Development and optimization of genetic toolboxes for a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973

The fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973) has been considered a good chassis candidate for "microbial cell factory" as it can perform oxygenic photosynthesis and its doubling time can be as short as 1.9 h. However, the limited genetic tools currently restrict its further research and application efforts using synthetic biology approaches. In this study, a series of genetic tools were systematically developed and optimized for Synechococcus 2973. First, the introduction of Tfp pilus assembly protein encoding gene pilN into Synechococcus 2973 successfully recovered its natural transformability, which greatly simplified the DNA transformation process. Second, a series of promoters with different strengths were evaluated and the super-strong promoters including P_cpc560 from Synechocystis sp. PCC 6803, native P_psbA2 and P_psbA3 of Synechococcus 2973 were found with the highest activity of β-galactosidase among those evaluated by miller values. Some promoters related to photosystems (i.e., P_psbA2, P_psbA3, P_6803psbA2 and P_cpc560) were also demonstrated to be induced by high intensity of light. Third, three lactose induction systems were evaluated, among which P_lac combined with lacI^q showed the best application prospect with great induction capacity, low leakage and middle induced expression. Fourth, the translational on riboswitch theoE* , the transcriptional off riboswitches theo/yitJ and xpt(C74U)/metE and an artificial inducing system combining theoE* with T7 RNA polymerase were successfully developed and characterized in Synechococcus 2973. Finally, by using T7 induction system to control the expression of both small RNA and chaperone Hfq, a small RNA regulatory tool was developed and optimized to be a strictly inducible off system for gene regulation in Synechococcus 2973. The work here presented valuable genetic toolboxes necessary for metabolic engineering and synthetic biology research in Synechococcus 2973, which will facilitate the future application of the fast growing cyanobacterial chassis.