Abstract: The present invention provides a method for synthesizing nicotinic acid from 3-picoline using transformed recombinant host cells with synthetically designed gene constructs as whole cell biocatalysts. Adaptive engineering of aromatic ring metabolizing genes isolated from microorganisms enables efficient metabolism of 3-picoline. Mutants with enhanced activity profiles are developed through gene-level modifications, ensuring superior catalytic efficiency and stability. Synthetic biology techniques generate tailored coding sequences for optimum expression. Synthetic constructs embedded with engineered genes, ribosomal binding sites, and spacers are co-expressed within one cellular unit. A one-pot reaction system utilizes versatile plasmid vectors like pET28a(+) for efficient co-expression, advancing the host microorganism matrix. The invention integrates immobilized whole-cell catalysts, addressing catalyst reusability, stability, and industrial scalability.

Abstract: Presented herein is a computer-implemented method for protein engineering that constructs a three-dimensional gridspace around a protein. Different probes, simulating the interactions of amino acids are iteratively placed within the grid. Pair Interaction Energy is calculated using FMO technique for probe-protein interactions. An algorithmic process calculates the sum of PIEs, extending from each grid point to its neighbors, until a higher cumulative PIE is obtained. Grid points are then grouped into patches and an alignment process matches a derived query probe pattern from each patch against existing patterns of an internal database. Mutations are made based on the highest PIE probe-amino acid pairs from the matched pattern, leading to a modified protein. This process can be iterated for generating optimal variants and can be localized to any part of the protein for identification of patches.

Abstract: The present invention provides an engineered nitrilase polypeptide capable of converting (1-cyanomethyl) cyclohexane-1-carbonitrile into (1-cyanocyclohexyl)-acetic acid. Utilizing advanced enzyme engineering techniques, the nitrilase exhibits enhanced stability and activity over natural variants. These engineered enzymes can hydrolyze a wide range of nitrile-containing compounds, including cyclic and aliphatic substrates. They handle higher substrate concentrations (200 g/L to 300 g/L), are thermostable above 50° C., and remain stable within a pH range of 5.5 to 8.0, making them suitable for various industrial applications. Their ability to convert substrates such as mandelonitrile, 2-(2-chlorophenyl)-2-hydroxyacetonitrile, 2-(6-methoxynaphthalen-2-yl)propanenitrile, and 2-[1-(aminomethyl)cyclohexyl]acetonitrile into corresponding carboxylic acids enables efficient and cost-effective production from diverse starting materials.

Abstract: The invention describes a method for utilizing the CRISPR-Cas system to edit any gene of interest present on a plasmid. The method uses CRISPR tool to engineer enzymes for better activity by allowing a cell to undergo specific and random mutations. Described methods include newly designed, engineered and modified vector systems, which encodes single or multiplex gene targets and Cas9/deaminase Cas9 proteins. The invention is useful for single or multiple gene editing for industrial applications such as to edit genes encoding antibiotics, therapeutic proteins or any important industrial enzymes. The invention is a quick and efficient tool for creating enzyme variant libraries containing a vast range of permutation and combination of mutation that will be assayed for highest activity. Hot spots will be identified on gene of interest which will aid in generating mutations in these places. These mutations can be rationalized in specific places or random single base substitutions.

Abstract: The present invention discloses a computer-implemented method for engineering fluorinase enzymes towards the synthesis of fluorophenyl compounds. Limited or no mechanistic details of fluorinase enzymes have hindered progress in understanding their catalytic mechanisms for synthesizing synthetic organofluorine compounds. Through a comprehensive computational screening process, specific methionine-sulfonium phenyl substrates, including [(3S)-3-amino-3-carboxypropyl][2,5-difluoro-4-(4-methoxy-2,4-dioxobutyl)phenyl]methylsulfonium, were designed and optimized using quantum chemical optimization techniques. This methodology uncovers crucial information on F— ion attack conformation and the catalytic mechanism of the substrate, leading to the formation of Methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate. Furthermore, a protein sequence and 3D modeling-based enzyme screening process was employed to identify the most suitable enzyme for this substrate.