Construction Of Recombinant Pichia Pastoris Efficiently Expressing Active Microbial Transglutaminase And Its Secretory Pathway Engineering

Transglutaminase (EC2.3.2.13, TGase or TG) catalyzes the acyl transfer reaction in whichy-carboxamide groups of peptide-bound glutamine residues serve as acyl donors, serving as crosslinker of proteins. TG is abundant in human beings, animals, plants and microbes. The microbial transglutaminase (MTG) is widely used in food industry for improving texture and nutritive value of protein foods. It also has applications in cosmetic, textile and leather industries and also showing promise in biomaterials in recent years.The main producers of MTG belong to Streptomyces spp., eg. Streptomyces mobaraense, Streptomyces hygroscopicus. However, due to the lack of genetic engineering tools and methods, their MTG production levels cannot be significantly improved by rational modification. In addition, these strains also serete a large amount of proteasase into culture media, which are difficult to remove. The application of heterologous expression systems to produce MTG becomes a burgeoning trend as a consequence. Despite of its great promise, up to now the MTG production levels of heterologous hosts are far from commercialization. Pichia pastoris is one of the most successful expression systems developed in the past decades and previous research has already demonstrated the possibility of using P. pastoris to directly express active MTG, although the production level was low. In this work, we are aiming to achieve high level expression of MTG using P. pastoris by means of strain construction and culture optimization.Firstly, genes encoding pro sequence and mature TG were cloned from genomic DNA of S. mobaraense, and then the recombinant vector pAOa-pro-MTG containing1-copy of pro/mtg co-expression cassette was constructed. After that, recombinant vectors containing2-copy and3-copy of pro/mtg co-expression cassettes were acquired. Secondly, recombinant P. pastoris strains GS1MTG, GS2MTG and GS3MTG containing one, two, and three copies of pro/mtg co-expression cassettes, respectively were constructed, and GS2MTG showed the highest MTG expression level when the three strains were compared for their MTG expression abilities in shaking-flask fermentation. Thirdly, optimization of culture conditions for GS2MTG showed that the best pH value was6.0-7.0and the best media was BMMY. Lastly, high density fermentation was performed and a MTG production level of7.3U/mL was finally reached in1-liter fermentor.In order to overcome the bottleneck in protein secretion and further enhance the MTG production level of recombinant P. pastoris strains, the chaperone co-expression strategy was adopted in this research. A chaperone gene library was established which contains13strains. Each strain has one of the following chaperone genes overexpressed:A674, SSO, A156, B27, KAR, B357, PDI, C467, FB40, B424, B230, FD002and B582. The13strains were then transformed with MTG expression vector one by one. Finally, seven strains were generated which have MTG expression vector and chaperone gene co-expressed:GS2MTG-SSO, GS2MTG-A156, GS2MTG-B27, GS2MTG-KAR, GS2MTG-B357, GS2MTG-PDI and GS2MTG-C467. With the shaking-flask fermentation, GS2MTG-C467and GS2MTG-KAR showed90.2%and21.5%of increase in MTG titer as compared with GS2MTG control.In summary, we successfully obtained a series of recombinant P. pasloris strains exhibiting remarkably high MTG production level, and considerably optimized culture conditions to improve their performances. Consequently, a MTG production of7.3U/mL was achieved, which to our best knowledge is the highest level ever reported for heterologous expression of active MTG. The results showed the potential of applying P. pastor is as the producer of MTG on an industrial scale, and laid a solid foundation for the commercialization of this technology.