Strain improvement for cephalosporin production by Acremonium chrysogenum using geneticin as a suitable transformation marker

Abstract

An Acremonium chrysogenum strain improvement program based on the transformation with cephalosporin biosynthetic genes was carried out to enhance cephalosporin C production. Best results were obtained with cefEF and cefG genes, selecting transformants with increased cephalosporin C production and lower accumulation of biosynthetic intermediates. Phleomycin resistant transformants, designated B1 and C1, showed a single copy random integration event, higher levels of cefEF transcript and, according to immunoblotting analyses, higher amounts of deacetylcephalosporin C acetyltransferase (DAC-AT) protein than their parental strains. Moreover, DAC-AT activity was higher in the transformants. Plasmids carrying geneticin resistance markers based on the nptII gene from Tn5 and the aphI gene from Tn903 were constructed to transform again B1 and C1, showing that the cassette Pgdh-nptII-trpC was able to confer geneticin resistance to A. chrysogenum and demonstrating that geneticin is a helpful selection marker.

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

“…Knowledge of the limiting steps in cephalosporin biosynthesis is very important for the development of new strains with improved cephalosporin production (Skatrud et al 1989;Rodrı´guez-Sa´iz et al 2004). Many attempts have been made to construct genetically manipulated fungal strains with improved b-lactam antibiotic production, but interpretation of the results has been hampered by the lack of basic knowledge about the real rate-limiting steps in the process.…”

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

“…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].…”

Nonribosomal Peptide Synthesis

“…However, as the number of fungal genome sequences continues to expand it is clear the Adenylation domain specificity code of eukaryotic NRP synthetases will become clearer. The prototype and one of the most widely exploited NRPS pathways remains the industrial production of cephalosporins by the filamentous fungus A. chrysogenum, where, as noted above, the NRP synthetase, ACV sythetase, directs the biosynthesis of the penicillin precursor ACV from the cognate substrate amino acids [9,10,26,27]. For a significant period this system provided the majority of information on fungal NRPS; however, Specifically, the high-affinity hydroxamate siderophores found in many fungal species, and which are responsible for environmental Fe 3 þ scavenging and storage in fungi, are NRPs [28][29][30][31].…”