Thomas Eng, Deepanwita Banerjee, A. Lau, E. Bowden, R. Herbert, Jessica Trinh, Jan-Philip Prahl, A. Deutschbauer, Deepti Tanjore, A. Mukhopadhyay
{"title":"高通量适应度分析揭示GacS-GacA调控缺失改善恶臭假单胞菌靛蓝苷生产","authors":"Thomas Eng, Deepanwita Banerjee, A. Lau, E. Bowden, R. Herbert, Jessica Trinh, Jan-Philip Prahl, A. Deutschbauer, Deepti Tanjore, A. Mukhopadhyay","doi":"10.1101/2021.02.02.429437","DOIUrl":null,"url":null,"abstract":"Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the ΔgacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2021-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"2","resultStr":"{\"title\":\"High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida\",\"authors\":\"Thomas Eng, Deepanwita Banerjee, A. Lau, E. Bowden, R. Herbert, Jessica Trinh, Jan-Philip Prahl, A. Deutschbauer, Deepti Tanjore, A. Mukhopadhyay\",\"doi\":\"10.1101/2021.02.02.429437\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the ΔgacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.\",\"PeriodicalId\":72407,\"journal\":{\"name\":\"bioRxiv : the preprint server for biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2021-02-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"2\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"bioRxiv : the preprint server for biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1101/2021.02.02.429437\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"bioRxiv : the preprint server for biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1101/2021.02.02.429437","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida
Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the ΔgacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.