黑曲霉发酵积累L-苹果酸的关键基因差异分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0512

摘要/Abstract

摘要：

【目的】微生物生产L-苹果酸是目前极为高效的一种生产方法，为揭示黑曲霉生产L-苹果酸的生物合成机制，借助转录组学分析探究关键基因在代谢调控过程中的差异变化。【方法】MA-1菌株在发酵144 h产酸水平达到88.27 g/L，选取产酸速率不同的3个时间点（48、72和120 h）进行转录组分析。【结果】GO分析显示，生物过程的调控、RNA结合与核糖体相关的二级条目下的显著基因差异性最大、数目最多。转录组分析发现发酵48-120 h过程中，HacA、Ace1、Rpn4等转录调控因子始终处于较高水平，FPKM值达到20 181.64-94 573.00，且葡萄糖转运蛋白（Rco-3）、酮戊二酸/苹果酸转运蛋白Yhm1呈现相对较高的转录水平，FPKM值达到4 971.83-6 575.46；糖酵解EMP途径中，其重要的限速酶丙酮酸激酶（FPKM值13 109.15-25 649.30）和6个己糖激酶中的ANI_1_1984024（FPKM值达4 111.68-7 325.43）转录水平相对较高，再者从葡萄糖转化为丙酮酸途径中的其他酶转录水平均在120 h呈下降趋势；TCA循环中整体基因转录水平均维持较高水平，为细胞代谢提供充足能量，rTCA途径中柠檬酸合酶表达水平最高，苹果酸脱氢酶次之，乙醛酸循环水平较低。【结论】推测黑曲霉MA-1菌株中L-苹果酸合成的主要途径为rTCA和丙酮酸羧化途径。

关键词: 黑曲霉, L-苹果酸, 转录组, 差异基因, 代谢调控

Abstract:

【Objective】 Microbial production of L-malic acid is currently an extremely efficient producing method. In order to reveal the biosynthetic mechanism of L-malic acid production by Aspergillus niger, the differential changes of key genes in the metabolic regulation process were explored with the help of transcriptomic analysis. 【Method】The acid production level of MA-1 strain reached 88.27 g/L at 144 h of fermentation, and three time points（48, 72, and 120 h）with different acid production rates were selected for transcriptome analysis. 【Result】GO analysis showed that the regulation of biological processes, RNA binding and ribosome-related secondary entries under the significant genes were the most differentiated and the largest number. Transcriptome analysis revealed that transcriptional regulators such as HacA, Ace1, and Rpn4 were consistently at high levels during fermentation from 48 to 120 h, with FPKM values reaching 20 181.64-94 573.00, and that glucose transporter protein（Rco-3）, and ketoglutarate/malate transporter protein Yhm1 showed relatively high transcript levels, with FPKM values reaching 4 971.83-6 575.46; in the glycolytic EMP pathway, its important rate-limiting enzyme pyruvate kinase（FPKM values of 13 109.15-25 649.30）and ANI_1_1984024 among the six hexokinase enzymes（FPKM values up to 4 111.68-7 325.43）showed relatively high transcript levels, and then the pathway for the conversion from glucose to pyruvate. The transcript levels of other enzymes in the glucose-to-pyruvate pathway showed a decreasing trend at 120 h. The transcript levels of the whole genes in the TCA cycle were maintained at a high level to provide sufficient energy for cellular metabolism, and the highest level of citrate synthase in the rTCA pathway, followed by malate dehydrogenase, and the level of the glyoxylate cycle was low.【Conclusion】The main pathways of L-malate synthesis in A. niger MA-1 strain were presumed to be rTCA and pyruvate carboxylation pathway.

Key words: Aspergillus niger, L-malic acid, transcriptome, differential genes, metabolic regulation

刘书彤, 武胜, 谭奕阳, 王德培, 薛鲜丽. 黑曲霉发酵积累L-苹果酸的关键基因差异分析[J]. 生物技术通报, 2024, 40(12): 227-238.

LIU Shu-tong, WU Sheng, TAN Yi-yang, WANG De-pei, XUE Xian-li. Differential Analysis of Key Genes Involved in the Accumulation of L-malic Acid by Aspergillus niger Fermentation[J]. Biotechnology Bulletin, 2024, 40(12): 227-238.

图/表 8

参考文献 33

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基因ID Gene ID	酶 Enzyme	MA_48 h	MA_72 h	MA_120 h
ANI_1_558134	葡萄糖转运蛋白Rco-3 Glucose transporter rco-3	17766.95	14805.64	20002.87
ANI_1_2730024	己糖载体蛋白 Hexose carrier protein	99.77	69.82	157.77
ANI_1_546054	高葡萄糖转运蛋白 High-affinity glucose transporter	7.80	11.33	5.38
ANI_1_2366024	葡萄糖苷转运蛋白 MFS alpha-glucoside transporter	137.39	233.78	309.00
ANI_1_1282124	葡萄糖苷转运蛋白 MFS alpha-glucoside transporter	149.08	85.48	91.51
ANI_1_1272144	葡萄糖苷转运蛋白 MFS alpha-glucoside transporter	2.92	0.00	0.00
ANI_1_572084	葡萄糖转运蛋白MFS glucose transporter	1531.73	1558.22	1556.82
ANI_1_486024	单糖转运蛋白 MFS monosaccharide transporter	770.19	578.17	554.32
ANI_1_1330064	单糖转运蛋白 MFS monosaccharide transporter	31.23	31.30	27.03
ANI_1_158044	单糖转运蛋白 MFS monosaccharide transporter	63.25	36.34	57.28
ANI_1_2012094	单糖转运蛋白 MFS monosaccharide transporter	8.57	3.87	21.48
ANI_1_52054	单糖转运蛋白 MFS monosaccharide transporter	4.79	3.13	2.29
ANI_1_404044	单糖转运蛋白 MFS monosaccharide transporter	1.70	2.06	2.29
ANI_1_390124	肌醇转运体 MFS myo-inositol transporter	5.32	0.67	1.06
ANI_1_1588124	奎宁酸转运蛋白 MFS quinate transporter	37.11	35.14	18.33
ANI_1_158064	糖转运蛋白 MFS sugar transporter	3.74	4.32	0.69
ANI_1_86024	糖转运蛋白 Sugar transporter	2.89	4.55	3.08
ANI_1_1080034	糖转运蛋白 Sugar transporter	6.85	17.83	11.66
ANI_1_1626094	糖转运蛋白 Sugar transporter	4.61	8.07	17.06
ANI_1_54124	C4-二羧酸/苹果酸转运蛋白 C4-dicarboxylate/malic acid transporter Mtp1	185.13	289.40	206.71
ANI_1_1316144	C4-二羧酸/苹果酸转运蛋白 C4-dicarboxylate/malic acid transport Mtp2	223.13	268.80	545.86
ANI_1_2040144	C4-二羧酸/苹果酸转运蛋白 C4-dicarboxylate/malic acid transport Mtp3	687.92	164.78	206.71
ANI_1_1474094	柠檬酸转运蛋白Citrate transporter protein CtpA	2408.95	2095.42	2202.19
ANI_1_926164	三羧酸盐转运蛋白 Tricarboxylate transport protein	10.55	6.04	6.22
ANI_1_874084	酮戊二酸转运蛋白 Ketoglutarate transporter protein Yhm1	6575.46	4971.83	6272.55
ANI_1_3028024	酮戊二酸转运蛋白 Ketoglutarate transporter protein Yhm2	1.92	2.01	6.22
ANI_1_2232014	2-酮葡萄糖酸转运蛋白2-Ketogluconate transporter	0.97	7.21	2.15
ANI_1_1286124	2-酮葡萄糖酸转运蛋白2-Ketogluconate transporter	4.87	0.00	1.08
ANI_1_942124	草酰乙酸转运蛋白 Oxaloacetate transport protein Oac	584.25	614.94	607.86
ANI_1_928144	羧酸转运蛋白 Carboxylic acid transport protein	186.12	74.48	141.07
ANI_1_478154	柠檬酸转运蛋白Citrate transporter protein CexA	488.17	711.65	2363.22

基因ID Gene ID	酶 Enzyme	MA_48 h	MA_72 h	MA_120 h
ANI_1_558134	葡萄糖转运蛋白Rco-3 Glucose transporter rco-3	17766.95	14805.64	20002.87
ANI_1_2730024	己糖载体蛋白 Hexose carrier protein	99.77	69.82	157.77
ANI_1_546054	高葡萄糖转运蛋白 High-affinity glucose transporter	7.80	11.33	5.38
ANI_1_2366024	葡萄糖苷转运蛋白 MFS alpha-glucoside transporter	137.39	233.78	309.00
ANI_1_1282124	葡萄糖苷转运蛋白 MFS alpha-glucoside transporter	149.08	85.48	91.51
ANI_1_1272144	葡萄糖苷转运蛋白 MFS alpha-glucoside transporter	2.92	0.00	0.00
ANI_1_572084	葡萄糖转运蛋白MFS glucose transporter	1531.73	1558.22	1556.82
ANI_1_486024	单糖转运蛋白 MFS monosaccharide transporter	770.19	578.17	554.32
ANI_1_1330064	单糖转运蛋白 MFS monosaccharide transporter	31.23	31.30	27.03
ANI_1_158044	单糖转运蛋白 MFS monosaccharide transporter	63.25	36.34	57.28
ANI_1_2012094	单糖转运蛋白 MFS monosaccharide transporter	8.57	3.87	21.48
ANI_1_52054	单糖转运蛋白 MFS monosaccharide transporter	4.79	3.13	2.29
ANI_1_404044	单糖转运蛋白 MFS monosaccharide transporter	1.70	2.06	2.29
ANI_1_390124	肌醇转运体 MFS myo-inositol transporter	5.32	0.67	1.06
ANI_1_1588124	奎宁酸转运蛋白 MFS quinate transporter	37.11	35.14	18.33
ANI_1_158064	糖转运蛋白 MFS sugar transporter	3.74	4.32	0.69
ANI_1_86024	糖转运蛋白 Sugar transporter	2.89	4.55	3.08
ANI_1_1080034	糖转运蛋白 Sugar transporter	6.85	17.83	11.66
ANI_1_1626094	糖转运蛋白 Sugar transporter	4.61	8.07	17.06
ANI_1_54124	C4-二羧酸/苹果酸转运蛋白 C4-dicarboxylate/malic acid transporter Mtp1	185.13	289.40	206.71
ANI_1_1316144	C4-二羧酸/苹果酸转运蛋白 C4-dicarboxylate/malic acid transport Mtp2	223.13	268.80	545.86
ANI_1_2040144	C4-二羧酸/苹果酸转运蛋白 C4-dicarboxylate/malic acid transport Mtp3	687.92	164.78	206.71
ANI_1_1474094	柠檬酸转运蛋白Citrate transporter protein CtpA	2408.95	2095.42	2202.19
ANI_1_926164	三羧酸盐转运蛋白 Tricarboxylate transport protein	10.55	6.04	6.22
ANI_1_874084	酮戊二酸转运蛋白 Ketoglutarate transporter protein Yhm1	6575.46	4971.83	6272.55
ANI_1_3028024	酮戊二酸转运蛋白 Ketoglutarate transporter protein Yhm2	1.92	2.01	6.22
ANI_1_2232014	2-酮葡萄糖酸转运蛋白2-Ketogluconate transporter	0.97	7.21	2.15
ANI_1_1286124	2-酮葡萄糖酸转运蛋白2-Ketogluconate transporter	4.87	0.00	1.08
ANI_1_942124	草酰乙酸转运蛋白 Oxaloacetate transport protein Oac	584.25	614.94	607.86
ANI_1_928144	羧酸转运蛋白 Carboxylic acid transport protein	186.12	74.48	141.07
ANI_1_478154	柠檬酸转运蛋白Citrate transporter protein CexA	488.17	711.65	2363.22

基因ID Gene ID	酶 Enzyme	MA_48 h	MA_72 h	MA_120 h
ANI_1_1686024	6-磷酸葡萄糖-1-脱氢酶 Glucose-6-phosphate-1-dehydrogenase	13100.61	12837.52	3969.57
ANI_1_1030104	己糖激酶 Hexokinase	2777.00	2356.38	2285.70
ANI_1_1498144	己糖激酶 Hexokinase	105.23	125.65	83.98
ANI_1_1984024	己糖激酶 Hexokinase	7325.43	6395.59	4111.68
ANI_1_66114	己糖激酶 Hexokinase	2803.31	812.58	3533.53
ANI_1_802134	己糖激酶 Hexokinase	1605.79	1858.94	1593.42
ANI_1_378054	己糖激酶 Hexokinase	50.67	33.99	176.57
ANI_1_2126094	6-磷酸葡萄糖酸脱氢酶 6-Phosphogluconate dehydrogenase	92.57	131.83	166.88
ANI_1_282094	6-磷酸葡萄糖酸脱氢酶 6-Phosphogluconate dehydrogenase	25702.34	28531.95	9040.53
ANI_1_1786144	葡萄糖-6-磷酸异构酶 Glucose-6-phosphate isomerase	278.67	252.32	338.06
ANI_1_754144	6-磷酸葡萄糖异构酶 Glucose-6-phosphate isomerase	9834.47	8628.38	2883.24
ANI_1_926074	转酮醇酶 Transketolase	22848.37	21614.21	6073.32
ANI_1_1068024	磷酸葡萄糖变位酶 Phosphoglucomutase	6415.35	6118.55	4245.19
ANI_1_1946064	磷酸葡萄糖变位酶 Phosphoglucomutase	5347.43	4869.3	4366.85
ANI_1_1646064	转醛酶 Transaldolase	585.61	699.29	576
ANI_1_472064	转醛酶 Transaldolase	15129.28	15500.81	5795.54
ANI_1_884184	果糖-1,6-二磷酸酶I Fuctose-1,6-bisphosphatase I	6298.43	4570.63	4615.55
ANI_1_204164	6-磷酸果糖激酶1 6-Phosphofructokinase 1	8831.83	7770.49	3201.92
ANI_1_1038024	果糖二磷酸醛缩酶II Fructose-bisphosphate aldolase,class II	27230.18	27948.01	11979.76
ANI_1_628124	果糖二磷酸醛缩酶II Fructose-bisphosphate aldolase,class II	73.08	96.81	116.28
ANI_1_1126064	丙酮酸激酶 Pyruvate kinase	25341.81	25649.30	13109.15

基因ID Gene ID	酶 Enzyme	MA_48 h	MA_72 h	MA_120 h
ANI_1_1686024	6-磷酸葡萄糖-1-脱氢酶 Glucose-6-phosphate-1-dehydrogenase	13100.61	12837.52	3969.57
ANI_1_1030104	己糖激酶 Hexokinase	2777.00	2356.38	2285.70
ANI_1_1498144	己糖激酶 Hexokinase	105.23	125.65	83.98
ANI_1_1984024	己糖激酶 Hexokinase	7325.43	6395.59	4111.68
ANI_1_66114	己糖激酶 Hexokinase	2803.31	812.58	3533.53
ANI_1_802134	己糖激酶 Hexokinase	1605.79	1858.94	1593.42
ANI_1_378054	己糖激酶 Hexokinase	50.67	33.99	176.57
ANI_1_2126094	6-磷酸葡萄糖酸脱氢酶 6-Phosphogluconate dehydrogenase	92.57	131.83	166.88
ANI_1_282094	6-磷酸葡萄糖酸脱氢酶 6-Phosphogluconate dehydrogenase	25702.34	28531.95	9040.53
ANI_1_1786144	葡萄糖-6-磷酸异构酶 Glucose-6-phosphate isomerase	278.67	252.32	338.06
ANI_1_754144	6-磷酸葡萄糖异构酶 Glucose-6-phosphate isomerase	9834.47	8628.38	2883.24
ANI_1_926074	转酮醇酶 Transketolase	22848.37	21614.21	6073.32
ANI_1_1068024	磷酸葡萄糖变位酶 Phosphoglucomutase	6415.35	6118.55	4245.19
ANI_1_1946064	磷酸葡萄糖变位酶 Phosphoglucomutase	5347.43	4869.3	4366.85
ANI_1_1646064	转醛酶 Transaldolase	585.61	699.29	576
ANI_1_472064	转醛酶 Transaldolase	15129.28	15500.81	5795.54
ANI_1_884184	果糖-1,6-二磷酸酶I Fuctose-1,6-bisphosphatase I	6298.43	4570.63	4615.55
ANI_1_204164	6-磷酸果糖激酶1 6-Phosphofructokinase 1	8831.83	7770.49	3201.92
ANI_1_1038024	果糖二磷酸醛缩酶II Fructose-bisphosphate aldolase,class II	27230.18	27948.01	11979.76
ANI_1_628124	果糖二磷酸醛缩酶II Fructose-bisphosphate aldolase,class II	73.08	96.81	116.28
ANI_1_1126064	丙酮酸激酶 Pyruvate kinase	25341.81	25649.30	13109.15

基因ID Gene ID	酶 Enzyme	定位^[18]Localization	MA_48 h	MA_72 h	MA_120 h
ANI_1_826184	2-氧戊二酸脱氢酶 2-Oxoglutarate dehydrogenase	线粒体 Mito	28679.09	23360.9	25247.17
ANI_1_1482094	2-氧戊二酸脱氢酶 2-Oxoglutarate dehydrogenase		7933.45	7173.15	10847.13
ANI_1_1410074	乌头酸水合酶 Aconitate hydratase	线粒体 Mito	81340.73	66682.01	56515.98
ANI_1_76094	ATP柠檬酸合成酶亚基ATP-citrate synthase subunit		34209.7	57561.34	26769.54
ANI_1_78094	ATP柠檬酸合成酶亚基ATP-citrate synthase subunit		21984.09	37616.57	19648.65
ANI_1_1226134	柠檬酸合酶 Citrate synthase	线粒体 Mito	2788.69	3390.38	1998.24
ANI_1_876084	柠檬酸合酶 Citrate synthase	线粒体 Mito	36133.14	36566.09	38547.96
ANI_1_952104	富马酸水合酶 Fumarate hydratase, class II	线粒体 Mito	8034.78	9005.32	10182.85
ANI_1_798074	异柠檬酸脱氢酶 Isocitrate dehydrogenase（NAD⁺）		11756.94	11619.17	14172.87
ANI_1_906164	异柠檬酸脱氢酶 Isocitrate dehydrogenase（NAD⁺）		15801.61	15039.42	17418.93
ANI_1_3136024	异柠檬酸脱氢酶 Isocitrate dehydrogenase icdA	线粒体 Mito	12236.33	9424.48	4141.83
ANI_1_914104	异柠檬酸裂解酶 Isocitrate lyase		6829.47	7628.36	4459.44
ANI_1_1256014	异柠檬酸裂解酶 Isocitrate lyase	乙醛酸循环体 Per	10619.83	4334.79	3883.43
ANI_1_1336134	异柠檬酸裂解酶 Isocitrate lyase/malate synthase	乙醛酸循环体 Per	96.46	153.45	181.95
ANI_1_268064	苹果酸脱氢酶 Malate dehydrogenase	线粒体 Mito	25629.26	28759.56	27775.12
ANI_1_320134	苹果酸合酶 Malate synthase	乙醛酸循环体 Per	5453.64	7402.82	3444.17
ANI_1_1750024	琥珀酸脱氢酶 Succinate dehydrogenase	线粒体 Mito	13360.77	13283.47	14462.48
ANI_1_230154	琥珀酰辅酶A合成酶α亚基 Succinyl-CoA synthetase alpha subunit		4877.77	5342.02	9288.16
ANI_1_58124	琥珀酰辅酶A合成酶α亚基 Succinyl-CoA synthetase alpha subunit		7810.67	7585.11	9026.54
ANI_1_1474074	柠檬酸合酶 Citrate synthase	胞质 Cyto	32971.26	70840.69	57437.58
ANI_1_2950014	柠檬酸合酶 Citrate synthase	胞质 Cyto	3.9	10.3	14
ANI_1_1826104	异柠檬酸裂解酶 Isocitrate lyase/malate synthase	胞质 Cyto	6.82	0	1.08
ANI_1_12134	苹果酸脱氢酶 Malate dehydrogenase	胞质 Cyto	19324.01	21774.87	12436.25
ANI_1_2230094	苹果酸脱氢酶 Malate dehydrogenase	胞质 Cyto	0	0	1.08
ANI_1_1422124	琥珀酸脱氢酶（泛醌）铁硫亚基 Succinate dehydrogenase（ubiquinone）iron-sulfur subunit		2900.75	2764.21	4988.07
ANI_1_436134	丙酮酸羧化酶 Pyruvate carboxylase	胞质 Cyto	34.1	62.82	38.76
ANI_1_440184	丙酮酸羧化酶 Pyruvate carboxylase	胞质 Cyto	58514.77	57300.78	17441.54