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

Liqiu Su,Tian Gao,Yanbing Shen,Wenkai Zhang,Min Wang,Zhihua Shang

doi:10.1186/s12934-017-0796-4

Liqiu Su, Tian Gao + Show 4 more

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https://doi.org/10.1186/s12934-017-0796-4

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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.

Highlights

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
Interaction between phytosterols degradation process and the intracellular N AD+/NADH ratio To better understand the connection of the intracellular NAD (H) with AD (D) production in M. neoaurum TCCC 11028 (MNR) Mycobacterium neoaurum Technology Culture Collection Center (TCCC) 11978 (M3), the intracellular NAD+/NADH ratio and AD (D) production during biotransformation were examined at regular intervals; on the other hand, nicotinic acid (NA) was added to the fermentation system to change the intracellular redox status
We preliminarily inferred a correlation between the NAD+/NADH ratio and the AD (D) productivity during the late biotransformation period in Mycobacterium neoaurum TCCC M3 (MNR M3)

Summary

Introduction

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. A subset of metabolic engineering, is defined as the manipulation of the cofactors in metabolic pathways and optimize dynamic control of the target metabolic flux. It has been successfully applied in Escherichia coli (E. coli) [2, 3], Lactococcus lactis [1], Saccharomyces cerevisiae [4], Klebsiella pneumoniae [5], Serratia marcescens [6], Torulopsis glabrata [7], and Colletotrichum lini [8]. Nicotinamide adenine dinucleotides (NADH and N AD+) is an important cofactor pair that acts in plenty of oxidation–reduction (redox) reactions and regulates various enzyme activities and genetic processes [5,6,7, 9]. The overexpression of the gene of pncB, which encodes NAPRTase, results in increased total NAD+ level and ratio of NAD+/NADH [2]

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