Transcription Variations of Key Genes in the Central Metabolism Pathways of the Sterols Transformation in Mycobacterium

doi:10.13560/j.cnki.biotech.bull.1985.2020-1497

Abstract

Abstract:

To investigate the regulation mechanism of central metabolism during the transformation of sterols to steroidal drug intermediates in Mycobacterium neoaurum,RNA sequencing and qRT-PCR analysis were employed to analyze the transcriptional levels of key genes in the central metabolism of steroid drug intermediate-producing strains. Meanwhile,the glucose consuming rate during the transformation process was assessed. The results showed that the transcription of pfkB and pyk in glycolytic pathway was downregulated by 1.96-fold and 1.49-fold. The transcription of zwf and gntZ in pentose phosphate pathway was downregulated by 3.57-fold and 2.43-fold. And the citA,icd2 and kdg transcription were downregulated about 2.5-fold,1.78-fold and 1.92-fold,respectively. In addition,the determination of glucose consumption rate revealed that the initial metabolic rate of the steroidal intermediate-producing strain was significantly lower than that of the wild-type strain. This indicated that the central metabolic pathway of M. neoaurum was inhibited to some extent during the conversion of sterols to value-added steroidal intermediates.

Key words: Mycobacterium neoaurum, steroidal drug intermediate, central metabolism, 9-OHAD, glucose

XIONG Liang-bin, SUN Ji, LIU Xian-zhou, QU Zhan-guo, JI Yu-qing, XU Yi-xin, WANG Feng-qing. Transcription Variations of Key Genes in the Central Metabolism Pathways of the Sterols Transformation in Mycobacterium[J]. Biotechnology Bulletin, 2021, 37(10): 120-127.

Figures/Tables 7

Table 1 Strains and plasmids used in this study

名称Name	描述Description	来源Source
菌株
Escherichia coli DH5α	用于质粒转化扩增	天根生化
Mycobacterium neoaurum ATCC 25795（Mn）	野生型菌株	ATCC
MnΔkstD1（MnΔk1）	在野生型Mn菌株中删除kstD1基因,具有转化甾醇积累9-OHAD的生产能力	姚抗等^[4]构建
质粒
p2NIL	用于分枝杆菌基因敲除的同源臂构建载体,Kan^R	Dr. Parish惠赠
p2N-kstD1	携带kstD1基因敲除同源臂的p2NIL重组质粒	姚抗等^[4]构建
pGOAL19	Hyg^R, Pag85-lacZ, Phsp60-sacB, Pac I cassette vector, Amp^R	Dr. Parish惠赠
p19-kstD1	p2NIL携带kstD1基因敲除同源臂及pGOAL19筛选表达框组成的重组质粒,Kan^R和Hyg^R	姚抗等^[4]构建

Table 2 Primers used in this study

用途 Purpose	名称Name	描述Description
基因删除引物 Primers for gene deletion	D-kstD1-UF-Hind III	CAGTaagcttCTTCTCAGCCATACGTGGCTCCTA
基因删除引物 Primers for gene deletion	D-kstD1-UR-Pst I	ttaactgcaggtcctgggcagtcatgtagaacac
	D-kstD1-DF-Pst I	tacactgcagttgcatctcgctggaaaggcctga
	D-kstD1-DR-Kpn I	TATAggtaccCGCGGTCAGCGTTCCGATGAACTT
qRT-PCR分析引物 Primers for qRT-PCR analysis	R-16S rRNA-F	CCTATGTTGCCAGCGGGTTATGC & GCGATTACTAGCGACTCCGACTTCA
qRT-PCR分析引物 Primers for qRT-PCR analysis	R-pfkA-F & R	CCAGGCTTGAACGCCGTGATCAG & GATCGGGATCAACACATCGATGC
	R-pfkB-F & R	TACCTGCTGCTGCCGACGATCCG & ACCACGTCGGCGTACCAGCTCGT
	R-pyk-F & R	GACCACGAAGAGTCCTATCGCCG & ACCAGTCCGACGTTGCCGTCGTC
	R-zwf-F & R	CTGATGCCGGCGATCTACGACCT & GTAGAACGCGTGATTGCCGTTGG
	R-gntZ-F & R	CTGACCGAAGGCTACGGATTGAT & GCAGCCAGCATCTCGTAACCCTC
	R-citA-F & R	GACATCGACGACCTGGTATCGGA & ACATAGGACAGCGCCATCGCCGA
	R-icd2-F & R	ATCAAGCTGCCGAACATCAGCGC & TGGCCCATGCTGTGCGGGTGCTT
	R-kdg-F & R	CGGCCAAGGCGATGATCGACAAC & CATCGTCTCGCACTTCTTGATGG

Fig. 1 Effect of deleting kstD1 on the Mycobacterium PCR verification and biotransformation of sterols A: Evidence for allelic replacement of kstD1 gene. Mn: The wild-type strain. MnΔk1: Deletion of kstD1 in the Mn strain. B: HPLC and TLC analysis results of the sterol biotransformation in MnΔk1 strain

Fig. 2 Transcriptional comparisons of genes related in central metabolism pathways during the sterol transformation in Mycobacterium A: Transcript profiles of the genes in wild-type Mn strain. B: Transcript profiles of the genes in MnΔk1 strain. Mn: The wild type strain was cultivated in fermentation medium without phytosterol addition. Mn+C: The wild type strain was cultured in fermentation medium with 1 g/L of phytosterol addition. MnΔk1+C: the strain MnΔk1 was cultured in fermentation medium with 1 g/L of phytosterol addition

Fig. 3 qRT-PCR analysis of key genes in central metabolism pfkA & pfkB: 6-phosphofructokinase; pyk: pyruvate kinase; zwf: glucose 6-phosphatedehydrogenase; gntZ: 6-phosphogluconate dehydrogenase; citA: citrate synthase; icd2: isocitric dehydrogenase; kdg: oxoglutarate dehydrogenase

Fig. 4 Metabolic rate of glucose during the sterol bioconv-ersion in Mycobacterium

Fig. 5 Schematic diagram of sterol metabolism pathway and central metabolism pathway in Mycobacterium CHO: cholesterol; CEO: cholesterone; AD: 4-androstene-3,17-dione; ADD: 1,4-androstadiene-3,17-dione; 9-OHAD: 9α-hydroxy-4-androstene-3,17-dione; 9-OHADD: 9 α-hydroxy-1,4-androstadiene-3,17-dione; Pro-CoA: propionyl-CoA; Hex: hexose; Hex-P: hexose phosphate; G-3-P: glyceraldehyde 3-phosphate; PEP: phosphoenolpyruvate; PYR: pyruvic acid; Ac-CoA: acetyl-CoA; (S)-MM-CoA: (S)-methylmalonyl-CoA; (R)-MM-CoA: (R)-methylmalonyl-CoA; 2-MC: 2-methycitrate; 2-MIC: 2-methylisocitrate; CIT: citrate; ACN: aconitic acid; ISOCIT: isocitrate; α-KG: α-ketoglutarate; SucCoA: succinyl-CoA; SUC: succinate; FMA: fumarate; MAL: malate; OAA: oxaloacetate

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