리파마이신 생합성을 위한 유전자의 클로닝

경원대학교 공과대학 화학공학과

강 익 중

Ⅰ. INTRODUCTION

The ansamycins have a very broad spectrum of biological activities with greate significance for both scientific and practical reasons. One number of the rifamycin, rifampicin, has proved to be an excellent, orally active antibiotic. It is now in widespread clinical use, especially an  antitubercular agent but also in the treatment of various other infective diseases. The unique mode of action of some of the ansamycin has aroused much interest1. Rifamycin and other ansamycin have been shown to inhibit bacterial transcription by interacting with DNA-dependent RNA polymerase. Maytansine and related compounds inhibit cell division by  interfering with the polymerization of tublin2. Streptovericins show antiviral activity, especially against the HIV virus3,4.

Floss and coworkers5 proposed a new route for the formation of AHBA which branches  from the shikimate pathway. A variety of antibiotics, notablely the ansamycin, e.g.,  rifamycin B6(1), and the mytomycins7, contain a biosynthetically unique moiety, called an mC7N unit, consisting of a six-membered carbonylic ring carring an extra carbon and a nitrogen in a meta arrangement. Extensive tracer7,8 and genetic experiments have demonstrated the  shikimate pathway origin of this mC7N unit, although neither skimate acid3,5,6 nor dehydroquinic acid8 were incoprated, and have identified 3-amino-5-hydroxybenzoic acid(AHBA, 4) as its proximate precusor7. the nitrogen of the mC7N unit is linked to the  carbon corresponding to C-5, not C-3 of, for example, dehydroshikimatic acid8,9, and its origin, in the case of 1, has been traced to the amide nitrogen of glutamine11-12. This and  mechanistic consideration led us to propose the pathway shown in scheme 1 for the formation of 4. All the previous results from biological and genetic studies on the AHBA  biosynthetic pathway led several groups to suggest that 5-amino-5-deoxy-dehydroquinic acid(aminoDHQ) would be good candidates as intermediate in the formation of AHBA. Enzyme formation  of  a radioactive compound co-migrating with aminoDHQ from 14C-PEP, erithrose 4-phosphate  and glutamine has been observed in cell-free extracts of N.mediterranei and of the ansatrienin producer, S.collinus.

DNA encoding an entire protein of interest can be obtained starting with only a limited amino acid sequence by utilizing recombinant DNA methods. Large amounts of the protein  can then be produced by introducing the DNA into a appropriate heterologous expression system. In addition, the complete amino acid sequence of a protein can be determined from the recombinant DNA clone. This information, coupled with comparison to previously characterized proteins can be used to gain tremendous insight into the structural and functional features of the protein. The large quantities of protein needed to define the  reaction mechanism of the protein at a molecular level are thus avaliable through recombinant technology. We designed degenerate oligonuceltides based on short sequences of amino acids from aminoDHQ synthase in order to use these as primers for amplification of genomic DNA by the polymerase chain reaction(PCR) and as probes for southern hybridization. A PCR  product was used to screen at very high stringency colonies, obtained from a cosmid library of genomic DNA containing the AminoDHQ synthase gene. A 396bp fragment prepared from total DNA. The subcloned DNA fragement will be sequenced.

Ⅱ.  EXPERIMENTAL

UV-Vis measurements were made with Hewlett-Packard 4854A diode array spectrophotometer. Shake flasks fermentation were carried out in New Brunswick G24 or G25 controlled environment incuber shaker, or in an Aldorf Kuhner ISF-4-V rotary shaker  cabinet. Centrifugations were done in a Sorvall(Du pont) RC5C refrigerated centrifuge  operating at 40C using SS-35, GSA, and GS-3 rotors. The cell cleavage was carried out in French Pressure cell press(American Instrument company) or Versonic sonificator   (Versonic  Cell Distributor). Scintillating counting were carried out in Beckmann Instruments, Inc. Scintillating Counter(LS 1801).

Total genomic DNA of N. mediterranie S699 was used to amplify an expected 396bp region.  Because of the high G+C content of the primers and the genomic DNA template, a modified  amplification cycl was used. 6mins melting step(950C) without tag DNA polymerase was  followed by 5mins combined step(700C) of annealing and polymerization after adding the polymerase. After this modified first cyles consisted of melting(950C, 1min), annealing(680C,  1min) and polymerization(740C, 1min) were run, the polymerization of the final cycle lasted  for 5mins. The modified amplification procedure led to an increased concentration of amplified DNA and a decrease in non-specific products produced by the degeneracy of primers.  Addition of 4 to 5% glycerol also helped to decrease non-specific products. The total  genomic DNA produced 396bP and 800bP PCR products respectively. Suggesting that the internal band 1 shoud be the expected band of aminoDHQ Synthase. The PCR products were purified by preparative low-melting agarose gel electrophoreisis.

The PCR product were purified by preparative low melting agarose gel electrophoresis.  The product were cloned for sequencing. PSK+ vector, which contains the lacZ gene to provide a-complementation for bule/white color selection of recombinant plasmid, was used as a cloning vector. The PCR products were filled in using Klenow enzyme(PCR products often  have non-complementary cohesive ends which could not be igated into the vector, digested  with a restriction enzyme, producing a blunt-end DNA). The resultant PCR products were  ligated into the EcoRV site of the PSK+vector and transformed into competent cells of E.coli  XL1-Blue. Since the efficiency of ligation between blunt-end DNAs was very low, the vector and insert were not dephosphorylated to improve ligation yield. Dephosphorylation of the  vector contained one or more inserts. After transformation of the ligation products, several  hundred white colonies were obtained. Two colonies for the clone containing the 396bP PCR product each of which had an insert ligated into PSK+ in two different direction, and one  colony for the clone containing 396bP PCR product were selected for sequencing. For obtaining of the pure PCR product and confirming the correct base pair, the transformated white colony was cultivated in the LB medium. The plasmid was isolated from the cell and identified by PvuⅡ restriction enzyme

Ⅲ. Results and Discussion

The PCR product, DNA of N. mediterranei, was cloned for sequencing. PSK+ Vector, which contains the lacZ gene to provide  -complementation for bule/white color selection of recombinant plasmid, was used as a cloning vector. The PCR products were filled in using  Klenow enzyme(PCR products often have non-complementary cohesive ends which could not  be ligated into the vector, digested with a restriction enzyme, producing a blunt-end DNA).  The resultant PCR products were ligated into the EcoRV site of the PSK+vector and transformed into competent cells of E.coli XL1-Blue. Since the efficiency of ligation between blunt-end DNAs was very low, the vector and insert were not dephosphorylated to improve ligation yield. Dephosphorylation of the vector contained one or more inserts. After transformation of the ligation products, several hundred white colonies were obtained. Two  colonies for the clone containing the 396bP PCR product each of which had an insert ligated into PSK+ in two different direction, and one colony for the clone containing 396bP PCR product will be selected for sequencing.

REFERENCES

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