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Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during ?-Lactam Production.

ABSTRACT: To produce high levels of ?-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 ?-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 ?-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.

SUBMITTER: Wu M

PROVIDER: S-EPMC6974646 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during β-Lactam Production.

Wu Min M Crismaru Ciprian G CG Salo Oleksandr O Bovenberg Roel A L RAL Driessen Arnold J M AJM

Applied and environmental microbiology 20200121 3

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 ...[more]

PMID: 31757830

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Project description:The multi-component global regulator Velvet complex has been identified as a key regulator of secondary metabolite production in Aspergillus and Penicillium species. Previous work indicated a massive impact of PcvelA and PclaeA deletions, two key components of the Velvet complex, on penicillin production in prolonged batch cultures of P. chrysogenum, as well as substantial changes in transcriptome. The present study investigates the impact of these mutations on product formation and genome-wide transcript profiles under glucose-limited, aerobic conditions, relevant for industrial production of ?-lactams. The gene-deletion cassette for PcvelA or PclaeA was integrated in a hdfA mutant of the penicillin high-producing strain P. chrysogenum DS17690. Predicted amino acid sequences of PcVelA and PcLaeA in this strain were identical to those in its ancestor Wisconsin54-1255. Controls were performed to rule out transformation-associated loss of penicillin-biosynthesis clusters which, in preliminary studies, led to a massive reduction of penicillin production in a PcvelA deletion mutant. The correct PcvelA and PclaeA deletion strains revealed a significant (up to 30 %) reduction of penicillin-G productivity relative to the reference strain, which is a much smaller reduction than previously reported for prolonged batch cultures of P. chrysogenum strains. Chemostat-based transcriptome analysis yielded only 23 genes with a consistent response in the PcvelA? and PclaeA? mutants when grown in the absence of the penicillin-G side-chain precursor phenylacetic acid. 11 of these genes belonged to two small gene clusters (with 5 and 6 genes, respectively), one of which contains a gene with high homology to an aristolochene synthase. These results provide a clear caveat that the impact of the Velvet complex on secondary metabolism in filamentous fungi may be strongly context dependent Previous studies on the impact of the Velvet complex in P. chrysogenum were performed in prolonged batch cultures. Time course analysis revealed that the impact of PcvelA and PclaeA mutations was most pronounced after prolonged incubation {Hoff, 2010 6 /id}, but the physiological status of these cultures was not precisely defined. Industrial production of ?-lactam antibiotics is performed in sugar-limited, aerobic fed-batch cultures {Menezes, 1994 76 /id}. The aim of the present study is to investigate the impact of the Velvet complex on physiology, penicilllin production and transcriptional regulation under industrially relevant conditions. To this end, we studied the impact of PcvelA and PclaeA deletions in aerobic, glucose-limited chemostat cultures of the penicillin high-producing strain P. chrysogenum DS17690.

Dataset Information

Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during ?-Lactam Production.

Publications

Impact of Classical Strain Improvement of <i>Penicillium rubens</i> on Amino Acid Metabolism during β-Lactam Production.

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