Harnessing novel chromosomal integration loci to utilize an organosolv‐derived hemicellulose fraction for isobutanol production with engineered Corynebacterium glutamicum

A successful bioeconomy depends on the manifestation of biorefineries that entirely convert renewable resources to valuable products and energies. Here, the poorly exploited hemicellulose fraction (HF) from beech wood organosolv processing was applied for isobutanol production with Corynebacterium glutamicum. To enable growth of C. glutamicum on HF, we integrated genes required for d‐xylose and l‐arabinose metabolization into two of 16 systematically identified and novel chromosomal integration loci. Under aerobic conditions, this engineered strain CArXy reached growth rates up to 0.34 ± 0.02 h(−1) on HF. Based on CArXy, we developed the isobutanol producer strain CIsArXy, which additionally (over)expresses genes of the native l‐valine biosynthetic and the heterologous Ehrlich pathway. CIsArXy produced 7.2 ± 0.2 mM (0.53 ± 0.02 g L(−1)) isobutanol on HF at a carbon molar yield of 0.31 ± 0.02 C‐mol isobutanol per C‐mol substrate (d‐xylose + l‐arabinose) in an anaerobic zero‐growth production process.