Genomic mutational analysis of the impact of the classical strain improvement program on β–lactam producing Penicillium chrysogenum

Abstract

BackgroundPenicillium chrysogenum is a filamentous fungus that is employed as an industrial producer of β–lactams. The high β–lactam titers of current strains is the result of a classical strain improvement program (CSI) starting with a wild-type like strain more than six decades ago. This involved extensive mutagenesis and strain selection for improved β–lactam titers and growth characteristics. However, the impact of the CSI on the secondary metabolism in general remains unknown.ResultsTo examine the impact of CSI on secondary metabolism, a comparative genomic analysis of β-lactam producing strains was carried out by genome sequencing of three P. chrysogenum strains that are part of a lineage of the CSI, i.e., strains NRRL1951, Wisconsin 54-1255, DS17690, and the derived penicillin biosynthesis cluster free strain DS68530. CSI has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. However, in this set of mutations, 8 out of 31 secondary metabolite genes (20 polyketide synthases and 11 non-ribosomal peptide synthetases) were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to β–lactam production. Additionally, key Velvet complex proteins (LeaA and VelA) involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during CSI. Using comparative metabolic profiling, the polyketide synthetase gene cluster was identified that is responsible for sorbicillinoid biosynthesis, a group of yellow-colored metabolites that are abundantly produced by early production strains of P. chrysogenum.ConclusionsThe classical industrial strain improvement of P. chrysogenum has had a broad mutagenic impact on metabolism and has resulted in silencing of specific secondary metabolite genes with the concomitant diversion of metabolism towards the production of β–lactams.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2154-4) contains supplementary material, which is available to authorized users.

Identification of a Polyketide Synthase Involved in Sorbicillin Biosynthesis by Penicillium chrysogenum

“…A related gene cluster was implicated in the biosynthesis of sorbicillactone A/B in the marine isolate E01-10/3 (7, 11), but direct evidence for the involvement of the PKS enzymes was not demonstrated. As shown in our previous work (16), this complete gene cluster is highly expressed in the parental strain NRRL1951 and is transcriptionally silenced in the improved ␤-lactam producer Wisconsin 54-1255 and its derivatives. The mechanism of the transcriptional silencing of this gene cluster is unknown, although the CSI-improved strains also accumulated mutations in the Velvet complex (16) that may have impacted the expression of these secondary metabolite genes.…”

“…S1 in the supplemental material). Both mutations occurred early during the CSI and have been inherited by the Wisconsin 54-1255 strain and thus also by later improved ␤-lactam producers (16). Since the CSI-derived strains of P. chrysogenum are not able to produce yellow pigments, the natural isolate NRRL1951 was chosen as the host in our study.…”

“…These pigments originate from the hexaketide sorbicillin, but the polyketide synthase responsible for its production has not been identified. Previously, we demonstrated that sorbicillin-related metabolites are produced early during fermentation, which is accompanied with the expression of a PKS gene cluster of unknown function (16 (Fig. 2A).…”

“…Transcriptional profiling of secondary metabolite genes in related strains of a lineage of improved ␤-lactam producers indicate the presence of a PKS gene cluster that was silenced during classical strain improvement (CSI) (log 2 fold change, Ϫ4.4 and Ϫ4.7 for pks12 and pks13, respectively) (16). In contrast, the progenitor strain NRRL1951, which still produces sorbicillinoids, exhibits the highest transcriptional level of the corresponding gene cluster (16). In addition, mutations emerged in each of the putative PKS enzymes that likely led to an inactivation.…”

Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi

“…In a comparison of the genomes of the wild-type strain NRRL1951 with two derived production strains, a total of 558 SNPs were identified in coding regions leading to changes in the amino acid sequences of the encoded proteins (Salo et al 2015). In this comparison, the authors identified SNPs in 29% of the secondary metabolite cluster.…”