Development of Synthetic Gene Circuits to Amplify the Production of Biomaterials, Cellulases, and Lentivirus

Positive feedback is one of the most common regulatory circuits in living organisms to enhance or amplify the intensity of signal. We implemented a positive feedback loop (PFL) to amplify the production of biomaterials in a filamentous fungus, Neurospora crassa and a lentivirus packaging system.In N. crassa, CLR-1 and CLR-2 are known as transcriptional activators, which regulate the expression of numerous cellulase genes. In order to develop N. crassa strains with higher degrading activity of cellulose, we constructed transgenic N. crassa with additional clr-1 or clr-2 genes under the control of a promoter from cbh-1, which is one of the CLR-1- and CLR-2-regulated cellulases. Therefore, the above new synthetic gene network creates a positive feedback circuit to amplify the expression of the transgenes. We observe that the mRNA expression of major cellulase genes (cbh-1, gh5-1, and gh6-2) is increased more than two orders of magnitude in the CLR-2 positive feedback strain compared to wild type.For development of lentivirus packaging system with positive feedback loop, we constructed a plasmid with tetracycline transactivator (tTA) under the control of tetracycline responsive element (TRE). tTA can bind to the TRE promoter and activate its own transcription. Therefore, the plasmid creates a synthetic gene network with PFL to amplify the expression of tTA and the other genes under the control of TRE promoter. Based on the PFL system, we developed a new lentivirus packaging system to amplify the expression of virus genes under the control of TRE promoter. We observe that the titer of virus with PFL is approximately 1,000 times higher compared to the ones without PFL.These results suggest that implementation of PFL into gene networks provide a feasible solution for the efficient production of biomaterials.