Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification

Table 3 ALA accumulation of mutant strains

Strain	genes knock-out	ALA accumulation (mg/L)	Standard Error	Relative Change	ALA production rate (g/g)	Significant Summary	P value
E. coli BW25113-T7 (D0)	Control	662.25	100.28	1.00	0.066
E. coli D1:F	ΔhemF	1361.22	100.90	2.06	0.136	****	< 0.0001
E. coli D1:D	ΔdppA	847.07	39.61	1.28	0.085	ns	0.4304
E. coli D1:M	ΔmppA	601.37	41.40	0.91	0.060	ns	0.9991
E. coli D2:FY	ΔhemF, ΔybdK	1280.62	207.11	1.93	0.128	****	< 0.0001
E. coli D2:FA	ΔhemF, ΔgdhA	1114.22	84.14	1.68	0.111	***	0.0008
E. coli D2:FB	ΔhemF, ΔgadB	1094.72	156.11	1.65	0.109	**	0.0014
E. coli D3:FYA	ΔhemF, ΔybdK, ΔgdhA	1473.67	18.11	2.23	0.147	****	< 0.0001
E. coli D3:FYB	ΔhemF, ΔybdK, ΔgadB	1526.32	25.79	2.30	0.153	****	< 0.0001
E. coli D3:FAB	ΔhemF, ΔgdhA, ΔgadB	1341.72	274.49	2.03	0.134	****	< 0.0001
E. coli D4:FYAB	ΔhemF, ΔybdK, ΔgdhA, ΔgadB	1434.02	137.53	2.17	0.143	****	< 0.0001
E. coli D5:FYABD	ΔhemF, ΔybdK, ΔgdhA, ΔgadB, ΔmppA	1601.72	40.62	2.42	0.160	****	< 0.0001
E. coli D5:FYABM	ΔhemF, ΔybdK, ΔgdhA, ΔgadB, ΔdppA	1278.67	161.32	1.93	0.128	****	< 0.0001
E. coli D6:FYABMD	ΔhemF, ΔybdK, ΔgdhA, ΔgadB, ΔmppA, ΔdppA	1014.77	122.83	1.53	0.101	*	0.0124

A 1% (v/v) inoculum from an overnight culture for 12 h was used. IPTG was added when OD600 reached 0.7. ALA production rate: yield of ALA (g) per g glucose. Samples were collected and measured until 24 h. Glucose (10 g/L) was added initially as the sole carbon source. Results are the average of three individual replications. P values were calculated using Dunnett’s multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

ISSN: 1754-1611