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Abstract
Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10–CcaS#10–CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10–CcaS#10–CcaR system was combined with a blue light-activated YF1–FixJ–PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene (gltA) and PHB synthesis gene (phbCAB) expression. Consequently, the strain RBS34, which has high gltA expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.
Highlights
Several natural cells have light-sensing proteins that respond to red, green, blue, ultraviolet, or near-infrared (NIR) [1,2,3]
In the RBS10–CcaS#10–CcaR system, expression of sfgfp was up and downregulated under green and red light with a dynamic range of approximately 10-fold but demonstrated a negligible response to blue light and dark conditions (Figure 3, Table 1). These results demonstrate that an RBS10–CcaS#10–CcaR system can be incorporated into a dual-switch system
The engineered strain GY demonstrated similar growth to that of E. coli TOP10 (Figure 4b). These findings indicate that the combination of optogenetic RBS10–CcaS#10–CcaR–sfGFP and YF1–FixJ–PhlF–RFP enables highly independent regulation
Summary
Several natural cells have light-sensing proteins that respond to red, green, blue, ultraviolet, or near-infrared (NIR) [1,2,3]. CcaR [7,8,9], YF1–FixJ [10], and Cph8–OmpR [11]) systems. A two-component system comprises a sensory histidine kinase (HK) and a response regulator (RR). The activities of the HK and RR are directed by the phosphorylation or dephosphorylation that occurs in response to different wavelengths of illumination [7,12]. CcaS belongs to the cyanobacteriochrome family of proteins, which respond to green and red light through the combination of the N-terminal GAF domain (cyclic GMP phosphodiesterase, adenylyl cyclase, FhlA). CcaR is a member of the OmpR class, and it contains
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