Multidirectional pathway engineering of Mycobacterium neoaurum LY-2 for the enhancement of androstenedione production from phytosterols.

Androstenedione (ADD), an important precursor for steroid hormone drug synthesis, can be obtained through the transformation of phytosterols by members of the Mycobacteria genus. In this study, multidirectional pathway engineering of Mycobacterium neoaurum LY-2 was applied to improve ADD production from phytosterols. Initially, the nonessential gene KasB encoding a cell wall β-ketoacyl acyl carrier protein synthase in Mycobacterium neoaurum LY-2 was knocked out, and the production of ADD was increased by 21.0%. Then, the key enzymes involved in ADD synthetic metabolism were systematically explored. These assays revealed that propionyl coenzyme A carboxylase (PCC) and Hsd4A played important roles in the synthesis of ADD. In addition, the heterologous expression of Vitreoscilla hemoglobin (VHb), a protein with oxygen-binding capacity, was successfully realized in Mycobacterium neoaurum LY-2, and the ADD concentration reached 4.42 g/L. Finally, the recombinant strain LY-2- ΔKasB-hsd4A-pcc-vgb with high ADD production was constructed by combining the above strategies. Through optimization of conversion conditions, the recombinant strain LY-2-Δ KasB - hsd4A - pcc-vgb produced an ADD concentration of 11.12 g/L when adding 20 g/L phytosterols. [Display omitted] • Mycobacterium neoaurum LY-2 efficiently catalyzes phytosterols to produce ADD. • Deletion of KasB enhanced the productivity of M. neoaurum LY-2. • PCC, Hsd4A, and VHb were co-expressed in the recombinant strain LY-2-Δ KasB. • The recombinant strain LY-2-Δ KasB - hsd4A - pcc-vgb produced 11.12 g/L of ADD. [ABSTRACT FROM AUTHOR]