Because of wide applications in food, feed, pharmaceutical and cosmetic industries, the carotenoid market is growing rapidly. Most carotenoids are hydrophobic, which limits their bioavailability. Glycosylation is a natural route that substantially increases the water solubility, as well as the bioavailability, photostability and biological activities of carotenoids. Here, we report metabolic engineering efforts (e.g., promoter and RBS engineering, optimization of carbon sources and supplementation of bottleneck genes) to produce glycosylated carotenoids in Escherichia coli. By fine-tuning the carotenoid-biosynthetic genes (crtX, crtZ and crtY), our strain produced up to 47.2 mg/L (~ 11,670 ppm) of zeaxanthin glucosides, ~ 78% of the total carotenoids produced. In another construct with mevalonate, astaxanthin pathway and crtX genes, the strain produced a mixture of carotenoid glucosides including astaxanthin and adonixanthin glucosides with a total yield of 8.1 mg/L (1774 ppm). Our work demonstrated a proof-of-concept study for the microbial biosynthesis of glycosylated carotenoids.

Background α-Ionone is highly valued in cosmetics and perfumery with a global usage of 100–1000 tons per year. Metabolic engineering by microbial fermentation offers a promising way to produce natural (R)-α-ionone in a cost-effective manner. Apart from optimizing the metabolic pathways, the approach is also highly dependent on generating a robust strain which retains productivity during the scale-up process. To our knowledge, no study has investigated strain robustness while increasing α-ionone yield. Results Built on our previous work, here, we further increased α-ionone yield to 11.4 mg/L/OD in 1 mL tubes by overexpressing the bottleneck dioxygenase CCD1 and re-engineering the pathway, which is  > 65% enhancement as compared to our previously best strain. However, the yield decreased greatly to 2.4 mg/L/OD when tested in 10 mL flasks. Further investigation uncovered an unexpected inhibition that excessive overexpression of CCD1 was accompanied with increased hydrogen peroxide (H2O2) production. Excessive H2O2 broke down lycopene, the precursor to α-ionone, leading to the decrease in α-ionone production in flasks. This proved that expressing too much CCD1 can lead to reduced production of α-ionone, despite CCD1 being the rate-limiting enzyme. Overexpressing the alkyl hydroperoxide reductase (ahpC/F) partially solved this issue and improved α-ionone yield to 5.0 mg/L/OD in flasks by reducing oxidative stress from H2O2. The strain exhibited improved robustness and produced  ~ 700 mg/L in 5L bioreactors, the highest titer reported in the literature. Conclusion Our study provides an insight on the importance of mediating the oxidative stress to improve strain robustness and microbial production of α-ionone during scaling up. This new strategy may be inspiring to the biosynthesis of other high-value apocarotenoids such as retinol and crocin, in which oxygenases are also involved.

Thermal preservation using the canning method is a promising alternative for retaining the maximum quality of the foods. Recently, the rising awareness of the nutritional value of canned products has received the attention of various researchers, as canned food can offer both healthy and convenient solutions to consumers. The present study reported the nutritional values and presence of food additives and preservatives in different Ayam BrandTM canned foods in tomato sauce namely sardines (CS), mackerel (CM) and baked beans (CB), canned tuna flakes in water (CTF) and canned tuna in mayonnaise (CTM) compared to the fresh and home-cooked food samples. The canned products, CS and CM contain significantly higher (p<0.05) amounts of Omega-3 but are lower in protein compared to their fresh counterparts. The calcium compositions of canned sardines and mackerels are 10-fold higher than fresh and home-cooked, contributed by the soft and brittles bones which enriched with calcium. The nutritional values of canned products were maintained throughout the storage period throughout the span of 3 years, with insignificant changes (p>0.05). Regardless of canned or fresh food samples, there was no total dietary fiber, benzoic acid, sorbic acid and sulfur dioxide detected in all samples except for the dietary fiber in the baked beans. CS and CTM contained significantly (p<0.05) higher amounts of glutamic acid than their fresh counterparts and the glutamic acid in all canned products ranging from 0.001-0.37 mg/g which is within the acceptable daily intake of 13 g/day. The natural glutamic acid was contributed by the raw materials of fish and tomato sauces themselves. No MSG was detected in all canned products. Therefore, it is safe to consume canned food products that greatly representing fresh and home-cooked foods without compromising with the nutritional values

Carotenoids have wide applications in food, feed, pharmaceutical and cosmetic industries. The fast-growing market demands highlight the importance of developing new routes for carotenoid biosynthesis. Meanwhile, a complementary need is to improve low bioavailability because of the hydrophobicity of carotenoids. One solution is glycosylation, which can substantially increase the water solubility of carotenoids, and enhance the bioavailability, photostability and biological activities as food supplements and medicines. Here, we report metabolic engineering efforts to produce glycosylated carotenoids in Escherichia coli . By fine tuning the 14 gene pathway, our strain produced up to 47.2 mg/L (~11670 ppm) of zeaxanthin glucosides, ~78% of the total carotenoids produced. In another construct with 15-gene pathway, the strain produced a mixture of carotenoid glucosides including astaxanthin and adonixanthin glucosides with a total yield of 8.1 mg/L (1774 ppm). Our work demonstrated a proof-of-concept study for the microbial biosynthesis of glycosylated carotenoids. (145 words)