Optimization Of Dual-PKS Expressions To Improve 10,11-dehydrocurvularin Production In Saccharomyces Cerevisiae

10,11-dehydrocurvularin（DCV）is a benzenediol lactones-type polyketide of fungal origin,whose anti-breast cancer activity has been reported in the previous researches.As a natural product derived from Aspergillus terreus,low productivity of DCV and lack of efficient tools for genetic modifications of its native host prevent strain engineering and hence large-scale production of this important polyketide compound.Instead,in the previous studies,our collaborating team developed the model eukaryotic organism Saccharomyces cerevisiae to synthesize DCV by heterologous expression of the PKS pairs from Aspergillus terreus required for DCV production.However,such yeast system hasn’t been well engineered,so that the productivity of DCV is still rather low,without any quantitative evaluation so far.Meanwhile,we also don’t know which PKS（highly-reducing PKS or non-reducing PKS）is the rate-limiting step hindering DCV production.Finally,as a promising chemotherapic drug candidate,its effect to other cancer cell lines besides breast cancer lines are still unknown.Thus in this thesis around the issues mentioned above,we conducted the following studies.（1）We tested the cytotoxity of DCV to two cancer cell lines:lymphoma cell U937 and leukemia cell K562,with human embryonic kidney cell HEK293 as the control.DCV showed noticeable specific toxicity to U937(IC50=2.03μmol·L^-1),as compared to HEK293(IC50=4.38μmol·L^-1)When incubated for 48 h.（2）We established a high-throughput method to produce DCV by yeast fermentation in deep-well plates,extraction of hydrophobic natural products via vacuum evaporator.This method allows robust fermentation of up to hundred yeast cultures and natural production exatraion in parallel,which is superior than the traditional method using flask-based fermentation and rotary evaporator-based extraction.（3）We built up a combinatorial duel-plasmid expression system to control highly-reducing PKS and non-reducing PKS via truncation of the selecting makers’promoters,which was used to optimize DCV produciton.We identified the best combinations of truncations（Kanmax and Hyg18）resulted in improved production of DCV to 0.38 mg·L^-1,which is 3.84-fold,compared with the full-length selection markers’promoters.（4）DCV production was further improved by optimization of the culture conditions and using the fed-batch fermentor,with the final titer ncreass to 5.02 mg·L^-1,which is another13.23-fold,compared with the un-optimized culture condition.The results in this thesis could serve as the basis for the further clinic study and strain engineering for industry-scale production.