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

Abstract

BackgroundCofactor 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.ResultsThrough 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.ConclusionsThe 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.

Biosynthesis and Industrial Production of Androsteroids

“…Propionyl-CoA is the major by-product during β-oxidation of the phytosterol side chains and is toxic in high concentrations [ 31 ]. Metabolism of one mole of β-sitosterol yields four moles of FADH2, two moles of Acetyl-CoA, and two moles of propionyl CoA [ 32 , 33 ]. Thus, engineering propionyl-CoA metabolism in mycobacteria through co-expression of the PCC subunit beta and type II NADH dehydrogenase (NDH-II) yielded an economical phytosterol biotransformation [ 27 ].…”

“…The maintenance of the redox balance by the NADH/NAD + levels within engineered mycobacteria intracellular environments is considered one of the rate-limiting factors in the phytosterols conversion process. Overexpression of the NADH:flavin oxidoreductase in mycobacteria, and a NADH oxidase from Lactobacillus brevis , has been shown as a useful strategy for industrial androstenedione production [ 32 ]. Similarly, the heterologous expression of Bacillus subtilis NADH oxidase in M. neoaurum , along with overexpression of the catalase gene, led to 80% higher yield of 1,4-androstadiene-3,17dione [ 29 ].…”

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum

“…During the fermentation of MNR, MNR-prpR and MNR-prpDBC, samples were taken at 24-hour intervals, and the samples were divided equally. One part was used to detect the dry cell weight (DCW) according to the previous method [40], and the other was used to analyze intracellular propionyl-CoA. Intracellular propionyl-CoA levels were detected using a modified method previously described by Xu et al [41].…”

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

“…Wei et al over-expressed 3-ketosteroid-Δ 1 -dehydrogenase (KSDD) in M. neoaurum to increase soybean phytosterol bioconversion [11]. Su et al used cofactor engineering to maintain the balance of redox to promote steroid biotransformation [12]. In our previous study, we used stepwise pathway engineering to strengthen the metabolic flux of the sterols for the improvement of ADD production [13].…”