MOESM1 of Ammonia production from amino acid-based biomass-like sources by engineered Escherichia coli

Additional file 1: Table S1. Primers

Folate Biofortification in Hydroponically Cultivated Spinach by the Addition of Phenylalanine

Folate is an important vitamin mainly ingested from vegetables, and folate deficiency causes various health problems. Recently, several studies demonstrated folate biofortification in plants or food crops by metabolic engineering through genetic modifications. However, the production and sales of genetically modified foods are under strict regulation. Here, we developed a new approach to achieve folate biofortification in spinach (Spinacia oleracea) without genetic modification. We hydroponically cultivated spinach with the addition of three candidate compounds expected to fortify folate. As a result of liquid chromatography tandem mass spectrometry analysis, we found that the addition of phenylalanine increased the folate content up to 2.0-fold (306 μg in 100 g of fresh spinach), representing 76.5% of the recommended daily allowance for adults. By measuring the intermediates of folate biosynthesis, we revealed that phenylalanine activated folate biosynthesis in spinach by increasing the levels of pteridine and <i>p</i>-aminobenzoic acid. Our approach is a promising and practical approach to cultivate nutrient-enriched vegetables

Metabolite profiling of the fermentation process of "yamahai-ginjo-shikomi" Japanese sake

<div><p>Sake is a traditional Japanese alcoholic beverage prepared by multiple parallel fermentation of rice. The fermentation process of “yamahai-ginjo-shikomi” sake is mainly performed by three microbes, <i>Aspergillus oryzae</i>, <i>Saccharomyces cerevisiae</i>, and <i>Lactobacilli</i>; the levels of various metabolites fluctuate during the fermentation of sake. For evaluation of the fermentation process, we monitored the concentration of moderate-sized molecules (m/z: 200–1000) dynamically changed during the fermentation process of “yamahai-ginjo-shikomi” Japanese sake. This analysis revealed that six compounds were the main factors with characteristic differences in the fermentation process. Among the six compounds, four were leucine- or isoleucine-containing peptides and the remaining two were predicted to be small molecules. Quantification of these compounds revealed that their quantities changed during the month of fermentation process. Our metabolomic approach revealed the dynamic changes observed in moderate-sized molecules during the fermentation process of sake, and the factors found in this analysis will be candidate molecules that indicate the progress of “yamahai-ginjo-shikomi” sake fermentation.</p></div

Quantification of peptides and associated compounds.

<p>(A) Quantification of Leu–Leu–Leu (left) and Phe–Pro–Leu by LC-TQMS. (B) Relative quantification of four factors (ID 1, 2, 6, and 7) that were identified by metabolic profiling using nanoLC-MS.</p

PC scores with Top 20 PC1 score.

<p>PC scores with Top 20 PC1 score.</p

Validation of peptide leucine or isoleucine compositions.

<p>(A) Chromatograms of sake samples with eight variations of synthetic [Leu/Ile]–[Leu/Ile]–[Leu/Ile] peptides. Extracted ion chromatograms (XIC) of m/z = 358.26–358.28 are presented. (B) Chromatograms of sake samples with two variations of synthetic Phe–Pro–[Leu/Ile] peptides. XIC of m/z = 376.22–376.23 are shown. Dashed lines with arrowheads indicate retention times of the main peaks of XIC for two samples. [Leu/Ile] indicates leucine or isoleucine residues that could not be determined by MS.</p

PCA analysis of metabolic profiles.

<p>(A) Score plot of the fermentation process. Metabolic profiles of samples at six time points were plotted using two analytical replicates from the same sample. (B) Loading scatter plot. Factors having values of PC1 from the top to the 20^th at each plot are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190040#pone.0190040.t001" target="_blank">Table 1</a>.</p

Identification of peptides.

<p>The factors that were predicted as peptides in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190040#pone.0190040.t002" target="_blank">Table 2</a> were analyzed using LC-MS/MS, and the amino acid sequences were identified using MS/MS spectra. [L/I] indicates leucine or isoleucine residue that could not be distinguished by MS analysis.</p