title: Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization.
abstract: Transglutaminases (TGase), which are synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in the food industry. Because this pro-peptide is essential for the correct folding of Streptomyces TGase, TGase is usually expressed in an inactive pro-TGase form, which is then converted to active TGase by the addition of activating proteases in vitro. In this study, Streptomyces hygroscopicus TGase was actively produced by Streptomyces lividans through promoter engineering and codon optimization.
  
  
  
  A gene fragment (tg1, 2.6 kb) that encoded the pro-TGase and its endogenous promoter region, signal peptide and terminator was amplified from S. hygroscopicus WSH03-13 and cloned into plasmid pIJ86, which resulted in pIJ86/tg1. After fermentation for 2 days, S. lividans TK24 that harbored pIJ86/tg1 produced 1.8 U/mL of TGase, and a clear TGase band (38 kDa) was detected in the culture supernatant. These results indicated that the pro-TGase was successfully expressed and correctly processed into active TGase in S. lividans TK24 by using the TGase promoter. Based on deletion analysis, the complete sequence of the TGase promoter is restricted to the region from -693 to -48. We also identified a negative element (-198 to -148) in the TGase promoter, and the deletion of this element increased the TGase production by 81.3 %, in contrast to the method by which S. lividans expresses pIJ86/tg1. Combining the deletion of the negative element of the promoter and optimization of the gene codons, the yield and productivity of TGase reached 5.73 U/mL and 0.14 U/mL/h in the recombinant S. lividans, respectively.
  
  
  
  We constructed an active TGase-producing strain that had a high yield and productivity, and the optimized TGase promoter could be a good candidate promoter for the expression of other proteins in Streptomyces.
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China. liusong@jiangnan.edu.cn.
authors: Song
authors: S
authors: Liu
authors: School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
authors: Miao
authors: M
authors: Wang
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China. gcdu@jiangnan.edu.cn.|Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China. gcdu@jiangnan.edu.cn.
authors: Guocheng
authors: G
authors: Du
authors: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, China.
authors: Jian
authors: J
authors: Chen
mesh_terms: D003062:Codon / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics*; D048170:Streptomyces lividans / Q000201:enzymology* / Q000235:genetics*; D011503:Transglutaminases / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D003062:Codon; D011503:Transglutaminases
keywords: Codon optimization; Endogenous promoter; Streptomyces hygroscopicus; Streptomyces lividans; Transglutaminase