| Saccharomyces cerevisiae,as a potential microbial cell factory,is widely used in the synthesis of various value-added compounds,including biofuels,bulk chemicals and pharmaceuticals.It is of great significance to construct the biosynthesis platform of bio-based chemicals and pharmaceutical products in S.cerevisiae by using synthetic biology technology.Until recently,the titers and yields of many compounds which are synthesized in S.cerevisiae are still low.And using conventional metabolic engineering strategies such as gene overexpression or knockout,transcriptional down-regulation to improve yeast productivity often result in metabolic flux imbalance and bring about metabolic burden on the host cell.These problems may lead to poor cell growth.Dynamic regulation of metabolic pathways can address these problems through adjusting the expression of genes to the appropriate levels at the right time point.Dynamic metabolic pathway control can be achieved by building genetic regulatory circuit that responds to the change of different signals,such as extracellular environment,specific metabolites in metabolic pathways,or cell population density.Quorum sensing-mediated dynamic control can sense the change of population density and it is the inducer-free and pathway-independent system.Quorum sensing-based dynamic regulation can be triggered when a certain cell density is reached.Although S.cerevisiae is one of the most widely used micro-organisms in metabolic engineering,the studies on quorum sensing-based genetic circuit which can be used for dynamic metabolic pathways regulation are still lackingTherefore,in this study,we constructed a cytokinin-mediated quorum sensing genetic circuit in S.cerevisiae,and optimized the system to reduce its basal expression level and increase its dynamic output range.The cellular localization of cytokinin receptor in yeast was also determined.The quorum sensing system was then coupled with the auxin induced protein degradation system to achieve dynamic and autonomous protein degradation controlled by population density.This cell density-controlled dynamic protein degradation approach was used to regulate the production pathways of ?-farnesene and geraniol.This strategy increased the titers of?-farnesene and geraniol.This is the first time to develop the population-regulated protein destabilizing strategy for dynamic metabolic pathway regulation in S.cerevisiae.The specific research contents of this study are as follows:(1)Construction and optimization of cytokinin-mediated quorum sensing genetic circuits in S.cerevisiaeIn this study,the heterologous proteins-AtIPT4 and AtCRE1 from Arabidopsis thaliana were introduced in S.cerevisiae.AtIPT4 catalyzes cytokinin(isopentenyladenine,IP)synthesis,and AtCREl is IP receptor.Through knocking out the endogenous osmotic response protein Slnl,the heterologous IP response pathway was combined with the endogenous Ypdl-Skn7 signal transduction pathway.The conditionally activated phosphorylation pathway of AtCRE1-Ypdl-Skn7 was obtained Then an Skn7 regulated synthetic promoter(SSRE promoter)was used to control the expression of the reporter protein encoded gene yEGFP and the IP synthase encoded gene AtIPT4 for positive-feedback circuit construction.In addition,Ptp2,as an inhibition protein of HOG pathway,was overexpressed in order to eliminate the effect caused by Slnl knockout.The endoplasmic reticulum membrane localization rather than plasma membrane localization of AtCRE1 was first confirmed through fluorescent colocalization.After constructing the synthetic circuits,we first determined whether synthetic receiver circuit can respond to IP signal molecules.By adding different concentrations of IP into receiver strain,which contained AtCRE1-Ypdl-Skn7 signal pathway,it was found that the fluorescence intensity of receiver strain increased with the increasing IP concentrations.It indicated that the synthetic receiver circuit can respond to IP signal molecules.Then the cell-cell communication experiment between sender strain and receiver strain was carried out GFP levels increased significantly when the receiver strain was treated with the medium of the sender strain at high population density(OD600? 0.5).It was confirmed that yeast cells could synthesize IP and secrete so that it acted as a communication signal molecule by triggering the activation of receiver pathway.Based on this,we integrated the IP synthesis pathway and IP-response pathway into a single strain to construct a population density-dependent positive-feedback circuit.When OD600 reached 0.6,the circuit began to response.However,this circuit had a high basal expression level and a low dynamic output range.Therefore,we designed a series of synthetic promoters which were activated by phosphorylated Skn7 to optimize the quorum sensing circuit.Among these optimized circuits,we chose 2SSREcore promoter to control the expression of AtIPT4.When the expression of yEGFP was controlled by 4SSREcore promoter,the basal expression level of the circuit was reduced by 50%,and the dynamic output range was increased by 22%.When the expression of yEGFP was controlled by SSRE promoter,the dynamic output range of the circuit was increased by 44%compared with 4SSREcore-yEGFP containing circuit.The fluorescence intensity increased by 3.5-fold.The performance of the quorum sensing system has been significantly improved.(2)Construction of dynamic protein degradation systemProtein degradation is a potential metabolic regulation strategy.Comparing with transcriptional approaches,protein degradation tools can regulate the concentration of target protein directly.However,there are few protein destabilized tools that can be applied for metabolic engineering in S.cerevisiae,especially the dynamic protein degradation approaches.In this part,we first constructed an auxin-inducible degron system(AID system)in S.cerevisiae.Through the addition of auxin(such as indole-3-acetic acid,IAA),the target protein fused with IAA tag,a degron from A.thaliana,binds to the auxin receptor protein,resulting in polyubiquitylation of the IAA degron-tagged protein.Then,the target protein is degraded by proteasome.We first compared the protein degradation rates with different IAA degrons and auxin receptors.We found that the protein degradation efficiency was better when using IAA17 degron and AFB2 receptor protein to construct the system,therefore,we selected AFB2 receptor protein IAA17 degron for further study.We then compared the degradation efficiency between the AID systems with different expression level of AFB2 or with different concentrations of IAA.We found that the concentration of IAA was the main factor affecting the protein degradation efficiency in AID system.The protein could be completely degraded within 110 min after adding the saturated concentration of IAA.When the heterologous Indole-3-acetamide hydrolase iaaH was constitutively expressed,the protein could be completely degraded within 120 min without IAA addition.In order to establish an inducer-free dynamic protein degradation system,we coupled the quorum sensing system with AID protein degradation system.We used the 4SSREcore promoter or SSRE promoter from the quorum sensing circuits to control the expression of iaaH,so as to control the concentration of synthesized IAA.We developed population density-regulated protein degradation systems with different degradation efficiencies.By using this dynamic approach,protein can be degraded autonomously.(3)Using quorum sensing-regulated protein degradation system for dynamic regulation of metabolic pathwaysTo verify whether the quorum sensing-regulated protein degradation approach can be implemented for metabolic pathway regulation,we applied this approach to the dynamic regulation of the sesquiterpene ?-farnesene and the monoterpene geraniol biosynthesis pathways.Erg9 and Erg20 are involved in the synthesis pathway of ergosterol,which is essential for growth.However,this ergosterol synthesis pathway is also a competitive pathway for the synthesis of sesquiterpenes and monoterpenes By tagging IAA 17 degron at the C-terminus of Erg9,we controlled Erg9 degradation to inhibit the ergosterol synthesis pathway from farnesyl pyrophosphate(FPP),the precursor for ?-farnesene synthesis.The titer of ?-farnesene in a Erg9 continuous degradation strain increased by 22%compared with it in the control strain without Erg9 degradation.When using 4SSREcore-iaaH-containing protein degradation system to dynamically regulate Erg9 degradation,the titer of ?-farnesene in this strain increased 80%compared with it in control strain,reaching 23.37 mg/L.The result demonstrated that dynamic Erg9 degradation was better than the continuous high-level Erg9 degradation.The titer of ?-farnesene in the 4SSREcore-iaaH-containing strain were still 105%and 71%higher than it in the control strain in batch and fed-batch cultivation,which indicated that this dynamic control approach was still effective at high cell density.We also applied the dynamic population density-regulated protein degradation system to regulate the degradation of Erg20,so as to regulate geraniol biosynthesis pathway.The geraniol titer was increased from an extremely low level to 0.51 mg/L by high-level contentious degradation of Erg20.When the degradation of Erg20 was dynamically regulated by SSRE-iaaH-containing system,the titer of geraniol was increased by 37%compared with high-level degradation of Erg20,reaching 0.7 mg/L.Meanwhile,using quorum sensing-regulated protein degradation system to control Erg20 could alleviate the growth inhibition caused by high-level degradation of erg20.In this paper,we constructed a quorum sensing system using cytokinin as a signal molecule in S.and coupled it with protein degradation system.It is the first time to establish a population density-regulated protein degradation approach in S.cerevisiae.The superiority of this method for metabolic pathway regulation was confirmed.This dynamic regulation method is inducer-free and pathway-independent It is a universal regulation approach,and has a great potential in the dynamic metabolic pathway regulation. |
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