Streptomyces clavuligerus is a gram-positive, filamentous bacterium which has a great ability to produce secondary metabolites including isopenicillin N, cephamycin C and a beta-lactamase inhibitor clavulanic acid. Clavulanic acid (CA) which is a bicyclic beta-lactam, inhibits most of class A beta-lactamases by binding irreversibly to the serine hydroxyl group at the active center of beta-lactamases and resulting in the stable acyl-enzyme complexes. Clavaminate synthase (CAS) is one of the best characterized enzymes in the CA biosynthesis pathway regarding its mechanism, activity and structure. It exists as two isoenzymes, CAS1 and CAS2. cas1 gene is located in clavam biosynthetic gene cluster and its expression is nutritionally regulated. cas2 gene encodes for the rate
limiting CAS2 isoenzyme which catalyzes the three distinct oxidative transformations. Conversion of deoxyguanidinoproclavaminic acid into guadinoproclavaminic acid takes part in early steps of CA biosynthesis and is
catalysed by CAS2. Similarly, proclaviminic acid is converted into claviminic acid by a two-step reaction and also catalysed by CAS2. Integration of an extra copy of the cas2 gene into the chromosome of a clavulanic acid over-producer industrial strain of Streptomyces clavuligerus by means of an integration vector to improve CA production capacity of the industrial strain is the
main goal of this study. Via comperative CA analysis based on HPLC, it was estimated that the recombinant strains produced higher amount of CA in comparison to the parental industrial S. clavuligerus strain. The highest CA production achieved
by the recombinant strain, namely S. clavuligerus GV61 (1583.3 &mu / g/g) corresponded to more than 2 fold increase in the maximal CA titer of the parental strain.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12612576/index.pdf |
| Date | 01 October 2010 |
| Creators | Vanli, Guliz |
| Contributors | Ozcengiz, Gulay |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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