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

Abstract

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.

Influence of oxygen concentration on the metabolism of Penicillium chrysogenum

“…Therefore, possible reasons for the lack of ACV accumulation are insufficient amino acid or energy supply for the conversion. It has been reported that amino acid synthesis, especially cysteine biosynthesis, can limit the pathway flux [51]; however, our measurements indicated steady cysteine and valine concentrations. In contrast to the 0.009 mmol L −1 DO step experiment, the intracellular AAA concentration decreased only slightly during oscillation experiment I.…”

Studying the Gene Expression of Penicillium rubens Under the Effect of Eight Essential Oils

“…Three AAs serve as building blocks, namely α-aminoadipate, L-cysteine and L-valine. A previous analysis demonstrated that the gene Pc20g04020 is considered to be unique for coding the threonine synthase in P. rubens [35]. It plays a key role in catalyzing the conversion of O-phospho-L-homoserine into L-threonine and phosphate.…”

“…Pc13g07730 is annotated as L-threonine deaminase, which removes an amino group from L-threonine, resulting in the production of 2-oxobutanoate and pyruvate. The corresponding enzyme that catalyzes the conversion of glycine and succinal-CoA into 5-aminolevulinate, CoA and CO 2 is 5-aminolevulinate synthase (Pc22g13500), also named as the pyridoxal 5'-phosphate-dependent (PLP) enzyme [35]. These findings suggested that the influence of applied EOs may play an important role in modulating these signaling pathways and, thus, may regulate processes involved in cysteine production, which is very important for beta lactam production [35].…”

“…According to current knowledge, gene Pc20g08350 is coding a cystathionine gamma synthase, which is a transsulfuration enzyme involved in the catalysis of the PLP-dependent γ-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-cystathione and acetate [35]. Hypothetically, the alteration of the β-lactam production can bring to the modification a synthesis of diverse compounds that can increase the vulnerability of the fungus against different environmental factors.…”

Strategies for the Development of Industrial Fungal Producing Strains

“…In addition, many T. reesei strains that are used in industry underwent point mutations leading to catabolite de-repression, resulting in increased extracellular enzyme and protein levels compared to their parent strain [ 24 ]. Point mutations also resulted in improved cysteine biosynthesis in P. rubens by the inactivation of enzymatic conversions that compete with the cysteine biosynthetic pathway, which plays a key role in penicillin production [ 248 ]. By site-directed mutagenesis, the thermostability of an A. niger xylanase has been improved, showing up to 80% of its maximal activity after incubation for 2 h at 50 °C in the presence of xylan, compared to only 15% activity for the wild-type enzyme [ 249 ].…”