Expression of cefD2 and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis

Abstract

The conversion of isopenicillin N into penicillin N in Acremonium chrysogenum is catalyzed by an epimerization system that involves an isopenicillin N-CoA synthethase and isopenicillin N-CoA epimerase, encoded by the genes cefD1 and cefD2. Several transformants containing two to seven additional copies of both genes were obtained. Four of these transformants (TMCD26, TMCD53, TMCD242 and TMCD474) showed two-fold higher IPN epimerase activity than the untransformed A. chrysogenum C10, and produced 80 to 100% more cephalosporin C and deacetylcephalosporin C than the parental strain. A second class of transformants, including TMCD2, TMCD32 and TMCD39, in contrast, showed a drastic reduction in cephalosporin biosynthesis relative to the untransformed control. These transformants had no detectable IPN epimerase activity and did not produce cephalosporin C or deacetylcephalosporin C. They also expressed both endogenous and exogenous cefD2 genes only after long periods (72-96 h) of incubation, as shown by Northern analysis, and were impaired in mycelial branching in liquid cultures. The negative effect of amplification of the cefD1 - cefD2 gene cluster in this second class of transformants is not correlated with high gene dosage, but appears to be due to exogenous DNA integration into a specific locus, which results in a pleiotropic effect on growth and cefD2 expression.

Solid-State and Submerged Fermentations Show Different Gene Expression Profiles in Cephalosporin C Production by <b><i>Acremonium chrysogenum</i></b>

“…Ullán et al [2004] reported that the reaction in which this enzyme is involved is a limiting step in CPC production in SmF. This limitation was also observed in our SmF versus SSF experiment, where the cefD2 gene showed from 2-to over 10-fold higher expression in SSF than in SmF, concurring with a better intermediate transformation in the CPC biosynthesis pathway during the culture ( fig.…”

“…6.4, but these same activities were faster in SSF at a higher pH. Larger amounts of penicillin N were observed only in SmF at the time of maximum CPC production rate, suggesting a better transformation of intermediates in SSF that helps to overcome the limiting step from penicillin N to deacetoxy-CPC, or the previous one from isopenicillin N to penicillin N [Rodríguez- Sáiz et al, 2004;Skatrud and Queener, 1989;Ullán et al, 2004;Zhou et al, 1992].…”

“…cefD2 Gene Expression This gene encodes a protein showing high similarity to acyl-CoA racemases, and its activity is involved in the epimerization of isopenicillin N to penicillin N in the CPC biosynthesis pathway [Ullán et al, 2004]. Results of expression analysis showed a higher expression of cefD2 in SSF than in SmF ( fig.…”

Utilization of glycerol as cysteine and carbon sources for cephalosporin C production by Acremonium chrysogenum M35 in methionine-unsupplemented culture

“…Recently, it was reported that there is a clear correlation between increased IPN-CoA activity and cephalosporin overproduction by amplification of the cefD2 gene in A. chrysogenum. In this study, when the cefD2 gene was amplified in A. chrysogenum, the level of IPN was decreased whereas the level of PN was increased during fermentation compared to the wild-type strain (Ullan et al, 2004). Therefore, it is expected that upregulation of pcbAB and cefD2 by glycerol during exponential phase affects cephalosporin C production by A. chrysogenum M35.…”

Characterization of a novel peroxisome membrane protein essential for conversion of isopenicillin N into cephalosporin C

“…Digested total DNA was separated by agarose gel electrophoresis and blotted onto nylon membranes (Hybond NX, Amersham Pharmacia Biotech) [20]. Southern blot hybridization was performed as previously described [21].…”