title: Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements.
abstract: Levan is a functional homopolymer of fructose with considerable applications in food, pharmaceutical and cosmetic industries. To improve the levan production in Bacillus amyloliquefaciens, the regulatory elements of sacB (encoding levansucrase) expression and levansucrase secretion were optimized. Four heterologous promoters were evaluated for sacB expression, and the Pgrac promoter led to the highest level for both sacB transcription and levansucrase enzyme activity. The levan production in the corresponding recombinant strain ΔLP-pHTPgrac reached 30.5 g/L, which was 114% higher than that of the control strain NK-ΔLP. In a further step, eight signal peptides were investigated (with Pgrac as the promoter for sacB expression) for their effects on the levansucrase secretion and levan production. The signal peptide yncM was identified as the optimal one, with a secretion efficiency of approximately 90%, and the levan production in the corresponding recombinant strain ΔLP-Y reached 37.4 g/L, which was 161% higher when compared with the control strains NK-ΔLP. Finally, fed-batch fermentation was carried out in 5-L bioreactors for levan production using the recombinant strain ΔLP-Y. A final levan concentration of 102 g/L was achieved, which is very close to the ever reported highest levan production level from the literature. To our best knowledge, this is the first report of the improvement of levan production through metabolic optimization for sacB expression and levansucrase secretion. The results from this study provided essential insights for systematically metabolic engineering of microbial cell factories for enhanced biochemical production.
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.|Biosystems Engineering Department, Auburn University, Auburn, AL, 36849, USA.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.
authors.forename: Jiayi
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.|Biosystems Engineering Department, Auburn University, Auburn, AL, 36849, USA.|State Key Laboratory of Medicinal Chemical Biology, Nankai University, 94 Weijin Road, Tianjin, 300071, China.|Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi, 710049, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Feng
authors.affiliation: Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, 50011, USA.
authors.forename: Mingfeng
authors.initials: M
authors.lastname: Cao
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.
authors.forename: Weixia
authors.initials: W
authors.lastname: Gao
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.
authors.forename: Yufen
authors.initials: Y
authors.lastname: Quan
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.
authors.forename: Yulei
authors.initials: Y
authors.lastname: Dang
authors.affiliation: Biosystems Engineering Department, Auburn University, Auburn, AL, 36849, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, Nankai University, 94 Weijin Road, Tianjin, 300071, China. wangshufang@nankai.edu.cn.
authors.forename: Shufang
authors.initials: S
authors.lastname: Wang
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China. songcj@nankai.edu.cn.
authors.forename: Cunjiang
authors.initials: C
authors.lastname: Song
mesh_terms: D000069977:Bacillus amyloliquefaciens / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D005285:Fermentation; D005630:Fructans / Q000096:biosynthesis* / Q000737:chemistry; D005632:Fructose / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006602:Hexosyltransferases / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D013395:Sucrose / Q000378:metabolism
chemical_list: D005630:Fructans; D021382:Protein Sorting Signals; D005632:Fructose; D013395:Sucrose; C072599:levan; D006602:Hexosyltransferases; C021513:levansucrase
keywords: Bacillus amyloliquefaciens; Levan; Levansucrase; Promoter; Signal peptide