Metabolic engineering of fungal strains for conversion of D-galacturonate to meso-galactarate.
Ontology highlight
ABSTRACT:
SUBMITTER: Mojzita D
PROVIDER: S-EPMC2798651 | biostudies-literature | 2010 Jan
REPOSITORIES: biostudies-literature
Publications
Metabolic engineering of fungal strains for conversion of D-galacturonate to meso-galactarate.
Mojzita Dominik D Wiebe Marilyn M Hilditch Satu S Boer Harry H Penttilä Merja M Richard Peter P
Applied and environmental microbiology 20091106 1
D-galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of D-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi D-galacturonate is catabolized through a reductive pathway with a D-galacturonate reductase as the first enzyme. Deleting the corresponding gene in the f ...[more]
Similar Datasets
Project description:BackgroundSynthetic L-ascorbic acid (vitamin C) is widely used as a preservative and nutrient in food and pharmaceutical industries. In the current production method, D-glucose is converted to L-ascorbic acid via several biochemical and chemical steps. The main source of L-ascorbic acid in human nutrition is plants. Several alternative metabolic pathways for L-ascorbic acid biosynthesis are known in plants. In one of them, D-galacturonic acid is the precursor. D-Galacturonic acid is also the main monomer in pectin, a plant cell wall polysaccharide. Pectin is abundant in biomass and is readily available from several waste streams from fruit and sugar processing industries.ResultsIn the present work, we engineered the filamentous fungus Aspergillus niger for the conversion of D-galacturonic acid to L-ascorbic acid. In the generated pathway, the native D-galacturonate reductase activity was utilized while the gene coding for the second enzyme in the fungal D-galacturonic acid pathway, an L-galactonate consuming dehydratase, was deleted. Two heterologous genes coding for enzymes from the plant L-ascorbic acid pathway--L-galactono-1,4-lactone lactonase from Euglena gracilis (EgALase) and L-galactono-1,4-lactone dehydrogenase from Malpighia glabra (MgGALDH)--were introduced into the A. niger strain. Alternatively, an unspecific L-gulono-1,4-lactone lactonase (smp30) from the animal L-ascorbic acid pathway was introduced in the fungal strain instead of the plant L-galactono-1,4-lactone lactonase. In addition, a strain with the production pathway inducible with D-galacturonic acid was generated by using a bidirectional and D-galacturonic acid inducible promoter from the fungus. Even though, the lactonase enzyme activity was not observed in the resulting strains, they were capable of producing L-ascorbic acid from pure D-galacturonic acid or pectin-rich biomass in a consolidated bioprocess. Product titers up to 170 mg/l were achieved.ConclusionsIn the current study, an L-ascorbic acid pathway using D-galacturonic acid as a precursor was introduced to a microorganism for the first time. This is also the first report on an engineered filamentous fungus for L-ascorbic acid production and a proof-of-concept of consolidated bioprocess for the production.
| S-EPMC4299797 | biostudies-literature
Project description:Background2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(-)-2,3-BD (purity >99 %) under low oxygen conditions.ResultsIn this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(-)-2,3-BD production was abolished by deleting d-(-)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L.ConclusionThis work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer.
| S-EPMC4837526 | biostudies-literature
Project description:Bacteria and yeast are being intensively used to produce biofuels and high-added-value products by using plant biomass derivatives as substrates. The number of microorganisms available for industrial processes is increasing thanks to biotechnological improvements to enhance their productivity and yield through microbial metabolic engineering and laboratory evolution. This is allowing the traditional industrial processes for biofuel production, which included multiple steps, to be improved through the consolidation of single-step processes, reducing the time of the global process, and increasing the yield and operational conditions in terms of the desired products. Engineered microorganisms are now capable of using feedstocks that they were unable to process before their modification, opening broader possibilities for establishing new markets in places where biomass is available. This review discusses metabolic engineering approaches that have been used to improve the microbial processing of biomass to convert the plant feedstock into fuels. Metabolically engineered microorganisms (MEMs) such as bacteria, yeasts, and microalgae are described, highlighting their performance and the biotechnological tools that were used to modify them. Finally, some examples of patents related to the MEMs are mentioned in order to contextualize their current industrial use.
| S-EPMC10535843 | biostudies-literature
Project description:BackgroundComputational modeling and analysis of metabolic networks has been successful in metabolic engineering of microbial strains for valuable biochemical production. Limitations of currently available computational methods for metabolic engineering are that they are often based on reaction deletions rather than gene deletions and do not consider the regulatory networks that control metabolism. Due to the presence of multi-functional enzymes and isozymes, computational designs based on reaction deletions can sometimes result in strategies that are genetically complicated or infeasible. Additionally, strains might not be able to grow initially due to regulatory restrictions. To overcome these limitations, we have developed a new approach (OptORF) for identifying metabolic engineering strategies based on gene deletion and overexpression.ResultsHere we propose an effective method to systematically integrate transcriptional regulatory networks and metabolic networks. This allows for the formulation of linear optimization problems that search for metabolic and/or regulatory perturbations that couple biomass and biochemical production, thus proposing adaptive evolutionary strain designs. Using genome-scale models of Escherichia coli, we have implemented the OptORF algorithm (which considers gene deletions and transcriptional regulation) and compared its metabolic engineering strategies for ethanol production to those found using OptKnock (which considers reaction deletions). Our results found that the reaction-based strategies often require more gene deletions to remove the identified reactions (2 more genes than reactions), and result in lethal growth phenotypes when transcriptional regulation is considered (162 out of 200 cases). Finally, we present metabolic engineering strategies for producing ethanol and higher alcohols (e.g. isobutanol) in E. coli using our OptORF approach. We have found common genetic modifications such as deletion of pgi and overexpression of edd, as well as chemical specific strategies for producing different alcohols.ConclusionsBy taking regulatory effects into account, OptORF can propose changes such as the overexpression of metabolic genes or deletion of transcriptional factors, in addition to the deletion of metabolic genes, that may lead to faster evolutionary trajectories. While biofuel production in E. coli is evaluated here, the developed OptORF approach is general and can be applied to optimize the production of different compounds in other biological systems.
| S-EPMC2887412 | biostudies-literature
Project description:The two metabolically versatile actinobacteria Rhodococcus opacus PD630 and R. jostii RHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway of d-xylose utilization was introduced into these two strains. This was accomplished by heterogenous expression of two well-selected genes, xylA, encoding xylose isomerase, and xylB, encoding xylulokinase from Streptomyces lividans TK23, under the control of the tac promoter with an Escherichia coli-Rhodococcus shuttle vector. The recombinant R. jostii RHA1 bearing xylA could grow on xylose as the sole carbon source, and additional expression of xylB further improved the biomass yield. The recombinant could consume both glucose and xylose in the sugar mixture, although xylose metabolism was still affected by the presence of glucose. The xylose metabolic pathway was also introduced into the high-lipid-producing strain R. opacus PD630 by expression of xylA and xylB. Under nitrogen-limited conditions, the fatty acid composition was determined, and lipid produced from xylose by recombinants of R. jostii RHA1 and R. opacus PD630 carrying xylA and xylB represented up to 52.5% and 68.3% of the cell dry weight (CDW), respectively. This work demonstrates that it is feasible to produce lipid from the sugars, including xylose, derived from renewable feedstock by genetic modification of rhodococcus strains.
| S-EPMC3406118 | biostudies-literature
Project description:Based on elementary mode analysis, an Escherichia coli strain was designed for efficient conversion of glycerol to ethanol. By using nine gene knockout mutations, the functional space of the central metabolism of E. coli was reduced from over 15,000 possible pathways to a total of 28 glycerol-utilizing pathways that support cell function. Among these pathways are eight aerobic and eight anaerobic pathways that do not support cell growth but convert glycerol into ethanol with a theoretical yield of 0.50 g ethanol/g glycerol. The remaining 12 pathways aerobically coproduce biomass and ethanol from glycerol. The optimal ethanol production depends on the oxygen availability that regulates the two competing pathways for biomass and ethanol production. The coupling between cell growth and ethanol production enabled metabolic evolution of the designed strain through serial dilution that resulted in strains with improved ethanol yields and productivities. In defined medium, the evolved strain can convert 40 g/liter of glycerol to ethanol in 48 h with 90% of the theoretical ethanol yield. The performance of the designed strain is predicted by the property space of remaining elementary modes.
| S-EPMC2772450 | biostudies-literature
Project description:BackgroundThe genus Eubacterium is quite diverse and includes several acetogenic strains capable of fermenting C1-substrates into valuable products. Especially, Eubacterium limosum and closely related strains attract attention not only for their capability to ferment C1 gases and liquids, but also due to their ability to produce butyrate. Apart from its well-elucidated metabolism, E. limosum is also genetically accessible, which makes it an interesting candidate to be an industrial biocatalyst.ResultsIn this study, we examined genomic, phylogenetic, and physiologic features of E. limosum and the closest related species E. callanderi as well as E. maltosivorans. We sequenced the genomes of the six Eubacterium strains 'FD' (DSM 3662T), 'Marburg' (DSM 3468), '2A' (DSM 2593), '11A' (DSM 2594), 'G14' (DSM 107592), and '32' (DSM 20517) and subsequently compared these with previously available genomes of the E. limosum type strain (DSM 20543T) as well as the strains 'B2', 'KIST612', 'YI' (DSM 105863T), and 'SA11'. This comparison revealed a close relationship between all eleven Eubacterium strains, forming three distinct clades: E. limosum, E. callanderi, and E. maltosivorans. Moreover, we identified the gene clusters responsible for methanol utilization as well as genes mediating chain elongation in all analyzed strains. Subsequent growth experiments revealed that strains of all three clades can convert methanol and produce acetate, butyrate, and hexanoate via reverse β-oxidation. Additionally, we used a harmonized electroporation protocol and successfully transformed eight of these Eubacterium strains to enable recombinant plasmid-based expression of the gene encoding the fluorescence-activating and absorption shifting tag (FAST). Engineered Eubacterium strains were verified regarding their FAST-mediated fluorescence at a single-cell level using a flow cytometry approach. Eventually, strains 'FD' (DSM 3662T), '2A' (DSM 2593), '11A' (DSM 2594), and '32' (DSM 20517) were genetically engineered for the first time.ConclusionStrains of E. limosum, E. callanderi, and E. maltosivorans are outstanding candidates as biocatalysts for anaerobic C1-substrate conversion into valuable biocommodities. A large variety of strains is genetically accessible using a harmonized electroporation protocol, and FAST can serve as a reliable fluorescent reporter protein to characterize genetically engineered cells. In total eleven strains have been assigned to distinct clades, providing a clear and updated classification. Thus, the description of respective Eubacterium species has been emended, improved, aligned, and is requested to be implemented in respective databases.
| S-EPMC10795377 | biostudies-literature
Project description:The genetic modification of microorganisms is conducive to the selection of high-yield producers of high-value-added chemicals, but a lack of genetic tools hinders the industrialization of most wild species. Therefore, it is crucial to develop host-independent gene editing tools that can be used for genetic manipulation-deprived strains. The Tn7-like transposon from Scytonema hofmanni has been shown to mediate homologous recombination-independent genomic integration after heterologous expression in Escherichia coli, but the integration efficiency of heterologous sequences larger than 5 kb remains suboptimal. Here, we constructed a versatile Cas12k-based genetic engineering toolkit (C12KGET) that can achieve genomic integration of fragments up to 10 kb in size with up to 100% efficiency in challenging strains. Using C12KGET, we achieved the first example of highly efficient genome editing in Sinorhizobium meliloti, which successfully solved the problem that industrial strains are difficult to genetically modify, and increased vitamin B12 production by 25%. In addition, Cas12k can be directly used for transcriptional regulation of genes with up to 92% efficiency due to its naturally inactivated nuclease domain. The C12KGET established in this study is a versatile and efficient marker-free tool for gene integration as well as transcriptional regulation that can be used for challenging strains with underdeveloped genetic toolkits.
| S-EPMC9410911 | biostudies-literature
Project description:Oleochemical activities (e.g. biodiesel production, fat saponification) generate annually very high amounts of concentrated glycerol-containing waters (called crude glycerol) as the principal residues of these processes. Crude glycerol is an industrial residue the valorization of which attracts remarkable and constantly increasing interest. In the current investigation, biodiesel-derived glycerol was employed as substrate for yeast and fungal strains cultivated under nitrogen-limited conditions in shake flasks. Glucose was employed as reference substrate. Several yeasts (Candida diddensiae, Candida tropicalis, Pichia ciferrii, Williopsis saturnus, Candida boidinii, and Candida oleophila) rapidly assimilated glucose and converted it into ethanol, despite aerobic conditions imposed, and were Crabtree-positive. None of these yeasts produced ethanol during growth on glycerol or accumulated significant quantities of lipid during growth on glucose or glycerol. Only Rhodosporidium toruloides produced notable lipid quantities from glucose and to lesser extent from glycerol. Yarrowia lipolytica LFMB 20 produced citrate ≈58 g/L growing on high-glucose media, while on high-glycerol media ≈42 g/L citrate and ≈18 g/L mannitol. During growth on glucose/glycerol blends, glycerol was assimilated first and thereafter glucose was consumed. Fungi produced higher lipid quantities compared with yeasts. High lipid quantities were produced by Mortierella ramanniana, Mucor sp., and mainly Mortierella isabellina, with glycerol being more adequate for M. ramanniana and glucose for Mucor sp. and M. isabellina. M. isabellina ATHUM 2935 produced lipids of 8.5 g/L, 83.3% w/w in dry cell weight (DCW) and conversion yield per unit of glucose consumed ≈0.25 g/g. The respective values on glycerol were 5.4 g/L, 66.6% w/w in DCW and ≈0.22 g/g. Lipids of all microorganisms were analyzed with regards to their fatty acid composition, and M. isabellina presented the closest similitude with rapeseed oil. Crude lipids produced by this fungus and extracted with chloroform/methanol blend, were composed mostly of triacylglycerols, thus indicating that these solvents are adequate for triacylglycerol extraction.
| S-EPMC6999228 | biostudies-literature
Project description:Background2,3-Butanediol (2,3-BD) can be used as a liquid fuel additive to replace petroleum oil, and as an important platform chemical in the pharmaceutical and plastic industries. Microbial production of 2,3-BD by Bacillus licheniformis presents potential advantages due to its GRAS status, but previous attempts to use this microorganism as a chassis strain resulted in the production of a mix of D-2,3-BD and meso-2,3-BD isomers.ResultsThe aim of this work was to develop an engineered strain of B. licheniformis suited to produce the high titers of the pure meso-2,3-BD isomer. Glycerol dehydrogenase (Gdh) was identified as the catalyst for D-2,3-BD biosynthesis from its precursor acetoin in B. licheniformis. The gdh gene was, therefore, deleted from the wild-type strain WX-02 to inhibit the flux of acetoin to D-2,3-BD biosynthesis. The acoR gene involved in acetoin degradation through AoDH ES was also deleted to provide adequate flux from acetoin towards meso-2,3-BD. By re-directing the carbon flux distribution, the double-deletion mutant WX-02ΔgdhΔacoR produced 28.2 g/L of meso-2,3-BD isomer with >99 % purity. The titer was 50 % higher than that of the wide type. A bench-scale fermentation by the double-deletion mutant was developed to further improve meso-2,3-BD production. In a fed-batch fermentation, meso-2,3-BD titer reached 98.0 g/L with a purity of >99.0 % and a productivity of 0.94 g/L-h.ConclusionsThis work demonstrates the potential of producing meso-2,3-BD with high titer and purity through metabolic engineering of B. licheniformis.
| S-EPMC4890260 | biostudies-literature