Coexpression of Vitreoscilla Hemoglobin Reduces the Toxic Effect of Expression of D-Amino Acid Oxidase in E. coli Liang-Jung Chien, † Jyh-Ming Wu, † I-Ching Kuan, ‡ and Cheng-Kang Lee* ,† Department of Chemical Engineering, National Taiwan University of Science and Technology, and Department of Bioengineering, Tatung University, Taipei, Taiwan Expression of the gene (daao) encoding D-amino acid oxidase (DAAO) in Escherichia coli typically results in a marked decrease of cell viability, and it has generally been assumed that the consumption of intracellular D-alanine by DAAO is responsible for this effect. Vitreoscilla hemoglobin (VHb) gene (vgb) was coexpressed with Rhodospo- ridium toruloides D-amino acid oxidase in E. coli BL21(DE3) and BL21(DE3)pLysS, expression hosts differing in the stringency of suppressing basal transcription. Not only was the toxic effect of DAAO on cell growth relieved but also the pronounced cell lysis of BL21(DE3)pLysS caused by the expression of DAAO was prevented by coexpressing VHb with DAAO. As a result of the higher cell density achieved, DAAO activity about 1.5-fold higher than that of DAAO-expressing control strains could be obtained by DAAO/VHb-coexpressing strains. The relieving effect of VHb on DAAO toxicity resulted from its oxygen-binding ability. The low availability of free intracel- lular oxygen reduced DAAO activity and consequently its toxicity. Introduction D-Amino acid oxidase (DAAO, EC 1.4.3.3) is a flavo- enzyme containing FAD as the prosthetic group. It catalyzes the oxidative deamination of D-amino acids to the corresponding 2-oxoacids and ammonia with the simultaneous reduction of molecular oxygen to hydrogen peroxide. DAAO has considerable industrial importance because it has been used for the production of 7-amino cephalosporanic acid (7-ACA) from cephalosporin C (CPC) in a process involving two enzymatic reaction steps (1). Several other potential biotechnological applications involving DAAO are the production of R-keto acids (2), resolution of D/L-amino acid (3), and preparation of biosensors (4). DAAOs were found in a wide variety of organisms, from microorganisms to vertebrates. DAAOs from pig kidney (5), Rhodotorula gracilis (6), Trigonopsis variabilis (7), and Fusarium solani (8) have been isolated, and their genes daao have been cloned, sequenced, and expressed successfully in yeast and E. coli (9-14). However, the expression of DAAO in E. coli encountered the problem of a marked decrease of cell viability and growth (12- 14). It has generally assumed that the consumption of the intracellular pool of D-alanine by the expressed DAAOs, which interferes with biosynthesis of the cell wall, is responsible for the toxic effect of DAAO on cell growth. Besides, the H 2 O 2 produced by oxidation of D-amino acids is also deleterious to the cells (15). Since there are many advantages of employing E. coli as an expressing host for the production of recombinant pro- teins on a large scale (16, 17), a strategy to relieve the toxic effect of DAAO in E. coli is needed if DAAO is to be produced on an economic scale. Vitreoscilla hemoglobin (VHb) is an oxygen-binding homodimeric protein (15,775 Da/subunit) that has been successfully expressed in several organisms to increase microaerobic cell growth and enhance oxygen-dependent cell metabolism (18-22). Because of its very effective oxygen-binding ability, the possibility of employing VHb to inhibit DAAO activity by reducing the oxygen concen- tration involved in DAAO reaction deserves exploration. Once DAAO activity is inhibited by the coexpressed VHb, the deleterious effect of DAAO on cell growth will be relieved. DAAO fused with VHb as a chimeric enzyme (DAAO-VHb) has been expressed in E. coli by Khang et al. (23). The fusion with VHb enhanced the immobilized DAAO activity and stability in the bioconversion process of cephalosporin C. However, the effect of expression the chimeric DAAO-VHb on cell viability and growth has never been discussed. In this work, the possibility to relieve the toxic effect of the overexpressed DAAO on cell growth by coexpressing VHb in E. coli was investigated. Expression hosts with different stringencies of suppress- ing basal transcription were employed to study the expression of the toxic daao gene. In vitro testing of the effect of VHb on DAAO and glucose oxidase activity was also carried out to demonstrate that the toxicity relieving effect of VHb is a result of its oxygen-binding ability. Materials and Methods Bacterial Strains and Plasmids. E. coli TOP10 [F - mcrA Δ(mrr-hsdRMS-mcrBC)Φ80lacZΔM15ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Str R ) endA1 nupG] (Invitrogen) was used as a cloning host. E. coli BL21(DE3) [F - ompT hsdS B (r B - m B - )gal dcm (DE3)] and BL21(DE3)pLysS [F - ompT hsdS B (r B - m B - )gal dcm (DE3)pLysS (Cm R )] (Novagen), which differ in stringency of suppressing basal transcription, were used as expression hosts. Plasmid pET 30b(+) (Novagen), which contains the IPTG inducible T7 promoter and the lacI repressor, was used as an expression vector. Plasmid * To whom correspondence should be addressed. Fax: +886-2- 27376629. E-mail: cklee@ch.ntust.edu.tw. † National Taiwan University of Science and Technology. ‡ Tatung University. 1359 Biotechnol. Prog. 2004, 20, 1359-1365 10.1021/bp0498589 CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers Published on Web 09/04/2004