Syed Shams Yazdani

Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals.

ABSTRACT Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ± 0.003 h−1. The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-13C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-13C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (∼95%) and redox (∼96%) balances. On the other hand, cultivation conditions that prevent H2 accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H2 is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.

Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co-products.

Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for producing reduced compounds via anaerobic fermentation. We recently identified environmental conditions enabling the fermentative metabolism of glycerol in E. coli, along with the pathways and mechanisms mediating this metabolic process. In this work, we used the knowledge base created in previous studies to engineer E. coli for the efficient conversion of crude glycerol to ethanol. Our strategy capitalized on the high degree of reduction of carbon in glycerol, thus enabling the production of not only ethanol but also co-products hydrogen and formate. Two strains were created for the co-production of ethanol-hydrogen and ethanol-formate: SY03 and SY04, respectively. High ethanol yields were achieved in both strains by minimizing the synthesis of by-products succinate and acetate through mutations that inactivated fumarate reductase (DeltafrdA) and phosphate acetyltransferase (Deltapta), respectively. Strain SY04, which produced ethanol-formate, also contained a mutation that inactivated formate-hydrogen lyase (DeltafdhF), thus preventing the conversion of formate to CO(2) and H(2). High rates of glycerol utilization and product synthesis were achieved by simultaneous overexpression of glycerol dehydrogenase (gldA) and dihydroxyacetone kinase (dhaKLM), which are the enzymes responsible for the conversion of glycerol to glycolytic intermediate dihydroxyacetone phosphate. The resulting strains, SY03 (pZSKLMgldA) and SY04 (pZSKLMgldA), produced ethanol-hydrogen and ethanol-formate from unrefined glycerol at yields exceeding 95% of the theoretical maximum and specific rates in the order of 15-30 mmol/gcell/h. These yields and productivities are superior to those reported for the conversion of glycerol to ethanol-H(2) or ethanol-formate by other organisms and equivalent to those achieved in the production of ethanol from sugars using E. coli.

A new model for the anaerobic fermentation of glycerol in enteric bacteria: Trunk and auxiliary pathways in Escherichia coli

Anaerobic fermentation of glycerol in the Enterobacteriaceae family has long been considered a unique property of species that synthesize 1,3-propanediol (1,3-PDO). However, we have discovered that Escherichia coli can ferment glycerol in a 1,3-PDO-independent manner. We identified 1,2-propanediol (1,2-PDO) as a fermentation product and established the pathway that mediates its synthesis as well as its role in the metabolism of glycerol. We also showed that the trunk pathway responsible for the conversion of glycerol into glycolytic intermediates is composed of two enzymes: a type II glycerol dehydrogenase (glyDH-II) and a dihydroxyacetone kinase (DHAK), the former of previously unknown physiological role. Based on our findings, we propose a new model for glycerol fermentation in enteric bacteria in which: (i) the production of 1,2-PDO provides a means to consume reducing equivalents generated in the synthesis of cell mass, thus facilitating redox balance, and (ii) the conversion of glycerol to ethanol, through a redox-balanced pathway, fulfills energy requirements by generating ATP via substrate-level phosphorylation. The activity of the formate hydrogen-lyase and F(0)F(1)-ATPase systems were also found to facilitate the fermentative metabolism of glycerol, and along with the ethanol and 1,2-PDO pathways, were considered auxiliary or enabling. We demonstrated that glycerol fermentation in E. coli was not previously observed due to the use of medium formulations and culture conditions that impair the aforementioned pathways. These include high concentrations of potassium and phosphate, low concentrations of glycerol, alkaline pH, and closed cultivation systems that promote the accumulation of hydrogen gas.

Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

Plasmodium vivax depends on interaction with the Duffy antigen/receptor for chemokines (DARC) for invasion of human erythrocytes. The 140 kDa P. vivax Duffy‐binding protein (PvDBP) mediates interaction with DARC. The receptor‐binding domain of PvDBP maps to its N‐terminal, cysteine‐rich region, region II (PvRII), which contains approximately 300 amino acid residues including 12 conserved cysteines. Using surface plasmon resonance, we show that binding of PvRII to DARC is a high‐affinity interaction with a binding constant (KD) of 8.7 nM. The minimal binding domain of PvRII has been previously mapped to a central 170‐amino‐acid stretch that includes cysteines 5–8. Here, we have used site‐directed mutagenesis and quantitative binding assays to map amino acid residues within PvRII that make contact with DARC. Of the seven alanine replacement mutations that had an effect on binding, five were mutations in hydrophobic residues suggesting that hydrophobic interactions play a major role in the interaction of PvDBP with DARC. Genetic diversity studies have shown that six of the seven binding residues identified in PvRII are conserved in P. vivax field isolates, which provides support for their role in interaction with DARC.

Definition of structural elements in Plasmodium vivax and P. knowlesi Duffy-binding domains necessary for erythrocyte invasion

Plasmodium vivax and P. knowlesi use the Duffy antigen as a receptor to invade human erythrocytes. Duffy-binding ligands belong to a family of erythrocyte-binding proteins that bind erythrocyte receptors to mediate invasion. Receptor-binding domains in erythrocyte-binding proteins lie in conserved cysteine-rich regions called Duffy-binding-like domains. In the present study, we report an analysis of the overall three-dimensional architecture of P. vivax and P. knowlesi Duffy-binding domains based on mild proteolysis and supportive-functional assays. Our proteolysis experiments indicate that these domains are built of two distinct subdomains. The N-terminal region from Cys-1-4 (C1-C4) forms a stable non-functional subdomain. The region spanning C5-C12 forms another subdomain, which is capable of binding Duffy antigen. These subdomains are joined by a protease-sensitive linker. Results from deletion constructs, designed for expression of truncated proteins on COS cell surface, show that regions containing C5-C8 of the Duffy-binding domains are sufficient for the binding receptor. Therefore the central region of Duffy-binding domains, which is flanked by two non-functional regions, is responsible for receptor recognition. Moreover, the minimal Duffy-binding region identified here is capable of folding into a functionally competent module. These studies pave the way for understanding the architecture of Duffy-binding domains and their interactions with host receptors.

Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein.

ABSTRACT Erythrocyte invasion by Plasmodium vivax is completely dependent on binding to the Duffy blood group antigen by the parasite Duffy binding protein (DBP). The receptor-binding domain of this protein lies within a cysteine-rich region referred to as region II (DBPII). To examine whether antibody responses to DBP correlate with age-acquired immunity to P. vivax, antibodies to recombinant DBP (rDBP) were measured in 551 individuals residing in a village endemic for P. vivax in Papua New Guinea, and linear epitopes mapped in the critical binding region of DBPII. Antibody levels to rDBPII increased with age. Four dominant linear epitopes were identified, and the number of linear epitopes recognized by semiimmune individuals increased with age, suggesting greater recognition with repeated infection. Some individuals had antibodies to rDBPII but not to the linear epitopes, indicating the presence of conformational epitopes. This occurred in younger individuals or subjects acutely infected for the first time with P. vivax, indicating that repeated infection is required for recognition of linear epitopes. All four dominant B-cell epitopes contained polymorphic residues, three of which showed variant-specific serologic responses in over 10% of subjects examined. In conclusion, these results demonstrate age-dependent and variant-specific antibody responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in populations in endemic areas.

Immunogenicity of Duffy Binding-Like Domains That Bind Chondroitin Sulfate A and Protection against Pregnancy-Associated Malaria

ABSTRACT Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is implicated in pathological outcomes of pregnancy-associated malaria (PAM). P. falciparum isolates that sequester in the placenta primarily bind chondroitin sulfate A (CSA). Following exposure to malaria during pregnancy, women in areas of endemicity develop immunity, and so multigravid women are less susceptible to PAM than primigravidae. Protective immunity to PAM is associated with the development of antibodies that recognize diverse CSA-binding, placental P. falciparum isolates. The epitopes recognized by such protective antibodies have not been identified but are likely to lie in conserved Duffy binding-like (DBL) domains, encoded by var genes, that bind CSA. Immunization of mice with the CSA-binding DBL3γ domain encoded by var1CSA elicits cross-reactive antibodies that recognize diverse CSA-binding P. falciparum isolates and block their binding to placental cryosections under flow. However, CSA-binding isolates primarily express var2CSA, which does not encode any DBLγ domains. Here, we demonstrate that antibodies raised against DBL3γ encoded by var1CSA cross-react with one of the CSA-binding domains, DBL3X, encoded by var2CSA. This explains the paradoxical observation made here and earlier that anti-rDBL3γ sera recognize CSA-binding isolates and provides evidence for the presence of conserved, cross-reactive epitopes in diverse CSA-binding DBL domains. Such cross-reactive epitopes within CSA-binding DBL domains can form the basis for a vaccine that provides protection against PAM.

Synthesis and Characterization of Chimeric Proteins Based on Cellulase and Xylanase from an Insect Gut Bacterium

ABSTRACT Insects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated a Paenibacillus strain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, β-1,4-endoglucanase (Endo5A) and β-1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species of Paenibacillus. Endo5A and Xyl11D were overexpressed in Escherichia coli, and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range.

Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates

ABSTRACT Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel β-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a β-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G4S)3-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for β-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.

Calcium-dependent permeabilization of erythrocytes by a perforin-like protein during egress of malaria parasites

Clinical malaria is associated with proliferation of blood-stage parasites. During the blood stage, Plasmodium parasites invade host red blood cells, multiply, egress and reinvade uninfected red blood cells to continue the life cycle. Here we demonstrate that calcium-dependent permeabilization of host red blood cells is critical for egress of Plasmodium falciparum merozoites. Although perforin-like proteins have been predicted to mediate membrane perforation during egress, the expression, activity and mechanism of action of these proteins have not been demonstrated. Here, we show that two perforin-like proteins, perforin-like protein 1 and perforin-like protein 2, are expressed in the blood stage. Perforin-like protein 1 localizes to the red blood cell membrane and parasitophorous vacuolar membrane in mature schizonts following its Ca(2+)-dependent discharge from micronemes. Furthermore, perforin-like protein 1 shows Ca(2+)-dependent permeabilization and membranolytic activities suggesting that it may be one of the effector proteins that mediate Ca(2+)-dependent membrane perforation during egress.