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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

Knockout of eqx genes. (A) The wild-type genecluster. (B) Previously, eqxC was deleted with hph. (C) Here, this eqxC knockout was extendedto delete peqxS and eqxC and todisrupt the 5′-regions of eqxS and eqxD, using the ble marker. This knockoutrequired the prior deletion of eqxC and did not workin the wild-type strain.
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fig2: Knockout of eqx genes. (A) The wild-type genecluster. (B) Previously, eqxC was deleted with hph. (C) Here, this eqxC knockout was extendedto delete peqxS and eqxC and todisrupt the 5′-regions of eqxS and eqxD, using the ble marker. This knockoutrequired the prior deletion of eqxC and did not workin the wild-type strain.

Mentions: We desiredto compare expression in the wild-type F. heterosporum with expression in an eqx knockout strain. Although eqxC has been previously deleted, eqxS wasdifficult to delete. Here, we created a knockout vector in which eqxS, eqxC, eqxD, andthe promoter region peqxS were deleted (Figure 2). The resulting strain, FusΔeqx5 was confirmedto be an eqx cluster knockout by PCR (Supporting Information Figure S4). Subsequentgenome sequencing of FusΔeqx5 revealed that the eqxS gene was knocked out in the 5′ end and that the deletionvector was inserted into a total of 3 locations in the genome (Supporting Information Figure S5).


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)

Knockout of eqx genes. (A) The wild-type genecluster. (B) Previously, eqxC was deleted with hph. (C) Here, this eqxC knockout was extendedto delete peqxS and eqxC and todisrupt the 5′-regions of eqxS and eqxD, using the ble marker. This knockoutrequired the prior deletion of eqxC and did not workin the wild-type strain.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Knockout of eqx genes. (A) The wild-type genecluster. (B) Previously, eqxC was deleted with hph. (C) Here, this eqxC knockout was extendedto delete peqxS and eqxC and todisrupt the 5′-regions of eqxS and eqxD, using the ble marker. This knockoutrequired the prior deletion of eqxC and did not workin the wild-type strain.
Mentions: We desiredto compare expression in the wild-type F. heterosporum with expression in an eqx knockout strain. Although eqxC has been previously deleted, eqxS wasdifficult to delete. Here, we created a knockout vector in which eqxS, eqxC, eqxD, andthe promoter region peqxS were deleted (Figure 2). The resulting strain, FusΔeqx5 was confirmedto be an eqx cluster knockout by PCR (Supporting Information Figure S4). Subsequentgenome sequencing of FusΔeqx5 revealed that the eqxS gene was knocked out in the 5′ end and that the deletionvector was inserted into a total of 3 locations in the genome (Supporting Information Figure S5).

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