Bioengineering of the plant culture of Capsicum frutescens with vanillin synthase gene for the production of vanillin

Production of vanillin by bioengineering has gained popularity due to consumer demand towards 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

3D

© 2021 Wiley Periodicals LLC.Fabrication of scaffolds using polymers and then cell seeding is a routine protocol of tissue engineering applications. Synthetic polymers have adequate mechanical properties to substitute for some bone tissue, but they are generally hydrophobic and have no specific cell recognition sites, which leads to poor cell affinity and adhesion. Some natural polymers, have high cell affinity but are mechanically weak and do not have the strength required as a bone supporting material. In the present study, 3D printed hybrid scaffolds were fabricated using PCL and GelMA carrying dental pulp stem cells (DPSCs), which is printed in the gaps between the PCL struts. This cell loaded GelMA was shown to support osteoinductivity, while the PCL provided mechanical strength needed to mimic the bone tissue. 3D printed PCL/GelMA and GelMA scaffolds were highly stable during 21 days of incubation in PBS. The compressive moduli of the hybrid scaffolds were in the range of the compressive moduli of trabecular bone. DPSCs were homogeneously distributed throughout the entire hydrogel component and exhibited high cell viability in both scaffolds during 21 days of incubation. Upon osteogenic differentiation DPSCs expressed two key matrix proteins, osteopontin and osteocalcin. Alizarin red staining showed mineralized nodules, which demonstrates osteogenic differentiation of DPSCs within GelMA. This construct yielded a very high cell viability, osteogenic differentiation and mineralization comparable to cell culture without compromising mechanical strength suitable for bone tissue engineering applications. Thus, 3D printed, cell loaded PCL/GelMA hybrid scaffolds have a great potential for use in bone tissue engineering applications