Enhancing the secretion pathway maximizes the effects of mixed feeding strategy for glucose oxidase production in the methylotrophic yeast Pichia pastoris

Abstract

Background: A mixed feeding strategy (co-feeding of complex carbon sources with methanol) has become a common practice for process development in Pichia pastoris to increase cell biomass and enzyme production levels. However, in some cases mixed feeding did not have a significant impact or even had a negative effect on specific enzyme productivity. We hypothesized that this may be due to a bottleneck in the protein secretion pathway caused by too strong protein expression as a result of mixed feeding operation. Results: Using glucose oxidase (Gox) as a model protein, the individual and synergistic effects of co-feeding of sorbitol or yeast extract (YE) with methanol and Hac1p overexpression on the secretory expression of Gox were investigated both in shake flasks and in a laboratory fermenter. The results showed that YE is superior to sorbitol in terms of stimulating protein expression and cell growth. Moreover, separate applications of the mixed feeding strategy and secretory pathway engineering only achieved limited success in enhancing Gox levels, while the combined use of the two strategies acted synergistically, leading to 297% increase of Gox production and the final enzyme titer reached 787.4 U/mL in GSgox-Pp on 1-L fermenter. Conclusions: Co-feeding of YE combined with secretion pathway engineering significantly improved glucose oxidase secretion, which can be also applied to improve secretory expression of other foreign proteins in P. pastoris system.

Microbial protein cell factories fight back?

“…Combining strain and process engineering in P. pastoris has yielded promising results, as showcased by two recent examples from Che and colleagues and Bankefa and colleagues [76,77]. In the former, a combination of codon optimization, gene dosage, and fermentation optimization resulted in the production of 12 690 U/ml of fibase, while the latter showed how a mixed-feeding strategy combined with secretory pathway engineering led to a threefold increase in glucose oxidase production to 787.4 U/ml.…”

Improved β-galactosidase Secretion in Fresh Water Fungi through Cultural Optimization

“…Although some proteins such as human serum albumin and antibody fragments can be highly secreted from native host [18]; however, majority of others are still being reported in low level secretion, owing to different bottlenecks such as; mis-folding, sub-optimal conditions and secretion limitation pathways which may result in accumulation of these proteins in the endoplasmic reticulum, among others [19]. Nevertheless, efforts had been directed towards overcoming this bottleneck [20,21], but unfortunately, the sources of these proteins are rarely investigated.…”

Enzyme production ofd-gluconic acid and glucose oxidase: successful tales of cascade reactions

“…19,66,[73][74][75][76][77][78][79][80] In addition, strategies of formulating the fermentation medium 73,81 and computational tools 82 have proven to be powerful tools to increase glucose oxidase productivity. As an example, Bankefa et al 73 reported a strategy of co-feeding complex carbon sources (e.g., yeast extract) with methanol, combining a secretory pathway engineering in a Pichia pastoris strain containing the gene encoding glucose oxidase from A. niger. Using this strategy, the volumetric extracellular activity of glucose oxidase in a 1 L fermenter was enhanced by around 3-fold compared to that reached when utilizing a recombinant P. pastoris strain containing only the gene encoding glucose oxidase.…”

“…Both glucose oxidases are 10-16 wt% glycosylated with high-mannose type carbohydrates. 14,16,18,75,[84][85][86][87] Bankefa et al 73 reported that the SDS-PAGE analyses of glucose oxidase from A. niger extracellularly expressed by a recombinant P. pastoris strain showed a protein band of approximately 80 kDa, while its theoretical size was 63.5 kDa. This higher molecular mass of the enzyme subunit was attributed to enzyme glycosylation (around 21 wt%).…”