Dataset Information

Living the sweet life: how Liquorilactobacillus hordei TMW 1.1822 changes its behavior in the presence of sucrose in comparison to glucose

ABSTRACT: Liquorilactobacillus (L.) hordei (formerly Lactobacillus hordei) is one of the dominating lactic acid bacteria within the water kefir consortium, being highly adapted to survive in this environment, while producing high molecular weight dextrans from sucrose. In this work, we extensively studied the physiological response of L. hordei TMW 1.1822 to sucrose compared to glucose, applying label-free, quantitative proteomics of cell lysates and exoproteomes. This revealed the differential expression of 53 proteins within cellular proteomes, mostly associated with carbohydrate uptake and metabolism. Supported by growth experiments, this suggests L. hordei TMW 1.1822 to favor fructose over other sugars. The dextransucrase was expressed irrespective of the present carbon source, while it was significantly more released in the presence of sucrose (log2FC = 3.09), being among the most abundant proteins within exoproteomes of sucrose-treated cells. Still, L. hordei TMW 1.1822 expressed other sucrose active enzymes, predictively competing with the dextransucrase reaction. While osmolysis appeared to be unlikely, sucrose led to an increased release of a multitude of cytoplasmic proteins, suggesting that biofilm formation in L. hordei is not only composed of a polysaccharide matrix, but also of proteinaceous nature. Therefore, our study highlights the intrinsic adaptation of water kefir-borne L. hordei to sucrose-rich habitats and provides fundamental knowledge for the use as starter culture in plant-based food fermentations with in situ dextran formation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Lactobacillus Hordei

SUBMITTER: Christina Ludwig

LAB HEAD: Christina Ludwig

PROVIDER: PXD020664 | Pride | 2021-01-07

REPOSITORIES: Pride

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Publications

Living the Sweet Life: How Liquorilactobacillus hordei TMW 1.1822 Changes Its Behavior in the Presence of Sucrose in Comparison to Glucose.

Bechtner Julia J Ludwig Christina C Kiening Michael M Jakob Frank F Vogel Rudi F RF

Foods (Basel, Switzerland) 20200821 9

Liquorilactobacillus (L.) hordei (formerly Lactobacillus hordei) is one of the dominating lactic acid bacteria within the water kefir consortium, being highly adapted to survive in this environment, while producing high molecular weight dextrans from sucrose. In this work, we extensively studied the physiological response of L. hordei TMW 1.1822 to sucrose compared to glucose, applying label-free, quantitative proteomics of cell lysates and exoproteomes. This r ...[more]

PMID: 32825547

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Project description:Lactobacillus (L.) sakei belongs to the dominating lactic acid bacteria in indigenous meat fermentations, while diverse strains of this species have also been isolated from plant fermentations. We could recently show, that L. sakei TMW 1.411 produces a high molecular weight dextran from sucrose, indicating its potential use as a dextran forming starter culture. However, the general physiological response of L. sakei to sucrose as carbohydrate source has not been investigated yet, especially upon simultaneous dextran formation. To address this lack of knowledge, we sequenced the genome of L. sakei TMW 1.411 and performed a label-free, quantitative proteomics approach to investigate the sucrose-induced changes in the proteomic profile of this strain in comparison to its proteomic response to glucose. In total, 21 proteins were found to be differentially expressed at the applied significance criteria (FDR ≤ 0.01). Among these, 14 were associated with the carbohydrate metabolism including several enzymes, which enable sucrose and fructose uptake as well as their subsequent intracellular metabolization, respectively. The plasmid-encoded, extracellular dextransucrase of L. sakei TMW 1.411 was expressed at high levels irrespective of the present carbohydrate and was predominantly responsible for sucrose consumption in growth experiments using sucrose as sole carbohydrate source, while the released fructose from the dextransucrase reaction was more preferably taken up and intracellularly metabolized than sucrose. Genomic comparisons revealed, that operons coding for uptake and intracellular metabolism of sucrose and fructose are chromosomally conserved among L. sakei, while plasmid-located dextransucrase genes are present only in few strains. In accordance with these findings, all 59 different L. sakei strains of our strain collection were able to grow on sucrose as sole carbohydrate source, while eight of them exhibited a mucous phenotype on agar plates indicating dextran formation from sucrose. Our study therefore highlights the intrinsic adaption of L. sakei to plant environments, where sucrose is abundant, and provides fundamental knowledge regarding the use of L. sakei as starter culture for sucrose-based food fermentation processes with in-situ dextran formation.

Project description:Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-H+ symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9 % higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5’ coding sequences of SUC2, resulting in relocation of invertase to the cytosol. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 95 generations of anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11 % higher ethanol yield on sucrose in chemostat and batch cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. Deletion of AGT1, which had been duplicated during laboratory evolution, restored the growth characteristics of the unevolved iSUC2 strain. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes. The goal of the present study was to investigate whether a relocation of sucrose hydrolysis to the cytosol can be used to improve ethanol yields on sucrose and which additional steps may be required to improve sucrose utilization by strains that only express intracellular invertase. To this end, the SUC2 gene was modified to cause an exclusive intracellular localization. Growth and product formation by the engineered strain were compared with that of the parental strain in anaerobic sucrose-limited chemostat cultures. Subsequently, evolutionary engineering was used to improve sucrose uptake kinetics and an evolved strain was characterized for growth and product formation in chemostat cultures. Transcriptome analysis and gene deletion studies were used to identify genetic changes in the evolved strain that contribute to its improved sucrose-uptake kinetics.

Dataset Information

Living the sweet life: how Liquorilactobacillus hordei TMW 1.1822 changes its behavior in the presence of sucrose in comparison to glucose

Publications

Living the Sweet Life: How <i>Liquorilactobacillus hordei</i> TMW 1.1822 Changes Its Behavior in the Presence of Sucrose in Comparison to Glucose.

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