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

Abstract

Secondary metabolism in Penicillium chrysogenum was intensively subjected to classical strain improvement (CSI), the resulting industrial strains producing high levels of β-lactams. During this process, the production of yellow pigments, including sorbicillinoids, was eliminated as part of a strategy to enable the rapid purification of β-lactams. Here we report the identification of the polyketide synthase (PKS) gene essential for sorbicillinoid biosynthesis in P. chrysogenum We demonstrate that the production of polyketide precursors like sorbicillinol and dihydrosorbicillinol as well as their derivatives bisorbicillinoids requires the function of a highly reducing PKS encoded by the gene Pc21g05080 (pks13). This gene belongs to the cluster that was mutated and transcriptionally silenced during the strain improvement program. Using an improved β-lactam-producing strain, repair of the mutation in pks13 led to the restoration of sorbicillinoid production. This now enables genetic studies on the mechanism of sorbicillinoid biosynthesis in P. chrysogenum and opens new perspectives for pathway engineering.

Importance: Sorbicillinoids are secondary metabolites with antiviral, anti-inflammatory, and antimicrobial activities produced by filamentous fungi. This study identified the gene cluster responsible for sorbicillinoid formation in Penicillium chrysogenum, which now allows engineering of this diverse group of compounds.

FIG 1

Sorbicillin-related compounds isolated from Penicillium species. The compounds detected in this study are shown by number in parentheses.

FIG 2

(A) Proposed cluster of genes involved in sorbicillinoid biosynthesis in P. chrysogenum. Abbreviations used for PKS domains: SAT, starter unit acyl-carrier protein transacylase domain; KS, ketosynthase; AT, acetyltransferase; ACP, acyl carrier protein; DH, dehydratase; KR, ketoreductase; ER, enoylreductase; MT, methyltransferase; TE/Red, thioester reductase domain. (B) Proposed mechanism of sorbicillin/dehydrosorbicillin biosynthesis (adapted from reference 14) involving PKS12 and PKS13.

FIG 3

Schematic representation of the pks13 gene deletion and its confirmation by Southern blot analysis. (A) Scheme of the deletion plasmid pKO13. Features include amdS, an A. nidulans acetoamidase gene for positive selection of the fungal transformants on medium supplemented with acetamide as the sole nitrogen source, bla, an ampicillin resistance gene for the selection in E. coli, ori, the pUC origin of replication, and attB3/4, the Gateway recombination sites. (B) Southern blot analysis. Genomic DNA was digested with NdeI endonuclease. Southern blot analysis was carried out with the pks13 3′ fragment (3′ FR) as a probe. Shown are the expected 7.3-kb DNA fragment of the parental strain NRRL1951 and a 3.6-kb signal confirming that the pks13 locus was replaced with amdS marker. (C) Scheme of the replacement of the Pc21905080 gene in P. chrysogenum NRRL1951 with the amdS cassette, with indicated lengths of the DNA fragments in the wild-type and Δpks13 strains detected by Southern blot analysis using the pks13 3′ FR as a probe.

FIG 4

Secondary metabolite profiling of liquid cultures of NRRL1951 and the deletion strain NRRL1951 Δpks13, the yellow pigmentless strain DS58630, and DS58630 Res13 carrying the restored native nucleotide sequences of the pks13 gene. (A) Cultures were grown for 3 days on liquid SMP medium in shaking flasks. (B) LC-MS elution profiles. The major compounds eliminated from the secondary metabolism of NRRL1951 Δpks13 and restored in the DS58630 Res13 strain are indicated: compound 2, sorbicillinol; compound 3, dihydrosorbicillinol; compound 6, bisvertinolone; compound 7, dihydrobisvertinolone; and compound 8, tetrahydrobisvertonolone.