Heterotypic stress-induced adaptive evolution enhances freeze-drying tolerance and storage stability of Leuconostoc mesenteroides WiKim33.

Lactic acid bacteria (LAB) are currently being investigated for their potential use as probiotics and starter cultures. Researchers have developed powdering processes for the commercialization of LAB. Previous studies have focused on identifying innovative cryoprotective agents and freeze-drying (FD) techniques to enhance the stability of LAB. In this study, adaptive laboratory evolution (ALE) was employed to develop a strain with high FD tolerance and enhanced storage stability. Leuconostoc mesenteroids WiKim33 was subjected to heterotypic shock (heat and osmosis shock) to induce the desired phenotype and genotype. An FD-tolerant enhanced Leu. mesenteroides WiKim33 strain (ALE50) was obtained, which harbored a modified fatty acid composition and cell envelope characteristics. Specifically, ALE50 showed a lower unsaturated fatty acid (UFA)/saturated fatty acid (SFA) ratio and a higher cyclic fatty acid (CFA) composition. Moreover, the exopolysaccharide (EPS) thickness increased significantly by 331% compared to that of the wild type (WT). FD tolerance, which was evaluated using viability testing after FD, was enhanced by 33.4%. Overall, we demonstrated the feasibility of ALE to achieve desirable characteristics and provided insights into the mechanisms underlying increased FD tolerance.

The mechanism of Leuconostoc mesenteroides subsp. IMAU:80679 in improving meat color: Myoglobin oxidation inhibition and myoglobin derivatives formation based on multi enzyme-like activities.

The Leuconostoc mesenteroides subsp. IMAU:80679 (LM) was chosen for its superior capability in enhancing redness, and was incubated in a broth system containing metmyoglobin (MetMb) to investigate its mechanisms for color improvement. The a* value of LM group reached its highest level of 52.75 ± 1.04 at 24 h, significantly higher than control of 19.75 ± 0.6 (p < 0.05). The addition of LM could inhibit myoglobin oxidation to some extent. Meanwhile, higher content of nitrosylmyoglobin (NOMb) and Zn-protoporphyrin (Znpp) were observed in LM samples during the whole incubation period. Furthermore, enzymatic activity and encoded genes related to MetMb reduction and pigment formation were determined to explain its possible mechanism on color enhancement. Finally, by extracting crude enzymes and adding them to meat batters, the redness of crude enzyme group was comparable to that achieved with 20 ppm nitrite, providing a potential method on compensating for nitrite/nitrate substitution in meat products.

A potential probiotic Leuconostoc mesenteroides TBE-8 for honey bee.

An isolated bacterium TBE-8, was identified as Leuconostoc mesenteroides according to the sequences of 16S rDNA and the 16S-23S rDNA intergenic spacer region. The probiotic properties of the L. mesenteroides TBE-8 strain were characterized and revealed that TBE-8 could utilize various carbohydrates, exhibited high tolerance to sucrose's osmotic pressure and acidic conditions, and could mitigate the impact of the bee pathogen Paenibacillus larvae. In addition, we found that the TBE-8 broth increased the expression of the nutrition-related genes major royal jelly protein 1 and vitellogenin in bees by approximately 1400- and 20-fold, respectively. The expression of genes encoding two antibacterial peptides, hymenoptaecin and apidaecin, in the bee abdomen was significantly increased by 17- and 7-fold in bees fed with the TBE-8 fermented broth. Furthermore, we fed four-frame bee colonies with 50% sucrose syrup containing TBE-8 and can detect the presence of approximately 2 × 106 16S rDNA copies of TBE-8 in the guts of all bees in 24 h, and the retention of TBE-8 in the bee gut for at least 5 days. These findings indicate that the L. mesenteroides TBE-8 has high potential as a bee probiotic and could enhance the health of bee colonies.

Enzymatically synthesized exopolysaccharide of a probiotic strain Leuconostoc mesenteroides NTM048 shows adjuvant activity to promote IgA antibody responses.

Leuconostoc mesenteroides strain NTM048 produces an exopolysaccharide (EPS; glucose polymers 94% and fructose polymers 6%) with adjuvanticity for mucosal vaccination. Strain NTM048 includes three putative EPS-synthesizing genes, gtf1 and gtf2 for synthesizing glucose polymers, and lvnS for synthesizing fructose polymer. To elucidate the key polymer structure for adjuvanticity, two genes, gtf1 and gtf2, which were annotated as glycoside hydrolase family 70 enzyme genes, were expressed in Escherichia coli. Glycosyl-linkage composition analysis and NMR analysis showed that the recombinant enzyme Gtf1 produced a soluble form of α-1,6-glucan, whereas the recombinant enzyme Gtf2 produced glucans with approximately equal percentages of α-1,6- and α-1,3-glucose residues both in the supernatant (S-glucan) and as a precipitate (P-glucan). Comparison of polysaccharides synthesized by Gtf1, Gtf2, and LvnS revealed that Gtf2-S-glucan, which was produced in the supernatant by Gtf2 and formed particles of 7.8 µm, possessed 1.8-fold higher ability to stimulate IgA production from murine Peyer's patch cells than native NTM048 EPS. Evaluation of adjuvanticity by intranasal administration of mice with an antigen (ovalbumin) and Gtf2-S-glucan or NTM048 EPS showed that Gtf2-S-glucan induced the production of higher antigen-specific antibodies in the airway mucosa and plasma, suggesting a pivotal role of Gtf2-S-glucan in the adjuvanticity of NTM048 EPS.

Functional Identification of the Dextransucrase Gene of Leuconostoc mesenteroides DRP105.

Leuconostoc mesenteroides DRP105 isolated from Chinese sauerkraut juice is an intensive producer of dextran. We report the complete genome sequence of Leu. mesenteroides DRP105. This strain contains a dextransucrase gene (dsr) involved in the production of dextran, possibly composed of glucose monomers. To explore the dextran synthesis mechanism of Leu. mesenteroides DRP105, we constructed a dsr-deficient strain derived from Leu. mesenteroides DRP105 using the Cre-loxP recombination system. The secondary structure prediction results showed that Leu. mesenteroides DRP105 dextransucrase (Dsr) was coded by dsr and contained 17.07% α-helices, 29.55% ß-sheets, 10.18% ß-turns, and 43.20% random coils. We also analyzed the dextran yield, monosaccharide change, organic acid, and amino-acid content of Leu. mesenteroides DRP105 and Leu. mesenteroides DRP105-Δdsr. The result showed that the lack of dsr changed the Leu. mesenteroides DRP105 sugar metabolism pathway, which in turn affected the production of metabolites.

Engineering Leuconostoc mesenteroides dextransucrase by inserting disulfide bridges for enhanced thermotolerance.

The disulfide bridge is a very important part of the peptide chain and plays an important role in stabilizing the protein structure and maintaining its active function. One hundred and fourteen potential disulfide bridges were determined by Disulfide by Design™, and 4 disulfide bridges were constructed for the purpose of obtaining new enzyme species with high thermotolerance. High thermotolerance is achieved by increasing the number of hydrogen bonds between amino acids. The optimum temperatures of mutant L838C-V887C and A948C-A1013C were improved by 10 °C compared to that of the original enzyme, which was beneficial to reduce the viscosity of the reaction system. Some of the mutations resulted in the alteration of catalytic specificity, and the products D739C-F932C and A948C-A1013C catalyzed synthesis of dextran containing a new α(1-4) glycosidic linkage and α(1-2) glycosidic linkage. This study may provide information valuable for increasing the reaction temperature of recombinant dextransucrase. The molecular docking study presents a plausible explanation for reaction specificity alteration and optimum temperature improvement for the mutants.

Comparative genome analysis provides shreds of molecular evidence for reclassification of Leuconostoc mesenteroides MTCC 10508 as a strain of Leu. suionicum.

This study presents the whole-genome comparative analysis of a Leuconostoc sp. strain, previously documented as Leu. mesenteroides MTCC 10508. The ANI, dDDH, dot plot, and MAUVE analyses suggested its reclassification as a strain of Leu. suionicum. Functional annotation identified a total of 1971 genes, out of which, 265 genes were mapped to CAZymes, evincing its carbohydrate transforming capability. The genome comparison with 59 Leu. mesenteroides and Leu. suionicum strains generated the core and pan-genome profiles, divulging the unique genes in Leuconostoc sp. MTCC 10508. For the first time, this study reports the genes encoding alpha-xylosidase and copper oxidase in a strain of Leu. suionicum. The genetic information for any possible allergenic molecule could not be detected in the genome, advocating the safety of the strain. The present investigation provides the genomic evidence for reclassification of the Leuconostoc sp. strain and also promulgates the molecular insights into its metabolic potential.

DNAzyme-based quantitative loop-mediated isothermal amplification for strain-specific detection of starter kimchi fermented with Leuconostoc mesenteroides WiKim32.

Leuconostoc spp. are generally utilized as kimchi starters because of their beneficial effects on kimchi fermentation and sensory characteristics. We developed a DNAzyme-based colorimetric method for measuring the abundance of the kimchi starter Leuconostoc mesenteroides WiKim32. A primer set for loop-mediated isothermal amplification and target-specific DNAzyme was designed based on the WiKim32 nucleotide sequence. In the presence of the target amplicon, DNAzyme bound to it, resulting in negligible amounts of green product. In contrast, with the addition of hemin and in the absence of the target amplicon, DNAzyme fragments not bound to the target amplicon formed G-quadruplex-hemin conjugates, generating a visible green product by oxidizing 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt. There was no cross-reaction with other strains. The method had high detection sensitivity and quantitative capacity in kimchi samples without a requirement for DNA isolation. This strategy provides a rapid, sensitive, and simple detection method with possible industrial applications.

Analysis of rpoB polymorphism and PCR-based approaches for the identification of Leuconostoc mesenteroides at the species and subspecies level.

Leuconostoc mesenteroides includes the subsp. cremoris, subsp. dextranicum, subsp. mesenteroides and subsp. jonggajibkimchii, but the identification at the subspecies level using current phenotypic and/or genotypic methods is still difficult. In this study, a polyphasic approach based on the analysis of rpoB gene polymorphism, Multiplex-PCR and phenotypic tests was optimised and used to identify a collection of Leuc. mesenteroides strains at the species and subspecies levels. The annotation of published Leuc. mesenteroides genomes was also revised. A polymorphic region of rpoB gene was effective in separating Leuc. mesenteroides strains at the species (rpoB-species-specific-PCR) and subspecies (phylogenetic comparison) levels. Multiplex-PCR discriminated the subsp. mesenteroides from subsp. cremoris, but strains of uncertain attribution were found among subsp. dextranicum and subsp. jonggajibkimchii. Most of phenotypic features were not suitable for subspecies discrimination. Our assays may provide a rapid and reliable identification of subsp. mesenteroides and subsp. cremoris strains in fermented foods. The discrimination of subsp. dextranicum and subsp. jonggajibkimchii suffered from several limitations (e.g. low number of available strains and genomes, phenotypic profile close to subsp. mesenteroides, discrepancy between genotypic and phenotypic traits) and further investigations are needed to clarify their delineation and taxonomical position.

Alginate-pectin co-encapsulation of dextransucrase and dextranase for oligosaccharide production from sucrose feedstocks.

The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497, respectively. Heterologous expression of genes was performed in Escherichia coli. The purified enzyme fractions were entrapped in the alginate-pectin beads. A high immobilization yield of dextransucrase (~ 96%), and dextranase (~ 85%) was achieved. Alginate-pectin immobilization did not affect the optimum temperature and pH of the enzymes; rather, the thermal tolerance and storage stability of the enzymes was improved. The repetitive batch experiments suggested substantially good operational stability of the co-immobilized enzyme system. The synergistic catalytic reactions of alginate-pectin co-entrapped enzyme system were able to produce 7-10 g L-1 oligosaccharides of a high degree of polymerization (DP 3-9) from sucrose (~ 20 g L-1) containing feedstocks, e.g., table sugar and cane molasses. The alginate-pectin-based co-immobilized enzyme system is a useful catalytic tool to bioprocess the agro-industrial bio-resource for the production of prebiotic biomolecules.

Determination of glucansucrase encoding gene in Leuconostoc mesenteroides.

A glucansucrase encoding gene was cloned into pET-28a(+) vector and expression in Escherichia coli BL21(DE3). An about 160 kDa recombinant glucansucrase was purified with a yield of 50.73% and a 4.02-fold increase in activity. The 1464 amino acid residue enzyme belongs to the GH70 subfamily and shares 90% similarity with Leuconostoc sp. glucansucrase. The optimal temperature and pH were 30 °C and pH 5.5, and 80% of activity was retained after incubation at 10-30 °C and pH 5-7. Enzyme activity was strongly activated by Ca2+ and Mn2+ and inhibited by various metal ions and chemical agents, and a high affinity for sucrose (Km = 11.6 mM, Vmax = 8.1 mmol/(mL·min)). Circular dichroism (CD) and Raman spectra collectively indicated a high proportion of random coil structure.

A genome-scale metabolic network of the aroma bacterium Leuconostoc mesenteroides subsp. cremoris.

Leuconostoc mesenteroides subsp. cremoris is an obligate heterolactic fermentative lactic acid bacterium that is mostly used in industrial dairy fermentations. The phosphoketolase pathway (PKP) is a unique feature of the obligate heterolactic fermentation, which leads to the production of lactate, ethanol, and/or acetate, and the final product profile of PKP highly depends on the energetics and redox state of the organism. Another characteristic of the L. mesenteroides subsp. cremoris is the production of aroma compounds in dairy fermentation, such as in cheese production, through the utilization of citrate. Considering its importance in dairy fermentation, a detailed metabolic characterization of the organism is necessary for its more efficient use in the industry. To this aim, a genome-scale metabolic model of dairy-origin L. mesenteroides subsp. cremoris ATCC 19254 (iLM.c559) was reconstructed to explain the energetics and redox state mechanisms of the organism in full detail. The model includes 559 genes governing 1088 reactions between 1129 metabolites, and the reactions cover citrate utilization and citrate-related flavor metabolism. The model was validated by simulating co-metabolism of glucose and citrate and comparing the in silico results to our experimental results. Model simulations further showed that, in co-metabolism of citrate and glucose, no flavor compounds were produced when citrate could stimulate the formation of biomass. Significant amounts of flavor metabolites (e.g., diacetyl and acetoin) were only produced when citrate could not enhance growth, which suggests that flavor formation only occurs under carbon and ATP excess. The effects of aerobic conditions and different carbon sources on product profiles and growth were also investigated using the reconstructed model. The analyses provided further insights for the growth stimulation and flavor formation mechanisms of the organism.

Catalytic biosynthesis of levan and short-chain fructooligosaccharides from sucrose-containing feedstocks by employing the levansucrase from Leuconostoc mesenteroides MTCC10508.

Levansucrase gene (LmLEVS) was cloned from Leuconostoc mesenteroides MTCC 10508. The heterologous expression and purification of the truncated (TrLmLEVS) gene, lacking the N-terminal signal peptide, was performed in Escherichia coli. The recombinant enzyme (TrLmLEVS) was physico-kinetically characterized using sucrose as substrate. TrLmLEVS exhibited the maximum activity at pH 6 and temperature 30 °C. Thin layer chromatography and high performance liquid chromatography analyses unveiled the biosynthesis of fructooligosaccharides and levan by TrLmLEVS using sucrose as substrate. The catalytically synthesized polymer was characterized by Fourier-Transform Infrared Spectroscopy and Nuclear Magnetic Resonance analyses, confirming it as levan. TrLmLEVS was capable of catalyzing the transformation of raffinose-derived molecules, besides sucrose, into fructans. Further, TrLmLEVS was employed for the genesis of non-digestible fructans from sucrose-containing feedstocks like table sugar, jaggery, cane molasses, and sweet sorghum juice. The results suggest that Leu. mesenteroides MTCC 10508 levansucrase is a potential candidate for the production of levan-type biomolecules in plant-based food products.

High Levels of CO2 Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes.

During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8 h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.

Effect of the pat, fk, stpk Gene Knock-out and mdh Gene Knock-in on Mannitol Production in Leuconostoc mesenteroides.

Leuconostoc mesenteroides can be used to produce mannitol by fermentation, but the mannitol productivity is not high. Therefore, in this study modify the chromosome of Leuconostoc mesenteroides by genetic methods to obtain high-yield strains of mannitol production. In this study, gene knock-out strains and gene knock-in strains were constructed by a two-step homologous recombination method. The mannitol productivity of the pat gene (which encodes phosphate acetyltransferase) deleteon strain (Δpat::amy), fk gene (which encodes fructokinase) deleteon strain (Δfk::amy) and stpk gene (which encodes serine-threonine protein kinase) deleteon strain (Δstpk::amy) were all increased compared to the wild type, and the productivity of mannitol for each strain was 84.8%, 83.5% and 84.1% respectively. The mannitol productivity of the mdh gene (which encodes mannitol dehydrogenase) knock-in strains (Δpat::mdh, Δfk::mdh and Δstpk::mdh) was increased to a higher level than that of the single-gene deletion strains, and the productivity of mannitol for each was 96.5%, 88% and 93.2%, respectively. The multi-mutant strain ΔdtsΔldhΔpat::mdhΔstpk::mdhΔfk::mdh had mannitol productivity of 97.3%. This work shows that multi-gene knock-out and gene knock-in strains have the greatest impact on mannitol production, with mannitol productivity of 97.3% and an increase of 24.7% over wild type. This study used the methods of gene knock-out and gene knock-in to genetically modify the chromosome of Leuconostoc mesenteroides. It is of great significance that we increased the ability of Leuconostoc mesenteroides to produce mannitol and revealed its broad development prospects.

Identification and Characterization of l-Malate Dehydrogenases and the l-Lactate-Biosynthetic Pathway in Leuconostoc mesenteroides ATCC 8293.

One putative l-lactate dehydrogenase gene (l- ldh) and three putative d- ldh genes from Leuconostoc mesenteroides ATCC 8293 were overexpressed, and their enzymatic properties were investigated. Only one gene showed d-LDH activity, catalyzing pyruvate and d-lactate interconversion, whereas the other genes displayed l- and d-malate dehydrogenase (MDH) activity, catalyzing oxaloacetate and l- and d-malate interconversion, suggesting that strain ATCC 8293 may not harbor an l- ldh gene. Putative phosphoenolpyruvate carboxylase (PEPC)- and malolactic enzyme (MLE)-encoding genes were identified from strain ATCC 8293, and sequence analysis showed that they could exhibit PEPC and MLE activities, respectively. l-Lactate production and transcriptional expression of the mle gene in this strain were highly increased in the presence of l-malate. We propose that in strain ATCC 8293, which lacks an l- ldh gene, l-lactate is produced through sequential enzymatic conversions from phosphoenolpyruvate to oxaloacetate, then l-malate, and finally l-lactate by PEPC, l-MDH, and MLE, respectively.

Lactobacillus curvatus KFP419 and Leuconostoc mesenteroides subsp. mesenteroides KDK411 Isolated from Kimchi Ameliorate Hypercholesterolemia in Rats.

Western-style diets increase the risk for cardiovascular diseases. It is suggested that the risk could be prevented by lowering cholesterol concentrations in blood. In the present study, hypocholesterolemic effects of the probiotics isolated from kimchi (Lactobacillus curvatus KFP419, Leuconostoc paramesenteroides KJP421, and Leuconostoc mesenteroide subsp. mesenteroides KDK411) were investigated in hypercholesterolemia-induced rats. There was no difference in growth performance between the rats fed high cholesterol diet (HCD) and normal diet (ND). However, blood total cholesterol, low-density lipoprotein cholesterol, and hepatic cholesterol were elevated by the HCD compared to ND, and those concentrations were decreased by dietary supplementation of KFP419 and KDK411. It was concomitant with an increase in fecal excretion of neutral sterols (cholesterol, coprostanol, and coprostanone) in the rats fed HCD compared to ND and was even greater with KDK411 supplementation. These findings indicate that probiotics L. curvatus KFP419 and L. mesenteroide subsp. mesenteroides KDK411 isolated from kimchi ameliorate hypercholesterolemia in rats by assimilating and excreting cholesterol in feces.

Genetic diversity analysis of Leuconostoc mesenteroides from Korean vegetables and food products by multilocus sequence typing.

In the present study, 35 Leuconostoc mesenteroides strains isolated from vegetables and food products from South Korea were studied by multilocus sequence typing (MLST) of seven housekeeping genes (atpA, groEL, gyrB, pheS, pyrG, rpoA, and uvrC). The fragment sizes of the seven amplified housekeeping genes ranged in length from 366 to 1414 bp. Sequence analysis indicated 27 different sequence types (STs) with 25 of them being represented by a single strain indicating high genetic diversity, whereas the remaining 2 were characterized by five strains each. In total, 220 polymorphic nucleotide sites were detected among seven housekeeping genes. The phylogenetic analysis based on the STs of the seven loci indicated that the 35 strains belonged to two major groups, A (28 strains) and B (7 strains). Split decomposition analysis showed that intraspecies recombination played a role in generating diversity among strains. The minimum spanning tree showed that the evolution of the STs was not correlated with food source. This study signifies that the multilocus sequence typing is a valuable tool to access the genetic diversity among L. mesenteroides strains from South Korea and can be used further to monitor the evolutionary changes.

Molecular and Functional Study of a Branching Sucrase-Like Glucansucrase Reveals an Evolutionary Intermediate between Two Subfamilies of the GH70 Enzymes.

Glucansucrases (GSs) in glycoside hydrolase family 70 (GH70) catalyze the synthesis of α-glucans from sucrose, a reaction that is widely seen in lactic acid bacteria (LAB). These enzymes have been implicated in many aspects of microbial life. Products of GSs have great commercial value as food supplements and medical materials; therefore, these enzymes have attracted much attention from both science and industry. Certain issues concerning the origin and evolution of GSs are still to be addressed, although an increasing number of GH70 enzymes have been characterized. This study describes a GS enzyme with the appearance of a branching sucrase (BrS). Structural analysis indicated that this GS enzyme produced a type of glucan composed of an α-(1→6) glucosidic backbone and α-(1→4) branches, as well as a considerable amount of α-(1→3) branches, distinguishing it from the GSs identified so far. Moreover, sequence-based analysis of the catalytic core of this enzyme suggested that it might be an evolutionary intermediate between the BrS and GS subgroups. These results provide an evolutionary link between these subgroups of GH70 enzymes and shed new light on the origination of GSs.IMPORTANCE GH70 GSs catalyze the synthesis of α-glucans from sucrose, a reaction that is widely seen in LAB. Products of these enzymes have great commercial value as food supplements and medical materials. Moreover, these enzymes have attracted much attention from scientists because they have potential in tailored synthesis of α-glucans with desired structures and properties. Although more and more GSs have been characterized, the origin and evolution of these enzymes have not been well addressed. This study describes a GS with the appearance of a BrS (i.e., high levels of similarity to BrSs in sequence analysis). Further analysis indicated that this enzyme synthesized a type of insoluble glucan composed of an α-(1→6) glucosidic backbone and many α-(1→4)- and α-(1→3)-linked branches, the linkage composition of which has rarely been reported in the literature. This BrS-like GS enzyme might be an evolutionary intermediate between BrS and GS enzymes.

Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae.

Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02h-1 (LmSPase) and 0.06 ± 0.01h-1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01h-1 and 0.08 ± 0.00h-1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.