Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus

Abstract

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter P_kasO* or P_neo . The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.

Fig. 1

Effect of salJ overexpression under the control of different promoters on salinomycin production. A. Construction of salJ overexpression strains. P, promoter. Sa‐PJ indicates salJ overexpression strains under the control of different promoters. B. Genomic analysis of salJ overexpression strains by PCR. M, DNA ladder; lanes 1, 4 and 7: PCR amplification products using the genomic DNA of Sa as template; lanes 2‐3, 5‐6 and 8‐9: PCR products using genomic DNA of strains Sa‐hrdBJ (primer pair P _hrdB ‐F/salJ‐R), Sa‐neoJ (primer pair P _neo ‐F/salJ‐R) or Sa‐kasO*J (primer pair P _kasO *‐F/salJ‐R) as template, and the sizes of the corresponding PCR products are 3333 bp, 3072 bp and 3002 bp respectively. C. HPLC analysis of salinomycin production from the fermentation broth of different strains. Data are presented as the averages of three independent experiments. Error bars indicate standard deviations (SD). Significant difference between the recombinant strains and wild‐type strain (Sa) was confirmed by Student’s t‐test (* and ** represent P < 0.05 and P < 0.01 respectively). D. Bioassays of salinomycin from the fermentation broth of different strains against B. cereus. Sa, Streptomyces albus CGMCC 4.5716 (wild‐type strain); Sa‐hrdBJ, Sa‐neoJ and Sa‐kasO*J are salJ overexpression strains.

Fig. 2

Effect of the overexpression of key gene modules on salinomycin production. A. Genetic organization of salinomycin biosynthetic gene cluster in S. albus. B. Proposed pathway of salinomycin biosynthesis. C. Construction of recombinant plasmid pSET152::D‐C, an assembly of salBIII‐E, salC, salD‐F and salH‐I (abbreviated as salD‐C) on pSET152. D. Agarose gel electrophoresis of restriction fragments from plasmid pSET152::D‐C. M, 1 kb DNA ladder; lanes 1‐4, plasmid pSET152::D‐C digested with EcoRV (14 415 bp), KpnI (9522 bp, 4893 bp), BamHI (8605 bp, 3005 bp, 2805 bp) and PvuII (6607 bp, 5394 bp, 2414 bp) respectively. The expected sizes of DNA fragments are shown in brackets above. E. HPLC analysis of salinomycin production in the fermentation broth of Sa and Sa‐D‐C strains. Data are presented as the averages of three independent experiments. Error bars indicate standard deviations (SD). Significant difference between the recombinant strains and wild‐type strain (Sa) was confirmed by Student’s t‐test (** represents P < 0.01). F. Bioassays of salinomycin from the fermentation broth of Sa and Sa‐D‐C strains against B. cereus. Sa, S. albus CGMCC 4.5716 (wild‐type strain); Sa‐D‐C, salD‐C overexpression strain.

Fig. 3

Construction and verification of the recombinant plasmid pSET152::mini‐cluster. A. Construction of the recombinant plasmid pSET152::mini‐cluster. B. Agarose gel electrophoresis of PCR amplification products from the plasmid pSET152::mini‐cluster. M, 1 kb DNA ladder; lanes 1–7, PCR products using primer pairs MCS‐F/qJ‐F (1491 bp), salJ‐in‐R/qccr‐R (1772 bp), ccr‐F/qD‐R (2056 bp), qD‐F/qT1‐F (2631 bp), qH‐R/qBI‐R (1269 bp), salE‐in‐F/qC‐F (1010 bp) or qC‐R/MCS–R (1431 bp) respectively. C. Agarose gel electrophoresis of restriction fragments from the plasmid pSET152::mini‐cluster. M, 1 kb DNA ladder; lane 1, the recombinant plasmid pSET152::mini‐cluster; lanes 2–4, the restriction fragments of plasmid pSET152::mini‐cluster digested with EcoRI (18 852 bp), EcoRI + KpnI (9529 bp, 4893 bp, 4430 bp) or NcoI (6553 bp, 6170 bp, 3306 bp, 1788 bp, 1035 bp) respectively. The expected sizes of DNA fragments are shown in brackets above.

Fig. 4

Effect of metK overexpression on salinomycin production. A. Transcriptional analysis of metK in Sa and Str‐99 strains. The transcriptional level of metK was normalized internally to that of hrdB transcription of Sa. Data are presented as the averages of three independent experiments. Error bars indicate standard deviations (SD). B. Construction of metK overexpression strains. C. HPLC analysis of salinomycin production from the fermentation broth of Sa, Str‐99 and Sa‐metK strains. Significant difference between the recombinant strains and wild‐type strain (Sa) was confirmed by Student’s t‐test (** represents P < 0.01). D. Proposed pleiotropic effect of rsmG mutation and metK overexpression on salinomycin production. Sa, S. albus CGMCC 4.5716 (wild‐type strain); Str‐99, the streptomycin‐resistant mutant of S. albus CGMCC 4.5716; Sa‐metK, metK overexpression strain; rsmG*, mutated rsmG gene with a base C insertion at position 26.

Fig. 5

Effect of the overexpression of mini‐cluster on salinomycin production. a whole gene cluster in a defineda whole gene cluster in a defined A. HPLC analyses of salinomycin production from the fermentation broth of different strains. Data are presented as the averages of three independent experiments. Error bars indicate standard deviations (SD). Significant difference between the recombinant strains and wild‐type strain (Sa) was confirmed by Student’s t‐test (** represents P < 0.01). a whole gene cluster in a defined B. Bioassays of salinomycin from the fermentation broth of different strains against B. cereus. a whole gene cluster in a defined C. Time‐course of salinomycin production in Sa and Str‐99‐mini‐cluster determined by HPLC. a whole gene cluster in a defined D. Growth curves of strains Sa and Str‐99‐mini‐cluster in liquid culture. Sa, S. albus CGMCC 4.5716 (wild‐type strain). Str‐99, a streptomycin‐resistant mutant of S. albus CGMCC 4.5716. Sa‐kasO*J and Str‐99‐kasO*J, Sa or Str‐99 contains salJ under the control of P _kasO* promoter for overexpression respectively. Sa‐mini‐cluster and Str‐99‐mini‐cluster, Sa or Str‐99 contains mini‐cluster respectively.

Fig. 6

Effect of the overexpression of sal gene cluster on salinomycin production. Sa, S. albus CGMCC 4.5716 (wild‐type strain). Str‐99, a streptomycin‐resistant mutant of S. albus CGMCC 4.5716. Sa‐sal and Str‐99‐sal, Sa or Str‐99 contains sal gene cluster respectively. Sa‐mini‐cluster and Str‐99‐mini‐cluster, Sa or Str‐99 contains mini‐cluster respectively. Error bars indicate standard deviations (SD). Significant difference between the recombinant strains and Sa or Str‐99 was confirmed by Student’s t‐test (* and ** represent P < 0.05 and P < 0.01 respectively).

Fig. 7

RT‐qPCR transcriptional analysis of salinomycin biosynthetic genes in Sa and Str‐99‐mini‐cluster strains. Sa, S. albus CGMCC 4.5716 (wild‐type strain). Str‐99‐mini‐cluster strain, Str‐99 strain contains the mini‐cluster for overexpression. Error bars show standard deviations (SD). Transcriptional levels of the genes were normalized internally to the level of hrdB transcription. Data are presented as means ± standard deviations (SD) of three independent experiments.

Fig. 8

Overview of sal mini‐cluster in Str‐99 chassis strain for promoting salinomycin production. This combinatorial optimization strategy consists of five routes. Route I involves the overexpression of tailoring genes driven by strong promoters (P _kasO* or P _neo ). Route II involves the overexpression of transporter genes (salH, salI) to enhance the tolerance of salinomycin. Route III involves the overexpression of the activator gene salJ to upregulate the transcription of pks operon. Route IV involves the overexpression of ccr (crotonyl‐CoA reductase gene) to improve the formation of ethylmalonyl‐CoA precursor from crotonyl‐CoA. Route V involves the acquirement of a host strain Str‐99 with rsmG mutation (here represented as rsmG*) by ribosome engineering for salinomycin production.