BACKGROUND
for the industrial production of β–lactams, the filamentous fungus penicillium chrysogenum has been subjected to an intense classical strain improvement  program that consisted of repeating rounds of mutagenesis and selection.
this program started in  <dig> with a natural isolated strain nrrl <dig> derived from an infected cantaloupe because of its high levels of β–lactam production under submerged fermentation conditions.
the spontaneous variant nrrl <dig> b <dig> showed almost three times the production levels of p. notatum strain nrrl  <dig> - the first industrial candidate adopted for the tank fermentation method of β–lactam production during the forties of last century.
to achieve production of increasing levels of β–lactams and to ease the purification of the product, a comprehensive mutagenesis was applied to the nrrl <dig> b <dig> strain  <cit> .
this included harsh mutagenic techniques like uv irradiation, x-ray, and nitrogen mustard  amine) treatment as well as selection of the desired spontaneous variants for production and fermentation characteristics.
the confidentiality of the industrial csi programmes prohibits a comprehensive view on the exact mutagenesis steps during the generation of improved penicillin producing strains.
only few intermediate strains can be mapped on the csi chart with the addressed method of treatment .
however, a major breakthrough in the csi of nrrl <dig> was the selection of the so-called wisconsin series of improved strains that were derived from the x- <dig>  mutant that was further evolved into the current international laboratory standard strain wisconsin 54- <dig> whose genome sequence became available in  <dig>  <cit> .
currently, wisconsin 54- <dig> is the reference strain for genome, proteome and transcriptome comparative studies .
the genetic studies and genome sequencing resulted in an understanding of the mechanisms underlying the enhanced β-lactam production, which included the amplification of the penicillin biosynthesis gene cluster  <cit> , an enhanced amino acid production and various aspects of cellular development  <cit> .fig.
<dig> schematic representation of main industrial β-lactam production improvement programs of p. chrysogenum.
the main lineages of strains subjected to industrial β-lactam production classical strain improvement programs are shown.
strains nrrl <dig> , wisconsin 54- <dig> , and ds <dig>  examined in this study are shown in red.
in brackets the production levels of penicillin v are indicated according to reference  <cit> .
the penicillin cluster free strain ds <dig> that no longer produces penicillins is not included but it is derived from strain ds <dig> by genetically removing the multiple penicillin biosynthetic gene clusters  <cit> .
when reported, the techniques used for mutagenesis are indicated according to newbert et al.
<cit> .
used abbreviations: s, selection of the spontaneous variant; x, x-ray irradiation; uv, ultraviolet irradiation; nm, nitrogen mustard  amine) treatment; mg, other molecular genetic manipulations.
also indicated are the periods  <dig> and  <dig> followed in this study in order to identify major steps in the csi
unlike β-lactam biosynthesis, little is known about the consequences of csi on the secondary metabolism in general.
the genome sequence of wisconsin 54- <dig> revealed that in addition to β-lactams, the genome encodes a range of other secondary metabolite gene clusters that have been characterized only to a limited extent.
more recently, advances in metabolome techniques systematically revealed a diversity of the secondary metabolites that can be produced by industrial variants of p. chrysogenum.
for more than  <dig> of these compounds the genes involved in biosynthesis have been identified and partially characterized.
this includes the non-ribosomal peptide synthetases involved in the production of toxins such as roquefortine and meleagrine , the pigment chrysogine , siderophores  and cyclic hydrophobic tetrapeptides  <cit> .
however, this analysis also revealed that during csi the diversity of the secondary metabolism by improved β-lactam producers has been significantly narrowed.
for instance, elimination of the production of yellow pigments like sorbicillin and penitrinic acid, that were contaminants during antibiotic purification, was already achieved at the early stage of csi  <cit> .
however, the genetic origin of the production of these pigments and their bioactive properties has remained unstudied for long.
in recent years many sorbicillin related bioactive compounds were re-identified in p. notatum  <cit> , trichoderma  <cit> , and other fungal species isolated from different environments  <cit> .
the broad spectra of activities of sorbicillins range from radical scavenging to cytotoxicity against l5178y leukemic cells, while the actual function and genetic basis for the production of these compounds in p. chrysogenum and other filamentous fungi are mostly unknown.
here we performed a systematic genomic, expression and extracellular metabolome analysis to investigate the impact of csi on secondary metabolism of p. chrysogenum.
we demonstrate that csi resulted in the silencing of many secondary metabolite pathways, whereas others remained unaffected.
in addition, we identified the polyketide synthases responsible for the production of a vast group of pigments termed sorbicillinoids that were mutated and transcriptionally silenced at the early stages of the csi programme.
RESULTS
mutational spread in the p. chrysogenum genome during classical strain improvement
to examine the mutational impact of the classical strain improvement  program on the genome of p. chrysogenum, we sequenced the genomes of four p. chrysogenum strains.
these are the early β-lactam producing strain p. chrysogenum nrrl <dig>  which acts as a reference and two strains in the lineage that signify two different periods of mutagenesis.
here, we define period  <dig> as the early stage of csi, using the wisconsin 54- <dig> strain as the hall mark.
during this period csi has had a major impact on the fermentation characteristics and pigment formation.
this strain is the current international reference as its genome was sequenced.
the second period entails strain ds <dig> that has undergone major mutagenesis which amongst others has led to the amplification of the penicillin biosynthetic gene cluster.
it is currently studied as a type strain.
in addition, the penicillin biosynthesis cluster free strain ds <dig> was sequenced that was derived from the latter strain.
the absence of the multiple penicillin clusters facilitates the analysis of β-lactam unrelated secondary metabolites present in the extracellular metabolome .
the genomes of the four strains were sequenced using the illumina hiseq  <dig> platform with near to complete coverage  .
based on a comparison of the respective genomes,  <dig> snps were detected between the nrrl <dig> and wisconsin 54- <dig> strain that accumulated during the first period of csi.
within this set,  <dig> mutations occurred within coding regions, while  <dig> non-synonymus,  <dig> synonymus,  <dig> termination mutations,  <dig> frame shifts and one nonsense mutation were identified.
during the second period of the csi the multicopy strain ds <dig> additionally obtained  <dig> snps.
this included  <dig> non-coding, and  <dig> mutations within coding regions including  <dig> synonymus and  <dig> non-synonymus mutations, three snps at the terminal region,  <dig> frame shifts and  <dig> nonsense mutations .table  <dig> quality statistics of the genome sequencing of p. chrysogenum strains within a csi lineage
amutations mapped relative to strain nrrl1951
the use of the advanced next generation sequencing technique to the reference strain wisconsin 54- <dig> resulted in the identification of mismatches with the previously published sequence of this strain  <cit> .
we identified  <dig> snps including  <dig> non-coding,  <dig> synonymus and  <dig> non-synonymus polymorphisms.
this included  <dig> frame shifts, one termination and one nonsense mutation.
these newly identified features are the result of the improved quality of the sequencing and correspondingly appeared in the genome assemblies of nrrl <dig>  ds <dig> and ds <dig> strain.
to simplify the following comparative analysis these snps were subtracted from the mutational profiles of the subjected strains.
an analysis using gasvpro  <cit>  did not reveal any structural variation between the strains.
in order to assess the distribution of snps across genes involved in different cellular processes, we performed a functional classification of the entire gene set of the p. chrysogenum wisconsin 54- <dig> reference genome.
in order to classify a maximal proportion of genes, we overlapped functional classifications from the cog, kog and kegg brite databases  into a single consensus prediction.
the complete sets of identified snps between nrrl  <dig> and wisconsin 5412- <dig> and between wisconsin 54- <dig> and ds <dig> were mapped to these functional categories.
subsequently, we performed a statistical comparison of the functional distribution of genes affected by the snps and the overall functional distribution of all genes in the genome, based on the hypergeometric distribution.
surprisingly, after bonferroni multiple-testing correction, there was no statistically significant overrepresentation or underrepresentation of mutations in secondary metabolism, amino acid metabolism, transcriptional regulation, or any other functional category .
hence, strain selection seems to have had only very limited control over the mutations accumulated during the csi, and it is likely that only a minority of the changes introduced by the mutagenesis procedure are responsible for the phenotypic improvements of the strains.
the next key challenge was therefore to identify this important minority.
distribution of mutations in secondary metabolism gene clusters
an inventory was made of mutations that were accumulated in key secondary metabolism genes during the csi using strain nrrl <dig> as a reference.
the genome contains  <dig> nrps and  <dig> pks genes likely involved in secondary metabolite formation.
in total,  <dig> putative secondary metabolite gene clusters accumulated mutational changes during both periods of the csi .
this affected  <dig> nrps gene and  <dig> pks genes.
the mutations were all verified by dna sequencing of the respective gene clusters.fig.
<dig> mutational impact of the csi program on secondary metabolite gene clusters of p. chrysogenum.
a mutations acquired by wisconsin 54- <dig> in period  <dig> of the csi programme are colored in red.
the mutagenic impact in period  <dig> is shown in green.
mutated pks and nrps domains are indicated.
the grey arrows represent other genes of the putative gene clusters.
abbreviations used for pks and nrps domains: ks, keto-synthase; at, acyltransferase; dh, dehydratase; mt, methyltransferase; er, enoyl reductase; kr, ketoreductase; acp, acyl carrier protein; te, thioesterase; te/red, thioester reductase; a, adenylation; t, thiolation; c, condensation domain.
b schematic representation of the velvet complex proteins vela, velb and laea with the mutations obtained during the csi
the partially reducing  pks <dig>  gene cluster is part of predicted cluster of nineteen orfs  <cit> .
pks <dig> shows 87 % identity to an unknown pks of p. digitatum and strong similarity  to the lovastatin diketide synthase lovf of a. terreus.
two snps occurred at the second period of csi and resulted in a glycine to glutamic acid substitution at position  <dig> within the ketosynthase domain of pks <dig> that likely has inactivated the enzyme.
the gene cluster of pr pks <dig>  is predicted to consist of  <dig> orfs  <cit> .
a homologous cluster of five genes is found in the genome of a. fumigatus, but has no assigned function.
pks <dig> carries the snp resulting in an alanine to aspartic acid substitution at position  <dig> located at the acyl transferase/dehydratase inter-domain region.
this mutation was acquired in the second csi period and presumably does not affect the function of the neighboring domains.
the highly reducing  pks <dig>  is homologous  to an uncharacterized pks enzyme of a. flavus and belongs to a putative cluster of  <dig> genes  <cit> .
a single mutation resulted in a phenylalanine to leucine substitution at position  <dig> in the c-/dehydratase inter-domain region and occurred in the second period of csi.
a further gene cluster contains two pkss encoding genes hr pks <dig>  and non-reducing  pks <dig>  and consists of  <dig> orfs  <cit> .
a mutation within the ketosynthase domain of pks <dig> occurred during the first csi period and resulted in isoleucine to phenylalanine substitution at position  <dig>  a nr pks encoded by pc21g <dig>  shows similarity to the a. nidulans wa and a. fumigatus pksp naphthopyrone synthases.
these enzymes are involved in the biosynthesis of a precursor for the formation of a conidial pigment  <cit> .
a similar gene cluster of  <dig> orfs is present in the genomes of a. niger and a. kawachii.
two nucleotide substitutions were detected within pks <dig> causing no effect on the amino acid sequence.
a third mutation is located within the terminator region of the pks gene and is represented by an a to c nucleotide substitution.
the actual impact of these mutations on pigment formation is unclear as the green coloring of the conidia was not affected during the csi.
the gene cluster of pr pks <dig>  shows 82 % identity to the 6-methylsalicylic acid synthase from p. griseofulvum and contains  <dig> orfs.
the p. griseofulvum homolog is involved in patulin biosynthesis and requires additional enzymatic conversions like decarboxylation, two hydroxylation and oxidation reactions  <cit> .
in p. chrysogenum these enzymatic steps are presumably provided by the aminohydrolase and two cytochrome p <dig> enzymes encoded within the cluster.
however, the essential gene encoding the isoepoxidon dehydrogenase cannot be identified in the genome of p. chrysogenum supporting the notion that this fungus does not produce patulin  <cit> .
a single mutation was obtained during the second period of csi and this affected a putative mfs transporter  encoding gene introducing a tyrosine to asparagine substitution at position  <dig>  finally, two neighboring, oppositely oriented nr pks <dig>  and hr pks <dig>  that belong to a gene cluster that potentially is related to sorbicillinoid biosynthesis, received snps in both coding sequences.
pks <dig> acquired an isoleucine to arginine substitution at c-/te inter-domain region, while pks <dig> obtained a mutation in the ks domain where leucine in position  <dig> is substituted by a phenylalanine.
both mutations were obtained at the first period of the csi and thus were inherited already by strain wisconsin 54- <dig>  the associated gene cluster is predicted to contain  <dig> orfs  <cit> .
the only nrps gene that accumulated snps concerns pssa  that is involved in siderophore coprogen b biosynthesis under the iron depleted growth conditions.
at the first csi period the pssa encoding gene was mutated causing a glutamic acid for aspartic acid substitution at position  <dig> within a non-conserved region.
pssa is involved in the production of coprogen that still can be produced by the improved β-lactam producing strains .
therefore, this mutation apparently did not inactivate the enzyme.
mutational changes within the velvet complex
the velvet complex is involved in the regulation of sexual development and secondary metabolism as demonstrated in various filamentous fungi including p. chrysogenum  <cit> .
since it is a multisubunit complex, we examined the impact of csi on the core component of the velvet complex.
the p. chrysogenum orthologs lrea , lreb , fpha , kapa , velb , and vosa  were all identical in the genomes of strains nrrl <dig>  wisconsin 54- <dig> and ds <dig>  however, mutational changes were found in pc16g <dig> and pc13g <dig> genes that encode leaa and vela, respectively.
in the first period, the leaa gene acquired a nucleotide substitution at position  <dig>  which resulted in a substitution of lysine for glutamic acid at position of  <dig> of the protein.
a second mutation occurred in period  <dig> and caused a nucleotide change at position  <dig>  causing a glycine to serine substitution at position  <dig>  in addition, a mutation was identified in the vela encoding gene that occurred at the second csi period.
a c to t substitution at nucleotide position  <dig> caused a stop codon resulted in the formation of a truncation of the vela protein from  <dig> to  <dig> amino acids long.
these data indicate that the velvet complex has been a major target in the csi, and this likely has impacted the expression of secondary metabolite encoding genes .
expression of the secondary metabolism gene clusters
since the velvet complex and many secondary metabolism genes acquired mutations during the csi, the expression of the latter genes may have vastly changed along the lineage of strains.
therefore, we examined the expression of all  <dig> pkss and  <dig> nrps encoding genes in strains nrrl <dig>  wisconsin 54- <dig>  ds <dig> and the penicillin cluster free strain ds <dig> .
herein, the fungi were grown for 5 days and total rna was isolated for qpcr analysis of the respective genes.
the expression of γ-actin gene  was used as reference for normalization.
the observed ct values for the amplified γ-actin gene from the cdna of nrrl <dig>  wisconsin 54- <dig>  ds <dig> and ds <dig> were  <dig>  ±  <dig> ;  <dig>  ±  <dig> ;  <dig>  ±  <dig>  and  <dig>  ±  <dig>  respectively.
this demonstrates nearly identical expression of the reference gene in the analyzed strains.
the obtained expression profiles clearly indicates that the expression of the acv synthetase, the key enzyme of β-lactam biosynthesis was dramatically enhanced upon csi of wisconsin 54- <dig> as expected from the amplification of the β-lactam biosynthesis genes in strain ds <dig> that contains  <dig> copies of this penicillin cluster.
with the other nrps and pks genes, there were no marked expression changes except for three pks genes.
the two highly expressed pks genes pc21g <dig>  and pc21g <dig>  were dramatically down regulated in wisconsin 54- <dig> and the derivatives ds <dig> and ds <dig>  the nr pks <dig> exhibits 43 % identity to citrinin polyketide synthase of monascus purpureus while hr pks <dig> is homologous to lovastatin diketide synthase lovf of a. terreus  <cit> .
in addition, the cluster contains genes encoding a putative regulator , a monooxygenase , a transcriptional factor , a mfs transporter  and an oxidoreductase .
all five genes of this putative cluster are also present in the genome of trichoderma reesei and arranged in one locus.
an identical gene cluster in the marine isolate of p. chrysogenum e01-10/ <dig> has been previously associated with the biosynthesis of sorbicillactones a and b and thus by analogy, the aforementioned gene cluster may encode the biosynthesis pathway of yellow pigments sorbicillinoids in p. chrysogenum  <cit> .
there is a significant decrease in expression of the seven genes of this cluster in the improved strains, whereas expression of flanking genes that are likely not part of the cluster was unaltered .
in addition, expression of pks <dig> was increased substantially upon csi of the nrll <dig> strain.
despite the significant overexpression of this pks in the improved penicillin producing strain, the polyketide product has not yet been identified.fig.
<dig> comparison of secondary metabolite gene expression in strains nrrl <dig>  wisconsin 54- <dig>  ds <dig> and ds <dig>  expression data was obtained with rna isolated after 5 days of fungal growth.
the error bars indicate the standard error of the mean of two biological samples including technical duplicates
fig.
<dig> relative expression of the putative gene cluster of sorbicillinoid biosynthesis in strains nrrl <dig>  wisconsin 54- <dig>  ds <dig> and ds <dig>  the transcriptional silencing of seven genes  belonging to the putative sorbicillinoid gene cluster is shown.
expression data was obtained with rna isolated after 5 days of fungal growth.
the error bars indicate the standard error of the mean of two biological samples including technical duplicates
comparative metabolomic profiling of p. chrysogenum strains
next, we examined the extracellular metabolome focusing on secondary metabolites excreted into the spent culture medium.
lc-ms and lc-ms/ms were used for identification.
in this analysis, five β-lactams were detected in the growth media of all strains, except for the penicillin biosynthesis cluster-free strain ds <dig> .
6-apa , penicillin g , v , isopenicillin n  and penicillin k  were identified by their expected molecular masses and on the basis of their calculated empirical formulas and by the use of standards, except for penicillin k. 6-apa and isopenicillin n were produced in the early stages of fermentation.
as expected, the levels of these compounds were relatively low for the nrrl <dig> strain, increased in strain wisconsin 54- <dig>  and dramatically increased in ds <dig>  in particular during the late growth phase.
6-apa, isopenicillin n and the naturally produced penicillin k were the main products.
since the growth conditions did not involve any feed with the commercially used side chain precursors, penicillin g and v were presented very low amounts compared to other penicillins.fig.
<dig> response ratio of secondary metabolite concentrations present in the culture broth of strains nrrl <dig>  wisconsin 54- <dig>  ds <dig> and ds <dig>  cell coloring corresponds to the internal standard corrected peak area of the detected metabolites in the culture broth after  <dig>   <dig> and 7 days of growth.
the mass to charge ratio  of the protonated metabolites, retention time  and empirical formulas are indicated.
abbreviations: htd, histidyltryptophanyldiketopiperazine; dhtd, dehydrohistidyltryptophanyldi-ketopiperazine; na, metabolites without structural data
another group of compounds are the chrysogine related metabolites.
this yellow pigment is produced in a biosynthetic pathway that has not yet been completely characterized, but at least four structurally resolved compounds can be identified including chrysogine , n-acetylalanylanthranilamide  , n-malonylalanylanthranilamide   and n-pyrovoylanthranilamide , as well as  <dig> related but structurally unresolved molecules .
these metabolites are already found in the culture broth after 3 days.
chrysogine was produced at high levels by the ds <dig> strain, but in particular in the related penicillin cluster free ds <dig> strain, production was at the highest levels.
a further class of compounds are the roquefortine and meleagrin related metabolites that are toxins .
the precursors for these toxins are   and dehydrohistidyltryptophanyldi-ketopiperazine   that are only produced at low levels by strain nrrl  <dig> and wisconsin, but at substantial higher levels during the late growth phases by the ds <dig> strain.
production further increased in the ds <dig> strain.
the same observation was made for the precursors of this pathway like glandicoline a-b , roquefortine c, d, f, m, n  and neoxalin , as well as the final product meleagrin .
it thus appears that like with the chrysogine, period  <dig> of the csi has resulted in an enhanced production of roquefortines.
in the analysis, a unique group of  <dig> metabolites were identified in the culture broth of nrrl <dig> that were absent in the other more evolved strains.
based on their exact masses and their ms spectra, empirical formulas could be assigned to these molecules with less than 2 ppm accuracy.
two groups of compounds could be discriminated .
the first group includes eleven molecules related to sorbicillins that are known to be produced by natural isolates of p. chrysogenum.
both, monomeric and dimeric sorbicillinoids were identified in the culture broth during seven days of culturing.
the identified compounds fit to the proposed biosynthesis pathway  <cit> .
major molecules are sorbicillin  and the derivative sorbicillinol  that already occur after 3 days of fermentation.
although dihydrosorbicillin was not detected, the accumulation of its oxidized derivative dihydrosorbicillinol  occurred after five days of cultivation.
oxosorbicillinol , and the oxidized compound bisvertinolon   were identified.
further dimers were bisorbicillinol , dyhydrobisvertinulone  and tetrahydrobisvertinolone .
in addition, two uncharacterized metabolites with a short  carbon chain  were observed.
the second group included four, presumably nitrogen containing compounds  .fig.
<dig> metabolites characterized in this study.
β-lactam related compounds , the intermediates of chrysogine pathway , roquefortine/meleagrin related compounds , the representative of the cyclic tetrapeptides - fungisporin  and the sorbicillinoids
discussion
here we have examined the effect of the classical strain improvement  program on secondary metabolism by p. chrysogenum.
combined metabolome, transcriptome and genome profiling approaches were applied to a lineage of β-lactam producing strains.
this concerned the natural isolate strain nrrl <dig>  the current laboratory reference wisconsin 54- <dig> and the high penicillin producer ds <dig> which carries multiple copies of the penicillin biosynthesis cluster.
in addition, a ds <dig> derivative, which lacks the penicillin clusters, i.e., strain ds <dig> was included in this analysis .
the genomes of the subjected strains were sequenced using the illumina next generation technique that allowed us to determine the mutational impact on p. chrysogenum during csi.
of the  <dig> pkss and  <dig> nrps present in p. chrysogenum, the majority remains uncharacterized and has not been associated to the production of specific secondary metabolites.
it is generally believed that many of these genes and gene clusters are transcriptionally silent or inactive.
the introduction of the parental isolate nrrl <dig> in our analysis revealed a number of mutations inherited by the current international laboratory standard strain wisconsin 54- <dig> during the first period of csi.
further comparative analysis showed the accumulation of additional mutations during the evolvement of the industrial penicillin cluster multi-copy strain ds <dig>  out of the  <dig> nrps and  <dig> pks associated gene clusters present in the genome, one nrps and  <dig> pks gene clusters accumulated mutational changes during both periods of the csi.
three amino acid substitutions were located within the essential ketosynthase domains of pks <dig>  pks <dig> and pks <dig> that presumably led to a functional inactivation .
significant changes of the secondary metabolite genes expression between the csi derived mutants as well as the mutational impact on transcriptional factor encoding genes indicates that the regulation of secondary metabolism has been intensively altered during the csi .
the three proteins vela, velb and laea of the velvet complex represent an important regulatory element in filamentous fungi.
in a. nidulans, this heterotrimer has been associated with both cell development and secondary metabolism providing the first evidence on the regulatory cross-talk between these phenomena in filamentous fungi.
the current structural model of the velvet complex is derived from protein interaction studies performed in a. nidulans  <cit> .
vela is a scaffolding protein essential for recruiting velb and laea.
velb interacts with the n-terminus of vela while the c-terminus interacts with laea.
it is important to emphasize that all recently published studies on the characterization of the velvet complex proteins in p. chrysogenum were performed in strains derived from the csi, i.e., the eight copy penicillin gene cluster strain ds <dig> derived from wisconsin 54- <dig> and the two copy penicillin gene cluster strain p2niad <dig> obtained during the alternative panlabs strain improvement program  <cit> .
both overproducers originate from the parental nrrl <dig> isolate but the p <dig> series diverged early at the csi prior the generation of wisconsin 54- <dig>  a pronounced phenotypic effect of the deletion of vela and laea on β-lactam titers  in conjunction with a dramatic transcriptional deregulation of up to 10 % of genes present in the genome was demonstrated for the panlabs derived p2niad <dig> strain  <cit> .
in contrast, the penicillin gene expression and secondary metabolite production in ds <dig> strain exhibited only moderate responses to the deletion of vela and laea.
the latter study, however, was performed under glucose-limited chemostat growth conditions, and it was suggested that the growth conditions weakened the phenotype  <cit> .
however, since the velvet complex has been repetitively targeted during the improvement of the ds <dig> strain  this likely caused the weak phenotypes of the respective deletion strains.
indeed, the p2niad <dig> strain  <cit>  shows no alterations of the velvet complex encoding genes and must therefore have remained functionally intact.
specifically, in leaa, we identified two mutations resulting in amino acids changes while in vela, in ds <dig> strain a stop codon is introduced resulting in a c-terminal truncation by about  <dig> amino acids.
vela plays a primary role in the complex assembly where the c-terminus that was lost, is required for interaction with laea.
therefore, the velvet complex of the improved ds <dig> is likely functionally impaired and this must have contributed to an improvement of the β-lactam production presumably as a collateral effect, increased or decreased expression of other secondary metabolite genes.
in addition, pathway specific transcriptional regulation of secondary metabolism is crucial.
we identified  <dig> mutated proteins specifying zn2cys <dig> binuclear cluster dna-binding domains typical found in transcription regulators like gal <dig> and six genes encoding proteins that are homologous to fungal specific transcription factors.
additionally,  <dig> transporter related genes were mutated during the csi, eight abc transporters and  <dig> transporters belonging to the major facilitator superfamily  .
one of the identified mfs pc22g <dig> belongs to the putative secondary metabolite gene cluster harboring a pks gene  that can be linked to the biosynthesis of patulin-like metabolites in p. chrysogenum  <cit> .
the mutational and expression effects of csi led to the significantly alterations in the secondary metabolite profile among the analyzed p. chrysogenum strains.
previously, genes and gene clusters have been identified for the production of roquefortine/meleagrine  <cit> , cyclic tetrapeptides  <cit>  and chrysogine  that all arise from the activity of nrpss.
by high-resolution mass spectrometry we generated a specific metabolite profile for each of the strains and demonstrate significant alterations in the pattern of the production of these secondary metabolites .
importantly, during the csi and with the onset of strain ds <dig>  the ability to produce toxins such as roquefortine/meleagrine and chrysogen increased.
however, the transcriptional levels of the roqa and chya genes was not significantly changed in the lineage of strains.
considering that the biosynthesis of these secondary metabolites is strongly dependent on nitrogen metabolism, it seems likely that the improvement in amino acid production  <cit>  in the higher β-lactam producers is the indirect cause of this phenomenon.
moreover, the highest levels of these secondary metabolites where found in the ds <dig> strain in which the multiple penicillin gene clusters have been removed  <cit> .
this strain is genomically identical to the parental strain ds <dig>  also showing the same expression levels and timing of the expression of the corresponding nrps encoding genes.
therefore, the observed increase in the aforementioned nonribosomal peptide products strongly supports our notion that their enhanced production is because of a re-diversion of nitrogen metabolism.
during the csi, also the production of a suite of secondary metabolites was lost.
early p. chrysogenum isolates produced yellow pigments that were eliminated early on during csi as they were found to be colored contaminants during antibiotic production.
however, the genetic origin of pigment formation has remained obscure.
our secondary metabolite profiling indicates a group of structurally related compounds that are specifically produced by the nrrl <dig> strain but are absent in the fermentation broth of wisconsin 54- <dig>  ds <dig> and ds <dig>  the empirical formulas generated based on mass accuracy  correspond to known hexaketide yellow pigments termed sorbicillinoids.
the production of sorbicillin, sorbicillinol, dihydrosorbicillinol, oxosorbicillinol, bisvertinolone, bisorbicillinol, and dihydrobisvertinolone was detected.
we found that accumulation of the pathway precursors sorbicillin and sorbicillinol by a three days grown culture correlates with high levels of expression of a single pks gene cluster in the nrrl <dig> strain.
qpcr analysis revealed the transcriptional silencing of the entire gene cluster of seven genes although expression of the flanking genes remained unchanged.
additionally, the genome sequencing analysis revealed two mutations accumulated in the two pks encoding genes of the aforementioned cluster at the beginning of the csi program.
nr-pks <dig> acquired an isoleucine to arginine substitution at the c-/dh inter-domain region.
the neighboring oppositely oriented hr-pks <dig> encoding gene obtained a mutation causing a leucine to a phenylalanine substitution in position  <dig> within the ks domain of the enzyme.
the proposed model for sorbicillin biosynthesis, as the hexaketide precursor for all sorbicillinoid derivatives, foresees the function of a pair of nr-pks and hr-pks enzymes.
the triacetate precursor is formed by hr-pks following the non-reductive carbon chain extension by the second nr-pks to form hexaketide.
taken together our results suggest this pks gene cluster is responsible for the biosynthesis of sorbicillinoids in the early producer nrrl <dig>  the mutations found within the pks gene cluster combined with the transcriptional silencing of the entire gene cluster shown for derivative strains wisconsin 54- <dig> and ds <dig>  demonstrates a multi-step complex eliminating effect of the csi on the biosynthesis of these yellow pigments in p. chrysogenum.
importantly, the functional characterization of the aforementioned gene cluster through direct targeting of the pks genes has not yet been achieved  <cit> , and will be subject of future work.
CONCLUSIONS
our study concerned a combined genome, transcriptome and metabolome analysis of improved β-lactam producing strains of p. chrysogenum with a focus on secondary metabolism.
whereas several secondary metabolite genes were targeted by mutagenesis, overall there was not a single specific functional group of genes that was specifically mutagenized.
mutations appear to be randomly spread across the chromosomes and this is likely the result of the rather undirected approach used during csi.
the results suggest that the improved titers of the β-lactams achieved during the csi are only partially reflected by the broad mutagenic impact of the csi program.
whereas mutagenesis was random, the selection was focused on specific features such as the increased β-lactam titers; morphological aspects as well the fermentation characteristics.
thus, the strains collected many mutations that do not contribute to these features.
the elevated production of nonribosomal peptide products such as chrysogine and roquefortine/meleagrin likely emerged from the enhanced nitrogen metabolic flux in improved penicillin producing strains.
a group of yellow pigments including sorbicillinoids has been eliminated at the early stages of csi.
production of sorbicillinoids is likely associated with a pks gene cluster of seven genes that was transcriptionally silenced during the csi and that acquired mutations at functionally essential pks domains.
these finding indicate a complex mechanism underlying the elimination of the production of sorbicillinoids from the secondary metabolism of p. chrysogenum and provide insight in the molecular basis of biosynthesis of this interesting group of bioactive compounds.

 for the industrial production of β–lactams, the filamentous fungus penicillium chrysogenum has been subjected to an intense classical strain improvement program that consisted of repeating rounds of mutagenesis and selection. this program started in with a natural isolated strain nrrl derived from an infected cantaloupe because of its high levels of β–lactam production under submerged fermentation conditions. the spontaneous variant nrrl b showed almost three times the production levels of p. notatum strain nrrl - the first industrial candidate adopted for the tank fermentation method of β–lactam production during the forties of last century. to achieve production of increasing levels of β–lactams and to ease the purification of the product, a comprehensive mutagenesis was applied to the nrrl b strain. this included harsh mutagenic techniques like uv irradiation, x-ray, and nitrogen mustard amine) treatment as well as selection of the desired spontaneous variants for production and fermentation characteristics. the confidentiality of the industrial csi programmes prohibits a comprehensive view on the exact mutagenesis steps during the generation of improved penicillin producing strains. only few intermediate strains can be mapped on the csi chart with the addressed method of treatment. however, a major breakthrough in the csi of nrrl was the selection of the so-called wisconsin series of improved strains that were derived from the x- mutant that was further evolved into the current international laboratory standard strain wisconsin 54- whose genome sequence became available in. currently, wisconsin 54- is the reference strain for genome, proteome and transcriptome comparative studies. the genetic studies and genome sequencing resulted in an understanding of the mechanisms underlying the enhanced β-lactam production, which included the amplification of the penicillin biosynthesis gene cluster, an enhanced amino acid production and various aspects of cellular development.fig. schematic representation of main industrial β-lactam production improvement programs of p. chrysogenum. the main lineages of strains subjected to industrial β-lactam production classical strain improvement programs are shown. strains nrrl, wisconsin 54-, and ds examined in this study are shown in red. in brackets the production levels of penicillin v are indicated according to reference. the penicillin cluster free strain ds that no longer produces penicillins is not included but it is derived from strain ds by genetically removing the multiple penicillin biosynthetic gene clusters. when reported, the techniques used for mutagenesis are indicated according to newbert et al.. used abbreviations: s, selection of the spontaneous variant; x, x-ray irradiation; uv, ultraviolet irradiation; nm, nitrogen mustard amine) treatment; mg, other molecular genetic manipulations. also indicated are the periods and followed in this study in order to identify major steps in the csi unlike β-lactam biosynthesis, little is known about the consequences of csi on the secondary metabolism in general. the genome sequence of wisconsin 54- revealed that in addition to β-lactams, the genome encodes a range of other secondary metabolite gene clusters that have been characterized only to a limited extent. more recently, advances in metabolome techniques systematically revealed a diversity of the secondary metabolites that can be produced by industrial variants of p. chrysogenum. for more than of these compounds the genes involved in biosynthesis have been identified and partially characterized. this includes the non-ribosomal peptide synthetases involved in the production of toxins such as roquefortine and meleagrine, the pigment chrysogine, siderophores and cyclic hydrophobic tetrapeptides. however, this analysis also revealed that during csi the diversity of the secondary metabolism by improved β-lactam producers has been significantly narrowed. for instance, elimination of the production of yellow pigments like sorbicillin and penitrinic acid, that were contaminants during antibiotic purification, was already achieved at the early stage of csi. however, the genetic origin of the production of these pigments and their bioactive properties has remained unstudied for long. in recent years many sorbicillin related bioactive compounds were re-identified in p. notatum, trichoderma, and other fungal species isolated from different environments. the broad spectra of activities of sorbicillins range from radical scavenging to cytotoxicity against l5178y leukemic cells, while the actual function and genetic basis for the production of these compounds in p. chrysogenum and other filamentous fungi are mostly unknown. here we performed a systematic genomic, expression and extracellular metabolome analysis to investigate the impact of csi on secondary metabolism of p. chrysogenum. we demonstrate that csi resulted in the silencing of many secondary metabolite pathways, whereas others remained unaffected. in addition, we identified the polyketide synthases responsible for the production of a vast group of pigments termed sorbicillinoids that were mutated and transcriptionally silenced at the early stages of the csi programme.  mutational spread in the p. chrysogenum genome during classical strain improvement to examine the mutational impact of the classical strain improvement program on the genome of p. chrysogenum, we sequenced the genomes of four p. chrysogenum strains. these are the early β-lactam producing strain p. chrysogenum nrrl which acts as a reference and two strains in the lineage that signify two different periods of mutagenesis. here, we define period as the early stage of csi, using the wisconsin 54- strain as the hall mark. during this period csi has had a major impact on the fermentation characteristics and pigment formation. this strain is the current international reference as its genome was sequenced. the second period entails strain ds that has undergone major mutagenesis which amongst others has led to the amplification of the penicillin biosynthetic gene cluster. it is currently studied as a type strain. in addition, the penicillin biosynthesis cluster free strain ds was sequenced that was derived from the latter strain. the absence of the multiple penicillin clusters facilitates the analysis of β-lactam unrelated secondary metabolites present in the extracellular metabolome. the genomes of the four strains were sequenced using the illumina hiseq platform with near to complete coverage. based on a comparison of the respective genomes, snps were detected between the nrrl and wisconsin 54- strain that accumulated during the first period of csi. within this set, mutations occurred within coding regions, while non-synonymus, synonymus, termination mutations, frame shifts and one nonsense mutation were identified. during the second period of the csi the multicopy strain ds additionally obtained snps. this included non-coding, and mutations within coding regions including synonymus and non-synonymus mutations, three snps at the terminal region, frame shifts and nonsense mutations.table quality statistics of the genome sequencing of p. chrysogenum strains within a csi lineage amutations mapped relative to strain nrrl1951 the use of the advanced next generation sequencing technique to the reference strain wisconsin 54- resulted in the identification of mismatches with the previously published sequence of this strain. we identified snps including non-coding, synonymus and non-synonymus polymorphisms. this included frame shifts, one termination and one nonsense mutation. these newly identified features are the result of the improved quality of the sequencing and correspondingly appeared in the genome assemblies of nrrl ds and ds strain. to simplify the following comparative analysis these snps were subtracted from the mutational profiles of the subjected strains. an analysis using gasvpro did not reveal any structural variation between the strains. in order to assess the distribution of snps across genes involved in different cellular processes, we performed a functional classification of the entire gene set of the p. chrysogenum wisconsin 54- reference genome. in order to classify a maximal proportion of genes, we overlapped functional classifications from the cog, kog and kegg brite databases into a single consensus prediction. the complete sets of identified snps between nrrl and wisconsin 5412- and between wisconsin 54- and ds were mapped to these functional categories. subsequently, we performed a statistical comparison of the functional distribution of genes affected by the snps and the overall functional distribution of all genes in the genome, based on the hypergeometric distribution. surprisingly, after bonferroni multiple-testing correction, there was no statistically significant overrepresentation or underrepresentation of mutations in secondary metabolism, amino acid metabolism, transcriptional regulation, or any other functional category. hence, strain selection seems to have had only very limited control over the mutations accumulated during the csi, and it is likely that only a minority of the changes introduced by the mutagenesis procedure are responsible for the phenotypic improvements of the strains. the next key challenge was therefore to identify this important minority. distribution of mutations in secondary metabolism gene clusters an inventory was made of mutations that were accumulated in key secondary metabolism genes during the csi using strain nrrl as a reference. the genome contains nrps and pks genes likely involved in secondary metabolite formation. in total, putative secondary metabolite gene clusters accumulated mutational changes during both periods of the csi. this affected nrps gene and pks genes. the mutations were all verified by dna sequencing of the respective gene clusters.fig. mutational impact of the csi program on secondary metabolite gene clusters of p. chrysogenum. a mutations acquired by wisconsin 54- in period of the csi programme are colored in red. the mutagenic impact in period is shown in green. mutated pks and nrps domains are indicated. the grey arrows represent other genes of the putative gene clusters. abbreviations used for pks and nrps domains: ks, keto-synthase; at, acyltransferase; dh, dehydratase; mt, methyltransferase; er, enoyl reductase; kr, ketoreductase; acp, acyl carrier protein; te, thioesterase; te/red, thioester reductase; a, adenylation; t, thiolation; c, condensation domain. b schematic representation of the velvet complex proteins vela, velb and laea with the mutations obtained during the csi the partially reducing pks gene cluster is part of predicted cluster of nineteen orfs. pks shows 87 % identity to an unknown pks of p. digitatum and strong similarity to the lovastatin diketide synthase lovf of a. terreus. two snps occurred at the second period of csi and resulted in a glycine to glutamic acid substitution at position within the ketosynthase domain of pks that likely has inactivated the enzyme. the gene cluster of pr pks is predicted to consist of orfs. a homologous cluster of five genes is found in the genome of a. fumigatus, but has no assigned function. pks carries the snp resulting in an alanine to aspartic acid substitution at position located at the acyl transferase/dehydratase inter-domain region. this mutation was acquired in the second csi period and presumably does not affect the function of the neighboring domains. the highly reducing pks is homologous to an uncharacterized pks enzyme of a. flavus and belongs to a putative cluster of genes. a single mutation resulted in a phenylalanine to leucine substitution at position in the c-/dehydratase inter-domain region and occurred in the second period of csi. a further gene cluster contains two pkss encoding genes hr pks and non-reducing pks and consists of orfs. a mutation within the ketosynthase domain of pks occurred during the first csi period and resulted in isoleucine to phenylalanine substitution at position a nr pks encoded by pc21g shows similarity to the a. nidulans wa and a. fumigatus pksp naphthopyrone synthases. these enzymes are involved in the biosynthesis of a precursor for the formation of a conidial pigment. a similar gene cluster of orfs is present in the genomes of a. niger and a. kawachii. two nucleotide substitutions were detected within pks causing no effect on the amino acid sequence. a third mutation is located within the terminator region of the pks gene and is represented by an a to c nucleotide substitution. the actual impact of these mutations on pigment formation is unclear as the green coloring of the conidia was not affected during the csi. the gene cluster of pr pks shows 82 % identity to the 6-methylsalicylic acid synthase from p. griseofulvum and contains orfs. the p. griseofulvum homolog is involved in patulin biosynthesis and requires additional enzymatic conversions like decarboxylation, two hydroxylation and oxidation reactions. in p. chrysogenum these enzymatic steps are presumably provided by the aminohydrolase and two cytochrome p enzymes encoded within the cluster. however, the essential gene encoding the isoepoxidon dehydrogenase cannot be identified in the genome of p. chrysogenum supporting the notion that this fungus does not produce patulin. a single mutation was obtained during the second period of csi and this affected a putative mfs transporter encoding gene introducing a tyrosine to asparagine substitution at position finally, two neighboring, oppositely oriented nr pks and hr pks that belong to a gene cluster that potentially is related to sorbicillinoid biosynthesis, received snps in both coding sequences. pks acquired an isoleucine to arginine substitution at c-/te inter-domain region, while pks obtained a mutation in the ks domain where leucine in position is substituted by a phenylalanine. both mutations were obtained at the first period of the csi and thus were inherited already by strain wisconsin 54- the associated gene cluster is predicted to contain orfs. the only nrps gene that accumulated snps concerns pssa that is involved in siderophore coprogen b biosynthesis under the iron depleted growth conditions. at the first csi period the pssa encoding gene was mutated causing a glutamic acid for aspartic acid substitution at position within a non-conserved region. pssa is involved in the production of coprogen that still can be produced by the improved β-lactam producing strains. therefore, this mutation apparently did not inactivate the enzyme. mutational changes within the velvet complex the velvet complex is involved in the regulation of sexual development and secondary metabolism as demonstrated in various filamentous fungi including p. chrysogenum. since it is a multisubunit complex, we examined the impact of csi on the core component of the velvet complex. the p. chrysogenum orthologs lrea, lreb, fpha, kapa, velb, and vosa were all identical in the genomes of strains nrrl wisconsin 54- and ds however, mutational changes were found in pc16g and pc13g genes that encode leaa and vela, respectively. in the first period, the leaa gene acquired a nucleotide substitution at position which resulted in a substitution of lysine for glutamic acid at position of of the protein. a second mutation occurred in period and caused a nucleotide change at position causing a glycine to serine substitution at position in addition, a mutation was identified in the vela encoding gene that occurred at the second csi period. a c to t substitution at nucleotide position caused a stop codon resulted in the formation of a truncation of the vela protein from to amino acids long. these data indicate that the velvet complex has been a major target in the csi, and this likely has impacted the expression of secondary metabolite encoding genes. expression of the secondary metabolism gene clusters since the velvet complex and many secondary metabolism genes acquired mutations during the csi, the expression of the latter genes may have vastly changed along the lineage of strains. therefore, we examined the expression of all pkss and nrps encoding genes in strains nrrl wisconsin 54- ds and the penicillin cluster free strain ds. herein, the fungi were grown for 5 days and total rna was isolated for qpcr analysis of the respective genes. the expression of γ-actin gene was used as reference for normalization. the observed ct values for the amplified γ-actin gene from the cdna of nrrl wisconsin 54- ds and ds were  ± ;  ± ;  ±  and  ±  respectively. this demonstrates nearly identical expression of the reference gene in the analyzed strains. the obtained expression profiles clearly indicates that the expression of the acv synthetase, the key enzyme of β-lactam biosynthesis was dramatically enhanced upon csi of wisconsin 54- as expected from the amplification of the β-lactam biosynthesis genes in strain ds that contains copies of this penicillin cluster. with the other nrps and pks genes, there were no marked expression changes except for three pks genes. the two highly expressed pks genes pc21g and pc21g were dramatically down regulated in wisconsin 54- and the derivatives ds and ds the nr pks exhibits 43 % identity to citrinin polyketide synthase of monascus purpureus while hr pks is homologous to lovastatin diketide synthase lovf of a. terreus. in addition, the cluster contains genes encoding a putative regulator, a monooxygenase, a transcriptional factor, a mfs transporter and an oxidoreductase. all five genes of this putative cluster are also present in the genome of trichoderma reesei and arranged in one locus. an identical gene cluster in the marine isolate of p. chrysogenum e01-10/ has been previously associated with the biosynthesis of sorbicillactones a and b and thus by analogy, the aforementioned gene cluster may encode the biosynthesis pathway of yellow pigments sorbicillinoids in p. chrysogenum. there is a significant decrease in expression of the seven genes of this cluster in the improved strains, whereas expression of flanking genes that are likely not part of the cluster was unaltered. in addition, expression of pks was increased substantially upon csi of the nrll strain. despite the significant overexpression of this pks in the improved penicillin producing strain, the polyketide product has not yet been identified.fig. comparison of secondary metabolite gene expression in strains nrrl wisconsin 54- ds and ds expression data was obtained with rna isolated after 5 days of fungal growth. the error bars indicate the standard error of the mean of two biological samples including technical duplicates fig. relative expression of the putative gene cluster of sorbicillinoid biosynthesis in strains nrrl wisconsin 54- ds and ds the transcriptional silencing of seven genes belonging to the putative sorbicillinoid gene cluster is shown. expression data was obtained with rna isolated after 5 days of fungal growth. the error bars indicate the standard error of the mean of two biological samples including technical duplicates comparative metabolomic profiling of p. chrysogenum strains next, we examined the extracellular metabolome focusing on secondary metabolites excreted into the spent culture medium. lc-ms and lc-ms/ms were used for identification. in this analysis, five β-lactams were detected in the growth media of all strains, except for the penicillin biosynthesis cluster-free strain ds. 6-apa, penicillin g, v, isopenicillin n and penicillin k were identified by their expected molecular masses and on the basis of their calculated empirical formulas and by the use of standards, except for penicillin k. 6-apa and isopenicillin n were produced in the early stages of fermentation. as expected, the levels of these compounds were relatively low for the nrrl strain, increased in strain wisconsin 54- and dramatically increased in ds in particular during the late growth phase. 6-apa, isopenicillin n and the naturally produced penicillin k were the main products. since the growth conditions did not involve any feed with the commercially used side chain precursors, penicillin g and v were presented very low amounts compared to other penicillins.fig. response ratio of secondary metabolite concentrations present in the culture broth of strains nrrl wisconsin 54- ds and ds cell coloring corresponds to the internal standard corrected peak area of the detected metabolites in the culture broth after and 7 days of growth. the mass to charge ratio of the protonated metabolites, retention time and empirical formulas are indicated. abbreviations: htd, histidyltryptophanyldiketopiperazine; dhtd, dehydrohistidyltryptophanyldi-ketopiperazine; na, metabolites without structural data another group of compounds are the chrysogine related metabolites. this yellow pigment is produced in a biosynthetic pathway that has not yet been completely characterized, but at least four structurally resolved compounds can be identified including chrysogine, n-acetylalanylanthranilamide, n-malonylalanylanthranilamide and n-pyrovoylanthranilamide, as well as related but structurally unresolved molecules. these metabolites are already found in the culture broth after 3 days. chrysogine was produced at high levels by the ds strain, but in particular in the related penicillin cluster free ds strain, production was at the highest levels. a further class of compounds are the roquefortine and meleagrin related metabolites that are toxins. the precursors for these toxins are and dehydrohistidyltryptophanyldi-ketopiperazine that are only produced at low levels by strain nrrl and wisconsin, but at substantial higher levels during the late growth phases by the ds strain. production further increased in the ds strain. the same observation was made for the precursors of this pathway like glandicoline a-b, roquefortine c, d, f, m, n and neoxalin, as well as the final product meleagrin. it thus appears that like with the chrysogine, period of the csi has resulted in an enhanced production of roquefortines. in the analysis, a unique group of metabolites were identified in the culture broth of nrrl that were absent in the other more evolved strains. based on their exact masses and their ms spectra, empirical formulas could be assigned to these molecules with less than 2 ppm accuracy. two groups of compounds could be discriminated. the first group includes eleven molecules related to sorbicillins that are known to be produced by natural isolates of p. chrysogenum. both, monomeric and dimeric sorbicillinoids were identified in the culture broth during seven days of culturing. the identified compounds fit to the proposed biosynthesis pathway. major molecules are sorbicillin and the derivative sorbicillinol that already occur after 3 days of fermentation. although dihydrosorbicillin was not detected, the accumulation of its oxidized derivative dihydrosorbicillinol occurred after five days of cultivation. oxosorbicillinol, and the oxidized compound bisvertinolon were identified. further dimers were bisorbicillinol, dyhydrobisvertinulone and tetrahydrobisvertinolone. in addition, two uncharacterized metabolites with a short carbon chain were observed. the second group included four, presumably nitrogen containing compounds.fig. metabolites characterized in this study. β-lactam related compounds, the intermediates of chrysogine pathway, roquefortine/meleagrin related compounds, the representative of the cyclic tetrapeptides - fungisporin and the sorbicillinoids discussion here we have examined the effect of the classical strain improvement program on secondary metabolism by p. chrysogenum. combined metabolome, transcriptome and genome profiling approaches were applied to a lineage of β-lactam producing strains. this concerned the natural isolate strain nrrl the current laboratory reference wisconsin 54- and the high penicillin producer ds which carries multiple copies of the penicillin biosynthesis cluster. in addition, a ds derivative, which lacks the penicillin clusters, i.e., strain ds was included in this analysis. the genomes of the subjected strains were sequenced using the illumina next generation technique that allowed us to determine the mutational impact on p. chrysogenum during csi. of the pkss and nrps present in p. chrysogenum, the majority remains uncharacterized and has not been associated to the production of specific secondary metabolites. it is generally believed that many of these genes and gene clusters are transcriptionally silent or inactive. the introduction of the parental isolate nrrl in our analysis revealed a number of mutations inherited by the current international laboratory standard strain wisconsin 54- during the first period of csi. further comparative analysis showed the accumulation of additional mutations during the evolvement of the industrial penicillin cluster multi-copy strain ds out of the nrps and pks associated gene clusters present in the genome, one nrps and pks gene clusters accumulated mutational changes during both periods of the csi. three amino acid substitutions were located within the essential ketosynthase domains of pks pks and pks that presumably led to a functional inactivation. significant changes of the secondary metabolite genes expression between the csi derived mutants as well as the mutational impact on transcriptional factor encoding genes indicates that the regulation of secondary metabolism has been intensively altered during the csi. the three proteins vela, velb and laea of the velvet complex represent an important regulatory element in filamentous fungi. in a. nidulans, this heterotrimer has been associated with both cell development and secondary metabolism providing the first evidence on the regulatory cross-talk between these phenomena in filamentous fungi. the current structural model of the velvet complex is derived from protein interaction studies performed in a. nidulans. vela is a scaffolding protein essential for recruiting velb and laea. velb interacts with the n-terminus of vela while the c-terminus interacts with laea. it is important to emphasize that all recently published studies on the characterization of the velvet complex proteins in p. chrysogenum were performed in strains derived from the csi, i.e., the eight copy penicillin gene cluster strain ds derived from wisconsin 54- and the two copy penicillin gene cluster strain p2niad obtained during the alternative panlabs strain improvement program. both overproducers originate from the parental nrrl isolate but the p series diverged early at the csi prior the generation of wisconsin 54- a pronounced phenotypic effect of the deletion of vela and laea on β-lactam titers in conjunction with a dramatic transcriptional deregulation of up to 10 % of genes present in the genome was demonstrated for the panlabs derived p2niad strain. in contrast, the penicillin gene expression and secondary metabolite production in ds strain exhibited only moderate responses to the deletion of vela and laea. the latter study, however, was performed under glucose-limited chemostat growth conditions, and it was suggested that the growth conditions weakened the phenotype. however, since the velvet complex has been repetitively targeted during the improvement of the ds strain this likely caused the weak phenotypes of the respective deletion strains. indeed, the p2niad strain shows no alterations of the velvet complex encoding genes and must therefore have remained functionally intact. specifically, in leaa, we identified two mutations resulting in amino acids changes while in vela, in ds strain a stop codon is introduced resulting in a c-terminal truncation by about amino acids. vela plays a primary role in the complex assembly where the c-terminus that was lost, is required for interaction with laea. therefore, the velvet complex of the improved ds is likely functionally impaired and this must have contributed to an improvement of the β-lactam production presumably as a collateral effect, increased or decreased expression of other secondary metabolite genes. in addition, pathway specific transcriptional regulation of secondary metabolism is crucial. we identified mutated proteins specifying zn2cys binuclear cluster dna-binding domains typical found in transcription regulators like gal and six genes encoding proteins that are homologous to fungal specific transcription factors. additionally, transporter related genes were mutated during the csi, eight abc transporters and transporters belonging to the major facilitator superfamily. one of the identified mfs pc22g belongs to the putative secondary metabolite gene cluster harboring a pks gene that can be linked to the biosynthesis of patulin-like metabolites in p. chrysogenum. the mutational and expression effects of csi led to the significantly alterations in the secondary metabolite profile among the analyzed p. chrysogenum strains. previously, genes and gene clusters have been identified for the production of roquefortine/meleagrine, cyclic tetrapeptides and chrysogine that all arise from the activity of nrpss. by high-resolution mass spectrometry we generated a specific metabolite profile for each of the strains and demonstrate significant alterations in the pattern of the production of these secondary metabolites. importantly, during the csi and with the onset of strain ds the ability to produce toxins such as roquefortine/meleagrine and chrysogen increased. however, the transcriptional levels of the roqa and chya genes was not significantly changed in the lineage of strains. considering that the biosynthesis of these secondary metabolites is strongly dependent on nitrogen metabolism, it seems likely that the improvement in amino acid production in the higher β-lactam producers is the indirect cause of this phenomenon. moreover, the highest levels of these secondary metabolites where found in the ds strain in which the multiple penicillin gene clusters have been removed. this strain is genomically identical to the parental strain ds also showing the same expression levels and timing of the expression of the corresponding nrps encoding genes. therefore, the observed increase in the aforementioned nonribosomal peptide products strongly supports our notion that their enhanced production is because of a re-diversion of nitrogen metabolism. during the csi, also the production of a suite of secondary metabolites was lost. early p. chrysogenum isolates produced yellow pigments that were eliminated early on during csi as they were found to be colored contaminants during antibiotic production. however, the genetic origin of pigment formation has remained obscure. our secondary metabolite profiling indicates a group of structurally related compounds that are specifically produced by the nrrl strain but are absent in the fermentation broth of wisconsin 54- ds and ds the empirical formulas generated based on mass accuracy correspond to known hexaketide yellow pigments termed sorbicillinoids. the production of sorbicillin, sorbicillinol, dihydrosorbicillinol, oxosorbicillinol, bisvertinolone, bisorbicillinol, and dihydrobisvertinolone was detected. we found that accumulation of the pathway precursors sorbicillin and sorbicillinol by a three days grown culture correlates with high levels of expression of a single pks gene cluster in the nrrl strain. qpcr analysis revealed the transcriptional silencing of the entire gene cluster of seven genes although expression of the flanking genes remained unchanged. additionally, the genome sequencing analysis revealed two mutations accumulated in the two pks encoding genes of the aforementioned cluster at the beginning of the csi program. nr-pks acquired an isoleucine to arginine substitution at the c-/dh inter-domain region. the neighboring oppositely oriented hr-pks encoding gene obtained a mutation causing a leucine to a phenylalanine substitution in position within the ks domain of the enzyme. the proposed model for sorbicillin biosynthesis, as the hexaketide precursor for all sorbicillinoid derivatives, foresees the function of a pair of nr-pks and hr-pks enzymes. the triacetate precursor is formed by hr-pks following the non-reductive carbon chain extension by the second nr-pks to form hexaketide. taken together our results suggest this pks gene cluster is responsible for the biosynthesis of sorbicillinoids in the early producer nrrl the mutations found within the pks gene cluster combined with the transcriptional silencing of the entire gene cluster shown for derivative strains wisconsin 54- and ds demonstrates a multi-step complex eliminating effect of the csi on the biosynthesis of these yellow pigments in p. chrysogenum. importantly, the functional characterization of the aforementioned gene cluster through direct targeting of the pks genes has not yet been achieved, and will be subject of future work.  our study concerned a combined genome, transcriptome and metabolome analysis of improved β-lactam producing strains of p. chrysogenum with a focus on secondary metabolism. whereas several secondary metabolite genes were targeted by mutagenesis, overall there was not a single specific functional group of genes that was specifically mutagenized. mutations appear to be randomly spread across the chromosomes and this is likely the result of the rather undirected approach used during csi. the results suggest that the improved titers of the β-lactams achieved during the csi are only partially reflected by the broad mutagenic impact of the csi program. whereas mutagenesis was random, the selection was focused on specific features such as the increased β-lactam titers; morphological aspects as well the fermentation characteristics. thus, the strains collected many mutations that do not contribute to these features. the elevated production of nonribosomal peptide products such as chrysogine and roquefortine/meleagrin likely emerged from the enhanced nitrogen metabolic flux in improved penicillin producing strains. a group of yellow pigments including sorbicillinoids has been eliminated at the early stages of csi. production of sorbicillinoids is likely associated with a pks gene cluster of seven genes that was transcriptionally silenced during the csi and that acquired mutations at functionally essential pks domains. these finding indicate a complex mechanism underlying the elimination of the production of sorbicillinoids from the secondary metabolism of p. chrysogenum and provide insight in the molecular basis of biosynthesis of this interesting group of bioactive compounds. 
additionally, key velvet complex proteins involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during csi. however, the impact of the csi on the secondary metabolism in general remains unknown. 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 nrrl wisconsin 54- ds and the derived penicillin biosynthesis cluster free strain ds csi has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. penicillium chrysogenumsecondary metabolismsorbicillinoidspolyketide synthasessequencingissue-copyright-statement© the author 2015 however, in this set of mutations, out of secondary metabolite genes were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to β–lactam production. the high β–lactam titers of current strains is the result of a classical strain improvement program starting with a wild-type like strain more than six decades ago. 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. 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. this involved extensive mutagenesis and strain selection for improved β–lactam titers and growth characteristics.
 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 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.  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 nrrl wisconsin 54- ds and the derived penicillin biosynthesis cluster free strain ds 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, out of secondary metabolite genes 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 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords penicillium chrysogenumsecondary metabolismsorbicillinoidspolyketide synthasessequencingissue-copyright-statement© the author 2015 