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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.
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.
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.
Seol, Eunhee,Sekar, Balaji Sundara,Raj, Subramanian Mohan,Park, Sunghoon WILEY‐VCH Verlag 2016 Biotechnology Journal Vol.11 No.2
<P><B>Abstract</B></P><P>Hydrogen (H<SUB>2</SUB>) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co‐production of H<SUB>2</SUB> and ethanol, both of which are good biofuels, has been suggested. To this end, using <I>Escherichia coli</I>, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden‐Meyhof‐Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co‐production of H<SUB>2</SUB> and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner‐Doudoroff (ED) pathway, was greatly enhanced. The maximum co‐production yields were 1.32 mol H<SUB>2</SUB> mol<SUP>−1</SUP> glucose and 1.38 mol ethanol mol<SUP>−1</SUP> glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co‐factors.</P>
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.