Project description:Investigation of azodyrecin biosynthesis through heterologous expression and knockout studies in wild type producer (Streptomyces mirabilis P8-A2). FBMN conducted and preprocessing with MS-DIAL.
Project description:Raw LC-MS/MS data and MS-DIAL processed file from the collaborative study with Prof. Jae-Seoun Hur, Sunchon National University., which performed a heterologous expression of the atranorin BGC.
Project description:Type I polyketide synthases (T1PKSs) hold an enormous potential as a rational production platform for the biosynthesis of speciality chemicals. However, despite the great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enables the production of an unnatural polyketide in each of the hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. Here, we not only highlight the significance of codon optimization but also establish the groundwork for high-throughput assembly and characterization of PKS pathways in alternative hosts.
Project description:Heterologous E. coli expression of AtABC1K6 tagged with maltose binding protein (MBP) and 6xHis tags revealed a spontaneous cleavage event of the recombinant protein during purification. Resulting bands were resolved on SDS-PAGE and excised for MS/MS identification. Mass spec-based identification of the individual bands determined the cleavage event as ocurring after Lysine-443. The results demonstrate that the resulting ca. 80 kD band, showing kinase activity, comprises the AtABC1K6-6xHis fragment lacking the first 71 residues of AtABC1K6.
Project description:Heterologous gene expression to expand the native genetic capability of E. coli is the backbone of protein expression and metabolic engineering. The goal of this study was to determine how the identity of the heterologous gene expressed affected the host cell transcriptome. We generated a library of E. coli expressing 46 heterologous genes through an identical rhamnose inducible expression system and perform high throughput RNAseq as well as independent component analysis to elucidate the major variances in the transcriptome. We find that the major variations during heterologous gene expression can be divided into 5 main cellular responses: Fear vs greed, metal homeostasis, respiration, protein folding and amino acid and nucleotide metabolism.
Project description:The goal of this study was to use heterologous microarray hybridization to determine genomic content shared among different vesicomyid symbionts. These symbionts are closely related and can be thought of as different strains of bacteria, facilitating the use of heterologous microarray hybridization to determine genomic content. Keywords: comparative genomic hybridization
Project description:Heterologous gene expression to expand the native genetic capability of E. coli is the backbone of protein expression and metabolic engineering. The goal of this study was to determine how the identity of the heterologous gene expressed affected the host cell transcriptome. We generated a library of E. coli expressing 46 heterologous genes through an identical rhamnose inducible expression system and perform high throughput ribosome profiling.
Project description:We used RNA-seq to profile E. coli K-12 MG1655 strains subjected to adaptive laboratory evolution after knockout of endogenous glucose-6-phosphate isomerase (pgi) and subsequent expression of heterologous version of the pgi gene from Pseudomonas aeruginosa and Bacillus megaterium.
Project description:The goal of this study was to use heterologous microarray hybridization to determine genomic content shared among different vesicomyid symbionts. These symbionts are closely related and can be thought of as different strains of bacteria, facilitating the use of heterologous microarray hybridization to determine genomic content. Keywords: comparative genomic hybridization Microarrays were built off the Ruthia magnifica genome and two replicate hybridizations to this organism were used as a baseline for comparisons. Genomic DNA from two other vesicomyid symbionts (Calyptogena kilmeri and C. pacifica symbionts) was also hybridized to the array with three biological replicates for each sample.
Project description:Transcriptome sequencing to understand the impact of integration copy number and genome location on heterologous gene expression in R. toruloides