Abstract
Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast that is widely used in industry as a host system for heterologous protein expression. Heterologous gene expression is typically facilitated by strongly inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters. However, protein production is usually accomplished by a fed-batch induction process, which is known to negatively affect cell physiology, resulting in limited protein yields and quality. To assess how yields of exogenous proteins can be increased and to further understand the physiological response of P. pastoris to the carbon conversion of glycerol and methanol, as well as the continuous induction of methanol, we analyzed recombinant protein production in a 10,000-L fed-batch culture. Furthermore, we investigated gene expression during the yeast cell culture phase, glycerol feed phase, glycerol-methanol mixture feed (GM) phase, and at different time points following methanol induction using RNA-Seq. We report that the addition of the GM phase may help to alleviate the adverse effects of methanol addition (alone) on P. pastoris cells. Secondly, enhanced upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was observed in P. pastoris following methanol induction. The MAPK signaling pathway may be related to P. pastoris cell growth and may regulate the alcohol oxidase1 (AOX1) promoter via regulatory factors activated by methanol-mediated stimulation. Thirdly, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways were not significantly upregulated during the methanol induction period. These results imply that the presence of unfolded or misfolded phytase protein did not represent a serious problem in our study. Finally, the upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes but not to the production of phytase. This work describes the metabolic characteristics of P. pastoris during heterologous protein production under high-cell-density fed-batch cultivation. We believe that the results of this study will aid further in-depth studies of P. pastoris heterologous protein expression, regulation, and secretory mechanisms.
Highlights
Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast species that has been shown to efficiently produce exogenous proteins
By comparing recent transcriptomics or proteomics studies pertaining to the expression of recombinant proteins in P. pastoris (Liang et al, 2012; Vanz et al, 2012; Hesketh et al, 2013), we identified studies where fermentation was conducted through direct methanol culture or the addition of methanol to glycerol-mediated growth cultures
In relation to carbon metabolism pathways, glycerol kinase (GUT1) and alcohol dehydrogenase (ADH) were remarkably upregulated during the adaptation period of GM, and alcohol oxidase1 (AOX1) was significantly upregulated during this stage (p-Value < 0.001 and log2 fold changes ≈ 6.30) (Figure 3)
Summary
Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast species that has been shown to efficiently produce exogenous proteins This species exhibits many of the advantages normally associated with Escherichia coli expression systems (Morton and Potter, 2000) while overcoming many of the deficiencies associated with Saccharomyces cerevisiae systems (Tran et al, 2017). It is imperative that further efforts are made to improve levels of expression exhibited by the analyzed strain With this in mind, many researchers have conducted experiments that have resulted in the improvement of protein stability through rational design (Han et al, 2017, 2018), the development of strong promoters (Portela et al, 2018; Prielhofer et al, 2018; Ergun et al, 2019; Wang J. et al, 2019), the optimization of protein secretion (Barrero et al, 2018; Nguyen et al, 2018), the alteration of protein glycation in P. pastoris (Pekarsky et al, 2018), and co-expression of transcription factors (Chang et al, 2018; Dey et al, 2018). Increasing productivity during scale-up of processes largely depends on trial and Abbreviations: 6PG, gluconate-6P; CTS, citrate synthase; DAK, triose/dihydroxyacetone kinase; DAS, dihydroxyacetone synthase; DHA, dihydroxyacetone; DHAP, dihydroxyacetone-P; DLD, dihydrolipoamide dehydrogenase; E4P, erytgrose-4P; ENOI, enolase I; F1,6BP, fructose-1, 6-P2; F6P, fructose-6P; FBAII, fructose-bisphosphate aldolase, class II; FBPI, fructose1,6-bisphosphatase I; FCH, S-formylglutathione hydrolase; FDH, formate dehydrogenase; FLD1, S-(hydroxymethyl)glutathione dehydrogenase; Form, formaldehyde; FUM, fumarate; G1,3P, glycerate-1,3P2; G2P, glycerate-2P; G3P, glycerate-3P; G6P, glucose-6P; GAP, glyceraldehyde-3P; GAPD, glyceraldehyde 3-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; GS-CH2OH, S-hydroxymethylglutathione; GS-CHO, S-formylglutathione; ISO, isocitrate; MAL, malate; MDH, malate dehydrogenase; ODE, 2-oxoglutarate dehydrogenase E2; OXA, oxaloacetate; OXAL, oxalosuccinate; PDE, pyruvate dehydrogenase E; PEP, phosphoenolpyrvate; PEX, peroxisomal; PGK, phosphoglycerate kinase; R5P, ribose-5P; RKI, ribose-5-phosphate ketol-isomerase; Rul5P, ribulose-5P; S7P, sedoheptulose-7p; SCS1, succinyl-CoA synthetase 1; SCS2, succinyl-CoA synthetase 2; SDH, succinate dehydrogenase; SUC, succinate; TAL, transaldolase; TK, transketolase; X5P, xylulose-5P
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