Green synthesis of ω-hydroxydodecanoic acid by engineering C. viswanathii with Cas13d

Abstract

ω-Hydroxydodecanoic acid (HDA) is a precursor for producing Nylon 12 but its chemical synthesis is environmentally unfriendly and bacterial production titers are only in the range of mg L⁻¹. Here we developed a novel HDA production platform by engineering the yeast Candida viswanathii that converts dodecane into dodecanedioic acid (DDA), with HDA as a metabolic intermediate. We established the RNA-guided Cas13d system for programmable knockdown of various genes involved in HDA conversion to DDA and proved that repressing these genes enhanced HDA production. Fine-tuning the repression module achieved multiplexed inhibition and increased the HDA titer. Genomic integration of the gene repression module with a new Split CRISPR system generated a strain that simultaneously suppressed all genes with repression efficiencies reaching 51–72% and sharply increased the HDA titer to 12.7 g L⁻¹ in the bioreactor, which tremendously exceeded those conferred by metabolically engineered bacteria. This study paves a new avenue for green HDA synthesis.