Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae.
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 [OD600]) 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 OD600 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.
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark cbj@biosustain.dtu.dk atn@biosustain.dtu.dk.
Christian Bille
http://orcid.org/0000-0002-1921-2164
CB
Jendresen
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Steen Gustav
SG
Stahlhut
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Mingji
M
Li
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Paula
P
Gaspar
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Solvej
S
Siedler
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Jochen
J
Förster
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Jérôme
J
Maury
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Irina
I
Borodina
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark cbj@biosustain.dtu.dk atn@biosustain.dtu.dk.
Alex Toftgaard
AT
Nielsen
D000642:Ammonia-Lyases / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006841:Hydrocarbons, Aromatic / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D006841:Hydrocarbons, Aromatic; D011994:Recombinant Proteins; D000642:Ammonia-Lyases; C023208:L-tyrosine ammonia-lyase