Expandase/hydroxylase

SM Samson, JE Dotzlaf, ML Slisz, GW Becker, RM Van Frank, LE Veal, WK Yeh, JR Miller, SW Queener, TD Ingolia. Cloning and expression of the fungal expandase/hydroxylase gene involved in cephalosporin biosynthesis. Bio/Technol-ogy 5 1207-1214, 1987. 

WK Yeh, JE Dotzlaf, GW Huffman. Biochemical characterization and evolutionary implication of (3-lactam expandase/hydroxylase, expandase and hydroxylase. In H Kleikauf, H vonDohren, eds. 50 Years of Penicillin Application History and Trends. Prague Public, 1994, pp 208-223. 

For monitoring the activity of the expandase/hydroxylase-system a method described by Jensen et al. [54] and modified by the authors of [49] was used. The assays contained PEN DAOC as substrate and a system with DTT, FeS04, KCl, MgS04, ascorbic acid, a-ketoglutarate and TRIS/HCl as buffer. After 25 min, at 25 °C, the reaction can be stopped by cool methanol. Also protease inhibitors were used as well and some disadvantages were observed. The PEN was difficult to locate because there are some inhibitor peaks at the same retention time, there was even DTT and some cosubstrates leaving the column at the same time. However, it is possible to measure the activity over the whole cultivation time if many tests are done. The detection of DAOC and DAC was carried out with the same method as the cultivation byproducts. 

The investigations indicated that the productivity in complex media is much higher than in synthetic and semi synthetic media. With 100 g I1 PF, the product concentration in the stirred tank was twice as high as with 30 g I1 PF and three times as high as one without PF. With another medium, the CPC concentration was twice as high with CSL than without it. The maximum CPC concentration was obtained with CSL and amino acid supplement. The medium compositions for optimal CPC concentration, CPC yield coefficient and CPC space time yield are different. ACVS is the bottleneck in the biosynthesis in the first phase of the cultivation. After 100 h, the expandase/hydroxylase becomes the bottleneck. In this phase, DAC and PEN concentrations considerably increase, but the CPC concentration varies only slightly. By the suppression of the PEN formation the production of DAOC, which impairs the purification of CPC, is reduced as well. 

When Penicillium chrysogenum contains an expandase/hydroxylase as well as an acetyltransferase from Acremonium chrysogenum, then 7-aminocephalosporan-ic acid is generated correspondingly. 

FIGURE 11 Pathways for the production of 7-ADCA and 7-ACA directly by fermentation. In this process the atyl-transferase converts isopenicillin N to adipoyl-6-APA, and when the expandase of S. clavuligerus is expressed in P. chrysogenum, this compound is directly converted to adlpoyl-7-ADCA. When P chrysogenum is transformed with the expandase/hydroxylase and the acyltransferase of Acremonium chrysogenum, adipoyl-6-APA is converted first to adipoyl-7-ADACA, which is further converted to adipoyl-7-ACA. 

S Kovacevic, JR Miller. Cloning and sequencing of the (3-lactam hydroxylase gene (ce/F) from Streptomyces clavuligerus gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes. J Bacteriol 173 398-400, 1991. 

The biosynthesis of CPC is well known. The first step of CPC synthesis is the formation of the tripeptide S-(L-a-aminoadipyl)-L-cysteinlyl-D-valine (LLD-ACV) from L-a-aminoadipinic acid (AAA), L-cystein (CYS ) and D-valine (VAL) by the LLD-ACV synthetase (ACVS). LLD-ACV is converted with the enzyme isopenicillin AT-synthetase (cyclase) to isopenicillin N (IPN) and then with isopenicillin N-epimerase to penicillin N (PEN). The 6-ring is formed by deacetoxycephalosporin C-synthase (expandase). Deacetoxycephalosporin C (DAOC) is converted with deacetoxycephalosporin C-hydroxylase to decacetylcephalosporin C (DAC) and the latter with deacetylcephalosporin C-acyltransferase to CPC (Fig. 1). Except the amino acid and ACV, all others were monitored by on-line HPLC. 

DAOC-expandase and DAC-hydroxylase activities in A. chrysogenum are exerted by the same enzyme (MW 41 kD), which like the IPNS belongs to the group of a-ketoglutarate-dependent dioxygenases. 

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