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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 [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 ?M 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.

SUBMITTER: Jendresen CB

PROVIDER: S-EPMC4475877 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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

Jendresen Christian Bille CB Stahlhut Steen Gustav SG Li Mingji M Gaspar Paula P Siedler Solvej S Förster Jochen J Maury Jérôme J Borodina Irina I Nielsen Alex Toftgaard AT

Applied and environmental microbiology 20150424 13

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 ...[more]

PMID: 25911487

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Project description:BackgroundDuring must fermentation thousands of volatile aroma compounds are formed, with higher alcohols, acetate esters and ethyl esters being the main aromatic compounds contributing to floral and fruity aromas. The action of yeast, in particular Saccharomyces cerevisiae, on the must components will build the architecture of the wine flavour and its fermentation bouquet. The objective of the present work was to better understand the molecular and metabolic bases of aroma production during a fermentation process. For such, comparative transcriptomic and metabolic analysis was performed at two time points (5 and 50 g/L of CO2 released) in fermentations conducted by four yeast strains from different origins and/or technological applications (cachaça, sake, wine, and laboratory), and multivariate factorial analyses were used to rationally identify new targets for improving aroma production.ResultsResults showed that strains from cachaça, sake and wine produced higher amounts of acetate esters, ethyl esters, acids and higher alcohols, in comparison with the laboratory strain. At fermentation time T1 (5 g/L CO2 released), comparative transcriptomics of the three S. cerevisiae strains from different fermentative environments in comparison with the laboratory yeast S288c, showed an increased expression of genes related with tetracyclic and pentacyclic triterpenes metabolism, involved in sterol synthesis. Sake strain also showed upregulation of genes ADH7 and AAD6, involved in the formation of higher alcohols in the Ehrlich pathway. For fermentation time point T2 (50 g/L CO2 released), again sake strain, but also VL1 strain, showed an increased expression of genes involved in formation of higher alcohols in the Ehrlich pathway, namely ADH7, ADH6 and AAD6, which is in accordance with the higher levels of methionol, isobutanol, isoamyl alcohol and phenylethanol observed.ConclusionsOur approach revealed successful to integrate data from several technologies (HPLC, GC-MS, microarrays) and using different data analysis methods (PCA, MFA). The results obtained increased our knowledge on the production of wine aroma and flavour, identifying new gene in association to the formation of flavour active compounds, mainly in the production of fatty acids, and ethyl and acetate esters.

Project description:DNA replication initiates from multiple replication origins (ORIs) in eukaryotes. Discovery and characterization of replication origins are essential for a better understanding of the molecular mechanism of DNA replication. In this study, the features of autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae have been comprehensively analyzed as follows. Firstly, we carried out the analysis of the ARSs available in S. cerevisiae S288C. By evaluating the sequence similarity of experimentally established ARSs, we found that 94.32% of ARSs are unique across the whole genome of S. cerevisiae S288C and those with high sequence similarity are prone to locate in subtelomeres. Subsequently, we built a non-redundant dataset with a total of 520 ARSs, which are based on ARSs annotation of S. cerevisiae S288C from SGD and then supplemented with those from OriDB and DeOri databases. We conducted a large-scale comparison of ORIs among the diverse budding yeast strains from a population genomics perspective. We found that 82.7% of ARSs are not only conserved in genomic sequence but also relatively conserved in chromosomal position. The non-conserved ARSs tend to distribute in the subtelomeric regions. We also conducted a pan-genome analysis of ARSs among the S. cerevisiae strains, and a total of 183 core ARSs existing in all yeast strains were determined. We extracted the genes adjacent to replication origins among the 104 yeast strains to examine whether there are differences in their gene functions. The result showed that the genes involved in the initiation of DNA replication, such as orc3, mcm2, mcm4, mcm6, and cdc45, are conservatively located adjacent to the replication origins. Furthermore, we found the genes adjacent to conserved ARSs are significantly enriched in DNA binding, enzyme activity, transportation, and energy, whereas for the genes adjacent to non-conserved ARSs are significantly enriched in response to environmental stress, metabolites biosynthetic process and biosynthesis of antibiotics. In general, we characterized the replication origins from the genome-wide and population genomics perspectives, which would provide new insights into the replication mechanism of S. cerevisiae and facilitate the design of algorithms to identify genome-wide replication origins in yeast.

Dataset Information

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

Publications

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

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