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

IMPORTANCE Penicillium rubens is an important industrial producer of β-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

为了产生高水平的β-内酰胺，丝状真菌青霉（Penicillium rurysogenum）在过去的几十年中经历了广泛的经典菌株改良（CSI）程序。这导致了许多分布在基因组上的突变的积累。详细的分析表明，几个突变针对的基因编码的氨基酸代谢中涉及的酶，特别是氨基酸的生物合成。升-半胱氨酸，一种用于生产β-内酰胺的氨基酸。为了检查突变对酶功能的影响，将具有和不具有突变的各个基因克隆并在大肠杆菌中表达，纯化并进行酶分析。突变严重破坏了苏氨酸和丝氨酸脱氨酶的活性，这使竞争竞争的代谢途径失活。升-半胱氨酸的生物合成。色氨酸合酶，可转换升-丝氨酸成 升-色氨酸通过突变而失活，而利用甘氨酸的5-氨基乙酰丙酸酯合酶中的突变没有作用。重要的是，CSI导致直接参与半胱氨酸生物合成的一组基因的表达水平提高。这些结果表明，CSI通过使直接与半胱氨酸生物合成途径竞争资源的酶促转化失活而改善了半胱氨酸生物合成，这与半胱氨酸是青霉素生产过程中的关键成分这一观念相一致。

重要事项 青霉素是β-内酰胺类抗生素的重要工业生产商。在70多年的经典菌株改良（CSI）计划中，通过广泛的诱变，高水平的青霉素生产得以实现。一些突变靶向氨基酸代谢并导致增强升-半胱氨酸的生物合成。这项工作为次级代谢产物的产生与氨基酸代谢之间的相互关系以及经典菌株的改良如何导致改良的生产菌株提供了分子解释。