Impact of classical strain improvement of penicillium rubens on amino acid metabolism during β-Lactam production

To produce high levels of β-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of L-cysteine, one of the amino acids used for β-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for L-cysteine biosynthesis. Tryptophan synthase, which converts L-serine into L-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production. IMPORTANCE Penicillium rubens is an important industrial producer of β-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced L-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains

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

BACKGROUND: Penicillium 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. RESULTS: To 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. CONCLUSIONS: The 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

Mechanism and regulation of sorbicillin biosynthesis by Penicillium chrysogenum

Penicillium chrysogenum is a filamentous fungus that is used to produce β-lactams at an industrial scale. At an early stage of classical strain improvement, the ability to produce the yellow-coloured sorbicillinoids was lost through mutation. Sorbicillinoids are highly bioactive of great pharmaceutical interest. By repair of a critical mutation in one of the two polyketide synthases in an industrial P. chrysogenum strain, sorbicillinoid production was restored at high levels. Using this strain, the sorbicillin biosynthesis pathway was elucidated through gene deletion, overexpression and metabolite profiling. The polyketide synthase enzymes SorA and SorB are required to generate the key intermediates sorbicillin and dihydrosorbicillin, which are subsequently converted to (dihydro)sorbillinol by the FAD-dependent monooxygenase SorC and into the final product oxosorbicillinol by the oxidoreductase SorD. Deletion of either of the two pks genes not only impacted the overall production but also strongly reduce the expression of the pathway genes. Expression is regulated through the interplay of two transcriptional regulators: SorR1 and SorR2. SorR1 acts as a transcriptional activator, while SorR2 controls the expression of sorR1. Furthermore, the sorbicillinoid pathway is regulated through a novel autoinduction mechanism where sorbicillinoids activate transcription

Pathway for the Biosynthesis of the Pigment Chrysogine by Penicillium chrysogenum

Chrysogine is a yellow pigment produced by Penicillium chrysogenum and other filamentous fungi. Although the pigment was first isolated in 1973, its biosynthetic pathway has so far not been resolved. Here, we show that deletion of the highly expressed nonribosomal peptide synthetase (NRPS) gene Pc21g12630 (chyA) resulted in a decrease in the production of chrysogine and 13 related compounds in the culture broth of P. chrysogenum. Each of the genes of the chyAcontaining gene cluster was individually deleted, and corresponding mutants were examined by metabolic profiling in order to elucidate their function. The data suggest that the NRPS ChyA mediates the condensation of anthranilic acid and alanine into the intermediate 2-(2-aminopropanamido) benzoic acid, which was verified by feeding experiments of a Delta chyA strain with the chemically synthesized product. The remainder of the pathway is highly branched, yielding at least 13 chrysogine-related compounds. IMPORTANCE Penicillium chrysogenum is used in industry for the production of Delta-lactams, but also produces several other secondary metabolites. The yellow pigment chrysogine is one of the most abundant metabolites in the culture broth, next to Delta-lactams. Here, we have characterized the biosynthetic gene cluster involved in chrysogine production and elucidated a complex and highly branched biosynthetic pathway, assigning each of the chrysogine cluster genes to biosynthetic steps and metabolic intermediates. The work further unlocks the metabolic potential of filamentous fungi and the complexity of secondary metabolite pathways

Secondary metabolism by industrially improved Penicillium chrysogenum strains

Penicillium chrysogenum is de filamenteuze schimmel die in de industrie gebruikt voor de productie van het antibioticum penicilline. Penicillines worden nog steeds veel gebruikt maar voor de productie worden tegenwoordig stammen gebruikt die een zeer hoge opbrengst van β-lactams vertonen en die zijn verkregen middels een uitgebreide klassieke stamverbetering (CSI) programma dat bijna zeven decennia heeft geduurd. Bij CSI worden cellen blootgesteld aan agressieve behandelingen zoals UV-straling en mostertgas waardoor er mutaties ontstaan waarvan sommige gunstig zijn voor β-lactam productie veroorzaakt. Echter, de invloed van CSI op de productie van andere secundaire metabolieten is onbekend gebleven. In dit proefschrift is een vergelijkende analyse uitgevoerd van P. chrysogenum mutanten uit het CSI-programma waarbij de genoomsequentie, het transcriptoom en de metaboliet productie is gemeten. De gegevens suggereren dat tijdens de CSI de meeste secundaire metaboliet routes zijn geïnactiveerd ten gunsten van de β-lactams. Dit betreft ondermeer een groep van bioactieve verbindingen, de sorbicillinoids. Dit zijn gele pigmenten die tevens antimicrobiële, anti-HIV en anti-kanker activiteiten vertonen. Met behulp van de genoomsequentie gegevens is een belangrijke mutatie in een van de sorbicillinoid biosynthese genen gerestaureerd in een industriële P. chrysogenum stam waardoor eenvoudig een hoge productie werd bereikt. Dit systeem kan nu worden gebruikt om het mechanisme van biosynthese van sorbicillinoids op te helderen en derivaten te produceren met verhoogde activiteit en specificiteit

Identification of a polyketide synthase involved in sorbicillin biosynthesis by Penicillium chrysogenum

Secondary metabolism in Penicillium chrysogenum was intensively subjected to classical strain improvement (CSI) 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 sorbicillinoids biosynthesis in P. chrysogenum We demonstrate that the production of polyketide precursors like sorbicillinol and dihydrosorbicillinol as well as their derivatives bisorbicillinoids require 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 sorbicillinoids production. This now enable genetic studies on the mechanism of sorbicillinoid biosynthesis in P. chrysogenum and opens new perspectives for pathway engineering. IMPORTANCE: Sorbicillinoids are secondary metabolites with anti-viral, anti-inflammatory and anti-microbial activity produced by filamentous fungi. This study identified the gene cluster responsible for sorbicillinoids formation in Penicillium chrysogenum now allow engineering of this diverse group of compounds

Additional file 2: Table S2. of Genomic mutational analysis of the impact of the classical strain improvement program on β–lactam producing Penicillium chrysogenum

The list of COG/KOG/BRITE functional categories and distribution of the mutated nucleotides. (XLSX 354 kb

Additional file 1: Table S1. of Genomic mutational analysis of the impact of the classical strain improvement program on β–lactam producing Penicillium chrysogenum

The list of coding mutations acquired by the strain lineage of P. chrysogenum. (XLSX 52 kb