“…A dramatic increase in coniferyl alcohol accumulation was recorded as the temperature increased to 30 °C, and then it decreased. This finding is consistent with a previous report demonstrating that the optimal temperature for CAD catalysis was 30 °C [35]. Consequently, pH 7.0 and 30 °C were chosen as the best parameters under the experimental conditions, and this value was used in the following experiments.Fig.…”
Section: Resultssupporting
confidence: 90%
“…In this study, we developed a novel, fast and highly efficient biological technique to convert diverse phenylpropanoic acids to their corresponding 4 - hydroxycinnamyl alcohols using immobilized whole cells of recombinant E. coli as the biocatalyst, along with the recombinant E. coli strain M15–4CL1–CCR and the recombinant E. coli strain M15–CAD expressing CAD from Populus tomentosa ( P. tomentosa ) [34, 35]. The goals of this study were: (1) to establish a rapid HPLC–PDA–ESI–MSn method for the characterization of 4 - hydroxycinnamyl alcohols; (2) to explore the feasibility of using immobilized whole cells of two recombinant E. coli to catalyze the conversion; (3) to investigate the optimum buffer pH and reaction temperature to improve the production; and (4) to evaluate the productivity of this novel biosynthesis system.…”
Background4-Hydroxycinnamyl alcohols are a class of natural plant secondary metabolites that include p-coumaryl alcohol, caffeyl alcohol, coniferyl alcohol and sinapyl alcohol, and have physiological, ecological and biomedical significance. While it is necessary to investigate the biological pathways and economic value of these alcohols, research is hindered because of their limited availability and high cost. Traditionally, these alcohols are obtained by chemical synthesis and plant extraction. However, synthesis by biotransformation with immobilized microorganisms is of great interest because it is environmentally friendly and offers high stability and regenerable cofactors. Therefore, we produced 4-hydroxycinnamyl alcohols using immobilized whole cells of engineered Escherichia coli as the biocatalyst.ResultsIn this study, we used the recombinant E. coli strain, M15–4CL1–CCR, expressing the fusion protein 4-coumaric acid: coenzyme A ligase and the cinnamoyl coenzyme A reductase and a recombinant E. coli strain, M15–CAD, expressing cinnamyl alcohol dehydrogenase from Populus tomentosa (P. tomentosa). High performance liquid chromatography and mass spectrometry showed that the immobilized whole cells of the two recombinant E. coli strains could effectively convert the phenylpropanoic acids to their corresponding 4-hydroxycinnamyl alcohols. Further, the optimum buffer pH and the reaction temperature were pH 7.0 and 30 °C. Under these conditions, the molar yield of the p-coumaryl alcohol, the caffeyl alcohol and the coniferyl alcohol was around 58, 24 and 60%, respectively. Moreover, the highly sensitive and selective HPLC–PDA–ESI–MSn method used in this study could be applied to the identification and quantification of these aromatic polymers.ConclusionsWe have developed a dual-cell immobilization system for the production of 4-hydroxycinnamyl alcohols from inexpensive phenylpropanoic acids. This biotransformation method is both simple and environmental-friendly, which is promising for the practical and cost effective synthesis of natural products.Graphical abstractBiotransformation process of phenylpropanoic acids by immobilized whole-cells
“…A dramatic increase in coniferyl alcohol accumulation was recorded as the temperature increased to 30 °C, and then it decreased. This finding is consistent with a previous report demonstrating that the optimal temperature for CAD catalysis was 30 °C [35]. Consequently, pH 7.0 and 30 °C were chosen as the best parameters under the experimental conditions, and this value was used in the following experiments.Fig.…”
Section: Resultssupporting
confidence: 90%
“…In this study, we developed a novel, fast and highly efficient biological technique to convert diverse phenylpropanoic acids to their corresponding 4 - hydroxycinnamyl alcohols using immobilized whole cells of recombinant E. coli as the biocatalyst, along with the recombinant E. coli strain M15–4CL1–CCR and the recombinant E. coli strain M15–CAD expressing CAD from Populus tomentosa ( P. tomentosa ) [34, 35]. The goals of this study were: (1) to establish a rapid HPLC–PDA–ESI–MSn method for the characterization of 4 - hydroxycinnamyl alcohols; (2) to explore the feasibility of using immobilized whole cells of two recombinant E. coli to catalyze the conversion; (3) to investigate the optimum buffer pH and reaction temperature to improve the production; and (4) to evaluate the productivity of this novel biosynthesis system.…”
Background4-Hydroxycinnamyl alcohols are a class of natural plant secondary metabolites that include p-coumaryl alcohol, caffeyl alcohol, coniferyl alcohol and sinapyl alcohol, and have physiological, ecological and biomedical significance. While it is necessary to investigate the biological pathways and economic value of these alcohols, research is hindered because of their limited availability and high cost. Traditionally, these alcohols are obtained by chemical synthesis and plant extraction. However, synthesis by biotransformation with immobilized microorganisms is of great interest because it is environmentally friendly and offers high stability and regenerable cofactors. Therefore, we produced 4-hydroxycinnamyl alcohols using immobilized whole cells of engineered Escherichia coli as the biocatalyst.ResultsIn this study, we used the recombinant E. coli strain, M15–4CL1–CCR, expressing the fusion protein 4-coumaric acid: coenzyme A ligase and the cinnamoyl coenzyme A reductase and a recombinant E. coli strain, M15–CAD, expressing cinnamyl alcohol dehydrogenase from Populus tomentosa (P. tomentosa). High performance liquid chromatography and mass spectrometry showed that the immobilized whole cells of the two recombinant E. coli strains could effectively convert the phenylpropanoic acids to their corresponding 4-hydroxycinnamyl alcohols. Further, the optimum buffer pH and the reaction temperature were pH 7.0 and 30 °C. Under these conditions, the molar yield of the p-coumaryl alcohol, the caffeyl alcohol and the coniferyl alcohol was around 58, 24 and 60%, respectively. Moreover, the highly sensitive and selective HPLC–PDA–ESI–MSn method used in this study could be applied to the identification and quantification of these aromatic polymers.ConclusionsWe have developed a dual-cell immobilization system for the production of 4-hydroxycinnamyl alcohols from inexpensive phenylpropanoic acids. This biotransformation method is both simple and environmental-friendly, which is promising for the practical and cost effective synthesis of natural products.Graphical abstractBiotransformation process of phenylpropanoic acids by immobilized whole-cells
“…Functional characterization has revealed variability of CADs in terms of substrate acceptance, with enzymes of gymnosperms showing specificity towards coniferaldehyde or a P. tremuloides CAD selectively targeting sinapaldehyde (SAD, Li et al 2001). While most investigated CADs exhibit high promiscuity among phenylpropenyl aldehyde derivatives, some CAD-like enzymes show no activity whatsoever (Chao et al 2014). Hence, divergent roles of the dehydrogenase family members, played in defense or in response to stress, have been postulated (Kim et al 2004).…”
Section: Discussionmentioning
confidence: 97%
“…It seems likely that VR2 has evolved in course of a gene duplication process within the CAD family, and a change in function recruited this paralog from the more basic involvement in lignification towards a defense-related capacity within MIA biosynthesis. Studies in genera like Arabidopsis (Kim et al 2004) or Populus (Chao et al 2014) have indicated that among the identified CAD enzymes, a few do not convert phenylpropenyl aldehydes and, therefore, might have acquired different functions as well.…”
Section: Discussionmentioning
confidence: 98%
“…CADs are involved in monolignol biosynthesis and are, therefore, essential in lignification processes and wood development (Lewis and Yamamoto 1990). Previous research on various species has shown that CADs are encoded by a multigene family, with numerous isoenzymes frequently present in a given representative of the plant kingdom (Kim et al 2004;Guo et al 2010;Chao et al 2014). Functional characterization has revealed variability of CADs in terms of substrate acceptance, with enzymes of gymnosperms showing specificity towards coniferaldehyde or a P. tremuloides CAD selectively targeting sinapaldehyde (SAD, Li et al 2001).…”
Based on findings described herein, we contend that the reduction of vomilenine en route to antiarrhythmic ajmaline in planta might proceed via an alternative, novel sequence of biosynthetic steps. In the genus Rauvolfia, monoterpenoid indole alkaloids (MIAs) are formed via complex biosynthetic sequences. Despite the wealth of information about the biochemistry and molecular genetics underlying these processes, many reaction steps involving oxygenases and oxidoreductases are still elusive. Here, we describe molecular cloning and characterization of three cinnamyl alcohol dehydrogenase (CAD)-like reductases from Rauvolfia serpentina cell culture and R. tetraphylla roots. Functional analysis of the recombinant proteins, with a set of MIAs as potential substrates, led to identification of one of the enzymes as a CAD, putatively involved in lignin formation. The two remaining reductases comprise isoenzymes derived from orthologous genes of the investigated alternative Rauvolfia species. Their catalytic activity consists of specific conversion of vomilenine to 19,20-dihydrovomilenine, thus proving their exclusive involvement in MIA biosynthesis. The obtained data suggest the existence of a previously unknown bypass in the biosynthetic route to ajmaline further expanding structural diversity within the MIA family of specialized plant metabolites.
Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Populus tomentosa were cloned and studied and active sites in CCRs were further identified based on sequence divergence, molecular simulation, and site-directed mutants. Cinnamoyl-coenzyme A (CoA) reductase (CCR) is the first committed gene in the lignin-specific pathway and plays a role in the lignin biosynthesis pathway. In this study, we cloned 11 genes encoding CCR or CCR-like proteins in Populus tomentosa. An enzymatic assay of the purified recombinant P. tomentosa (Pto) CCR and PtoCCR-like proteins indicated that only PtoCCR1 and PtoCCR7 had detectable activities toward hydroxycinnamoyl-CoA esters. PtoCCR1 exhibited specificity for feruloyl-CoA, with no detectable activity for any other hydroxycinnamoyl-CoA esters. However, PtoCCR7 catalyzed p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA with a preference for feruloyl-CoA. Site-directed mutations of selected amino acids divergent between PtoCCR1 and 7, combined with modeling and docking, showed that A132 in CCR7 combined with the catalytic triad might comprise the catalytic center. In CCR7, L192, F155, and H208 were identified as the substrate-binding sites, and site-directed mutations of these amino acids showed obvious changes in catalytic efficiency with respect to both feruloyl-CoA and sinapoyl-CoA. Mutant F155Y exhibited greater catalytic efficiency for sinapoyl-CoA compared with that of wild-type PtoCCR7. Finally, recent genome duplication events provided the foundation for CCR divergence. This study further identified the active sites in CCRs and the evolutionary process of CCRs in terrestrial plants.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
