title: Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production.
abstract: In previous work, we constructed a recombinant Bacillus subtilis strain for microbial production of N-acetylglucosamine (GlcNAc), which has applications in nutraceuticals and pharmaceuticals. In this work, we improve GlcNAc production through modular engineering of B. subtilis. Specifically, the GlcNAc synthesis-related metabolic network in B. subtilis was divided into three modules-GlcNAc synthesis, glycolysis, and peptidoglycan synthesis. First, two-promoter systems with different promoter types and strengths were used for combinatorial assembly of expression cassettes of glmS (encoding GlcN-6-phosphate synthase) and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) at transcriptional levels in the GlcNAc synthesis module, resulting in a 32.4% increase in GlcNAc titer (from 1.85g/L to 2.45g/L) in shake flasks. In addition, lactate and acetate synthesis were blocked by knockout of ldh (encoding lactate dehydrogenase) and pta (encoding phosphotransacetylase), leading to a 44.9% increase in GlcNAc production (from 2.45g/L to 3.55g/L) in shake flasks. Then, various strengths of the glycolysis and peptidoglycan synthesis modules were constructed by repressing the expression of pfk (encoding 6-phosphofructokinase) and glmM (encoding phosphoglucosamine mutase) via the expression of various combinations of synthetic small regulatory RNAs and Hfq protein. Next, GlcNAc, glycolysis, and peptidoglycan synthesis modules with various strengths were assembled and optimized via a module engineering approach, and the GlcNAc titer was improved to 8.30g/L from 3.55g/L in shake flasks. Finally, the GlcNAc titer was further increased to 31.65g/L, which was 3.8-fold that in the shake flask, in a 3-L fed-batch bioreactor. This work significantly enhanced GlcNAc production through modular pathway engineering of B. subtilis, and the engineering strategies used herein may be useful for the construction of versatile B. subtilis cell factories for the production of other industrially important chemicals.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China.
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Yanqiu
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China.
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.affiliation: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta 30332, USA.
authors.forename: Hyun-dong
authors.initials: HD
authors.lastname: Shin
authors.affiliation: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta 30332, USA.
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Chen
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China. Electronic address: longliu@jiangnan.edu.cn.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China. Electronic address: jchen@jiangnan.edu.cn.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D000117:Acetylglucosamine / Q000096:biosynthesis / Q000235:genetics; D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D010709:Phosphate Acetyltransferase / Q000235:genetics / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism; D010733:Phosphoglucomutase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D007770:L-Lactate Dehydrogenase; D000123:Acetyltransferases; D010709:Phosphate Acetyltransferase; D010732:Phosphofructokinase-1; C097631:phosphoglucosamine mutase; D010733:Phosphoglucomutase; D000117:Acetylglucosamine
keywords: Bacillus subtilis; Modular pathway engineering; N-acetylglucosamine; Synthetic small regulatory RNAs