NADPH-dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: influence on global gene expression and role of oxygen supply.
An Escherichia coli ΔpfkA 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 (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation. DNA microarray analysis revealed amongst other things increased expression of soxS, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of soxS. Furthermore, several target genes of the ArcAB two-component system showed a lower mRNA level in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2 /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 mmolMHB  h(-1)  gcdw (-1) ) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h(-1)  gcdw (-1) ) and of the ΔpfkA mutant (11.0 mmol h(-1)  gcdw (-1) ) 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 ΔpfkA 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.
Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, D-52425, Jülich, Germany.
Solvej
S
Siedler
Stephanie
S
Bringer
Tino
T
Polen
Michael
M
Bott
D000090:Acetoacetates / Q000378:metabolism; D001711:Biotransformation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism; D059467:Transcriptome
D000090:Acetoacetates; D006885:Hydroxybutyrates; C016579:2-methyl-3-hydroxybutyric acid; D009249:NADP; C005217:methyl acetoacetate; D010732:Phosphofructokinase-1; D005947:Glucose; D010100:Oxygen
DNA microarrays; Escherichia coli; SthA transhydrogenase; phosphofructokinase; reductive whole-cell biotransformation; resting cells