Biological machinery for the production of biosurfactant and their potential applications.

The growing biotechnology industry has focused a lot of attention on biosurfactants because of several advantages over synthetic surfactants. These benefits include worldwide public health, environmental sustainability, and the increasing demand from sectors for environmentally friendly products. Replacement with biosurfactants can reduce upto 8% lifetime CO2 emissions avoiding about 1.5 million tons of greenhouse gas released into the atmosphere. Therefore, the demand for biosurfactants has risen sharply occupying about 10% (∼10 million tons/year) of the world production of surfactants. Biosurfactants' distinct amphipathic structure, which is made up of both hydrophilic and hydrophobic components, enables these molecules to perform essential functions in emulsification, foam formation, detergency, and oil dispersion-all of which are highly valued characteristic in a variety of sectors. Today, a variety of biosurfactants are manufactured on a commercial scale for use in the food, petroleum, and agricultural industries, as well as the pharmaceutical and cosmetic industries. We provide a thorough analysis of the body of knowledge on microbial biosurfactants that has been gained over time in this research. We also discuss the benefits and obstacles that need to be overcome for the effective development and use of biosurfactants, as well as their present and future industrial uses.

An atlas of protein homo-oligomerization across domains of life.

Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. Our results suggest that approximately 45% of an archaeal proteome and a bacterial proteome and 20% of two eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of homo-oligomer types, including three confirmed experimentally by structure determination. Integrating these datasets with omics information suggests that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations and reveal coiled-coil regions as major enablers of quaternary structure evolution in human. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes.

CC+ : A searchable database of validated coiled coils in PDB structures and AlphaFold2 models.

α-Helical coiled coils are common tertiary and quaternary elements of protein structure. In coiled coils, two or more α helices wrap around each other to form bundles. This apparently simple structural motif can generate many architectures and topologies. Coiled coil-forming sequences can be predicted from heptad repeats of hydrophobic and polar residues, hpphppp, although this is not always reliable. Alternatively, coiled-coil structures can be identified using the program SOCKET, which finds knobs-into-holes (KIH) packing between side chains of neighboring helices. SOCKET also classifies coiled-coil architecture and topology, thus allowing sequence-to-structure relationships to be garnered. In 2009, we used SOCKET to create a relational database of coiled-coil structures, CC+ , from the RCSB Protein Data Bank (PDB). Here, we report an update of CC+ following an update of SOCKET (to Socket2) and the recent explosion of structural data and the success of AlphaFold2 in predicting protein structures from genome sequences. With the most-stringent SOCKET parameters, CC+ contains ≈12,000 coiled-coil assemblies from experimentally determined structures, and ≈120,000 potential coiled-coil structures within single-chain models predicted by AlphaFold2 across 48 proteomes. CC+ allows these and other less-stringently defined coiled coils to be searched at various levels of structure, sequence, and side-chain interactions. The identified coiled coils can be viewed directly from CC+ using the Socket2 application, and their associated data can be downloaded for further analyses. CC+ is available freely at http://coiledcoils.chm.bris.ac.uk/CCPlus/Home.html. It will be updated automatically. We envisage that CC+ could be used to understand coiled-coil assemblies and their sequence-to-structure relationships, and to aid protein design and engineering.

De novo design of discrete, stable 310-helix peptide assemblies.

The α-helix is pre-eminent in structural biology1 and widely exploited in protein folding2, design3 and engineering4. Although other helical peptide conformations do exist near to the α-helical region of conformational space-namely, 310-helices and π-helices5-these occur much less frequently in protein structures. Less favourable internal energies and reduced tendencies to pack into higher-order structures mean that 310-helices rarely exceed six residues in length in natural proteins, and that they tend not to form normal supersecondary, tertiary or quaternary interactions. Here we show that despite their absence in nature, synthetic peptide assemblies can be built from 310-helices. We report the rational design, solution-phase characterization and an X-ray crystal structure for water-soluble bundles of 310-helices with consolidated hydrophobic cores. The design uses six-residue repeats informed by analysing 310-helical conformations in known protein structures, and incorporates α-aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 310-helical folding, and identify features required for stabilizing assemblies of 310-helices. This work provides principles and rules to open opportunities for designing into this hitherto unexplored region of protein-structure space.

Diversity of the Tryptophanase Gene and Its Evolutionary Implications in Living Organisms.

Tryptophanase encoded by the gene tnaA is a pyridoxal phosphate-dependent enzyme that catalyses the conversion of tryptophan to indole, which is commonly used as an intra- and interspecies signalling molecule, particularly by microbes. However, the production of indole is rare in eukaryotic organisms. A nucleotide and protein database search revealed tnaA is commonly reported in various Gram-negative bacteria, but that only a few Gram-positive bacteria and archaea possess the gene. The presence of tnaA in eukaryotes, particularly protozoans and marine organisms, demonstrates the importance of this gene in the animal kingdom. Here, we document the distribution of tnaA and its acquisition and expansion among different taxonomic groups, many of which are usually categorized as non-indole producers. This study provides an opportunity to understand the intriguing role played by tnaA, and its distribution among various types of organisms.

Socket2: a program for locating, visualizing and analyzing coiled-coil interfaces in protein structures.

MOTIVATION: Protein-protein interactions are central to all biological processes. One frequently observed mode of such interactions is the α-helical coiled coil (CC). Thus, an ability to extract, visualize and analyze CC interfaces quickly and without expert guidance would facilitate a wide range of biological research. In 2001, we reported Socket, which locates and characterizes CCs in protein structures based on the knobs-into-holes (KIH) packing between helices in CCs. Since then, studies of natural and de novo designed CCs have boomed, and the number of CCs in the RCSB PDB has increased rapidly. Therefore, we have updated Socket and made it accessible to expert and nonexpert users alike. RESULTS: The original Socket only classified CCs with up to six helices. Here, we report Socket2, which rectifies this oversight to identify CCs with any number of helices, and KIH interfaces with any of the 20 proteinogenic residues or incorporating nonnatural amino acids. In addition, we have developed a new and easy-to-use web server with additional features. These include the use of NGL Viewer for instantly visualizing CCs, and tabs for viewing the sequence repeats, helix-packing angles and core-packing geometries of CCs identified and calculated by Socket2. AVAILABILITY AND IMPLEMENTATION: Socket2 has been tested on all modern browsers. It can be accessed freely at http://coiledcoils.chm.bris.ac.uk/socket2/home.html. The source code is distributed using an MIT licence and available to download under the Downloads tab of the Socket2 home page.

Diverse roles of microbial indole compounds in eukaryotic systems.

Indole and its derivatives are widespread across different life forms, functioning as signalling molecules in prokaryotes and with more diverse roles in eukaryotes. A majority of indoles found in the environment are attributed to bacterial enzymes converting tryptophan into indole and its derivatives. The involvement of indoles among lower organisms as an interspecies and intraspecies signal is well known, with many reports showing that inter-kingdom interactions involving microbial indole compounds are equally important as they influence defence systems and even the behaviour of higher organisms. This review summarizes recent advances in our understanding of the functional properties of indole and indole derivatives in diverse eukaryotes. Furthermore, we discuss current perspectives on the role of microbial indoles in human diseases such as diabetes, obesity, atherosclerosis, and cancers. Deciphering the function of indoles as biomarkers of metabolic state will facilitate the formulation of diet-based treatments and open unique therapeutic opportunities.

Fatty Acids as Antibiofilm and Antivirulence Agents.

Fatty acids (FAs) have emerged as a potential alternative to conventional antibiotics. Since many microbes respond differently to a variety of natural and synthetic FAs, substantial efforts have been made to understand the unique features of FAs that function as antimicrobials at high doses and biofilm inhibitors at low doses. Here, we provide an overview of the emerging antibiofilm properties of FAs and their relations to virulence and quorum sensing, such as diffusible signal factors (DSFs), acyl-homoserine lactones, and autoinducer-2 systems. Several FAs mimic DSFs and control motility, fimbriae, hyphae, and biofilm development as well as virulence characteristics of diverse microbes. The findings of FAs as antibiofilm and antivirulence agents will provide a new paradigm to cope with recalcitrant pathogens.

Lipid production by Cryptococcus albidus using biowastes hydrolysed by indigenous microbes.

The efficiency of Cryptococcusalbidus was evaluated for its abilities to assimilate onion and apple hydrolysates as a medium for lipid production. Onion waste (OW) and apple waste (AW) were hydrolysed at an organic load of 2% total solids by indigenous microbes under mesophilic conditions. The indigenous microbes effectively hydrolysed both wastes giving the highest reducing sugar content of 4.8 g/L and 10.8 g/L with OW and AW hydrolysates, respectively. The microbiome analysis revealed that most of the indigenous microbes belonged to genus Bacillus and a significant population of α-proteobacteria and γ-proteobacteria were also present. Cell retention culture of C. albidus at a dilution rate of 0.01 h-1 resulted in a total dry cell weight (DCW) of 13.5 g/L with an intracellular lipid content of 20.0% at 168 h, corresponding to an enhancement of 3.48-folds and 2.37-folds in DCW and lipid concentration, respectively, as compared to batch fermentation.

Bioconversion of lignin and its derivatives into polyhydroxyalkanoates: Challenges and opportunities.

Renewable energy resources are considered to be promising for the development of a sustainable circular economy. Among various alternatives, the microbial route for various biofuels production is quite lucrative. Use of cellulose and lignocellulose for methane, H2 , organic acids, ethanol, and cellulase has been explored a lot in the past few decades. The major leftover or a coproduct of these processes belongs to lignin-an aromatic cross-link polymer and one of the most abundant complex compounds on earth. A successful bioconversion route of lignin into high-value products is highly desirable for biorefinery perspective. It requires a complex set of enzymes/catalysts to decompose lignin through depolymerization and oxygen removal leading to its monomers that can be metabolized by engineered organisms to synthesize muconic acids, polyhydroxyalkanoates (PHAs), methane, and other high-value products. This article will focus on the opportunities and challenges in the bioconversion of lignin and its derivatives into PHAs.

Valorization of polyhydroxyalkanoates production process by co-synthesis of value-added products.

Polyhydroxyalkanoates (PHAs) are the only polyesters that are completely synthesized biologically and possess features equivalent to petroleum-based plastics besides being biodegradable. PHA based materials may certainly prove helpful in addressing the concerns caused due to the indiscriminate use of synthetic plastics. However, the cost of producing these polymers on a large scale is still uneconomical. Various approaches have been developed to tackle this issue through usage of agro-industrial wastes, co-production of high market value products, polymer extraction using green solvents, etc. The advent of recombineering and CRISPR technologies has broadened the scope of constructing a microbe capable of synthesizing multiple products with economic feasibility. Quite a few high-market value chemicals are possible to synthesize along with the favorable accumulation of PHA. The present article attempts to review all PHA polymer co-production processes with other chemicals reported till date and discusses the opportunities for their large-scale operation in future.

Microbial Conversion of Vegetable Oil to Hydroxy Fatty Acid and Its Application to Bio-Based Polyurethane Synthesis.

New polyurethanes were synthesized based on dihydroxy fatty acid obtained by the microbial conversion of olive oil. Monounsaturated 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced from olive oil by Pseudomonas aeruginosa PR3 and reacted with hexamethylene diisocyanate (HMDI) at different ratios to form polyurethanes. Fourier transform infrared spectroscopy and gas chromatography/mass spectrometry confirmed the synthesis of DOD. The thermal and tensile properties of the polyurethanes were investigated by differential scanning calorimetry, thermogravimetric analysis, and a universal testing machine. At an isocyanate/hydroxyl ratio of 1.4, the polyurethane exhibited an elongation at break of 59.2% and a high tensile strength of 37.9 MPa. DOD was also mixed with polycaprolactone diol or polyethylene glycol at different weight ratios and then reacted with HMDI to produce new polyurethanes of various properties. These polyurethanes displayed higher elongation at break and good thermal stability. This is the first report on the synthesis of polyurethanes based on DOD produced by the microbial conversion of vegetable oil.

Co-production of polyhydroxyalkanoates and carotenoids through bioconversion of glycerol by Paracoccus sp. strain LL1.

Polyhydroxyalkanoates (PHA) have emerged as a potential alternative to synthetic plastics, although its large-scale production is limited by the expenditure incurred in feed materials. Co-production of various valuable bioproducts along with PHA has been proposed to alleviate the overall production cost. Here, high-yield co-production of PHA and carotenoids was achieved in single step fermentation by Paracoccus sp. LL1. The halophilic bacterial strain could metabolize a wide range of substrates including methanol, lactose, galactose, glycerol, fructose, mannitol, etc. Under batch fermentation using mineral media supplemented with 2% glycerol, Paracoccus sp. LL1 synthesized 3.77gL-1 PHA with concomitant production of 3.6mgL-1 of carotenoids after 96h. An enhancement of 2.2-folds in total dry cell weight (DCW) was achieved through cell retention culture of Paracoccus sp. LL1, resulting in a maximum DCW (24.2gL-1) containing PHA (39.3%, ww-1) with concomitant enhancement in the production of total carotenoids. Paracoccus sp. LL1 could convert glycerol into carotenoids and PHA to a level of 7.14mgL-1 and 9.52gL-1, respectively. It is the first report showing value addition in PHA production process through co-production of high value carotenoids by Paracoccus sp. using glycerol as substrate.

RNAHelix: computational modeling of nucleic acid structures with Watson-Crick and non-canonical base pairs.

Comprehensive analyses of structural features of non-canonical base pairs within a nucleic acid double helix are limited by the availability of a small number of three dimensional structures. Therefore, a procedure for model building of double helices containing any given nucleotide sequence and base pairing information, either canonical or non-canonical, is seriously needed. Here we describe a program RNAHelix, which is an updated version of our widely used software, NUCGEN. The program can regenerate duplexes using the dinucleotide step and base pair orientation parameters for a given double helical DNA or RNA sequence with defined Watson-Crick or non-Watson-Crick base pairs. The original structure and the corresponding regenerated structure of double helices were found to be very close, as indicated by the small RMSD values between positions of the corresponding atoms. Structures of several usual and unusual double helices have been regenerated and compared with their original structures in terms of base pair RMSD, torsion angles and electrostatic potentials and very high agreements have been noted. RNAHelix can also be used to generate a structure with a sequence completely different from an experimentally determined one or to introduce single to multiple mutation, but with the same set of parameters and hence can also be an important tool in homology modeling and study of mutation induced structural changes.

Structural and functional analyses of PolyProline-II helices in globular proteins.

PolyProline-II (PPII) helices are defined as a continuous stretch of a protein chain in which the constituent residues have backbone torsion angle (φ, ψ) values of (-75°, 145°) and take up an extended left handed helical conformation, without any intra-chain hydrogen bonds. They are found to occur quite frequently in protein structures, with their number exceeding that of π-helices, though it is considerably less than that of α-helices and ß-strands. A relatively new procedure, ASSP, for the identification of regular secondary structures using Cα trace identifies 3597 PPII-helices in 3582 protein chains, solved at resolution ⩽2.0Å. Taking advantage of this significantly expanded database of PPII-helices, we have analyzed their structural and functional roles as well as determined the amino acid propensity within and around them. Though Pro residues are highly preferred, their presence is not a mandatory requirement for the formation of PPII-helices, since ∼40% PPII-helices were found to contain no Pro residues. Aromatic amino acids are avoided within this helix, while Gly, Asn and Asp residues are preferred in the proximal flanking regions. The PPII-helices range from 3 to 13 residues in length with the average twist and rise being -121.2°±9.2° and 3.0ű0.1Å respectively. A majority (∼72%) of PPII-helices were found to occur in conjunction with α-helices and ß-strands, and serve as linkers as well. The analysis of various intra-helical non-bonded interactions revealed frequent presence of CH⋯O H-bonds. PPII-helices participate in maintaining the three-dimensional structure of proteins and are important constituents of binding motifs involved in various biological functions.

A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for Streptococcus.

The use of rrs (16S rRNA) gene is widely regarded as the "gold standard" for identifying bacteria and determining their phylogenetic relationships. Nevertheless, multiple copies of this gene in a genome is likely to give an overestimation of the bacterial diversity. In each of the 50 Streptococcus genomes (16 species, 50 strains), 4-7 copies of rrs are present. The nucleotide sequences of these rrs genes show high similarity within and among genomes, which did not allow unambiguous identification. A genome-wide search revealed the presence of 27 gene sequences common to all the Streptococcus species. Digestion of these 27 gene sequences with 10 type II restriction endonucleases (REs) showed that unique RE digestion in purH gene is sufficient for clear cut identification of 30 genomes belonging to 16 species. Additional gene-RE combinations allowed identification of another 15 strains belonging to S. pneumoniae, S. pyogenes, and S. suis. For the rest 5 strains, a combination of 2 genes was required for identifying them. The proposed strategy is likely to prove helpful in proper detection of pathogens like Streptococcus.

Searching Biomarkers in the Sequenced Genomes of Staphylococcus for their Rapid Identification.

Bacterial identification using rrs (16S rRNA) gene is widely reported. Bacteria possessing multiple copies of rrs lead to overestimation of its diversity. Staphylococcus genomes carries 5-6 copies of rrs showing high similarity in their nucleotide sequences, which lead to ambiguous results. The genomes of 31 strains of Staphylococcus representing 7 species were searched for the presence of common genes. In silico digestion of 34 common genes using 10 restriction endonucleases (REs) lead to select gene-RE combinations, which could be used as biomarkers. RE digestion of recA allowed unambiguous identification of 13 genomes representing all the 7 species. In addition, a few more genes (argH, argR, cysS, gyrB, purH, and pyrE) and RE combinations permitted further identification of 12 strains. By employing additional RE and genes unique to a particular strain, it was possible to identify the rest 6 Staphylococcus aureus strains. This approach has the potential to be utilized for rapid detection of Staphylococcus strains.

Production of co-polymers of polyhydroxyalkanoates by regulating the hydrolysis of biowastes.

Production of polyhydroxyalkanoate (PHA) co-polymers by Bacillus spp. was studied by feeding defined volatile fatty acids (VFAs) obtained through controlled hydrolysis of various wastes. Eleven mixed hydrolytic cultures (MHCs) each containing 6 strains could generate VFA from slurries of (2% total solids): pea-shells (PS), potato peels (PP), apple pomace (AP) and onion peels (OP). PS hydrolysates (obtained with MHC2 and MHC5) inoculated with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45 produced co-polymers of PHA at the rate of 15-60mg/L with a 3HV content of 1%w/w. An enhancement in PHA yield of 3.66-fold, i.e. 205-550mg/L with 3HV content up to 7.5%(w/w) was observed upon addition of OP hydrolysate and 1% glucose (w/v) to PS hydrolysates. This is the first demonstration, where PHA co-polymer composition, under non-axenic conditions, could be controlled by customizing VFA profile of the hydrolysate by the addition of different biowastes.

Genome Wide Search for Biomarkers to Diagnose Yersinia Infections.

Bacterial identification on the basis of the highly conserved 16S rRNA (rrs) gene is limited by its presence in multiple copies and a very high level of similarity among them. The need is to look for other genes with unique characteristics to be used as biomarkers. Fifty-one sequenced genomes belonging to 10 different Yersinia species were used for searching genes common to all the genomes. Out of 304 common genes, 34 genes of sizes varying from 0.11 to 4.42 kb, were selected and subjected to in silico digestion with 10 different Restriction endonucleases (RE) (4-6 base cutters). Yersinia species have 6-7 copies of rrs per genome, which are difficult to distinguish by multiple sequence alignments or their RE digestion patterns. However, certain unique combinations of other common gene sequences-carB, fadJ, gluM, gltX, ileS, malE, nusA, ribD, and rlmL and their RE digestion patterns can be used as markers for identifying 21 strains belonging to 10 Yersinia species: Y. aldovae, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, Y. rohdei, Y. ruckeri, and Y. similis. This approach can be applied for rapid diagnostic applications.