Effects of glucose availability in <i>Lactobacillus sakei</i>; metabolic change and regulation of the proteome and transcriptome

<div><p>Effects of glucose availability were investigated in <i>Lactobacillus sakei</i> strains 23K and LS25 cultivated in anaerobic, glucose-limited chemostats set at high (<i>D</i> = 0.357 h^-1) and low (<i>D</i> = 0.045 h^-1) dilution rates. We observed for both strains a shift from homolactic towards more mixed acid fermentation when comparing high to low growth rates. However, this change was more pronounced for LS25 than for 23K, where dominating products were lactate>formate>acetate≥ethanol at both conditions. A multivariate approach was used for analyzing proteome and transcriptome data from the bacterial cultures, where the predictive power of the omics data was used for identifying features that can explain the differences in the end-product profiles. We show that the different degree of response to the same energy restriction revealed interesting strain specific regulation. An elevated formate production level during slow growth, more for LS25 than for 23K, was clearly reflected in correlating pyruvate formate lyase expression. With stronger effect for LS25, differential expression of the Rex transcriptional regulator and NADH oxidase, a target of Rex, indicated that maintainance of the cell redox balance, in terms of the NADH/NAD⁺ ratio, may be a key process during the metabolic change. The results provide a better understanding of different strategies that cells may deploy in response to changes in substrate availability.</p></div

PCA of proteins and gene transcripts after variable selection towards the phenome.

<p>Proteins and gene transcripts selected by elastic net (left), where <i>L</i>. <i>sakei</i> strains 23K and LS25 are shown in blue and red, respectively. Squares indicate high growth rate and high glucose availability. Open triangles indicate low growth rate and restricted glucose availability. The selected proteins and transcripts were assigned to 4 groups with similar pattern (right), indicated in green, orange, black and red for groups 1–4, respectively.</p

<i>L</i>. <i>sakei</i> end-product formation at high and low glucose availability.

<p>Mol end-product (lactate; blue, formate; red, acetate; green, ethanol; orange) per mol glucose for <i>L</i>. <i>sakei</i> strains 23K and LS25 grown at high (<i>D</i> = 0.357 h^-1) and low (<i>D =</i> 0.045 h^-1) dilution rates in continuous glucose-limited chemostats. Percent (%) change in mol of end-product per mol of glucose between the growth rates is indicated.</p

Metabolite production, carbon flux and carbon flux/growth rate in <i>L</i>. <i>sakei</i> during continuous cultivation in glucose-limited CDM-LAB medium.

<p>Metabolite production, carbon flux and carbon flux/growth rate in <i>L</i>. <i>sakei</i> during continuous cultivation in glucose-limited CDM-LAB medium.</p

Gene transcripts explaining <i>L</i>. <i>sakei</i> strain differences in phenome end-product profiles from high to low growth rate.

<p>Fold change from high to low growth rate and simple correlation coefficient (<i>r</i>) between selected transcript and each end-product are shown. The transcripts are assigned to 4 groups with similar pattern (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187542#pone.0187542.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187542#pone.0187542.g005" target="_blank">5</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187542#pone.0187542.s009" target="_blank">S5 Fig</a>).</p

PCA on the phenome.

<p>PCA on the phenome end-products mean centered and standardized to unit variance. Score plot (left) and loading plot (right) on PC1 (x-axis) vs PC2 (y-axis). <i>L</i>. <i>sakei</i> strains 23K and LS25 are shown in blue and red, respectively. Squares indicate high growth rate and high glucose availability. Open triangles indicate low growth rate and restricted glucose availability.</p

PCA on all features of the proteome and transcriptome.

<p>PCA of the proteome (all 643 variables) and transcriptome (all 1632 variables) mean centered and standardized to unit variance. Score plots (left) and loading plots (right) on PC1 (x-axis) vs PC2 (y-axis). <i>L</i>. <i>sakei</i> strains 23K and LS25 are shown in blue and red, respectively. Squares indicate high growth rate and high glucose availability. Open triangles indicate low growth rate and restricted glucose availability.</p

Mean expression of selected proteins and gene transcripts that responded to changed glucose availability in <i>L</i>. <i>sakei</i>.

<p>Expression of selected proteins (P) and gene transcripts (T) as mean values within each group (1–4) are according to Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187542#pone.0187542.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187542#pone.0187542.t003" target="_blank">3</a>, respectively. Groups are defined by the PCs in the PCA shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187542#pone.0187542.g004" target="_blank">Fig 4</a>. <i>L</i>. <i>sakei</i> strain 23K is shown in blue and strain LS25 in red. Squares indicate high growth rate and high glucose availability (H). Open triangles indicate low growth rate and restricted glucose availability (L).</p

Nonhierarchical Flux Regulation Exposes the Fitness Burden Associated with Lactate Production in <i>Synechocystis</i> sp. PCC6803

Cyanobacteria are mostly engineered to be sustainable cell-factories by genetic manipulations alone. Here, by modulating the concentration of allosteric effectors, we focus on increasing product formation without further burdening the cells with increased expression of enzymes. Resorting to a novel 96-well microplate cultivation system for cyanobacteria, and using lactate-producing strains of <i>Synechocystis</i> PCC6803 expressing different l-lactate dehydrogenases (LDH), we titrated the effect of 2,5-anhydro-mannitol supplementation. The latter acts in cells as a nonmetabolizable analogue of fructose 1,6-bisphosphate, a known allosteric regulator of one of the tested LDHs. In this strain (SAA023), we achieved over 2-fold increase of lactate productivity. Furthermore, we observed that as carbon is increasingly deviated during growth toward product formation, there is an increased fixation rate in the population of spontaneous mutants harboring an impaired production pathway. This is a challenge in the development of green cell factories, which may be countered by the incorporation in biotechnological processes of strategies such as the one pioneered here