Development And Application Reaserch Of Blue Light Induced Expression System Based On EL222 Photoreceptors

At present,most heterologous protein expression systems are still regulated by traditional biochemical inducers.However,this method has some defects such as poor reversibility,rough regulation,transport delay and off-target effect,which makes it difficult to achieve accurate and rapid dynamic regulation.Therefore,it is urgent to develop dynamic regulatory switches with fast response,efficient reversible,and high spatiotemporal resolution.Optogenetics is dedicated to studying the effects of light stimulation,fully exploiting reversibility,low toxicity and precision to realize the combination of gene regulation and optics,which is expected to become a new dynamic regulatory switch.However,optogenetics still has some problems such as fewer hosts,exogenous cofactors and complex components.The author has carried out a series of researches to get closer to the ideal gene regulation system.The main research contents and results are as follows:The blue light induced expression system was constructed with EL222 blue light photosensitive protein from Erythrobacter litoralis as the core element.The applicability of EL222 in prokaryotic Escherichia coli was explored which laid a foundation for the subsequent development of EL222 in yeast.In order to achieve blue light induction in yeast,the photosensitive transcription factor SVEL was constructed by fusion expression of nuclear localization signal SV40 and transcription activation domain VP16 into the Nterminal of EL222 to adapt eukaryotic transcription.And the expression of fusion protein was driven by a component promoter.Then,the EL222 binding site(C120)was constructed as the upstream activating sequence and the core of common promoter was constructed as the driving region,so as to obtain the blue light induced promoter regulated by EL222.Finally,the single-component blue light induction systems,p Bctrl system and the p YLBI system were constructed for Pichia pastoris and Yarrowia lipolytica,respectively.The induction efficiency,biological toxicity,spatiotemporal resolution,response speed,uniformity,reversibility,and multiple copy strategy of the developed system were studied.It was proved that 100 μW/cm2 continuous blue light irradiation does not have toxic effects on the host in this research.The highest induction ratios were 79.7 times of p Bctrl and 45.8times of p YLBI.The positive correlation between the blue light induction system and the blue light irradiation duration,intensity and period was verified.The system can achieve centimeter-level spatial resolution and exhibits a uniform response to blue light regulation at the single-cell level.The reversible regulation can be achieved at the m RNA level within minutes and at the protein level within hours.In addition,the recombinant strains with 1 to4 copies of the blue light responsive fragment were constructed in Y.lipolytica.The induction ratio was positively correlated with the copy number.Among them,the highest induction ratio of 128.5-fold was achieved in the in 4-copy strain.Then,the ability of p YLBI to express of active proteins in Y.lipolytica was verified by using the bleomycin resistance gene as a reporter gene.The blue light induction system has a good application prospect in industrial production and metabolic engineering.Lipase,pectin lyase and endo-β-1,4-glucanase were expressed with p Bctrl system,and the above 3 enzymes obtained showed good activity and reversibility.Then,a modification pathway of human-like N-glycosylation regulated by p Bctrl was introduced into P.pastoris ΔOCH1 which α-1,6-mannosyltransferase gene is knocked out.Thus,the high-mannose structure was transformed into human-like complex Glc NAc Man5 Glc NAc2 structure.On this basis,the thiamine-repressed OCH1p-PSD was introduced to avoid the the growth delay and hypersensitivity,thus achieving blue light switching between high-mannose N-glycochain and human-like complex N-glycochain,which is conducive to the implementation of biphasic fermentation process.Through the development and verification of the blue light induction system,a set of universal blue light induction promoter design ideas and methods are summarized.The regulation mode and component library of P.pastoris and Y.lipolytica were expanded.And this research contributed to the application of optogenetics in synthetic biology and metabolic engineering.