Construction of the co-expression plasmids of fostriecin polyketide synthases and heterologous expression in Streptomyces

Figures & data

Table 1. Oligonucleotide primers used in this study.

Primes	Template	Sequence (5′→3′)
HA1-F	Cfos-F4	AGTCTAGATTAATTAAGAAGGAGATATACATATGCCGGGTGTTTTAGGG
HA1-R		TTCTGCAGTCGCCATCAGCGCCTGTG
HA2-F	Cfos-F2	AACTGCAGAAAGTACTGCCGGATGCAGGCGGTCA
HA2-R		TACTCGAGACTAGTCAGGGCCGGGTGAGGCC
NA-F	Cfos-F3	TGTCTAGATTAATTAAGAAGGAGATATACATATGATGCCGTCCTGCCCG
NA-R		AAGCTAGCTCAGCAGCCGCCAGCCCGT
NAB-F	S.ATCC 31906	TTGCTAGCCAGGACCGACACCCCCGA
NAB-R	Chromosome	TAGGATCCGGACGAGAACAGTACGAAAG
NB-F	Cfos-F1	AAGGATCCCCCACGTGGCGCAGCTCA
NB-R		ATAAGCTTTCACGACGTGGTCGGCCG
Rc-A	pRC	GCCGGCGGCTCCGGACCCTGCGACTTTCTGGCGGCTGCTGAGGCCTGGTGATGATGGCGG
Rc-AB		TGCCGGGCGGCGACCGCCTCGGAGTCGGGGGTGTCGGTCCTGTTACGCCCCGCCCTGCCA
46F-F	p124	AATCTAGATCGGCCGTGGCGCCGCC
46F-R		AACATATGTGTCCTCCTTAATTAATC
4R-F	p126	AAACTAGTGACCGAGAAGGCCAGCTCC
4R-R		AAACTAGTCAGGCGACCTCCAGCAGCA
6R-F	p126	AAAAGCTTGAATTCGTAATCATGTCATA
6R-R		AAGCGGCCGCTTTTGCTCACATGTTCTTTC
FosD-F		CTGCCATGGACCCGCAGC
FosD-R		CGACGTCGACGTCTGCGG
FosE-F		CGGCGCCGAACGGTCCGT
FosE-R		CGTCACCCCGCGCAACCC

The bold sequences are the homologous arms.

Figure 1. Strategy of Fos-PKS biosynthetic gene cluster cloning by Red/ET recombination and confirmation of constructed expression plasmids: (a) distribution of sequenced cosmids, organization of the fostriecin biosynthetic gene cluster, and strategy of Fos-PKS biosynthetic gene cluster cloning using Red/ET recombination. Each arrow represents an open reading frame (ORF). The direction of transcription and the relative sizes of ORFs are indicated; (b) maps of plasmids for the expression of Fos-PKS biosynthetic gene cluster in Streptomyces. The expression plasmid p6-fosAB containing fosAB and the expression plasmid p4-fosCDEF containing fosCDEF; (c) restriction analysis of the constructed plasmid p6-fosAB by digestion with Xho I. M: λ-Hind III digest Marker and DNA Marker III; p126, F1, and F3: vector p126, cosmid Cfos-F1, and Cfos-F3 digestion with Xho I for compared; 1, 2, 3 and 4: four correct constructs of plasmid p8-fosAB digestion with Xho I; (d) restriction analysis of the constructed plasmid p4-fosCDEF by digestion with Mlu I. M: λ-Hind III digest Marker and DNA Marker III; p124, and F2: vector p124 and cosmid Cfos-F2 digestion with Mlu I for compared; 1 and 2: two correct constructs of plasmid pM-fosCDEF digestion with Mlu I.

Figure 2. Analysis of heterologous expression of Fos-PKS biosynthetic genes cluster in Streptomyces: (A) phenotype of the Streptomyces heterologous expression strains. TK24-V: S. lividans TK24/p126–p124 in FST12 liquid fermentation medium, TK24-G: S. lividans TK24/p6-fosAB–p4-fosCDEF in FST12 liquid fermentation medium, TK24-P: S. lividans TK24/p6-fosAB–p4-fosCDEF on MS medium; M512-V: S. coelicolor M512/p126–p124 in FST12 liquid fermentation medium, M512-G: S. coelicolor M512/p6-fosAB–p4-fosCDEF in FST12 liquid fermentation medium, M512-P: S. coelicolor M512/p6-fosAB–p4-fosCDEF on MS medium; (B) RT-PCR of S. coelicolor M512/p6-fosAB–p4-fosCDEF. A′–F′: the RNA as the template; A–F: the cDNA as the template.

Figure 3. Proposed post-PKS modification in a fostriecin polyketide biosynthetic pathway. ACP, acyl carrier protein; TE, thioesterase domain; “a”, FosM might play a role here.