Study On The Heterologous Expression Of Streptomyces Hygroscopicus Transglutaminase

Microbial transglutaminase （EC 2.3.2.13, TGase） is an enzyme that catalyzes the transfer of acyl group, resulting in the crosslinking among the proteins or polypeptides, but further research revealed that the substrates of the TGase are also non-protein materials. Because of the specific enzyme reaction, TGase has been widely used in food industry, pharmaceutical industry, textile and leather processing, and other non-food industry, suggesting a huge market potential. Therefore, much interest has been on the production of TGase domestically as well as globally.Although TGase producing Streptomyces strains were isolated and the fermentation process was optimized, the yield of TGase was very low and the enzyme was unstable. To solve the problem, many works have been oriented toward the production of TGase by the engineered strains. It was found that the TGase was synthesized as inactive enzyme in wild type strain, and direct expression of the mature enzyme gene often produced insoluble or inactive form. In this research, TGase from Streptomyces hygroscopicus WSH03-13 was successfully expressed in Streptomyces lividans and Escherichia coli by optimizing the expression rate at the gene level. The recombinant TGase was further modified to improve the catalytic properties and efficiencies.（1） Cloning and sequence analysis of S. hygroscopicus TGase geneA TGase-related gene fragment named as tgh （GenBank accession No. HM231108） was cloned from S. hygroscopicus WSH03-13. tgh contained an open reading frame （ORF） of the TGase encoding 418 amino acids （aa）. The ORF was composed of the signal peptide region （22 aa）, pro-region （57 aa）, and TGase region （332）. S. hygroscopicus TGase shared high identity （79.2-84.9%） with TGases from other Streptomyces species. Although the conservation of the pro-region was lower than that of the mature TGase, several highly conserved amino acids were found in the pro-region of the TGases from different Streptomyces species. The signal peptide exhibited approximately 34 to 72% identity with TGases from other Streptomyces species. A putative promoter region was found 500 upstream of the TGase ORF, and this region was well conserved in the upstream sequence of TGase genes from different Streptomyces species. In addition, an inverse repeat sequence which may function as a transcription terminator was identified 12 bp downstream of the ORF.（2） Cloning and expression of S. hygroscopicus TGase in S. lividansA gene fragment containing the S. hygroscopicus TGase ORF, native promoter and terminator was inserted into pIJ86 to express TGase in S. lividans TK24. In shaking flasks, the recombinant strain produced 3.2 U/mL of the TGase when it was cultured at 30 oC for 48 h in the Streptomyces seed medium developed by our lab. The yield of the recombinant TGase was 2 times higher than that of wild type TGase under the same condition. Under the same fermentation condition in 3L fermentor, the recombinant strain produed 2.3 U/mL of the TGase at the pH-stat culture at 7.5. These results suggested that the TGase promoter and signal peptide can be recognized by S. lividans, and the pro-TGase was activated by the protease from the host strain. The recombinant strain thus may have a potential for TGase production at industrial scale. Gene deletions were performed to identify the TGase promoter. It was found that the TGase promoter region ranges from -693 to -98 and the sequence between -148 and -198 might be the negative regulatory element of the promoter.（3） Secretion of S. hygroscopicus pro-TGase in E. coliS. hygroscopicus pro-TGase gene was inserted into the downstream of pelB signal peptide gene in pET-22b（+） to express pro-TGase in E. coli BL21（DE3）. The optimized condition for secretion of pro-TGase was that the recombinant E. coli was cultured in TB medium containing 0.5% （w/v） glucose and induced with 0.4 mmol/L IPTG at 20 oC for 40 h. In this case, the yield of the secreted pro-TGase was up to 6.8 U/mL. Under the same condition, S. hygroscpicus TGase signal peptide could also mediate the secretion of the pro-TGase in E. coli. This is the first report describing the pro-TGase secretion in E. coli. N-terminal deletion of pro-TGase indicated that the first 6 amino acids of the pro-region were responsible for TGase secretion and the following 10 amino acids related to the correct folding of the TGase. Structure modeling showed that Tyr12 of the pro-region interacted with Asp362 and Asn334 through hydrogen bonds. Mutation of Tyr12 to Ala12 resulted in insoluble pro-TGase in E. coli, which further confirmed the role of the pro-region in the expression and secretion of TGase in E. coli.（4） Expression of active TGase from S. hyroscopicus in E. colipET22b （+） was used to construct the co-expression of the S. hygroscopicus TGase and its pro-region. The vector’s features were described as follow: the TGase gene and its pro-region gene were derived from the same T7 promoter; lac operator was downstream of the T7 promoter; TGase and its pro-region were proceeded with a ribosome binding site, respectively; the TGase and its pro-region were fused with or without the pelB signal peptide. Therefore, the order of gene expression could be altered by changing the order of gene inserted in downstream of the T7 promoter. The co-expression vector was transformed into E. coli BL21（DE3）, and then induced with 0.4 mmol/L IPTG at 20 oC for 40 h in TB medium. When both the pro-region and TGase were fused with the pelB signal peptide and sequentially expressed, the recombinant strain yielded 0.13 U/mL/OD600. However, a change in the expression order of the two regions led to insoluble TGase. These findings indicated that the expression order was important in production of active form in E. coli using the co-expression system. This is the first report describing that the pro-region assisted the correct folding of TGase intermolecularly in E. coli.（5） Purification and characterization of the wild type and recombinant TGaseThe wild type TGase, the recombinant pro-TGase and TGase were purified. The wild type TGase, the recombinant TGase, and the pro-TGase activated by dispase had the similar catalytic properties. The optimal reaction temperature and pH of the three enzyme preparations were 40 oC and 6.0, respectively. The common heavy-metal ion inhibited all three enzymes significantly. Ca²⁺, Mg²⁺, Ba²⁺ and Mn²⁺ activated the activity of three preparations. The Km value of the wild type TGase, the recombinant TGase and the activated pro-TGase were 54.7, 57.1, and 56.6 mmol/L, respectively. In order to improve the enzymatic properties of the TGase, phthalic anhydride （PA） was applied to modify lysines of the TGase. The optimal conditions for the modification of the TGase were at 20 oC and pH 8 for 2 h. In the presence of 10 mmol/L of cystamine which is an competitive inhibitor of the TGase, the thermostability of TGase was increased by 31.3% after the PA modification, whereas specific activity of the TGase increased 48.3% in the absence of the cystamine. The surface hydrophobicity analysis indicated that PA modification enhanced the hydrophobicity of the enzyme. This finding provided a basis for further modification of TGase.