A genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis

A novel mechanism is proposed for ring formation in the biosynthetic pathway to thiotetronate antibiotics thiolactomycin and Tü 3010.


Contents of Supporting
. 1 H and 13   End-repaired DNA was then ligated with EcoRV linearized and FastAP dephosphorylated vector pJTU2554. 3 The ligation mixture was packaged using MaxPlax Lambda Packaging Extracts and plated with E. coli EPI300/pUZ8002. Screening of the S. thiolactonus genomic library for the stu cluster was performed by PCR using primer pairs stuE-up/stuE-dn, stuH-up/stuH-dn, stuA-up/stuA-dn, and stuC-up/stuC-dn.

Genetic manipulation of thiotetronate-producing strains.
For Lentzea sp. ATCC 31319, in-frame deletions of the tlmA and tlmD1 genes were carried out. The recombinant plasmids pMYtlmA and pMYtlmD1 were constructed by amplifying regions upstream and downstream of tlmA (1.5 kbp on each) and tlmD1 (2 kbp on each) using Lentzea sp. genomic DNA and the primer pairs: tlmA-1F/tlmA-1R, tlmA-2F/tlmA-2R, tlmD1-1F/tlmD1-1R, tlmD1-2F/tlmD1-2R. The vector pYH7 4 was digested with BglII and HindIII, treated with alkaline phosphatase, and purified by gel electrophoresis. Ligation of cut pYH7 and the amplified fragments was accomplished by the isothermal Gibson assembly method according to the manufacturer's protocol. The assembly mixture was incubated at 50°C for 60 min, and then was used to transform E. coli DH10B. Plasmids pMYtlmA and pMYtlmD1 were confirmed by sequencing and were each introduced by conjugation into Lentzea sp. ATCC 31319 through donor strain E. coli ET12567/pUZ8002. After incubation at 30°C for 16 hours, exconjugants were selected with 5 μg/mL apramycin and 25 μg/mL nalidixic acid. Antibiotic resistance was confirmed through transfer of exconjugants to a SFM plate containing 50 μg/mL apramycin and 25 μg/mL nalidixic acid. To form the double cross-over mutants, loss of pMYtlmA and pMYtlmD1 was obtained by several rounds of non-selective growth of the exconconjugants on SFM agar medium. The integrity of the Lentzea sp. tlmA and tlmD1 mutants were checked by PCR analysis using the primer pairs: tlmA-3F/tlmA-3R and tlmD1-3F/tlmD1-3R.
For S. thiolactonus NRRL 15439, gene replacement of stuJ,K,S, and four respective gene in-frame deletions of stuK, stuS, stuH, stuB were carried out.
Taking replacement of stuJ,K,S as an example, the recombinant plasmid pWHU2617 was constructed by amplifying 2 kbp regions upstream and downstream of the stuJ,K,S gene, using S. thiolactonus genomic DNA and the primer pairs: stuJ,K,S-L1/stuJ,K,S-L2 and stuJ,K,S-R1/stuJ,K,S-R2. The pYH7 4 fragment was prepared by digesting with BglII and NdeI, and subsequently treating with alkaline phosphatase. Amplified fragments (2 kbp) after digestion were cloned into BglII and NdeI sites of pYH7 to give an intermediate recombinant. A 827 bp cat resistance gene cassette was amplified using primer pairs: cat-up/cat-dn. Plasmid pWHU2617 was created by inserting the cat cassette between the two 2 kbp amplified fragments.
Restriction digestion and sequencing of inserts were performed to confirm the recombinant plasmid pWHU2617, which was then introduced into S. thiolactonus NRRL 15439 by conjugation. Screening for gene replacement mutant of stuJ,K,S was performed as described for tlmA mutant screening.
Trans-complementation was carried out by introducing the complementation plasmids pIB-tlmA, pWHU2698, pWHU2699, pWHU2700 and pWHU2701 into the corresponding gene-null mutant. The integrative vector pIB139 was used for inserting tlmA, stuJ, stuK, stuS, stuJ+S under the control of the ermE* promoter, respectively. Genes including tlmA (2 kbp), stuJ (1 kbp), stuK (666 bp) and stuS (1 kbp), were amplified using primer pairs: tlmA-NdeI/tlmA-XbaI, stuJ-up/stuJ-dn, stuK-up/stuK-dn, stuS-up/stuS-dn, respectively. The resulting products were digested with NdeI and XbaI, and then cloned into the corresponding site of pIB139, except for stuS which was digested with NdeI and EcoRI and inserted into the NdeI and EcoRI site of pIB139. After sequencing confirmation, each plasmid was introduced into the appropriate gene-null mutant and analyzed for the production of thiotetronate antibiotic.          The arrows indicate the expected size of the fragments from the wild-type and mutant chromosomal DNA, respectively. has been confirmed to be an artifact, unrelated to thiolactomycin.