Methanol Independent Expression by <em>Pichia Pastoris</em> Employing De-repression Technologies

Abstract

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters P DC and P DF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Secretion of Pseudomonas aeruginosa Lipoxygenase by Pichia pastoris upon Glycerol Feed

“…The use of methanol is considered hazardous (toxic and flammable) and in addition, removal of methanol contaminated cells can be problematic for a lot of branches. [ 30,35,39,40 ] This is one rare example in which glycerol as a sole carbon source (an inexpensive and safe compound) clearly outcompetes methanol in terms of activity yields. Methanol‐free cultivation of P. pastoris might be more than an alternative, here.…”

“…(2019) discovered a novel promoter system, that is, the promoter of the P. pastoris CAT1 gene. [ 35 ] Using this system, a methanol‐induced, but also a methanol‐free expression approach can be established, providing flexibility in terms of application. The CAT1 promoter is repressed in the presence of glucose or glycerol, and de‐repressed upon depletion of these carbon sources.…”

“…The CAT1 promoter is repressed in the presence of glucose or glycerol, and de‐repressed upon depletion of these carbon sources. [ 35 ] Here, we report Pa LOX production in P. pastoris for the first time. As secreted protein, downstream processes and analysis of the enzyme are tremendously simplified.…”

Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9

“…On the other hand, the expression of the P PDC promoter (native catalase gene promoter) is repressed in the presence of glucose or glycerol and derepressed upon the depletion of these carbon sources. Its expression can further be induced with methanol and remarkably also with oleic acid [ 39 ]. Besides the P PDC , an orthologous promoter, i.e.…”

Amino Benzamidoxime (ABAO)‐Based Assay to Identify Efficient Aldehyde‐Producing Pichia pastoris Clones

“…All constructs with high 1 d concentrations expressed Mm CAR via an equally strong or stronger promoter ( P ) compared to Mm PPTase. Construct #2 is based on the strongest methanol‐inducible promoter P AOX1 ( alcohol oxidase 1 ) [27] for Mm CAR expression and P CAT1 ( catalase ) [28] for Mm PPTase expression (Table 1). Construct #5 expressed Mm CAR via P CAT1 , also known as P DC , and is a promoter of medium strength, [18] whereas Mm PPTase was expressed by the weak methanol‐inducible promoter P PEX5 [29] .…”