Construction Of Recombinant Pichia Pastoris Strains With High Carotenoid Production Level

The methylotrophic yeast Pichia pastoris as a high-level expression system is widely used in the high-cell-density batch fermentation of enzyme industry.However,the large amounts of yeast cells produced during the fermentation process are low values and represent great loss.It is of great importance and economic value to fully utilize the yeast cells.Carotenoid plays an important role in the human health and life,and it has been widely used in food,feed,health products and other fields.The present study aims to introduce the key genes involved in theβ-carotene biosynthesis into P.pastoris GS115 to construct an engineering strain with high yield of intracellular carotenoid and high-level expression of exogenous enzymes,and consequently achieves the efficient utilization of yeast cells.To construct the engineering strain with high-level and genetically-stable production of carotenoid,six constitutive promoters of P_GCW14,P_TEF,P_PGK1,P_GAP,P_ILV5 and P_AOD and corresponding terminators were used to express the key genes（idi,crtE,crtYB,crtI,crtS and crtR）of Xanthophyllomyces dendrorhous involved in the biosynthesis ofβ-carotene and astaxanthin.These genes expression cassettes having no homologous sequences facilitated the genetic stability of recombinant strains.By constructing recombinant plasmids p PICZαA-idi-crtE-crtYB-crtI and p PICZαA-idi-crtE-crtYB-crtI-crtS-crtR and transforming them into the competent cells of strain GS115,positive transformants with high yields of intracellularβ-carotene（GS115-CARO,up to 28 mg/g dry cell at 72 h）and detectable astaxanthin were obtained.The extracellular mannanase gene man5T from Talaromyces leycettanus JCM12802 was then selected to compare the expression efficiency in strains GS115 and GS115-CARO.After transformation and activity screening,both strains showed similar mannanase yields（280 U/mL vs.286 U/mL,P>0.05）at flask level,but the yield of intracellularβ-carotene was up to 31.27 mg/g dry cell in P.pastoris GS115-CARO.The results showed the strain constructed herein not only achieved both the high-level expression of exogenous enzymes and high yield of intracellular carotenoids,but also promoted the utilization value of yeast resources.In summary,the engineering strain P.pastoris GS115-CARO has great genetic stability and high yield of intracellular carotenoid,and has no difference in the production of exogenous enzymes.This strain will be much value to be used in the high-cell-density fermentation process for high-level expression of both exogenous enzymes and intracellular carotenoids.This mode of production can effectively improve the utilization value of biomass resources and turn waste into treasure.