Development of a Transcription Factor-Based Lactam Biosensor

Lactams are an important class of commodity chemicals used in the manufacture of nylons, with millions of tons produced every year. Biological production of lactams could be greatly improved by high-throughput sensors for lactam biosynthesis. To identify biosensors of lactams, we applied a chemoinformatic approach inspired by small molecule drug discovery. We define this approach as analogue generation toward catabolizable chemicals or AGTC. We discovered a lactam biosensor based on the ChnR/Pb transcription factor-promoter pair. The microbial biosensor is capable of sensing ε-caprolactam, δ-valerolactam, and butyrolactam in a dose-dependent manner. The biosensor has sufficient specificity to discriminate against lactam biosynthetic intermediates and therefore could potentially be applied for high-throughput metabolic engineering for industrially important high titer lactam biosynthesis

Application of an Acyl-CoA Ligase from Streptomyces aizunensis for Lactam Biosynthesis

ε-Caprolactam and δ-valerolactam are important commodity chemicals used in the manufacture of nylons, with millions of tons produced annually. Biological production of these highly valued chemicals has been limited due to a lack of enzymes that cyclize ω-amino fatty acid precursors to corresponding lactams under ambient conditions. In this study, we demonstrated production of these chemicals using ORF26, an acyl-CoA ligase involved in the biosynthesis of ECO-02301 in Streptomyces aizunensis. This enzyme has a broad substrate spectrum and can cyclize 4-aminobutyric acid into γ-butyrolactam, 5-aminovaleric acid into δ-valerolactam and 6-aminocaproic acid into ε-caprolactam. Recombinant E. coli expressing ORF26 produced valerolactam and caprolactam when 5-aminovaleric acid and 6-aminocaproic acid were added to the culture medium. Upon coexpressing ORF26 with a metabolic pathway that produced 5-aminovaleric acid from lysine, we were able to demonstrate production of δ-valerolactam from lysine

Engineered polyketides: Synergy between protein and host level engineering

Metabolic engineering efforts toward rewiring metabolism of cells to produce new compounds often require the utilization of non-native enzymatic machinery that is capable of producing a broad range of chemical functionalities. Polyketides encompass one of the largest classes of chemically diverse natural products. With thousands of known polyketides, modular polyketide synthases (PKSs) share a particularly attractive biosynthetic logic for generating chemical diversity. The engineering of modular PKSs could open access to the deliberate production of both existing and novel compounds. In this review, we discuss PKS engineering efforts applied at both the protein and cellular level for the generation of a diverse range of chemical structures, and we examine future applications of PKSs in the production of medicines, fuels and other industrially relevant chemicals

Calibración a nivel de Hardware de un detector Cherenkov de agua (Chitaga) en el arreglo GUANE para estudios de clima espacial

Water Cherenkov Detectors (WCDs) play an essential role in space weather and cosmic rays studies around the world. In this article, we present a general methodology to calibrate a WCD based finding its optimum operation point and counting the delay time of the transmission cables. The dynode charge histogram of the WCD and CORSIKA simulations validate this methodology. Finally, to illustrate the capability of WCD tomeasure fluctuations in the secondary CR flux, we studied the 07.09.2017 Forbush and compared our measurements with data obtained from the Neutron Flux Monitor at UNAM and the Kyoto Dst index service.Los detectores Cherenkov de agua (WCD, por sus siglas en inglés) desempeñan un papel esencial en el estudio del clima espacial y los rayos cósmicos en todo el mundo. En este artículo, presentamos una metodología general para calibrar un WCD basado en encontrar su punto de operación óptimo y contarel tiempo de retardo de los cables de transmisión. El histograma de carga de dinodos de las simulaciones WCD y CORSIKA valida esta metodología. Finalmente, para ilustrar la capacidad de WCD para medir las fluctuaciones en el flujo de CR secundario, estudiamos el Forbush ocuurido el dia 07.09.2017 y comparamosnuestras mediciones con los datos obtenidos del Monitor de flujo de neutrones en la UNAM y el servicio de índice Dst de Kyoto

Calibración a nivel de Hardware de un detector Cherenkov de agua (Chitaga) en el arreglo GUANE para estudios de clima espacial

Water Cherenkov Detectors (WCDs) play an essential role in space weather and cosmic rays studies around the world. In this article, we present a general methodology to calibrate a WCD based finding its optimum operation point and counting the delay time of the transmission cables. The dynode charge histogram of the WCD and CORSIKA simulations validate this methodology. Finally, to illustrate the capability of WCD tomeasure fluctuations in the secondary CR flux, we studied the 07.09.2017 Forbush and compared our measurements with data obtained from the Neutron Flux Monitor at UNAM and the Kyoto Dst index service.Los detectores Cherenkov de agua (WCD, por sus siglas en inglés) desempeñan un papel esencial en el estudio del clima espacial y los rayos cósmicos en todo el mundo. En este artículo, presentamos una metodología general para calibrar un WCD basado en encontrar su punto de operación óptimo y contarel tiempo de retardo de los cables de transmisión. El histograma de carga de dinodos de las simulaciones WCD y CORSIKA valida esta metodología. Finalmente, para ilustrar la capacidad de WCD para medir las fluctuaciones en el flujo de CR secundario, estudiamos el Forbush ocuurido el dia 07.09.2017 y comparamosnuestras mediciones con los datos obtenidos del Monitor de flujo de neutrones en la UNAM y el servicio de índice Dst de Kyoto

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B₂ Biosynthesis

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. Although β-amino acid loading in the fluvirucin B2 polyketide pathway was proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. Here we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferases to selectively transfer β-amino acids onto a polyketide synthase (PKS) loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms

Chemoinformatic-Guided Engineering of Polyketide Synthases.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKS module to produce a saturated β-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates  into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structure similarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced by the host Streptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products

Massively parallel fitness profiling reveals multiple novel enzymes in Pseudomonas putida lysine metabolism

P. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida. We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged random barcode transposon sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both l- and d-lysine metabolism. We first describe three pathway enzymes that catabolize l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research