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.

Qingxiang ZHENG. 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 41

1	MYERS C R, NEALSON K H. Bacterial Manganese reduction and growth with manganese oxide as the sole electron acceptor[J]. Science, 1988, 240(4857): 1319-1321.
2	LARSEN I, LITTLE B, NEALSON K H, et al. Manganite reduction by Shewanella putrefaciens MR-4[J]. American Mineralogist, 1998, 83(11/12): 1564-1572.
3	URRUTIA M M, RODEN E E, FREDRICKSON J K, et al. Microbial and surface chemistry controls on reduction of synthetic Fe(Ⅲ) oxide minerals by the dissimilatory iron-reducing bacterium Shewanella alga [J]. Geomicrobiology Journal, 1998, 15(4): 269-291.
4	CACCAVO F, BLAKEMORE R P, LOVLEY D R. A hydrogen-oxidizing, Fe(Ⅲ)-reducing microorganism from the Great Bay Estuary, New Hampshire[J]. Applied and Environmental Microbiology, 1992, 58(10): 3211-3216.
5	WIELINGA B, MIZUBA M M, HANSEL C M, et al. Iron promoted reduction of chromate by dissimilatory iron-reducing bacteria[J]. Environmental Science & Technology, 2001, 35(3): 522-527.
6	FARRENKOPF A M, DOLLHOPF M E, NI CHADHAIN S, et al. Reduction of iodate in seawater during Arabian Sea shipboard incubations and in laboratory cultures of the marine bacterium Shewanella putrefaciens strain MR-4[J]. Marine Chemistry, 1997, 57(3/4): 347-354.
7	WILDUNG R E, GORBY Y A, KRUPKA K M, et al. Effect of electron donor and solution chemistry on products of dissimilatory reduction of technetium by Shewanella putrefaciens [J]. Applied and Environmental Microbiology, 2000, 66(6): 2451-2460.
8	LLOYD J R, YONG P, MACASKIE L E. Biological reduction and removal of Np(Ⅴ) by two microorganisms[J]. Environmental Science & Technology, 2000, 34(7): 1297-1301.
9	SALTIKOV C W, WILDMAN R A JR, NEWMAN D K. Expression dynamics of arsenic respiration and detoxification in Shewanella sp. strain ANA-3[J]. Journal of Bacteriology, 2005, 187(21): 7390-7396.
10	BOUKHALFA H, ICOPINI G A, REILLY S D, et al. Plutonium(Ⅳ) reduction by the metal-reducing bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1[J]. Applied and Environmental Microbiology, 2007, 73(18): 5897-5903.
11	KLONOWSKA A, HEULIN T, VERMEGLIO A. Selenite and tellurite reduction by Shewanella oneidensis [J]. Applied and Environmental Microbiology, 2005, 71(9): 5607-5609.
12	CARPENTIER W, SANDRA K, DE SMET I, et al. Microbial reduction and precipitation of vanadium by Shewanella oneidensis [J]. Applied and Environmental Microbiology, 2003, 69(6): 3636-3639.
13	PERRY K A, KOSTKA J E, LUTHER G W 3 RD,et al. Mediation of sulfur speciation by a Black Sea facultative anaerobe[J]. Science, 1993, 259(5096): 801-803.
14	KRAUSE B, NEALSON K H. Physiology and enzymology involved in denitrification by Shewanella putrefaciens [J]. Applied and Environmental Microbiology, 1997, 63(7): 2613-2618.
15	RINGØ E, STENBERG E, STRØM A R. Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735[J]. Applied and Environmental Microbiology, 1984, 47(5): 1084-1089.
16	GRALNICK J A, VALI H, LIES D P, et al. Extracellular respiration of dimethyl sulfoxide by Shewanella oneidensis strain MR-1[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(12): 4669-4674.
17	MYERS C R, MYERS J M. Fumarate reductase is a soluble enzyme in anaerobically grown Shewanella putrefaciens MR-1[J]. FEMS Microbiology Letters, 1992, 98(1/2/3): 13-19.
18	ZHAO J S, MANNO D, BEAULIEU C, et al. Shewanella sediminis sp nov., a novel Na⁺-requiring and hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading bacterium from marine sediment[J]. International Journal of Systematic and Evolutionary Microbiology, 2005, 55(Pt 4): 1511-1520.
19	ZHAO J S, MANNO D, THIBOUTOT S, et al. Shewanella canadensis sp nov. and Shewanella atlantica sp. nov., manganese dioxide- and hexahydro-1,3,5-trinitro-1,3,5-triazine-reducing, psychrophilic marine bacteria[J]. International Journal of Systematic and Evolutionary Microbiology, 2007, 57(9): 2155-2162.
20	HAU H H, GRALNICK J A. Ecology and biotechnology of the genus Shewanella [J]. Annual Review of Microbiology, 2007, 61: 237-258.
21	肖翔, 吴勇民, 徐灿灿, 等. 金属还原菌希瓦氏菌厌氧呼吸能力及其在环境修复中的研究进展[J]. 微生物学通报, 2012, 39(11): 1677-1686.
	XIAO X, WU Y M, XU C C, et al. Anaerobic respiratory capabilities of a metal-reducing microorganism Shewanella and its application in environmental remediation[J]. Microbiology China, 2012, 39(11): 1677-1686.
22	TANG Y J J, HWANG J S, WEMMER D E, et al. Shewanella oneidensis MR-1 fluxome under various oxygen conditions[J]. Applied and Environmental Microbiology, 2007, 73(3): 718-729.
23	TANG Y J J, MARTIN H G, DEUTSCHBAUER A, et al. Invariability of central metabolic flux distribution in Shewanella oneidensis MR-1 under environmental or genetic perturbations[J]. Biotechnology Progress, 2009, 25(5): 1254-1259.
24	TANG Y J J, MARTIN H G, DEHAL P S, et al. Metabolic flux analysis of Shewanella spp. reveals evolutionary robustness in central carbon metabolism[J]. Biotechnology and Bioengineering, 2009, 102(4): 1161-1169.
25	TANG Y J J, MEADOWS A L, KIRBY J, et al. Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling[J]. Journal of Bacteriology, 2007, 189(3): 894-901.
26	STOOKEY L L. Ferrozine— a new spectrophotometric reagent for iron[J]. Analytical Chemistry, 1970, 42(7): 779-781.
27	ALEF K, KLEINER D. Rapid and sensitive determination of microbial activity in soils and in soil aggregates by dimethylsulfoxide reduction[J]. Biology and Fertility of Soils, 1989, 8(4): 349-355.
28	MASUKO T, MINAMI A, IWASAKI N, et al. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format[J]. Analytical Biochemistry, 2005, 339(1): 69-72.
29	ZAMBONI N, FENDT S M, RÜHL M, et al. ¹³C-based metabolic flux analysis[J]. Nature Protocols, 2009, 4(6): 878-892.
30	PINCHUK G E, HILL E A, GEYDEBREKHT O V, et al. Constraint-based model of Shewanella oneidensis MR-1 metabolism: a tool for data analysis and hypothesis generation[J]. PLoS Computational Biology, 2010, 6(6): e1000822.
31	NANCHEN A, FUHRER T, SAUER U. Determination of metabolic flux ratios from ¹³C-experiments and gas chromatography-mass spectrometry data[M/OL]//Methods in molecular biology: metabolomics. Totowa, NJ, USA: Humana Press. 2007, 358: 177-197 [2022-09-01]. .
32	AYYASAMY P M, CHUN S H, LEE S H. Desorption and dissolution of heavy metals from contaminated soil using Shewanella sp. (HN-41) amended with various carbon sources and synthetic soil organic matters[J]. Journal of Hazardous Materials, 2009, 161(2/3): 1095-1102.
33	ONG W K, VU T T, LOVENDAHL K N, et al. Comparisons of Shewanella strains based on genome annotations, modeling, and experiments[J]. BMC Systems Biology, 2014, 8: 31.
34	CRUZ-GARCÍA C, MURRAY A E, KLAPPENBACH J A, et al. Respiratory nitrate ammonification by Shewanella oneidensis MR-1[J]. Journal of Bacteriology, 2007, 189(2): 656-662.
35	INOHANA Y, KATSUYA S, KOGA R, et al. Shewanella algae relatives capable of generating electricity from acetate contribute to coastal-sediment microbial fuel cells treating complex organic matter[J]. Microbes and Environments, 2020, 35(2): ME19161.
36	REWATKAR P, GOEL S. Shewanella putrefaciens powered microfluidic microbial fuel cell with printed circuit board electrodes and soft-lithographic microchannel[J]. Chemosphere, 2022, 286: 131855.
37	DAI H N, DUONG NGUYEN T A, LE L P MY, et al. Power generation of Shewanella oneidensis MR-1 microbial fuel cells in bamboo fermentation effluent[J]. International Journal of Hydrogen Energy, 2021, 46(31): 16612-16621.
38	CHAUDHARY S, BANERJEE D K. Metal phase association of chromium in contaminated soils from an industrial area in Delhi[J]. Chemical Speciation & Bioavailability, 2004, 16(4): 145-151.
39	FREDRICKSON J K, ROMINE M F, BELIAEV A S, et al. Towards environmental systems biology of Shewanella [J]. Nature Reviews Microbiology, 2008, 6(8): 592-603.
40	LEMAIRE O N, MÉJEAN V, IOBBI-NIVOL C. The Shewanella genus: ubiquitous organisms sustaining and preserving aquatic ecosystems[J]. FEMS Microbiology Reviews, 2020, 44(2): 155-170.
41	FAN G, DUNDAS C M, GRAHAM A J, et al. Shewanella oneidensis as a living electrode for controlled radical polymerization[J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(18): 4559-4564.

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%