*3.5. Phototaxis-Based Screening*

Each colony on agar plates was transferred into 1 mL of NIES-C medium in each well of a 24-well microplate and cultured under low light condition (40 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1) at 23 ◦C. All cultures of 10,000 transformants were then mixed, centrifuged, and resuspended in 6 mL of NIES-C medium to a cell density of 6.0 × 10<sup>8</sup> cells mL−1. For screening of the mixture of 10,000 transformants, a microfluidic device with enlarged chambers (8 mm in diameter) and a microchannel (width from 8 mm to 0.4 mm, height 100 μm) was used. A 2.75 mL aliquot of dark-adapted cell mixture was loaded into the inlet chamber and exposed to blue LED light (70 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1) for 10 min to isolate cells showing fast phototaxis. The cells arrived at the outlet chamber within 10 min and were collected by pipetting and recovered in NIES-C medium for 2 h. The cells were then used for the next cycle of phototaxis-assisted screening. After five cycles of screening, we obtained the cell mixture, and a 100 μL aliquot of the culture was plated onto NIES-C agar for the isolation of mutants as separated colonies. To compare the growth of mutant mixtures between each cycle of phototaxis-based screening, cells were inoculated to a density of 10,000 cells mL−<sup>1</sup> and grown photo-autotrophically.
