Nonlinear Biosynthetic Gene Cluster Dose Effect on Penicillin Production by Penicillium chrysogenum

Abstract

Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the ␤-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [␦-(L-␣-aminoadipyl)-L-cysteinyl-D-valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.

Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a β-Lactam High-ProducingPenicillium chrysogenumStrain

“…The vector and the fragment were recombined with BP clonase yielding pDONR23-3¢FR 16g14010. pDONRÔ 221 Gateway Ò vector (Invitrogen) was used to clone the A. nidulans acetamidase-gene (amdS) gene under control of the A. nidulans gpdA promoter and penDE terminator, which were amplified from plasmid pBlueAMDS (Nijland et al, 2010) with primers attB1F AMDS and attB2R AMDS. The three fragments were then combined with the pDESTÔ P4-R3 in the LR reaction of the Multisite Gateway …”

“…However, subsequent analysis showed that this reduced productivity coincided with the loss of all but one of its six to eight penicillin gene clusters. Previous studies have shown the instability of the number of penicillin gene clusters in high-producing strains, caused by recombination of the direct repeats in these clusters (Fierro et al, 1995;Nijland et al, 2010). Indeed, protoplast transformationassociated loss of penicillin gene cluster amplicons has been used in the construction of a ''cluster-free'' P. chrysogenum strain (DS50661) (Harris et al, 2009a).…”

Increased Penicillin Production in Penicillium chrysogenum Production Strains via Balanced Overexpression of Isopenicillin N Acyltransferase

“…While the protein levels of ACVS and IPNS increased accordingly (4-to 5-fold), the IAT protein level increased only 2-fold and saturated already at low gene copy numbers. This suggested that IAT might be the limiting factor in penicillin V production by high-yielding strains (19). To examine this phenomenon, a targeted overexpression of the penDE gene was initiated using the P. chrysogenum strains DS47274 (one cluster copy), DS47273 (three clusters), and DS17690 (seven or eight clusters) (9).…”

“…Previously, we have studied a series of isogenic strains that differed in the copy numbers of the penicillin biosynthesis cluster to reveal potential bottlenecks in high-yielding strains (19). The study showed the accumulation of substantial levels of isopenicillin N in strains harboring high numbers of the biosynthetic gene cluster.…”

“…This remarkable lack of correlation between the protein and transcript levels of the IAT enzyme suggested that the amount of active IAT is limiting in high-yielding strains. This was further evidenced by the accumulation of isopenicillin N in strains harboring high copy numbers (19). Alternatively, side chain activation could be a limiting factor in these high-yielding strains, but under those circumstances the intracellular accumulation of 6-aminopenicillanic acid (6-APA) is expected rather than the formation of isopenicillin N because of the hydrolase properties of the IAT enzyme.…”