Lynchrose2013

Lipoic acid is a valuable organosulfur compound used as an antioxidant for dietary supplementation, and potentially anti-diabetic and anti-cancer. Currently, lipoic acid is obtained mainly through chemical synthesis, which requires toxic reagents and organic solvents, thus causing environmental issues. Moreover, chemically synthesized lipoic acid is conventionally a racemic mixture. To obtain enantiomerically pure R-lipoic acid, which has superior bioactivity than the S form, chiral resolution and asymmetric synthesis methods require additional reagents and solvents, and often lead to wastage of S-lipoic acid or precursors with undesired chirality. Toward sustainable production of R-lipoic acid, we aim to develop a synthetic biology-based method using engineered yeast. Here, we deepened mechanistic understanding of lipoic acid biosynthesis and protein lipoylation in the model yeast Saccharomyces cerevisiae to facilitate metabolic engineering of the microbe for producing free R-lipoic acid. In brief, we studied the biosynthesis and confirmed the availability of protein-bound lipoate in yeast cells through LC-MS/MS. We then characterized in vitro the activity of a lipoamidase from Enterococcus faecalis for releasing free R-lipoic acid from lipoate-modified yeast proteins. Overexpression of the lipoamidase in yeast mitochondria enabled de novo free R-lipoic acid production in vivo. By overexpressing pathway enzymes and regenerating the cofactor, the production titer was increased ∼2.9-fold. This study represents the first report of free R-lipoic acid biosynthesis in S. cerevisiae. We envision that these results could provide insights into lipoic acid biosynthesis in eukaryotic cells and drive development of sustainable R-lipoic acid production.A large variety of microorganisms produces biosurfactants with the potential for a number of diverse industrial applications. To identify suitable wild-type or engineered production strains, efficient screening methods are needed, allowing for rapid and reliable quantification of biosurfactants in multiple cultures, preferably at high throughput. To this end, we have established a novel and sensitive assay for the quantification of biosurfactants based on the dye Victoria Pure Blue BO (VPBO). The assay allows the colorimetric assessment of biosurfactants directly in culture supernatants and does not require extraction or concentration procedures. Working ranges were determined for precise quantification of different rhamnolipid biosurfactants; titers in culture supernatants of recombinant Pseudomonas putida KT2440 calculated by this assay were confirmed to be the same ranges detected by independent high-performance liquid chromatography (HPLC)-charged aerosol detector (CAD) analyses. The assay was successfully applied for detection of chemically different anionic or non-ionic biosurfactants including mono- and di-rhamnolipids (glycolipids), mannosylerythritol lipids (MELs, glycolipids), 3-(3-hydroxyalkanoyloxy) alkanoic acids (fatty acid conjugates), serrawettin W1 (lipopeptide), and N-acyltyrosine (lipoamino acid). In summary, the VPBO assay offers a broad range of applications including the comparative evaluation of different cultivation conditions and high-throughput screening of biosurfactant-producing microbial strains.In this study, we have developed a mild and effective method to prepare a metal-organic framework (MOF)-based microcapsule by the self-assembly of pre-synthesized zeolite imidazolate framework-8 (ZIF-8) nanoparticles at the oil-water interface combined with deposition of a dense ZIF-8 coating outside the capsule. By introducing the enzyme Candida antarctica lipase B (CalB) directly into the stabilizer ZIF-8 or the water phase of Pickering emulsion during the preparation process, we achieved that the enzyme was immobilized within the shell (CalB@ZIF-8@cap) or in the cavity (ZIF-8@cap-CalB) of the microcapsules, respectively. The resulting CalB-loaded microcapsules were robust and had a core-shell structure proved by scanning electron microscopy. Meanwhile, Fourier transform infrared spectroscopy was conducted to confirm the encapsulation of enzymes in the microcapsules and their position in the microcapsules was confirmed by fluorescence microscopy. Furthermore, through the comparison of transesterification reactions between a pair of small substrates and a pair of larger ones, the two types of CalB-loaded microcapsules showed great catalytic activity, stability and size selectivity, and the catalytic activity of CalB@ZIF-8@cap was slightly higher than that of ZIF-8@cap-CalB. Selleckchem Citarinostat Importantly, due to the large size of the microcapsules, the catalyst could be separated from the reaction system by sedimentation, thereby reducing the energy consumption for separation. These kinds of multifunctional MOF-enzyme composites may open up new opportunities for the biocatalysis and microreactor.Teaching the processes of transcription and translation is challenging due to the intangibility of these concepts and a lack of instructional, laboratory-based, active learning modules. Harnessing the genetic code in vitro with cell-free protein synthesis (CFPS) provides an open platform that allows for the direct manipulation of reaction conditions and biological machinery to enable inquiry-based learning. Here, we report our efforts to transform the research-based CFPS biotechnology into a hands-on module called the "Genetic Code Kit" for implementation into teaching laboratories. The Genetic Code Kit includes all reagents necessary for CFPS, as well as a laboratory manual, student worksheet, and augmented reality activity. This module allows students to actively explore transcription and translation while gaining exposure to an emerging research technology. In our testing of this module, undergraduate students who used the Genetic Code Kit in a teaching laboratory showed significant score increases on transcription and translation questions in a post-lab questionnaire compared with students who did not participate in the activity. Students also demonstrated an increase in self-reported confidence in laboratory methods and comfort with CFPS, indicating that this module helps prepare students for careers in laboratory research. Importantly, the Genetic Code Kit can accommodate a variety of learning objectives beyond transcription and translation and enables hypothesis-driven science. This opens the possibility of developing Course-Based Undergraduate Research Experiences (CUREs) based on the Genetic Code Kit, as well as supporting next-generation science standards in 8-12th grade science courses.