Vegan leather: a sustainable reality or a marketing gimmick?

The textile industry is the only one which has utilised all kinds of resources available in nature, and the evolution of textile materials has drastically hampered nature as well. Leather and fur are a few of the classic examples of materials derived from animals that have attracted dialogues about animal rights and ethical sourcing. To substitute animal-based leather, numerous materials have been manufactured synthetically and semi-synthetically. This review article discusses various types of leather, viz., bovine leather, poromerics, leatherette, plant-based vegan leather, and the sustainable alternatives available in the market as well as at the inductive research phase. The article is a comprehensive review of the leather and its commercially available alternatives along with their marketing strategy, and technical details. The article also compiles insight into the processing, and the components of vegan leather and the environmental issues related to them. The sustainability and circularity of processing in manufacturing vegan leather have also been discussed, with biodegradability as the focal point.

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.

Rebooting life: engineering non-natural nucleic acids, proteins and metabolites in microorganisms.

The surging demand of value-added products has steered the transition of laboratory microbes to microbial cell factories (MCFs) for facilitating production of large quantities of important native and non-native biomolecules. This shift has been possible through rewiring and optimizing different biosynthetic pathways in microbes by exercising frameworks of metabolic engineering and synthetic biology principles. Advances in genome and metabolic engineering have provided a fillip to create novel biomolecules and produce non-natural molecules with multitude of applications. To this end, numerous MCFs have been developed and employed for production of non-natural nucleic acids, proteins and different metabolites to meet various therapeutic, biotechnological and industrial applications. The present review describes recent advances in production of non-natural amino acids, nucleic acids, biofuel candidates and platform chemicals.

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.

Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae.

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.

Activation of alternative metabolic pathways diverts carbon flux away from isobutanol formation in an engineered Escherichia coli strain.

OBJECTIVE: Metabolic engineering efforts are guided by identifying gene targets for overexpression and/or deletion. Isobutanol, a biofuel candidate, is biosynthesized using the valine biosynthesis pathway and enzymes of the Ehrlich pathway. Most reported studies for isobutanol production in Escherichia coli employ multicopy plasmids, an approach that suffers from disadvantages such as plasmid instability, increased metabolic burden, and use of antibiotics to maintain selection pressure. Cofactor imbalance is another issue that may limit production of isobutanol, as two enzymes of the pathway utilize NADPH as a cofactor. RESULTS: To address these issues, we constructed E. coli strains with chromosomally-integrated, codon-optimized isobutanol pathway genes (ilvGM, ilvC, kivd, adh) selected on the basis of their cofactor preferences. Genes involved in diverting pyruvate flux toward fermentation byproducts were deleted. Metabolite analyses of the constructed strains revealed extracellular accumulation of significant amounts of isobutyraldehyde, a pathway intermediate, and the overflow metabolites 2,3-butanediol and acetol. CONCLUSIONS: These results demonstrate that the genetic modifications carried out led to activation of alternative pathways that diverted carbon flux toward formation of unwanted metabolites. The present study highlights how precursor metabolites can be metabolized through enzymatic routes that have not been considered important in previous studies due to the different strategies employed therein. The insights gained from the present study will allow rational genetic modification of host cells for production of metabolites of interest.

A shortened, two-enzyme pathway for 2,3-butanediol production in Escherichia coli.

The platform chemical 2,3-butanediol (2,3-BDO) is produced by a number of microorganisms via a three-enzyme pathway starting from pyruvate. Here, we report production of 2,3-BDO via a shortened, two-enzyme pathway in Escherichia coli. A synthetic operon consisting of the acetolactate synthase (ALS) and acetoin reductase (AR) genes from Enterobacter under control of the T7 promoter was cloned in an episomal plasmid. E. coli transformed with this plasmid produced 2,3-BDO and the pathway intermediate acetoin, demonstrating that the shortened pathway was functional. To assemble a synthetic operon for inducer- and plasmid-free production of 2,3-BDO, ALS and AR genes were integrated in the E. coli genome under control of the constitutive ackA promoter. Shake flask-level cultivation led to accumulation of ~1 g/L acetoin and ~0.66 g/L 2,3-BDO in the medium. The novel biosynthetic route for 2,3-BDO biosynthesis described herein provides a simple and cost-effective approach for production of this important chemical.

A series of template plasmids for Escherichia coli genome engineering.

Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated.

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.

Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production.

Mannitol is a six carbon sugar alcohol that finds applications in the pharmaceutical and food industries. A novel Escherichia coli strain capable of converting D-glucose to D-mannitol has been constructed, wherein native mannitol-1-phosphate dehydrogenase (MtlD) and codon-optimized Eimeria tenella mannitol-1-phosphatase (M1Pase) have been overexpressed. Codon-optimized Pseudomonas stutzeri phosphite dehydrogenase (PtxD) was overexpressed for cofactor (NADH) regeneration with the concomitant oxidation of phosphite to phosphate. Whole-cell biotransformation using resting cells in a medium containing D-glucose and equimolar sodium phosphite resulted in d-mannitol yield of 87 mol%. Thus, production of an industrially relevant biochemical without using complex media components and elaborate process control mechanisms has been demonstrated.

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.

Biochemical changes in erythrocyte membrane in uncontrolled type 2 diabetes mellitus.

The levels of lipid peroxidation and alterations in lipid composition and ATPase activities were determined in erythrocyte plasma membrane of uncontrolled type 2 diabetes mellitus (DM) patients. The study groups consisted of 30 patients (16 males, 14 females) attending the Out Patients' Department of Lokmanya Tilak Municipal General Hospital, Mumbai, and 23 age- and sex-matched control subjects (15 males, 8 females). Glycated haemoglobin (an index of long-term glycaemic control), erythrocyte membrane cholesterol, phospholipid and cholesterol to phospholipid molar ratio, lipid peroxidation products in the form of thiobarbituric acid-reactive substances (TBARS), and Na(+)-K(+)-ATPase activity were found to be significantly increased, and Mg(2+)-ATPase activity significantly decreased, in the diabetic subjects, as compared to controls. The study suggests that biochemical changes in the erythrocyte membrane may be involved in the pathophysiology of type 2 DM.