Discovery of a rhamnose utilization pathway and rhamnose-inducible promoters in Pichia pastoris

L-rhamnose can be utilized for growth by multitudinous microorganisms as a sole carbon and energy source1,2,3,4,5,6,7,8, and to date two different L-rhamnose metabolism pathways have been clarified in prokaryotes and eukaryotes9,10. It was further found that expression of some genes related to L-rhamnose metabolism in some microorganisms, such as Escherichia coli, Listeria monocytogenes, and Scheffersomyces (Pichia) stipitis, was strictly induced by L-rhamnose and strongly repressed by D-glucose1,10,11,12. Hence, an abundance of rhamnose inducible promoters were exploited and adopted to drive the expression of target genes, for example, the E. coli rhaPBAD promoter performed excellently in the production of heterologous proteins in the presence of rhamnose4,13,14, and the commercial Expresso Rhamnose Cloning and Expression System was developed by the Lucigen Corporation. Therefore, rhamnose inducible promoters offer great potential for the production of target proteins and many more promoters of this kind should be exploited to satisfy the demand in basic research and for practical applications. Pichia pastoris, a methylotrophic yeast, is an outstanding host for heterologous protein production. Several strong methanol-inducible promoters of genes related to the methanol utilization pathway, especially the well-characterized alcohol oxidase 1 promoter (PAOX1), have been frequently used to express recombinant proteins15,16,17. However, this promoter is not perfect for recombinant protein production due to two disadvantages of the inducer, methanol: it is highly flammable and hazardous to health. Thus, other inducible strong promoters are in high demand for controllable expression of heterologous genes in P. pastoris. P. pastoris can grow on rhamnose as a sole carbon source, but the genes associated with L-rhamnose metabolism have not been identified. We hypothesized that the promoters of L-rhamnose metabolism-related genes would also perform well in realizing massive production of heterologous proteins. This study aimed to disclose the putative genes involved in rhamnose metabolism pathways in P. pastoris and to exploit the promoters of these genes for controllable production of heterologous proteins. As expected, a series of putative genes involved in L-rhamnose metabolism in P. pastoris was preliminarily identified and the promoters isolated from these genes, especially PAS_chr4_0341 and PAS_chr1-4_0075 designated as LRA4 and LRA3, exhibited obvious inducibility by rhamnose. The two promoters could be exploited to enable efficient production of target proteins, especially food-grade and therapeutically important recombinant proteins, in P. pastoris.