|Wu M, Crismaru CG, Salo O, Bovenberg RAL, Driessen AJ
|Impact of classical strain improvement of Penicillium rubens on amino acid metabolism during ß-lactam production
|Appl Environ Microbiol
|To produce high levels of β-lactams, the filamentous fungus Penicillium rubens (previously named P. 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 statistically spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular L-cysteine biosynthesis, one of the amino acids used for β-lactam production. To examine the impact of the mutations of the enzyme function, the respective genes with and without the mutations were cloned and expressed in E. 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 the 5-aminolevulinate synthase, which utilizes glycine was without an effect. Importantly, the CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that the CSI has resulted in improved cysteine biosynthesis by inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is key component during penicillin production.IMPORTANCE Penicillium rubens is an important industrial producer of β-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism, and resulted in enhanced L-cysteine biosynthesis. This work provides a molecular explanation on the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.