Excavation Of The Rhamnose Metabolic Pathway And Rhamnose-inducible Promoters Inpichia Pastoris

As the demand increases, specific proteins isolated from natural strains can hardly meet the industrial demand. Therefore, developing new strategy of strain improvement and efficient system for heterologous protein expression is in great demand. At present, many expression systems are used for producing heterologous protein effectively, including the most extensively used Pichia pastoris expression system, which has a clear genetic background, a high growth rate, ability to produce large amount of protein and is easy to be cultivated. The methanol-inducible andstrongpromoter PAOX1 and the strong constitutive promoter PGAP are the most widely used promoters in the P. pastoris expression system. However, the use of PAOX1 in both large-scale fermentation process and production of proteins used for food and medicine is disadvantageous since methanol is flammable and toxic. While the target proteinis continuously expressed during the whole fermentation under the control of PGAP, making it difficult to control the process. At the same time, target protein which is poisonous for the host cell can not be massively produced using PGAP. In conclusion, it is necessary to improve P. pastoris expression system via excavation of strong, nontoxic compound inducible promotersIn this study, four predicted genes, PAS_chr₀338, PAS_chr₀339, PAS_chr₀340, LRA4（PAS_chr₀341）, which are associated with rhamnose metabolism in P. pastoris and a previously identified gene LRA3 have been studied. The location of these genes is determined. PAS_chr₀338, PAS_chr₀339, PAS_chr₀340 and LRA4 are in the same gene cluster, but LRA3 is independent. The expression of these genes, especially LRA4 and LRA3, was significantly increased in the presences of rhamnose while decreased in the presence of glucose. LRA4 encoding a putative L-2-keto-3-deoxyrhamnonate aldolase, was further confirmed via gene disruption and gene complementation toparticipate in rhamnose metabolism. Using β-galactosidase and green fluorescent protein as reporters, the promoters of LRA4 and LRA3 performed well in driving expression of heterologous proteins. By using food-grade rhamnose instead of the toxic methanol as the inducer, the two promoters would be excellent candidates for driving the expression of food-grade and therapeutically important recombinant proteins.