Tyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family. Replacing the active site residue His89 with Phe in TAL completely switched its substrate selectivity from tyrosine to phenylalanine, thereby converting it into a highly active PAL. When a corresponding mutation was made in PAL, the enzyme lost PAL activity and gained TAL activity. The discovered substrate selectivity switch is a rare example of a complete alteration of substrate specificity by a single point mutation. We also show that the identity of the amino acid at the switch position can serve as a guide to predict substrate specificities of annotated aromatic amino acid lyases in genome sequences.
Previously, we utilized in vitro evolution to alter the catalytic functions of several carotenoid enzymes and produce the novel carotenoids tetradehydrolycopene and torulene in Escherichia coli. Here we report on the successful extension of these pathways and the C(30) carotenoid diaponeurosporene pathway with additional carotenoid genes. Extension of the known acyclic C(30) pathway with C(40) carotenoid enzymes-spheroidene monooxygenase and lycopene cyclase-yielded new oxygenated acylic products and the unnatural cyclic C(30) diapotorulene, respectively. Extension of acyclic C(40) pathways with spheroidene monooxygenase generated novel oxygenated carotenoids including the violet phillipsiaxanthin. Extension of the torulene biosynthetic pathway with carotene hydroxylase, desaturase, glucosylase, and ketolase yielded new torulene derivatives. These results demonstrate the utility of extending an in vitro evolved central metabolic pathway with catalytically promiscuous downstream enzymes in order to generate structurally novel compounds.
Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28 degrees C showed better production of torulene than 37 degrees C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2xYT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and beta-carotene, in E. coli. In contrast, glycerol-containing LB, 2xYT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1-3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.
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