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The trisaccharide 1-kestose, a major constituent of commercial fructooligosaccharide (FOS) formulations, shows a superior prebiotic effect compared to higher-chain FOS. The plant sucrose:sucrose 1-fructosyltransferases (1-SST) are extensively used for selective synthesis of lower chain FOS. In this study, enhanced recombinant (r) 1-SST production was achieved in Komagataella phaffii (formerly Pichia pastoris) containing three copies of a codon-optimized Festuca arundinacea 1-SST gene. R1-SST production reached 47 U/mL at the shake-flask level after a 96-h methanol induction phase. A chemostat-based strain characterization methodology was adopted to assess the influence of specific growth rate (µ) on cell-specific r1-SST productivity (Qp) and cell-specific oxygen uptake rate (Qo) under two different feeding strategies across dilution rates from 0.02 to 0.05 h-1. The methanol-sorbitol co-feeding strategy significantly reduced Qo by 46 ± 2.4% compared to methanol-only feeding without compromising r1-SST productivity. Based on the data, a dilution rate of 0.025 h-1 was applied for continuous cultivation of recombinant cells to achieve a sustained r1-SST productivity of 5000 ± 64.4 U/L/h for 15 days.

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BACKGROUND: The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. RESULTS: We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. CONCLUSIONS: Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.

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Transglutaminase (TG, EC 2.3.2.13) is widely used to modify functional properties in food systems, which can catalyze cross-linking reaction of proteins. In this work, microbial transglutaminase (MTG) from Streptomyces netropsis was heterologously expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris). The specific activity of recombinant microbial transglutaminase (RMTG) was 26.17 ± 1.26 U/mg, and the optimum pH and temperature were measured as 7.0 and 50 °C, respectively. Bovine serum albumin (BSA) was used as a substrate to evaluate the effect of cross-linking reaction, and we found that RMTG had significant (p < 0.05) cross-linking effect for more than 30 min reactions. RMTG was further utilized in the investigation of plant-based chicken nuggets. Results showed that the hardness, springiness and chewiness of nuggets increased, and the adhesiveness decreased after RMTG treatment, which can prove that RMTG has the potential to improve the texture properties of plant-based chicken nuggets.

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The expression of recombinant proteins by the AOX1 promoter of Komagataella phaffii is typically induced by adding methanol to the cultivation medium. Since growth on methanol imposes a high oxygen demand, the medium is often supplemented with an additional secondary carbon source which serves to reduce the consumption of methanol, and hence, oxygen. Early research recommended the use of glycerol as the secondary carbon source, but more recent studies recommend the use of sorbitol because glycerol represses P AOX1 expression. To assess the validity of this recommendation, we measured the steady state concentrations of biomass, residual methanol, and LacZ expressed from P AOX1 over a wide range of dilution rates (0.02-0.20 h-1) in continuous cultures of the Mut+ strain fed with methanol + glycerol (repressing) and methanol + sorbitol (non-repressing). We find that under these conditions, the specific P AOX1 expression rate (measured as either specific LacZ productivity or specific AOX productivity) is completely determined by the specific methanol consumption rate regardless of the type (repressing/non-repressing) of the secondary carbon source. In both cultures, the specific P AOX1 expression rate is proportional to the specific methanol consumption rate, provided that the latter is below 0.15 g/(gdw-h); beyond this threshold consumption rate, the specific P AOX1 expression rate of both cultures saturates to the same value. Analysis of the data in the literature shows that the same phenomenon also occurs in continuous cultures of Escherichia coli fed with mixtures of lactose plus repressing/non-repressing carbon sources. The specific P lac expression rate is completely determined by the specific lactose consumption rate, regardless of the type of secondary carbon source, glycerol or glucose.

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Komagataella phaffii (syn. Pichia pastoris), a methylotrophic yeast, has many advantages as a protein expression system, but has the disadvantage of hazardous methanol as an inducer and carbon source. To enable substitution of formate for methanol, a formate assimilation pathway was constructed by the co-expression of acetyl-CoA synthase, acetaldehyde dehydrogenase, and transcription factor Mit1, resulting in a 103.5 ± 12.5% increase in xylanase production. Recombinant K. phaffii was able to use formate as a carbon source, indicating successful substitution of formate for methanol. Xylanase production, using the safe and sustainable formate as an inducer and carbon source, is a major advance in the field of industrial enzyme production. KEY POINTS: • Change to formate assimilation by recombinant K. phaffii instead of methanol • K. phaffii expressed xylanase by formate induction instead of methanol induction • Increased xylanase expression by transcription factor co-expression.

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The present supply of prebiotics is entirely inadequate to meet their demand. To produce novel prebiotics, a d-mannose isomerase (XpMIaseA) from Xanthomonas phaseoli was first produced in Komagataella phaffii (Pichia pastoris). XpMIaseA shared the highest amino acid sequence identity (58.0%) with the enzyme from Marinomonas mediterranea. Efficient secretory production of XpMIaseA (282.0 U mL-1) was achieved using high cell density fermentation. The optimal conditions of XpMIaseA were pH 7.5 and 55 °C. It showed a broad substrate specificity, which isomerized d-mannose, d-talose, mannobiose, epilactose, and mannotriose. XpMIaseA was employed to construct a one-pot three-enzyme system for the production of mannosyl-ß-(1 → 4)-fructose (MF) using mannan (5%, w/v) as the substrate. The equilibrium yield of MF was 58.2%. In in vitro fermentations, MF significantly stimulated (≤3.2-fold) the growth of 12 among 15 tested Bifidobacterium and Lactobacillus strains compared with fructo-oligosaccharides. Thus, the novel d-mannose isomerase provides a one-pot bioconversion strategy for efficiently producing novel prebiotics.

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Alpha-lactalbumin (α-LA; the most abundant whey protein in human milk) contributes to infant development, providing bioactive peptides and essential amino acids. Here, Komagataella phaffii (K. phaffii) was selected as the production host. We found that the K. phaffii host X33 was suitable for expressing the target protein, yielding 5.2 mg·L-1 α-LA. Thereafter, several secretory signal peptides were applied to obtain a higher titer of α-LA. The strain with α-factor secretory signal peptide secreted the highest extracellular titer. Additionally, promoters AOX1, GAP, and GAP(m) were compared and applied. The strain with the promoter AOX1 produced the highest extracellular titer. In addition, coexpressing human protein disulfide isomerase A3 (hPDIA3) increased the titer by 27%. Human α-LA production by the strain X33-pPICZαA-hLALBA-hPDIA3 reached 56.3 mg·L-1 in a 3 L bioreactor. This is the first report of successful secretory human α-LA expression in K. phaffii and lays foundations for the simulation of human milk for infant formulas and further development of bioengineered milk.

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BACKGROUND: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. RESULTS: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. CONCLUSIONS: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.

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BACKGROUND: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (PAOX1), and the constitutive GAP promoter (PGAP). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (PPDF, PUPP) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. RESULTS: Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. CONCLUSIONS: As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.

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The methylotrophic yeast Komagataella phaffii is among the most popular hosts for recombinant protein synthesis. Most recombinant proteins have been expressed in the wild-type Mut+ host strain from the methanol-inducible alcohol oxidase (AOX) promoter PAOX1. Since methanol metabolism has undesirable consequences, two additional host strains, Muts (Δaox1) and Mut- (Δaox1Δaox2), were introduced which consume less methanol and reportedly also express recombinant protein better than Mut+. Both results follow from a simple model based on two widespread assumptions, namely methanol is transported by diffusion and the sole inducer of PAOX1. To test this model, we studied 14C-methanol uptake in the Mut- strain and ß-galactosidase expression in all three strains. We confirmed that methanol is transported by diffusion, but in contrast to the literature, Mut+ expressed ß-galactosidase 5- and 10-fold faster than Muts and Mut-. These results imply that methanol is not the sole inducer of PAOX1-metabolites downstream of methanol also induce PAOX1. We find that formate or/and formaldehyde are probably true inducers since both induce PAOX1 expression in Mut- which cannot synthesize intracellular methanol from formate or formaldehyde. Formate offers a promising substitute for methanol since it does not appear to suffer from the deficiencies that afflict methanol. KEY POINTS: • This is the first study to systematically compare all three Mut phenotypes as host strains. • Mut+ strain expresses 5- and 10-fold faster than Muts and Mut- strains. • Methanol is transported by diffusion in Komagataella phaffii. • Formate and formaldehyde are true and strong inducers of PAOX1 expression.

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