Abstract
Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.
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Acknowledgements
This work was funded by the Malaysian Ministry of Science, Technology and Innovation through the eScience Fund (02-02-12-SF0130), and by the Malaysian Ministry of Education through the MyBrain15 PhD scholarship programme.
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12033_2016_9986_MOESM1_ESM.tif
Fig. S1 Representation of colony PCR of VpVAN gene from thirteen E. coli colonies transformed with pcDNA6.2::(35Sp-VpVAN-V5)ΔccdB expression vector. M—1 kb DNA ladder; (+)—PCR positive control using synthesized holding vector, pIDT-AMP:35Sp-VpVAN-V5, as the template; (−)—no template PCR control. (TIFF 1049 kb)
12033_2016_9986_MOESM2_ESM.tif
Fig. S2 (i) PCR of VpVAN gene from pcDNA6.2::(35Sp-VpVAN-V5)ΔccdB expression vector extracted from E. coli, and (ii) DNA profile of the extracted expression vector after double restriction digest with PstI and BamHI restriction endonucleases (REs). M—1 kb DNA ladder; (+)—PCR positive control using synthesized holding vector, pIDT-AMP:35Sp-VpVAN-V5, as the template; (−)—no template PCR control. (TIFF 1303 kb)
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Chee, M.J.Y., Lycett, G.W., Khoo, TJ. et al. Bioengineering of the Plant Culture of Capsicum frutescens with Vanillin Synthase Gene for the Production of Vanillin. Mol Biotechnol 59, 1–8 (2017). https://doi.org/10.1007/s12033-016-9986-2
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DOI: https://doi.org/10.1007/s12033-016-9986-2