Resumen de Systems metabolic engineering for recombinant protein production in pichia pastoris
The methylotrophic yeast Pichia pastoris (Komagataella sp.) is one of the most attractive expression systems for heterologous protein production, which constitutes a continuously expanding market. The strong alcohol oxidase gene 1 promoter (PAOX1), induced by methanol but repressed by glucose, glycerol or ethanol, is one of the most used for this purpose. Nevertheless, there still exist several physiological bottlenecks limiting the process.
In this context, several strategies have been proposed and tested in order to improve the heterologous production of many different types of proteins. Common approaches include increasing heterologous gene copy number, promoter engineering and modification of the folding and secretory mechanisms. The aim of this thesis has been the development of new strategies to increase recombinant protein yields, using the Rhizopus oryzae lipase (Rol) as model protein in a PAOX1-based expression system.
Firstly, the PAOX1 transcription factor genes MXR1 and MIT1 were constitutively overexpressed aiming at improving ROL transcription. This was confirmed by an improved methanol assimilation capacity and an increase in relative mRNA levels of ROL and several genes related with methanol metabolism, i.e. reverting the titration effect caused by the transcription of multiple ROL expression cassettes. Despite such improvements, extracellular lipase activity levels did not increase significantly in chemostat cultures, pointing out to additional bottlenecks limiting Rol production.
Second, possible metabolic engineering targets in P. pastoris’ cell metabolism were explored using the consensus genome-scale metabolic model (GEM) iMT1026 v3.0. This in silico step provided several promising knock-outs which were going to be experimentally tested using the CRISPR/Cas9 genome editing system. The simulations pointed to NADPH availability and limited supply of some amino acids (serine and cysteine) as potential Rol production limiting factors. A reduction in cell fitness affecting the viability of the obtained strains impeded to verify most of the proposed knock-outs.
Finally, since our in silico analyses and previously published studies identified NADPH as an important limiting cofactor in recombinant protein production, our efforts were geared towards increasing its availability through gene knock-in strategies. Specifically, we overexpressed two genes encoding redox enzymes, a NADH kinase and a NADH oxidase, with the aim to directly perturb the cell’s redox balance. Further, we tested the physiological effect of these enzymes using different co-substrate/methanol mixtures as carbon source. In short, we observed an increase in recombinant protein production with different degrees of improvement depending on the carbon source(s) tested. We also performed a transcriptomic analysis and an in silico evaluation of our results in order to provide a better interpretation of the cell physiological state. To our knowledge, this is the first study aiming to increase NADPH generation in the PAOX1-based expression system, under methanol growth conditions.
Overall, novel strain engineering strategies have been proposed and tested during the execution of this study. Furthermore, GEMs and related systems biology approaches were applied, proving to be promising powerful tools for rational engineering of industrial microorganisms.
