{"title":"Engineering cascade biocatalysis in whole cells for syringic acid bioproduction.","authors":"Xin Liu, Yi An, Haijun Gao","doi":"10.1186/s12934-024-02441-x","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells.</p><p><strong>Results: </strong>An O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA<sup>**</sup>) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 μM (26.2 mg/L).</p><p><strong>Conclusion: </strong>Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11143566/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Microbial Cell Factories","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1186/s12934-024-02441-x","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Background: Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells.
Results: An O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 μM (26.2 mg/L).
Conclusion: Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.
背景:丁香酸(SA)是一种高价值的天然化合物,具有多种生物活性和广泛用途,通常存在于水果、蔬菜和草药中。丁香酸主要通过化学合成法生产,然而,这些化学方法存在许多缺点,如设备要求高、反应条件苛刻、催化剂昂贵和副产物多等。因此,本研究利用工程化全细胞设计并开发了一种生产 SA 的新型生物转化途径:结果:从醋酸脱硫单胞菌(Desulfuromonas acetoxidans,DesAOMT)中发现了一种O-甲基转移酶(O-methyltransferase),它能优先催化儿茶酚类似物的元羟基上的甲基转移反应。当以 S-腺苷蛋氨酸(SAM)为甲基供体时,表达 DesAOMT 的全细胞可将没食子酸(GA)转化为 SA。我们在大肠杆菌中构建了一个多酶级联反应,其中包含内源莽草酸激酶(AroL)和氯氨酸裂解酶(UbiC),以及荧光假单胞菌的对羟基苯甲酸羟化酶突变体(PobA**)和DesAOMT;通过全细胞催化,从莽草酸(SHA)中生物合成了SA。底盘细胞的代谢系统也会影响 SA 的生物合成效率,阻断络氨酸代谢途径会提高 SA 的产量。当优化辅助因子 NADPH 的供应时,SA 的滴度达到 133 μM(26.2 mg/L):总之,我们在大肠杆菌中设计了一个多酶级联,利用静止或生长的全细胞进行 SA 生物合成。这项工作发现了一种 O-甲基转移酶(DesAOMT),它能催化 GA 的甲基化作用,从而产生 SA。在改造的大肠杆菌中表达了包含四种酶的多酶级联,用于从 SHA 合成 SA。菌株的代谢系统和生物转化条件影响催化效率。这项研究为 SA 的生物合成提供了一条新的绿色途径。
期刊介绍:
Microbial Cell Factories is an open access peer-reviewed journal that covers any topic related to the development, use and investigation of microbial cells as producers of recombinant proteins and natural products, or as catalyzers of biological transformations of industrial interest. Microbial Cell Factories is the world leading, primary research journal fully focusing on Applied Microbiology.
The journal is divided into the following editorial sections:
-Metabolic engineering
-Synthetic biology
-Whole-cell biocatalysis
-Microbial regulations
-Recombinant protein production/bioprocessing
-Production of natural compounds
-Systems biology of cell factories
-Microbial production processes
-Cell-free systems