Rewiring of metabolic pathways in yeasts for sustainable production of biofuels.

The ever-increasing global energy demand has led world towards negative repercussions such as depletion of fossil fuels, pollution, global warming and climate change. Designing microbial cell factories for the sustainable production of biofuels is therefore an active area of research. Different yeast cells have been successfully engineered using synthetic biology and metabolic engineering approaches for the production of various biofuels. In the present article, recent advancements in genetic engineering strategies for production of bioalcohols, isoprenoid-based biofuels and biodiesels in different yeast chassis designs are reviewed, along with challenges that must be overcome for efficient and high titre production of biofuels.

Mutations in SARS-CoV-2 nsp7 and nsp8 proteins and their predicted impact on replication/transcription complex structure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) has been identified to be a mutation hot spot, with the P323L mutation being commonly observed in viral genomes isolated from North America. RdRp forms a complex with nonstructural proteins nsp7 and nsp8 to form the minimal replication/transcription machinery required for genome replication. As mutations in RdRp may affect formation of the RdRp-nsp7-nsp8 supercomplex, we analyzed viral genomes to identify mutations in nsp7 and nsp8 protein sequences. Based on in silico analysis of predicted structures of the supercomplex comprising of native and mutated proteins, we demonstrate that specific mutations in nsp7 and nsp8 proteins may have a role in stabilization of the replication/transcription complex.

Utilization of Bacillus subtilis cells displaying a glucose-tolerant ß-glucosidase for whole-cell biocatalysis.

The microbial production of industrial enzymes requires a large number of complex biochemical steps for purification which increases their production cost. Additionally, poor thermo-stability of the purified enzymes under the operational conditions along with the challenges in their recovery and subsequent reuse, limit their usage in an industrial bioprocess. Surface display of heterologous enzymes on bacterial cells appear to be a suitable alternative. Bacillus subtilis, the most well characterized Gram-positive bacterium, is being increasingly studied as a host for surface display. We displayed a glucose-tolerant ß-glucosidase (UnBgl1A) on the surface of B. subtilis cells using CWBb as the anchor protein. These cells displaying UnBgl1A (SD-01) were directly employed for biocatalysis without cell lysis and enzyme purification. The SD-01 cells elicited ∼2 times more catalytic activity compared to the cells expressing the enzyme intracellularly (IN-01). The displayed enzyme and the purified enzyme elucidated similar glucose tolerance (IC50 ∼0.9 M glucose), temperature optima (∼50 °C), and pH optima (∼6.0). The surface displayed UnBgl1A retained ∼50% activity after 4 h when stored at 50 °C whereas the purified UnBgl1A lost all its activity by the 4th hour. Additionally, the SD-01 cells could be efficiently reused for 3 sets of reactions. Further, supplementation of a cellulase cocktail with the cells of the SD-01 strain resulted in ∼2 times more glucose release from sugarcane bagasse compared to supplementation with the purified UnBgl1A. Therefore, displaying enzymes on the B. subtilis cell surface could be an attractive platform for the commercial production of industrial enzymes.

Evaluation of autoantibody signatures in meningioma patients using human proteome arrays.

Meningiomas are one of the most common tumors of the Central nervous system (CNS). This study aims to identify the autoantibody biomarkers in meningiomas using high-density human proteome arrays (~17,000 full-length recombinant human proteins). Screening of sera from 15 unaffected healthy individuals, 10 individuals with meningioma grade I and 5 with meningioma grade II was performed. This comprehensive proteomics based investigation revealed the dysregulation of 489 and 104 proteins in grades I and II of meningioma, respectively, along with the enrichment of several signalling pathways, which might play a crucial role in the manifestation of the disease. Autoantibody targets like IGHG4, CRYM, EFCAB2, STAT6, HDAC7A and CCNB1 were significantly dysregulated across both the grades. Further, we compared this to the tissue proteome and gene expression profile from GEO database. Previously reported upregulated proteins from meningioma tissue-based proteomics obtained from high-resolution mass spectrometry demonstrated an aggravated autoimmune response, emphasizing the clinical relevance of these targets. Some of these targets like SELENBP1 were tested for their presence in tumor tissue using immunoblotting. In the light of highly invasive diagnostic modalities employed to diagnose CNS tumors like meningioma, these autoantibody markers offer a minimally invasive diagnostic platform which could be pursued further for clinical translation.

Prediction of Loops in G Protein-Coupled Receptor Homology Models: Effect of Imprecise Surroundings and Constraints.

In the present study, we explored the extent to which inaccuracies inherent in homology models of the transmembrane helical cores of G protein-coupled receptors (GPCRs) can impact loop prediction. We demonstrate that loop prediction in homology models is much more difficult than loop reconstruction in crystal structures because of the imprecise positioning of loop anchors. Deriving information from 17 recently available GPCR crystal structures, we estimated all of the possible errors that could occur in loop anchors as the result of comparative modeling. Subsequently, we performed an exhaustive analysis to decipher the effect of these errors on loop modeling using ICM High Precision Sampling. The influence of the presence of other extracellular loops was also explored. Our results reveal that the error space of modeled loop residues is much larger than that of the anchor residues, although modeling a particular extracellular loop in the presence of other extracellular loops provides constraints that help in predicting near-native loop conformations observed in crystal structures. This implies that errors in loop anchor positions introduce increased uncertainty in the modeled loop coordinates. Therefore, for the success of any GPCR structure prediction algorithm, minimizing errors in the helical end points is likely to be critical for successful loop modeling.

Evaluation of risedronate as an antibiofilm agent.

Escherichia coli cra null mutants have been reported in the literature to be impaired in biofilm formation. To develop E. coli biofilm-inhibiting agents for prevention and control of adherent behaviour, analogues of a natural Cra ligand, fructose-1,6-bisphosphate, were identified based on two-dimensional similarity to the natural ligand. Of the analogues identified, those belonging to the bisphosphonate class of drug molecules were selected for study, as these are approved for clinical use in humans and their safety has been established. Computational and in vitro studies with purified Cra protein showed that risedronate sodium interacted with residues in the fructose-1,6-bisphosphate-binding site. Using a quantitative biofilm assay, risedronate sodium, at a concentration of 300-400 µM, was found to decrease E. coli and Salmonella pullorum biofilm formation by >60 %. Risedronate drastically reduced the adherence of E. coli cells to a rubber Foley urinary catheter, demonstrating its utility in preventing the formation of biofilm communities on medical implant surfaces. The use of risedronate, either alone or in combination with other agents, to prevent the formation of biofilms on surfaces is a novel finding that can easily be translated into practical applications.

Autoantibody Profiling of Glioma Serum Samples to Identify Biomarkers Using Human Proteome Arrays.

The heterogeneity and poor prognosis associated with gliomas, makes biomarker identification imperative. Here, we report autoantibody signatures across various grades of glioma serum samples and sub-categories of glioblastoma multiforme using Human Proteome chips containing ~17000 full-length human proteins. The deduced sets of classifier proteins helped to distinguish Grade II, III and IV samples from the healthy subjects with 88, 89 and 94% sensitivity and 87, 100 and 73% specificity, respectively. Proteins namely, SNX1, EYA1, PQBP1 and IGHG1 showed dysregulation across various grades. Sub-classes of GBM, based on its proximity to the sub-ventricular zone, have been reported to have different prognostic outcomes. To this end, we identified dysregulation of NEDD9, a protein involved in cell migration, with probable prognostic potential. Another subcategory of patients where the IDH1 gene is mutated, are known to have better prognosis as compared to patients carrying the wild type gene. On a comparison of these two cohorts, we found STUB1 and YWHAH proteins dysregulated in Grade II glioma patients. In addition to common pathways associated with tumourigenesis, we found enrichment of immunoregulatory and cytoskeletal remodelling pathways, emphasizing the need to explore biochemical alterations arising due to autoimmune responses in glioma.

Engineering of yeast pyruvate decarboxylase for enhanced selectivity towards carboligation.

The aim of the study was to increase production of (R)-PAC by altering carboligation activity of Pdc in Saccharomyces cerevisiae. Pdc1 activity was modified by over-expression as well as changing the rate of decarboxylation and carboligation by site specific mutation in Pdc1. Over-expression of mutant Pdc1 resulted in 50 ± 2.5% increase in levels of (R)-PAC in wild-type and further 30-40% in pdc null background. The combination of mutant Pdc1 in pdc null background was successfully evaluated for production of (R)-PAC at industrial scale. This is the first report of enhancing (R)-PAC product in yeast by recombinant technology with capability of commercial production.

(R)-PAC biosynthesis in [BMIM][PF6]/aqueous biphasic system using Saccharomyces cerevisiae BY4741 cells.

(R)-phenylacetylcarbinol or (R)-PAC is a pharmaceutical precursor of (1R, 2S) ephedrine and (1S, 2S) pseudoephedrine. Biotransformation of benzaldehyde and glucose by pyruvate decarboxylase produces (R)-PAC. This biotransformation suffers from toxicity of the substrate, product [(R)-PAC] and by-product (benzyl alcohol). In the present study, ionic liquid/aqueous biphasic system was employed to enhance (R)-PAC production. Fermented broth was the reaction medium in which Saccharomyces cerevisiae BY4741 was the source of pyruvate decarboxylase. Hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) was the non-aqueous phase in which toxic compounds reside. Biocompatibility of [BMIM][PF6] and adequate distribution coefficients of benzaldehyde, (R)-PAC and benzyl alcohol were determined. A Box-Behnken design and response surface methodology were used for the optimization of biotransformation variables in order to maximize (R)-PAC yield and productivity. The results showed higher (R)-PAC yield and productivity of ∼1.5-fold each in the biphasic biotransformation of phase volume ratio 0.05 as compared to the monophasic (conventional) biotransformation. Moreover, the level of major by-product benzyl alcohol was also 3.5-fold lower in biphasic biotransformation. [BMIM][PF6]/aqueous biphasic system is a new approach which could intensify the (R)-PAC production.

Production, characterization, and immunogenicity of a secreted form of Plasmodium falciparum merozoite surface protein 4 produced in Bacillus subtilis.

Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.

Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris).

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.

Co-production of two recombinant proteins in Escherichia coli using the promoter region of the divergently expressed curli genes.

Expression of multiple proteins in a single host is desirable in biotechnological processes. The curli intergenic region in Escherichia coli contains promoter elements for the expression of the divergent csgBAC and csgDEFG operons. Using this bidirectionally active promoter region, we demonstrate high level production of two different recombinant proteins. The curli intergenic region may thus be used to produce multiple enzymes involved in biosynthetic pathways and biotransformations in a cost-effective manner.

Biofilm formation in Escherichia coli cra mutants is impaired due to down-regulation of curli biosynthesis.

Cra is a pleiotropic regulatory protein that controls carbon and energy flux in enteric bacteria. Recent studies have shown that Cra also regulates other cell processes and influences biofilm formation. The purpose of the present study was to investigate the role of Cra in biofilm formation in Escherichia coli. Congo red-binding studies suggested that curli biosynthesis is impaired in cra mutants. Microarray analysis of wild-type and mutant E. coli cultivated in conditions promoting biofilm formation revealed that the curli biosynthesis genes, csgBAC and csgDEFG, are poorly expressed in the mutant, suggesting that transcription of genes required for curli production is regulated by Cra. Four putative Cra-binding sites were identified in the curli intergenic region, which were experimentally validated by performing electromobility shift assays. Site-directed mutagenesis of three Cra-binding sites in the promoter region of the csgDEFG operon suggests that Cra activates transcription of this operon upon binding to operator regions both downstream and upstream of the transcription start site. Based on the Cra-binding sites identified in this and other studies, the Cra consensus sequence is refined.

Analyzing metabolic variations in different bacterial strains, historical perspectives and current trends--example E. coli.

The analysis of metabolic differences in bacterial strains is a useful tool for the development of strains with desired growth and production properties. Several methods are available for the evaluation and understanding of the differences: Biochemical methods to measure metabolites concentration and enzyme activity, mathematical methods to analyze metabolic fluxes through the various pathways, proteomic methods to identify expressed proteins, and genomic methods to detect and measure gene expression. A combination of the various methods is required to obtain a comprehensive understanding of metabolic activities. The genomic methods provide substantial amount information on global gene expression but do not always reflect the actual activity of the individual components. The review focuses on the different methodologies and their use, as well as historical overview of the evaluation of the differences between Escherichia coli K and E. coli B.

Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses.

In a series of previous reports it was established by implementing metabolic flux, NMR/MS, and Northern blot analysis that the glyoxylate shunt, the TCA cycle, and acetate uptake by acetyl-CoA synthetase are more active in Escherichia coli BL21 than in Escherichia coli JM109. These differences were accepted as the reason for the differences in the glucose metabolism and acetate excretion of these two strains. Examination of the bacterial metabolism by microarrays and time course Northern blot showed that in addition to the glyoxylate shunt, the TCA cycle and the acetate uptake, other metabolic pathways are active differently in the two strains. These are gluconeogenesis, sfcA shunt, ppc shunt, glycogen biosynthesis, and fatty acid degradation. It was found that in E. coli JM109, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase-acetate kinase (Pta-AckA) system which uses acetyl-CoA. The inactivation of the gluconeogenesis enzyme phosphoenolpyruvate synthetase (ppsA), the activation of the anaplerotic sfcA shunt, and low and stable pyruvate dehydrogenase (aceE, aceF) cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs, and aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose concentration was high, no transcription of these genes was observed and acetate concentration increased, but at low glucose concentrations these genes were expressed and the acetate concentration decreased. It is possible that there is a major regulatory molecule that controls not only ppsA and aceBAK but also acs. The gluconeogenesis pathway (fbp, pckA, and ppsA) which leads to glycogen accumulation is constitutively active in E. coli BL21 regardless of glucose feeding strategy.