The Heterologous Expression Of Trehalose Synthase In Pichia Pastoris

Recently,the methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production for it can use methanol as sole carbon source.Besides,owning the abilities of post-translation,secret the protein to the medium,reach high enzyme activity and achieve high density fermentation, pichia pastoris has been a good choice for lab research and industry.This study focus on the different resources of trehalose synthase gene express in Pichia pastoris through different expression plasmid.Screening recombinant strain with high trehalose synthase expression, using the corresponding detection methods to achieve measured enzymatic properties.In this study, improving enzyme activity is with the start of improving the gene copy number. Using 2A-system to construct single promoter double gene copies plasmid or screening the multiple copies of recombinant strain. Then, directly using the replace or mutant AOX1 promoter to express trehalose systhase. Meanwhile,overexpression of central metabolism pathway key gene increase trehalose synthase activity. The results are as follows:1. In this work,three different sources of trehalose synthase are from Corynebacterium glutamicum ATCC 13032,Thermus thermophilus HB27, Thermotoga maritima MSB8,respectively combine with the secretory plasmid pPICZaA, pPIC9k, pGAPZaA and the intracellular plasmid pPIC3.5k,and then transformed pichia gives 12 different recombinant trehalose synthase strain. Screening the high enzyme activity strain get the best expression strain is thermus thermophilus HB27 gene resource with the pPIC3.5k plasmid,the enzyme activity can reach 2748.3U.The sources of trehalose synthase detecting enzymatic properties, which Km is 194.57mM, Vmax is 3.68×10-2mM·S-1, Kcat is 223.43S-1, Kcat/Km is 1.152S-1·mM-1.2. With the copy number as the starting point, on the one hand, by the eukaryotic expression 2A-System,single promoter double trehalose synthase gene in Pichia has been constructed; on the other hand, through a multi-copy recombinant Pichia strain screening different gene copy number, and using the droplet digital PCR assay the absolute copy number. Compared the two methods, multiple copies can be screened for the gene copy number strains 4, enzyme activity was measured up to 3054.1U, activity increase by 11%.3. with the promoter as a starting point,the PAOX1 has been replaced of the strong constitutive PGAP, so that we can use glucose as carbon source to produce trehalose. Expression of enzyme activity averaged 1363.3U, less than using PAOX1. Therefore, by directed mutate PAOX1, to obtain a higher promoter activity,.when combined the deletion mutation(?-777bp to-712) and replication (2×-203bp to-190bp),the PAOX1 activity enhanced. At that time, the trehalose synthase activity reached 5103.1U,the enzyme activity increased by 44% compared to the origin enzyme activity.4. It is going to have a influence on basic metabolic pathway in pichia pastoris that producing heterologous protein. In this paper, by overexpression the key enzyme gene in central metabolic pathway(pentose phosphate pathway and TCA cycle) in pichia pastoris and co-expression with trehalose synthase to improve the enzyme activity. Separate Co-express ZWF1, SOL3 (from PPP pathway), MDH1, GDH3 (from TCA cycle) can improve trehalose synthase activity. When co-expressed with MDH1, enzyme expression is 20% higher than control group, activity reached the highest value 5680.3U, compared with the control group increased by 11%,while compared with the original group increased by 60%.