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Native promoter strategy for high-yielding synthesis and engineering of fungal secondary metabolites.

Kakule TB, Jadulco RC, Koch M, Janso JE, Barrows LR, Schmidt EW - ACS Synth Biol (2014)

Bottom Line: As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L(-1), leading to the practical synthesis of a selective antituberculosis agent.Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi.These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites.

View Article: PubMed Central - PubMed

Affiliation: §Natural Products, Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, Connecticut 06355, United States.

ABSTRACT
Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus Fusarium heterosporum natively synthesizes the polyketide equisetin at >2 g L(-1) in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native eqxS promoter and eqxR regulator in F. heterosporum, synthesizing heterologous natural products in yields of ∼1 g L(-1). As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L(-1), leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites.

No MeSH data available.


Related in: MedlinePlus

Trichosetin synthesis requires EqxC and unmodified EqxS.(A) Expressionvector FH-3 designed with complete intergenic sequence peqxS, for dual expression of genes. Also shown are elements that permitcloning by recombination in S. cerevisiae and selectionin E. coli. (B) Analytical HPLC of crude extractsof PDB cultures of FusΔeqx5 transformed with eqxSgfp together with either eqxC or noncognate trans-ER lovC. Also shown is nontagged eqxS coexpressed with eqxC and the trichosetinstandard. Trichosetin is only produced in the presence of eqxC and unmodified eqxS. (C) LC/MS analysisof crude extracts shows that a gfp tag on the C-terminus of EqxS interfereswith formation of trichosetin and instead results in production ofonly the ring-open form 4.
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fig5: Trichosetin synthesis requires EqxC and unmodified EqxS.(A) Expressionvector FH-3 designed with complete intergenic sequence peqxS, for dual expression of genes. Also shown are elements that permitcloning by recombination in S. cerevisiae and selectionin E. coli. (B) Analytical HPLC of crude extractsof PDB cultures of FusΔeqx5 transformed with eqxSgfp together with either eqxC or noncognate trans-ER lovC. Also shown is nontagged eqxS coexpressed with eqxC and the trichosetinstandard. Trichosetin is only produced in the presence of eqxC and unmodified eqxS. (C) LC/MS analysisof crude extracts shows that a gfp tag on the C-terminus of EqxS interfereswith formation of trichosetin and instead results in production ofonly the ring-open form 4.

Mentions: In initial experiments,only one side of peqxS was used, comprising 1.5 kbpof gene sequence. To express two genes, peqxS wassynthesized, containing the entire ∼2.5kbp of the divergent promoter, to generate vector FH-3 (Figure 5A). Previously, we showed by knockout mutagenesisthat eqxC was critical for the production of equisetin 2 and trichosetin 3, and we proposed that itwas the trans-acting ER for the equisetin pathway.11 Here, we showed the direct involvement of eqxS in the biosynthesis of trichosetin 3. Both eqxS and eqxC were cloned into vector FH-3under control of the divergent eqx promoter. In initialexperiments, eqxS was fused with a C-terminal sGFPtag so that we could readily confirm protein expression. The resultingvector, hpheqxC+eqxSgfp, was transformed into FusΔeqx5. Theisolated transformants were brightly fluorescent, demonstrating appropriategene expression under control of the divergent promoter. In addition,the fluorescence was constitutively obtained on PDA, further demonstratingthe constitutive regulation of the pathway under control of leaky palcA.


Native promoter strategy for high-yielding synthesis and engineering of fungal secondary metabolites.

Kakule TB, Jadulco RC, Koch M, Janso JE, Barrows LR, Schmidt EW - ACS Synth Biol (2014)

Trichosetin synthesis requires EqxC and unmodified EqxS.(A) Expressionvector FH-3 designed with complete intergenic sequence peqxS, for dual expression of genes. Also shown are elements that permitcloning by recombination in S. cerevisiae and selectionin E. coli. (B) Analytical HPLC of crude extractsof PDB cultures of FusΔeqx5 transformed with eqxSgfp together with either eqxC or noncognate trans-ER lovC. Also shown is nontagged eqxS coexpressed with eqxC and the trichosetinstandard. Trichosetin is only produced in the presence of eqxC and unmodified eqxS. (C) LC/MS analysisof crude extracts shows that a gfp tag on the C-terminus of EqxS interfereswith formation of trichosetin and instead results in production ofonly the ring-open form 4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487227&req=5

fig5: Trichosetin synthesis requires EqxC and unmodified EqxS.(A) Expressionvector FH-3 designed with complete intergenic sequence peqxS, for dual expression of genes. Also shown are elements that permitcloning by recombination in S. cerevisiae and selectionin E. coli. (B) Analytical HPLC of crude extractsof PDB cultures of FusΔeqx5 transformed with eqxSgfp together with either eqxC or noncognate trans-ER lovC. Also shown is nontagged eqxS coexpressed with eqxC and the trichosetinstandard. Trichosetin is only produced in the presence of eqxC and unmodified eqxS. (C) LC/MS analysisof crude extracts shows that a gfp tag on the C-terminus of EqxS interfereswith formation of trichosetin and instead results in production ofonly the ring-open form 4.
Mentions: In initial experiments,only one side of peqxS was used, comprising 1.5 kbpof gene sequence. To express two genes, peqxS wassynthesized, containing the entire ∼2.5kbp of the divergent promoter, to generate vector FH-3 (Figure 5A). Previously, we showed by knockout mutagenesisthat eqxC was critical for the production of equisetin 2 and trichosetin 3, and we proposed that itwas the trans-acting ER for the equisetin pathway.11 Here, we showed the direct involvement of eqxS in the biosynthesis of trichosetin 3. Both eqxS and eqxC were cloned into vector FH-3under control of the divergent eqx promoter. In initialexperiments, eqxS was fused with a C-terminal sGFPtag so that we could readily confirm protein expression. The resultingvector, hpheqxC+eqxSgfp, was transformed into FusΔeqx5. Theisolated transformants were brightly fluorescent, demonstrating appropriategene expression under control of the divergent promoter. In addition,the fluorescence was constitutively obtained on PDA, further demonstratingthe constitutive regulation of the pathway under control of leaky palcA.

Bottom Line: As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L(-1), leading to the practical synthesis of a selective antituberculosis agent.Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi.These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites.

View Article: PubMed Central - PubMed

Affiliation: §Natural Products, Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, Connecticut 06355, United States.

ABSTRACT
Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus Fusarium heterosporum natively synthesizes the polyketide equisetin at >2 g L(-1) in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native eqxS promoter and eqxR regulator in F. heterosporum, synthesizing heterologous natural products in yields of ∼1 g L(-1). As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L(-1), leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites.

No MeSH data available.


Related in: MedlinePlus