Part:BBa_K857000

acetaldehyde dehydrogenase

Coding protein for acetaldehyde dehydrogenase convert acetaldehyde into acetyl-CoA.

CH3CHO + NAD+ + CoA → acetyl-CoA + NADH + H+

This part was designed by the 2012 iGEM UC Merced team and it is a gene that is involved in the E. Coli glucose metabolism pathway. Our team characterized this part by performing an in silico knockout of this gene. Using quantitative analysis, we discovered that its deletion does not affect the growth of E. Coli when under optimal conditions to produce a 4:1 hydrogen production to glucose uptake ratio. More importantly, we identified possible non-lethal gene deletions that may help maximize hydrogen production in the dark fermentation process.

First, we measured the growth of E. Coli bacteria under the conditions in which the insertion of mhpF allows the production of hydrogen to glucose uptake ratio to be 4:1. This was done using the Constraint Model Based Reconstruction and Analysis (COBRA) toolbox on Matlab platform which allowed us to constrain the uptake of glucose and hydrogen release to a ratio of 4:1 on our E. Coli glucose metabolism model. Our results were performed using Flux Balance Analysis (FBA) on the Core E. Coli Model by the Systems Biology Research Group at the University of California generated from the current gene expression data on E. coli.

Figure 1. Demonstrates the growth of E.coli when subject to the hydrogen release to glucose uptake requirement.

Our results showed that when the MhpF gene is introduced, E. Coli growth is higher under anaerobic conditions than aerobic conditions when under stress requirement to produce a 4:1 hydrogen to glucose ratio. This was achieved by setting oxygen flux values to both unlimited and zero for aerobic and anaerobic conditions respectively, whilst measuring E. coli’s growth in rate per hour. Growth measurements were done as a quantitative measure of how E.coli performs against the gene modification added to its metabolism.

Furthermore, our analysis from Flux Balance Analysis (FBA) validated team UC Merced hypothesis that removing IdhA, pfIB and adhE from E. coli fermentation pathway and replacing it with mhpF, pyruvate decarboxylase and ferredoxin oxidoreductase. This was achieved by performing in silico knockouts of IdhA, pfIB and adhE on the E. coli model and multiple gene insertions of mhpF, pyruvate decarboxylate and ferredoxin and looking at how the shift in metabolism network improve hydrogen yield under anaerobic condition.

Figure 2.Shift in metabolism flux network when E. coli model is subjected to hydrogen release to glucose uptake 4:1 ratio under aerobic conditions (left) and anaerobic conditions(right).

Next, we analyzed the effect of deleting the mhpF gene on the growth rate of E. Coli (in biomass/hour). The quantitative analysis shows that the growth remained the same, which indicates that the mhpF gene does not affect the growth rate of E. Coli. This served as motivation to perform more single gene deletions on the E.coli model. We analyzed the effect of creating knockouts of all the genes involved in glucose metabolism on the bacteria by examining the lethality of deleting each gene. Team UC Merced attempted to create knockouts of three genes, and below we present suggestions regarding the genes in the dark fermentation process that can be deleted without being lethal. Deletions of genes in the dark fermentation process, a form of anaerobic conversion would facilitate the conversion of organic substrate to biohydrogen. (see Table 1) For instance our analysis from the gene deletion suggested that the deletion zwf gene( glucose-6-phosphate 1-dehydrogenase) which is principal in the pentose pathway would be lethal to the E. coli. It turns out that zwf and mphF gene is overexpressed in the dark fermentation process. Even though both are very necessary for hydrogen production, mphF deletion would not result in much difference in the flux. Further our metabolic network flux predicts that the overexpression of zwf leads to the diversion of carbon flux through the PP pathway and hence result in an increase in the efficiency of the production of H2. Finally, a robustness analysis was performed to verify the validity of the results by setting fixed amounts of glucose and hydrogen and measuring the growth as a function of the oxygen concentration. This analysis was performed to demonstrate the strength of our model's prediction. The linear graph predicts a linear rise in growth with an intermittent increase in glucose intake. This agrees and supports our previous data which suggested that mphF is implied in the increase in growth of E.coli when subject to the condition stipulated above.



In sum , using metabolic modeling allow for in silico gene knockouts and metabolic analysis that unveiled non lethal gene deletions and insertion that would prove useful for the generation of biohydrogen from E.coli. Using metabolic modeling, it is easier to generate genetic changes that would optimize hydrogen production from E.coli. graph robustness

Finally, a robustness analysis was performed to verify the validity of the results by setting fixed amounts of glucose and hydrogen and measuring the growth as a function of the oxygen concentration. Table 1: Possible gene deletions and their measure effects on E. coli.

 

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LOCUS	GENE	PRODUCT	FUNCTION	NOTE	LETHALITY
b0008	talB	transaldolase B	enzyme; Central intermediary metabolism:Non-oxidative branch, pentose pathway		not lethal
b0118	acnB	aconitate hydratase 2; aconitase B;2-methyl-cis-aconitate hydratase	enzyme; Energy metabolism, carbon: TCA cycle	aconitate hydrase B	not lethal
b0351	mhpF	acetaldehyde-CoA dehydrogenase II, NAD-binding	enzyme; Degradation of small molecules: Carboncompounds	acetaldehyde dehydrogenase	not lethal
b0356	frmA	alcohol dehydrogenase class III;glutathione-dependent formaldehyde dehydrogenase	enzyme; Energy metabolism, carbon:Fermentation	alcohol dehydrogenase class III; formaldehydedehydrogenase, glutathione-dependent	not lethal
b0451	amtB	ammonium transporter	putative transport; Central intermediarymetabolism: Pool, multipurpose conversions	probable ammonium transporter	not lethal
b0474	adk	adenylate kinase	enzyme; Purine ribonucleotide biosynthesis	adenylate kinase activity; pleiotropic effects onglycerol-3-phosphate acyltransferase activity	not lethal
b0485	glsA	glutaminase 1	enzyme; l-glutamine catabolism	putative glutaminase	not lethal
b0733	cydA	cytochrome d terminal oxidase, subunit I	enzyme; Energy metabolism, carbon: Electrontransport	cytochrome d terminal oxidase, polypeptide subunitI	not lethal
b0734	cydB	cytochrome d terminal oxidase, subunit II	enzyme; Energy metabolism, carbon: Electrontransport	cytochrome d terminal oxidase polypeptide subunitII	not lethal
b0755	gpmA	phosphoglyceromutase 1	enzyme; Energy metabolism, carbon: Glycolysis		not lethal
b0809	glnQ	glutamine transporter subunit	transport; Transport of small molecules: Aminoacids, amines	ATP-binding component of glutamine high-affinitytransport system	not lethal
b0810	glnP	glutamine transporter subunit	transport; Transport of small molecules: Aminoacids, amines	glutamine high-affinity transport system; membranecomponent	not lethal
b0811	glnH	glutamine transporter subunit	transport; Transport of small molecules: Aminoacids, amines	periplasmic glutamine-binding protein; permease	not lethal
b0875	aqpZ	aquaporin Z	transport; Transport of small molecules: Other	transmembrane water channel; aquaporin Z	not lethal
b0902	pflA	pyruvate formate-lyase 1-activating enzyme;[formate-C-acetyltransferase 1]-activating enzyme; PFLactivase	enzyme; Energy metabolism, carbon: Anaerobicrespiration		not lethal
b0903	pflB	formate C-acetyltransferase 1, anaerobic;pyruvate formate-lyase 1	enzyme; Energy metabolism, carbon: Anaerobicrespiration	formate acetyltransferase 1	not lethal
b0904	focA	formate channel	putative transport; Degradation of smallmolecules: Carbon compounds	probable formate transporter (formate channel 1)	not lethal
b0978	cbdA	cytochrome bd-II oxidase, subunit I	putative enzyme; Energy metabolism, carbon:Electron transport		not lethal
b0979	cbdB	cytochrome bd-II oxidase, subunit II	putative enzyme; Energy metabolism, carbon:Electron transport	probable third cytochrome oxidase, subunit II	not lethal
b1101	ptsG	fused glucose-specific PTS enzymes: IIBcomponent/IIC component	transport; Transport of small molecules:Carbohydrates, organic acids, alcohols	PTS system, glucose-specific IIBC component	not lethal
b1241	adhE	fused acetaldehyde-CoAdehydrogenase/iron-dependent alcoholdehydrogenase/pyruvate-formate lyase deactivase	enzyme; Energy metabolism, carbon:Fermentation	CoA-linked acetaldehyde dehydrogenase andiron-dependent alcohol dehydrogenase;pyruvate-formate-lyase deactivase	not lethal
b1276	acnA	aconitate hydratase 1; aconitase A	enzyme; Energy metabolism, carbon: TCA cycle	aconitate hydrase 1	not lethal
b1297	puuA	glutamate--putrescine ligase	enzyme; Degradation of small molecules: Carboncompounds	putative glutamine synthetase	not lethal
b1380	ldhA	fermentative D-lactate dehydrogenase,NAD-dependent	enzyme; Energy metabolism, carbon:Fermentation		not lethal
b1478	adhP	ethanol-active dehydrogenase/acetaldehyde-activereductase	enzyme; Energy metabolism, carbon: Anaerobicrespiration	alcohol dehydrogenase	not lethal
b1479	maeA	malate dehydrogenase, decarboxylating,NAD-requiring; malic enzyme	enzyme; Central intermediary metabolism:Gluconeogenesis	NAD-linked malate dehydrogenase (malic enzyme)	not lethal
b1524	glsB	glutaminase 2	enzyme; l-glutamine catabolism	putative glutaminase	not lethal
b1602	pntB	pyridine nucleotide transhydrogenase, betasubunit	enzyme; Central intermediary metabolism: Pool,multipurpose conversions		not lethal
b1603	pntA	pyridine nucleotide transhydrogenase, alphasubunit	enzyme; Central intermediary metabolism: Pool,multipurpose conversions		not lethal
b1611	fumC	fumarate hydratase (fumarase C),aerobic ClassII	enzyme; Energy metabolism, carbon: TCA cycle	fumarase C	not lethal
b1612	fumA	fumarate hydratase (fumarase A), aerobic ClassI	enzyme; Energy metabolism, carbon: TCA cycle	fumarase A isozyme	not lethal
b1621	malX	maltose and glucose-specific PTS enzyme IIBcomponent and IIC component	transport; Transport of small molecules:Carbohydrates, organic acids, alcohols	PTS system, maltose and glucose-specific IIABCcomponent	not lethal
b1676	pykF	pyruvate kinase I	enzyme; Energy metabolism, carbon: Glycolysis	pyruvate kinase I (formerly F), fructosestimulated	not lethal
b1702	ppsA	phosphoenolpyruvate synthase	enzyme; Central intermediary metabolism:Gluconeogenesis		not lethal
b1723	pfkB	6-phosphofructokinase II	enzyme; Energy metabolism, carbon: Glycolysis	6-phosphofructokinase II; suppressor of pfkA	not lethal
b1773	ydjI	putative aldolase	putative enzyme; Not classified		not lethal
b1812	pabB	aminodeoxychorismate synthase, subunit I	enzyme; Biosynthesis of cofactors, carriers:Folic acid	p-aminobenzoate synthetase, component I	not lethal
b1817	manX	fused mannose-specific PTS enzymes: IIAcomponent/IIB component	enzyme; Transport of small molecules:Carbohydrates, organic acids, alcohols	PTS enzyme IIAB, mannose-specific	not lethal
b1818	manY	mannose-specific enzyme IIC component of PTS	transport; Transport of small molecules:Carbohydrates, organic acids, alcohols	PTS enzyme IIC, mannose-specific	not lethal
b1819	manZ	mannose-specific enzyme IID component of PTS	transport; Transport of small molecules:Carbohydrates, organic acids, alcohols	PTS enzyme IID, mannose-specific	not lethal
b1849	purT	phosphoribosylglycinamide formyltransferase 2	enzyme; Purine ribonucleotide biosynthesis		not lethal
b1854	pykA	pyruvate kinase II	enzyme; Energy metabolism, carbon: Glycolysis	pyruvate kinase II, glucose stimulated	not lethal
b2097	fbaB	fructose-bisphosphate aldolase class I			not lethal
b2133	dld	D-lactate dehydrogenase, FAD-binding, NADHindependent	enzyme; Energy metabolism, carbon: Aerobicrespiration	D-lactate dehydrogenase, FAD protein, NADHindependent	not lethal
b2296	ackA	acetate kinase A and propionate kinase 2	enzyme; Energy metabolism, carbon: Electrontransport	acetate kinase	not lethal
b2297	pta	phosphate acetyltransferase	enzyme; Degradation of small molecules: Carboncompounds	phosphotransacetylase	not lethal
b2417	crr	glucose-specific enzyme IIA component of PTS	enzyme; Transport of small molecules:Carbohydrates, organic acids, alcohols	PTS system, glucose-specific IIA component	not lethal
b2458	eutD	phosphate acetyltransferase	putative enzyme; Degradation of smallmolecules: Amines	ethanolamine utilization; homolog of Salmonellaacetyl/butyryl P transferase	not lethal
b2463	maeB	malic enzyme: putativeoxidoreductase/phosphotransacetylase	putative enzyme; Not classified	putative multimodular enzyme	not lethal
b2464	talA	transaldolase A	enzyme; Central intermediary metabolism:Non-oxidative branch, pentose pathway		not lethal
b2465	tktB	transketolase 2, thiamine triphosphate-binding	enzyme; Central intermediary metabolism:Non-oxidative branch, pentose pathway	transketolase 2 isozyme	not lethal
b2492	focB	putative formate transporter	putative transport; Transport of smallmolecules: Carbohydrates, organic acids, alcohols	probable formate transporter (formate channel 2)	not lethal
b2579	grcA	autonomous glycyl radical cofactor	putative enzyme; Energy metabolism, carbon:Anaerobic respiration	putative formate acetyltransferase	not lethal
b2587	kgtP	alpha-ketoglutarate transporter	transport; Transport of small molecules:Carbohydrates, organic acids, alcohols	alpha-ketoglutarate permease	not lethal
b2914	rpiA	ribose 5-phosphate isomerase, constitutive	enzyme; Central intermediary metabolism:Non-oxidative branch, pentose pathway	ribosephosphate isomerase, constitutive	not lethal
b2925	fbaA	fructose-bisphosphate aldolase, class II	enzyme; Energy metabolism, carbon: Glycolysis		not lethal
b2935	tktA	transketolase 1, thiamine triphosphate-binding	enzyme; Central intermediary metabolism:Non-oxidative branch, pentose pathway	transketolase 1 isozyme	not lethal
b2975	glcA	glycolate transporter	putative transport; Not classified	putative permease	not lethal
b2976	glcB	malate synthase G	enzyme; Central intermediary metabolism:Glyoxylate bypass		not lethal
b2987	pitB	phosphate transporter	transport; Transport of small molecules:Anions	low-affinity phosphate transport	not lethal
b3114	tdcE	pyruvate formate-lyase 4/2-ketobutyrateformate-lyase	putative enzyme; Energy metabolism, carbon:Anaerobic respiration	probable formate acetyltransferase 3	not lethal
b3115	tdcD	propionate kinase/acetate kinase C, anaerobic	putative enzyme; Not classified	propionate kinase/acetate kinase II, anaerobic	not lethal
b3212	gltB	glutamate synthase, large subunit	enzyme; Central intermediary metabolism: Pool,multipurpose conversions		not lethal
b3213	gltD	glutamate synthase, 4Fe-4S protein, smallsubunit	enzyme; Central intermediary metabolism: Pool,multipurpose conversions	glutamate synthase, small subunit	not lethal
b3386	rpe	D-ribulose-5-phosphate 3-epimerase	enzyme; Central intermediary metabolism:Non-oxidative branch, pentose pathway		not lethal
b3403	pck	phosphoenolpyruvate carboxykinase [ATP]	enzyme; Central intermediary metabolism:Gluconeogenesis		not lethal
b3493	pitA	phosphate transporter, low-affinity; telluriteimporter	transport; Transport of small molecules:Anions	low-affinity phosphate transport	not lethal
b3528	dctA	C4-dicarboxylic acid, orotate and citratetransporter	transport; Transport of small molecules:Carbohydrates, organic acids, alcohols	uptake of C4-dicarboxylic acids	not lethal
b3603	lldP	L-lactate permease	transport; Transport of small molecules:Carbohydrates, organic acids, alcohols		not lethal
b3612	gpmM	phosphoglycero mutase III, cofactor-independent	putative enzyme; Not classified	putative 2,3-bisphosphoglycerate-independentphosphoglycerate mutase	not lethal
b3739	atpI	ATP synthase, membrane-bound accessory factor	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, dispensable protein,affects expression of atpB	not lethal
b3870	glnA	glutamine synthetase	enzyme; Amino acid biosynthesis: Glutamine		not lethal
b3916	pfkA	6-phosphofructokinase I	enzyme; Energy metabolism, carbon: Glycolysis		not lethal
b3925	glpX	fructose 1,6-bisphosphatase II	phenotype; Not classified	not required for growth on glycerol	not lethal
b3951	pflD	putative glycine radical domain-containingpyruvate formate-lyase	enzyme; Energy metabolism, carbon: Anaerobicrespiration	formate acetyltransferase 2	not lethal
b3952	pflC	putative [formate-C-acetyltransferase2]-activating enzyme; pyruvate formate-lyase 1-activatingenzyme	putative enzyme; Energy metabolism, carbon:Anaerobic respiration	probable pyruvate formate lyase activating enzyme2	not lethal
b3962	sthA	pyridine nucleotide transhydrogenase, soluble	putative enzyme; Not classified		not lethal
b4014	aceB	malate synthase A	enzyme; Central intermediary metabolism:Glyoxylate bypass		not lethal
b4015	aceA	isocitrate lyase	enzyme; Central intermediary metabolism:Glyoxylate bypass		not lethal
b4077	gltP	glutamate/aspartate:proton symporter	transport; Transport of small molecules: Aminoacids, amines	glutamate-aspartate symport protein	not lethal
b4090	rpiB	ribose 5-phosphate isomerase B/allose6-phosphate isomerase	enzyme; Central intermediary metabolism:Non-oxidative branch, pentose pathway	ribose 5-phosphate isomerase B	not lethal
b4122	fumB	anaerobic class I fumarate hydratase (fumaraseB)	enzyme; Energy metabolism, carbon: TCA cycle	fumarase Bisozyme	not lethal
b4151	frdD	fumarate reductase (anaerobic), membrane anchorsubunit	enzyme; Energy metabolism, carbon: Anaerobicrespiration	fumarate reductase, anaerobic, membrane anchorpolypeptide	not lethal
b4152	frdC	fumarate reductase (anaerobic), membrane anchorsubunit	enzyme; Energy metabolism, carbon: Anaerobicrespiration	fumarate reductase, anaerobic, membrane anchorpolypeptide	not lethal
b4153	frdB	fumarate reductase (anaerobic), Fe-S subunit	enzyme; Energy metabolism, carbon: Anaerobicrespiration	fumarate reductase, anaerobic, iron-sulfur proteinsubunit	not lethal
b4154	frdA	anaerobic fumarate reductase catalytic andNAD/flavoprotein subunit	enzyme; Energy metabolism, carbon: Anaerobicrespiration	fumarate reductase, anaerobic, flavoproteinsubunit	not lethal
b4232	fbp	fructose-1,6-bisphosphatase I	enzyme; Central intermediary metabolism:Gluconeogenesis		not lethal
b4301	sgcE	putative epimerase	putative enzyme; Not classified		not lethal
b4395	ytjC	phosphatase	enzyme; Not classified	phosphoglyceromutase 2	not lethal
b0114	aceE	pyruvate dehydrogenase, decarboxylase componentE1, thiamine triphosphate-binding	enzyme; Energy metabolism, carbon: Pyruvatedehydrogenase	pyruvate dehydrogenase (decarboxylase component)	lethal
b0115	aceF	pyruvate dehydrogenase,dihydrolipoyltransacetylase component E2	enzyme; Energy metabolism, carbon: Pyruvatedehydrogenase	pyruvate dehydrogenase (dihydrolipoyltransacetylasecomponent)	lethal
b0116	lpd	dihydrolipoyl dehydrogenase; E3 component ofpyruvate and 2-oxoglutarate dehydrogenases complexes;glycine cleavage system L protein; dihydrolipoamidedehydrogenase	enzyme; Energy metabolism, carbon: Pyruvatedehydrogenase	lipoamide dehydrogenase (NADH); component of2-oxodehydrogenase and pyruvate complexes; L-protein ofglycine cleavage complex	lethal
b0720	gltA	citrate synthase	enzyme; Energy metabolism, carbon: TCA cycle		lethal
b0721	sdhC	succinate dehydrogenase, membrane subunit, bindscytochrome b556	enzyme; Energy metabolism, carbon: TCA cycle	succinate dehydrogenase, cytochrome b556	lethal
b0722	sdhD	succinate dehydrogenase, membrane subunit, bindscytochrome b556	enzyme; Energy metabolism, carbon: TCA cycle	succinate dehydrogenase, hydrophobic subunit	lethal
b0723	sdhA	succinate dehydrogenase, flavoprotein subunit	enzyme; Energy metabolism, carbon: TCA cycle		lethal
b0724	sdhB	succinate dehydrogenase, FeS subunit	enzyme; Energy metabolism, carbon: TCA cycle	succinate dehydrogenase, iron sulfur protein	lethal
b0726	sucA	2-oxoglutarate decarboxylase, thiaminetriphosphate-binding	enzyme; Energy metabolism, carbon: TCA cycle	2-oxoglutarate dehydrogenase (decarboxylasecomponent)	lethal
b0727	sucB	dihydrolipoyltranssuccinase	enzyme; Energy metabolism, carbon: TCA cycle	2-oxoglutarate dehydrogenase(dihydrolipoyltranssuccinase E2 component)	lethal
b0728	sucC	succinyl-CoA synthetase, beta subunit	enzyme; Energy metabolism, carbon: TCA cycle		lethal
b0729	sucD	succinyl-CoA synthetase, NAD(P)-binding, alphasubunit	enzyme; Energy metabolism, carbon: TCA cycle	succinyl-CoA synthetase, alpha subunit	lethal
b0767	pgl	6-phosphogluconolactonase	orf; Not classified	putative isomerase	lethal
b1136	icd	isocitrate dehydrogenase; e14 prophageattachment site; tellurite reductase	enzyme; Energy metabolism, carbon: TCA cycle;Phage or Prophage Related	isocitrate dehydrogenase, specific for NADP+	lethal
b1761	gdhA	glutamate dehydrogenase, NADP-specific	enzyme; Amino acid biosynthesis: Glutamate	NADP-specific glutamate dehydrogenase	lethal
b1779	gapA	glyceraldehyde-3-phosphate dehydrogenase A	enzyme; Energy metabolism, carbon: Glycolysis		lethal
b1852	zwf	glucose-6-phosphate 1-dehydrogenase	enzyme; Energy metabolism, carbon: Oxidativebranch, pentose pathway		lethal
b2029	gnd	6-phosphogluconate dehydrogenase,decarboxylating	enzyme; Energy metabolism, carbon: Oxidativebranch, pentose pathway		lethal
b2276	nuoN	NADH:ubiquinone oxidoreductase, membrane subunitN	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain N	lethal
b2277	nuoM	NADH:ubiquinone oxidoreductase, membrane subunitM	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain M	lethal
b2278	nuoL	NADH:ubiquinone oxidoreductase, membrane subunitL	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain L	lethal
b2279	nuoK	NADH:ubiquinone oxidoreductase, membrane subunitK	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain K	lethal
b2280	nuoJ	NADH:ubiquinone oxidoreductase, membrane subunitJ	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain J	lethal
b2281	nuoI	NADH:ubiquinone oxidoreductase, chain I	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain I	lethal
b2282	nuoH	NADH:ubiquinone oxidoreductase, membrane subunitH	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain H	lethal
b2283	nuoG	NADH:ubiquinone oxidoreductase, chain G	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain G	lethal
b2284	nuoF	NADH:ubiquinone oxidoreductase, chain F	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain F	lethal
b2285	nuoE	NADH:ubiquinone oxidoreductase, chain E	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain E	lethal
b2286	nuoC	NADH:ubiquinone oxidoreductase, fused CDsubunit	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain C, D	lethal
b2287	nuoB	NADH:ubiquinone oxidoreductase, chain B	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain B	lethal
b2288	nuoA	NADH:ubiquinone oxidoreductase, membrane subunitA	enzyme; Energy metabolism, carbon: Aerobicrespiration	NADH dehydrogenase I chain A	lethal
b2415	ptsH	phosphohistidinoprotein-hexosephosphotransferase component of PTS system (Hpr)	enzyme; Transport of small molecules:Carbohydrates, organic acids, alcohols	PTS system protein HPr	lethal
b2416	ptsI	PEP-protein phosphotransferase of PTS system(enzyme I)	enzyme; Transport of small molecules:Carbohydrates, organic acids, alcohols	PEP-protein phosphotransferase system enzyme I	lethal
b2779	eno	enolase	enzyme; Energy metabolism, carbon: Glycolysis		lethal
b2926	pgk	phosphoglycerate kinase	enzyme; Energy metabolism, carbon: Glycolysis		lethal
b3236	mdh	malate dehydrogenase, NAD(P)-binding	enzyme; Energy metabolism, carbon: TCA cycle	malate dehydrogenase	lethal
b3731	atpC	F1 sector of membrane-bound ATP synthase,epsilon subunit	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, F1 sector,epsilon-subunit	lethal
b3732	atpD	F1 sector of membrane-bound ATP synthase, betasubunit	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, F1 sector,beta-subunit	lethal
b3733	atpG	F1 sector of membrane-bound ATP synthase, gammasubunit	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, F1 sector,gamma-subunit	lethal
b3734	atpA	F1 sector of membrane-bound ATP synthase, alphasubunit	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, F1 sector,alpha-subunit	lethal
b3735	atpH	F1 sector of membrane-bound ATP synthase, deltasubunit	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, F1 sector,delta-subunit	lethal
b3736	atpF	F0 sector of membrane-bound ATP synthase,subunit b	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, F0 sector, subunit b	lethal
b3737	atpE	F0 sector of membrane-bound ATP synthase,subunit c	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, F0 sector, subunit c	lethal
b3738	atpB	F0 sector of membrane-bound ATP synthase,subunit a	enzyme; ATP-proton motive forceinterconversion	membrane-bound ATP synthase, F0 sector, subunit a	lethal
b3919	tpiA	triosephosphate isomerase	enzyme; Energy metabolism, carbon: Glycolysis		lethal
b3956	ppc	phosphoenolpyruvate carboxylase	enzyme; Energy metabolism, carbon:Fermentation		lethal
b4025	pgi	glucosephosphate isomerase	enzyme; Energy metabolism, carbon: Glycolysis		lethal

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 349
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 765
  
• 1000
  COMPATIBLE WITH RFC[1000]