Construction of Carbon Sources Synergetic Utilization Mechanism and Its Application on Trehalose Biosynthesis

Author:Wu Yi Fei

Supervisor:yuan qi peng

Database:Doctor

Degree Year:2017

Download:63

Pages:122

Size:10255K

Keyword:

Selection and utilization of carbon sources are always the basic issues during the biosynthesis via metabolic engineering technology.How to high-efficiently utilize carbon sources affects the productivity and cost directly.However,instead of the basic metabolic issues of carbon sources,the researchers focused on improving the activity of key enzymes or introducing efficient pathways to achieve the high-level production.It leads most carbon sources flow into biomass rather than the biosynthesis pathway of target product.Though many efforts were made to solve the issues on carbon sources utilization,the essential connections between metabolic pathways of different carbon sources have not been constructed so that the conversion effienciency cannot be improved.Hence,to solve the problems on carbon source utilization,we have studied the following four aspects based on metabolic characteristics in E.coli:1)construct synergetic utilization platform by using glucose and glycerol as mixed carbon sources;2)introduce trehalose biosynthesis model to verify the synergetic platform strain;3)achieve the production of six-carbon sugar derived compounds from five-carbon sugars by using xylose as sole carbon source;4)construct platform strain of synergetic utilization of glucose and xylose which come from lignocellusic sugars,then achieve trehalose biosynthesis by using that platform successfully.Firstly,to realize high-efficiently conversion of glucose to sugar derived compounds,we constructed glucose non-catabolic pathway.Meanwhile,glycerol as the second carbon source was introduced for suppoting cell growth.In the media with glucose and glycerol as mixed carbon sources,carbon catabolite repression between the utilization of glucose and glycerol was relieved.Then,to connect the metabolic pathways of two carbon sources,we modified the pathway of phosphoenolpyruvate(PEP)to pyruvate so that PEP could be converted into pyruvate only through phosphotransferase system which was involved in glucose transportation.Thus,the uptake of glucose could be coupled with the catabolism of glycerol so that the synergetic utilization mechanism of carbon sources was constucted.However,the lack of consuming pathway of glucose might affect the consumption of glucose,which suggested that this platform needed to be further verified.Then we verified the synergetic utilization platform strain and detected the utilization of carbon sources by introducing trehalose biosynthesis model as the glucose consuming pathway.Firstly,we stepwise constructed trehalose biosynthesis model to evaluate the synergetic utilization platform.Second,we realized the high-level production of trehalose by strengthening the synergetic utilization mechanism and blocking the competing pathways of glucose.Finally,8.2 g/L trehalose was produced in shake flasks.The conversion ratio of glucose to trehalose was 0.86 g trehalose/g glucose,which reached 91%of the theoretical maximum yield.Meanwhile,the utilization ratio of glycerol to trehalose was 0.53 g trehalose/g glycerol.The results above demonstrated that the synergetic utilization platform was available which provided a new idea for trehalose biosynthesis.What’s more,we attempted to study how to efficiently utilize lignocellulosic sugars according to the achievement of carbon sources synergetic utilization platform.In the lignocellulosic hydrolysates,xylose is an abundant five-carbon sugar.However,to our best knowledge,no attempt was made to produce the six-carbon sugar derived compound from five-carbon sugars.Hence,we explored the strategy that converted xylose to trehalose for the first time.To redirect the carbon flux to trehalose biosynthesis pathway instead of biomass,we blocked the competing pathway of fructose-6-phosphate in the glycolysis so that more fructose-6-phosphate could flow into trehalose synthesis pathway.From the results,the attempt on conversion of xylose to trehalose provided a new thought for utilization of five-carbon sugars and lignocellulosic sugars.Therefore,we utilized lignocellulosic sugars as carbon sources in the following research.That is,we constructed a synergetic utilization platform by using glucose and xylose as mixed carbon sources.The mechanism of this platform was similar with the previous platform,which suggested glucose was used as the substrate of trehalose and xylose was used as the second carbon source to support glucose uptake and cell growth.Likewise,trehalose biosynthesis model was introduced to verify this synergetic utilization platform.After enhancement of the synergetic utilization mechanism and optimization of trehalose biosynthesis pathway,we could achieve 5.5 g/L trehalose in the shake flask media.Notablely,the conversion ratio of glucose or xylose to trehalose was around 4-fold higher than that of the control strain.Hence,this work not only provided a new method for trehalose bioproduction,but also proposed a novel strategy for lignocellulosic sugars high-level utilization.In summary,attempt and construction of the novel synergetic utilization platform.opened a new way for high-efficiently utilization of carbon sources and sugar-derived compounds production.This also laid a good foundation for the low-cost industrial bioproduction.