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Review
. 2024 Feb 7;12(2):346.
doi: 10.3390/microorganisms12020346.

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Tamara M Khlebodarova  1   2 , Natalia V Bogacheva  1   2 , Andrey V Zadorozhny  1   2 , Alla V Bryanskaya  1   2 , Asya R Vasilieva  1   2 , Danil O Chesnokov  3 , Elena I Pavlova  3 , Sergey E Peltek  1   2
Affiliations
Review

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Tamara M Khlebodarova et al. Microorganisms. .

Abstract

In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker's yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species' several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.

Keywords: Komagataella phaffii; expression; post-translational modifications; promoters of heterologous proteins; protein production; signal sequence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The development of methanol-free expression system based on a heat-shock gene promoter (PDH) using glycerol as sole carbon source. (a,b) The selection of the shortest putative sequence of the HPSP12 gene promoter from the K. phaffii genome, demonstrating the expression level comparable to that of the full-length promoter; (c) comparative analysis of the PDH-C promoter efficiency with the reference GAP-C, UPP-C, and PDF-C promoters; and (d) the impact of osmolarity levels on the efficiency of the PDH-C promoter. Adapted from [49].
Figure 2
Figure 2
The development of methanol-free expression system based on the glucose transporter gene promoter (pGTH1) [83]. (a) Schematic of the workflow for generating a K. phaffii GTH1 promoter library by random mutagenesis; (b,c) impact of single point mutations on expression properties of PGTH1: (b) random mutagenesis (the red colored mutations are located outside of a transcription factor binding sites), (c) systematic introduction of single point mutations into the transcription factor binding site; (d) impact of the segmental deletions introduced to the −200 to −400 bp region of pGTH1 on its expression properties; (e) impact of the segmental duplications on expression properties of pGTH1; and (f) impact of the duplication of the main regulatory promoter region and point mutations on expression properties of pGTH1. Designations: Bars represent mean values and closed circles the calculated RF values of the individual clones; the horizontal dotted line highlights the average expression level of the native PGTH1 control strain (set to 100% for each condition); **—p ≤ 0.05; ***—p ≤ 0.005. Adapted from [83].
Figure 3
Figure 3
Schematic diagram of N-linked glycan structure in a mammalian cell, S. cerevisiae, and K. phaffii. (A) N-linked glycan structure in mammalian cells commonly generates complex terminally sialylated structures. (B) In S. cerevisiae, the N-linked glycan structure is typically hypermannosylated (Man > 50GlcNAc2). (C) N-linked glycan structure in K. phaffii typically is of the Man8-14GlcNAc2 type with a triantennary-branched structure. (D) In Pichia GlycoSwitch® strains (SuperMan5), N-linked glycan structure is typically hypomannosylated (with a mannose-5 structure). Adapted from [29].
Figure 4
Figure 4
The common scheme of producing recombinant strains of enzyme producers based on the K. phaffii genome. (a) _target gene cloning; (b) analysis of recombinant clones for the presence of protein and enzyme activity; (c) electrophoretic analysis of the mannanase preparation from the culture liquid of recombinant clone; (d) analysis accumulation of biomass and protein during cultivation of selected clones in a bioreactor; (e) full genomic sequencing of the nucleotide sequence of the genome of the strain producing the _target protein; (f) analysis of the culture fluid of the producer line (general view of the MS1 mass spectrogram containing the peptide mixture obtained from the culture liquid); (g) the fragment peptide sequence founding in culture fluid samples of the recombinant strain K. phaffii T07 identifying the structure with the specified putative amino acid sequence with a convergence of 58.22%; (h) protein and enzyme preparations by tangential diffusion and liquid chromatography methods; (i) characteristics of purified and lyophilized proteinase K, dependence of enzyme activity on pH and temperature. Adapted from [246,330,331,332].
Figure 4
Figure 4
The common scheme of producing recombinant strains of enzyme producers based on the K. phaffii genome. (a) _target gene cloning; (b) analysis of recombinant clones for the presence of protein and enzyme activity; (c) electrophoretic analysis of the mannanase preparation from the culture liquid of recombinant clone; (d) analysis accumulation of biomass and protein during cultivation of selected clones in a bioreactor; (e) full genomic sequencing of the nucleotide sequence of the genome of the strain producing the _target protein; (f) analysis of the culture fluid of the producer line (general view of the MS1 mass spectrogram containing the peptide mixture obtained from the culture liquid); (g) the fragment peptide sequence founding in culture fluid samples of the recombinant strain K. phaffii T07 identifying the structure with the specified putative amino acid sequence with a convergence of 58.22%; (h) protein and enzyme preparations by tangential diffusion and liquid chromatography methods; (i) characteristics of purified and lyophilized proteinase K, dependence of enzyme activity on pH and temperature. Adapted from [246,330,331,332].
Figure 4
Figure 4
The common scheme of producing recombinant strains of enzyme producers based on the K. phaffii genome. (a) _target gene cloning; (b) analysis of recombinant clones for the presence of protein and enzyme activity; (c) electrophoretic analysis of the mannanase preparation from the culture liquid of recombinant clone; (d) analysis accumulation of biomass and protein during cultivation of selected clones in a bioreactor; (e) full genomic sequencing of the nucleotide sequence of the genome of the strain producing the _target protein; (f) analysis of the culture fluid of the producer line (general view of the MS1 mass spectrogram containing the peptide mixture obtained from the culture liquid); (g) the fragment peptide sequence founding in culture fluid samples of the recombinant strain K. phaffii T07 identifying the structure with the specified putative amino acid sequence with a convergence of 58.22%; (h) protein and enzyme preparations by tangential diffusion and liquid chromatography methods; (i) characteristics of purified and lyophilized proteinase K, dependence of enzyme activity on pH and temperature. Adapted from [246,330,331,332].
Figure 4
Figure 4
The common scheme of producing recombinant strains of enzyme producers based on the K. phaffii genome. (a) _target gene cloning; (b) analysis of recombinant clones for the presence of protein and enzyme activity; (c) electrophoretic analysis of the mannanase preparation from the culture liquid of recombinant clone; (d) analysis accumulation of biomass and protein during cultivation of selected clones in a bioreactor; (e) full genomic sequencing of the nucleotide sequence of the genome of the strain producing the _target protein; (f) analysis of the culture fluid of the producer line (general view of the MS1 mass spectrogram containing the peptide mixture obtained from the culture liquid); (g) the fragment peptide sequence founding in culture fluid samples of the recombinant strain K. phaffii T07 identifying the structure with the specified putative amino acid sequence with a convergence of 58.22%; (h) protein and enzyme preparations by tangential diffusion and liquid chromatography methods; (i) characteristics of purified and lyophilized proteinase K, dependence of enzyme activity on pH and temperature. Adapted from [246,330,331,332].
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