Combinatorial strain engineering in Pichia pastoris using Golden Gate cloning

Abstract

this final project, we show the results of a strain engineering strategy for heterologous protein production in the host organism Pichia pastoris. P. pastoris is a yeast with a huge industrial potential due to its set of strong and inducible promoters and its low endogenous protein secretion rate. Moreover, P. pastoris has the capability to secrete a recombinant protein with mostly all post-translational modifications and can be grown to high cell densities compared to other eukaryotic organisms. In a previous project, a similar strain engineering approach was performed using the Cre/loxP technique, which enables the integration of several genes by multiple consecutive DNA transformations. However, using Golden Gate technique, multiple genes can be simultaneously assembled and introduced by just one DNA transformation. The aim of this project was the study of protein secretion improvement using different helper factor gene combinations created by Golden Gate assembly. Based on omics data, we rationally selected 4 endogenous gene candidates related with the secretion pathway to be co-overexpressed. These genes were denominated X, Y, Z and K*, and different combinations of transcription units thereof were assembled on one vector. These combinations were K+X, K+X+Y and X+Z+Y+K. Surprisingly, during transformation not all the constructs were totally integrated into the genome as verified by gene copy number determination. Only strains that integrated the whole construct and that remained stable were used for further characterization. First screening results revealed that all engineered strains increased product titer and yield when compared to the parental strain. This result was statistically significant with a p-value below 10-4 for all the strains tested. The best gene combination in terms of product titer was K+X+Y while X+Z+Y+K achieved the best yield. Selected colonies were rescreened and showed a significant improvement with a 2.3-fold increased product titer in combination K+X+Y and an almost 2.9-fold increased product yield in combination X+Z+Y+K when compared to the parental strain, with a p-value also below 10-4. These results are in good agreement with those obtained by Cre/loxP cloning. With this project, we have not only shown the efficacy of Golden Gate assembly for its use in P. pastoris, but also identified some factors to be improved for future applications. Moreover, the four genes that have been successfully tested in two different strain engineering approaches seem to have a considerable industrial potential.