NADPH‐dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: Influence on global gene expression and role of oxygen supply. Issue 10 (4th June 2014)

• Record Type: Journal Article
• Authors: Siedler, Solvej; Bringer, Stephanie; Polen, Tino; Bott, Michael
• Is Part Of: Biotechnology and bioengineering. Volume 111:Issue 10(2014:Oct.)
• Page Start: 2067
• Page End: 2075
• Publication Date: 2014-06-04
• Subjects: Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.25271
• Languages: English
• ISSNs: 0006-3592

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>ABSTRACT</title> <sec id="bit25271-sec-0001" sec-type="section"> <p>An <italic>Escherichia coli</italic> Δ<italic>pfkA</italic> mutant lacking the major phosphofructokinase possesses a partially cyclized pentose phosphate pathway leading to an increased NADPH per glucose ratio. This effect decreases the amount of glucose required for NADPH regeneration in reductive biotransformations, such as the conversion of methyl acetoacetate (MAA) to (<italic>R</italic>)‐methyl 3‐hydroxybutyrate (MHB) by an alcohol dehydrogenase from <italic>Lactobacillus brevis</italic>. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation. DNA microarray analysis revealed amongst other things increased expression of <italic>soxS</italic>, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of <italic>soxS</italic>. Furthermore, several target genes of the ArcAB two‐component system showed a lower mRNA level in the reference strain than in the Δ<italic>pfkA</italic> mutant, pointing to an increased QH<sub>2</sub>/Q ratio in the reference strain. This prompted us to analyze yields and productivities of MAA reduction to MHB under different oxygen regimes in a bioreactor. Under anaerobic conditions, the specific MHB production rates of both strains were comparable (7.4 ± 0.2 mmol<sub>MHB</sub> h<sup>−1</sup> g<sub>cdw</sub><sup>−1</sup>) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h<sup>−1</sup> g<sub>cdw</sub><sup>−1</sup>) and of the Δ<italic>pfkA</italic> mutant (11.0 mmol h<sup>−1</sup> g<sub>cdw</sub><sup>−1</sup>) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference strain increased after the addition of MAA, presumably due to the oxidation of the acetate accumulated before MAA addition, the OTR of the Δ<italic>pfkA</italic> strain strongly decreased, indicating a very low respiration rate despite sufficient oxygen supply. The latter effect can likely be attributed to a restricted conversion of NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is outcompeted in the presence of MAA by the recombinant NADPH‐dependent alcohol dehydrogenase. The differences in respiration rates can explain the suggested higher ArcAB activity in the reference strain. Biotechnol. Bioeng. 2014;111: 2067–2075. © 2014 Wiley Periodicals, Inc.</p> </sec> </abstract>