Hyung-Min Seo

Production of itaconate by whole-cell bioconversion of citrate mediated by expression of multiple cis-aconitate decarboxylase ( cadA ) genes in Escherichia coli

Itaconate, a C5 unsaturated dicarboxylic acid, is an important chemical building block that is used in manufacturing high-value products, such as latex and superabsorbent polymers. Itaconate is produced by fermentation of sugars by the filamentous fungus Aspergillus terreus. However, fermentation by A. terreus involves a long fermentation period and the formation of various byproducts, resulting in high production costs. E. coli has been developed as an alternative for producing itaconate. However, fermentation of glucose gives low conversion yields and low productivity. Here, we report the whole-cell bioconversion of citrate to itaconate with enhanced aconitase and cis-aconitate decarboxylase activities by controlling the expression of multiple cadA genes. In addition, this bioconversion system does not require the use of buffers, which reduces the production cost and the byproducts released during purification. Using this whole-cell bioconversion system, we were able to catalyze the conversion of 319.8 mM of itaconate (41.6 g/L) from 500 mM citrate without any buffer system or additional cofactors, with 64.0% conversion in 19 h and a productivity of 2.19 g/L/h. Our bioconversion system suggests very high productivity for itaconate production.

Application of a non-halogenated solvent, methyl ethyl ketone (MEK) for recovery of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] from bacterial cells

Conventional solvent-based methods are still the most practical approaches for recovery of polyhydroxyalkanoate (PHA) polymer from cellular biomass, even though potential alternatives exist, including chemical, mechanical, and enzymatic methods. It is still necessary, however, to avoid dangerous and environmentally unfriendly solvents (e.g., chloroform and dichloromethane) in the polymer recovery process. In the work presented here, we applied various solvent systems to recover PHA from Ralstonia eutropha and recombinant Escherichia coli cells. It was demonstrated that methyl ethyl ketone (MEK) is a promising solvent for PHA recovery from bacterial cells, particularly for the copolymer poly(hydroxybutyrate-cohydroxyvalerate) [P(HB-co-HV)], exhibiting > 90% polymer recovery. Even though MEK did not solubilize PHAs to the same extent as chloroform, it can recover a comparable amount of polymer because of its processing advantages, such as the low viscosity of the MEK/PHA solution, and the lower density of MEK as compared to cellular components. MEK was found to be the best alternative, non-halogenated solvent among examined candidates for recovery of P(HB-co-HV) from cells. The MEK treatment of PHAcontaining cells further allowed us to eliminate several costly and lengthy steps in the extraction process, such as cell lysis, centrifugation, and filtration.

A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay.

A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl-CoA synthetase and anaplerotic enzymes in engineered Escherichia coli

Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source. This article is protected by copyright. All rights reserved.