Characterization And Application Of SUT2 In Pichia Pastoris

At present,methylotrophic yeast Pichia pastoris has been used for the expression of many exogenous proteins as one of the widely used eukaryotic expression systems.Methanol utilization（MUT）pathway is the main pathway of expression proteins in P.pastoris.Alcohol oxidase 1 is the first and rate-limiting enzyme in MUT pathway,and the efficient promoter of alcohol oxidase 1(P_AOX1)can be strongly induced by methanol and inhibited by other carbon sources（such as glycerol）.The regulation of methanol metabolism is a complex biological process involving multiple transcription factors and signaling pathways,and specific molecular mechanism remains unclear.Previous transcriptome studies revealed that the expression of P.pSUT2 positively responds to carbon source changes and SUT2 has a high expression level with methanol as carbon source.SUT2 is a potential transcription factor and is likely involved in methanol metabolism.SUT2 was identified and applied in this study and the main results are as follows:（1）P.pSUT2 is highly expressed in glycerol-depletion medium or methanol medium,which means SUT2 shared a similar transcriptional profile with AOX1 in P.pastoris.Via knockout and overexpression of AOX1,we found that the expression level of SUT2 was positively correlated with AOX1.（2）Methanol may be main inducer of SUT2 expression.By comparing the transcription levels of SUT2 with methanol or formaldehyde as carbon source,it was found that the induction effect of methanol on SUT2 expression was significantly higher than that of formaldehyde.Moreover,the changes of SUT2 transcription level in wild-type under different induction conditions were similar to AOX1.（3）P.pSUT2 may be involved in methanol metabolism by regulating the biosynthesis of ergosterol.Via knockout and overexpression of SUT2,it was found that overexpression of SUT2 could increase the transcription and expression level of AOX1,and deletion of SUT2could lead to a significant decrease in the transcription level of AOX1.Furthermore,the content of ergosterol in P.pastoris was positively correlated with the expression level of SUT2.Combined with the reported literature and the data of this study,we speculate a regulatory pathway SUT2 involved in methanol metabolism,which provides a reference for exploration of key genes in methanol metabolism.（4）P_SUT2,a new methanol-inducible promoter,was isolated and identified.The transcriptional start site of SUT2,“A”at the 22^nd bp upstream of ATG,was determined with 5′-rapid amplification of cDNA ends.Via truncation of the putative SUT2 promoter at diverse loci,the-973 base pair（bp）to-547 bp region to the ATG was shown to be the core element of the inducible promoter P_SUT2,which strongly responds to the methanol signal.And the TATA box、CAAT box of P_SUT2 were predicted.（5）A forward-loop cassette was constructed with P_SUT2,enabling moderate elevation in activity of P_AOX1,which optimizes P.pastoris expression system for extrinsic proteins.This study successfully increased the expression of extrinsic proteins per unit of OD₆₀₀ by 18%,representing a promising direction for industry production.