| Cordyceps militaris has many physiological functions,of which antiinflammatory,anti-cancer and antioxidation are related to carotenoids.At present,four watersoluble carotenoids are found in C.militaris.As a rare water-soluble carotenoid discovered so far,theoretically it has stronger potential physiological activity than traditional carotenoids.Currently,metabolically engineered microorganisms and fungi are widely used in the production of carotenoids to meet the high global demand.Therefore,molecular identification of carotenoid production in C.militaris can promote the accumulation of a large number of highly bioavailable carotenoids in heterologous expression systems.However,due to inadequate clarification of the role of carotenoids in edible fungi,the carotenoid production pathway of C.militaris is not yet clear.In this study,in order to explore the carotenoids biosynthetic pathway of C.militaris,the cDNAs of Cmcao-2 and Cmylo-1 were cloned,and then characterized and analyzed by a series of bioinformatics tools,and heterologous expression was carried out in host cell Escherichia coli.The main research results of this article are as follows:(1)For the first time,the cDNA sequences of C.militaris Cmcao-2 and Cmylo-1 were completely cloned.In this study,the cDNAs of Cmcao-2 and Cmylo-1 were isolated from C.militaris for the first time.The full-length cDNA sequences of Cmcao-2 and Cmylo-1 are 2049 bp and 1587 bp,encoding 682 amino acid and 582 amino acid deduced polypeptides,with molecular weights of 76.5 kDa and 58.8 kDa respectively.(2)The heterologous expression of C militaris Cmcao-2 and Cmylo-1 was realized.The pET32a-CAO-2 and pET32a-YLO-1 expression engineering strains were successfully constructed to achieve the expression of C.militaris Cmcao-2 and Cmylo-1 in E.coli,and the target protein was detected by SDS-PAGE.(3)Validation the function of key enzyme genes for carotenoid biosynthesis.The plasmid pAC-BETA was successfully modified to achieve heterologous expression of Neurospora crassa al-1 and al-2,which ultimately resulted in the production of β-carotene in E.coli,and the replacement of al-1 caused the E.coli to produce more products,and pACAL1 has more abundant functions than pAC-BETA. |
