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

Summary 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 CA rXy reached growth rates up to 0.34 ± 0.02 h −1 on HF . Based on CA rXy, we developed the isobutanol producer strain CI sArXy, which additionally (over)expresses genes of the native l ‐valine biosynthetic and the heterologous Ehrlich pathway. CI sArXy 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.