Annals of Microbiology, 58 (2) 275-280 (2008) Physiologic roles of soluble pyridine nucleotide transhydrogenase in Escherichia coli as determined by homologous recombination Hanjun ZHAO, Peng WANG, Enqi HUANG, Yadong GE, Guoping ZHU* The Key Laboratory of Molecular Evolution and the Institute of Molecular Biology and Biotechnology, Anhui Normal University, 1 Beijing Road, Wuhu, Anhui 241000, P.R. China Received 28 January 2008 / Accepted 15 April 2008 Abstract - The soluble transhydrogenase is an energy-independent flavoprotein and important in cofactor regenerating system. In order to understand its physiologic roles, the recombinant strain with the deletion of soluble transhydroge- nase gene (ΔudhA)in Escherichia coli was constructed using homologous recombination. Then the different genetic back- grounds containing either icdNADP or icdNAD, which encodes NADP-dependent isocitrate dehydrogenase (IDH) or engineered NAD-dependent IDH, were transduced into ΔudhA, creating two strains (icdNADP/ΔudhA, icdNAD/ΔudhA). During growth on acetate, icdNADP/ΔudhA grew poorly and its growth rate was remarkably reduced by 75% as compared with the wild type. However, icdNAD/ΔudhA showed significantly better growth than icdNADP/ΔudhA. Its growth rate was about 3.7 fold of icdNADP/ΔudhA, which was equivalent to the wild type. These results indicated that UdhA is an essential NADH resource for acetate-grown E. coli and is a dominant factor for bacteria to adapt to the stress environment. Furthermore, when UdhA was absence, icdNAD/ΔudhA displayed about 1.5 fold increase in the IDH activity after switching the carbon source from glucose to acetate. And RT-PCR showed that the expression of NADH dehydrogenase II (NDH-2) in icdNAD/ΔudhA was remarkably up-regulated by about 2.8 fold as compared with icdNADP/ΔudhA. The increase of IDH activity and NDH-2 expression can be explained by the reducing excess NADPH production and restoring higher levels of NADH generation in cells. Key words: soluble transhydrogenase; homologous recombination; growth rate; isocitrate dehydrogenase; NADH dehy- drogenase II; real-time PCR. INTRODUCTION the enzyme activity was strongly activated by NADPH and 2’-AMP and inhibited by NADP+.The More and more attention is paid to the utilisations of inhibition of STH by NADP+ implies that its physio- the soluble pyridine nucleotide transhydrogenase logical role is the conversion of NADPH to NADH that (STH) in the metabolic engineering and industrial can enter the respiratory chain for energy genera- biocatalysis today. STH is found in the cytoplasm of tion (Voordouw et al., 1983). some heterotrophic bacteria. It is a kind of redox Although the functions have not been determined enzyme which catalyses the reversible transfer of definitely, the soluble transhydrogenase has been reducing equivalents between two major cofactors, extensively applied in the metabolic engineering nicotinamide adenine dinucleotide (NAD+)and and industrial biocatalysis today. STH of P. fluo- nicotinamide adenine dinucleotide phosphate rescens was used to a cell-free system for efficient (NADP+). STH specifically transfers the 4B hydrogen cofactor cycling, resulting in high yields of the from the nicotinamide ring of both NADH and important semisynthetic opiate drug hydromor- NADPH to NADP+ and NAD+, respectively (Boonstra phone. In addition, an enzymatic system for the et al., 2000). The enzyme has been studied in some regeneration of redox cofactors NADH and NADPH detail from Pseudomonas fluorescens, Pseudomonas was established in nanostructural reverse micelles aeruginosa and Azotobacter vinelandii. It is a soluble using the soluble transhydrogenase of E. coli cou- flavoprotein containing flavin adenine dinucleotide pled with the expression of glycerol dehydrogenase (FAD+) and is remarkable for its formation of large (Ichinose et al., 2005). polymers. The kinetic behavior of STH showed that In E. coli, STH is encoded by an udhA gene. The protein, UdhA, is composed of 466 amino acids and was found to be 59% identical and 77% similar to * Corresponding author. Phone/Fax: +86-553-3883592; E-mail: [email protected] P. fluorescens STH (Boonstra et al., 1999). 276 H. ZHAO et al. However, its physiological role remains obscure and enzymes were purchased from New England little is known about its three dimensional structure. Biolabs. QIAquick gel extraction kit (Qiagen) was Recently, it was reported that a mutant E. coli strain used to purify DNA from agarose gels. Plasmids and with the deletion of pgi gene (encoding phosphoglu- genomic DNA were extracted using Wizard® purifi- cose isomerase) and udhA gene did not grow on cation kits (Promega). HerculaseTM enhanced DNA glucose, but the growth could be recovered upon polymerase (Stratagene) was used in all PCR tests. plasmid-based expression of UdhA, seeming that Total RNA was extracted using SV Total RNA UdhA catalyses the reoxidation of NADPH. Isolation System (Promega). The reverse transcrip- Additionally, the UdhA mutant did not grow on tion was performed with Superscript II reverse tran- acetate at all, presumably because extensive catab- scriptase (Invitrogen). LightCycler-FastStart DNA olism of acetate through the tricarboxylic acid (TCA) Master SYBR Green I Kit (Roche Applied Science) cycle generated more NADPH than was required for was used for RT-PCR tests. biosynthesis (Sauer et al., 2004). During growth on a rich carbon source, such as glu- Plasmid cloning. A 2.4-kb XhoI-KpnI fragment, cose, E. coli can proliferate from a tiny population to spanning 0.4 kb of upstream sequence (flanking a high density speedily. However, on acetate, E. coli sequence a, Fs-a) and 0.6 kb of downstream has to force the flux of isocitrate through the gly- sequence (flanking sequence b, Fs-b) of udhA gene, oxylate bypass for preventing the quantitative loss was generated by PCR from the genomic DNA of E. of the acetate as CO2 in the TCA cycle (Miller et al., coli MG1655 with the forward primer 5’-TTGTCTC- 2000). When transferred from glucose to acetate, GAGTTCCAGATAGTCCGCAAGTTCCGC-3’ and the the redistribution of NADH/NADPH ratio in E. coli reverse primer 5’-TGTAGGTACCACATTGGTTTGATTC- was required for cell growth because NADH and CCACAGTTG-3’. The product was then digested and NADPH play as major cofactors in energy production ligated to pSP72 (Promega) XhoIand KpnIback- and biosynthesis. UdhA is one of two transhydroge- bone fragment, creating pUW (~ 4.8 kb) (Fig. 1). nases for maintaining the balance between NAD(H) Next, the MfeI-XmaI fragment carrying the kan pool and NADP(H) pool in cells. gene was amplified from pKD13 (Datsenko et al., In this work, we were interested in elucidating the 2000) using the forward primer 5’-TAGTTAGT- important function of UdhA for acetate-grown E. CAATTGTGAGTTGAGCGATTGTGTAGGCTGGAG-3’ coli. We used homologous recombination to con- and the reverse primer 5’-ATAGTATTCCCGGGT- struct recombinant strains with the deletion of udhA TATAGTGATAAGCTGTCAAACATGAG-3’. The resulting gene in different genetic background. The growth product was inserted into MfeIand AgeIsites of rates, activity assay and RT-PCR analysis were pUW, creating pUK (~ 5.1 kb) (Fig.1). employed to investigate the physiologic roles of UdhA in E. coli during growth on acetate. The pres- ent paper is, to our knowledge, the first report Strain construction by chromosomal homolo- about the effect of the absence of UdhA on the isoc- gous recombination. The udhA gene was disrupt- itrate dehydrogenase (IDH) activity and NADH ed using the phage λ recombination (Red) system dehydrogenase II (NDH-2) expression in E. coli. (Datsenko and Wanner, 2000). Linear PCR product containing kan gene and flanking sequence on either side of udhA gene from pUK was introduced MATERIALS AND METHODS into competent E. coli MG1655 carrying pKD46 by CaCl2 transformation (Johnsonans Brooker, 1999). Bacterial strains. Escherichia coli MG1655 provid- Cells were shocked at 42 °C and recovered at 37 °C ed the wild type background for the experiments. in SOC (it is identical to SOB medium, except that it Strain MM294 (glnV44(AS), λ¯, rfbD1?, endA¯, contains 20 mM glucose.), and then spread on LB spoT1?, thi¯, hsdR17, recA¯) was used as host for plates to select kanamycin-resistant transformants. routine cloning. Escherichia coli strains containing The disruption of udhA gene was confirmed by natural NADP-dependent IDH or engineered NAD- sequencing the PCR products whose regions dependent IDH (Zhu et al., 2005) were a gift from spanned 1 kb of flanking DNA on either side of kan Antony M. Dean’s laboratory (BioTechnology gene. The kanr cassette replacing udhA gene was Institute, University of Minnesota, MN 55108, USA). transduced using P1 (cml, clr100) into a fresh E. coli MG1655 genetic background, resulting in strain Media and reagents. LB, SOB, SOC and MOPS- ΔudhA. Then the different genetic backgrounds con- based minimal medium were prepared as described taining either icdNADP or icdNAD, which encodes NADP- elsewhere (Neidhardt et al., 1974; Stueland et al., dependent IDH or engineered NAD-dependent IDH, 1988; Miller, 1992; Zhu et al., 2005). All media were introduced into ΔudhA by cotransduction with were supplemented with 100 µg/ml ampicillin, 15 the tetr cassettes, respectively (Zhu et al., 2005). µg/ml tetracycline, 30 µg/ml kanamycin, and 20 The resulting strains were denoted icdNADP/ΔudhA NAD µg/ml chloramphenicol as required. Restriction and icd /ΔudhA (Fig. 1). The chromosome-inte- Ann. Microbiol., 58 (2) 275-280 (2008) 277 pUW (~ 4.8 kb) amp Fs-a udhA Fs-b XhoI MfeI AgeI KpnI Digestion of MfeI+AgeI MfeI XmaI Ligation kan cassette pUK (~ 5.1 kb) amp Fs-a kan cassette Fs-b Chromosome of wild type Fs-a udhA Fs-b Double crossover Chromosome of udhA kan cassette P1 Transduction of icdNADP and icdNAD Chromosome of icdNADP/udhA kan cassette icdNADP Chromosome of icdNAD/udhA kan cassette icdNAD FIG. 1 - Schematic illustration of the disruption of udhA gene. The disruption of udhA gene was constructed in wild type Escherichia coli strain MG1655 (Datsenkoand Wanner, 2000). Then the kan cassette was transduced using P1 (cml, clr100) into a fresh E.