{"title":"通过并行进化谷氨酸棒状杆菌直接硫化途径中的两种酶来工程化蛋氨酸辅助营养大肠杆菌,使其能够在最小培养基中复原","authors":"Matan Gabay , Inbar Stern , Nadya Gruzdev , Adi Cohen , Lucia Adriana-Lifshits , Tamar Ansbacher , Itamar Yadid , Maayan Gal","doi":"10.1016/j.mec.2024.e00236","DOIUrl":null,"url":null,"abstract":"<div><p>Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. <em>Escherichia coli</em>, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph <em>Escherichia</em> <em>coli</em> strain (MG1655) with deletion of <em>metA</em>, encoding for L-homoserine O-succinyl transferase, and <em>metB</em>, encoding for cystathionine gamma synthase, could be complemented by introducing the genes <em>metX</em>, encoding for L-homoserine O-acetyltransferases and <em>metY</em>, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the <em>Escherichia coli</em> methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing <em>metX</em> and <em>metY</em> from <em>Corynebacterium glutamicum</em> failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the <em>metX</em> and <em>metY</em> of <em>Corynebacterium glutamicum</em> and transformed it into a methionine-auxotrophic <em>Escherichia coli</em> strain lacking the <em>metA</em> and <em>metB</em> genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant <em>metX</em> mutations in the evolved methionine-autotrophs <em>Escherichia coli</em> were L315P and H46R. Interestingly, we found that a <em>metY</em> gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph <em>Escherichia coli</em> validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the <em>Escherichia coli</em> methionine direct-sulfurylation pathway, leading to efficient complementation.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000051/pdfft?md5=13216db11f331277ec4bffeddfb976eb&pid=1-s2.0-S2214030124000051-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium\",\"authors\":\"Matan Gabay , Inbar Stern , Nadya Gruzdev , Adi Cohen , Lucia Adriana-Lifshits , Tamar Ansbacher , Itamar Yadid , Maayan Gal\",\"doi\":\"10.1016/j.mec.2024.e00236\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. <em>Escherichia coli</em>, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph <em>Escherichia</em> <em>coli</em> strain (MG1655) with deletion of <em>metA</em>, encoding for L-homoserine O-succinyl transferase, and <em>metB</em>, encoding for cystathionine gamma synthase, could be complemented by introducing the genes <em>metX</em>, encoding for L-homoserine O-acetyltransferases and <em>metY</em>, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the <em>Escherichia coli</em> methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing <em>metX</em> and <em>metY</em> from <em>Corynebacterium glutamicum</em> failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the <em>metX</em> and <em>metY</em> of <em>Corynebacterium glutamicum</em> and transformed it into a methionine-auxotrophic <em>Escherichia coli</em> strain lacking the <em>metA</em> and <em>metB</em> genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant <em>metX</em> mutations in the evolved methionine-autotrophs <em>Escherichia coli</em> were L315P and H46R. Interestingly, we found that a <em>metY</em> gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph <em>Escherichia coli</em> validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the <em>Escherichia coli</em> methionine direct-sulfurylation pathway, leading to efficient complementation.</p></div>\",\"PeriodicalId\":18695,\"journal\":{\"name\":\"Metabolic Engineering Communications\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2024-05-10\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S2214030124000051/pdfft?md5=13216db11f331277ec4bffeddfb976eb&pid=1-s2.0-S2214030124000051-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Metabolic Engineering Communications\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2214030124000051\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic Engineering Communications","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2214030124000051","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium
Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichiacoli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.
期刊介绍:
Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.