Synthesis of Crocin I and Crocin II by Multigene Stacking in Nicotiana benthamiana

Abstract

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer’s disease. Crocins are mainly biosynthesized in the stigmas of food–medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco.

Research progress on the pharmacological activity, biosynthetic pathways, and biosynthesis of crocins

“…Xie et al introduced a multigene expression vector ( GjCCD4a - GjALDH2C3 - GjUGT74F8 - GjUGT94E13 ) into N. benthamiana by using the fusion and 2A polypeptides strategy and obtained a crocin yield of 78,362 ng/g (fresh weight). The content of crocin-I ( 2a ) and crocin-II ( 2b ) accounted for 99% of the total crocin content [ 114 ]. The supply of UDP-glucose is a rate-limiting step in crocin biosynthesis.…”

Research Progress in Heterologous Crocin Production

“…Wang et al [7] introduced Cs-CCD2L and different UGTs genes into Escherichia coli and synthesized crocetin of 4.42 mg/L. Xie et al [102] for the first time transformed Gj-ALDH2C3 and glycosyltransferase Gj-UGT74F8 and Gj-UGT94E13 genes into Nicotiana benthamiana, resulting in a yield of 105.8945 mg/g DW of crocins, of which crocin I and crocin II accounted for 99%. It is the highest recorded heterologous synthesis of crocin.…”

“…According to available reports, many microorganisms have been successfully transformed to synthesize crocetin or crocetin, including E. coli [7], yeast [62], Nicotiana glauca [108], Chlorella vulgaris [109], and Dunaliella salina [104]. Recently, the transient transformation of N. benthamiana to synthesize crocins was reported [102]. The above cases provide solid theoretical support and practical basis for further heterologous production of crocin.…”

“…It was found that Gj-CCD4a had higher substrate specificity and catalytic efficiency in the leaves, demonstrating that a single enzyme, Gj-CCD4a, could drive the synthesis of crocin [98]. Xie et al [102] combined the strategy of fusion with the 2A polypeptide connection and successfully constructed a multi-gene vector containing four genes to N. benthamiana, which transformed GjCCD4a and two downstream glycosyltransferase genes Gj-UGT74F8 and Gj-UGT94E13, to achieve higher substrate conversion efficiency that solved the problem of the low proportion of the main active components crocin I and crocin II, especially crocin I, as evidenced in previous research for synthesizing crocin in transgenic tobacco, and transformed ALDH introduced into tobacco for the first time.…”

Enhancing Nicotiana benthamiana as chassis for Molecular Farming: targeting flowering time for increased biomass and recombinant protein production

“…benthamiana has been a powerful chassis for the production of metabolites, such as taxadiene (Hasan et al, 2014), casbene (Forestier et al, 2023), crocins (L. Xie et al, 2023), moth sex pheromones for sustainable pest control (Mateos-Fernández et al, 2021), and enzymes, as glucocerebrosidase (Limkul et al, 2015), glucose oxidase (Talens-Perales et al, 2023), a bacterial laccase (van Eerde et al, 2022) and fungal enzymes for the degradation of lignin (Khlystov et al, 2021). Despite the advantages and examples mentioned above, N. benthamiana is not yet an ideal platform for molecular farming.…”