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

Characterization of Sortase BBa_K3925009 (and comparison to BBa_K857000)

pSB-BBa_K857000 only contains the alcohol dehydrogenase of BBa_K857000. In order to improve its enzyme activity, we modified BBa_K3925009. In addition to the alcohol dehydrogenase of BBa_K857000, pSB-BBa_K3925009 also has BBa_K3925005-NAD synthetase gene (nadE) and BBa_K3925007-NADH oxidase gene (nox).

In order to more intuitively reflect the expression of functional genes of the engineered strains E.coli pSB-BBa_K857000 and E.coli pSB-BBa_K3925009, we tested the enzymatic activity of various exogenous enzymes and the coenzyme NAD+ under the conditions of in vitro culture of different strains. content.

The results are shown in the figure. In the crude enzyme extracts of bacteria, E. coli 1917 basically did not show alcohol dehydrogenase, acetaldehyde dehydrogenase and NADH oxidase activities, and the content of the coenzyme NAD+ was extremely low; E. The ADH activity of coli pSB-BBa_K857000 was 47.91 ± 3.12 U/mL, the ALDH activity was 33.57 ± 2.59 U/mL, and NADH oxidase activity was not detected; while the ADH activity of E. coli pSB-BBa_K3925009 was 81.41 ± 3.64 U/mL mL, ALDH activity is 57.56 ± 1.48 U/mL, nox activity is 14.4 ± 2.29 U/mL, bacterial expression of ADH and ALDH enzyme activity is greatly increased, and the content of coenzyme NAD+ is also greatly increased compared with strain E.coli pSB-BBa_K857000. It shows that the expression of NAD synthase gene nadE and NADH oxidase gene nox can help increase the content of dehydrogenase coenzyme NAD in bacterial cells, thereby enhancing the degradation ability of alcohol dehydrogenase and acetaldehyde dehydrogenase.

literuture1

Alcohol use disorder (AUD) is a major global health concern (Peacock et al., 2018). There are considerable efforts being made to identify new molecular targets within the brain for the development of more effective therapies to treat this addictive disorder. As an approach in this direction, new research by Mews et al., 2019 published recently in Nature has demonstrated that the tertiary metabolite of alcohol (acetyl-CoA) is important in regulating ethanol’s effects in the brain and serves as a substrate in directly promoting histone acetylation, thereby regulating gene expression in the neurons and alcohol-related behaviors

Ethanol metabolism has previously been identified as an important factor in the toxic effects produced by ethanol consumption (Cederbaum, 2012). Ethanol is metabolized to acetyl-CoA through a three-step process, starting first with the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase IB in the liver (Cederbaum, 2012). Other tissue-specific enzymes, such as alcohol dehydrogenase IA, are also important in this step. Acetaldehyde is then converted to acetic acid by aldehyde dehydrogenase 2, which is the rate-limiting step of alcohol metabolism. Finally, acetic acid is converted to Acetyl-CoA, where it enters the citric cycle and can be released as H2O and CO2. Acetaldehyde is a highly reactive species that induces oxidative stress, mitochondrial damage, and cytokine formation (Guo and Ren, 2010). Furthermore, build-up of acetaldehyde is often found in heavy drinkers, and individuals without functioning aldehyde dehydrogenase 2 have vasodilation, nausea, and dysphasia when alcohol is consumed (Cederbaum, 2012, Guo and Ren, 2010). Efforts to capitalize on this effect as a deterrent to alcohol consumption resulted in the use of disulfiram (Antabuse) in the clinic to limited efficacy. Furthermore, ethanol has been shown to affect a staggeringly large number of different organ systems (notably the liver and brain) and a substantial number of different cell-transduction pathways, including epigenetic processes (Cederbaum, 2012, Choudhury and Shukla, 2008, Pandey et al., 2017). Chronic ethanol exposure is known to contribute to many of the negative physiological symptoms of withdrawal (Koob and Volkow, 2016, Pandey et al., 2017).

Histone acetylation is one of the most studied epigenetic processes that controls gene transcription (Pandey et al., 2017). The effects of acute ethanol on the epigenome via histone acetylation appear to be involved in anxiolytic effects (Pandey et al., 2017). Several previous studies have demonstrated that acute ethanol has the ability to activate histone acetyltransferases (HATs) and inhibit histone deacetylases (HDACs) in the brain (Pandey et al., 2017; Figure 1). These properties may lead to increased histone acetylation (active epigenetic marks) that produces chromatin remodeling and changes in gene expression in the amygdala, a brain region responsible for comorbidity of anxiety and AUD (Pandey et al., 2008, Pandey et al., 2017). Previous research has demonstrated that different alcohols and their metabolites can also induce changes to histone acetylation in hepatocytes (Choudhury and Shukla, 2008).

Mews et al. have suggested that acetate coming from the liver directly binds to neuronal chromatin (Figure 1) and is then converted to acetyl-CoA via chromatin-bound acetyl-CoA synthetase 2 (ACSS2) (Mews et al., 2019). This appears to be another fascinating epigenetic mechanism involved in the action of ethanol. The current research elegantly combines metabolomics and RNA and ChIP sequencing to show that alcohol contributes to histone acetylation in the mouse brain. Using liquid-chromatography mass spectrometry, the authors demonstrate that a single intraperitoneal injection of deuterated ethanol into mice rapidly increases histone acetylation in the dorsal hippocampus (dHPC) and prefrontal cortex (as well as the liver), and this increase lasts about 8 h. The authors further demonstrate that this is regulated by chromatin-bound acetyl-CoA synthetase 2 (ACSS2). Interestingly, alcohol exposure produces more H3K9ac and H3K27ac peaks in dHPC at the genome-wide level, which is prevented by the knockdown of ACSS2. This manipulation also prevents ethanol-conditioned place preference, suggesting that ACSS2 is required for ethanol reward learning, and provides more experimental evidence that epigenetic mechanisms are required for learning and memory. Previous studies have shown that learning and memory crucially rely on histone deacetylase-2-mediated epigenetic processes (Guan et al., 2009), and this new evidence creates a clearer picture about how ethanol facilitates its rewarding properties via ACSS2-dependent histone acetylation (Mews et al., 2019).

Since both ethanol and its secondary metabolite are known to be involved in changes in gene expression, the authors also evaluated if there was significant overlap between genes upregulated by the addition of acetate (ex vivo) and ethanol (in vivo) using RNA sequencing. There was substantial overlap between genes induced by acetate and ethanol (830 genes). An additional 1,010 genes were upregulated by ethanol and 2,783 upregulated by acetate, suggesting that there are also effects of ethanol that are independent of an acetate-induced mechanism. While the authors did perform gene ontology (GO) analysis on genes induced by acetate, it would also be interesting to see what GO analysis was involved for genes that overlapped with exposure to ethanol and exposure to acetate. Also, it would be interesting to examine if acetate has any direct effects on other epigenetic players responsible for histone acetylation changes in the brain, such as HATs and HDACs.

Transgenerational epigenetic regulation by drugs of abuse has been an area of intense study, particularly in the field of alcohol abuse (Chastain and Sarkar, 2017). Fetal alcohol spectrum disorder (FASD) occurs when the fetus is harmed during prenatal alcohol exposure. The authors demonstrate that maternal exposure to alcohol also causes changes in histone acetylation in the fetal brain, and this may contribute to the distinctive facial features, learning disabilities, and psychiatric symptoms that characterize this disorder by causing epigenetic dysregulation of tightly controlled developmental pathways (Mews et al., 2019). In summary, this study helps to answer questions regarding how acute ethanol increases histone acetylation and contributes significantly to our understanding of epigenetic regulation produced by alcohol drinking. Previous studies have indicated that histone deacetylase pathways are crucial in alcohol withdrawal and dependence and could be acting as a responsive mechanism for epigenetic reprogramming (Pandey et al., 2008, Pandey et al., 2017). Several key questions remain, specifically how an ACSS2-dependent histone acetylation mechanism contributes to the withdrawal aspects and ethanol tolerance that are observed in both animal models of alcohol dependence and in clinical populations. It would be interesting to see an expansion of this research to explore these topics.

Acknowledgments

SCP is supported by National Institute on Alcohol Abuse and Alcoholism grants UO1AA-019971 , U24AA-024605 ( Neurobiology of Adolescent Drinking in Adulthood [NADIA] project), RO1AA-010005 , P50AA-022538 ( Center for Alcohol Research in Epigenetics ), and by the Department of Veterans Affairs (VA merit grant I01BX004517 & Senior Research Career Scientist award). J.P.B. is supported by a fellowship F32 AA027410 grant.

Reference

Pandey SC, Bohnsack JP. Alcohol Makes Its Epigenetic Marks. Cell Metab. 2020 Feb 4;31(2):213-214. doi: 10.1016/j.cmet.2020.01.008. PMID: 32023443; PMCID: PMC7162615.

Literature 2

The robustness of Saccharomyces cerevisiae in industrial fermentation processes, combined with fast developments in yeast synthetic biology and systems biology, have made this microorganism a popular platform for metabolic engineering (Hong and Nielsen, 2012). Many natural and heterologous compounds whose production from sugars is under investigation or already implemented in industry require acetyl-coenzyme A (acetyl-CoA) as a key precursor. Examples of such products include n-butanol, isoprenoids, lipids and flavonoids (Dyer et al., 2002, Koopman et al., 2012, Shiba et al., 2007, Steen et al., 2008, Veen and Lang, 2004).

Acetyl-CoA metabolism in S. cerevisiae is compartmented (Pronk et al., 1996, van Roermund et al., 1995). During respiratory growth on sugars, a substantial flux through acetyl-CoA occurs via the mitochondrial pyruvate dehydrogenase complex (Pronk et al., 1994). However, mutant analysis has shown that mitochondrial acetyl-CoA cannot meet the extramitochondrial requirement for acetyl-CoA in the yeast cytosol, which includes, for example, its use as a precursor for synthesis of lipids and lysine (Flikweert et al., 1999, van den Berg and Steensma, 1995). In this respect, it is relevant to note that S. cerevisiae does not contain ATP-citrate lyase, an enzyme that plays a major role in translocation of acetyl-CoA across the mitochondrial membrane in mammalian cells and in several non-Saccharomyces yeasts (Boulton and Ratledge, 1981). When sugars are used as the carbon source, cytosolic acetyl-CoA synthesis in S. cerevisiae occurs via the concerted action of pyruvate decarboxylase (Pdc1, 5 and 6), acetaldehyde dehydrogenase (Ald2, 3, 4, 5 and 6) and acetyl-CoA synthetase (Acs1 and 2) (Pronk et al., 1996). Heterologous, acetyl-CoA-dependent product pathways expressed in the S. cerevisiae cytosol exclusively depend on this ‘pyruvate dehydrogenase bypass’ for provision of acetyl-CoA. Indeed, overexpression of acetyl-CoA synthetase (ACS) from Salmonella enterica has been shown to lead to increased productivities of the isoprenoid amorphadiene by engineered S. cerevisiae strains (Shiba et al., 2007).

The ACS reaction involves the hydrolysis of ATP to AMP and pyrophosphate (PPi): Together with the subsequent hydrolysis of PPi to inorganic phosphate (Pi), this ATP consumption is equivalent to the hydrolysis of 2 ATP to 2 ADP and 2Pi. The resulting ATP expenditure for acetate activation can have a huge impact on the maximum theoretical yields of acetyl-CoA derived products. For example, the production of a C16 lipid from sugars requires 8 acetyl-CoA, whose synthesis via ACS requires 16 ATP. At an effective P/O ratio of respiration in S. cerevisiae of 1 (Verduyn, 1991), this ATP requirement for acetyl-CoA synthesis corresponds to 1 mol of glucose that needs to be respired for the synthesis of 1 mol of product.

In addition to the pyruvate-dehydrogenase complex, other reactions have been described in nature that enable the ATP-independent conversion of pyruvate into acetyl-CoA (Powlowski et al., 1993, Rudolph et al., 1968, Smith and Kaplan, 1980). Many prokaryotes contain an acetylating acetaldehyde dehydrogenase (A-ALD; EC 1.2.1.10) which catalyses the reversible reaction:

Although functional expression of bacterial genes encoding A-ALD in S. cerevisiae has been described in the literature, these studies focused on reductive conversion of acetyl-CoA to ethanol as part of a phosphoketolase pathway for pentose fermentation (Sonderegger et al., 2004) or as part of a metabolic engineering strategy to convert acetic acid to ethanol (Guadalupe Medina et al., 2010). Complete replacement of the native acetaldehyde dehydrogenases and/or ACS of S. cerevisiae by A-ALD, thereby bypassing ATP hydrolysis in the ACS reaction, has not been demonstrated. In many anaerobic bacteria and some eukaryotes (Stairs et al., 2011), pyruvate can be converted into acetyl-CoA and formate in the non-oxidative, ATP-independent reaction catalysed by pyruvate-formate lyase (PFL; EC 2.3.1.54):

PFL and PFL-activating enzyme (PFL-AE; EC 1.97.1.4) from Escherichia coli have previously been expressed in S. cerevisiae (Waks and Silver, 2009). Although formate production by this oxygen-sensitive enzyme system was demonstrated in anaerobic yeast cultures, its impact on cytosolic acetyl-CoA metabolism has not been investigated.

To gain the full potential benefit of ATP-independent cytosolic acetyl-CoA synthesis, the implemented heterologous pathways expressed in S. cerevisiae should, ideally, completely replace the ACS reaction. In wild-type strain backgrounds, deletion of both ACS1 and ACS2 is lethal (van den Berg and Steensma, 1995) and, during batch cultivation on glucose, presence of a functional ACS2 gene is essential (van den Berg and Steensma, 1995) because ACS1 is subject to glucose repression (van den Berg et al., 1996) and its product is inactivated in the presence of glucose (de Jong-Gubbels et al., 1997). Moreover, it has been proposed that Acs2, which was demonstrated to be partially localized in the yeast nucleus, is involved in histone acetylation (Takahashi et al., 2006). Involvement of Acs isoenzymes in the acetylation of histones and/or other proteins might present an additional challenge in replacing them with heterologous reactions, if this includes another mechanism than merely the provision of extramitochondrial acetyl-CoA.

The goal of this study is to investigate whether the heterologous ATP-independent A-ALD and PFL pathways can support the growth of acs1 acs2 mutants of S. cerevisiae by providing extramitochondrial acetyl-CoA and to study the impact of such an intervention on growth, energetics and cellular regulation. To this end, several heterologous genes encoding A-ALD and PFL were screened by expression in appropriate yeast genetic backgrounds, followed by detailed analysis of S. cerevisiae strains in which both ACS1 and ACS2 were replaced by either of the alternative reactions. The resulting strains were studied in batch and chemostat cultures. Furthermore, genome-wide transcriptional responses to these modifications were studied by chemostat-based transcriptome analysis of engineered and reference strains.

Reference

Nakazawa M. C2 metabolism in Euglena. Adv Exp Med Biol. 2017;979:39-45. doi: 10.1007/978-3-319-54910-1_3. PMID: 28429316.

Sequence and Features

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