Impact of classical strain improvement of penicillium rubens on amino acid metabolism during β-Lactam production

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

Metabolism of β-valine via a CoA-dependent ammonia lyase pathway

Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.

Engineering of an enantioselective tyrosine aminomutase by mutation of a single active site residue in phenylalanine aminomutase

By replacing a single active-site residue Cys107 with Ser in phenylalanine aminomutase (PAM), the enzyme gained tyrosine aminomutase (TAM) activity while retaining PAM activity and high enantioselectivity. This engineered enantioselective TAM also catalyzed formation of β-tyrosine from p-coumaric acid and may prove to be useful for the synthesis of enantiopure β-tyrosine and its derivatives.

Biochemical Properties and Crystal Structure of a beta-Phenylalanine Aminotransferase from Variovorax paradoxus:a Novel Zinc-Regulated Expression System for Lactococcus lactis

<p>By selective enrichment, we isolated a bacterium that can use beta-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg(-1) for (S)-beta-phenylalanine at 30 degrees C and 33 U mg(-1) at the optimum temperature of 55 degrees C. The beta-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted beta-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-beta-phenylalanine (enantioselectivity [E], > 100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic beta-amino acids to yield (R)-beta-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the beta-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo-and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic beta-amino acid-converting aminotransferases may be identified.</p>