Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-L-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters.
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ABSTRACT: 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.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.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.
Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-L-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters.
<h4>Background</h4>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.<h4>Results</h4>In this study, Ketogulonicigenium ro ...[more]
Project description:The acetic acid bacterium Gluconobacter oxydans is known for its unique incomplete oxidation and therefore widely applied in the industrial production of many compounds, e.g., 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. However, few molecular tools are available for metabolically engineering G. oxydans, which greatly limit the strain development. Promoters are one of vital components to control and regulate gene expression at the transcriptional level for boosting production. In this study, the low activity of SDH was found to hamper the high yield of 2-KLG, and enhancing the expression of SDH was achieved by screening the suitable promoters based on RNA sequencing data. We obtained 97 promoters from G. oxydans's genome, including two strong shuttle promoters and six strongest promoters. Among these promoters, P3022 and P0943 revealed strong activities in both Escherichia coli and G. oxydans, and the activity of the strongest promoter (P2703) was about threefold that of the other reported strong promoters of G. oxydans. These promoters were used to overexpress SDH in G. oxydans WSH-003. The titer of 2-KLG reached 3.7 g/L when SDH was under the control of strong promoters P2057 and P2703. This study obtained a series of gradient promoters, including two strong shuttle promoters, and expanded the toolbox of available promoters for the application in metabolic engineering of G. oxydans for high-value products.
Project description:Currently, the establishment of synthetic microbial consortia with rational strategies has gained extensive attention, becoming one of the important frontiers of synthetic biology. Systems biology can offer insights into the design and construction of synthetic microbial consortia. Taking the high-efficiency production of 2-keto-l-gulonic acid (2-KLG) as an example, we constructed a synthetic microbial consortium "Saccharomyces cerevisiae-Ketogulonigenium vulgare" based on systems biology analysis. In the consortium, K. vulgare was the 2-KLG producing strain, and S. cerevisiae acted as the helper strain. Comparative transcriptomic analysis was performed on an engineered S. cerevisiae (VTC2) and a wild-type S. cerevisiae BY4741. The results showed that the up-regulated genes in VTC2, compared with BY4741, were mainly involved in glycolysis, TCA cycle, purine metabolism, and biosynthesis of amino acids, B vitamins, and antioxidant proteases, all of which play important roles in promoting the growth of K. vulgare. Furthermore, Vitamin C produced by VTC2 could further relieve the oxidative stress in the environment to increase the production of 2-KLG. Therefore, VTC2 would be of great advantage in working with K. vulgare. Thus, the synthetic microbial consortium "VTC2-K. vulgare" was constructed based on transcriptomics analyses, and the accumulation of 2-KLG was increased by 1.49-fold compared with that of mono-cultured K. vulgare, reaching 13.2 ± 0.52 g/L. In addition, the increased production of 2-KLG was accompanied by the up-regulated activities of superoxide dismutase and catalase in the medium and the up-regulated oxidative stress-related genes (sod, cat and gpd) in K. vulgare. The results indicated that the oxidative stress in the synthetic microbial consortium was efficiently reduced. Thus, systems analysis confirmed a favorable symbiotic relationship between microorganisms, providing guidance for further engineering synthetic consortia.
Project description:High-throughput screening is a powerful tool for discovering strains in the natural environment that may be suitable for target production. Herein, a novel enzyme-based high-throughput screening method was developed for rapid screening of strains overproducing 2-keto-L-gulonic acid (2-KLG). The screening method detects changes in the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) at 340 nm using a microplate reader when 2-KLG is degraded by 2-KLG reductase. In this research, three different 2-KLG reductases were expressed, purified, and studied. The 2-KLG reductase from Aspergillus niger were selected as the best appropriate reductase to establishment the method for its high activity below pH 7. Using the established method, and coupled with fluorescence-activated cell sorting, we achieved a high 2-KLG-producing strain of Gluconobacter oxydans WSH-004 from soil. When cultured with D-sorbitol as the substrate, the 2-KLG yield was 2.5 g/L from 50 g/L D-sorbitol without any side products. Compared with other reported screening methods, our enzyme-based method is more efficient and accurate for obtaining high-producing 2-KLG strains, and it is also convenient and cost-effective. The method is broadly applicable for screening keto acids and other products that can be oxidized via nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+).
Project description:The solid-liquid equilibrium (SLE) in the ternary system 2-keto-L-gulonic acid (HKGA) + L-ascorbic acid (vitamin C) + water was investigated experimentally at temperatures between 276 K and 308 K at ambient pressure, i.e., under conditions that are of particular interest for industrial applications. Phase diagrams with one eutonic point were obtained for all temperatures. The dissociation constant and the solubility constant of vitamin C were determined as a function of temperature. Based on an extended version of the Debye-Hückel theory, a physicochemical model was developed that describes the SLE in the ternary system. The agreement between experimental data and results from the model is excellent.
Project description:MicroRNAs (miRNAs) are RNA sequences of approximately 22 nucleotides that mediate post-transcriptional regulation of specific mRNAs. miRNA sequences are dispersed throughout the genome and are classified as intergenic (between genes) or intronic (embedded into a gene). Intergenic miRNAs are expressed by their own promoter, and until recently, it was supposed that intronic miRNAs are transcribed from their host gene. Here, we performed a genomic analysis of currently known intronic miRNA regions and observed that approximately 35% of intronic miRNAs have upstream regulatory elements consistent with promoter function. Among all intronic miRNAs, 30% have associated Pol II regulatory elements, including transcription start sites, CpG islands, expression sequence tags, and conserved transcription factor binding sites, while 5% contain RNA Pol III regulatory elements (A/B box sequences). We cloned intronic regions encompassing miRNAs and their upstream Pol II (miR-107, miR-126, miR-208b, miR-548f-2, miR-569, and miR-590) or Pol III (miR-566 and miR-128-2) sequences into a promoterless plasmid, and confirmed that miRNA expression occurs independent of host gene transcription. For miR-128-2, a miRNA overexpressed in acute lymphoblastic leukemia, ChIP analysis suggests dual regulation by both intronic (Pol III) and host gene (Pol II) promoters. These data support complex regulation of intronic miRNA expression, and have relevance to disregulation in disease settings.
Project description:Salt stress has long been a prominent obstacle that restricts crop growth, and increasing the L-ascorbic acid (ASA) content of crops is an effective means of alleviating this stress. 2-Keto-L-gulonic acid (2KGA) is a precursor used in industrial ASA production as well as an ASA degradation product in plants. However, to date, no study has investigated the effects of 2KGA on ASA metabolism and salt stress. Here, we evaluated the potential of using 2KGA to improve crop resistance to salt stress (100mM NaCl) through a cultivation experiment of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). The results showed that the leaf and root biomass were significantly improved by 2KGA application. The levels of metabolites and enzymes related to stress resistance were increased, whereas the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents were decreased. Lipid peroxidation and cell membrane damage were alleviated following 2KGA treatment. Positive correlations were found between photosynthetic pigments and organic solutes, ASA and photosynthetic pigments, and ASA and antioxidant enzymes. In contrast, negative correlations were observed between antioxidant enzymes and H2O2/MDA. Moreover, the expression levels of L-gulono-1,4-lactone oxidase, GDP-mannose pyrophosphorylase, dehydroascorbate reductase-3, and ascorbate peroxidase were increased by 2KGA treatment. These results suggested that exogenous 2KGA application can relieve the inhibitory effect of salt stress on plant growth, and the promotion of ASA synthesis may represent a critical underlying mechanism. Our findings have significant implications for the future application of 2KGA or its fermentation residue in agriculture.
Project description:L-ascorbic acid (ASA) is a micronutrient that is essential for reproduction, growth, and immunity in animals. Due to the loss of enzyme L-gulono-1,4-lactone oxidase (GLO), most aquatic animals lack the capacity for ASA biosynthesis and therefore require supplementation with exogenous ASA. Recent studies have shown that 2-keto-L-gulonic acid (2KGA), a novel potential precursor of ASA, can enhance plant growth and improve stress resistance by promoting the synthesis and accumulation of ASA. Our hypothesis is that 2-keto-L-gulonic acid (2KGA) plays a similar role in aquatic animals. To investigate this, we conducted an in vivo trial to examine the effects of exogenous 2KGA supplementation on ASA metabolism and growth of zebrafish (Danio rerio). Zebrafish were categorized into groups based on their dietary intake, including a basal diet (CK group), a basal diet supplemented with 800 mg/kg ASA (ASA group), and 800 mg/kg 2KGA-Na (2KGA group) for a duration of three weeks. The results demonstrated a significant increase in ASA content in zebrafish treated with 2KGA (34.82% increase, p < 0.05) compared to the CK group, reaching a consistent level with the ASA group (39.61% increase, p < 0.05). Furthermore, the supplementation of 2KGA significantly improved growth parameters relevant to zebrafish (specific growth rate increased by 129.04%, p < 0.05) and enhanced feed utilization (feed intake increased by 15.65%, p < 0.05). Positive correlations were observed between growth parameters, feed utilization, whole-body chemical composition, and ASA content. Our findings suggest that supplementation with exogenous 2KGA can serve as a novel approach for elevating ASA synthesis in aquatic animals, and further investigation of its underlying mechanism is required.
Project description:This work represents an innovative study that, for the first time, explores the possibility to use waste flours to produce thermoplastic polymeric bio-films. To the best of our knowledge, this is the first time that waste flours, derived from bakeries, pizzerias or pasta factories, have been proposed for the production of bio-polymers, as a replacement of neat starch. To this aim, durum waste flour derived from a pasta factory, soft waste flour derived from pizzerias and neat maize starch used as control material were firstly analyzed from dimensional, morphological and chemical points of view. Afterwards, waste flour films were produced by the addition of a nature-based plasticizer, glycerol. Mechanical characterization of the plasticized thermoplastic films, produced by compression molding, evidenced low performances, even in the case of the neat maize starch. In order to improve the mechanical properties, the possibility to include polylactic acid and cardanol-based plasticizer was also investigated. Mass transport properties of all the produced bio-films were investigated by measuring their water vapor permeability and hygroscopic absorption. The durability properties of the bio-films were assessed by accelerated ageing tests, while the bio-degradability of the waste-based films was evaluated by measuring the solubility and the degradation in water. The physicochemical analyses of the novel bio-films evidenced good mechanical properties; specifically, the waste-based films showed a lower hygroscopic absorption and water solubility than those of the blends containing neat starch.
Project description:The nuclear retinoic acid (RA) receptor alpha (RARalpha) is a transcriptional transregulator that controls the expression of specific gene subsets through binding at response elements and dynamic interactions with coregulators, which are coordinated by the ligand. Here, we highlighted a novel paradigm in which the transcription of RARalpha target genes is controlled by phosphorylation cascades initiated by the rapid RA activation of the p38MAPK/MSK1 pathway. We demonstrate that MSK1 phosphorylates RARalpha at S369 located in the ligand-binding domain, allowing the binding of TFIIH and thereby phosphorylation of the N-terminal domain at S77 by cdk7/cyclin H. MSK1 also phosphorylates histone H3 at S10. Finally, the phosphorylation cascade initiated by MSK1 controls the recruitment of RARalpha/TFIIH complexes to response elements and subsequently RARalpha target gene activation. Cancer cells characterized by a deregulated p38MAPK/MSK1 pathway, do not respond to RA, outlining the essential contribution of the RA-triggered phosphorylation cascade in RA signalling.
