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Liu S.,Beijing Forestry University | Qi Q.,Beijing Forestry University | Chao N.,Beijing Forestry University | Hou J.,Beijing Forestry University | And 9 more authors.
Microbial Cell Factories | Year: 2015

Background: 4-Hydroxycinnamaldehydes are important intermediates in several secondary metabolism pathways, including those involved in the biosynthesis of phenolic acids, flavonoids, terpenoids and monolignols. They are also involved in the biosynthesis and degradation of lignins, which are important limiting factors during the processes of papermaking and biofuel production. Access to these aromatic polymers is necessary to explore the secondary biometabolic pathways they are involved in. Coniferaldehyde, sinapaldehyde, p-coumaraldehyde and caffealdehyde are members of the 4-hydroxycinnamaldehyde family. Although coniferaldehyde and sinapaldehyde can be purchased from commercial sources, p-coumaraldehyde and caffealdehyde are not commercially available. Therefore, there is increasing interest in producing 4-hydroxycinnamaldehydes. Here, we attempted to produce 4-hydroxycinnamaldehydes using engineered Escherichia coli. Results: 4-Coumaric acid: coenzyme A ligase (4CL1) and cinnamoyl coenzyme A reductase (CCR) were fused by means of genetic engineering to generate an artificial bifunctional enzyme, 4CL1-CCR, which was overexpressed in cultured E. coli supplemented with phenylpropanoic acids. Three 4-hydroxycinnamaldehydes, p-coumaraldehyde, caffealdehyde and coniferaldehyde, were thereby biosynthesized and secreted into the culture medium. The products were extracted and purified from the culture medium, and identically characterized by the HPLC-PDA-ESI-MSn. The productivity of this new metabolic system were 49 mg/L for p-coumaraldehyde, 19 mg/L for caffealdehyde and 35 mg/L for coniferaldehyde. Extracellular hydroxycinnamoyl-coenzyme A thioesters were not detected, indicating that these thioesters could not pass freely through the cellular membrane. The fusion enzyme 4CL1-CCR can catalyze sequential multistep reactions, thereby avoiding the permeability problem of intermediates, which reveals its superiority over a mixture of individual native enzymes. Moreover, we have described a highly sensitive and selective method for separation and identification of phenylpropanoic acids and their corresponding cinnamaldehydes in the present paper. The feasibility of this method has been proven in the application of the method to the analysis of the metabolites of whole-cell catalysts. Conclusions: We have established a bioconversion pathway for the microbial production of valuable 4-hydroxycinnamaldehydes from phenylpropanoic acids. This biotransformation method is both convenient and environmentally friendly, and provides new insights into the biosynthesis of natural plant secondary products. © 2015 Liu et al.

Xie J.,Beijing Forestry University | Liu S.,Beijing Forestry University | Qi Q.,Beijing Forestry University | Hou J.,Beijing Forestry University | And 4 more authors.
Pakistan Journal of Botany | Year: 2014

Various protein extraction methods have been used to investigate Chinese white poplar (Populus tomentosa) proteomics. However, extracting and characterizing proteins from woody plants remains a challenge. Two-dimensional gel electrophoresis is a powerful, widely used method for the analysis of complex protein mixtures extracted from biological samples. The technique separates mixtures of proteins along two dimensions, by isoelectric point and molecular weight, and can resolve thousands of different proteins. Here, we report a new application of two-dimensional gel electrophoresis to investigate the proteomics of P. tomentosa cambium tissues over the course of a growing season. Of three protein extraction methods that we compared (the Tris-phenol method, trichloroacetic acid-acetone method, and trichloroacetic acid-acetonephenol method), trichloroacetic acid-acetone was the most efficient approach for protein extraction from cambium tissues of P. tomentosa. After extraction, the proteins were separated using two-dimensional gel electrophoresis. The protein quantities of six spots changed over the course of the growing season from February to July. Five spots were identified using matrixassisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry, and the sixth spot was identified by liquid chromatography-mass spectrometry. The proteins included enolase, class Ia chitinase, and four unnamed proteins. Our results show the best approach to proteomics in P. tomentosa and reveal trends in protein activities during a growing season in this tree species.

Chao N.,Beijing Forestry University | Li N.,Beijing Forestry University | Qi Q.,Beijing Forestry University | Li S.,Beijing Forestry University | And 5 more authors.
Planta | Year: 2016

Main conclusion: Two distinct cinnamoyl-coenzyme A reductases (CCRs) fromPopulus tomentosawere 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. © 2016 Springer-Verlag Berlin Heidelberg

Chao N.,Beijing Forestry University | Liu S.-X.,Beijing Forestry University | Liu B.-M.,Beijing Forestry University | Li N.,Beijing Forestry University | And 5 more authors.
Planta | Year: 2014

Main conclusion: Nine CAD/CAD-like genes inP. tomentosawere classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD.Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa. © 2014, Springer-Verlag Berlin Heidelberg.

Han B.,Beijing Forestry University | Liu S.,Beijing Forestry University | Gai Y.,Beijing Forestry University | Jiang X.,Beijing Forestry University | Jiang X.,The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry Administration
Journal of Plant Biochemistry and Biotechnology | Year: 2014

Cinnamoyl-CoA reductase (CCR, EC, which catalyzes the reduction of cinnamoyl-CoA esters to their respective cinnamaldehydes, is considered as a key enzyme in lignin formation. The substrates of CCR, cinnamoyl-CoA esters, are products of 4-Coumarate-CoA ligase (4CL, EC, which is an enzyme upstream of CCR. The PtCCR and Pt4CL were isolated from Populus tomentosa and expressed in E. coli. Results showed that 4CL can catalyze the conversion of hydroxycinnamic acids to cinnamoyl-CoA esters, with high efficiency. The purification of esters using SPE cartridges suggested that 40 % methanol with 0.1 M of acetic acid was the optimal elution buffer for cinnamoyl-CoA esters. The optimization of prokaryotic expression demonstrated that the best expression conditions for recombinant PtCCR was 6 h of 0.4 mM IPTG induction at 37 °C. PtCCR enzyme assay illustrated that the recombinant protein can catalyze the reduction of cinnamoyl-CoA esters. Kinetics analysis showed that feruloyl-CoA has higher affinity to PtCCR with faster reaction speed (Vmax), indicating that feruloyl-CoA was the most favorable substrate for PtCCR catalysis. The recombinant protein was expressed in E. coli, purified through affinity column chromatography, and characterized by SDS-PAGE. SPE cartridges were used to purify the ester products of the Pt4CL reaction. HPLC-MS was used to analyze the structure of esters and evaluate their purity or quantity. Furthermore, the enzyme activity of recombinant CCR to feruloyl-CoA at different pHs indicated that compartmentalization may be an important factor in lignin monomer formation. © 2013, Society for Plant Biochemistry and Biotechnology.

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