Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor

Penicillium chrysogenum was successfully engineered to produce a novel carbamoylated cephalosporin that can be used as a synthon for semi-synthetic cephalosporins. To this end, structural genes for Acremonium chrysogenum expandase/hydroxylase and Streptomyces clavuligerus carbamoyltransferase were expressed in a penicillinG high-producing strain of P. chrysogenum. Growth of the engineered strain in the presence of the side-chain precursor adipic acid resulted in production of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA) and of several adipoylated pathway intermediates. A combinatorial chemostat-based transcriptome study, in which the ad7-ACCCA- producing strain and a strain lacking key genes in β-lactam synthesis were grown in the presence and absence of adipic acid, enabled the dissection of transcriptional responses to adipic acid per se and to ad7-ACCCA production. In chemostat cultures of both strains, adipic acid served as an additional carbon source. Transcriptome analysis supported an earlier proposal, based on 13C-labelling studies, that adipic acid catabolism in P. chrysogenum occurs via β-oxidation and enabled the identification of putative genes for enzymes involved in mitochondrial and peroxisomal β-oxidation pathways. Several of the genes that showed a specifically altered transcript level in ad7-ACCCA-producing cultures were previously implicated in oxidative stress responses. As strain improvement programmes lead to increased specific productivity and yields, a deeper understanding of these stress responses is likely to be important to also achieve high ad7-ACCCA titers with engineered strains of P. chrysogenum. The goal of the present study was to explore the metabolic engineering of P. chrysogenum for the production of the important semisynthetic cephalosporin precursor ad7-ACCCA. In this way, new fermentation processes for the production of this compound can be designed, resulting in a reduction of the use of environmentally unfriendly chemicals as well as a cost reduction. Genetic engineering for this purpose involved the introduction into P. chrysogenum of the cefEF gene from A. chrysogenum and the cmcH gene of S. clavuligerus. To investigate the physiological impact of the introduction of this novel pathway, physiological and genome-wide transcriptional studies were performed in batch and chemostat cultures of the engineered P. chrysogenum strains grown in the absence and presence of the side-chain precursor adipic acid. To distinguish between effects of the side-chain precursor per se and effects of ad7-ACCCA production, parallel experiments were run with a congenic strain that lacks three key genes for beta-lactam biosynthesis.