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Background  Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD + ) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by  Mycobacterium . Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD + -dependent process, this work utilized cofactor engineering in  Mycobacterium neoaurum  and investigated the effect on cofactor and phytosterols metabolism.    Results  Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD + /NADH ratio and the AD (D) production of  M. neoaurum  TCCC 11978 ( MNR  M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of  MNR  cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from  Lactobacillus brevis , were successfully overexpressed and heterologously expressed in  MNR  M3 to improve the intracellular ratio of NAD + /NADH. After 96 h of cultivation, the expression of these two enzymes in  MNR  M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD + /NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by  MNR  M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols.    Conclusions  The ratio of NAD + /NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in  MNR  improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant  MNR  strain may be useful in industrial production.

Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation