Heterologous biosynthesis of costunolide in Escherichia coli and yield improvement.

Costunolide, the main bioactive compound of the medicinal plant, Radix Aucklandiae, is a sesquiterpene lactone (SL) and has a broad range of biological activities. It is also a precursor of many biologically-active SLs and is a branching point in the biosynthesis of SLs. Here we have reconstituted the costunolide biosynthetic pathway in Escherichia coli by co-expression of three genes (GAS, GAO, LsCOS) involved in costunolide biosynthesis and eight genes involved in converting acetyl-CoA into farnesyl diphosphate from mevalonate pathway. Costunolide production was then detected. By screening and optimization of cultured medium and inducing temperature, costunolide yield was up to 100 mg l(-1) in E. coli.

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.).

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l(-1) 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l(-1) hygromycin and 200 mg l(-1) cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato.