Intracellular Environment Improvement of Mycobacterium neoaurum for Enhancing Androst-1,4-Diene-3,17-Dione Production by Manipulating NADH and Reactive Oxygen Species Levels

Abstract

As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD has received extensive attention in recent years. In a previous study, Mycobacterium neoaurum JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from Bacillus subtilis to balance the intracellular NAD+ availability in order to strengthen the ADD yield. Then, the catalase gene from M. neoaurum was also over-expressed to simultaneously scavenge the generated H2O2 and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12yodC-katA produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.

Improving the Production of 9,21-di​hydroxy-​20-​methyl-pregna-​4-​en-​3-​one from Phytosterols in Mycobacterium Neoaurum by Modifying Multiple Genes and Improving the Intracellular Environment

“…Regeneration of NAD + and enhancement of the NAD + /NADH ratio have been proven to be able to enhance the ability of microorganisms to transform phytosterols. Overexpression of NADH oxidase in M. neoaurum JC-12 increased ADD production by 43% [17]. Overexpression of avin oxidoreductase and NADH oxidase from Lactobacillus brevis in M. neoaurum TCCC 11978 increased the NAD + /NADH ratio by 113% and 192%, respectively, and signi cantly enhanced the conversion ratio of AD(D) [18].…”

“…In addition, hydrogen peroxide (H2O2) is produced due to incomplete oxidation during aerobic metabolism and the regeneration of avin adenine dinucleotide (FAD) during the phytosterol transformation process [17]. A high level of H2O2 can damage proteins, DNA, and lipids in cells, resulting in inhibition of cell growth and metabolite yield [25].…”

“…Manipulation of NAD + /NADH contents could enhance the production of AD and ADD to various degrees [17][18][19]. Overexpression of NADH oxidase in M. neoaurum JC-12 increased ADD production by 43% [17], and an increase in the amounts of the ratio of NAD + /NADH in M. neoaurum TCCC 11978 enhanced the productivity of ADD by 93%. Thus, the elimination of H 2 O 2 and regeneration of NAD + could contribute to higher concentrations of phytosterol metabolites.…”

“…Toxic steroid intermediates cause cells to produce reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ), during aerobic metabolism. A high level of H 2 O 2 might harm cell growth, hence slowing the rate of phytosterol degradation and decreasing the yield of metabolites [17], and vice versa. For example, elimination of H 2 O 2 from M. neoaurum JC-12 increased the 4-HP yield by 24%.…”

“…In addition, during the phytosterol degradation process, intracellular nicotinamide adenine dinucleotides (NAD + and NADH) are consumed, which participate in multistep reactions during phytosterol degradation, such as when dehydrogenation occurs. NAD + /NADH regeneration and maintenance of the redox balance are considered the rate-limiting factors in the steroid degradation pathway [17,18]. Manipulation of NAD + /NADH contents could enhance the production of AD and ADD to various degrees [17][18][19].…”