Improvement of bioactive metabolite production in microbial cultures—A systems approach by OSMAC and deconvolution‐based ¹HNMR quantification

Abstract

Traditionally, the screening of metabolites in microbial matrices is performed by monocultures. Nonetheless, the absence of biotic and abiotic interactions generally observed in nature still limit the chemical diversity and leads to "poorer" chemical profiles. Nowadays, several methods have been developed to determine the conditions under which cryptic genes are activated, in an attempt to induce these silenced biosynthetic pathways. Among those, the one strain, many compounds (OSMAC) strategy has been applied to enhance metabolic production by a systematic variation of growth parameters. The complexity of the chemical profiles from OSMAC experiments has required increasingly robust and accurate techniques. In this sense, deconvolutionbased 1 HNMR quantification have emerged as a promising methodology to decrease complexity and provide a comprehensive perspective for metabolomics studies. Our present work shows an integrated strategy for the increased production and rapid quantification of compounds from microbial sources.Specifically, an OSMAC design of experiments (DoE) was used to optimize the microbial production of bioactive fusaric acid, cytochalasin D and 3nitropropionic acid, and Global Spectral Deconvolution (GSD)-based 1 HNMR quantification was carried out for their measurement. The results showed that OSMAC increased the production of the metabolites by up to 33% and that GSD was able to extract accurate NMR integrals even in heavily coalescence spectral regions. Moreover, GSD-1 HNMR quantification was reproducible for all species and exhibited validated results that were more selective and accurate than comparative methods. Overall, this strategy up-regulated important metabolites using a reduced number of experiments and provided fast analyte monitor directly in raw extracts.

A Simple and Feasible Quantification of Metabolites in the Human Follicular Fluid Using 1H HR-MAS NMR Spectroscopy

“…In their study, the accuracy of the internal standard and ERETIC2 methodologies was determined by the quantification of standard benzyl benzoate solutions of different concentrations. The values obtained for 1 mM benzyl benzoate solution were reported as 0.96 mM for the classical internal standard method and 1.20 mM for the ERETIC2 (36).…”

Microorganisms: the secret agents of the biosphere, and their key roles in biotechnology

“…An inhouse database was created hosting spectroscopic and spectrometric data (MS and 1 H, 13 C NMR) of the isolated molecules from diverse microorganisms, nowadays integrated into a much larger database called NuBBEDB (Pilon et al 2017) (https://nubbe.iq.unesp.br/portal/nubb). The formulation and conception of algorithms to analyze highly complex mixtures using NMR5-7 (Selegato et al 2019, Valli et al 2019, Valli et al 2019) allowed the deconvolution and dereplication of spectra from OSMAC (One Strain, Many Compounds) cultures. As a result of these efforts, a patent was deposited through the Patent Cooperation Treaty (PCT) (Freire & Castro-Gamboa 2015) describing the algorithms and their functionality towards the dereplication of NMR spectra sets.…”

Metabolômica De Plantas: Métodos E Desafios

“…(3) Normalização por quociente probabilístico: esse método leva em consideração a relação probabilística dos sinais de uma amostra frente a uma referência, ou seja, cada sinal de uma amostra é dividido pelo correspondente sinal de amostra referência gerando um quociente. 247 e mostra resultados satisfatórios para normalização de conjunto de dados de matrizes de produtos naturais.…”