Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

Abstract

ABSTRACT Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p -coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p -coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus , a bacterial-type enzyme from the amoeba Dictyostelium discoideum , and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD 600 ]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p -coumaric acid in Escherichia coli (producing 440 μM p -coumaric acid OD 600 unit −1 in the medium) and in Lactococcus lactis . The enzymes were also efficient in Saccharomyces cerevisiae , where p -coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.