基因工程外文翻譯--由共表達碳酰還原酶和葡萄糖脫氫酶的大腸桿菌轉(zhuǎn)化（英文）

1、Abstract The asymmetric reduction of ethyl 4-chloro-3- oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxy- butanoate ((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene f

2、rom Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the catalyst. In an organic-solvent-water two-phase system, (S)-CHBE formed in the organic phase amounted to 2.58 M (43

3、0 g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulat- ed 1.25 M (208 g/l) (S)-CHBE in an aqueous mono- phase system by continuously feeding on COBE, which is unstable in an aqueo

4、us solution. In this case, the calcu- lated turnover of NADP+ (the oxidized form of nicotina- mide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomer

5、ic excess in both systems. The aqueous system used for the reduction reaction in- volving E. coli HB101 cells carrying a plasmid contain- ing the S1 and GDH genes as a catalyst is simple. Fur- thermore, the system does n

6、ot require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.IntroductionOptically active 4-chloro-3-hyd

7、roxybutanoic acid esters are useful chiral building blocks for the synthesis of pharmaceuticals. The (R)-enantiomer is a precursor of L-carnitine (Zhou et al. 1983), and (S)-enantiomer is an important starting material f

8、or hydroxymethylglutaryl- CoA (HMG-CoA) reductase inhibitors (Karanewsky et al. 1990). Many studies have described the microbial or enzymatic asymmetric reduction of 4-chloro-3-oxobuta- noic acid esters (Aragozzini and V

9、alenti 1992; Bare et al. 1991; Hallinan et al. 1995; Patel et al. 1992; Shimizu et al. 1990; Wong et al. 1985) based on the reduction by baker’s yeast (Zhou et al. 1983). We have previously showed that Candida magnoliae

10、 AKU4643 cells reduced ethyl 4-chloro-3-oxobutanoate (COBE) to (S)-CHBE with an optical purity of 96% enantiomeric excess (e.e.) (Yasohara et al. 1999). As this yeast has at least three different stereoselective reduct-

11、ases (Wada et al. 1998, 1999a, b), the (S)-CHBE pro- duced by this yeast was not optically pure. From among these three enzymes, an NADPH-dependent carbonyl re- ductase, designated as S1, was purified and characterized i

12、n some detail (Wada et al. 1998). We cloned and se- quenced the gene encoding S1 and overexpressed it in Escherichia coli cells. This E. coli transformant reduced COBE to optically pure (S)-CHBE in the presence of glucos

13、e, NADP+, and commercially available glucose dehydrogenase (GDH) as a cofactor generator (Yasohara et al. 2000). Here, we describe the construction of three E. coli transformants coexpressing the S1 from C. magnoliae and

14、 GDH from Bacillus megaterium genes and analyze the reduction of COBE catalyzed by these strains. Previ- ous reports on the enzymatic reduction of COBE to (R)-CHBE with an optical purity of 92% e.e. (Kataoka et al. 199

15、9; Shimizu et al. 1990) recommended an orga- nic-solvent two-phase system reaction for an enzymatic or microbial reduction, because the substrate (COBE) is unstable in an aqueous solvent and inactivates enzymes. We exami

16、ned the reduction of COBE to optically pureN. Kizaki (?) · Y. Yasohara · J. Hasegawa Fine Chemicals Research Laboratories, Kaneka Corporation, 1–8 Miyamae, Takasago, Hyogo 676–8688, Japan e-mail: Noriyuki.Kiza

17、ki@kaneka.co.jp Tel.: +81-794-452415, Fax: +81-794-452668M. Wada · M. Kataoka · S. Shimizu Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 61

18、6–8502, JapanPresent address: M. Wada, Department of Bioscience, Fukui Prefectural University, 4–1-1 Kenjyojima, Matsuoka-cho, Fukui 910–1195, JapanAppl Microbiol Biotechnol (2001) 55:590–595 DOI 10.1007/s002530100599O R

19、 I G I N A L PA P E RN. Kizaki · Y. Yasohara · J. Hasegawa · M. Wada M. Kataoka · S. Shimizu Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli transformant cells

20、 coexpressing the carbonyl reductase and glucose dehydrogenase genesReceived: 7 June 2000 / Received revision: 11 November 2000 / Accepted: 8 December 2000 / Published online: 7 April 2001 © Springer-Verlag 2001592

21、various concentrations of CHBE, was incubated at 30 °C for 24 h in order to study the enzyme’s stability in the presence of CHBE. The remaining enzyme activities were assayed as described above.COBE reduction with E

22、. coli cells expressing the S1 gene and E. coli cells expressing GDH genes in a two-phase system reactionThe reaction mixture comprised 15 ml culture broth of E. coli HB101 carrying pNTG, 17 ml culture broth of E. coli H

23、B101 car- rying pNTS1, 1.6 mg NADP+, 4 g glucose, 2.5 g COBE, 25 ml n-butyl acetate, and about 25 mg Triton X-100. The pH of the re- action mixture was controlled at 6.5 with 5 M sodium hydroxide. At 2 h, 1.25 g COBE and

24、 2.5 g glucose were added to the reaction mixture. To compare the reaction by E. coli transformant coex- pressing the GDH and S1 genes, 30 ml culture broth of E. coli HB101 carrying pNTS1G was used instead of culture bro

25、th of E. coli HB101 carrying pNTG and E. coli HB101 carrying pNTS1. Other components and the procedure were the same as de- scribed above.COBE reduction to (S)-CHBE in a two-phase system reactionThe reaction mixture cont

26、ained 50 ml of culture broth of an E. coli HB101 transformant, 3.2 mg NADP+, 11 g glucose, 10 g COBE, 50 ml n-butyl acetate, and about 50 mg Triton X-100. The reaction mixture was stirred at 30 °C, and the pH was co

27、ntrolled at 6.5 with 5 M sodium hydroxide. Five grams of COBE/5.5 g glucose and 10 g COBE/11 g glucose were added to the reaction mixture at 3 h and 7 h, respectively; 3.2 mg NADP+ was added at 26 h.COBE reduction to (S)

28、-CHBE in an aqueous system reactionThe reaction mixture was made up of 50 ml of culture broth of an E. coli HB101 transformant, 3.1 mg NADP+, 11 g glucose, and about 50 mg Triton X-100. The reaction mixture was stirred a

29、t 30 °C. Fifteen grams of COBE was fed continuously by means of a micro-feeding machine at a rate of about 0.02 g/min for about 12 h. The pH of the reaction mixture was controlled at 6.5 with 5 M sodium hydroxide. T

30、he reaction mixture was extracted with 100 ml ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and then evaporated in vacuo.AnalysisThe organic layer was obtained on centrifugation of the reaction

31、 mixture and was assayed for CHBE and COBE by gas chromatog- raphy. Optical purity of CHBE was analyzed by high-performance liquid chromatography (HPLC), as described previously (Yasohara et al. 1999).Enzymes and chemica

32、lsRestriction enzymes and DNA polymerase were purchased from Takara Shuzo (Japan). COBE (molecular weight: 164.59) was pur- chased from Tokyo Kasei Kogyo (Japan). Racemic CHBE (molec- ular weight: 166.60) was synthesized

33、 by reduction of COBE with NaBH4. All other chemicals used were of analytical grade and commercially available.ResultsConstruction of E. coli transformants overproducing S1 and GDHTo express the carbonyl reductase S1 and

34、 GDH genes in the same E. coli cells, four expression vectors were con-structed (Fig. 1). Plasmids pNTS1G and pNTGS1 con- tain the S1 gene from C. magnoliae, the GDH gene from B. megaterium, the lac promoter derived from

35、 pUC19, and the terminator derived from pTrc99A. Plasmid pNTS1 contains the S1 gene, the lac promoter derived from pUC19, and the terminator derived from pTrc99A. The enzyme activities in cell-free extracts of the E. col

36、i transformants are shown in Table 1. E. coli HB101 cells carrying the vector plasmid pUCNT had no detectable S1 or GDH activity. E. coli HB101 carrying either pNTS1G or pNTGS1 showed S1 and GDH activity with- out isopro

37、pyl-β-D-thiogalactopyranoside (IPTG) induc- tion. The S1 activities of these two transformants were lower than the GDH activities. To obtain a transformant whose S1 activity was equal to or greater than the level of GDH

38、activity, we used a lower copy vector, pSTV28 (Homma et al. 1995; Takahashi et al. 1995), to express the GDH gene. It may be possible to raise the S1 activity by lowering the GDH activity. Plasmid pSTVG contains the GDH

39、gene, the lac promoter, the chloramphenicol re- sistance gene, and the replicative origin derived from pACYC184 for compatibility with the plasmid pNTS1. In E. coli HB101 carrying pNTS1 and pSTVG, the S1 activity was hig

40、her than the GDH activity, but this GDH level may be too low to regenerate in a COBE reduction reaction as described below.Study of enzyme stabilityThe stability of S1 in the presence of various organic sol- vents is sho

41、wn in Table 2. S1 was stable in the presence of n-butyl acetate, 1-octanol, and isopropyl ether. GDHTable 1 S1 and GDH activities in cell-free extracts of E. coli transformantsPlasmid transformed Specific activity (U/mg)

42、S1 GDHpUCNT <0.01 <0.01 pNTS1 16.0 <0.01 pNTG <0.01 121 pNTS1G 11.5 77.2 pNTGS1 4.90 117 pNTS1 and pSTVG 13.5 1.60Table 2 Stability of S1 in various organic solvents. For other con- ditions, see the textOrga