction rates (fluxes) play a crucial role in comprehending cellular phenotypes and are essential in areas such as metabolic engineering, biotechnology, and biomedical research. The state-of-the-art technique for estimating fluxes is metabolic flux analysis using isotopic labelling (<sup>13</sup>C-MFA), which uses a dataset-model combination to determine the fluxes. Bayesian statistical methods are gaining popularity in the field of life sciences, but the use of <sup>13</sup>C-MFA is still dominated by conventional best-fit approaches. The slow take-up of Bayesian approaches is, at least partly, due to the unfamiliarity of Bayesian methods to metabolic engineering researchers. To address this unfamiliarity, we here outline similarities and differences between the two approaches and highlight particular advantages of the Bayesian way of flux analysis. With a real-life example, re-analysing a moderately informative labelling dataset of <em>E. coli,</em> we identify situations in which Bayesian methods are advantageous and more informative, pointing to potential pitfalls of current <sup>13</sup>C-MFA evaluation approaches. We propose the use of Bayesian model averaging (BMA) for flux inference as a means of overcoming the problem of model uncertainty through its tendency to assign low probabilities to both, models that are unsupported by data, and models that are overly complex. In this capacity, BMA resembles a tempered Ockham's razor. With the tempered razor as a guide, BMA-based <sup>13</sup>C-MFA alleviates the problem of model selection uncertainty and is thereby capable of becoming a game changer for metabolic engineering by uncovering new insights and inspiring novel approaches.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140559145","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}
Pub Date : 2024-04-04DOI: 10.1016/j.ymben.2024.04.001
Hui-Zhong Sun , Qing Li , Wei Shang , Bin Qiao , Qiu-Man Xu , Jing-Sheng Cheng
Polymyxin is a lipopeptide antibiotic that is effective against multidrug-resistant Gram-negative bacteria. However, its clinical development is limited due to low titer and the presence of homologs. To address this, the polymyxin gene cluster was integrated into Bacillus subtilis, and sfp from Paenibacillus polymyxa was expressed heterologously, enabling recombinant B. subtilis to synthesize polymyxin B. Regulating NRPS domain inhibited formation of polymyxin B2 and B3. The production of polymyxin B increased to 329.7 mg/L by replacing the native promoters of pmxA, pmxB, and pmxE with PfusA, C2up, and PfusA, respectively. Further enhancement in this production, up to 616.1 mg/L, was achieved by improving the synthesis ability of 6-methyloctanoic acid compared to the original strain expressing polymyxin heterologously. Additionally, incorporating an anikasin-derived domain into the hybrid nonribosomal peptide synthase of polymyxin increased the B1 ratio in polymyxin B from 57.5% to 62.2%. Through optimization of peptone supply in the fermentation medium and fermentation in a 5.0-L bioreactor, the final polymyxin B titer reached 962.1 mg/L, with a yield of 19.24 mg/g maltodextrin and a productivity of 10.02 mg/(L·h). This study demonstrates a successful approach for enhancing polymyxin B production and increasing the B1 ratio through combinatorial metabolic engineering.
多粘菌素是一种脂肽类抗生素,对具有多重耐药性的革兰氏阴性菌有效。然而,由于滴度低和存在同源物,其临床开发受到限制。为了解决这个问题,多粘菌素基因簇被整合到了Ⅳ-Ⅴ基因中,并通过异源表达,使重组体能够合成多粘菌素 B。用 P、C2up 和 P 分别取代原生启动子 、 、 和 ,多粘菌素 B 的产量增加到 329.7 mg/L。与异源表达多粘菌素的原始菌株相比,通过提高 6-甲基辛酸的合成能力,产量进一步提高到 616.1 毫克/升。此外,在多粘菌素的杂交非核糖体肽合成酶中加入安乃近衍生结构域,可将多粘菌素 B 中的 B1 比率从 57.5% 提高到 62.2%。通过优化发酵培养基中蛋白胨的供应和在 5.0 升生物反应器中发酵,最终多粘菌素 B 的滴度达到了 962.1 mg/L,产量为 19.24 mg/g麦芽糊精,生产率为 10.02 mg/(L-h)。这项研究展示了一种通过组合代谢工程提高多粘菌素 B 产量和增加 B1 比率的成功方法。
{"title":"Combinatorial metabolic engineering of Bacillus subtilis for de novo production of polymyxin B","authors":"Hui-Zhong Sun , Qing Li , Wei Shang , Bin Qiao , Qiu-Man Xu , Jing-Sheng Cheng","doi":"10.1016/j.ymben.2024.04.001","DOIUrl":"10.1016/j.ymben.2024.04.001","url":null,"abstract":"<div><p>Polymyxin is a lipopeptide antibiotic that is effective against multidrug-resistant Gram-negative bacteria. However, its clinical development is limited due to low titer and the presence of homologs. To address this, the polymyxin gene cluster was integrated into <em>Bacillus subtilis</em>, and <em>sfp</em> from <em>Paenibacillus polymyxa</em> was expressed heterologously, enabling recombinant <em>B. subtilis</em> to synthesize polymyxin B. Regulating NRPS domain inhibited formation of polymyxin B2 and B3. The production of polymyxin B increased to 329.7 mg/L by replacing the native promoters of <em>pmxA</em>, <em>pmxB</em>, and <em>pmxE</em> with P<em>fusA</em>, C2up, and P<em>fusA</em>, respectively. Further enhancement in this production, up to 616.1 mg/L, was achieved by improving the synthesis ability of 6-methyloctanoic acid compared to the original strain expressing polymyxin heterologously. Additionally, incorporating an anikasin-derived domain into the hybrid nonribosomal peptide synthase of polymyxin increased the B1 ratio in polymyxin B from 57.5% to 62.2%. Through optimization of peptone supply in the fermentation medium and fermentation in a 5.0-L bioreactor, the final polymyxin B titer reached 962.1 mg/L, with a yield of 19.24 mg/g maltodextrin and a productivity of 10.02 mg/(L·h). This study demonstrates a successful approach for enhancing polymyxin B production and increasing the B1 ratio through combinatorial metabolic engineering.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140534661","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}