The molecular basis of sugar detection by an insect taste receptor.

Animals crave sugars because of their energy potential and the pleasurable sensation of tasting sweetness. Yet all sugars are not metabolically equivalent, requiring mechanisms to detect and differentiate between chemically similar sweet substances. Insects use a family of ionotropic gustatory receptors to discriminate sugars1, each of which is selectively activated by specific sweet molecules2-6. Here, to gain insight into the molecular basis of sugar selectivity, we determined structures of Gr9, a gustatory receptor from the silkworm Bombyx mori (BmGr9), in the absence and presence of its sole activating ligand, D-fructose. These structures, along with structure-guided mutagenesis and functional assays, illustrate how D-fructose is enveloped by a ligand-binding pocket that precisely matches the overall shape and pattern of chemical groups in D-fructose. However, our computational docking and experimental binding assays revealed that other sugars also bind BmGr9, yet they are unable to activate the receptor. We determined the structure of BmGr9 in complex with one such non-activating sugar, L-sorbose. Although both sugars bind a similar position, only D-fructose is capable of engaging a bridge of two conserved aromatic residues that connects the pocket to the pore helix, inducing a conformational change that allows the ion-conducting pore to open. Thus, chemical specificity does not depend solely on the selectivity of the ligand-binding pocket, but it is an emergent property arising from a combination of receptor-ligand interactions and allosteric coupling. Our results support a model whereby coarse receptor tuning is derived from the size and chemical characteristics of the pocket, whereas fine-tuning of receptor activation is achieved through the selective engagement of an allosteric pathway that regulates ion conduction.

Rare sugar L-sorbose exerts antitumor activity by impairing glucose metabolism.

Rare sugars are monosaccharides with low natural abundance. They are structural isomers of dietary sugars, but hardly be metabolized. Here, we report that rare sugar L-sorbose induces apoptosis in various cancer cells. As a C-3 epimer of D-fructose, L-sorbose is internalized via the transporter GLUT5 and phosphorylated by ketohexokinase (KHK) to produce L-sorbose-1-phosphate (S-1-P). Cellular S-1-P inactivates the glycolytic enzyme hexokinase resulting in attenuated glycolysis. Consequently, mitochondrial function is impaired and reactive oxygen species are produced. Moreover, L-sorbose downregulates the transcription of KHK-A, a splicing variant of KHK. Since KHK-A is a positive inducer of antioxidation genes, the antioxidant defense mechanism in cancer cells can be attenuated by L-sorbose-treatment. Thus, L-sorbose performs multiple anticancer activities to induce cell apoptosis. In mouse xenograft models, L-sorbose enhances the effect of tumor chemotherapy in combination with other anticancer drugs. These results demonstrate L-sorbose as an attractive therapeutic reagent for cancer treatment.

Engineering Gluconobacter cerinus CGMCC 1.110 for direct 2-keto-L-gulonic acid production.

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9 g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9 g/L.

Intestinal absorption of D-fructose isomers, D-allulose, D-sorbose and D-tagatose, via glucose transporter type 5 (GLUT5) but not sodium-dependent glucose cotransporter 1 (SGLT1) in rats.

D-allulose, D-sorbose and D-tagatose are D-fructose isomers that are called rare sugars. These rare sugars have been studied intensively in terms of biological production and food application as well as physiological effects. There are limited papers with regard to the transporters mediating the intestinal absorption of these rare sugars. We examined whether these rare sugars are absorbed via sodium-dependent glucose cotransporter 1 (SGLT1) as well as via GLUT type 5 (GLUT5) using rats. High-fructose diet fed rats, which express more intestinal GLUT5, exhibited significantly higher peripheral concentrations, Cmax and AUC0­180 min when D-allulose, D-sorbose and D-tagatose were orally administrated. KGA-2727, a selective SGLT1 inhibitor, did not affect the peripheral and portal vein concentrations and pharmacokinetic parameters of these rare sugars. The results suggest that D-allulose, D-sorbose and D-tagatose are likely transported via GLUT5 but not SGLT1 in rat small intestine.

Adaptation to the dietary sugar D-tagatose via genome instability in polyploid Candida albicans cells.

The opportunistic fungal pathogen Candida albicans undergoes an unusual parasexual cycle wherein diploid cells mate to form tetraploid cells that can generate genetically diverse progeny via a nonmeiotic program of chromosome loss. The genetic diversity afforded by parasex impacts clinically relevant features including drug resistance and virulence, and yet the factors influencing genome instability in C. albicans are not well defined. To understand how environmental cues impact genome instability, we monitored ploidy change following tetraploid cell growth in a panel of different carbon sources. We found that growth in one carbon source, D-tagatose, led to high levels of genomic instability and chromosome loss in tetraploid cells. This sugar is a stereoisomer of L-sorbose which was previously shown to promote karyotypic changes in C. albicans. However, while expression of the SOU1 gene enabled utilization of L-sorbose, overexpression of this gene did not promote growth in D-tagatose, indicating differences in assimilation of the two sugars. In addition, genome sequencing of multiple progenies recovered from D-tagatose cultures revealed increased relative copy numbers of chromosome 4, suggestive of chromosome-level regulation of D-tagatose metabolism. Together, these studies identify a novel environmental cue that induces genome instability in C. albicans, and further implicate chromosomal changes in supporting metabolic adaptation in this species.

Enhanced 2-keto-L-gulonic acid production by a mixed culture of Ketogulonicigenium vulgare and Bacillus megaterium using three-stage temperature control strategy.

As a key precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG) was mainly produced from L-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain (Bacillus spp.) with a low conversion rate for decades. The aim of this study was to enhance the 2-KLG production by co-culturing K. vulgare and Bacillus megaterium using three-stage temperature control (TSTC) strategy. By investigating the temperature effect on the 2-KLG fermentation, the optimum temperatures for the growths of K. vulgare and B. megaterium were 32 °C and 29 °C, respectively, while the optimum temperature for 2-KLG production was 35 °C. We developed a TSTC process: the temperature was kept at 32 °C during the first 16 h of fermentation, then decreased to 29 °C for the following 14 h, and maintained at 35 °C to the end of fermentation. By using this new process, the productivity and yield of 2-KLG from L-sorbose were obtained at 2.19 ± 0.19 g/L/h and 92.91 ± 1.02 g/L in 20-L fermentors for 5 batches, respectively, which were 22.35% and 6.02% higher than that of the control treatment (the single temperature of 29 °C). The increased cell density of K. vulgare during the exponential phase and the enhanced SDH activity (increased by 25.18% at 36 h, 17.14% at 44 h) in the production stage might be the reasons for enhanced 2-KLG conversion rate and yield. Our results demonstrated the feasibility of the TSTC strategy for 2-KLG production.

CSU57 encodes a novel repressor of sorbose utilization in opportunistic human fungal pathogen Candida albicans.

Human fungal pathogen Candida albicans cannot utilize L-sorbose as a sole carbon source. However, chromosome 5 monosomic strains can grow on sorbose as repressors present on this chromosome get diminished allowing the expression of sorbose utilization gene (SOU1) located on chromosome 4. Functional identification of these repressors has been a difficult task as they are scattered on a large portion of the right arm of chromosome 5. Herein, we have applied the telomere-mediated chromosomal truncation approach to identify a novel repressor for sorbose utilization in this pathogen. Multiple systematic chromosomal truncations were performed on the right arm of Chr5 in the background of csu51∆/CSU51 to minimize the functional region to 6-kb chromosomal stretch. Further, truncation that removes the part of Orf19.3942 strongly suggested its role in sorbose utilization. However, compelling evidence comes from the observation that truncation at 1,044.288-kb position of Chr5 in the strain csu51∆/CSU51 orf19.3942∆/Orf.19.3942 produced Sou+ phenotype; otherwise, the strain remains Sou- . This confirms beyond doubt the role of Orf.19.3942 in the regulation of sorbose utilization and designated as CSU57. Comparison of SOU1 gene expression of Sou+ strains with wild type suggested its role at transcriptional level. Strain carrying double disruption of CSU57 remains Sou- . Co-overexpression of SOU1 and CSU57 together does not make the recipient strain Sou- ; however, multiple tandem copies of CSU57 produced diminished growth compared with control suggesting that it is a weak repressor. Taken together, we report that CSU57 encodes a novel repressor of L-sorbose utilization in this pathogen. TAKE AWAY: CSU57 encodes a repressor for L-sorbose utilization in Candida albicans. Csu57p acts in combination with Csu51p and other regulators. Csu57p exerts its repressing effect at transcriptional level of SOU1 gene. Utilization of sorbose positively correlates to the expression of SOU1 gene. Multiple copies of CSU57 can partially suppress Sou+ phenotype.

Gas chromatography - Mass spectrometry as a preferred method for quantification of insect hemolymph sugars.

Insects, due to their small size, have limited energy storage space, but they also have high metabolic rate, so their hemolymph sugars are incredibly dynamic and play a number of important physiological functional roles in maintaining energetic homeostasis. In contrast to vertebrates, trehalose is generally the primary sugar found in insect hemolymph, which is followed by glucose and fructose. Many analytical chemistry methods exist to measure sugars, yet a direct comparison of methods that can measure all three simultaneously, and trehalose in particular, from low sample volumes, are sparse. Using the honey bee as a model, we directly compare the leading current methods of using High Performance Liquid Chromatography (HPLC) with an evaporative light-scattering detector and Gas Chromatography coupled with Mass Spectrometry (GC-MS) to determine which method would be better for measuring trehalose, glucose, and fructose in terms of reproducibility, accuracy, and sensitivity. Furthermore, we injected the enzyme inhibitors trehalozin (a trehalase inhibitor) and sorbose (a trehalase p-synthase inhibitor) to manipulate the trehalose levels in honey bee foragers as a proof of concept that this sugar can be altered independently of hemolymph glucose and fructose levels. Overall the HPLC method was less reproducible for measuring fructose and glucose, and it also had lower sensitivity for measuring trehalose. Consequently, significant differences in trehalose levels within the forager class were only detected with the GC-MS and not the HPLC method. Lastly, using the GC-MS method in the follow up study we found that trehalozin and sorbose causes a significant increase and decrease of trehalose levels respectively, in forager honey bees, independent of the glucose and fructose levels, ten minutes after injection. Taken together, these methods will provide useful tools for future studies exploring the many different physiological functional roles that trehalose can play in maintaining insect energetic homeostasis.

The membrane-bound sorbosone dehydrogenase of Gluconacetobacter liquefaciens is a pyrroloquinoline quinone-dependent enzyme.

Membrane-bound sorbosone dehydrogenase (SNDH) of Gluconacetobacter liquefaciens oxidizes l-sorbosone to 2-keto-l-gulonic acid (2KGLA), a key intermediate in vitamin C production. We constructed recombinant Escherichia coli and Gluconobacter strains harboring plasmids carrying the sndh gene from Ga. liquefaciens strain RCTMR10 to identify the prosthetic group of SNDH. The membranes of the recombinant E. coli showed l-sorbosone oxidation activity, only after the holo-enzyme formation with pyrroloquinoline quinone (PQQ), indicating that SNDH is a PQQ-dependent enzyme. The sorbosone-oxidizing respiratory chain was thus heterologously reconstituted in the E. coli membranes. The membranes that contained SNDH showed the activity of sorbosone:ubiquinone analogue oxidoreductase. These results suggest that the natural electron acceptor for SNDH is membranous ubiquinone, and it functions as the primary dehydrogenase in the sorbosone oxidation respiratory chain in Ga. liquefaciens. A biotransformation experiment showed l-sorbosone oxidation to 2KGLA in a nearly quantitative manner. Phylogenetic analysis for prokaryotic SNDH homologues revealed that they are found only in the Proteobacteria phylum and those of the Acetobacteraceae family are clustered in a group where all members possess a transmembrane segment. A three-dimensional structure model of the SNDH constructed with an in silico fold recognition method was similar to the crystal structure of the PQQ-dependent pyranose dehydrogenase from Coprinopsis cinerea. The structural similarity suggests a reaction mechanism under which PQQ participates in l-sorbosone oxidation.

Cascade synthesis of rare ketoses by whole cells based on L-rhamnulose-1-phosphate aldolase.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases demonstrate important values in the production of rare ketoses due to their unique stereoselectivities. As a specific example, we developed an efficient Escherichia coli whole-cell biocatalytic cascade system in which rare ketoses were produced from abundant glycerol and catalyzed by four enzymes based on L-rhamnulose-1-phosphate aldolase (RhaD). For the semicontinuous bioconversion in which D-glyceraldehyde was continuously added, once D-glyceraldehyde was consumed, the final yields of D-sorbose and D-psicose were 15.30 g/L and 6.35 g/L, respectively. Moreover, the maximum conversion rate and productivity of D-sorbose and D-psicose were 99% and 1.11 g/L/h at 8 h, respectively. When L-glyceraldehyde was used instead of the D-isomer, the final yield of L-fructose was 16.80 g/L. Furthermore, the maximum conversion rate and productivity of L-fructose were 95% and 1.08 g/L/h at 8 h, respectively. This synthetic platform was also compatible with other various aldehydes, which allowed the production of many other high-value chemicals from glycerol.

Systematic characterization of sorbose/sorbosone dehydrogenases and sorbosone dehydrogenases from Ketogulonicigenium vulgare WSH-001.

2-Keto-L-gulonic acid (2-KLG) is the direct precursor of vitamin C in industrial synthesis. 2-KLG is mainly produced via the classical two-step fermentation route. In the two-step fermentation process, 2-KLG can be synthesized from L-sorbose by Ketogulonicigenium vulgare aided by Bacillus megaterium. There are five sorbose/sorbosone dehydrogenases (SSDHs), SSDA1, SSDA1-P, SSDA2, SSDA3 and SSDB, and two sorbosone dehydrogenases (SNDHs), glucose/sorbosone dehydrogenase (GSNDH) and sorbosone dehydrogenase (SNDH), in K. vulgare, which could play crucial roles in transforming L-sorbose or L-sorbosone to 2-KLG. However, confusion about the catalytic characteristics of the individual SSDHs and SNDHs makes construction of a recombinational strain for the purpose of enhancing 2-KLG production difficult. In this study, the five SSDHs and two SNDHs from K. vulgare WSH-001 were purified, and their optimal pH values and reaction temperatures, kinetic properties, thermostabilities, substrate spectra and effects of electron acceptors on their performances were systematically determined. Among these dehydrogenases, only SSDA1 and SSDA3 have high activity for catalyzing L-sorbose to 2-KLG directly. These data provide more clues for ways to achieve enhanced conversion of L-sorbose in K. vulgare, which could facilitate both the construction of a more efficient one-step fermentation 2-KLG producer and the reconstruction of a one-step fermentation process.

Continuous co-production of biomass and bio-oxidized metabolite (sorbose) using Gluconobacter oxydans in a high-oxygen tension bioreactor.

High-efficiency cell proliferation of Gluconobacter oxydans was carried out in a compressed oxygen supply and sealed bioreactor to co-produce biomass and corresponding metabolite (sorbose). Higher cell density of Gluconobacter oxydans was achieved by implementing high-oxygen tension supply strategy resulting into high sorbose production from bio-oxidation of sorbitol. The highest biomass of 8.8 g/L and highest sorbose production of 432.8 g/L were simultaneously obtained after 72 h of fed-batch operation. Moreover, continuous co-production of biomass and sorbose was successfully conducted with retaining 10% sorbitol fermentation broth as the seed of next stage culture. The key features of this bioprocess application would enable cost-competitive Gluconobacter oxydans industrial applications.

NADH oxidase from Lactobacillus reuteri: A versatile enzyme for oxidized cofactor regeneration.

Pyridine nucleotide cofactors play important roles in biocatalytic processes that generate value-added chemicals for the pharmaceutical and food industries. Because of the high price of these pyridine cofactors, cofactor regeneration is highly desirable. However, recycling the oxidized form of cofactors, especially NADP+, remains a challenge. Here, we cloned and characterized an NADH oxidase from Lactobacillus reuteri (LreNox) which can oxidize both NADH and NADPH. Unlike many other Noxs, LreNox showed equal catalytic efficiency towards NADH and NADPH. To the best our knowledge, LreNox has the highest activity towards NADPH as a substrate compared to other wild type Noxs. Homology modeling and substrate docking studies provided insights into the dual substrate specificity of LreNox. Gly155, Ser179, and His184 in the LreNox substrate binding pocket, which are absent in other Noxs structures, are crucial for NADPH recognition, providing more space for interactions with the additional phosphate group present in NADPH. We also explored the utility of LreNox for NADP+ regeneration in l-sorbose production by coupling it with a sorbitol dehydrogenase. The turn over number (TTN) improved ~53-fold after using LreNox as the NADP+ recycling enzyme. This study demonstrates that LreNox could potentially be used for the regeneration of NAD(P)+ in commercial applications.

Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-L-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters.

BACKGROUND: 2-Keto-L-gulonic acid (2-KGA), the precursor of vitamin C, is currently produced by two-step fermentation. In the second step, L-sorbose is transformed into 2-KGA by the symbiosis system composed of Ketogulonicigenium vulgare and Bacillus megaterium. Due to the different nutrient requirements and the uncertain ratio of the two strains, the symbiosis system significantly limits strain improvement and fermentation optimization. RESULTS: In this study, Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that of K. vulgare in a mono-culture system. The complete genome of K. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reported K. vulgare genomes, K. robustum SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. Moreover, the amino acid biosynthesis pathways were more complete. Two species-specific internal promoters, P1 (orf_01408 promoter) and P2 (orf_02221 promoter), were predicted and validated by detecting their initiation activity. To efficiently produce 2-KGA with decreased CO2 release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced into K. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp) and phosphotransacetylase (pta) initiated by internal promoters. After gene optimization, the recombinant strain K. robustum/pBBR-P1_xfp2502-P2_pta2145 enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strain K. robustum/pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (pgd) and pyruvate dehydrogenase genes (pdh) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (sdh) and sorbosone dehydrogenase gene (sndh), were up-regulated to different degrees in the recombinant strain. CONCLUSIONS: The genome-based functional analysis of K. robustum SPU_B003 provided a new understanding of the specific metabolic characteristics. The new XFP-PTA pathway was an efficient route to enhance acetyl-CoA levels and to therefore promote 2-KGA production.

A Single-Nucleotide Insertion in a Drug Transporter Gene Induces a Thermotolerance Phenotype in Gluconobacter frateurii by Increasing the NADPH/NADP+ Ratio via Metabolic Change.

Thermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adapted Gluconobacter frateurii strain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The ΔotsAB strain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The ΔotsAB mutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP+ ratio was higher in strain Wild-G, and much higher in the ΔotsAB strain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance in G. frateuriiIMPORTANCE The biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show that Gluconobacterfrateurii, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP+ ratios and reductions in ROS levels. We concluded that in G. frateurii, trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP+ ratio to enhance thermotolerance.

Rare sugars, d-allulose, d-tagatose and d-sorbose, differently modulate lipid metabolism in rats.

BACKGROUND: Rare sugars including d-allulose, d-tagatose, and d-sorbose are present in limited quantities in nature; some of these rare sugars are now commercially produced using microbial enzymes. Apart from the anti-obesity and anti-hyperglycaemic activities of d-allulose, effects of these sugars on lipid metabolism have not been investigated. Therefore, we aimed to determine if and how d-tagatose and d-sorbose modulate lipid metabolism in rats. After feeding these rare sugars to rats, parameters on lipid metabolism were determined. RESULTS: No diet-related effects were observed on body weight and food intake. Hepatic lipogenic enzyme activity was lowered by d-allulose and d-sorbose but increased by d-tagatose. Faecal fatty acid excretion was non-significantly decreased by d-allulose, but significantly increased by d-sorbose without affecting faecal steroid excretion. A trend toward reduced adipose tissue weight was observed in groups fed rare sugars. Serum adiponectin levels were decreased by d-sorbose relative to the control. Gene expression of cholesterol metabolism-related liver proteins tended to be down-regulated by d-allulose and d-sorbose but not by d-tagatose. In the small intestine, SR-B1 mRNA expression was suppressed by d-sorbose. CONCLUSION: Lipid metabolism in rats varies with rare sugars. Application of rare sugars to functional foods for healthy body weight maintenance requires further studies. © 2017 Society of Chemical Industry.

A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production

Abstract A new plate method was developed for rapid screening of Ketogulonicigenium vulgare mutants overproducing 2-keto-l-gulonic acid (2-KLG). The screening methodology took the advantage of the acidity caused by 2-KLG, which changes the color of bromothymol blue (pH indicator) from blue to yellow. Using the proposed method, a mutant, K. vulgare 65, was selected from 20,000 colonies produced by a strain subjected to spaceflight mutagenesis. When co-cultured with Bacillus megaterium 2980 in 20-L fermenters, K. vulgare 65 showed a high conversion rate (94.45%) of l-sorbose to 2-KLG. In contrast to the traditional screening method, this one significantly improved the frequency of obtaining positive mutants. The proposed plate screening method is cost-effective and easy to run and is thus useful for the isolation and screening of K. vulgare mutants overproducing 2-KLG.

Reconstruction of amino acid biosynthetic pathways increases the productivity of 2-keto-L-gulonic acid in Ketogulonicigenium vulgare-Bacillus endophyticus consortium via genes screening.

Defect in the amino acid biosynthetic pathways of Ketogulonicigenium vulgare, the producing strain for 2-keto-L-gulonic acid (2-KGA), is the key reason for its poor growth and low productivity. In this study, five different strains were firstly reconstructed by expressing absent genes in threonine, proline and histidine biosynthetic pathways for better 2-KGA productivity. When mono-cultured in the shake flasks, the strain SyBE_Kv02080002 expressing hsk from Gluconobacter oxydans in threonine biosynthetic pathway achieved the highest biomass and the titer increased by 25.13%. When co-cultured with Bacillus endophyticus, the fermentation cycle decreased by 28.57% than that of the original consortium in 5-L fermenter. Furthermore, reconstruction of threonine biosynthetic pathway resulted in up-regulation of genes encoding sorbosone dehydrogenase and idonate-dehydrogenase, which increased the 2-KGA productivity in SyBE_Kv02080002. This study shows that reconstruction of absent biosynthetic pathways in bacteria is an effective way to enhance the productivity of target products.

A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production.

A new plate method was developed for rapid screening of Ketogulonicigenium vulgare mutants overproducing 2-keto-l-gulonic acid (2-KLG). The screening methodology took the advantage of the acidity caused by 2-KLG, which changes the color of bromothymol blue (pH indicator) from blue to yellow. Using the proposed method, a mutant, K. vulgare 65, was selected from 20,000 colonies produced by a strain subjected to spaceflight mutagenesis. When co-cultured with Bacillus megaterium 2980 in 20-L fermenters, K. vulgare 65 showed a high conversion rate (94.45%) of l-sorbose to 2-KLG. In contrast to the traditional screening method, this one significantly improved the frequency of obtaining positive mutants. The proposed plate screening method is cost-effective and easy to run and is thus useful for the isolation and screening of K. vulgare mutants overproducing 2-KLG.

Prebiotic potential of L-sorbose and xylitol in promoting the growth and metabolic activity of specific butyrate-producing bacteria in human fecal culture.

Dietary low-digestible carbohydrates (LDCs) affect gut microbial metabolism, including the production of short-chain fatty acids. The ability of various LDCs to promote butyrate production was evaluated in in vitro human fecal cultures. Fecal suspensions from five healthy males were anaerobically incubated with various LDCs. L-Sorbose and xylitol markedly promoted butyrate formation in cultures. Bacterial 16S rRNA gene-based denaturing gradient gel electrophoresis analyses of these fecal cultures revealed a marked increase in the abundance of bacteria closely related to the species Anaerostipes hadrus or A. caccae or both, during enhanced butyrate formation from L-sorbose or xylitol. By using an agar plate culture, two strains of A. hadrus that produced butyrate from each substrate were isolated from the feces of two donors. Furthermore, of 12 species of representative colonic butyrate producers, only A. hadrus and A. caccae demonstrated augmented butyrate production from L-sorbose or xylitol. These findings suggest that L-sorbose and xylitol cause prebiotic stimulation of the growth and metabolic activity of Anaerostipes spp. in the human colon.