Periodic Reporting for period 1 - RG-II (Development and exploitation of new molecular tools to understand rhamnogalacturonan-II metabolism in plants)
Reporting period: 2022-07-01 to 2024-06-30
Most plants cells have a wall protecting them from environmental stress and diseases caused by pathogens. The cell wall is made up of a network of complex carbohydrates including rhamnogalacturonan II (RG-II), a major component of pectin. RG-II is the most complex carbohydrate known and has been shown to play a critical role in plant growth, development, and protection from environmental stresses. Mutations altering the structure and composition of RG-II for example have been shown to lead to strong developmental phenotypes or lethality and decreased crop yields. RG-II components have been shown to be targeted by plant pathogens responsible for significant crop losses in agriculture and recently in the human diet by beneficial species of the human gut microbiota, a key player in host-microbial interactions. Despite its importance and potential, we know very little about RG-II metabolism and function, largely due to its complex nature and lack of appropriate tools. This project aims to generate and exploit new molecular tools to understand the biosynthesis of RG-II in plants. The specific objectives include a) Generation and expansion of RG-II oligosaccharides and RGII-producing microbial libraries b) development and validation of high-throughput RG-II glycan arrays c) screening of plant encoded proteins for RG-II glycosyltransferase activity. Data from this work will not only enhance our understanding of RG-II metabolism but also facilitate research in other aspects of RG-II biology and application including its role in plant growth, development and protection from environmental stress as well as its potential as a next generation prebiotic to improve human or animal health.
RG-II purification and analyses: RG-II was purified from wine (w1RGII) and analysed alongside a previously purified wine RGII sample (w2RGII) and apple juice RG-II (aRGII) by agarose gel electrophoresis (ref), thin layer chromatography (TLC) of RG-II degradation products after growth with the RG-II utilising bacterium B. thetaiotaomicron and mass spectrometry (MS) using FITDOG-MS approach (for w1RGII and w2RGII) (ref). RG-II from all sources shared many features based on staining, migration properties and degradation products. FITDOG-MS analyses however revealed a small amount of mass peaks likely representing unique modifications in w1RGII and w2RGII. w2RGII degradation products including meXyl-Fuc and meFuc also showed slightly weaker staining compared to w1RGII and aRGII. aRGII was used for subsequent experiments based on availability and more clarity around its composition from the current and previous studies.
Expanding the RG-II oligosaccharide (RGDO) producing library: Several genetically modified B. thetaiotaomicron mutants capable of generating and secreting RGDOs had been produced from previous studies through the deletion of specific RG-II degrading enzymes (by allelic exchange (Ndeh et al., 2017)) especially those targeting side chain A. To expand the RGDO library, mostly side-chain B enzymes were targeted including the L-arabinofuranosidase/glucuronidase enzyme BT0996, methylfocusidase BT0984, rhamnosidase BT1019 and L-arabinopyranosidase BT0983. An apiosidase mutant ΔBT1012 had been generated earlier but its RGDO products had not been characterised. Cloning experiments to generate recombinant knockout plasmid constructs for allelic exchange experiments were successful for all genes except BT0983 which failed. Conjugation experiments were only successful for BT0984 after several attempts and trialling different stocks of the conjugating strain E. coli s17 lambda phage. The BT0984 knockout construct was finally conjugated into B. thetaiotaomicron wild type and ΔBT01012 strains yielding two new B. thetaiotaomicron deletion mutant strains namely ΔBT0984 and ΔBT0984/BT1012.
Generating and characterising RGDOs: To generate RGDOs, several RDGO generating B. thetaiotaomicron mutant strains including ΔBT0984, ΔBT0984/BT1012, ΔBT1012, ΔBT0986, ΔBT0997, ΔBT1003, ΔBT1010, were independently cultured with aRGII and product profiles analysed by TLC to identify new RDGOs. RDGOs were successfully detected for all strains compared to wild type B. thetaiotaomicron. ΔBT0984, ΔBT0984/BT1012 and ΔBT1012 strains also produced more than one unique RDGO each. At least one from each of the latter mutants was successfully purified by size exclusion chromatography on a Biogel P2 resin. Atleast seven sugars were purified including ΔBT0984oligoI, ΔBT0984oligoII, ΔBT0984/BT1012oligoI, ΔBT0984/BT1012oligoII, ΔBT1012, ΔBT0997 and ΔBT0986, and subjected to mass spectrometry (MALDI TOF). Analyses of the MS data has so far detected mass peaks for the new sugar ΔBT0984oligoI and the previously characterised sugar ΔBT0997oligo. NMR studies on BT0984oligo has successfully confirmed anomeric H1 signals for various monosaccharide components and the structure of the sugar.
Expanding the RG-II oligosaccharide (RGDO) producing library: Several genetically modified B. thetaiotaomicron mutants capable of generating and secreting RGDOs had been produced from previous studies through the deletion of specific RG-II degrading enzymes (by allelic exchange (Ndeh et al., 2017)) especially those targeting side chain A. To expand the RGDO library, mostly side-chain B enzymes were targeted including the L-arabinofuranosidase/glucuronidase enzyme BT0996, methylfocusidase BT0984, rhamnosidase BT1019 and L-arabinopyranosidase BT0983. An apiosidase mutant ΔBT1012 had been generated earlier but its RGDO products had not been characterised. Cloning experiments to generate recombinant knockout plasmid constructs for allelic exchange experiments were successful for all genes except BT0983 which failed. Conjugation experiments were only successful for BT0984 after several attempts and trialling different stocks of the conjugating strain E. coli s17 lambda phage. The BT0984 knockout construct was finally conjugated into B. thetaiotaomicron wild type and ΔBT01012 strains yielding two new B. thetaiotaomicron deletion mutant strains namely ΔBT0984 and ΔBT0984/BT1012.
Generating and characterising RGDOs: To generate RGDOs, several RDGO generating B. thetaiotaomicron mutant strains including ΔBT0984, ΔBT0984/BT1012, ΔBT1012, ΔBT0986, ΔBT0997, ΔBT1003, ΔBT1010, were independently cultured with aRGII and product profiles analysed by TLC to identify new RDGOs. RDGOs were successfully detected for all strains compared to wild type B. thetaiotaomicron. ΔBT0984, ΔBT0984/BT1012 and ΔBT1012 strains also produced more than one unique RDGO each. At least one from each of the latter mutants was successfully purified by size exclusion chromatography on a Biogel P2 resin. Atleast seven sugars were purified including ΔBT0984oligoI, ΔBT0984oligoII, ΔBT0984/BT1012oligoI, ΔBT0984/BT1012oligoII, ΔBT1012, ΔBT0997 and ΔBT0986, and subjected to mass spectrometry (MALDI TOF). Analyses of the MS data has so far detected mass peaks for the new sugar ΔBT0984oligoI and the previously characterised sugar ΔBT0997oligo. NMR studies on BT0984oligo has successfully confirmed anomeric H1 signals for various monosaccharide components and the structure of the sugar.
We have generated for the first time a genetic strain capable of producing scalable quantities of the side chain B sugars ΔBT0984oligoI and ΔBT0984/BT1012oligoI sugars and carried out their purification. The NMR structure of ΔBT0984oligo is also reported. We have also reported the purification of ΔBT1012oligoI and partial purification of ΔBT1012oligoII. These results set a stage for the production of various side chain B sugars linked to the backbone of GalA units which are rare.
Our work contributes solutions to a major bottleneck in glycoscience research which is the limited availability of chemically defined glycan oligosaccharides. This is particularly a major problem for highly complex glycans such as RG-II. Generating and expanding the range of RDGOs opens up opportunities for further research to better understand the structure, function and metabolism of this complex and enigmatic plant cell wall sugar for which very little is known about.
Our work contributes solutions to a major bottleneck in glycoscience research which is the limited availability of chemically defined glycan oligosaccharides. This is particularly a major problem for highly complex glycans such as RG-II. Generating and expanding the range of RDGOs opens up opportunities for further research to better understand the structure, function and metabolism of this complex and enigmatic plant cell wall sugar for which very little is known about.