A novel cultivation strategy, "heterotrophy - stress" two-step cultivation was developed to solve the problem of asynchronism between biomass and lipid production in Chlorella protothecoides research. The C. protothecoides was first cultivated in optimized heterotrophic medium to achieve high cell density with low lipid content. Then, the algal cells were washed, condensed and transferred to stress medium without nitrogen source in order to accumulate high lipid content. With this strategy, the C. protothecoides biomass in 500ml flask achieved 5.32 g/L dry-weight which was close to the level of traditional heterotrophic cultivation mode, but the lipid content increased from 15.40% to 34.81%, and the algal polysaccharide content of the residue after lipid extraction increased from 9.57% to 18.06%. Furthermore, a 3L fermenter experiment showed a consistent pattern. In nitrogen rich medium, the biomass reached 14.1 g/L dry-weight, lipid content reached 17.16%, and the algal polysaccharide content was 10.16%, while in " heterotrophy - stress" two-step cultivation, the biomass attained 13.2 g/L dry-weight, the lipid attained 40.15%, and the algal polysaccharide content was 24.74%. Therefore, this study indicates that the proposed strategy may provide an effective approach for microalgal biomass production with high lipid content.
The ‘Golden Gate’ cloning is based on the ability of type IIS restriction enzymes and DNA ligase enzymes to assemble multiple DNA fragments in a defined linear order in one tube and one step. The method is efficient and yields recombinant vectors that do not contain unwanted sequences in the final construct after just a short time restriction-ligation. To edit mouse β casein gene, recombination targeting vectors of the mouse β casein gene targeting locus were constructed by the ‘Golden Gate’ cloning.
