Resumen de Combining bioprocess and strain engineering strategies as efficient tools for the optimization of recombinant protein production in pichia pastoris
Microbial cell factories can be used to produce a wide range of bioproducts of interest for the biotechnological industry, which comprises mainly the production of recombinant proteins and metabolites. Pichia pastoris (Komagataela phaffii), emerges as a promising host for recombinant protein production (RPP) due to it shares many features with Saccharomyces cerevisiae, however, displays some advantages in terms of oxygen consumption, simpler glycosylation pattern, and lower endogenous protein secretion. For these reasons, great efforts have been performed with the objective to optimize the efficiency of this host which can be grouped in two main and complementary approaches: the strain and bioprocess engineering. The present PhD thesis was focused in the use of both approaches, in order to improve the production bioprocess of recombinant lipases with industrial interest.
At first, it was demonstrated the importance of the knowledge of production kinetics as strong tool to design optimal strategies for RPP through the characterization of two clones with contrasting production performance, due to its different gene dosage, expressing Candida rugosa lipase 1 (Crl1) regulated under GAP promoter (PGAP) using chemostat and fed-batch cultures. The results, also supported by transcriptional analysis of some target genes as marked novelty, demonstrated that production kinetics depends on the intrinsic characteristics of each clone used. Therefore, the selection of adequate µ for each case enables, in a different way, the rational process development to optimize RPP bioprocesses.
Later, it was also evaluated the potential implementation of carbon-starving as innovative strategy to enhance the Crl1 production rates and yields on fed-batch cultures with a previous physiological characterization on chemostat cultivation. Results showed that positive effects observed using this strategy are highly dependent on the specific features of the clone used. An additional transcriptomic analysis (RNAseq) was carried out with chemostat samples, pointing out the difference on the transcription of all the genes of the yeast.
In addition, bioprocess performance of the GAP promoter (PGAP) was compared with the inducible AOX1 promoter (PAOX1) by carrying out chemostat cultures producing Crl1. Although PAOX1 displayed higher production, an economical evaluation should be necessary before scale-up of the bioprocess, considering the numerous drawbacks of using methanol as substrate.
Finally, following the strain engineering approach, it was characterized the use of two alternative methanol free novel promoters on the expression of lipase B from Candida antarctica (CalB) as strong tool that allows to exploit P. pastoris potential on RPP. Both promoters displayed much better production parameters than the observed with PGAP although the production pattern between promoters were significantly different on each case.
Overall, the results presented along the different chapters of this current thesis support the usefulness of bioprocess and strain engineering through the different studies performed, which gave significant improvements in RPP efficiency. The knowledge of key factors involved on recombinant expression opens a window of new opportunities that allows P. pastoris to be established as a robust platform for RPP and showing it as highly competitive to conventional systems.
