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.

Evaluation of two Plasmodium vivax sexual stage antigens as transmission-blocking vaccine candidates

Background Plasmodium vivax transmission-blocking vaccines (TBVs) are receiving increasing attention. Based on excellent transmission-blocking activities of the PbPH (PBANKA_0417200) and PbSOP26 (PBANKA_1457700) antigens in Plasmodium berghei, their orthologs in P. vivax, PVX_098655 (PvPH) and PVX_101120 (PvSOP26), were selected for the evaluation of their potential as TBVs. Methods Fragments of PvPH (amino acids 22–304) and PvSOP26 (amino acids 30–272) were expressed in the yeast expression system. The recombinant proteins were used to immunize mice to obtain antisera. The transmission-reducing activities of these antisera were evaluated using the direct membrane feeding assay (DMFA) using Anopheles dirus mosquitoes and P. vivax clinical isolates. Results The recombinant proteins PvPH and PvSOP26 induced robust antibody responses in mice. The DMFA showed that the anti-PvSOP26 sera significantly reduced oocyst densities by 92.0 and 84.1% in two parasite isolates, respectively, whereas the anti-PvPH sera did not show evident transmission-reducing activity. The variation in the DMFA results was unlikely due to the genetic polymorphisms of the two genes since their respective sequences were identical in the clinical P. vivax isolates. Conclusion PvSOP26 could be a promising TBV candidate for P. vivax, which warrants further evaluation. Graphical Abstract

The functional expression in yeast of two unusual acidic peroxygenases from Candolleomyces aberdarensis by adopting evolved secretion mutations

Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression in yeast of two new unusual acidic peroxygenases from Candolleomyces ( Psathyrella ) aberdarensis ( Pab UPO) and their production at large scale in bioreactor. Our strategy was based on adopting secretion mutations from Agrocybe aegerita UPO mutant −PaDa-I variant− designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as Pab UPOs –long-type UPOs− and that shares 65% sequence identity. After replacing the native signal peptides by the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries yielding two recombinant Pab UPOs with expression levels of 5.4 and 14.1 mg/L in S. cerevisiae . These variants were subsequently transferred to P. pastoris for overproduction in fed-batch bioreactor, boosting expression levels up to 290 mg/L with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/L, with veratryl alcohol as substrate). With a broad pH activity profile, ranging from 2.0 to 9.0, these highly secreted, active and stable peroxygenases are promising tools for future engineering endeavors, as well as for their direct application in different industrial and environmental settings. IMPORTANCE In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction in a ∼300 mg/L scale, is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.

Genome-wide identification and expression analysis of aquaporin family in Canavalia rosea and their roles in the adaptation to saline-alkaline soils and drought stress

Background Canavalia rosea (Sw.) DC. (bay bean) is an extremophile halophyte that is widely distributed in coastal areas of the tropics and subtropics. Seawater and drought tolerance in this species may be facilitated by aquaporins (AQPs), channel proteins that transport water and small molecules across cell membranes and thereby maintain cellular water homeostasis in the face of abiotic stress. In C. rosea, AQP diversity, protein features, and their biological functions are still largely unknown. Results We describe the action of AQPs in C. rosea using evolutionary analyses coupled with promoter and expression analyses. A total of 37 AQPs were identified in the C. rosea genome and classified into five subgroups: 11 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, 11 Nod26-like intrinsic proteins, 4 small and basic intrinsic proteins, and 1 X-intrinsic protein. Analysis of RNA-Seq data and targeted qPCR revealed organ-specific expression of aquaporin genes and the involvement of some AQP members in adaptation of C. rosea to extreme coral reef environments. We also analyzed C. rosea sequences for phylogeny reconstruction, protein modeling, cellular localizations, and promoter analysis. Furthermore, one of PIP1 gene, CrPIP1;5, was identified as functional using a yeast expression system and transgenic overexpression in Arabidopsis. Conclusions Our results indicate that AQPs play an important role in C. rosea responses to saline-alkaline soils and drought stress. These findings not only increase our understanding of the role AQPs play in mediating C. rosea adaptation to extreme environments, but also improve our knowledge of plant aquaporin evolution more generally.

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.

Divalent metal transporter-related protein restricts animals to marine habitats

Utilization and regulation of metals from seawater by marine organisms are important physiological processes. To better understand metal regulation, we searched the crown-of-thorns starfish genome for the divalent metal transporter (DMT) gene, a membrane protein responsible for uptake of divalent cations. We found two DMT-like sequences. One is an ortholog of vertebrate DMT, but the other is an unknown protein, which we named DMT-related protein (DMTRP). Functional analysis using a yeast expression system demonstrated that DMT transports various metals, like known DMTs, but DMTRP does not. In contrast, DMTRP reduced the intracellular concentration of some metals, especially zinc, suggesting its involvement in negative regulation of metal uptake. Phylogenetic distribution of the DMTRP gene in various metazoans, including sponges, protostomes, and deuterostomes, indicates that it originated early in metazoan evolution. However, the DMTRP gene is only retained in marine species, and its loss seems to have occurred independently in ecdysozoan and vertebrate lineages from which major freshwater and land animals appeared. DMTRP may be an evolutionary and ecological limitation, restricting organisms that possess it to marine habitats, whereas its loss may have allowed other organisms to invade freshwater and terrestrial habitats.

Evaluation of two Plasmodium vivax sexual-stage antigens as transmission-blocking vaccine candidates

Background: Plasmodium vivax transmission-blocking vaccines (TBVs) have received high attention. PVX_098655 (PvPH) and PVX_101120 (PvSOP26) were predicted to be potential TBV antigens based on the studies of their orthologs in Plasmodium berghei. Methods: Fragments of PvPH (amino acids 22–304) and PvSOP26 (amino acids 30–272) were expressed in the yeast expression system. The recombinant proteins were used to immunize mice to obtain the antisera. The transmission-reducing activities of these antisera were evaluated using the standard membrane feeding assay (SMFA) using Anopheles dirus mosquitoes and P. vivax clinical isolates. Results: The recombinant proteins PvPH and PvSOP26 induced robust antibody responses in mice. With SMFA, the anti-PvSOP26 sera significantly reduced oocyst densities by 92.0% and 84.1% in two parasite isolates, while the anti-PvPH sera did not show evident transmission-reducing activity. Both PvPH and PvSOP26 showed limited gene polymorphisms in the clinical P. vivax isolates. Conclusion: PvSOP26 could be a promising TBV candidate for P. vivax.

Post-transcriptional regulation of glutamate metabolism of Pichia pastoris and development of a glutamate-inducible yeast expression system

Pichia pastoris harbours a unique glutamate utilization pathway in which glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the conversion of glutamate to α-ketoglutarate, oxaloacetate, and phosphoenolpyruvate respectively in the cytosol. GDH2 and PEPCK are glutamate-inducible enzymes and their synthesis is regulated post-transcriptionally by Rtg1p, a cytosolic basic helix-loop-helix protein via Rtg1p response elements located downstream of TATA box of GDH2 and PEPCK promoters. Glutamate-inducible synthesis of PEPCK is abrogated in Δgdh2 and Δaat2. α-ketoglutarate induces PEPCK synthesis in Δgdh2 but not Δaat2. We propose that oxaloacetate derived from glutamate is the inducer of PEPCK synthesis. Enzymes of glutamate utilization pathway are synthesized during carbon starvation and they enable P. pastoris to overcome nutritional stress. Finally, green fluorescent protein can be synthesized efficiently from GDH2 and PEPCK promoters using food-grade monosodium glutamate as inducer indicating that the post-transcriptional regulatory circuit described here can be exploited for the development of glutamate-inducible P. pastoris expression system.

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation

Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.