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

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 M 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 M 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. Small organic molecules of biotechnological interest include aromatic structures that are derived from p-coumaric acid (pHCA). pHCA can be formed from phenylalanine either through deamination to cinnamic acid (CA) by phenylalanine ammonialyase (PAL; EC 4.3.1.24) and subsequent hydroxylase activity by trans-cinnamate-4-monooxygenase or through deamination of tyrosine by tyrosine ammonia-lyase (TAL; EC 4.3.1.23) (Fig. 1). Considering that the route from phenylalanine requires activity of a P450 enzyme, which has been shown to be the limiting step in previous engineering strategies (1, 2), deamination of tyrosine for production of pHCA may be preferred in microbial cell factories. Tyrosine ammonia-lyases (TAL) have been employed for the production of plant biochemicals and aromatic compounds, e.g., for pHCA itself (3-5), or in the production of stilbenes, such as resveratrol (6, 7), flavonoids, such as naringenin (8, 9), cinnamoyl anthranilates (10), or plastic precursors, such as p-hydroxystyrene (11, 12), yet the TAL reaction may be the limiting step (10, 13).Due to the significant interest in production of phenolic compounds in biotechnological processes, we aimed at increasing the range of characterized phenylalanine ammonia-lyases (PAL) and tyrosine ammonia-lyases (TAL), and in particular at identifying the optimal TAL for use in microbial cell factories...

Production of clovamide and its analogues inSaccharomyces cerevisiaeandLactococcus lactis

“…Functional expression of bacterial tyrosine ammonia‐lyases (TAL) has recently been demonstrated in L . lactis for conversion of tyrosine into micromolar quantities of p ‐coumarate (Jendresen et al ). Therefore, co‐expression of TAL, 4CL5 and HDT1 in L .…”

Fine‐tuning the (2S)‐naringenin synthetic pathway using an iterative high‐throughput balancing strategy

“…To improve the titer of target chemicals, researchers first focused on screening for high‐activity enzymes in plants, animals, fungi, and bacteria (Jendresen et al, ; Santos et al, ). If the screened enzymes cannot satisfy present requirements of metabolic engineering, then protein engineering strategies, like directed evolution (S. Zhou & Alper, ; S. Zhou et al, ) and computer‐aided methods (Ebert & Pelletier, ; Fang, Zhang, Du, & Chen, ; Verma, Schwaneberg, & Roccatano, ), are typically performed to further improve the properties of the target enzymes.…”

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

“…Here, taking advantage of 4CL5 substrate’s promiscuity, several BAHD acyltransferases were selected and co-expressed with 4CL5 in an attempt to synthesize in yeast novel hydroxycinnamate and benzoate esters and amides. Furthermore, although the yields of product formation were typically two to fivefold lower compared to those achieved with p -coumarate feedings, additional expression of tyrosine ammonia-lyase from Flavobacterium johnsoniae (FjTAL), which converts tyrosine into p -coumarate [23], enabled the synthesis of p -coumarate esters and amides directly from the endogenous tyrosine pool.…”