Differential gene expression associated with the acid resistance systems in response to n-heptanoic acid stress.

<p>Differential gene expression associated with the acid resistance systems in response to n-heptanoic acid stress.</p

The relationship between GadA/B activity and cultivation pH (A) or n-heptanoic acid concentration (B).

<p>The GadA/B activities of the recombinant <i>E</i>. <i>coli</i> BL21(DE3) pCOLA (empty vector) (<i>grey bar</i>), <i>E</i>. <i>coli</i> BL21(DE3) pCOLA-RcsB-DsrA (<i>grey bar with diagonal line</i>) and <i>E</i>. <i>coli</i> K-12 MG1655 pCOLA (<i>dark grey bar</i>) were determined when cells were cultivated at different pH and at different n-heptanoic acid concentration. Values are the mean of more than three independent samples. Bars represent standard error of the mean.</p

The survival frequency of the recombinant <i>E</i>. <i>coli</i> strains after 1 h exposure to acidified and n-heptanoic acid added culture medium.

<p>The survival frequencies of <i>E</i>. <i>coli</i> BL21(DE3) pCOLA (empty vector) (<i>grey bar</i>), <i>E</i>. <i>coli</i> BL21(DE3) pCOLA-RcsB-DsrA (<i>grey bar with diagonal line</i>) and <i>E</i>. <i>coli</i> K-12 MG1655 pCOLA (<i>dark grey bar</i>) were determined under different cultivation pH (A) and at different n-heptanoic acid concentration (B) as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.ref034" target="_blank">34</a>]. Values are the mean of more than three independent samples. Bars represent the standard error of the mean.</p

DataSheet1_DoE-based medium optimization for improved biosurfactant production with Aureobasidium pullulans.pdf

Polyol lipids (a.k.a. liamocins) produced by the polyextremotolerant, yeast-like fungus Aureobasidium pullulans are amphiphilic molecules with high potential to serve as biosurfactants. So far, cultivations of A. pullulans have been performed in media with complex components, which complicates further process optimization due to their undefined composition. In this study, we developed and optimized a minimal medium, focusing on biosurfactant production. Firstly, we replaced yeast extract and peptone in the best-performing polyol lipid production medium to date with a vitamin solution, a trace-element solution, and a nitrogen source. We employed a design of experiments approach with a factor screening using a two-level-factorial design, followed by a central composite design. The polyol lipid titer was increased by 56% to 48 g L−1, and the space-time yield from 0.13 to 0.20 g L−1 h−1 in microtiter plate cultivations. This was followed by a successful transfer to a 1 L bioreactor, reaching a polyol lipid concentration of 41 g L−1. The final minimal medium allows the investigation of alternative carbon sources and the metabolic pathways involved, to pinpoint targets for genetic modifications. The results are discussed in the context of the industrial applicability of this robust and versatile fungus.</p

Tn7-Based Device for Calibrated Heterologous Gene Expression in <i>Pseudomonas putida</i>

The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida’s chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments and flow cytometry with single-copy promoter–GFP constructs revealed a robust, constitutive behavior of these promoters, whose strengths and properties could be faithfully compared. This standardized expression device significantly extends the repertoire of tools available for reliable metabolic engineering and other genetic enhancements of P. putida

Schematic diagram to show the ratios of expression levels of the genes.

<p>The gene products are involved in the acid resistance systems of <i>E</i>. <i>coli</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.ref041" target="_blank">41</a>] (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.s012" target="_blank">S3 Table</a>). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.g002" target="_blank">Fig 2</a> for description of the bars.</p

Time course of the biotransformation of ricinoleic acid.

<p>Ricinoleic acid (<i>triangle</i>) was converted into n-heptanoic acid (<b>5</b>) and 11-hydroxyundec-9-enoic acid (<b>4</b>) (<i>solid triangle</i>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.s009" target="_blank">S1 Scheme</a>) by the recombinant <i>E</i>. <i>coli</i> BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA (A) and <i>E</i>. <i>coli</i> BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA-RcsB-DsrA (B). The 12-ketooleic acid (<i>triangle down</i>) and ester (<i>solid square</i>) are intermediates. The biotransformation was initiated by adding 15 mM of ricinoleic acid, 0.5 g L⁻¹ Tween 80, and esterase of <i>P</i>. <i>fluorescens</i> SIK WI into the culture broth of the strains at the early stationary growth phase (pH 8.0). The cell density was 3.2 g dry cells L⁻¹. Values are the mean of more than three independent samples. Bars represent standard error of the mean.</p

Internal carbon flux distribution in <i>E</i>. <i>coli</i> BL21(DE3) and K-12 MG1655 strains.

<p>The cells were grown in the presence of different concentrations of n-heptanoic acid, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.g001" target="_blank">Fig 1</a>. The upper values and third upper values indicate the internal carbon flux distribution in <i>E</i>. <i>coli</i> BL21(DE3) and K-12 MG1655 strains growing in the absence of n-heptanoic acid. The second upper values and lower values indicate the internal carbon flux distribution in <i>E</i>. <i>coli</i> BL21(DE3) and K-12 MG1655 strains growing in the presence of n-heptanoic acid. The BL21(DE3) was cultivated with 3 mM n-heptanoic acid, while the MG1655 was with 10 mM n-heptanoic acid. The heptanoic acid concentration is the concentration, in which the specific growth rate of the <i>E</i>. <i>coli</i> strains reduced to ca. half of the maximum (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.g001" target="_blank">Fig 1A</a>). Carbon flux distribution was estimated based on stoichiometric constraints using a metabolic network model implemented in MetaFluxNet [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.ref030" target="_blank">30</a>]. The flux data were not normalized to highlight the difference between <i>E</i>. <i>coli</i> BL21(DE3) and MG1655 strains at the specific glucose uptake rates and carbon fluxes. The normalized data were presented in the Supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163265#pone.0163265.s004" target="_blank">S4 Fig</a>). The bars indicate ratios of the expression level of the genes, which products are correspond to the enzymes involved in the reaction step. The first bars indicate the ratio of the gene expression in the BL21(DE3) strain in the absence and presence (3 mM) of n-heptanoic acid. The second bars indicate the ratio of the gene expression between the BL21(DE3) and MG1655 strains in the absence of n-heptanoic acid. The third bars indicate the ratio of the gene expression in the BL21(DE3) strain in the absence of n-heptanoic acid to that in the MG1655 in the presence (10 mM) of n-heptanoic acid.</p

The specific growth rates (A), specific glucose uptake rates (B), and specific metabolite production rates (C) of the <i>E</i>. <i>coli</i> strains.

<p><i>E</i>. <i>coli</i> BL21(DE3) (<i>grey</i>) and K-12 MG1655 (<i>dark grey</i>) were cultivated with different concentrations of n-heptanoic acid at 37°C in a shaking incubator (200 rpm). Values are the mean of more than three independent samples. Bars represent standard error of the mean.</p

Discovery and Evaluation of Biosynthetic Pathways for the Production of Five Methyl Ethyl Ketone Precursors

The limited supply of fossil fuels and the establishment of new environmental policies shifted research in industry and academia toward sustainable production of the second generation of biofuels, with methyl ethyl ketone (MEK) being one promising fuel candidate. MEK is a commercially valuable petrochemical with an extensive application as a solvent. However, as of today, a sustainable and economically viable production of MEK has not yet been achieved despite several attempts of introducing biosynthetic pathways in industrial microorganisms. We used BNICE.ch as a retrobiosynthesis tool to discover all novel pathways around MEK. Out of 1325 identified compounds connecting to MEK with one reaction step, we selected 3-oxopentanoate, but-3-en-2-one, but-1-en-2-olate, butylamine, and 2-hydroxy-2-methylbutanenitrile for further study. We reconstructed 3 679 610 novel biosynthetic pathways toward these 5 compounds. We then embedded these pathways into the genome-scale model of <i>E. coli</i>, and a set of 18 622 were found to be the most biologically feasible ones on the basis of thermodynamics and their yields. For each novel reaction in the viable pathways, we proposed the most similar KEGG reactions, with their gene and protein sequences, as candidates for either a direct experimental implementation or as a basis for enzyme engineering. Through pathway similarity analysis we classified the pathways and identified the enzymes and precursors that were indispensable for the production of the target molecules. These retrobiosynthesis studies demonstrate the potential of BNICE.ch for discovery, systematic evaluation, and analysis of novel pathways in synthetic biology and metabolic engineering studies