ABSTRACT
Degradation of xylan requires several enzymes. Two chimeric enzymes, xyln-ara and xyln-xylo, were constructed by linking the catalytic portion of a xylanase (xyln) to either an arabinofuranosidase (ara) or a xylosidase (xylo) with a flexible peptide linker. The recombinant parental enzymes and chimeras were produced in E. coli at high levels and purified for characterization of their enzymatic and kinetic properties as well as activities on natural substrates. The chimeras closely resemble the parental enzymes or their mixtures with regard to protein properties. They share similar temperature profiles and have similar catalytic efficiencies as the parental enzymes when assayed using substrates 4-nitrophenyl-alpha-L-arabinofuranoside or 2-nitrophenyl- beta-D-xylopyranoside. The chimeras also show unique enzymatic characteristics. In xylanase activity assays using Remazol Brilliant Blue-xylan, while the parental xylanase has a pH optimum of pH 8, the chimeras showed shifted pH optima as a consequence of significantly increased activity at pH 6 (the optimal pH for ara and xylo). Both chimeras exhibited additive effects of the parental enzymes when assayed at wide ranges of pH and temperatures. The xyln-xylo chimera had the same activities as the xyln/xylo mixture in hydrolyzing the natural substrates oat spelt xylan and wheat arabinoxylan. Compared to the xyln/ara mixture, the xyln-ara chimera released the same amounts of xylose from oat spelt xylan and approximately 30% more from wheat arabinoxylan at pH 6. Our results demonstrate the feasibility and advantages of generating bifunctional enzymes for the improvement of xylan bioconversion.
Subject(s)
Glycoside Hydrolases/metabolism , Xylans/metabolism , Xylosidases/metabolism , Base Sequence , Biomass , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Glycoside Hydrolases/genetics , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Temperature , Xylosidases/geneticsABSTRACT
Myc-RP from Perilla frutescens and Delila from Antirrhinum majus, two plant basic helix-loop-helix transcription factors (bHLH TFs) involved in the flavonoid biosynthetic pathway, have been used for the improvement of transactivational properties by directed evolution. Through two rounds of DNA shuffling, Myc-RP variants with up to 70-fold increase in transcriptional activities have been identified using a yeast transactivation system. In a tobacco protoplast transient expression assay, one of the most improved variants, M2-1, also shows significant increase of transactivation. The majority of resulting mutations (approximately 53%) are localized in the acidic (activation) domains of the improved Myc-RP variants. In variant M2-1, three of the four mutations (L301P/N354D/S401F) are in the acidic domain. The fourth mutation (K157M) is localized to a helix within the N-terminal interaction domain. Combinatorial site-directed mutagenesis reveals that, while the acidic domain mutations contribute modestly to the increase in activity, the K157M substitution is responsible for 80% of the improvement observed in variant M2-1. The transactivation activity of the K157M/N354D double mutant is equal to that of M2-1. These results suggest that the interaction domain plays a critical role in transactivation of these bHLH TFs. Delila variants have also been screened for increased activities toward the Arabidopsis chalcone synthase (CHS) promoter, a pathway promoter that responds weakly to the bHLH TFs. Variants with increased activity on the CHS promoter, while maintaining wildtype-level activities on the naturally responsive dihydroflavonol reductase promoter, have been obtained. This study demonstrates that functional properties of TFs can be modified by directed evolution.
Subject(s)
Directed Molecular Evolution/methods , Plant Proteins/genetics , Plant Proteins/metabolism , Plants/genetics , Plants/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Acyltransferases/genetics , Amino Acid Sequence , Antirrhinum/genetics , Antirrhinum/metabolism , Arabidopsis/genetics , Base Sequence , DNA, Plant/genetics , Genes, Plant , Helix-Loop-Helix Motifs/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Perilla frutescens/genetics , Perilla frutescens/metabolism , Plant Proteins/chemistry , Plants, Genetically Modified , Promoter Regions, Genetic , Sequence Homology, Amino Acid , Nicotiana/genetics , Nicotiana/metabolism , Transcription Factors/chemistry , Transcriptional Activation , Two-Hybrid System TechniquesABSTRACT
The major cluster of resistance genes in lettuce cv. Diana contains approximately 32 nucleotide binding site-leucine-rich repeat encoding genes. Previous molecular dissection of this complex region had identified a large gene, RGC2B, as a candidate for encoding the downy mildew resistance gene, Dm3. This article describes genetic and transgenic complementation data that demonstrated RGC2B is necessary and sufficient to confer resistance with Dm3 specificity. Ethylmethanesulphonate was used to induce mutations to downy mildew susceptibility in cv. Diana (Dm1, Dm3, Dm7, and Dm8). Nineteen families were identified with a complete loss of resistance in one of the four resistance specificities. Sequencing revealed a variety of point mutations in RGC2B in the six dm3 mutants. Losses of resistance were due to single changes in amino acid sequence or a change in an intron splice site. These mutations did not cluster in any particular region of RGC2B. A full-length genomic copy of RGC2B was isolated from a lambdaphage library and introduced into two genotypes of lettuce. Transgenics expressing RGC2B exhibited resistance to all isolates expressing Avr3 from a wide range of geographical origins. In a wildtype Dm3-expressing genotype, many of the RGC2 family members are expressed at low levels throughout the plant.