Design,Construction And Application Of A Stable Self-Regulating Microbial Co-Culture System

Microbial co-culture had been widely used in synthetic biology.Compared to monoculture,microbial co-culture offers significant advantages in terms of labor partitioning to reduce metabolic burden,pathway compartmentalization to provide a suitable enzyme-catalyzed environment,and facilitating the utilization of complex substrates such as lignocellulose.However,despite the great success of co-cultures,two great challenges remain.One is that previous co-cultures were established mainly based on cross-feeding of single metabolites,with loose relationships between strains and high vulnerability to population stability.The second is that the previous co-culture system could not actively sense the accumulation of intermediates,let alone autonomously regulate the population ratio to achieve the maximum production of target products.To solve these two major problems,an extremely stable and mutualistic co-culture system was constructed in this study,and the dynamic regulation of the co-culture population ratio and the efficient production of target products were further achieved by introducing biosensors.Both TCA cycle and amino acid synthesis are necessary for cell life activities,and their defects are simultaneous defects of multiple substances that will lead to cell growth arrest.In this study,we constructed two defective strains in TCA cycle and amino acid synthesis,respectively,and co-cultured these two strains to form a novel mutualistic co-culture system with offering each other the defective multi-substances.Due to the close and strong interactions between the co-culture members,the system achieved the population ratio change and cell growth no longer dependent on the initial inoculation ratio,and a 3:1 ratio could be reached within 48 h at any inoculation ratio.Further,we constructed a biosynthetic pathway for salidroside and hydroxytyrosol,and achieved their efficient biosynthesis by static co-culture(the population ratio remained almost stable at any inoculation ratio,called static).Firstly,through this static co-culture system,we successfully achieved a shake flask titer of 1550 mg/L in 48 h and a fed batch fermentation titer of 12.52 g/L in 84 h for the de novo biosynthesis of salidroside.we demonstrated the excellent robustness of the designed mutualistic co-culture system in long-term fermentation production through continuous passaged culture.In addition,this study achieved the first de novo synthesis of hydroxytyrosol with a titer of 550mg/L.Further,we achieved the titer of 647 mg/L by using a biphasic culture strategy to mitigate the biotoxicity of hydroxytyrosol.Finally,the titer of hydroxytyrosol was increased to 1634 mg/L by the static co-culture.In addition,a Dmp R biosensor with good responsiveness to caffeate was selected based on the principle of structural similarity in order to establish a dynamic co-culture system that can regulate the population ratio on demand.By introducing the Dmp R biosensor into the static co-culture system,an efficient dynamic co-culture production system of coniferol that can automatically sense the intermediate caffeate to adjust the population ratio on demand was successfully established.Through dynamic co-culture,the population ratio was automatically adjusted from5:1 to 1:1,and the titer of coniferol was increased from 95 mg/L to 258mg/L.Finally,we selected silybin as the target compound,and demonstrated the application of dynamic co-culture in the production of long pathway compounds.In this study,we successfully constructed the pathways for the de nove synthesis of silybin by two and three bacteria co-culture,and achieved the titer of 0.28 mg/L and 2.02 mg/L,respectively,which is the first de nove synthesis of silymarin.The static and dynamic co-culture systems designed in this study,not only realized the efficient production of various high-value compounds,but also laid the foundation for the industrial application of microbial co-culture.