Development Of A New Biocontainment Strategy In Model Cyanobacterium Synechococcus Strains

Cyanobacteria are autotrophic microorganisms capable of utilizing light energy and carbon dioxide for growth.With the development and application of synthetic biology,the role of cyanobacteria as a"photosynthetic microbial cell factory"has become increasingly prominent.So far,dozens of biofuels and fine chemicals have been successfully synthesized using the cyanobacteria chassis.However,the characteristics of cyanobacteria such as strong vitality and high adaptability in natural hydrosphere as well as easily horizontal gene transfer also determine that once the engineering cyanobacteria are released into the natural environment,they may cause a series of biological safety issues such as species invasion and genetic pollution.To address these problems,we used model cyanobacteria Synechococcus elongatus PCC 7942(hereafter Synechococcus 7942)and the fast-growing cyanobacteria Synechococcus elongatus UTEX 2973(hereafter Synechococcus 2973)as host strains to conduct a biocontainment strategy study.In order to develop an automatic inducible biological storage system in cyanobacteria,this paper carried out the following work:i)We selected six metal ion-inducible promoters derived from cyanobacteria and used gus A as a reporter gene to measure the activity of?-D-glucuronidase in two kind of host bacteria.We identified that iron-repressed promoter Pisi AB,derived from Synechococcus 7942,has low leakage and high induction fold(induction fold of 5.4 in Synechococcus 7942 and 7.9 in Synechococcus 2973).ii)Using Pisi AB as a promoter,we introduced the Holin-endolysin system P22 and the nuclease Nuc A system,and the growth curve measurement proved that both can inhibit the growth of Synechococcus but cannot completely kill the cells.Immediately after,we tested four kind of suicide programs based on the toxin/antitoxin cassette(TA).Among them,the TA cassette sep A1/T1,derived from Synechococcus 7942,had a significant effect on Synechococcus 2973,and caused the host strain to die quickly after induction of iron deficiency,while sep A2/T2 was suitable for both hosts in this article.iii)We measured the escape frequency for these two biocontainment systems and found that the escape rate was below the detection limit of 10^-9 after 3 days,which was up to NIH's standards for biological storage(<10^-8/CFU).In addition,we compared the growth of engineered strains and their wild-type strains in tap water,and found that the wild strains could grow but the engineered bacteria failed to form visible colonies,proving that the biosafe strategy of integrating the iron-repressed promoter Pisi AB and the toxin/antitoxin module in Synechococcus has practical significance for large-scale application,which laid the foundation for further study for solving the cyanobacterial biosecurity problems.