分枝杆菌转化甾醇过程的中心代谢关键基因转录差异分析

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

摘要/Abstract

摘要：

为探究新金色分枝杆菌代谢甾醇积累甾体药物中间体过程的中心代谢调节机制,采用转录组测序结合qRT-PCR技术,对比分析甾药中间体生产菌株中心代谢的关键基因转录水平,测定转化过程的葡萄糖消耗速率。结果显示,糖酵解途径的pfkB、pyk转录下调1.96倍及1.49倍;磷酸戊糖途径的zwf、gntZ转录下调3.57倍及2.43倍;三羧酸循环的citA、icd2和kdg转录分别下调2.5倍、1.78倍及1.92倍。对葡萄糖消耗速率的测定显示,中间体生产菌株的初始代谢速率显著低于野生型菌株。研究结果表明在转化甾醇积累甾药中间体过程中,分枝杆菌的中心代谢途径受到一定程度的抑制性调节。

关键词: 新金色分枝杆菌, 甾体药物中间体, 中心代谢, 9-OHAD, 葡萄糖

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

熊亮斌, 孙吉, 刘显舟, 屈占国, 计雨情, 徐一新, 王风清. 分枝杆菌转化甾醇过程的中心代谢关键基因转录差异分析[J]. 生物技术通报, 2021, 37(10): 120-127.

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.

图/表 7

表1 本研究涉及的菌株及质粒

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

表2 本研究涉及的引物

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

图1 删除kstD1基因的分枝杆菌PCR验证及转化甾醇结果 A:PCR扩增验证结果。Mn:野生型菌株的PCR扩增结果;MnΔk1:缺失kstD1基因菌株的PCR验证结果。B:MnΔk1菌株转化甾醇发酵液的HPLC和TLC分析结果

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

图2 分枝杆菌转化甾醇过程中心代谢相关基因的转录对比分析 A:野生型菌株Mn代谢甾醇的转录变化。B:MnΔk1代谢甾醇过程的转录变化。Mn:野生型菌株接种于不含甾醇的发酵培养液;Mn+C:野生型菌株接种于添加1 g/L甾醇的发酵培养液;MnΔk1+C:9-OHAD初始转化菌株接种于添加1 g/L甾醇的发酵培养液

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

图3 中心代谢关键基因转录的qRT-PCR分析结果 pfkA & pfkB,6-磷酸果糖激酶;pyk,丙酮酸激酶;zwf,葡萄糖-6-磷酸脱氢酶基因;gntZ,6-磷酸葡萄糖酸脱氢酶;citA,柠檬酸合酶;icd2,异柠檬酸脱氢酶;kdg,酮戊二酸脱氢酶

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

图4 分枝杆菌转化甾醇过程的葡萄糖代谢速率变化

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

图5 分枝杆菌甾醇代谢与中心代谢途径的关联简图 CHO:胆甾醇;CEO:胆甾酮;AD:雄甾-4-烯-3,17-二酮;ADD:雄甾-1,4-二烯-3,17-二酮;9-OHAD,9α-羟基化-雄甾-4-烯-3,17-二酮;9-OHADD:9α-羟基化-雄甾-1,4-二烯-3,17-二酮;Pro-CoA:丙酰辅酶A;Hex:己糖;Hex-P:磷酸己糖;G-3-P:甘油醛3-磷酸;PEP:磷酸烯醇式丙酮酸;PYR:丙酮酸;Ac-CoA:乙酰辅酶A;（S）-MM-CoA:（S）-甲基丙二酰-CoA;（R）-MM-CoA:（R）-甲基丙二酰-CoA;2-MC:2-甲基柠檬酸;2-MIC:2-甲基异柠檬酸;CIT:柠檬酸;CAN:乌头酸;ISOCIT:异柠檬酸;α-KG:α-酮戊二酸;SucCoA:琥珀酰-CoA;SUC:琥珀酸;FMA:延胡索酸;MAL:苹果酸;OAA:草酰乙酸

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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