The Fitness Of Optimal GGPPS Module Identified By Docking Strategy With Saccharomyces Cerevisiae Chassis

Producing heterologous compounds in bacteria or yeast using synthetic biologymethod have attracted more and more attention. Many strategies have been used foryield improving in previous researches. However, the key point for improving theyield of heterologous compounds is the fitness of chassis and functional modulesintroduced into it. Therefore, the study of fitness is very important in future research.Firstly, six geranylgeranyl diphosphate synthases(GGPPS) were selected for theanalysis of catalytic capabilities by docking with FPP. The simulating data showedthat there was significant difference among their catalytic capabilities. In order toverify the simulation data, three GGPPS which ranked1,4,6in the docking resultwere selected for further experiment.The three selected GGPPSs were originated from Taxus baccata x Taxuscuspidata, Erwinia herbicola and Saccharomyces cerevisiae. They constructed theheterologous functional modules together with TS from Taxus brevifolia. After studyof fitness of key enzyme-GGPPS, chassis and the expressing level of heterologousgenes, the highest titer of taxadiene reached72.8mg/L. And a more stablegenome-transformed strain was also obtained for more efficient taxadiene production.Application of fusion protein strategy had not further improved the yield of taxadiene.But after protein structural simulation, we discovered the reason was the damage ofGGPPS structure. So the structures of fusion proteins must be complete when weapply this strategy. This part of study showed the fitness of chassis and heterologousmodules is the key problem we must solved in heterologous production of targetcompounds.Metabolites are the end products of regulating process in yeast，so the changes ofmetabolites are usually regarded as the cells’ response to the heterologous genes orcellular stress. In this study, the metabolomics analysis of two chassis provided usmore evidence on why YSG50was more capable for taxadiene production. And thechanges of cellular metabolites during fermentation process of artificial functionalyeasts were determined by metabolomics method. The results represented thatcompared to W303-1A which was a blank control, several metabolites related to TCAcycle and glycolytic pathway were influenced. The metabolomics analysis is benefitfor understand the influencing of taxadiene modules introduced into chassis. The fitness of chassis and heterologous modules is the key point in heterologousproduction of target compounds. In this study, rational design of taxadienebiosynthetic yeast was carried out based on predicting the structures and catalyticcapabilities of six key enzymes-GGPPS from different sources usingenzyme-substrate docking method. The computational identification method can beuseful for our rational design in application of synthetic biology. While themetabolomics analysis can give us more informations on the interactions of chassisand heterologous modules. So that it can be developed into an universal method in theresearch of compounds heterologous production. The new strategies or methodsapplied in this study can provide more informations on the further research of fitnessbetween chassis and heterologous modules.