title: Metabolic engineering of Enterobacter cloacae for high-yield production of enantiopure (2R,3R)-2,3-butanediol from lignocellulose-derived sugars.
abstract: Biotechnological production of biofuels is restricted by toxicity of the products such as ethanol and butanol. As its low toxicity to microbes, 2,3-butanediol (2,3-BD), a fuel and platform bio-chemical, could be a promising alternative for biofuel production from renewable bioresources. In addition, no bacterial strains have been reported to produce enantiopure 2,3-BD using lignocellulosic hydrolysates. In this study, Enterobacter cloacae strain SDM was systematically and metabolically engineered to construct an efficient biocatalyst for production of the fuel and enantiopure bio-chemical-(2R,3R)-2,3-BD. First, the various (2R,3R)-2,3-BD dehydrogenase encoding genes were expressed in a meso-2,3-BD dehydrogenase encoding gene disrupted E. cloacae strain under native promoter Pb of the 2,3-BD biosynthetic gene cluster of E. cloacae. Then, carbon catabolite repression was eliminated via inactivation of the glucose transporter encoding gene ptsG and overexpression of a galactose permease encoding gene galP. The resultant strain could utilize glucose and xylose simultaneously. To improve the efficiency of (2R,3R)-2,3-BD production, the byproduct-producing genes (ldh and frdA) were knocked out, thereby enhancing the yield of (2R,3R)-2,3-BD by 16.5% in 500-mL Erlenmeyer flasks. By using fed-batch fermentation in a 5-L bioreactor, 152.0 g/L (2R,3R)-2,3-BD (purity>97.5%) was produced within 44 h with a specific productivity of 3.5 g/[Lh] and a yield of 97.7% from a mixture of glucose and xylose, two major carbohydrate components in lignocellulosic hydrolysates. In addition, when a lignocellulosic hydrolysate was used as the substrate, 119.4 g/L (2R,3R)-2,3-BD (purity>96.0%) was produced within 51 h with a productivity of 2.3g/[Lh] and a yield of 95.0%. These results show that the highest records have been acquired for enantiopure (2R,3R)-2,3-BD production by a native or engineered strain from biomass-derived sugars. In addition to producing the 2,3-BD, our systematic approach might also be used in the production of other important chemicals by using lignocellulose-derived sugars.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People׳s Republic of China; State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, People׳s Republic of China.
authors.forename: Lixiang
authors.initials: L
authors.lastname: Li
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People׳s Republic of China.
authors.forename: Kun
authors.initials: K
authors.lastname: Li
authors.affiliation: State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, People׳s Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People׳s Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People׳s Republic of China.
authors.forename: Youqiang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People׳s Republic of China.
authors.forename: Lijie
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People׳s Republic of China.
authors.forename: Binbin
authors.initials: B
authors.lastname: Han
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People׳s Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, People׳s Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Tao
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People׳s Republic of China. Electronic address: macq@sdu.edu.cn.
authors.forename: Cuiqing
authors.initials: C
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, People׳s Republic of China. Electronic address: pingxu@sjtu.edu.cn.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D056804:Biofuels; D002072:Butylene Glycols / Q000378:metabolism*; D016972:Enterobacter cloacae / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D008031:Lignin / Q000737:chemistry*; D060847:Metabolic Engineering; D014994:Xylose / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D056804:Biofuels; D002072:Butylene Glycols; C036909:lignocellulose; D008031:Lignin; D014994:Xylose; D005947:Glucose
keywords: (2R,3R)-2,3-Butanediol; 2,3-Butanediol dehydrogenase; Carbon catabolite repression; Enterobacter cloacae; Metabolic engineering