High-level Cell-surface Display Of β-glucosidase From Dalbergia Cochinchinensis Pierre In Pichia Pastoris And The Primary Application On The Synthesis Of Alkyl Polyglycoside

Pichia pastoris surface display system is one of the rapidly growing microbial surfacedisplay systems, that the cells after methanol induction and lyophilization can be used aswhole-cell biocatalyst. Alkyl polyglucoside, APG, is widely used as low-toxic and greensurfactant. Traditional chemical method to synthesize APG is inefficient with numerous stepsand many by-products. These disadvantages can be avoided when the glycosyl transferasesand glycosyl hydrolases are used as the catalyst because of its regioselectivity andstereoselectivity. DCBGL (dalcochinase) with both β-glucosidase and β-fucosidase activitieswas isolated and purified from the seeds of Dalbergia cochinchinensis Pierre. It couldcatalyze the synthesis of homo-and hetero-oligosaccharides. Besides, it showed higher APGyields than almond β-glucosidase.Besides，DCBGL showed higher transglucoslation abilitythan almond β-glucosidase with medium-chain-lenghtg alcohols.In this study, we got β-glucosidase gene (dcbgl) from Dalbergia cochinchinensisPierreby gene synthesis after being optimized as the Pichia pastoris preferred codons. And19recombinant Pichia pastoris stains were constructed, which successfully display DCBGL onthe cell surface by using the GPI-modified cell wall protein Gcwnp (n=12,19,21,49,51,61)and in vitro multi-copy technique. Then we conducted the preliminary study of the synthesisof APG using the column reactor. The specific research content is listed below:(1) construction of Pichia pastoris displaying Dalbergia cochinchinensis Pierre β-glucosidase(DCBGL)Cell-surface display vectors pKDCBGL-GCWn （n=12,19,21,49,51,61） andrecombinant Pichia pastoris GS115/pKDCBGL-GCWn （n=12,19,21,49,51,61） wereconstructed. The fusion of DCBGL and cell wall protein does not influence the growth. Flowcytometry and fluorescence microscope assay revealed that DCBGL was successfullydisplayed on the cell surface via the6GPI-modified cell wall proteins. The highesthydrolytic activity of DCBGL displayed on the cell surface were found in the recombinantsbased on Gcw61p, Gcw51p and Gcw21p proteins, reaching212.67U/g,190U/g and176U/g。 (2)Construction and application of the co-displaying systemTwo methods are used to generate multi-copy strains, one is generate multipleexpression cassettes in a single vector pPICZαA before integrating into Pichia pastoris, theother one is using two different selection marker in Pichia pastoris.11strains harboring2to5copies were obtained based on several cell wall proteins. A strain based on differentanchoring proteins harboring3copies (GS115/DCBGL-GCW(61×2+51)) had the bestefficiency of DCBGL, reaching747.90U/g。It was almost3.52fold than that of GS115/pKDCBGL-GCW12. In this study, the growth conditions were inhibited when the copynumber of DCBGL reached4. Recombinant strains harboring3copies DCBGL showedhigher hydrolytic ability than other strains harboring1,2,4,5copy/copies DCBGL, andPichia pastoris cell does not have enough space for all displayed enzyme molecules to foldcorrectly.Further increasing copy number will lead to decreasing effct on the active enzymemolecules and bad growth condition of recombinant strains.The whole-cell catalyst GS115/DCBGL-GCW(61×2+51) was obtained after168hinduction of methanol, and lyophilization. They were added to the column PBR to synthesizethe APG. When the glass wool was used to separate the whole-cell biocatalyst, the conversionrate at24h was12.67%, corresponding with the level without interlayers at48h. At60h, theconversion rate reached23.995%. Additionally, the residual activity of the whole-cellbiocatalyst remained almost unchanged. These result indicated that whole-cell biocatalyst canbe used in reactor to realize synthetic reaction.