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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.

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Dual expression with peqxS promoter reconstitutespathway to lovastatin precursor. (A) Analytical HPLC of crude extractsof PDB cultures of FusΔeqx5 transformed with lovBgfp together with lovC or a noncognate trans-ER eqxC. In the presence of eqxC, lovB produces the polyene pyrone 5 and ketone 6; and more reduced pyrone 7 with lovC coexpression. (B) LC/MS analysis of crude extracts shows formationof the lovastatin precursor, dihydromonacolin L acid 8 and the lactone 9 when lovBgfp iscoexpressed with lovC. EqxC is not able to complementLovB to form 8 or 9.
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fig6: Dual expression with peqxS promoter reconstitutespathway to lovastatin precursor. (A) Analytical HPLC of crude extractsof PDB cultures of FusΔeqx5 transformed with lovBgfp together with lovC or a noncognate trans-ER eqxC. In the presence of eqxC, lovB produces the polyene pyrone 5 and ketone 6; and more reduced pyrone 7 with lovC coexpression. (B) LC/MS analysis of crude extracts shows formationof the lovastatin precursor, dihydromonacolin L acid 8 and the lactone 9 when lovBgfp iscoexpressed with lovC. EqxC is not able to complementLovB to form 8 or 9.

Mentions: To establishthe utility of this new expression strategy for coexpression of heterologousgenes, we cloned the well-studied lovastatin nonaketide synthase (lovB) from Aspergillus terreus togetherwith its cognate trans-ER (lovC)19 to make the hphlovC+lovBgfp plasmid. We alsocloned only lovB into FH-3 to make the plasmid hpheqxC+lovBgfp.Transformation of these constructs independently into FusΔeqx5resulted in production of the expected products. Without its cognate trans-ER, the expressed LovB synthesized the previouslyreported truncated intermediates, 5 and 6 (Figure 6A).19 Coexpression of lovB with lovC produced the expected reduced metabolites 7 (Figure 6A), the dihydromonacolin L acid 8,and the lovastatin precursor lactone 9 (Figure 6B).19,29 These metabolites were characterizedby comparing their liquid chromatography/mass spectroscopy (LC/MS),ultraviolet (UV), and 1H NMR data to previous reports (Supporting Information Figures S8–S10, S12–S14). This confirmed that, indeed, the intergenic sequence between eqxS and eqxC could guide transcriptiondivergently to heterologously coexpress two genes. The purified yieldof 9 was 130 mg kg–1, and 8 was produced in about equal amounts, indicating that the initialunoptimized yield should exceed ∼300 mg kg–1.


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)

Dual expression with peqxS promoter reconstitutespathway to lovastatin precursor. (A) Analytical HPLC of crude extractsof PDB cultures of FusΔeqx5 transformed with lovBgfp together with lovC or a noncognate trans-ER eqxC. In the presence of eqxC, lovB produces the polyene pyrone 5 and ketone 6; and more reduced pyrone 7 with lovC coexpression. (B) LC/MS analysis of crude extracts shows formationof the lovastatin precursor, dihydromonacolin L acid 8 and the lactone 9 when lovBgfp iscoexpressed with lovC. EqxC is not able to complementLovB to form 8 or 9.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4487227&req=5

fig6: Dual expression with peqxS promoter reconstitutespathway to lovastatin precursor. (A) Analytical HPLC of crude extractsof PDB cultures of FusΔeqx5 transformed with lovBgfp together with lovC or a noncognate trans-ER eqxC. In the presence of eqxC, lovB produces the polyene pyrone 5 and ketone 6; and more reduced pyrone 7 with lovC coexpression. (B) LC/MS analysis of crude extracts shows formationof the lovastatin precursor, dihydromonacolin L acid 8 and the lactone 9 when lovBgfp iscoexpressed with lovC. EqxC is not able to complementLovB to form 8 or 9.
Mentions: To establishthe utility of this new expression strategy for coexpression of heterologousgenes, we cloned the well-studied lovastatin nonaketide synthase (lovB) from Aspergillus terreus togetherwith its cognate trans-ER (lovC)19 to make the hphlovC+lovBgfp plasmid. We alsocloned only lovB into FH-3 to make the plasmid hpheqxC+lovBgfp.Transformation of these constructs independently into FusΔeqx5resulted in production of the expected products. Without its cognate trans-ER, the expressed LovB synthesized the previouslyreported truncated intermediates, 5 and 6 (Figure 6A).19 Coexpression of lovB with lovC produced the expected reduced metabolites 7 (Figure 6A), the dihydromonacolin L acid 8,and the lovastatin precursor lactone 9 (Figure 6B).19,29 These metabolites were characterizedby comparing their liquid chromatography/mass spectroscopy (LC/MS),ultraviolet (UV), and 1H NMR data to previous reports (Supporting Information Figures S8–S10, S12–S14). This confirmed that, indeed, the intergenic sequence between eqxS and eqxC could guide transcriptiondivergently to heterologously coexpress two genes. The purified yieldof 9 was 130 mg kg–1, and 8 was produced in about equal amounts, indicating that the initialunoptimized yield should exceed ∼300 mg kg–1.

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