生物合成产品途径的优化策略

1. 生物合成产品途径的优化策略 王长松 2012.9

3. Systems metabolic engineering Recently, metabolic engineering has been upgraded to systems level (thus, systems Metabolic engineering ) by the integrated use of global technologies of systems biology fine design capabilities of synthetic biology rational–random mutagenesis through evolutionary engineering

4. systems biology Unravel the underlying principles of biological systems through profiling the whole cellular characteristics using high-throughput technologies together with computational methods synthetic biology Creat novel biologically functional parts, modules and systems by employing various molecular biology and synthetic DNA tools together with mathematical methodologies evolutionary engineering Expression levels of multiple genes are tuned and adapted simultaneously and autonmously by following the rules of natural selection for the desired cellular properties

6. Ways to increase the efficiency of engineered strain • Allowing the creation of novel enzymes and metabolic and gene regulatory circuits • Fine-tuning and optimization of the metabolic fluxes that lead to increased yield and concentration of a desired product • Increased tolerance of cells to the product and harmful medium components • Formation of fewer or no byproducts • Enhanced utilization of desired carbon substrates • Development of cost-effective fermentation and downstream processes Based on several criteria such as the availability of native producer, biosynthetic pathways, enzymes, and others, the starting point of systems metabolic engineering can be decided.

8. MEP operon GT/TG operons dxs idi ispD ispF G/T T/G dxs、idi、ispDF基因克隆于E. coli K12 GGPPS和TS基因分别去除N端98和60个氨基酸，进行密码子优化后全合成。

9. Regulating the upstream and down stream modular pathways p5-pSC101 plasmid p10-p15A plasmid p20-pBR322 plasmid Downstream: p20TrcGT(20) p20TrcTG(24) p20T5GT(39) p20T5GTTrcT(59) Upstream: Native(1) Ch1TrcMEP(2) p5TrcMEP(6) p10TrcMEP(11) Ch1-1copy in chromosome Trc-Trc promoter T5-T5 promoter T7-T7 promoter

10. 红色为菌株编号，单位为mg/L

11. Upstream: Native(1) Ch1TrcMEP(2) Ch1T5MEP(3) Ch1T7MEP(6) Upstream: p10TrcMEP(11) p20TrcMEP(21) p20T5MEP(40) p20T7MEP(100) Downstream: p5T7TG(31) P10T7TG(61) Downstream: p5T7TG(31) P10T7TG(61)

12. Upstream and downstream pathway strength correlates to transcriptional gen expression levels Downstream TS gene Upstream DXS gene

13. Unknown metabolite that correlates inversely with taxadiene taxadiene Through CG-MS、1HNMR、13CNMR identified as indole Unknown metabolite

14. Fed-batch cultivation of engineered strains Cultivation: 0.5% yeast extract , 20%(v/v) dodecane Dodecane: 13.3g/L KH2HPO4, 4g/L (NH4)2HPO4, 1.7 g/L citric acid, 0.0084g/L EDTA, 0.0025 g/L CoCl2, 0.015 g/L MnCl2, 0.0015 g/L CuCl2, 0.003 g/L H3BO3, 0.0025 g/LNaMoO4, 0.008 g/L Zn(CH3COO)2, 0.06g/L Fe(Ⅲ) citrate, 0.0045 g/L thiamine, 1.3 g/L MgSO4, 10 g/L glycerol, 5 g/L yeast extract, pH=7.0) Biphasic liquid-liquid fermentation using a dodecane overlay DO 30% pH was controlled at 7.0 using 10% NaOH 30℃ until OD600=0.8 , temperature was reduced to 22 ℃ and induced 0.1mM IPTG 3g/L glycerol was introduced when concentration depleted to 0.5-1 g/L

15. 210mg/L 174mg/L 1020mg/L Strain 17: EDE3p10TrcMEP p5T7TG 125mg/L Strain 22: EDE3p20TrcMEP p10T7TG 59mg/L Strain 26: EDE3Ch1TrcMEP p5T7TG 297mg/L

16. 对大肠杆菌MEP途径限速酶过表达； 调整表达强度 将GGPPS与TS基因N端短，进行密码子优化，表达温度为22℃ 培养基优化； 两相发酵

17. Thanks for your attention