Komagaella phaffii has become the preferred yeast cell factory for recombinant protein and biomolecule production in recent years. Developing a new bioprocess mainly requires two phases: the generation of the heterologous strain and the optimization of the production process, also known as bioprocess engineering. However, so far only the first stage has been extensively studied in K. phaffii. In bioprocess engineering, it is crucial that the cultivation conditions are adapted to the selected promoter, which drives the expression of the recombinant protein. To avoid the use of methanol and promote more a carbon-source-independent regulation, alternative derepressed and/or growth-decoupled promoters have recently gained much relevance, such as the orthologous PDF promoter. Therefore, this thesis has focused on the identification of growth-decoupled and methanol-free promoters, as well as the development of innovative cultivation strategies to fully exploit the promoter's potential. The final goal was to apply the acquired knowledge to develop a scalable bioprocess for the production of an unspecific peroxygenase (UPO). The PDH promoter was identified through transcriptomic analysis of shake-flask cultivations under de-repressed conditions. Preliminary cultures showed that the PDH promoter was highly activated under severe carbon restriction conditions. Accordingly, a growth-decoupled bioreactor strategy was designed, performing first a fed-batch phase at a high specific growth rate to achieve medium cell densities. Then, a severe carbon source restriction was applied by maintaining a very low constant feeding rate, resulting in the so-called pseudo-starving conditions. Although biomass did not increase in this second phase, the production of Candida antarctica lipase B (CalB) remarkably increased, doubling the CalB levels produced by a strain based on the classic GAP promoter. Due to the relevance of PDH regulation, two promoter engineering campaigns were carried out to increase its strength. As a result, MV1 and MV2 emerged as the best variants for CalB expression in shale-flask cultivations, nearly doubling CalB production compared to the PDH-based strain. After expandint the number of growth-decoupled promoters available for K. phaffii (PDH, MV1, MV2, and PDF), bioreactor cultivations were conducted using different feeding strategies with glucose, glycerol, and methanol, studying the effects of carbon restriction to CalB production. In all cultures, PDF showed the highest production levels, as well as better robustness and reproducibility, becoming the selected promoter for scaling up an UPO production process to pilot plant level. Additionally, the transcriptional analysis of 26 genes enabled the identification of 12 genes up-regulated under pseudo-starvating conditions, suggesting that their promoters would be promising candidates to drive recombinant protein expression. Finally, a 100-fold scale-up of the production bioprocess of the UPO from Hypoxylon sp. EC38 (HspUPO) was carried out, maintaining the same production levels as at the laboratory scale, besides two downstream campaigns. In total, two cultivations with different methanol-free feeding strategies, previously designed in the lab, were successfully scaled up. The best of the two conditions obtained the highest UPO productivity ever reported.
Providing growth-decoupled strategies for recombinant protein production in Komagataella phaffii : from promoter identification to bioprocess scale-up
Bernat Camps, N. (Author). 29 Nov 2024
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis