Drought stress is one of the major environmental factors affecting crop growth and productivity.Cuticular wax plays essential roles in protecting plants from environmental stress via forming a hydrophobic barrier on l...Drought stress is one of the major environmental factors affecting crop growth and productivity.Cuticular wax plays essential roles in protecting plants from environmental stress via forming a hydrophobic barrier on leaf epidermis.In this study,we analyzed nine members(OsCUT1‒OsCUT9)ofβ-ketoacyl-CoA synthase,the rate-limiting key enzyme for cuticular wax synthesis in rice by homology search and domain prediction.The expression levels of OsCUT genes under different abiotic stresses were investigated and OsCUT1 down-regulated by abiotic stress was selected for further function validation.Compared to the wild type,overexpression of OsCUT1(OX-OsCUT1)exhibited significantly increased drought resistance.Epicuticular wax was increased on the leaf surface of OX-OsCUT1 and the chlorophyll leaching experiment showed that the cuticular permeability was decreased in the OX-OsCUT1 plants.Moreover,overexpression of OsCUT1 didn’t result in the significant changes of major agronomic traits.In total,these results suggested that OsCUT1 is a promising gene for engineering rice plants with enhanced drought tolerance.展开更多
Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA...Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.展开更多
The 3-ketoacyl-CoA thiolase is the rate-limiting enzyme for linear dicarboxylic acids production.However,the promiscuous substrate specificity and suboptimal catalytic performance have restricted its application.Here ...The 3-ketoacyl-CoA thiolase is the rate-limiting enzyme for linear dicarboxylic acids production.However,the promiscuous substrate specificity and suboptimal catalytic performance have restricted its application.Here we present both biochemical and structural analyses of a high-efficiency 3-ketoacyl-CoA thiolase Tfu_0875.Notably,Tfu_0875 displayed heightened activity and substrate specificity for succinyl-CoA,a key precursor in adipic acid production.To enhance its performance,a deep learning approach(DLKcat)was employed to identify effective mutants,and a computational strategy,known as the greedy accumulated strategy for protein engineering(GRAPE),was used to accumulate these effective mutants.Among the mutants,Tfu_0875N249W/L163H/E217L exhibited the highest specific activity(320%of wild-type Tfu_0875),the greatest catalytic efficiency(kcat/KM=1.00 min-1mM-1),the highest succinyl-CoA specificity(KM=0.59 mM,28.1%of Tfu_0875)and dramatically reduced substrate binding energy(-30.25 kcal mol^(-1)v.s.-15.94 kcal mol^(-1)).A structural comparison between Tfu_0875N249W/L163H/E217L and the wild type Tfu_0875 revealed that the increased interaction between the enzyme and succinyl-CoA was the primary reason for the enhanced enzyme activity.This interaction facilitated rapid substrate anchoring and stabilization.Furthermore,a reduced binding pocket volume improved substrate specificity by enhancing the complementarity between the binding pocket and the substrate in stereo conformation.Finally,our rationally designed mutant,Tfu_0875N249W/L163H/E217L,increased the adipic acid titer by 1.35-fold compared to the wild type Tfu_0875 in shake flask.The demonstrated enzymatic methods provide a promising enzyme variant for the adipic acid production.The above effective substrate binding pocket engineering strategy can be beneficial for the production of other industrially competitive biobased chemicals when be applied to other thiolases.展开更多
Production of b-ketoacyl-Co A, which is catalyzed by 3-ketoacyl-CoA synthase（KCS）, is the first step in very long chain fatty acid（VLCFA） biosynthesis. Here we identified 58 KCS genes from Gossypium hirsutum, 31 f...Production of b-ketoacyl-Co A, which is catalyzed by 3-ketoacyl-CoA synthase（KCS）, is the first step in very long chain fatty acid（VLCFA） biosynthesis. Here we identified 58 KCS genes from Gossypium hirsutum, 31 from G. arboreum and 33 from G. raimondii by searching the assembled cotton genomes. The gene family was divided into the plant-specific FAE1-type and the more general ELO-type. KCS transcripts were widely expressed and 32 of them showed distinct subgenome-specific expressions in one or more cotton tissues/organs studied. Six Gh KCS genes rescued the lethality of elo2Δelo3Δ yeast double mutant,indicating that this gene family possesses diversified functions.Most KCS genes with GA-responsive elements（GAREs） in the promoters were significantly upregulated by gibberellin A_3（GA）.Exogenous GA_3 not only promoted fiber length, but also increased the thickness of cell walls significantly. GAREs present also in the promoters of several cellulose synthase（CesA） genes required for cell wall biosynthesis and they were all induced significantly by GA_3. Because GA treatment resulted in longer cotton fibers with thicker cell walls and higher dry weight per unit cell length, we suggest that it may regulate fiber elongation upstream of the VLCFA-ethylene pathway and also in the downstream steps towards cell wall synthesis.展开更多
基金the National Natural Science Foundation of China(Grant Nos.32071918 and 32000227).
文摘Drought stress is one of the major environmental factors affecting crop growth and productivity.Cuticular wax plays essential roles in protecting plants from environmental stress via forming a hydrophobic barrier on leaf epidermis.In this study,we analyzed nine members(OsCUT1‒OsCUT9)ofβ-ketoacyl-CoA synthase,the rate-limiting key enzyme for cuticular wax synthesis in rice by homology search and domain prediction.The expression levels of OsCUT genes under different abiotic stresses were investigated and OsCUT1 down-regulated by abiotic stress was selected for further function validation.Compared to the wild type,overexpression of OsCUT1(OX-OsCUT1)exhibited significantly increased drought resistance.Epicuticular wax was increased on the leaf surface of OX-OsCUT1 and the chlorophyll leaching experiment showed that the cuticular permeability was decreased in the OX-OsCUT1 plants.Moreover,overexpression of OsCUT1 didn’t result in the significant changes of major agronomic traits.In total,these results suggested that OsCUT1 is a promising gene for engineering rice plants with enhanced drought tolerance.
基金supported by grants from China Na-tional Basic Research Program (NO. 2004CB117302)National Natural Science Foundation of China (No.30470171)the Sigrid Jusélius Foundation Finland and the Academy of Finland
文摘Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulatedduring early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoAreductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed duringthe cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAswere cloned and expressed in yeast haploid ybr159w? mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w? cells that expressed either GhKCR1 or 2. Further analysis showed thatGhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to theyeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductaseactivity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest thatGhKCR1 and 2 are functional orthologues of ScYbr159p.
基金supported by the National Key R&D Program of China(2022YFC2104600)National Natural Science Foundation of China(22378170)+1 种基金the Distinguished Young Scholars of Jiangsu Province(BK20220089)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-015).
文摘The 3-ketoacyl-CoA thiolase is the rate-limiting enzyme for linear dicarboxylic acids production.However,the promiscuous substrate specificity and suboptimal catalytic performance have restricted its application.Here we present both biochemical and structural analyses of a high-efficiency 3-ketoacyl-CoA thiolase Tfu_0875.Notably,Tfu_0875 displayed heightened activity and substrate specificity for succinyl-CoA,a key precursor in adipic acid production.To enhance its performance,a deep learning approach(DLKcat)was employed to identify effective mutants,and a computational strategy,known as the greedy accumulated strategy for protein engineering(GRAPE),was used to accumulate these effective mutants.Among the mutants,Tfu_0875N249W/L163H/E217L exhibited the highest specific activity(320%of wild-type Tfu_0875),the greatest catalytic efficiency(kcat/KM=1.00 min-1mM-1),the highest succinyl-CoA specificity(KM=0.59 mM,28.1%of Tfu_0875)and dramatically reduced substrate binding energy(-30.25 kcal mol^(-1)v.s.-15.94 kcal mol^(-1)).A structural comparison between Tfu_0875N249W/L163H/E217L and the wild type Tfu_0875 revealed that the increased interaction between the enzyme and succinyl-CoA was the primary reason for the enhanced enzyme activity.This interaction facilitated rapid substrate anchoring and stabilization.Furthermore,a reduced binding pocket volume improved substrate specificity by enhancing the complementarity between the binding pocket and the substrate in stereo conformation.Finally,our rationally designed mutant,Tfu_0875N249W/L163H/E217L,increased the adipic acid titer by 1.35-fold compared to the wild type Tfu_0875 in shake flask.The demonstrated enzymatic methods provide a promising enzyme variant for the adipic acid production.The above effective substrate binding pocket engineering strategy can be beneficial for the production of other industrially competitive biobased chemicals when be applied to other thiolases.
基金supported by grants from the China National Basic Research Program (2010CB126000)the National Natural Science Foundation of China (90717009)
文摘Production of b-ketoacyl-Co A, which is catalyzed by 3-ketoacyl-CoA synthase（KCS）, is the first step in very long chain fatty acid（VLCFA） biosynthesis. Here we identified 58 KCS genes from Gossypium hirsutum, 31 from G. arboreum and 33 from G. raimondii by searching the assembled cotton genomes. The gene family was divided into the plant-specific FAE1-type and the more general ELO-type. KCS transcripts were widely expressed and 32 of them showed distinct subgenome-specific expressions in one or more cotton tissues/organs studied. Six Gh KCS genes rescued the lethality of elo2Δelo3Δ yeast double mutant,indicating that this gene family possesses diversified functions.Most KCS genes with GA-responsive elements（GAREs） in the promoters were significantly upregulated by gibberellin A_3（GA）.Exogenous GA_3 not only promoted fiber length, but also increased the thickness of cell walls significantly. GAREs present also in the promoters of several cellulose synthase（CesA） genes required for cell wall biosynthesis and they were all induced significantly by GA_3. Because GA treatment resulted in longer cotton fibers with thicker cell walls and higher dry weight per unit cell length, we suggest that it may regulate fiber elongation upstream of the VLCFA-ethylene pathway and also in the downstream steps towards cell wall synthesis.
