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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

Expression of CpaS andproduction of cAATrp (1). (A)Expression in the wild-type F. heterosporum strain.HPLC-diode array detection (DAD) analysis of crude extracts of PDBcultures of Peqx:Cpas mutant and the Palc:eqxR control. The eqxS promoterdrives expression to avail expected product 1. Coproductionof equisetin 2 is observed. (B) Expression in eqx knockout strain. Production of 1 is improved,and equisetin 2 is no longer observed.
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fig4: Expression of CpaS andproduction of cAATrp (1). (A)Expression in the wild-type F. heterosporum strain.HPLC-diode array detection (DAD) analysis of crude extracts of PDBcultures of Peqx:Cpas mutant and the Palc:eqxR control. The eqxS promoterdrives expression to avail expected product 1. Coproductionof equisetin 2 is observed. (B) Expression in eqx knockout strain. Production of 1 is improved,and equisetin 2 is no longer observed.

Mentions: To determinewhether the peqxS′ could lead to biosynthesisof new compounds, the well-characterized cpaS genefrom Aspergillus flavus was cloned into FH-1 to makepHygB-Cpas. The resulting F. heterosporum mutant,Peqx:Cpas, was then cultured in potato dextrose broth (PDB) for 7days. The predicted product, cAATrp 1 was identifiedby high pressure liquid chromatography (HPLC) (Figure 4A) and isolated. Comparison of the 1H NMR spectrumand the molecular formula of 1 with those of the previouslyreported cAATrp showed that they were identical (Supporting Information Figure S13).23,27 This confirmed the value of peqxS′ in producingheterologous compounds. However, high levels of equisetin 2 were coproduced (Figure 4A).


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)

Expression of CpaS andproduction of cAATrp (1). (A)Expression in the wild-type F. heterosporum strain.HPLC-diode array detection (DAD) analysis of crude extracts of PDBcultures of Peqx:Cpas mutant and the Palc:eqxR control. The eqxS promoterdrives expression to avail expected product 1. Coproductionof equisetin 2 is observed. (B) Expression in eqx knockout strain. Production of 1 is improved,and equisetin 2 is no longer observed.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Expression of CpaS andproduction of cAATrp (1). (A)Expression in the wild-type F. heterosporum strain.HPLC-diode array detection (DAD) analysis of crude extracts of PDBcultures of Peqx:Cpas mutant and the Palc:eqxR control. The eqxS promoterdrives expression to avail expected product 1. Coproductionof equisetin 2 is observed. (B) Expression in eqx knockout strain. Production of 1 is improved,and equisetin 2 is no longer observed.
Mentions: To determinewhether the peqxS′ could lead to biosynthesisof new compounds, the well-characterized cpaS genefrom Aspergillus flavus was cloned into FH-1 to makepHygB-Cpas. The resulting F. heterosporum mutant,Peqx:Cpas, was then cultured in potato dextrose broth (PDB) for 7days. The predicted product, cAATrp 1 was identifiedby high pressure liquid chromatography (HPLC) (Figure 4A) and isolated. Comparison of the 1H NMR spectrumand the molecular formula of 1 with those of the previouslyreported cAATrp showed that they were identical (Supporting Information Figure S13).23,27 This confirmed the value of peqxS′ in producingheterologous compounds. However, high levels of equisetin 2 were coproduced (Figure 4A).

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