Pub Date : 2024-01-01DOI: 10.1016/j.ymben.2023.12.006
Helen O. Masson , Mojtaba Samoudi , Caressa M. Robinson , Chih-Chung Kuo , Linus Weiss , Km Shams Ud Doha , Alex Campos , Vijay Tejwani , Hussain Dahodwala , Patrice Menard , Bjorn G. Voldborg , Bradley Robasky , Susan T. Sharfstein , Nathan E. Lewis
Understanding protein secretion has considerable importance in biotechnology and important implications in a broad range of normal and pathological conditions including development, immunology, and tissue function. While great progress has been made in studying individual proteins in the secretory pathway, measuring and quantifying mechanistic changes in the pathway's activity remains challenging due to the complexity of the biomolecular systems involved. Systems biology has begun to address this issue with the development of algorithmic tools for analyzing biological pathways; however most of these tools remain accessible only to experts in systems biology with extensive computational experience. Here, we expand upon the user-friendly CellFie tool which quantifies metabolic activity from omic data to include secretory pathway functions, allowing any scientist to infer properties of protein secretion from omic data. We demonstrate how the secretory expansion of CellFie (secCellFie) can help predict metabolic and secretory functions across diverse immune cells, hepatokine secretion in a cell model of NAFLD, and antibody production in Chinese Hamster Ovary cells.
{"title":"Inferring secretory and metabolic pathway activity from omic data with secCellFie","authors":"Helen O. Masson , Mojtaba Samoudi , Caressa M. Robinson , Chih-Chung Kuo , Linus Weiss , Km Shams Ud Doha , Alex Campos , Vijay Tejwani , Hussain Dahodwala , Patrice Menard , Bjorn G. Voldborg , Bradley Robasky , Susan T. Sharfstein , Nathan E. Lewis","doi":"10.1016/j.ymben.2023.12.006","DOIUrl":"10.1016/j.ymben.2023.12.006","url":null,"abstract":"<div><p>Understanding protein secretion has considerable importance in biotechnology and important implications in a broad range of normal and pathological conditions including development, immunology, and tissue function. While great progress has been made in studying individual proteins in the secretory pathway, measuring and quantifying mechanistic changes in the pathway's activity remains challenging due to the complexity of the biomolecular systems involved. Systems biology has begun to address this issue with the development of algorithmic tools for analyzing biological pathways; however most of these tools remain accessible only to experts in systems biology with extensive computational experience. Here, we expand upon the user-friendly CellFie tool which quantifies metabolic activity from omic data to include secretory pathway functions, allowing any scientist to infer properties of protein secretion from omic data. We demonstrate how the secretory expansion of CellFie (secCellFie) can help predict metabolic and secretory functions across diverse immune cells, hepatokine secretion in a cell model of NAFLD, and antibody production in Chinese Hamster Ovary cells.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717623001799/pdfft?md5=fe08091f516bdd1ac5ce48a3328eea21&pid=1-s2.0-S1096717623001799-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138887191","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}
Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.
{"title":"Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization","authors":"Guoqiang Xu , Jiyue Wang , Jiancheng Shen , Yaxin Zhu , Wanjing Liu , Yuhang Chen , Jian Zha , Xiaomei Zhang , Xiaojuan Zhang , Jinsong Shi , Mattheos A.G. Koffas , Zhenghong Xu","doi":"10.1016/j.ymben.2023.12.008","DOIUrl":"10.1016/j.ymben.2023.12.008","url":null,"abstract":"<div><p>Previously, a novel <em>Corynebacterium glutamicum</em> strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in <em>Bacillus</em> spp. In the present study, PgsBCA was reconstituted and overexpressed in <em>C. glutamicum</em> to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from <em>B. licheniformis</em> are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in <em>C. glutamicum</em>. Next, the expression level of each <em>pgsB</em>, <em>pgsC</em>, and <em>pgsA</em> was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of <em>pgsB</em> or <em>pgsC</em> and maintaining a medium-level transcription level of <em>pgsA</em> led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of <em>pgsC</em> had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by <em>pgsB</em>. Next, genes encoding for PgsC from four different sources (<em>Bacillus subtilis</em>, <em>Bacillus anthracis</em>, <em>Bacillus</em> methylotrophicus<em>, and Bacillus amyloliquefacien</em>s) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from <em>B. licheniformis</em>. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717623001817/pdfft?md5=2b16e0c55fd2e5054d639d7facb55cd7&pid=1-s2.0-S1096717623001817-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139059584","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 : 2023-12-29DOI: 10.1016/j.ymben.2023.12.012
Helena Schulz-Mirbach , Beau Dronsella , Hai He , Tobias J. Erb
Synthetic biology aims at designing new biological functions from first principles. These new designs allow to expand the natural solution space and overcome the limitations of naturally evolved systems. One example is synthetic CO2-fixation pathways that promise to provide more efficient ways for the capture and conversion of CO2 than natural pathways, such as the Calvin Benson Bassham (CBB) cycle of photosynthesis. In this review, we provide a practical guideline for the design and realization of such new-to-nature CO2-fixation pathways. We introduce the concept of “synthetic CO2-fixation”, and give a general overview over the enzymology and topology of synthetic pathways, before we derive general principles for their design from their eight naturally evolved analogs. We provide a comprehensive summary of synthetic carbon-assimilation pathways and derive a step-by-step, practical guide from the theoretical design to their practical implementation, before ending with an outlook on new developments in the field.
{"title":"Creating new-to-nature carbon fixation: A guide","authors":"Helena Schulz-Mirbach , Beau Dronsella , Hai He , Tobias J. Erb","doi":"10.1016/j.ymben.2023.12.012","DOIUrl":"10.1016/j.ymben.2023.12.012","url":null,"abstract":"<div><p>Synthetic biology aims at designing new biological functions from first principles. These new designs allow to expand the natural solution space and overcome the limitations of naturally evolved systems. One example is synthetic CO<sub>2</sub>-fixation pathways that promise to provide more efficient ways for the capture and conversion of CO<sub>2</sub><span> than natural pathways, such as the Calvin Benson Bassham (CBB) cycle of photosynthesis. In this review, we provide a practical guideline for the design and realization of such new-to-nature CO</span><sub>2</sub>-fixation pathways. We introduce the concept of “synthetic CO<sub>2</sub><span>-fixation”, and give a general overview over the enzymology and topology of synthetic pathways, before we derive general principles for their design from their eight naturally evolved analogs. We provide a comprehensive summary of synthetic carbon-assimilation pathways and derive a step-by-step, practical guide from the theoretical design to their practical implementation, before ending with an outlook on new developments in the field.</span></p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2023-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139074510","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}
Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2′-fucosyllactose (2′-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2′-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2′-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2′-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2′-FL production. Therefore, to decrease the formation of byproduct 2′-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2′-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154–171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154–171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2′-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2′-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2′-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.
Lacto-N-fucopentaose I(LNFP I)是母乳中含量仅次于 2′-岩藻糖聚糖(2′-FL)的岩藻糖聚糖化母乳寡糖(HMO)。研究表明,LNFP I 对 B 组链球菌具有抗菌活性,对肠道病毒和诺瓦克病毒具有抗病毒作用。LNFP I 是由α1,2-岩藻糖基转移酶(α1,2-FucT)催化,通过α1,2-岩藻糖基转移反应合成乳-N-四糖骨架的 N-乙酰葡糖胺分子。然而,α1,2-岩藻糖基转移酶会竞争性地将岩藻糖转移到乳糖上,从而形成副产物 2′-FL。在这项研究中,我们构建了具有各种 α1,2-FucTs的大肠杆菌 LNFP I 生产菌株,并观察到在饲料批量发酵过程中出现了不希望出现的 2′-FL 积累,尽管在试管试验中,一些菌株产生的 LNFP I 不含 2′-FL。我们推测,α1,2-FucT 的杂合底物选择性是产生 2′-FL 的原因。因此,为了减少副产物 2′-FL 的生成,我们通过合理和半合理设计方法设计了 15 个来自弗朗西斯菌 FSC1006 的 FsFucT 变体。与野生型 FsFucT 相比,其中五个 FsFucT 变体的 2′-FL 产量降低了两倍多,同时还保持了相当水平的 LNFP I 产量。这些设计包括酶环区(残基 154-171)或直接或间接影响底物结合的特定残基(Q7、H162 和 L164)的置换。其中,表达 FsFucT_S3 变体的大肠杆菌菌株在 Fed-bach 发酵过程中积累了 19.6 克/升的 LNFP I 和 0.04 克/升的 2′-FL,而表达野生型 FsFucT 的大肠杆菌菌株则积累了 12.2 克/升的 LNFP I 和 5.85 克/升的 2′-FL。因此,我们通过对α1,2-FucT进行硅学结构建模和与各种配体的对接模拟,设计出α1,2-FucT的蛋白质工程,并结合宿主细胞的代谢工程,成功地证明了选择性地高效生产五糖LNFP I而不产生副产物2′-FL。
{"title":"Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases","authors":"Shun Endo , Tomotoshi Sugita , Sayaka Kamai , Kazuki Nakamura , Fuhito Yamazaki , Sotaro Sampei , Gustautas Snarskis , Audronė Valančiūtė , Masoud Kazemi , Irmantas Rokaitis , Kento Koketsu","doi":"10.1016/j.ymben.2023.12.009","DOIUrl":"10.1016/j.ymben.2023.12.009","url":null,"abstract":"<div><p>Lacto-<em>N</em><span>-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide<span> (HMO) in breast milk after 2′-fucosyllactose (2′-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B </span></span><em>Streptococcus</em> and antiviral effects against <span><em>Enterovirus</em></span> and <span><em>Norovirus</em></span>. Microbial production of HMOs by engineered <em>Escherichia coli</em> is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the <em>N</em>-acetylglucosamine moiety of the lacto-<em>N</em><span>-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2′-FL. In this study, we constructed LNFP I-producing strains of </span><em>E. coli</em> with various α1,2-fucTs, and observed undesired 2′-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2′-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2′-FL production. Therefore, to decrease the formation of byproduct 2′-FL, we designed 15 variants of FsFucT from <span><em>Francisella</em></span><span> sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2′-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154–171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the </span><em>E. coli</em> strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154–171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2′-FL, while the <em>E. coli</em> strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2′-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2′-FL by combining protein engineering of α1,2-FucT designed through <em>in silico</em><span> structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.</span></p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2023-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138887265","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 : 2023-12-14DOI: 10.1016/j.ymben.2023.12.005
Jie Liu , Yuanxiu Zhao , Jingmeng Zhang , Yu Kong , Pan Liu , Yumin Fang , Mengying Cui , Tianlin Pei , Xin Zhong , Ping Xu , Wenqing Qiu , Dongfeng Yang , Cathie Martin , Qing Zhao
Anthocyanins are widely distributed pigments in flowering plants with red, purple or blue colours. Their properties in promoting heath make anthocyanins perfect natural colourants for food additives. However, anthocyanins with strong colour and stability at neutral pH, suitable as food colourants are relatively rare in nature. Acylation increases anthocyanin stability and confers bluer colour. In this study, we isolated two anthocyanin regulators SbMyb75 and SbDel from S. baicalensis, and showed that constitutive expression of the two TFs led to accumulation of anthocyanins at high levels in black carrot hairy roots. However, these hairy roots had severe growth problems. We then developed a β-estradiol inducible system using XVE and a Lex-35S promoter, to initiate expression of the anthocyanin regulators and induced this system in hairy roots of black carrot, tobacco and morning glory. Anthocyanins with various decorations were produced in these hairy roots without any accompanying side-effects on growth. We further produced highly acylated anthocyanins with blue colour in a 5 L liquid culture in a bioreactor of hairy roots from morning glory. We provide here a strategy to produce highly decorated anthocyanins without the need for additional engineering of any of the genes encoding decorating enzymes. This strategy could be transferred to other species, with considerable potential for natural colourant production for the food industries.
{"title":"Production of species-specific anthocyanins through an inducible system in plant hairy roots","authors":"Jie Liu , Yuanxiu Zhao , Jingmeng Zhang , Yu Kong , Pan Liu , Yumin Fang , Mengying Cui , Tianlin Pei , Xin Zhong , Ping Xu , Wenqing Qiu , Dongfeng Yang , Cathie Martin , Qing Zhao","doi":"10.1016/j.ymben.2023.12.005","DOIUrl":"10.1016/j.ymben.2023.12.005","url":null,"abstract":"<div><p>Anthocyanins are widely distributed pigments in flowering plants with red, purple or blue colours. Their properties in promoting heath make anthocyanins perfect natural colourants for food additives. However, anthocyanins with strong colour and stability at neutral pH, suitable as food colourants are relatively rare in nature. Acylation increases anthocyanin stability and confers bluer colour. In this study, we isolated two anthocyanin regulators SbMyb75 and SbDel from <em>S. baicalensis</em>, and showed that constitutive expression of the two TFs led to accumulation of anthocyanins at high levels in black carrot hairy roots. However, these hairy roots had severe growth problems. We then developed a β-estradiol inducible system using XVE and a Lex-35S promoter, to initiate expression of the anthocyanin regulators and induced this system in hairy roots of black carrot, tobacco and morning glory. Anthocyanins with various decorations were produced in these hairy roots without any accompanying side-effects on growth. We further produced highly acylated anthocyanins with blue colour in a 5 L liquid culture in a bioreactor of hairy roots from morning glory. We provide here a strategy to produce highly decorated anthocyanins without the need for additional engineering of any of the genes encoding decorating enzymes. This strategy could be transferred to other species, with considerable potential for natural colourant production for the food industries.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2023-12-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717623001787/pdfft?md5=03751cec0875a63d9fdda172c4529597&pid=1-s2.0-S1096717623001787-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138658098","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 : 2023-12-09DOI: 10.1016/j.ymben.2023.12.003
Niklas Berndt Thalén , Mona Moradi Barzadd , Magnus Lundqvist , Johanna Rodhe , Monica Andersson , Gholamreza Bidkhori , Dominik Possner , Chao Su , Joakim Nilsson , Peter Eisenhut , Magdalena Malm , Alice Karlsson , Jeanette Vestin , Johan Forsberg , Erik Nordling , Adil Mardinoglu , Anna-Luisa Volk , Anna Sandegren , Johan Rockberg
Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.
罕见病虽然名为罕见病,但其实是一种常见病,每天都有数百万人受到罕见病的影响,而这些疾病往往无法得到治疗。硫酸酯酶是一个庞大的活化酶家族,与其中几种疾病有关。硫酸酯酶的遗传变异可能导致其活性受损,溶酶体内大分子分解减少,从而引发多种严重的致命疾病。虽然治疗方法很少,但可以通过重组酶替代治疗某些硫酸酯酶缺乏症。这种硫酸酯酶的重组生产存在产品活性和产量都很低的问题,进一步限制了患者群体的使用。为了缓解产品活性低的问题,我们通过对不同培养基条件下的培养物进行计算评估,并对两种具有不同活性硫酸酯酶变体水平的 CHO 克隆进行比较,从而对细胞特性进行了研究。转录组分析确定了分泌途径中与硫酸酯酶产量增加相关的 18 个基因。通过上调一组三个关键基因进行实验验证,另一种硫酸酶变体的特定酶活性得到了不同程度的提高,最高可达 150 倍,这说明生产普遍受益。我们还发现了产品 mRNA 水平与硫酸亚铁酶活性之间的相关性,当使用较弱的启动子表达时,硫酸亚铁酶活性会增加。此外,我们还建议,我们提出的解决细胞机器瓶颈的工作流程也可用于改进其他生物制剂的细胞工厂。
{"title":"Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold","authors":"Niklas Berndt Thalén , Mona Moradi Barzadd , Magnus Lundqvist , Johanna Rodhe , Monica Andersson , Gholamreza Bidkhori , Dominik Possner , Chao Su , Joakim Nilsson , Peter Eisenhut , Magdalena Malm , Alice Karlsson , Jeanette Vestin , Johan Forsberg , Erik Nordling , Adil Mardinoglu , Anna-Luisa Volk , Anna Sandegren , Johan Rockberg","doi":"10.1016/j.ymben.2023.12.003","DOIUrl":"10.1016/j.ymben.2023.12.003","url":null,"abstract":"<div><p>Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2023-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717623001763/pdfft?md5=5a0ae2bfd352b7b4d9f49d481e7be367&pid=1-s2.0-S1096717623001763-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138561884","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 : 2023-12-09DOI: 10.1016/j.ymben.2023.12.004
Julian Stegmüller , Marta Rodríguez Estévez , Wei Shu , Lars Gläser , Maksym Myronovskyi , Christian Rückert-Reed , Jörn Kalinowski , Andriy Luzhetskyy , Christoph Wittmann
Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus, making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 μg L−1 of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common Perm* promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that Perm* failed to drive expression during the product synthesis phase but that use of two synthetic promoters (PkasOP* and P41) enabled strong constitutive expression during the entire process. Using PkasOP*, several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L−1 in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3′-end nyb of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L−1 nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin.
{"title":"Systems metabolic engineering of the primary and secondary metabolism of Streptomyces albidoflavus enhances production of the reverse antibiotic nybomycin against multi-resistant Staphylococcus aureus","authors":"Julian Stegmüller , Marta Rodríguez Estévez , Wei Shu , Lars Gläser , Maksym Myronovskyi , Christian Rückert-Reed , Jörn Kalinowski , Andriy Luzhetskyy , Christoph Wittmann","doi":"10.1016/j.ymben.2023.12.004","DOIUrl":"10.1016/j.ymben.2023.12.004","url":null,"abstract":"<div><p>Nybomycin is an antibiotic compound with proven activity against multi-resistant <em>Staphylococcus aureus</em>, making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory <em>S. albidoflavus</em> 4N24, previously derived from the cluster-free chassis <em>S. albidoflavus</em> Del14, produced 860 μg L<sup>−1</sup> of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common P<sub>erm*</sub> promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that P<sub>erm*</sub> failed to drive expression during the product synthesis phase but that use of two synthetic promoters (<em>P</em><sub><em>kasOP*</em></sub> and <em>P</em><sub><em>41</em></sub>) enabled strong constitutive expression during the entire process. Using <em>P</em><sub><em>kasOP*</em></sub>, several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L<sup>−1</sup> in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of <em>nyb</em> genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator <em>nybW</em> enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the <em>nyb</em> gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3′-end <em>nyb</em> of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, <em>S. albidoflavus</em> NYB-11, accumulated up to 12 mg L<sup>−1</sup> nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2023-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717623001775/pdfft?md5=86ba2bdf60efc2296a6829636c79209c&pid=1-s2.0-S1096717623001775-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138561630","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 : 2023-12-08DOI: 10.1016/j.ymben.2023.12.002
Kwanghyun Park, Ji-Sook Hahn
Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.
油酸(C18:1-OH,RA)是一种宝贵的羟基脂肪酸,用途广泛。目前 RA 的工业来源主要依靠水解蓖麻油。然而,由于有毒化合物蓖麻毒素与这种植物的供应不稳定并存,人们开始探索有前景的替代品:在异源植物或微生物中生成 RA。在这项研究中,我们改造了含油酵母亚罗酵母(Yarrowia lipolytica),使其能以游离脂肪酸(FFA)的形式产生 RA。首先,我们过量表达了来自Claviceps purpurea的真菌Δ12油酸羟化酶基因(CpFAH12),同时删除了与脂肪酸降解(MEF1和PEX10)和油酸脱饱和(FAD2)相关的基因。由于Δ12 油酸羟化酶能转化位于磷脂酰胆碱(PC)sn-2 位的油酸(C18:1),我们接下来的重点是增加含有油酸的 PC 池。为了实现这一目标,我们实施了旨在加强 PC 和 C18 脂肪酸生物合成的代谢工程策略。为了增加 PC 池,我们通过删除分别参与生产二酰甘油和三酰甘油的磷脂酸磷酸酶基因(PAH1 和 APP1)和二酰甘油酰基转移酶基因(DGA1),将通量转向磷脂的生物合成。此外,通过过表达 CDS1、PSD1、CHO2 和 OPI3 基因,通过 CDP-DAG 途径的 PC 生物合成也得到了增强。随后,为了增加 PC 中的油酸含量,我们过表达了参与将 C16 脂肪酸转化为 C18 脂肪酸的异源脂肪酸伸长酶基因(MaC16E)。随着 RA 生成滴度的增加,生成的 RA 主要以 FFA 形式存在,从而导致细胞生长受到抑制。通过 Triton X-100 处理诱导 RA 分泌可减轻生长抑制,这一过程通过将通量重新导向 RA 合成,同时扩大了 RA 的产量。最终的工程菌株 JHYL-R146 在用 5% Triton X-100 处理的培养基中产生了 2.061 克/升的游离 RA,占游离脂肪酸总产量的 74%。产生游离 RA 的额外好处是绕过了使用蓖麻油作为 RA 来源时所需的水解阶段。这一成果代表了迄今为止从葡萄糖中合成 RA 的最高水平,凸显了脂肪溶解酵母作为宿主可持续生产 RA 的潜力。
{"title":"Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis","authors":"Kwanghyun Park, Ji-Sook Hahn","doi":"10.1016/j.ymben.2023.12.002","DOIUrl":"10.1016/j.ymben.2023.12.002","url":null,"abstract":"<div><p>Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast <em>Yarrowia lipolytica</em> to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (<em>CpFAH12</em>) from <em>Claviceps purpurea</em> while deleting genes related to fatty acid degradation (<em>MEF1</em> and <em>PEX10</em>) and oleic acid desaturation (<em>FAD2</em>). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the <em>sn</em>-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (<em>PAH1</em> and <em>APP1</em>) and diacylglycerol acyltransferase gene (<em>DGA1</em>), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of <em>CDS1</em>, <em>PSD1</em>, <em>CHO2</em>, and <em>OPI3</em> genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (<em>MaC16E</em>) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of <em>Y. lipolytica</em> as a host for sustainable RA production.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2023-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717623001751/pdfft?md5=b6f87d62a2e6305fdfc435ddd1a02e62&pid=1-s2.0-S1096717623001751-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138561625","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 : 2023-12-04DOI: 10.1016/j.ymben.2023.11.009
Bridget A. Luckie , Meera Kashyap , Allison N. Pearson , Yan Chen , Yuzhong Liu , Luis E. Valencia , Alexander Carrillo Romero , Graham A. Hudson , Xavier B. Tao , Bryan Wu , Christopher J. Petzold , Jay D. Keasling
Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.
{"title":"Development of Corynebacterium glutamicum as a monoterpene production platform","authors":"Bridget A. Luckie , Meera Kashyap , Allison N. Pearson , Yan Chen , Yuzhong Liu , Luis E. Valencia , Alexander Carrillo Romero , Graham A. Hudson , Xavier B. Tao , Bryan Wu , Christopher J. Petzold , Jay D. Keasling","doi":"10.1016/j.ymben.2023.11.009","DOIUrl":"10.1016/j.ymben.2023.11.009","url":null,"abstract":"<div><p>Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. <em>Corynebacterium glutamicum,</em> a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in <em>C. glutamicum</em> have remained relatively limited. In this study, we report a further expansion of the <em>C. glutamicum</em> biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in <em>C. glutamicum</em> to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that <em>C. glutamicum</em> first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717623001738/pdfft?md5=029fda2f52c17ebb474c7d79aad3dfa9&pid=1-s2.0-S1096717623001738-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138485703","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 : 2023-12-02DOI: 10.1016/j.ymben.2023.11.008
Komal Sharma , Mohammad Rifqi Ghiffary , GaRyoung Lee , Hyun Uk Kim
Kynurenine pathway has a potential to convert L-tryptophan into multiple medicinal molecules. This study aims to explore the biosynthetic potential of kynurenine pathway for the efficient production of actinocin, an antitumor precursor selected as a proof-of-concept target molecule. Kynurenine pathway is first constructed in Escherichia coli by testing various combinations of biosynthetic genes from four different organisms. Metabolic engineering strategies are next performed to improve the production by inhibiting a competing pathway, and enhancing intracellular supply of a cofactor S-adenosyl-L-methionine, and ultimately to produce actinocin from glucose. Metabolome analysis further suggests additional gene overexpression targets, which finally leads to the actinocin titer of 719 mg/L. E. coli strain engineered to produce actinocin is further successfully utilized to produce 350 mg/L of kynurenic acid, a neuroprotectant, and 1401 mg/L of 3-hydroxyanthranilic acid, an antioxidant, also from glucose. These competitive production titers demonstrate the biosynthetic potential of kynurenine pathway as a source of multiple medicinal molecules. The approach undertaken in this study can be useful for the sustainable production of molecules derived from kynurenine pathway, which are otherwise chemically synthesized.
{"title":"Efficient production of an antitumor precursor actinocin and other medicinal molecules from kynurenine pathway in Escherichia coli","authors":"Komal Sharma , Mohammad Rifqi Ghiffary , GaRyoung Lee , Hyun Uk Kim","doi":"10.1016/j.ymben.2023.11.008","DOIUrl":"10.1016/j.ymben.2023.11.008","url":null,"abstract":"<div><p>Kynurenine pathway has a potential to convert L-tryptophan into multiple medicinal molecules. This study aims to explore the biosynthetic potential of kynurenine pathway for the efficient production of actinocin, an antitumor precursor selected as a proof-of-concept target molecule. Kynurenine pathway is first constructed in <em>Escherichia coli</em> by testing various combinations of biosynthetic genes from four different organisms. Metabolic engineering strategies are next performed to improve the production by inhibiting a competing pathway, and enhancing intracellular supply of a cofactor <em>S</em>-adenosyl-L-methionine, and ultimately to produce actinocin from glucose. Metabolome analysis further suggests additional gene overexpression targets, which finally leads to the actinocin titer of 719 mg/L. <em>E. coli</em> strain engineered to produce actinocin is further successfully utilized to produce 350 mg/L of kynurenic acid, a neuroprotectant, and 1401 mg/L of 3-hydroxyanthranilic acid, an antioxidant, also from glucose. These competitive production titers demonstrate the biosynthetic potential of kynurenine pathway as a source of multiple medicinal molecules. The approach undertaken in this study can be useful for the sustainable production of molecules derived from kynurenine pathway, which are otherwise chemically synthesized.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":null,"pages":null},"PeriodicalIF":8.4,"publicationDate":"2023-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1096717623001726/pdfft?md5=b230d3b45d678246b960bd7968581529&pid=1-s2.0-S1096717623001726-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138473728","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}