Building AraBAD Toolkit For Controllable Regulation Of Gene Expression And Photonixotrophic Cultivation In S.Elongatus

Cyanobacteria have gained considerable popularity as a bio-energy synthetic platform,which can directly capture carbon dioxide(CO2)and solar energy for bioconversions to decrease the release of CO2 and mitigate global warming.The engineered cyanobacteria Synechococcus elongatus is the main using moldel bacteria.However,genetic control systems especially reliable inducible modules,which can boost production through precise regulation of heterologous gene expression,are limited in S.elongatus.Meanwhile,S.elongatus is a type of obligate photoautotrophs,and biomass accumulation is strongly dependent on the availability of light,resulting in low working efficiency.Natural conditions containing the light and dark cycle should be explored to maximize productivity and lead to efficient utilization of resources to remain competitive.To engineer S.elongatus as an ideal bio-energy synthetic platform the inducible promoter PTrc promoter has been refined from Escherichia coli to work in S.elongatus,but the inducer isopropyl-?-D-thiogalactoside is expensive and toxic.In the meantime,to promote the efficiency,potomixotrophic cultivation has been applied in S.elongatus with the additional organic carbon sources.Arabinose is an ideal heterologous organic carbon sources for its availability and economic and environmental benefits.Thus,in this study,we developed arabinose based AraBAD toolkit,which consisted of PBAD promoter that using arabinose as inducer and the arabinose metabolic module in S.elongatus.The AraBAD toolkit can achieve reliable regulation of gene expression and photomixotrophic cultivation.The main contents are as follows:(1)The research first introduced the PBAD promoter from E.coli in S.elongatus.By comparing the PBAD and PTrc promoters,we demonstrated the relative advanta ges of PBAD promoter.The PBAD promoter in S.elongatus could realize no leaky expression,linear regulation of the downstream genes,and also,the homogeneous gene expression.However,the leaky expression of PTrc promoter in S.elongatus was significant,and could hardly achieve linear regulation and homogeneous gene expression.Apart from that,high arabinose concentration did no harm to morphological change while high IPTG concentration would lead the cell elongated.Thus,the results showed that the PBAD promoter can be an ideal inducible module in S.elongatus.(2)Arabinose would suppress the growth of S.elongatus and cause the metabolic burden in S.elongates.To override the metabolic stress and realize photomixotrophic cultivation,the research applied arabinose as organic carbon source to acquire the growth of S.elongatus in diurnal condition.To optimize to choose the key exogenous genes,we first explored the arabinose transport ability of S.elongatus and found that S.elongatus owned strong ability of arabinose transport.Therefore,the research introduced the araBAD genes from E.coli to engineer S.elongatus to metabolize arabinose to enter pentose phosphate pathway to achieve photomixo trophic cultivation.(3)The research built AraBAD toolkit in S.elongatus consisting of both PBAD inducible and arabinose metabolic modules.The dynamic investigation indicated that AraBAD toolkit S.elongatus could realize gene expression and photomixotrophic cultivation simultaneously.No matter in continuous light and diurnal conditions,the PBAD promoter could achieve both reliably control gene expression.Thus,with the only additive arabinose,the strategy may generate a win-win scenario for both regulation and metabolism for autotrophic bio-energy synthetic platforms.