Qi Wang, Yongheng Liu, Tengteng Zhu, Wei Zhao, Jianyu Su
{"title":"Advancing VB<sub>12</sub> Production: Insights into Enhancing VB<sub>12</sub> Titer in <i>Ensifer adhaerens</i> Casida A through ARTP Mutagenesis and Multiomics Analysis.","authors":"Qi Wang, Yongheng Liu, Tengteng Zhu, Wei Zhao, Jianyu Su","doi":"10.1021/acssynbio.4c00884","DOIUrl":null,"url":null,"abstract":"<p><p><i>Ensifer adhaerens</i>, a microorganism recognized for its capacity to synthesize vitamin B<sub>12</sub> (VB<sub>12</sub>), has garnered significant attention in recent years. Nonetheless, its practical application has been limited by low production yields. Atmospheric and room-temperature plasma (ARTP) mutagenesis was utilized to improve VB<sub>12</sub> production and examine the associated mechanisms. Three high-yielding mutant strains─BCA-24, BCB-14, and BCC-27─were isolated through multiple rounds of mutagenesis. The VB<sub>12</sub> titer of the highly productive mutant strain, BCA-24, rose significantly from 65.64 mg/L to 104.54 mg/L. Genome resequencing identified 14 mutated genes, of which seven (<i>atpA</i>, <i>gntR</i>, <i>fusA</i>, <i>cobQ</i>, <i>ribD</i>, <i>cirA</i>, and <i>UP</i>) were functionally validated through overexpression in wild-type strains and found to positively influence VB<sub>12</sub> biosynthesis. Notably, coexpression of the <i>cobQ</i> and <i>UP</i> mutant genes in strain BCA-24 resulted in a VB<sub>12</sub> titer of 163.68 mg/L. Transcriptomic analysis indicated that critical pathways related to energy metabolism, S-adenosylmethionine (SAM), 5-aminolevulinic acid (5-ALA), and riboflavin synthesis were significantly upregulated in BCA-24 relative to the wild type. A multiomics approach clarified the mechanisms through which these mutations increase VB<sub>12</sub> production, including enhanced transcription and translation, optimized energy supply, and improved product efflux. The identification of novel candidate genes in <i>Ensifer adhaerens</i>, which have not been previously studied, provides valuable resources for future genetic engineering aimed at enhancing VB<sub>12</sub> production efficiency. This study offers practical improvements in microbial VB<sub>12</sub> production while also delivering essential insights into the genetic and metabolic regulation of this important biosynthetic pathway.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7000,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Synthetic Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acssynbio.4c00884","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Ensifer adhaerens, a microorganism recognized for its capacity to synthesize vitamin B12 (VB12), has garnered significant attention in recent years. Nonetheless, its practical application has been limited by low production yields. Atmospheric and room-temperature plasma (ARTP) mutagenesis was utilized to improve VB12 production and examine the associated mechanisms. Three high-yielding mutant strains─BCA-24, BCB-14, and BCC-27─were isolated through multiple rounds of mutagenesis. The VB12 titer of the highly productive mutant strain, BCA-24, rose significantly from 65.64 mg/L to 104.54 mg/L. Genome resequencing identified 14 mutated genes, of which seven (atpA, gntR, fusA, cobQ, ribD, cirA, and UP) were functionally validated through overexpression in wild-type strains and found to positively influence VB12 biosynthesis. Notably, coexpression of the cobQ and UP mutant genes in strain BCA-24 resulted in a VB12 titer of 163.68 mg/L. Transcriptomic analysis indicated that critical pathways related to energy metabolism, S-adenosylmethionine (SAM), 5-aminolevulinic acid (5-ALA), and riboflavin synthesis were significantly upregulated in BCA-24 relative to the wild type. A multiomics approach clarified the mechanisms through which these mutations increase VB12 production, including enhanced transcription and translation, optimized energy supply, and improved product efflux. The identification of novel candidate genes in Ensifer adhaerens, which have not been previously studied, provides valuable resources for future genetic engineering aimed at enhancing VB12 production efficiency. This study offers practical improvements in microbial VB12 production while also delivering essential insights into the genetic and metabolic regulation of this important biosynthetic pathway.
期刊介绍:
The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism.
Topics may include, but are not limited to:
Design and optimization of genetic systems
Genetic circuit design and their principles for their organization into programs
Computational methods to aid the design of genetic systems
Experimental methods to quantify genetic parts, circuits, and metabolic fluxes
Genetic parts libraries: their creation, analysis, and ontological representation
Protein engineering including computational design
Metabolic engineering and cellular manufacturing, including biomass conversion
Natural product access, engineering, and production
Creative and innovative applications of cellular programming
Medical applications, tissue engineering, and the programming of therapeutic cells
Minimal cell design and construction
Genomics and genome replacement strategies
Viral engineering
Automated and robotic assembly platforms for synthetic biology
DNA synthesis methodologies
Metagenomics and synthetic metagenomic analysis
Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction
Gene optimization
Methods for genome-scale measurements of transcription and metabolomics
Systems biology and methods to integrate multiple data sources
in vitro and cell-free synthetic biology and molecular programming
Nucleic acid engineering.