Rapid Gene Target Tracking for Enhancing β-Carotene Production Using Flow Cytometry-Based High-Throughput Screening in Yarrowia lipolytica
Mengmeng Liu, Jin Zhang, Xiaoqin Liu, Jin Hou, Qingsheng Qi
Abstract
β-Carotene is a high-value-added product that is widely used in the aquaculture, food, cosmetic, and pharmaceutical industries. In our previous study, Yarrowia lipolytica has been engineered extensively to produce β-carotene. To further improve its production, high-throughput screening and the identification of new beneficial gene targets are required. Herein, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-β-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased β-carotene significantly. By engineering these targets in a high-β-carotene production, a strain that produced 9.4 g/L β-carotene was constructed.