title: Comparative proteomics analysis of high n-butanol producing metabolically engineered Clostridium tyrobutyricum.
abstract: The acidogenic Clostridium tyrobutyricum has recently been metabolically engineered to produce n-butanol. The objective of this study was to obtain a comprehensive understanding as to how butanol production was regulated in C. tyrobutyricum to guide the engineering of next-generation strains. We performed a comparative proteomics analysis, covering 78.1% of open reading frames and 95% of core enzymes, using wild type, ACKKO mutant (Δack) producing 37.30 g/L of butyrate and ACKKO-adhE2 mutant (Δack-adhE2) producing 16.68 g/L of butanol. In ACKKO-adhE2, the expression of most glycolytic enzymes was decreased, the thiolase (thl), acetyl-CoA acetyltransferase (ato), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt) that convert acetyl-CoA to butyryl-CoA were increased, and the heterologous bifunctional acetaldehyde/alcohol dehydrogenase (adhE2) catalyzing butanol formation was highly expressed. The apparent imbalance of energy and redox was observed due to the downregulation of acids production and the addition of butanol synthesis pathway, which also resulted in increased expression of chaperone proteins and glycerol-3-phosphate dehydrogenase (glpA) and the silence of sporulation transcription factor Spo0A (spo0A) as the cellular responses to butanol production. This study revealed the mechanism of carbon redistribution, and limiting factors and rational metabolic cell and process engineering strategies to achieve high butanol production in C. tyrobutyricum.
authors.affiliation: Department of Chemical and Biological Engineering, The University of Alabama, 245 7th Avenue, Tuscaloosa, AL 35401, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Ma
authors.affiliation: Departments of Medicine and Biomedical Engineering, The University of Alabama at Birmingham, 703 19th Street South and 1530 3rd Avenue South, Birmingham, AL 35294, USA.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Kojima
authors.affiliation: Department of Chemical and Biological Engineering, The University of Alabama, 245 7th Avenue, Tuscaloosa, AL 35401, USA.
authors.forename: Ningning
authors.initials: N
authors.lastname: Xu
authors.affiliation: Departments of Medicine and Biomedical Engineering, The University of Alabama at Birmingham, 703 19th Street South and 1530 3rd Avenue South, Birmingham, AL 35294, USA.
authors.forename: James
authors.initials: J
authors.lastname: Mobley
authors.affiliation: Departments of Medicine and Biomedical Engineering, The University of Alabama at Birmingham, 703 19th Street South and 1530 3rd Avenue South, Birmingham, AL 35294, USA.
authors.forename: Lufang
authors.initials: L
authors.lastname: Zhou
authors.affiliation: William G Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA. Electronic address: yang.15@osu.edu.
authors.forename: Shang-Tian
authors.initials: ST
authors.lastname: Yang
authors.affiliation: Department of Chemical and Biological Engineering, The University of Alabama, 245 7th Avenue, Tuscaloosa, AL 35401, USA. Electronic address: mliu@eng.ua.edu.
authors.forename: Xiaoguang Margaret
authors.initials: XM
authors.lastname: Liu
mesh_terms: D020001:1-Butanol / Q000032:analysis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D018533:Biomass; D048014:Clostridium tyrobutyricum / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D020001:1-Butanol; D005947:Glucose
keywords: Butanol production; Carbon balance; Clostridium tyrobutyricum; Comparative proteomics; Metabolite analysis; Redox balance