As one of the most popular expression systems, recombinant protein expression in Pichia pastoris relies on the AOX1 promoter (P AOX1 ) which is strongly induced by methanol. However, the toxic and inflammatory nature of methanol restricts its application, especially in edible and medical products. Therefore, constructing a novel methanol-free system becomes necessary. The kinases involved in P AOX1 activation or repression by different carbon sources may be promising targets. We identified two kinase mutants: Δgut1 and Δdak, both of which showed strong alcohol oxidase activity under non-methanol carbon sources. Based on these two kinases, we constructed two methanol-free expression systems: Δgut1-HpGCY1-glycerol (P AOX1 induced by glycerol) and Δdak-DHA (P AOX1 induced by DHA). By comparing their GFP expression efficiencies, the latter one showed better potential. To further test the Δdak-DHA system, three more recombinant proteins were expressed as examples. We found that the expression ability of our novel methanol-free Δdak-DHA system was generally better than the constitutive GAP promoter, and reached 50–60 % of the traditional methanol induced system. We successfully constructed a novel methanol-free expression system Δdak-DHA. This modified expression platform preserved the favorable regulatable nature of P AOX1 , providing a potential alternative to the traditional system.

Background: Protein synthesis is one of the extremely important anabolic pathways in the yeast expression system Pichia pastoris. Codon optimization is a commonly adopted strategy for improved protein expression, although unexpected failures did appear sometimes waiting for further exploration. Recently codon bias has been studied to regulate protein folding and activity in many other organisms. Results: Here the codon bias profile of P. pastoris genome was examined first and a direct correlation between codon translation efficiency and usage frequency was identified. By manipulating the codon choices of both endogenous and heterologous signal peptides, secretion abilities of N-terminal signal peptides were shown to be tolerant towards codon changes. Then two gene candidates with different levels of structural disorder were studied, and full-length codon optimization was found to affect their expression profiles differentially. Finally, more evidences were provided to support possible protein conformation change brought by codon optimization in structurally disordered proteins. Conclusion: Our results suggest that codon bias regulates gene expression by modulating several factors including transcription and translation efficiency, protein folding and activity. Because of sequences difference, the extent of affection may be gene specific. For some genes, special codon optimization strategy should be adopted to ensure appropriate expression and conformation.

In many prokaryotes but limited eukaryotic species, the combination of transposon mutagenesis and high-throughput sequencing has greatly accelerated the identification of essential genes. Here we successfully applied this technique to the methylotrophic yeast Pichia pastoris and classified its conditionally essential/non-essential gene sets. Firstly, we showed that two DNA transposons, TcBuster and Sleeping beauty, had high transposition activities in P. pastoris. By merging their insertion libraries and performing Tn-seq, we identified a total of 202,858 unique insertions under glucose supported growth condition. We then developed a machine learning method to classify the 5,040 annotated genes into putatively essential, putatively non-essential, ambig1 and ambig2 groups, and validated the accuracy of this classification model. Besides, Tn-seq was also performed under methanol supported growth condition and methanol specific essential genes were identified. The comparison of conditionally essential genes between glucose and methanol supported growth conditions helped to reveal potential novel targets involved in methanol metabolism and signaling. Our findings suggest that transposon mutagenesis and Tn-seq could be applied in the methylotrophic yeast Pichia pastoris to classify conditionally essential/non-essential gene sets. Our work also shows that determining gene essentiality under different culture conditions could help to screen for novel functional components specifically involved in methanol metabolism.

As one of the most commonly used eukaryotic recombinant protein expression systems, P. pastoris relies heavily on the AOX1 promoter (P_AOX1), which is strongly induced by methanol but strictly repressed by glycerol and glucose. However, the complicated signaling pathways involved in P_AOX1 regulation when supplemented with different carbon sources are poorly understood. Here we constructed a kinase deletion library in P. pastoris and identified 27 mutants which showed peculiar phenotypes in cell growth or P_AOX1 regulation. We analyzed both annotations and possible functions of these 27 targets, and then focused on the MAP kinase Hog1. In order to locate its potential downstream components, we performed the phosphoproteome analysis on glycerol cultured WT and Δhog1 strains and identified 157 differentially phosphorylated proteins. Our results identified important kinases involved in P. pastoris cell growth and P_AOX1 regulation, which could serve as valuable targets for further mechanistic studies.