Reprogramming Engineered Populations By A Programmed Lysis System To Improve Chemical Production

Microbial cell factories can produce high value-added chemicals in a green and economical way,through using renewable resources as substrates.However,production performance of cell factories affect economic viability of chemical production.Therefore,how to improve production performance of cell factories is a major challenge in biotechnology.In order to solve this problem,this paper took Escherichia coli as research model,took regulation of cell lysis as starting point.Status and structure of engineered Escherichia coli population were regulated by programmed lysis systems,through the application of synthetic biology and protein engineering technology.Thus,downstream processing of macromolecular chemical production was simplified and performance of cell factories carrying large exogenous pathway was improved.The main results are as follows:1.Excavate and obtain units of programmed lysis system:Firstly,Colicin M with the quickest response and the highest efficiency was obtained,through analysis of lysis performance of five lysis proteins.Secondly,combined with protein engineering,it was transformed into lysis unit,whose activity could be controlled by protease.Based on this,OD₆₀₀value of Escherichia coli decreased to 0.27 after 2 h,and protein concentration in supernatant increased to 0.93 mg/mL.Then,programmed switches were constructed by protein engineering,through the utilization of proteases,growth phase promoter and stationary phase promoter.Finally,switch times of programmed switches were delayed from10 h to 32 h,through the utilization of three regulatory strategies,namely changing RBS,changing start codons and adding degradation labels.2.Construct programmed lysis systems to regulate state and structure of engineered population:Firstly,programmed lysis systems namely PLS031,PLS041,PLS161 and PLS361were obtained,through combining lysis unit with programmed switches,which could lyse Escherichia coli at 20 h,28 h,28 h and 32 h,respectively.Then,the universality and lysis performance of programmed lysis systems were verified in seven different subtypes of Escherichia coli.Cell mortality rates at 38 h were all more than 90%.The morphology of Escherichia coli cells presented broken,irregular and filamentous form.In addition,release rate of cell contents were all more than 73%.After adjusting seeding ratio to 10:1,programmed lysis system PLS361 regulated structure of engineered population by lysing original dominant strain,and then realized stable reversal of engineered population structure.3.Automatic release of poly（lactate-co-3-hydroxybutyrate）was achieved by regulating state of engineered population:Firstly,based on literature mining,metabolic pathway for production of poly（lactate-co-3-hydroxybutyrate）was designed.By knocking out competing metabolic pathway in E.coli B0031,consumption of acetyl-Co A was blocked,and chassis strain B0032 for poly（lactate-co-3-hydroxybutyrate）production was obtained.Then,population state transition from"PLH production"to"PLH release"was achieved in fermentation process of poly（lactate-co-3-hydroxybutyrate）,through a programmed lysis system.After comparing and analyzing effects of different programmed lysis system on titer of poly（lactate-co-3-hydroxybutyrate）,PLS361 was confirmed as the most suitable programmed lysis system for releasing poly（lactate-co-3-hydroxybutyrate）.Finally,titer of free poly（lactate-co-3-hydroxybutyrate）was increased by 335%,and yield and productivity were increased by 368%and 383%,respectively.4.Structure of engineered population was regulated to improve the performance of butyrate production from fatty acid:Firstly,based on literature mining,metabolic pathways for fatty acid degradation and butyrate production were designed.Secondly,after improving transport capacity of fatty acid and blocking competing metabolic pathway,E.coli BUT002for fatty acid degradation was obtained.After improving level of intracellular NADPH,strengthening production capacity of butyrate,and improving tolerance of acetate,E.coli BUT004 for butyrate production was obtained.Then,through a programmed lysis system,dominant strain of engineered population turnned over from"fatty acid degradation E.coli"to"butyrate production E.coli",in process of producing butyrate from fatty acid.After comparing and analyzing the effects of different seeding ratios and programmed lysis systems on fatty acid degradation and butyrate production,the most suitable engineered population POP8 for butyrate production was confirmed.Finally,titer,yield and productivity of butyrate were increased by 115%,48%and 115%,respectively,in a 5-L fermenter.