Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum

Table 1 Bacterial strains and plasmids

Strain or plasmid	Relevant characteristics	Source or reference
E.coli XL1-Blue-G	Fpr (donor of ppsA, pckA)	Ref. 28
E.coli P2392	(strain for expression)	Stored by our lab
B. flavum 311	Nxr, Fpr (strain for expressionand fermentation)	Stored by our lab
pλPR	Apr(E. coli expressing vector)	Ref. 28
pλPR-pps	pλPR inserted with ppsA	This study
pλPR-pck	pλPR inserted with pckA	This study
pλPR-2p	pλPR inserted with ppsA and pckA tandemly	This study
pSK-PBF	pBluscript SK- inserted with promoter PBF	Structured by our lab, unpublished
pCZ	Kmr (B. Flavum-E.coli shuttle vector)	Stored by our lab
pCZ-GAB	pCZ inserted with tandem aroG pheA tyrB	Ref. 1
pJN2	pCZinserted with tandem ppsA pckA	This study
pJN5	pCZ-GAB inserted with tandem ppsA pckA	This study

Fp^r, resistance to fluorophenylalanine; Nx^r, resistance to nalidixic acid; Ap^r, resistance to ampicillin; Km^r, resistance to kanamycin.

Table 2 The relative specific enzymatic activities in E. coli P2392 harboring different constructs

Strain/plasmid	Relative enzymatic activities
	PpsA	PckA	DS
E coli P2392/pλPR	1	1	1
E coli P2392/pλPR-pps	5.2	1.0	1.8
E coli P2392/pλPR-pck	1.0	2.5	1.5
E coli P2392/pλPR-2p	3.1	2.3	2.1

When we looked at the enzymatic activities of DS in different transformants, we found that either single-gene expression or co-expression of ppsA and pckA genes could induce elevated expression of DS by 0.5 to 1.1-fold.

Table 3 Relative specific enzymatic activities in B. flavum 311 harboring different constructs

Constructs	Relative specific enzymatic activities
	PpsA	PckA	DS	CM/PD	AT
pCZ	1	1	1	1/1	1
pJN2	4.2	4.5	1.9	1.0/1.2	1.2
pCZ-GAB	0.9	1.1	4.2	4.1/2.2	4.7
pJN5	2.8	3.3	6.3	4.9/2.5	5.5

Table 4 Phenylalanine production of B. flavum harboring different constructs

Constructs	Yield (g/L)	Relative yield
None	1.64 ± 0.27	1
pCZ	1.59 ± 0.29	1
pJN2	2.04 ± 0.25	1.3
pCZ-GAB	4.83 ± 0.18	3.0
pJN5	5.39 ± 0.32	3.4