| The microbial cell surface display technology is an important biotechnology rised with the genetic engineering development, in which the Pichia pastoris cell surface display system have become a rapid developing and widely used of all eukaryotic systems. The P. pastoris cell surface display system can used as whole-cell biocatalysts through display various heterogenous proteins, this biocatalysts not only have features and advantages that exist in immobilized enzymes, but also preparation method is simple and they are easy to recycle and reuse. Based on these features the biocatalysts have extensive prospects for utilization. The anchor proteins play an important role in cell surface display systems. It determines the display possibility of target protein, level of display efficiency and the activity of target protein. As the multiple anchor manners between pir proteins and target proteins, pir proteins can meet the display requirements of various target proteins. In this study, Pirlp was used as anchor protein to display 12 enzymes of glycolytic and ethanol metabolism on the surface of P. pastoris, the first aim of this artical is to study display function of Pirlp, the second is to establish the theoretical basis for ethanol production outside the cell.Main contents and results as follows:(1) Contruction of basal vectorsTwo recombinant pPIC9k of PDC and ADH were contructed firstly, which were named pPDC-9k and pADH-9k respectively. These two vectors will be the basal vectors to construct other recombinant vectors in the next experiments.(2) The contruction of display vectors of more target proteinsOn the basis of pPDC-9k and pADH-9k, the other 10 recombinant pPIC9k were contructed through gene replaced manner. The recombinant vectors of hexokinase(HXK), aldolase(ALD), phosphofructokinase(PFK), aldolase, triose phosphate isomerase(TIM), glyceraldehydes 3-phosphatedehydrogenase(GAPDH), phosphaglycerate kinase(PGK), phosphoglycerate mutase(PM), enolase(ENO) and pyruvate kinase(PK) were contructed based on the pPDC-9k, while recombinant phosphoglucose isomerase(PGI) was contructed from pADH-9k.(3) Analysis and identify of integration and expression of recombinant pPIC9k vectors with P.pastorisAll the 12 recombinant pPIC9k vectors were linerized and then introduced into the yeast cells. The multi-copy transformants were obtained by MD auxotrophic plates and G418 resistant plates. The genetic stability of transformants were verified by subculture experiments.In conclusion, several proteins including kinase, dehydrogenase, isomerase, aldolase, enolase and decarboxylase were displayed on the cell surface of P. pastoris by using Pirlp as anchor protein. The length of target proteins are varied from 247-988aa, the molecular weight of these proteins are between from 61kDa to 246 kDa, the length of protein coding gene are form 741bp to 2964bp. These results displayed the anchor ability of Pirlp. At the same time, all enzymes within an intact metabolic pathway for ethanol production were displayed on the cell surface of P. pastoris. These biocatalysts can be the basis for the ethanol production outside the cell. |
