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박성훈 ( Balaji Sundara Sekar ),설은희 ( Satish Kumar Ainala ) 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1
To overcome the low yield of H<sub>2</sub> production by dark fermentation, coproduction of H<sub>2</sub> and ethanol by Escherichia coli from glucose, was suggested. In order to increase the cofactor availability for ethanol production, pentose phosphate (PP) pathway was suggested as the major glycolytic route and its activation was studied by constructing various genetic backgrounds in the base strain (E. coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB). Resulting strains could increase the co-production yields (~ 1.7 mol/mol for H<sub>2</sub>, 1.38 mol/mol for ethanol) and significantly reduce by-products formation such as acetate or pyruvate by confirming the activation of PP pathway. The highest achieved co-production yield corresponds to ~ 85% energy recovery from glucose. Finally, in this study, we would like to discuss the possibility of operating PP pathway under anaerobic condition and its potential for further applicable studies.
Co-production of hydrogen and ethanol from glucose by the modification of glycolytic pathway
( Sundara Sekar Balaji ),설은희,( Satish Kumar Ainala ),박성훈 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1
In our previous study, co-production of H<sub>2</sub> and ethanol was studied by deleting pfkA and pta-ackA. Deletion of these genes accumulated substantial amount of pyruvate as by-products due to the shortage of NADH. To supply more NAD(P)H for ethanol production by activating PP pathway, we overexpressed G6PDH and 6PGDH which are known as limiting enzymes in PP pathway. Over-expression of these enzymes increased co-production yield by reducing pyruvate secretion significantly. This result confirms that carbon flux was diverted to PP pathway thus increasing NAD(P)H availability for co-production. RT-PCR and MFA supported this observed results. Final co-production yield of 1.21 H<sub>2</sub>/ mol glucose and 1.25 mol ethanol/mol glucose was achieved.
설은희,( Sundara Sekar Balaji ),( Satish Kumar Ainala ),박성훈 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1
To address low H<sub>2</sub> production yield by dark fermentation, co-production of H<sub>2</sub> and ethanol by E. coli from glucose, was suggested in this study. Moreover, ethanol is one of the attractive biofuel producible through the fermentation. In order to increase the cofactor availability for ethanol production, operation of Pentose-Phosphate (PP) pathway was tried by deleting pgi in the base strain (E. coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB). Additional over-expression of G6PHD and 6PGDH recovered the cell growth of Δpgi mutant as well as increased co-production yield by decreasing acetate formation. Resulting strain increased co-production yield (1.65 for H<sub>2</sub> and 1.38 mol/mol for ethanol) which corresponds to ~85% energy recovery from glucose showing the possibility of operating PP pathway under anaerobic condition.
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.
Zhengbin Li,노수문,Balaji Sundara Sekar,설은희,Suman Lama,이선구,Guangyi Wang,박성훈 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.6
1,3-propanediol oxidoreductase (DhaT), which catalyzes the conversion of 3-hydroxypropionaldehyde (3- HPA) to 1,3-propanediol (1,3-PD) with the oxidation of NADH to NAD+, is a key enzyme in the production of 1,3- PD from glycerol. DhaT is known to be severely inactivated by its physiological substrate, 3-HPA, due to the reaction of 3-HPA with the thiol group of the cysteine residues. In this study, using site-directed mutagenesis, four cysteine residues in Klebsiella pneumoniae J2B DhaT were substituted to alanine, the amino acid commonly found in cysteine’s positions in other DhaT, individually and in combination. Among the total of 15 mutants developed, a double mutant (C28A_C107A) and a triple mutant (C28A_C93A_C107A) exhibited approximately 50 and 16% higher activity than the wild-type counterpart, respectively, after 1 h incubation with 10 mM 3-HPA. According to detailed kinetic studies, the double mutant had slightly better kinetic properties (Vmax, Kcat, and Km for both 3-HPA and NADH) than wild-type DhaT. This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal, albeit to a limited extent.
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.
Isolation of a Novel Pseudomonas Species SP2 Producing Vitamin B12 under Aerobic Condition
Mariadhas Valan Arasu,Ritam Sarkar,Balaji Sundara Sekar,Vinod Kumar,Chelladurai Rathnasingh,Jin-dal-rae Choi,Hyohak Song,Doyoung Seung,박성훈 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.1
Vitamin B12 is a complex biomolecule that acts as a cofactor for a variety of enzymes in microbial metabolism. Pseudomonas denitrificans is exclusively used as an industrial strain for the production of vitamin B12under aerobic conditions. However, only a few strains of Pseudomonas have been reported to possess the capability of producing this vitamin and they are strongly patentprotected. To improve the applicability of the vitamin B12-producing microorganisms, a new isolate was obtained from municipal waste samples and characterized for its biological properties. The new isolate, designated as SP2,was identified to be a Pseudomonas species based on the sequence homology of its 16S rDNA. Pseudomonas species SP2 had essential genes for vitamin B12 synthesis such as cobB and cobQ and produced a similar amount of vitamin B12 (10.6 ± 0.05 μg/mL) as P. denitrificans ATCC 13867 in 24 h flask culture. SP2 grew well under aerobic condition with the maximum specific growth rate (μmax) of 0.91 +0.03/h, but showed a poor growth under micro-aerobic conditions. SP2 was resistant to antibiotics like streptomycin,carbenicillin, ampicillin, cefpodoxime, colistin, nalidixic acid and sparfloxacin. The ability of SP2 to grow faster and produce vitamin B12 under aerobic conditions makes it a promising host for the production of some biochemicals requiring a coenzyme B12-dependent enzyme, such as glycerol dehydratase.
새로이 분리된 Klebsiella pneumoniae 균주들의 글리세롤 기반 3-hydroxypropionic acid 및 1,3-propanediol 동시 생산성 평가
고연주(Yeounjoo Ko),설은희(Eunhee Seol),순달아 세칼 바라지(Balaji Sundara Sekar),권성진(Seongjin Kwon),이재현(Jaehyeon Lee),박성훈(Sunghoon Park) 한국생물공학회 2016 KSBB Journal Vol.31 No.4
Co-production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) was suggested as an innovative strategy to overcome several limitations occurring in the single production of 3-HP from glycerol. In this study, two new isolates of Klebsiella pneumoniae, which produce less lipopolysaccharide (LPS) thus considered less pathogenic than K. pneumoniae DSM 2026, were compared and evaluated for their potential for the co-production of 3-HP and 1,3-PDO. The newly isolated strains showed significantly faster sedimentation rate than DSM, which should be beneficial for downstream processing. Analysis of genome sequences of the isolates confirmed the presence of all genes necessary for glycerol assimilation, 1,3-PDO production and biosynthesis of coenzyme B12. Co-production yield was highest under anaerobic condition while cell growth was highest under aerobic condition. Both strains showed similarly good performance for the co-production although J2B gave the slightly higher co-production yield of 0.80 mol/mol than GSC021 (0.75 mol/mol). The evaluation of the newly developed strains presented here should be useful in designing similar evaluation experiments for other microorganisms.