Regulation of NAD (H) Pool by Overexpression of Nicotinic Acid Phosphoribosyltransferase for AD (D) Production in Mycoba
Phytosterol (PS) biotransformation to androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) was proposed a NAD+-dependence process in Mycobacterium neoaurum (MNR). To improve the PS biotransformation, overexpression of nicotinic acid phosph
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1 Introduction Androst-4-ene-3,17-dione (AD) and androst-1 4-diene-3 17-dione (ADD) are two versatile biological intermediates, kinds of strains have been reported capable of converting phytosterol (PS) to AD (D) via the side chain cleavage [1]. Mycobacterium is one of the most efficient AD (D) producer still [2]. It was reported by many studies that manipulation of the enzyme activities which included in steroid nucleus oxidation and the side chain cleavage pathways could enhance the AD (D) production, such as the deletion of 3-ketosteroid-9a-hydroxylase (Ksh) and 3-ketosteroid-1-dehydrogenase (KstD), or the overexpression of cholesterol oxidase and 3b-hydroxysteroid dehydrogenases (3b-HSDs) [3]. But little known the cofactor engineering could be used in Mycobacterium biocatalysis. It has been known that cofactors (NAD (H), NADP (H), FAD (H2), ATP, coenzyme A and its derivatives) are very important in the production of fermentation products. In the PS biotransformation process, it was speculated that side chain degradation of sitosterol resulted in the formation of propionyl-SCoA, FADH2, NADH, and acetic acid. The propionates and acetates are utilized through the tricarboxylic acid cycle (TCA). The complete breakdown of sitosterol to AD (D) gave 21 molecules of NADH and 10 molecules of FADH2 [4, 5]. It is crucially important for PS biotransformation that NADH can be oxidized to NAD+. Nicotinic acid phosphoribosyltransferase (NAPRTase), encoded by the pncB gene, is rate limiting in the salvage synthesis of NAD (H) from nicotinic acid (NA) [6]. Helena IMB [7] proved that the salvage pathway to synthesise NAD+ was included in L. Su Y. Shen T. Gao L. Cui J. Luo M. Wang (&) Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, TEDA, No. 29, 13th Avenue, Tianjin 300457, People’s Republic of China e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2018 H. Liu et al. (eds.), Advances in Applied Biotechnology, Lecture Notes in Electrical Engineering 444, https://doi.org/10.1007/978-981-10-4801-2_36
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Mycobacterium tuberculosis, and NAPRTase played an important role in cofactor salvage pathway. NAPRTase uses NA and a-D-5-phosphoribosyl-1-pyrophosphate (PRPP) with ATP hydrolysis to form nicotinate mononucleotide (NAMN), which is a precursor of NAD+. Liang LY [6, 8] had reported that overexpression of the pncB gene in Escherichia coli (E. coli) could increase the NAD (H) levels and the NAD+/NADH ratio. By overexpression of NAPRTase in E. coli NZN111, the NAD+ concentration increased 6.2-fold [6]. After overexpression of the pncB and nadE (encoding NAD synthetase) in E. coli, 7-fold and 2-fold increased intracellular concentrations of NAD (H) and NADP (H), respectively [9]. In this work, to increase the availability of intracellular NAD+ for AD (D) production, we constructed a recombinant strain, MNR M3-pMV306-pncB, and investigated the i
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