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Production of 1,3-Propanediol from glucose using Klebsiella pneumoniae
( Suman Lama ),설은희,정광현,박성훈 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.0
With the increase in demand for biodegradable polyester composed of 1,3 propanediol (1,3-PD), the microbial production of 1,3-PD has received much attention. However, till now, native microorganisms directly producing 1,3-PD from glucose was not identified and/or studied. Most microorganisms prefer glucose and glucose takes most portion in lignocellulosic biomass composed of various carbohydrates. Although using glucose is beneficial, there are only few reports on the production of 1,3-PD from glucose. In this study, we focus on development of strains that can convert glucose to 1,3-PD. For these purpose, two genes encoding glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase from Saccharomyces cerevisiae were overexpressed. Further, the produced glycerol was converted to 1,3-PD using native enzymes in Klebsiella pneumoniae J2B. Efficient recombinant strains developed in our study could lead to economical production of 1,3-PD.
Characterization of 1, 3-PD oxidoreductase (DhaT) from Klebsiella pneumoniae J2B
( Lama Suman ),설은희,( Sundara Sekar Balaji ),박성훈 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1
With the development of a new biodegradable and biocompatible polyester composed of 1, 3 propanediol (1, 3-PD), the bioconversion of glycerol and/or glucose to 1, 3-PD has gain much interest. In this biological system, glycerol is dehydrated to 3-HPA by glycerol dehydratase and 3-HPA is then reduced to 1, 3-PD by NADH dependent 1, 3-PD oxidoreductase encoded by dhaT gene. Despite practical importance of DhaT for 1, 3-PD production, there are few studies characterizing DhaT in terms of toxicity of 3-HPA intermediate and reversibility of DhaT. This work aims at kinetic characterization of DhaT from K. pneumoniae. The dhaT gene encoding 1, 3-PD oxidoreductase was cloned from the recently isolated K. pneumoniae J2B, and expressed and purified. Then, the reaction kinetics of DhaT for the forward and backward reactions were investigated using several aldehydes and alcohol as substrates.
( Lama Suman ),박성훈 한국공업화학회 2017 한국공업화학회 연구논문 초록집 Vol.2017 No.1
1,3-propanediol (1,3-PDO) is an industrially important chemical with increasing demand as an intermediate to fibers and resins. No natural microorganism can produce 1,3-PDO directly from glucose and only few scientific details on glucose-based 1,3-PDO production were disclosed. This study aims to provide information on glucose-based 1,3-PDO production using Klebsiella pneumoniae J2B as a microbial cell factory which can convert glycerol to 1,3-PDO and synthesize an essential coenzyme B12. K. pneumoniae J2B was engineered to have synthetic pathway which connect glycolytic pathway with that of 1,3-PDO synthesis from glycerol, thus to directly produce the diol from glucose. In addition, glycerol dissimilation pathways were disrupted. Furthermore, the metabolic flux toward 1,3-PDO was improved by modulation of byproduct formation pathway and fulfilling the demand of NADH. The engineered strains successfully produce 1,3-PDO from glucose.
Characterization of 1,3-Propanediol Oxidoreductase (DhaT) from Klebsiella pneumoniae J2B
Suman Lama,노수문,설은희,Balaji Sundara Sekar,Satish Kumar Ainala,Jayaraman Thangappan,송효학,승두영,박성훈 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.6
1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (kcat, 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (Km, mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD+) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag+ and Hg2+. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.