Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a β-Lactam High-Producing<i>Penicillium chrysogenum</i>Strain

Veiga, Tânia; Nijland, Jeroen G.; Driessen, Arnold J.M.; Bovenberg, Roel A. L.; Touw, Hesselein; Berg, M. A.; Pronk, Jack T.; Daran, Jean‐Marc

doi:10.1089/omi.2011.0153

Cited by 25 publications

(17 citation statements)

References 53 publications

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“…This is different to a previous report on another P. chrysogenum producer strain (DS17690), showing that strain improvement programs have targeted the velvet gene, and thus leading to a truncated version of Velvet [10]. A subsequent complete deletion of the velvet gene in DS17690 background did not result in a major reduction of the penicillin titer [84]. …”

Section: Resultscontrasting

confidence: 93%

Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs

2017

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BackgroundCephalosporins and penicillins are the most frequently used β-lactam antibiotics for the treatment of human infections worldwide. The main industrial producers of these antibiotics are Acremonium chrysogenum and Penicillium chrysogenum, two taxonomically unrelated fungi. Both were subjects of long-term strain development programs to reach economically relevant antibiotic titers. It is so far unknown, whether equivalent changes in gene expression lead to elevated antibiotic titers in production strains.ResultsUsing the sequence of PcbC, a key enzyme of β-lactam antibiotic biosynthesis, from eighteen different pro- and eukaryotic microorganisms, we have constructed a phylogenetic tree to demonstrate the distant relationship of both fungal producers. To address the question whether both fungi have undergone similar genetic adaptions, we have performed a comparative gene expression analysis of wild-type and production strains. We found that strain improvement is associated with the remodeling of the transcriptional landscape in both fungi. In P. chrysogenum, 748 genes showed differential expression, while 1572 genes from A. chrysogenum are differentially expressed in the industrial strain. Common in both fungi is the upregulation of genes belonging to primary and secondary metabolism, notably those involved in precursor supply for β-lactam production. Other genes not essential for β-lactam production are downregulated with a preference for those responsible for transport processes or biosynthesis of other secondary metabolites. Transcriptional regulation was shown to be an important parameter during strain improvement in different organisms. We therefore investigated deletion strains of the major transcriptional regulator velvet from both production strains.We identified 567 P. chrysogenum and 412 A. chrysogenum Velvet target genes. In both deletion strains, approximately 50% of all secondary metabolite cluster genes are differentially regulated, including β-lactam biosynthesis genes. Most importantly, 35-57% of Velvet target genes are among those that showed differential expression in both improved industrial strains.ConclusionsThe major finding of our comparative transcriptome analysis is that strain improvement programs in two unrelated fungal β-lactam antibiotic producers alter the expression of target genes of Velvet, a global regulator of secondary metabolism. From these results, we conclude that regulatory alterations are crucial contributing factors for improved β-lactam antibiotic titers during strain improvement in both fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3663-0) contains supplementary material, which is available to authorized users.

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Section: Resultscontrasting

confidence: 93%

Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs

2017

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“…The Velvet complex is involved in the regulation of sexual development and secondary metabolism as demonstrated in various filamentous fungi including P. chrysogenum [ 3 , 6 , 7 ]. Since it is a multisubunit complex, we examined the impact of CSI on the core component of the Velvet complex.…”

Section: Resultsmentioning

confidence: 99%

“…However, a major breakthrough in the CSI of NRRL1951 was the selection of the so-called Wisconsin series of improved strains that were derived from the X-1612 (implying X-ray treatment) mutant that was further evolved into the current international laboratory standard strain Wisconsin 54-1255 whose genome sequence became available in 2008 [ 2 ]. Currently, Wisconsin 54-1255 is the reference strain for genome, proteome and transcriptome comparative studies [ 3 – 7 ]. 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 [ 8 ], an enhanced amino acid production and various aspects of cellular development [ 2 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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.

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“…A significant downregulation and several morphological changes in the penicillin-biosynthesizing genes were observed in prolonged batch cultures of two different P. chrysogenum strains (Kosalková et al, 2009; Butchko et al, 2012). When the laeA deletion mutant was grown in chemostat cultures, only moderate transcriptional response and penicillin production was observed even at glucose-limited conditions (Veiga et al, 2012). Interestingly, Fekete et al observed that the growth-rate dependence of LaeA function in T. reesei was in the opposite direction.…”

Section: Discussionmentioning

confidence: 99%

The Different Roles of Penicillium oxalicum LaeA in the Production of Extracellular Cellulase and β-xylosidase

Zheng

Zhang

et al. 2016

Front. Microbiol.

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Cellulolytic enzyme hydrolysis of lignocellulose biomass to release fermentable sugars is one of the key steps in biofuel refining. Gene expression of fungal cellulolytic enzymes is tightly controlled at the transcriptional level. Key transcription factors such as activator ClrB/CLR2 and XlnR/XYR1, as well as repressor CreA/CRE1 play crucial roles in this process. The putative protein methyltransferase LaeA/LAE1 has also been reported to regulate the gene expression of the cellulolytic enzyme. The formation and gene expression of the cellulolytic enzyme was compared among Penicillium oxalicum wild type (WT) and seven mutants, including ΔlaeA (deletion of laeA), OEclrB (clrB overexpression), OEclrBΔlaeA (clrB overexpression with deletion of laeA), OExlnR (xlnR overexpression), OExlnRΔlaeA (xlnR overexpression with deletion of laeA), ΔcreA (deletion of creA), and ΔcreAΔlaeA (double deletion of creA and laeA). Results revealed that LaeA extensively affected the expression of glycoside hydrolase genes. The expression of genes that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA. Cellulase synthesis of four mutants ΔlaeA, OEclrBΔlaeA, OExlnRΔlaeA, and ΔcreAΔlaeA was repressed remarkably compared with their parent strains WT, OEclrB, OExlnR, and ΔcreA, respectively. The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent, suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR. In contrast to LaeA positive roles in regulating prominent cellulase and hemicellulase, the extracellular β-xylosidase formation was negatively regulated by LaeA. The extracellular β-xylosidase activities improved over 5-fold in the OExlnRΔlaeA mutant compared with that of WT, and the expression of prominent β-xylosidase gene xyl3A was activated remarkably. The cumulative effect of LaeA and transcription factor XlnR has potential applications in the production of more β-xylosidase.

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Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a β-Lactam High-ProducingPenicillium chrysogenumStrain

Cited by 25 publications

References 53 publications

Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs

Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs

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

The Different Roles of Penicillium oxalicum LaeA in the Production of Extracellular Cellulase and β-xylosidase

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