The Impact Of Promoters And Vectors On The Amorphadiene Production In Synthetic Yeast Cells

Synthetic Biology is the repurposing of living systems for useful ends.SyntheticBiology, philosophically rooted in the engineering paradigm, aims to reduce complex‘natural’（i.e.evolved）systems to simplified, reliable, quality-controlled modules, or‘parts’, that can be mathematically modeled, manipulated by computer aided design（CAD）‘,abstracted’（passed between loosely coupled design and production layers）,bolted together to achieve predictable results, and fabricated on an industrial scale. Tobuild the key artemisinin precursor amorphadiene synthesis route in thechassis cells:First introducing amorphadiene synthetic gene module, amorphadiene syntheticenzyme（ADS）module, converting precursor FPP into amorphadiene.After obtainingfunctional product preliminarily, the steady improving of amorphadiene productioncan be achieved by fine-tuningof the original and exogenous modules. Through aserials of regulatory fine-tuning of the exogenous module by using a mix and matchof different strength promoters and the adaptation between modules and chassis cells,the amorphadiene production can be steadily improved.To optimize the constructing strategy of synthetic yeast cells producingamorphadiene in this study, Promoter Engineering and Adaptation betweenExogenous Module and Chassis Cells were utilized to alter the synthetic yeast cellsproducing amorphadine.In this paper, two chassis cells（W303and YSG50）and three kind of exogenousmodules（including free replicated module, integrated module and centromericmodule）were utilized to construct synthetic yeast cells producing amorphadiene. Fourdifferent strength promoters（TDH3,ADH1,HXK2and TDH1） were replaced in theyeast cells with free replicated exogenous module. A strong promoter（TDH3） and aweak promoter（TDH1） were replaced in the yeast cells with integrated or centromericmodule. GC-TOF/MS analysis was utilized to detect and compare fermentationproducts concentration of the above cells.The results showed that all of theconstructed yeast cells could produce amorphadiene,and the centromeric cellSyBE₀01243（W-tH-20[pRS316/TDH3/ADS]） with the highest yield of44.7mg/L.Further comparison of the foregoing results showed that concentration of theamorphadiene in the fermentation broth was positively correlated to the strength ofthe promoters. Moreover, for each chassis cell, concentration of the amorphadiene in the fermentation broth was related to the type of vector of the exogenous module. Andfor the stong promoter, concentration of the amorphadiene in the fermentation brothwas in large differences among engineered cells with different vector types ofexogenous module, whereas for the weak promoter, the concentration was in smalldifferences among engineered cells with different vector types of exogenous module,which was much lower than the strong promoter.These studies provide a source of inspiration for researchers engaged in relatedwork to select chassis cells, vectors and promoters to reconstruct the exogenous genemodule and optimize strategy of construction of synthetic cells. Also provide atheoretical guidance for strain reconstruction and the industrial production ofterpenoids using synthetic biology.