RESUMO
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.
Assuntos
Amônia-Liases/metabolismo , Bactérias/enzimologia , Bactérias/metabolismo , Hidrocarbonetos Aromáticos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Amônia-Liases/genética , Bactérias/genética , Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Análise de Sequência de DNARESUMO
Yeast Saccharomyces cerevisiae was engineered for de novo production of six different flavonoids (naringenin, liquiritigenin, kaempferol, resokaempferol, quercetin, and fisetin) directly from glucose, without supplementation of expensive intermediates. This required reconstruction of long biosynthetic pathways, comprising up to eight heterologous genes from plants. The obtained titers of kaempferol 26.57±2.66mgL-1 and quercetin 20.38±2.57mgL-1 exceed the previously reported titers in yeast. This is also the first report of de novo biosynthesis of resokaempferol and fisetin in yeast. The work demonstrates the potential of flavonoid-producing yeast cell factories.
Assuntos
Fermentação , Engenharia Metabólica , Saccharomyces cerevisiae , Vias Biossintéticas , FlavonoidesRESUMO
KEY CLINICAL MESSAGE: Community-acquired Klebsiella pneumoniae primary liver abscess (KLA) has been emerging worldwide over the past two decades and with high incidence in Asia. The presence of specific virulence characteristics is a risk factor for a syndrome with metastatic complications. This report signals an increasing emergence in Northern Europe.