Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, β-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC–ESI–MS. A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Graphene quantum dots (GQDs) have shown great promise in a variety of medical applications. Recently, it has been found that GQDs are also beneficial for photodynamic therapy (PDT). However, the findings of GQDs as PDT agents have been controversial in the literature. Herein, we investigate the photoactivity of single-atomic-layered GQDs by examining their ability to generate singlet oxygen (¹O₂) under irradiation and their effects on the photoactivity of photosensitizers. We demonstrate that the GQDs with lateral sizes of ∼5 or 20 nm are photo-inactive for they cannot generate ¹O₂ under irradiation of either a 660 nm laser (105 mW cm⁻²) or a halogen light. Moreover, the GQDs inhibit the photoactivity of two classical photosensitizers, namely, methylene blue and methylene violet. The stronger interaction between the GQDs and the photosensitizer results in greater inhibition of GQDs. Besides, the large-sized GQDs exhibit stronger inhibition than the small-sized GQDs. The inhibitory effect of the GQDs on the photoactivity of photosensitizers is consistent with their photo-cytotoxicity. These results indicate that the single-atomic-layered GQDs are not potential PDT agents, but they may be helpful for photosensitizers by delivering them into the cells. The discrepancy between the current work and the literature is probably associated with the GQDs used.