Construction Of Light-controlled Gene Expression Regulation System And Its Application In Metabolic Engineering

In order to meet the increasing demands of industrialization,it is an effective and environmentally friendly method to produce high value compounds by microbial fermentation.Metabolic engineering is the key technology to optimize and improve the microbial fermentation process.In metabolic engineering,natural cell factories are constructed by introducing exogenous metabolic pathways or optimizing endogenous metabolic networks,so as to realize the transformation and optimization of microbial fermentation.The contradiction between the growth of strain and product production has always existed.Initially,researchers used static regulation methods to solve metabolic problems,such as gene knockout or introduction of exogenous genes.However,in the fermentation process,product production changes with cell density,dissolved oxygen,substrate concentration and other conditions.It is necessary to use synthetic biological methods to achieve dynamic regulation.Adding chemical inducers can precisely regulate genes,but they are costly and toxic.Specific biosensors can not only accurately regulate gene expression but also realize dynamic regulation,but they are lack of universality,and it takes a lot of time to screen suitable sensors.Quorum sensing(QS)can dynamically regulate gene expression according to population cell density,showing enormous potential in metabolic engineering,but the flexibility of QS system is poor.Therefore,it is necessary to construct a dynamic genetic switch with low toxicity,versatility and flexibility.The light-controlled gene expression system can satisfy the above advantages well.In this study,we first characterized of the single light-controlled gene expression system CcaS/R in Escherichia coli.Then,we combined the single light-controlled gene expression system with the endogenous I-E CRISPRI system in E.coli and applied it to the production of poly-β-hydroxybutyrate(PHB).The PHB production pathway competes with the tricarboxylic acid(TCA)cycle for the common substrate acetyl-CoA,so we designed the green light to activate I-E CRISPRi system.This system targeted the gene gltA,which is the key gene of the TCA cycle.We inhibited the expression of gltA at different growth stages to balance the metabolic flux between growth and product production,so as to achieve the purpose of improving product yield.The results showed that the PHB content of the experimental groups was 2-3 folds higher than control groups.In order to develop a more flexible and versatile genetic switch,we constructed dual light-controlled gene expression system.First,we constructed four light-controlled gene expression systems GX1,YF1,EL222,and LexRO in E.coli TOP 10.GX1 is activated and expressed under green light at 535 nm and inhibited under red light at 650 nm.YF1,EL222,and LexRO are activated under blue light at 440-470 nm and inhibited under dark conditions.We combined GX1 components with YF1,EL222,and LexRO components respectively to construct three different dual light control systems(referred to as G+Y,G+E and G+L).Among them,G+Y and G+E can regulate the expression under green and blue light,respectively.While G+Y has less crosstalk,that is,the GX1 element only activates the expression under green light,and the leakage is low under blue light and dark conditions.YF1 component is the same.So,we applied the G+Y dual light control system to PHB production.The results showed that the PHB content of the 34-1 TOP10 and 33-0.01 TOP10 strains increased by 2 folds and over 3 folds,compared with the control group.By adjusting the time differences between turning off gltA and turning on phbCAB gene,we constructed 5 experimental groups.The results showed that 34-1 TOP 10 had the highest PHB content under the condition of experimental group D(the downregulation of gltA was 3 hours later than phbCAB upregulation),while 33-0.01 TOP10 had the highest PHB content in experimental group C(The downregulation of gltA occurred at the same time as the upregulation of phbCAB).The PHB content of the two strains showed a trend of increasing first and then decreasing with the increase of gltA downregulation time.We found that the PHB content is higher when the time interval is 0-3 h between downregulation of gltA and phbCAB upregulation,and the time interval is too long to increase the PHB content.In addition,we also explored the application potential of dual-light-control gene expression system in strain co-culture.In metabolic engineering,the inhibitory effect of intermediates or products on enzymes and the growth burden caused by complex metabolic networks cannot often be solved in a single cell.Co-cultivation of two or more strains is an effective strategy to overcome the above difficulties.Based on two orthogonal quorum sensing systems,we constructed dual-light-controlling co-cultured strains,and they performed well in microplate reader and fluorescence microscope characterizations.In this study,we combined of four different light-controlled elements and constructed three different dual light-controlled gene expression systems.Both single and double light control gene expression systems were applied to PHB production.The result proved that our light-controlled switch is an effective tool for realizing dynamic regulation of gene expression.We further explored the application of dual-light-control gene expression system in bacterial co-culture,which provided a new method to solve the metabolic engineering problem.It is the first time that the dual light control system has been applied to co-culture.In general,as a new and flexible regulation method in synthetic biology,dual-light regulation has shown great advantages and is expected to make good progress in future research.