Production of the polyketide 6-deoxyerythronolide B in the heterologous host Bacillus subtilis

Polyketides, such as erythromycin, are complex natural products with diverse therapeutic applications. They are synthesized by multi-modular megaenzymes, so-called polyketide synthases (PKSs). The macrolide core of erythromycin, 6-deoxyerythronolide B (6dEB), is produced by the deoxyerythronolide B synthase (DEBS) that consists of three proteins each with a size of 330–370 kDa. We cloned and investigated the expression of the corresponding gene cluster from Saccharopolyspora erythraea, which comprises more than 30 kb, in Bacillus subtilis. It is shown that the DEBS genes are functionally expressed in B. subtilis when the native eryAI–III operon was separated into three individual expression cassettes with optimized ribosomal binding sites. A synthesis of 6dEB could be detected by using the acetoin-inducible acoA promoter and a fed-batch simulating EnBase-cultivation strategy. B. subtilis was capable of the secretion of 6dEB into the medium. In order to improve the 6dEB production, several genomic modifications of this production strain were tested. This included the knockout of the native secondary metabolite clusters of B. subtilis for the synthesis of surfactin (26 kb), bacillaene (76 kb), and plipastatin (38 kb). It is revealed that the deletion of the prpBD operon, responsible for propionyl-CoA utilization, resulted in a significant increase of the 6dEB product yield when exogenous propionate is provided. Although the presented B. subtilis 6dEB production strain is not competitive with established Escherichia coli 6dEB production strains, the results of this study indicate that B. subtilis is a suitable heterologous host for the secretory production of a complex polyketide. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6990-6) contains supplementary material, which is available to authorized users.


Introduction
Fig. S1 Biosynthesis of 6dEB. The three eryAI-III genes (A) are coding for the DEBS1-3 proteins (B). These consist of one loading didomain (LDD), six modules for elongation and a termination (TE) domain (C) and are organized in catalytic domains (D). Biosynthesis (E) involves the assembling of the starter unit propionyl-CoA with six (2S)-methylmalonyl-CoA extender units (F). Depending on the presence of auxiliary domains, the keto-function remains unaltered or is partially or entirely reduced. The termination domain catalyzes the release of the metabolite from the enzyme by intra-molecular lactonization resulting in the macrolide 6dEB. Within the native host, S. erythraea 6dEB is subsequently modified to the antibiotic erythromycin (G). Catalytic domains: AT, acyl transferase; ACP, acyl carrier protein; KS, ketosynthase; KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; TE, thioesterase.

Construction of plasmids
All plasmids for integration of the eryAI-III genes are presented in Fig. S2. In order to chromosomally integrate the modified eryAI gene in the srfA gene locus, the Kan R -cassette of the srfA deletion plasmid pJK93 was replaced by a Spec R -cassette, amplified from pAMY-Spec with the primers 5158 and 2355. Both, pJK93 and the resulting PCR product were digested with XhoI and SalI and ligated to give pJK94. This plasmid was then cut with SpeI and the 5'-region of eryAI was integrated via a "sequence and ligation independent cloning" (SLIC) method as described elsewhere (Li and Elledge 2007) after its amplification from cosmid P1394 with the oligonucleotides 5082 and 5206. Hereafter, the resulting plasmid was digested with PmeI and the acoA promoter, amplified from B. subtilis 168 with the primers 5076 and 5097, was integrated via the same method. In a next step, this plasmid was cut with SpeI and StuI and the 3'-region of eryAI, obtained from P1394 by using the primers 5072 and 5207, was integrated. The resulting plasmid now contained a PacoA-5'-eryAI-StuI-3'-eryAI-TT7-cassette. Restriction of this plasmid with StuI led to a linear product with homologous regions to the eryAI gene allowing recombination with cosmid P1394 via the Red/ET method (gene bridges) according to the manufacturer's protocol. The resulting plasmid was named pJK119. In order to use the previously described optimized cloning protocol for rapid and multiple genome modification in B. subtilis, the lox-SSS-cassette (in which the Spec R -cassette is flanked by two so called six-sites, which are in turn surrounded by two mutated lox sites) for marker removal and an enhanced transformation efficiency (Kumpfmüller et al. 2013) was used to replace the Spec R -cassette. To this purpose, plasmid pJK119 was digested with EcoRI and SwaI and plasmid pJET-lox-SSS was cut with MfeI and EcoRV. Ligation of the intended fragments resulted in plasmid pJK119c (Fig. S3). For the integration of the modified eryAII gene in the srfA locus containing the PacoA-eryAI-TT7-operon, the Spec R -cassette from pJK119 had to be replaced by a Kan R -cassette. Since Red/ET cloning requires a selection marker switch and cosmid P1394 already contained a Kan R -cassette, an additional Cm R -cassette for selection in E. coli was amplified from pCUlac_pB with the primers 5273 and 5274 and integrated in pJK123, cut with AatII, via SLIC to give pJK134. The following modifications of this plasmid were all performed via the SLIC method. First the 3'-region of eryAI was amplified using P1394 and the oligonucleotides 5276 and 5288 and integrated in pJK134, cut with AatII and SpeI, thereby replacing the 5'-srfA-region (landing pad). Hereafter, this plasmid was digested with PmeI and fused with the 3'-region of the eryAII gene, obtained from P1394 after amplification with the primers 5277 and 5278. The resulting plasmid was cut either with PmeI/SmaI or with SpeI/SmaI. The PmeI/SmaI digested plasmid was ligated with the 5'-region of the eryAII gene, amplified from P1394 using the primer pair 5081/5279. The resulting plasmid was digested with PmeI for integration of the acoA-promoter, obtained from B. subtilis 168 with the primers 5016 and 5017. Hereafter, this plasmid was linearized with SmaI and fused with the eryAII gene using P1394 and the Red/ET method to give pJK139 harboring the final 3'-eryAI-TT7-PacoA-eryAII-cassette. In analogue, the plasmid restricted with SpeI/SmaI was again ligated with the 5'-region of the eryAII gene, but amplified from P1394 using the primer pair 5077/5279. This resulted in a 3'-eryAI-RBS-5'-eryAII-SmaI-3'-eryAII-cassette that could be used for Red/ET cloning with P1394 and gave pJK140 containing the final 3'-eryAI-RBS-eryAII-cassette. For chromosomal integration it was necessary to add the lox-SSS-cassette. Hence, pJK139 and pJK140 were digested with EcoRI and ligated with pJET-lox-SSS, cut with MfeI, to give pJK139a and pJK140a, respectively. The plasmid pJK191 was digested with ScaI and SpeI. Hereafter, this plasmid was cut with PmeI, dephosphorylated and ligated with the 3'region of eryAIII after its amplification from P1394 with the primer pair 5451/5452, thereby adding the T7-terminator, and its phosphorylation. Analogous to the construction of pJ139 and pJK140, the resulting plasmid was digested with either PmeI/SmaI or SpeI/SmaI. Again, the PmeI/SmaI digested plasmid was ligated to the 5'-region of the eryAIII gene, amplified from P1394 using the primers 5453/5454. The resulting plasmid was digested with PmeI for integration of the acoApromoter, obtained from B. subtilis 168 with the primers 5090 and 5017. Hereafter, this plasmid was also linearized with SmaI and fused with the eryAIII gene using P1394 and the Red/ET method to give pJK245 harboring the final 3'-eryAII-TT7-PacoA-eryAIII-TT7-cassette.

Fig. S2
Vector maps of the plasmids for the (stepwise) chromosomal integration of the eryAI-III genes.
The unmodified eryAI-III gene cluster was integrated in a single step. For marker removal a simple SSS-cassette (Spec R -cassette flanked by two six-sites), obtained from pAMY-SSS with the primers 5160 and 5161 and cut with XhoI, was ligated into the srfA deletion plasmid pJK93, cut with XhoI and SalI, thereby replacing the Kan R -cassette to give pJK111. Hereafter, this plasmid was digested with SpeI and fused with the 3'-region of the eryAIII gene, amplified from P1394 with the primers 5308 and 5309, via SLIC. In a next step, the PacoA-5'-eryAI-cassette was amplified using pJK119 as template and the primer pair 5097/5310 and integrated into the obtained plasmid, cut with StuI. This plasmid was then used for Red/ET cloning with P1394 to give pJK155 which contained the final PacoA-eryAI-AIIInat-TT7 operon. In order to simplify future expression studies, a plasmid for deletion of the srfA operon containing both a PacoA-TT7-and the lox-SSS-cassette was constructed. To this purpose, the srfA deletion plasmid pJK126, which already harbored the T7-terminator was digested with BamHI and ligated with the lox-SSS-cassette obtained from pJET-lox-SSS, cut likewise, to give pJK218, thereby replacing the Nm R -cassette. Hereafter, this plasmid was linearized with XhoI and fused with the acoA-promoter, amplified from B. subtilis using the primers 5413/5414, via SLIC to give pJK219. This plasmid was then used for the ery-orf5 cloning. Therefore, the CDS was amplified from Saccharopolyspora erythraea DNA using the primer pair 5471/5472, digested with NdeI and SpeI and ligated with pJK219, cut in the same manner, to give pJK257. In order to chromosomally integrate the PacoA-ery-orf5-TT7 operon behind the eryAI-III cluster located in the srfA gene locus, pJK257 was digested with AatII/NheI and ligated with the 3'-region of the eryAIII gene, amplified from pJK245 with the primers 5473/5286 and cut with AatII/XbaI thereby replacing the 5'-srfA-region and resulting in plasmid pJK258. For the deletion of the ppsABCDE operon, responsible for synthesis of the nonribosomal peptide plipastatin, plasmid construction started from pAMY-lox-SSS. To this purpose, the 3'region of the operon was amplified from B. subtilis with the primers 5464/5465, digested with MscI and BamHI and ligated with pAMY-lox-SSS, which was cut likewise. Hereafter, this plasmid (digested with AatII/SpeI) was ligated with the 5'-region of the pps operon, obtained with the oligonucleotides 5466/5467 and cut in the same manner to give pJK254. The plasmid for deletion of the prpBD operon, responsible for propionyl-CoA utilization, was constructed likewise. pAMY-lox-SSS was cut with MscI/BamHI and ligated with the 5'-region of the prp operon, obtained from B. subtilis with the primer pair 5474/5475 and cut in the same manner. This plasmid was then digested with AatII/XbaI and ligated with the 3'-region of the prp operon, amplified with the primers 5476/5477 and cut with AatII/SpeI to finally give pJK260. Furthermore, a plasmid for integration of a remaining Kan R -cassette together with a removable, transformation-enhancing lox-SSS-cassette was constructed as follows. Plasmid pJK191 was digested with XbaI and ligated with the Kan R -cassette, amplified from pMSE3 with primers 5411/5412 and cut likewise, to give pJK206.

Fig. S3
Scheme of plasmid construction for the chromosomal integration of eryAI. Endonucleases for plasmid digestion, templates and oligonucleotides for PCR as well as the cloning method are specified.  subtilis JK70 (C). Finally, the frame shift mutated sfp 0 gene was replaced by the active sfp + gene by using pJK64a to give B. subtilis JK71 (D).

Fig. S6
Detection of eryAI-III transcription by slot-blot hybridization with specific probes and acoA as positive control in all three recombinant strains. The wt strain without the ery genes was used as negative control.