The optimization of penicillin biosynthesis in fungi

126

102

The Aspergillus nidulans velvet (veA) gene encodes a global regulator of gene expression controlling sexual development as well as secondary metabolism. We have identified the veA homologue AcveA from Acremonium chrysogenum, the major producer of the ␤-lactam antibiotic cephalosporin C. Two different disruption strains as well as the corresponding complements were generated as a prelude to detailed functional analysis. Northern hybridization and quantitative real-time PCR clearly indicate that the nucleus-localized AcVEA polypeptide controls the transcriptional expression of six cephalosporin C biosynthesis genes. The most drastic reduction in expression is seen for cefEF, encoding the deacetoxycephalosporine/deacetylcephalosporine synthetase. After 120 h of growth, the cefEF transcript level is below 15% in both disruption strains compared to the wild type. These transcriptional expression data are consistent with results from a comparative and time-dependent high-performance liquid chromatography analysis of cephalosporin C production. Compared to the recipient, both disruption strains have a cephalosporin C titer that is reduced by 80%. In addition to its role in cephalosporin C biosynthesis, AcveA is involved in the developmentally dependent hyphal fragmentation. In both disruption strains, hyphal fragmentation is already observed after 48 h of growth, whereas in the recipient strain, arthrospores are not even detected before 96 h of growth. Finally, the two mutant strains show hyperbranching of hyphal tips on osmotically nonstabilized media. Our findings will be significant for biotechnical processes that require a defined stage of cellular differentiation for optimal production of secondary metabolites.

Section: -Amino-5-carboxypentanoyl)-l-cysteinyl-d-valine Synthase (Amentioning

confidence: 85%

A Homologue of the Aspergillus velvet Gene Regulates both Cephalosporin C Biosynthesis and Hyphal Fragmentation in Acremonium chrysogenum

Dreyer

Eichhorn²,

Friedlin³

et al. 2007

126

102

“…During the last 70 years, the increase in penicillin production by industrial strains of the filamentous fungus Penicillium chrysogenum has been based mainly on classical strain improvement (1,14). The pathway of the biosynthesis of penicillin and the cellular localization of most of the critical enzymatic steps are known (12).…”

mentioning

confidence: 99%

“…Side chain activation is catalyzed by a group of enzymes termed CoA ligases. One of the enzymes involved in the activation of phenylacetic acid and phenoxyacetic acid in the biosynthesis of penicillins G and V is phenoxyacetic acid CoA ligase (PCL), which is encoded by the phl gene (Pc22g14900) (9,14). The phl gene is located outside the biosynthetic gene cluster elsewhere in the genome.…”

mentioning

confidence: 99%

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

Nijland

Ebbendorf

Woszczynska

et al. 2010

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.

“…A second obvious application for gene expression systems is the improvement of strains of commercially employed producers of pharmaceuticals, such as P. chrysogenum. In this respect, promoters with various characteristics are needed for increasing the transcript levels of the penicillin biosynthetic genes or for directed metabolic engineering in order to improve specific cellular properties (39,40). In contrast to Aspergillus, only a few homologous promoter systems have been analyzed in P. chrysogenum so far.…”

mentioning

confidence: 99%

xylP Promoter-Based Expression System and Its Use for Antisense Downregulation of the Penicillium chrysogenum Nitrogen Regulator NRE

Zadra¹,

Abt²,

Parson

et al. 2000

113

124

A highly inducible fungal promoter derived from the Penicillium chrysogenum endoxylanase (xylP) gene is described. Northern analysis and the use of a ␤-glucuronidase (uidA) reporter gene strategy showed that xylP expression is transcriptionally regulated. Xylan and xylose are efficient inducers, whereas glucose strongly represses the promoter activity. Comparison of the same expression construct as a single copy at the niaD locus in P. chrysogenum and at the argB locus in Aspergillus nidulans demonstrated that the xylP promoter is regulated similarly in these two species but that the level of expression is about 80 times higher in the Aspergillus species. The xylP promoter was found to be 65-fold more efficient than the isopenicillin-N-synthetase (pcbC) promoter in Penicillium and 23-fold more efficient than the nitrate reductase (niaD) promoter in Aspergillus under induced conditions. Furthermore, the xylP promoter was used for controllable antisense RNA synthesis of the nre-encoded putative major nitrogen regulator of P. chrysogenum. This approach led to inducible downregulation of the steady-state mRNA level of nre and consequently to transcriptional repression of the genes responsible for nitrate assimilation. In addition, transcription of nreB, which encodes a negative-acting nitrogen regulatory GATA factor of Penicillium, was found to be subject to regulation by NRE. Our data are the first direct evidence that nre indeed encodes an activator in the nitrogen regulatory circuit in Penicillium and indicate that cross regulation of the controlling factors occurs.