Determination of Putative Vacuolar Proteases, PEP4 and PRB1 in a Novel Yeast Expression Host Meyerozyma guilliermondii Strain SO Using Bioinformatics Tools

Meyerozyma guilliermondii strain SO, a newly isolated yeast species from spoilt orange, has been used as a host to express the recombinant proteins using methylotrophic yeast promoters. However, as a novel yeast expression system, the vacuolar proteases of this yeast have not been determined, which may have contributed to the low level of heterologous protein secretions. Thus, this study aimed to determine intra- and extracellular proteolytic activity and identify the putative vacuolar proteases using bioinformatics techniques. A clear zone was observed from the nutrient agar skimmed milk screening plate. Proteolytic activity of 117.30 U/ml and 75 U/ml were obtained after 72 h of cultivation for both extracellular and intracellular proteins, respectively. Next, the Hidden Markov model (HMM) was used to detect the presence of the vacuolar proteases (PEP4 and PRB1) from the strain SO proteome. Aspartyl protease (PEP4) with 97.55% identity to Meyerozyma sp. JA9 and a serine protease (PRB1) with 70.91% identity to Candida albicans were revealed. The homology with other yeast vacuolar proteases was confirmed via evolutionary analysis. PROSPER tool prediction of cleavage sites postulated that PEP4 and PRB1 might have caused proteolysis of heterologous proteins in strain SO. In conclusion, two putative vacuolar proteases (PEP4 and PRB1) were successfully identified in strain SO. Further characterization can be done to understand their specific properties, and their effects on heterologous protein expression can be conducted via genome editing.

Advantages of yeast-based recombinant protein technology as vaccine products against infectious diseases

The yeast expression system is widely used to produce functional recombinant proteins in the biopharmaceutical industry, such as vaccine products. The expression system choices using yeast as the host has many advantages. Various vaccines have been produced commercially using yeast expression systems. This review aims to explore the advantages of the yeast expression system in Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha, which emphasize vaccine products to prevent human infectious diseases. Selection of the appropriate expression system is carried out by identification at the genetic and fermentation levels, taking into account host features, vectors and expression strategies. We also demonstrate the development of a yeast expression system that can produce recombinant proteins, virus-like particles and yeast surface displays as a novel vaccine strategy against infectious diseases. The recombinant protein produced as a vaccine in the yeast system is cost-effective, immunogenic, and safe. In addition, this system has not introduced new microbe variants in nature that will be safe for the environment. Thus, it has the potential to become a commercial product used in vaccination programs to prevent human infectious diseases.

Codon usage bias regulates gene expression and protein conformation in yeast expression system P. pastoris

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.

Discovery and evaluation of a novel step in the flavonoid biosynthesis pathway regulated by F3H gene using a yeast expression system

Kaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. These metabolites are produced in low quantities in plants, therefore engineering microbial factory is a favorable strategy for the production of these metabolites. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in Saccharomyces cerevisiae using naringenin as a substrate. The results elucidated a novel step for the first time in kaempferol and quercetin biosynthesis directly from naringenin catalyzed by flavonol 3-hydroxylase (F3H). F3H gene from rice was cloned into pRS42K yeast episomal plasmid (YEP) vector using BamH1 and Xho1 restriction enzymes. We analyzed our target gene activity in engineered and in empty strains. The results were confirmed through TLC followed by Western blotting, nuclear magnetic resonance (NMR), and LC-MS. TLC showed positive results on comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed lack of Oryza sativa flavonol 3-hydroxylase (OsF3H) activity in empty strains while high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for naringenin conversion to both kaempferol and quercetin. These results concluded that rice F3H catalyzes naringenin metabolism via hydroxylation and synthesizes kaempferol and quercetin.HighlightsCurrent study is a discovery of a novel step in flavonoid biosynthesis pathway of rice plant.In this study F3H gene from rice plant was functionally expressed in yeast expression system.Results confirmed that, F3H gene is responsible for the canalization of naringenin and converted into kaempferol and quercetin.The results were confirmed through, western blotting, TLC, HPLC and NMR analysis.