Computational structural optimization enhances IL13Rα2 - B7-H3 tandem CAR T cells to overcome antigen-heterogeneity-mediated tumor escape.

Chimeric antigen receptor (CAR) T cell therapy is a highly effective treatment for multiple malignancies. However, one limitation is tumor antigen-heterogeneity and downregulation, which allows tumor cells to evade conventional, monospecific CAR T cells. One approach to overcome this tumor escape is by utilizing a tandem CAR recognizing two antigens.

Integration of metabolism and regulation reveals rapid adaptability to growth on non-native substrates.

Engineering synthetic heterotrophy is a key to the efficient bio-based valorization of renewable and waste substrates. Among these, engineering hemicellulosic pentose utilization has been well-explored in Saccharomyces cerevisiae (yeast) over several decades-yet the answer to what makes their utilization inherently recalcitrant remains elusive. Through implementation of a semi-synthetic regulon, we find that harmonizing cellular and engineering objectives are a key to obtaining highest growth rates and yields with minimal metabolic engineering effort.

Towards universal synthetic heterotrophy using a metabolic coordinator.

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3p), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates.

Flow cytometric evaluation of yeast-bacterial cell-cell interactions.

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting to enrich a specific yeast-displayed nanobody within a mixed yeast-display population.

A Silk-Based Platform to Stabilize Phenylalanine Ammonia-lyase for Orally Administered Enzyme Replacement Therapy.

Phenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects ∼1 in 15 000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from , which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix.

In-depth Sequence-Function Characterization Reveals Multiple Pathways to Enhance Enzymatic Activity.

Deep mutational scanning (DMS) has recently emerged as a powerful method to study protein sequence-function relationships but it has not been well-explored as a guide to enzyme engineering and identifying pathways by which their catalytic cycle may be improved. We report such a demonstration in this work using a Phenylalanine ammonia-lyase (PAL), which deaminates L-phenylalanine to -cinnamic acid and has widespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from (AvPAL*) has garnered significant attention as the active ingredient in Pegvaliase, the only FDA-approved drug treating classical Phenylketonuria (PKU).

Cheating the Cheater: Suppressing False-Positive Enrichment during Biosensor-Guided Biocatalyst Engineering.

Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false-positive/cheater cells in the enriched population.

Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities.

There is broad interest in engineering phenylalanine ammonia-lyase (PAL) for its biocatalytic applications in industry and medicine. While site-specific mutagenesis has been employed to improve PAL stability or substrate specificity, combinatorial techniques are poorly explored. Here, we report development of a directed evolution technique to engineer PAL enzymes.

Nitrogen-dependent induction of atrazine degradation pathway in Pseudomonas sp. strain AKN5.

Soil isolate Pseudomonas sp. strain AKN5 degrades atrazine as the sole source of nitrogen. The strain showed expeditious growth on medium containing citrate as the carbon source and ammonium chloride as the nitrogen source as compared to citrate plus atrazine or cyanuric acid.

Insights into metabolism and sodium chloride adaptability of carbaryl degrading halotolerant Pseudomonas sp. strain C7.

Pseudomonas sp. strain C7 isolated from sediment of Thane creek near Mumbai, India, showed the ability to grow on glucose and carbaryl in the presence of 7.5 and 3.

Insights into functional and evolutionary analysis of carbaryl metabolic pathway from Pseudomonas sp. strain C5pp.

Carbaryl (1-naphthyl N-methylcarbamate) is a most widely used carbamate pesticide in the agriculture field. Soil isolate, Pseudomonas sp. strain C5pp mineralizes carbaryl via 1-naphthol, salicylate and gentisate, however the genetic organization and evolutionary events of acquisition and assembly of pathway have not yet been studied.

Draft Genome Sequence of Carbaryl-Degrading Soil Isolate Pseudomonas sp. Strain C5pp.

We report the draft genome sequence of carbaryl-degrading Pseudomonas sp. strain C5pp. Genes encoding salicylate and gentisate metabolism, large amounts of oxygenase, nitrogen metabolism, and heavy metal tolerance were identified.

Analysis of preference for carbon source utilization among three strains of aromatic compounds degrading Pseudomonas.

Soil isolates Pseudomonas putida CSV86, Pseudomonas aeruginosa PP4 and Pseudomonas sp. C5pp degrade naphthalene, phthalate isomers and carbaryl, respectively. Strain CSV86 displayed a diauxic growth pattern on phenylpropanoid compounds (veratraldehyde, ferulic acid, vanillin or vanillic acid) plus glucose with a distinct second lag-phase.

Kinetic and spectroscopic characterization of 1-naphthol 2-hydroxylase from Pseudomonas sp. strain C5.

1-Naphthol 2-hydroxylase (1-NH) catalyzes the conversion of 1-naphthol to 1,2-dihydroxynaphthalene. 1-NH from carbaryl degrading Pseudomonas strain C5 was purified and characterized for its kinetic and spectroscopic properties. The enzyme was found to be NAD(P)H-dependent external flavin monooxygenase.

Purification and characterization of NAD+ -dependent salicylaldehyde dehydrogenase from carbaryl-degrading Pseudomonas sp. strain C6.

NAD+-dependent salicylaldehyde dehydrogenase (SALDH) which catalyzes the oxidation of salicylaldehyde to salicylate was purified form carbaryl-degrading Pseudomonas sp. strain C6. The enzyme was found to be a functional homotrimer (150 kDa) with subunit molecular mass of 50 kDa and contained calcium (1.

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6.

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into 'upper' (carbaryl to salicylate), 'middle' (salicylate to gentisate) and 'lower' (gentisate to TCA cycle) pathway.