The potential of R. toruloides mevalonate pathway genes in increasing isoprenoid yields in S. cerevisiae: Evaluation of GGPPS and HMG-CoA reductase.

The enzymes of the mevalonate pathway need to be improved to achieve high yields of isoprenoids in the yeast Saccharomyces cerevisiae. The red yeast Rhodosporidium toruloides produces high levels of carotenoids and may have evolved to carry a naturally high flux of isoprenoids. Enzymes from such yeasts are likely to be promising candidates for improvement. Towards this end, we have systematically investigated the various enzymes of the mevalonate pathway of R. toruloides and custom synthesized, expressed, and evaluated six key enzymes in S. cerevisiae. The two nodal enzymes geranyl pyrophosphate synthase (RtGGPPS) and truncated HMG-CoA reductase (RttHMG) of R. toruloides showed a significant advantage to the cells for isoprenoid production as seen by a visual carotenoid screen. These two were analyzed further, and attempts were also made at further improvement. RtGGPPS was confirmed to be superior to the S. cerevisiae enzyme, as seen from in vitro activity determinations and in vivo production of the heterologous diterpenoid sclareol. Four mutants were created through rational mutagenesis but were unable to improve the activity further. In the case of RttHMG, functional evaluation of the enzyme revealed that it was very unstable despite functioning very well in S. cerevisiae. We succeeded in stabilizing the enzyme through mutation of a conserved serine in the catalytic region, which did not alter the enzyme activity per se. In vivo evaluation of the mutant revealed that it could enable better sclareol yields. Therefore, these two enzymes from the red yeast are excellent candidates for heterologous isoprenoid production.

Designing dual inhibitors against potential drug targets of Plasmodium falciparum -M17 Leucyl Aminopeptidase and Plasmepsins.

Malaria is one of the major diseases of concern worldwide, especially in the African regions. According to a recent WHO report, 95% of deaths that occur due to malaria are in the African regions. Resistance to present antimalarial drugs is increasing rapidly and becoming a problem of concern. M17 Leucyl Aminopeptidase (PfM17LAP) and vacuolar Plasmepsins (PfPM) are two important enzymes involved in the haemoglobin degradation pathway of Plasmodium falciparum. PfM17LAP regulates the release of amino acids and PfPM mediates the conversion of haemoglobin proteins to oligopeptides. These enzymes thus play an essential role in the survival of malaria parasites inside the human body. In the present study, we used in-silico molecular docking, simulation and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) studies to find potential dual inhibitors of PfPM and PfM17LAP using the ChEMBL antimalarial library. Absorption, distribution, metabolism, excretion and toxicity (ADMET) profiling of the top ten ranked molecules was done using the BIOVIA Discovery Studio. The present investigation revealed that the compound CHEMBL426945 is stable in the binding site of both PfPM and PfM17LAP. In this study, we have reported novel dual-inhibitors that may act better than the present antimalarial drugs.Communicated by Ramaswamy H. Sarma.

Investigating the disordered regions (MoRFs, SLiMs and LCRs) and functions of mimicry proteins/peptides in silico.

Microbial mimicry of the host proteins/peptides can elicit host auto-reactive T- or B-cells resulting in autoimmune disease(s). Since intrinsically disordered protein regions (IDPRs) are involved in several host cell signaling and PPI networks, molecular mimicry of the IDPRs can help the pathogens in substituting their own proteins in the host cell-signaling and PPI networks and, ultimately hijacking the host cellular machinery. Thus, the present study was conducted to discern the structural disorder and intrinsically disordered protein regions (IDPRs) like, molecular recognition features (MoRFs), short linear motifs (SLiMs), and low complexity regions (LCRs) in the experimentally verified mimicry proteins and peptides (mimitopes) of bacteria, viruses and host. Also, functional characteristics of the mimicry proteins were studied in silico. Our results indicated that 78% of the bacterial host mimicry proteins and 45% of the bacterial host mimitopes were moderately/highly disordered while, 73% of the viral host mimicry proteins and 31% of the viral host mimitopes were moderately/highly disordered. Among the pathogens, 27% of the bacterial mimicry proteins and 13% of the bacterial mimitopes were moderately/highly disordered while, 53% of the viral mimicry proteins and 21% of the viral mimitopes were moderately/highly disordered. Though IDPR were frequent in host, bacterial and viral mimicry proteins, only a few mimitopes overlapped with the IDPRs like, MoRFs, SLiMs and LCRs. This suggests that most of the microbes cannot use molecular mimicry to modulate the host PPIs and hijack the host cell machinery. Functional analyses indicated that most of the pathogens exhibited mimicry with the host proteins involved in ion binding and signaling pathways. This is the first report on the disordered regions and functional aspects of experimentally proven host and microbial mimicry proteins.

Heterologous expression of novel SUMO proteases from Schizosaccharomyces pombe in E. coli: Catalytic domain identification and optimization of product yields.

Small ubiquitin-related modifier (SUMO) proteins are efficiently used to target the soluble expression of various difficult-to-express proteins in E. coli. However, its utilization in large scale protein production is restricted by the higher cost of Ulp, which is required to cleave SUMO fusion tag from protein-of-interest to generate an authentic N-terminus. This study identified and characterized two novel SUMO proteases i.e., Ulp1 and Ulp2 from Schizosaccharomyces pombe. Codon-optimized gene sequences were cloned and expressed in E. coli. The sequence and structure of SpUlp1 and SpUlp2 catalytic domains were deduced using bioinformatics tools. Protein-protein interaction studies predicted the higher affinity of SpUlp1 towards SUMO compared to its counterpart from Saccharomyces cerevisiae (ScUlp1). The catalytic domain of SpUlp1 was purified using Ni-NTA chromatography with 83.33% recovery yield. Moreover, In vitro activity data further confirmed the fast-acting nature of SpUlp1 catalytic domain, where a 90% cleavage of fusion proteins was obtained within 1 h of incubation, indicating novelty and commercial relevance of S. pombe Ulp1. Biophysical characterization showed 8.8% α-helices, 36.7% ß-sheets in SpUlp1SD. From thermal CD and fluorescence data, SpUlp1SD Tm was found to be 45 °C. Further, bioprocess optimization using fed-batch cultivation resulted in 3.5 g/L of SpUlp1SD production with YP/X of 77.26 mg/g DCW and volumetric productivity of 205.88 mg/L/h.