Project description:Background Lactococcus lactis is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth. Results Using glucose limited continuous cultivations, specific growth rate dependent metabolism of L. Lactis including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of L. lactis became more efficient with rising specific growth rate in the range 0.10 – 0.60 h-1, indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. L. lactis global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in L. lactis were presumably under allosteric control. Conclusions The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of Lactococcus lactis which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models.

Project description:Background Lactococcus lactis is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth. Results Using glucose limited continuous cultivations, specific growth rate dependent metabolism of L. Lactis including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of L. lactis became more efficient with rising specific growth rate in the range 0.10 – 0.60 h-1, indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. L. lactis global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in L. lactis were presumably under allosteric control. Conclusions The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of Lactococcus lactis which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models. For microarray analysis, steady state chemostat culture of L. lactis IL1403 was used as reference (? = 0.10 1/h). Subsequent quasi steady state points from A-stat experiment at specific growth rates 0.52 ± 0.03; 0.42 ± 0.02; 0.29 ± 0.01 1/h in biological duplicates and 0.17 1/h were compared to the reference sample.

Project description:Its characteristic rose-like aroma makes phenylethanol a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbor an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source such as glucose provides an economically attractive alternative for phenylalanine bioconversion. In this study, a Synthetic Genetic Array screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to cause a transcriptional upregulation of ARO10 during growth with ammonium sulfate as the sole nitrogen source. Physiological characterization revealed that the aro8M-oM-^AM-^D mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8 mutation also stimulated phenylethanol production when combined with other, previously documented mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced over 3 mM of phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products. The goal of the present study was to identify genes that influence the transcriptional (de)repression of the Ehrlich pathway during growth with ammonium as the nitrogen source. With the aid of Synthetic Genetic Array technology, we constructed a strain collection in which deletions in the non-essential genes in the S. cerevisiae genome were combined with a reporter plasmid comprising the ARO10 promoter fused to a reporter gene (egfp) encoding a fluorescent reporter protein. After screening by flow cytometry, deletion of ARO8 led to a deregulated expression from the ARO10 promoter. The impact of this deletion was further studied by transcriptome and intracellular metabolite analyses. Furthermore, phenylethanol production was measured in strains that combined the aro8 mutation with mutations that were previously shown to deregulate aromatic amino acid biosynthesis.

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