Prodrug-conjugated tumor-seeking commensals for targeted cancer therapy.

Prodrugs have been explored as an alternative to conventional chemotherapy; however, their target specificity remains limited. The tumor microenvironment harbors a range of microorganisms that potentially serve as tumor-targeting vectors for delivering prodrugs. In this study, we harness bacteria-cancer interactions native to the tumor microbiome to achieve high target specificity for prodrug delivery.

In vivo biosynthesis of tyrosine analogs and their concurrent incorporation into a residue-specific manner for enzyme engineering.

Herein we report the development of an efficient cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins by the residue-specific approach. This system makes use of common phenol derivatives and the tyrosine phenol lyase machinery to create various tyrosine analogues that impart desired properties on the target proteins. Biosynthesized 2-fluorotyrosine was incorporated into three industrially important enzymes which resulted in enhanced thermostability.

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms.

Living organisms have evolved over millions of years to fine tune their metabolism to create efficient pathways for producing metabolites necessary for their survival. Advancement in the field of synthetic biology has enabled the exploitation of these metabolic pathways for the production of desired compounds by creating microbial cell factories through metabolic engineering, thus providing sustainable routes to obtain value-added chemicals. Following the past success in metabolic engineering, there is increasing interest in diversifying natural metabolic pathways to construct non-natural biosynthesis routes, thereby creating possibilities for producing novel valuable compounds that are non-natural or without elucidated biosynthesis pathways.

Manganese and cobalt recovery by surface display of metal binding peptide on various loops of OmpC in Escherichia coli.

In a cell-surface display (CSD) system, successful display of a protein or peptide is highly dependent on the anchoring motif and the position of the display in that anchoring motif. In this study, a recombinant bacterial CSD system for manganese (Mn) and cobalt (Co) recovery was developed by employing OmpC as an anchoring motif on three different external loops. A portion of Cap43 protein (TRSRSHTSEG) was employed as a manganese and cobalt binding peptide (MCBP), which was fused with OmpC at three different external loops.

Engineering an FMN-based iLOV protein for the detection of arsenic ions.

Over the past few decades, genetically encoded fluorescent proteins have been widely used as efficient probes to explore and investigate the roles of metal ions in biological processes. The discovery of small FMN-based fluorescent proteins, such as iLOV and FbFP, has enabled researchers to exploit these fluorescent reporter proteins for metal-sensing applications. In this study, we report the inherent binding properties of iLOV towards arsenic ions.

Biotransformation of β-keto nitriles to chiral (S)-β-amino acids using nitrilase and ω-transaminase.

Objective: To enzymatically synthesize enantiomerically pure β-amino acids from β-keto nitriles using nitrilase and ω-transaminase.

Results: An enzyme cascade system was designed where in β-keto nitriles are initially hydrolyzed to β-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently β-keto acids were converted to β-amino acids using ω-transaminases. Five different ω-transaminases were tested for this cascade reaction, To enhance the yields of β-amino acids, the concentrations of nitrilase and amino donor were optimized.

Culture scale-up and immobilisation of a mixed methanotrophic consortium for methane remediation in pilot-scale bio-filters.

Robust methanotrophic consortia for methane (CH) remediation and by-product development are presently not readily available for industrial use. In this study, a mixed methanotrophic consortium (MMC), sequentially enriched from a marine sediment, was assessed for CH removal efficiency and potential biomass-generated by-product development. Suitable packing material for bio-filters to support MMC biofilm establishment and growth was also evaluated.

Biochemical characterization of thermostable ω-transaminase from Sphaerobacter thermophilus and its application for producing aromatic β- and γ-amino acids.

An (S)-ω-transaminase from the thermophilic eubacterium Sphaerobacter thermophilus was expressed and functionally characterized. The enzyme showed good stability at high temperature and in the presence of various substrates. Substrate specificity analysis showed that the enzyme had activity towards a broad range of substrates including amines, β- and γ-amino acids.

Influence of nutrients on oxidation of low level methane by mixed methanotrophic consortia.

Low-level methane emissions from coal mine ventilation air (CMV-CH4; i.e., 1 % CH4) can significantly contribute to global climate change, and therefore, treatment is important to reduce impacts.

FMN-Based Fluorescent Proteins as Heavy Metal Sensors Against Mercury Ions.

Bacterial light-oxygen-voltage-sensing photoreceptor-derived flavin mononucleotide (FMN)- based fluorescent proteins act as a promising distinct class of fluorescent proteins utilized for various biomedical and biotechnological applications. The key property of its independency towards oxygen for its chromophore maturation has greatly helped this protein to outperform the other fluorescent proteins such as GFP and DsRed for anaerobic applications. Here, we describe the feasibility of FMN-containing fluorescent protein FbFP as a metal-sensing probe by measuring the fluorescence emission changes of a protein with respect to the concentration of metal ions.

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties.

Effect of CH4/O2 ratio on fatty acid profile and polyhydroxybutyrate content in a heterotrophic-methanotrophic consortium.

Understanding the role of heterotrophic-methanotrophic (H-Meth) communities is important for improvement of methane (CH4) oxidation capacities (MOC) particularly in conjunction with bio-product development in industrial bio-filters. Initially, a H-Meth consortium was established and enriched from marine sediments and characterized by next generation sequencing of the 16s rDNA gene. The enriched consortium was subjected to 10-50% CH4 (i.

Construction of a high efficiency copper adsorption bacterial system via peptide display and its application on copper dye polluted wastewater.

For the construction of an efficient copper waste treatment system, a cell surface display strategy was employed. The copper adsorption ability of recombinant bacterial strains displaying three different copper binding peptides were evaluated in LB Luria-Bertani medium (LB), artificial wastewater, and copper phthalocyanine containing textile dye industry wastewater samples. Structural characteristics of the three peptides were also analyzed by similarity-based structure modeling.

Unnatural amino acid mutagenesis-based enzyme engineering.

Traditional enzyme engineering relies on substituting one amino acid by one of the other 19 natural amino acids to change the functional properties of an enzyme. However, incorporation of unnatural amino acids (UAAs) has been harnessed to engineer efficient enzymes for biocatalysis. Residue-specific and site-specific in vivo incorporation methods are becoming the preferred approach for producing enzymes with altered or improved functions.

Fungal cytochrome P450 monooxygenases of Fusarium oxysporum for the synthesis of ω-hydroxy fatty acids in engineered Saccharomyces cerevisiae.

Background: Omega hydroxy fatty acids (ω-OHFAs) are multifunctional compounds that act as the basis for the production of various industrial products with broad commercial and pharmaceutical implications. However, the terminal oxygenation of saturated or unsaturated fatty acids for the synthesis of ω-OHFAs is intricate to accomplish through chemocatalysis, due to the selectivity and controlled reactivity in C-H oxygenation reactions. Cytochrome P450, the ubiquitous enzyme is capable of catalyzing the selective terminal omega hydroxylation naturally in biological kingdom.

Production of chiral β-amino acids using ω-transaminase from Burkholderia graminis.

Optically pure β-amino acids are of high pharmacological significance since they are used as key ingredients in many physiologically active compounds. Despite a number of enzymatic routes to these compounds, an efficient synthesis of β-amino acids continues to pose a major challenge for researchers. ω-Transaminase has emerged as an important class of enzymes for generating amine compounds.

Temperature sensing using red fluorescent protein.

Genetically encoded fluorescent proteins are extensively utilized for labeling and imaging proteins, organelles, cell tissues, and whole organisms. In this study, we explored the feasibility of mRFP1 and its variants for measuring intracellular temperature. A linear relationship was observed between the temperature and fluorescence intensity of mRFP1 and its variants.

Engineering lead-sensing GFP through rational designing.

Lead is one of the most hazardous metals ubiquitous in the environment, causing serious health hazards to organisms. Recently, fluorescent proteins such as GFP and Dsred were utilized for the development of reagent-less rapid metal sensors. Here, we demonstrate the development of a lead-sensing GFP that is highly sensitive to lead at micro molar concentrations.

A New-Generation Fluorescent-Based Metal Sensor - iLOV Protein.

The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a lightoxygen- voltage sensing domain with a noncovalently bound flavin mononucleotide (FMN) as its chromophore. Owing to advantages such as its small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions.

Enhancing the biophysical properties of mRFP1 through incorporation of fluoroproline.

Here we enhanced the stability and biophysical properties of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. The global replacement of proline residue with (2S, 4R)-4-fluoroproline [(4R)-FP] into mRFP1 led to soluble protein but lost its fluorescence, whereas (2S, 4S)-4-fluoroproline [(4S)-FP] incorporation resulted in insoluble protein. The bioinformatics analysis revealed that (4R)-FP incorporation at Pro63 caused fluorescence loss due to the steric hindrance of fluorine atom of (4R)-FP with the chromophore.

An in silico approach to evaluate the polyspecificity of methionyl-tRNA synthetases.

Residue-specific incorporation is a technique used to replace natural amino acids with their close structural analogs, unnatural amino acids (UAAs), during protein synthesis. This is achieved by exploiting the substrate promiscuity of the wild type amino acyl tRNA synthetase (AARS) towards the close structural analogs of their cognate amino acids. In the past few decades, seleno-methionine was incorporated into proteins, using the substrate promiscuity of wild type AARSs, to resolve their crystal structures.