Production of Astaxanthin Using CBFD1/HFBD1 from Adonis aestivalis and the Isopentenol Utilization Pathway in Escherichia coli

Abstract

Astaxanthin is a powerful antioxidant and is used extensively as an animal feed additive and nutraceutical product. Here, we report the use of the β-carotene hydroxylase (CBFD1) and the β-carotene ketolase (HBFD1) from Adonis aestivalis, a flowering plant, to produce astaxanthin in E. coli equipped with the P. agglomerans β-carotene pathway and an over-expressed 4-methylerythritol-phosphate (MEP) pathway or the isopentenol utilization pathway (IUP). Introduction of the over-expressed MEP pathway and the IUP resulted in a 3.2-fold higher carotenoid content in LB media at 36 h post-induction compared to the strain containing only the endogenous MEP. However, in M9 minimal media, the IUP pathway dramatically outperformed the over-expressed MEP pathway with an 11-fold increase in total carotenoids produced. The final construct split the large operon into two smaller operons, both with a T7 promoter. This resulted in slightly lower productivity (70.0 ± 8.1 µg/g·h vs. 53.5 ± 3.8 µg/g·h) compared to the original constructs but resulted in the highest proportion of astaxanthin in the extracted carotenoids (73.5 ± 0.2%).

Keywords: Adonis aestivalis; Escherichia coli; carotenoids; hydrophobic products; isopentenol utilization pathway (IUP).

Figure A1

HPLC analysis of strains ASTA 3,6, and 9 at 475 nm. Peaks corresponding to astaxanthin, isozeaxanthin*, canthaxanthin, and β-carotene are labelled. * Isozeaxanthin was assumed to co-elute with zeaxanthin which was used as the standard for detection.

Figure 1

Biosynthesis pathways for heterologous production of astaxanthin in E. coli. E. coli possesses an endogenous MEP pathway starting from G3P and Pyr to form both IPP and DMAPP. The IUP uses the enzymes CK and IPK to produce IPP from isoprenol. DMAPP and IPP ratios are modulated by IDI. IspA and GGPP synthase convert IPP and DMAPP into GGPP. Two GGPP are converted into lycopene (pink) by CrtB and CrtI. β-carotene (yellow) is formed by cyclases CrtY or LycB. Bacterial ketolase CrtW and hydroxylase CrtZ produce astaxanthin (red) via isozeaxanthin (orange) and canthaxanthin (orange) intermediates. Flowering plants use CBFD and HBFD to produce astaxanthin via isozeaxanthin (orange). Enzymes used in this work are shown in blue. Modified from [11,16] G3P: glyceraldhyde-3-phoshpate; Pyr: pyruvate; IPP: isopentenyl diphosphate; DMAPP: dimethylallyl diphosphate; FPP: farnesyl diphosphate; GGPP: geranylgeranyl diphosphate; CK: choline kinase; IPK: isopentenyl phosphate kinase; IDI: isopentenyl diphosphate isomerase; IspA: FPP synthase; CrtE/GGPPS: GGPP synthase.

Figure 2

Plasmid designs used in this study for astaxanthin production. Gene organization in each operon are shown in the lefthand boxes while the biosynthesis precursors and products are shown in the righthand boxes. Upstream operons are located either on a plasmid or in the chromosome. Plasmids that form carotenoid intermediates can be transformed together to complete the pathway.

Figure 3

Total carotenoid production in strains 1–3 containing the wild-type (1), trcMEP (2), or IUP (3) pathway and the pAC-BETAipi and pCBFD1 plasmids. Cultures were grown in (A) LB media or (B) M9 media and induced with 0.1 mM IPTG. Cell growth by dry cell weight is plotted on the lefthand side. Total carotenoids were quantified, and carotenoid concentration (solid lines) and carotenoid content (dashed lines) are shown on the righthand plots.

Figure 4

Total carotenoid production in strains 4–6 containing the wild-type (4), trcMEP (5), or IUP (6) pathway and the p5T7-Astaipi plasmid. Cultures were grown in (A) LB media or (B) M9 media and induced with 0.1 mM IPTG. Cell growth by dry cell weight is plotted on the lefthand side. Total carotenoids were quantified, and carotenoid concentration (solid lines) and carotenoid content (dashed lines) are shown on the righthand plots.

Figure 5

Total carotenoid production in ASTA strains containing the wild-type (7), trcMEP (8), or IUP (9) pathway and the p5T7-lycipi-ispA and pAC-ASTA plasmids. Cultures were grown in M9 media and induced with 0.1 mM IPTG. Cell growth by dry cell weight is plotted on the lefthand side. Total carotenoids were quantified, and carotenoid concentration (solid lines) and carotenoid content (dashed lines) are shown on the righthand plots.

Figure 6

Productivity and carotenoid composition of ASTA strains grown in M9 media. (A) Total productivity of each strain over a 48 h cultivation period. (B) Percent composition of carotenoids extracted from strains 3, 6, and 9 based on HPLC analysis.

Figure 7

Stepwise improvement of astaxanthin production and purity in the course of this study.