希瓦氏菌Shewanella sp. MR-4在不同末端电子受体条件下的代谢通量分析

doi:10.12211/2096-8280.2022-052

摘要/Abstract

摘要：

本研究解析了Shewanella sp. MR-4在厌氧条件下以Fe³⁺、延胡索酸、硝酸和二甲基亚砜（DMSO）为末端电子受体时代谢通量分布，发现在各种不同末端电子受体条件下细胞内代谢通量分布发生显著改变。与延胡索酸相比，在Fe³⁺和硝酸为电子受体条件下，乙酸生成速率明显降低，而三羧酸循环代谢通量增加。同时，三羧酸循环回补反应存在显著差异：在延胡索酸为电子受体条件下，磷酸烯醇式丙酮酸羧化酶反应是唯一回补反应，而在Fe³⁺和硝酸条件下苹果酸酶催化成为唯一回补反应。本研究在代谢通量分析基础上定量解析了还原力NADH合成和消耗，分析了电子流向变化，发现在不同末端电子受体条件下，NADH产生途径和反应发生明显变化。研究结果为希瓦氏菌代谢工程改造提供了一定理论依据。

关键词: 希瓦氏菌, 代谢通量分析, 末端电子受体, 三羧酸循环, NADH

Abstract:

Shewanella is ubiquitous in natural environments, known to show various unique activities, such as metal reduction and trimethylamine production. The metal reduction capability expands the range of electron acceptors and enables its application in producing current in microbial fuel cells. Previous work primarily focused on the model organism Shewanella oneidensis MR-1, based on which the metabolic models were constructed for three other strains including Shewanella sp. MR-4. Interestingly, the genetic similarity does not always coincide with metabolic phenotype similarity between these two strains, suggesting that potential regulatory effects affecting cellular phenotypes are present in Shewanella sp.MR-4. This study analyzed the metabolic flux distribution of Shewanella sp. MR-4 when Fe³⁺, fumaric acid, nitric acid and dimethyl sulfoxide were used as electron acceptors under anaerobic conditions. The intracellular metabolic flux distribution was significantly changed with different electron acceptors. The formation rate of acetic acid was decreased, while the metabolic flux of the tricarboxylic acid (TCA) cycle was increased with Fe³⁺ and nitric acid as electron acceptors, compared to that with fumaric acid. The anaplerotic reaction in the TCA cycle was also dramatically altered: the phosphoenolpyruvate carboxylase-catalyzed reaction or malic enzyme-catalyzed reaction is the only anaplerotic reaction with fumaric acid or Fe³⁺ and nitric acid as electron acceptor, respectively. Based on the metabolic flux map, quantitative analysis was performed to derive the synthesis and consumption rates of reducing agent NADH, and the change of electron transfer. It was found that NADH metabolism varies significantly with the electron acceptors tested. The results provide a theoretical basis for the future metabolic engineering of Shewanella and experimental evidences that can be used for future study of different phenotypes unaccounted for in previous models of Shewanella sp. MR-4 and MR-1.

Key words: Shewanella, metabolic flux analysis, terminal electron receptor, tricarboxylic acid cycle, NADH

中图分类号:

Q816

郑庆祥. 希瓦氏菌Shewanella sp. MR-4在不同末端电子受体条件下的代谢通量分析[J]. 合成生物学, 2023, 4(6): 1300-1320.

ZHENG Qingxiang. Metabolic flux analysis of Shewanella sp. MR-4 with different terminal electron receptors[J]. Synthetic Biology Journal, 2023, 4(6): 1300-1320.

图/表 9

参考文献 16

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碱性溶液 Basic solution
成分 Component		浓度/（mmol/L） Concentration/（mmol/L）
PIPES		30
NH₄Cl		28.04
KCl		1.34
NaH₂PO₄·2H₂O		4.35
NaCl		30
Na₂SO₄		0.7
矿物溶液 Mineral solution
成分 Component		浓度/（µmol/L） Concentration/ (µmol/L)
C₆H₉NO₆		78.49
MgSO₄·7H₂O		121.71
MnSO₄·H₂O		29.58
FeSO₄·7H₂O		3.6
CaCl₂·2H₂O		6.8
CoCl₂·6H₂O		4.2
ZnCl₂		9.54
CuSO₄·5H₂O		0.4
AlK(SO₄)₂·12H₂O		0.21
H₃BO₃		1.62
Na₂MoO₄·2H₂O		1.03
NiCl₂·6H₂O		1.01
Na₂WO₄·2H₂O		0.76
维生素溶液 Vitamin solution
成分 Component		浓度/（µmol/L） Concentration/（µmol/L）
D-生物素	D-biotin	81.87
叶酸	folic acid	45.34
盐酸吡哆醇	pyridoxine HCl	486.38
核黄素	riboflavin	132.84
盐酸硫胺素	thiamine HCl	140.73
烟碱酸	nicotinic acid	406.17
D-泛酸，半钙盐	D-pantothenic acid, hemi calcium salt	209.82
维生素B₁₂	VB₁₂	7.4
对氨基苯甲酸	p-amino benzoic acid	364.62
硫辛酸	thioctic acid	242.37
氯化胆碱	choline chloride	14.3246

碱性溶液 Basic solution
成分 Component		浓度/（mmol/L） Concentration/（mmol/L）
PIPES		30
NH₄Cl		28.04
KCl		1.34
NaH₂PO₄·2H₂O		4.35
NaCl		30
Na₂SO₄		0.7
矿物溶液 Mineral solution
成分 Component		浓度/（µmol/L） Concentration/ (µmol/L)
C₆H₉NO₆		78.49
MgSO₄·7H₂O		121.71
MnSO₄·H₂O		29.58
FeSO₄·7H₂O		3.6
CaCl₂·2H₂O		6.8
CoCl₂·6H₂O		4.2
ZnCl₂		9.54
CuSO₄·5H₂O		0.4
AlK(SO₄)₂·12H₂O		0.21
H₃BO₃		1.62
Na₂MoO₄·2H₂O		1.03
NiCl₂·6H₂O		1.01
Na₂WO₄·2H₂O		0.76
维生素溶液 Vitamin solution
成分 Component		浓度/（µmol/L） Concentration/（µmol/L）
D-生物素	D-biotin	81.87
叶酸	folic acid	45.34
盐酸吡哆醇	pyridoxine HCl	486.38
核黄素	riboflavin	132.84
盐酸硫胺素	thiamine HCl	140.73
烟碱酸	nicotinic acid	406.17
D-泛酸，半钙盐	D-pantothenic acid, hemi calcium salt	209.82
维生素B₁₂	VB₁₂	7.4
对氨基苯甲酸	p-amino benzoic acid	364.62
硫辛酸	thioctic acid	242.37
氯化胆碱	choline chloride	14.3246

菌种	乳酸盐（lactate）氧气（oxygen）有氧（aerobic）	乳酸盐（lactate）延胡索酸（fumarate）厌氧（anaerobic）	葡萄糖（glucose）延胡索酸（fumarate）厌氧（anaerobic）	葡萄糖（glucose）次氮基三乙酸铁（Fe-NTA）厌氧（anaerobic）
Shewanella putrefaciens CN-32	＋	＋	＋	－
Shewanella loihica PV-4	＋	＋	＋	＋
Shewanella sp. MR-4	＋	＋	＋	＋
Shewanella amazonensis SB2B	＋	＋	＋	＋
Shewanella sp. W3-18-1	＋	＋	＋	－
Shewanella baltica OS155	＋	＋	＋	＋
Shewanella denitrificans OS217	＋	＋	－	－
Shewanella oneidensis MR-1	＋	＋	－	－
Shewanella sp. MR-7	＋	＋	－	＋
Shewanella sp. ANA-3	＋	＋	＋	＋
Shewanella pealeana ANG-SQ1	＋	－	－	－
Shewanella frigidimarina NCIMB400	＋	＋	－	－

菌种	乳酸盐（lactate）氧气（oxygen）有氧（aerobic）	乳酸盐（lactate）延胡索酸（fumarate）厌氧（anaerobic）	葡萄糖（glucose）延胡索酸（fumarate）厌氧（anaerobic）	葡萄糖（glucose）次氮基三乙酸铁（Fe-NTA）厌氧（anaerobic）
Shewanella putrefaciens CN-32	＋	＋	＋	－
Shewanella loihica PV-4	＋	＋	＋	＋
Shewanella sp. MR-4	＋	＋	＋	＋
Shewanella amazonensis SB2B	＋	＋	＋	＋
Shewanella sp. W3-18-1	＋	＋	＋	－
Shewanella baltica OS155	＋	＋	＋	＋
Shewanella denitrificans OS217	＋	＋	－	－
Shewanella oneidensis MR-1	＋	＋	－	－
Shewanella sp. MR-7	＋	＋	－	＋
Shewanella sp. ANA-3	＋	＋	＋	＋
Shewanella pealeana ANG-SQ1	＋	－	－	－
Shewanella frigidimarina NCIMB400	＋	＋	－	－

电子受体（Electron acceptor）	用于生物质合成的葡萄糖的比例（Percentage of glucose for biomass synthesis）	比率/[mmol/（gCDW·h）] Specific rate/[mmol/（gCDW·h）]				碳平衡（Carbon balance）
电子受体（Electron acceptor）		葡萄糖摄取（Glucose uptake）	醋酸盐分泌（Acetate secretion）	EPS形成（EPS formation）	CO₂净生成（Net CO₂ formation）	碳平衡（Carbon balance）
硝酸盐（Nitrate）	12.2% ± 0.5%	8.27 ± 0.24	4.19 ± 0.21	2.49 ± 0.11	17.47 ± 0.94	94.5%
Fe	5.1% ± 0.2%	4.86 ± 0.20	3.74 ± 0.19	1.28 ± 0.04	10.72 ± 0.35	93.9%
延胡索酸（Fumarate）	11.7%± 0.4%	5.04 ± 0.17	8.33 ± 0.54	0.31 ± 0.01	7.23 ± 0.38	96.8%
DMSO	5.9% ± 0.2%	5.38 ± 0.15	9.48 ± 0.47	0.47 ± 0.02	6.92 ± 0.21	94.9%