Directed evolution towards bioengineering of fatty acid-activating natural product pathways
脂肪酸激活天然产物途径的生物工程定向进化
基本信息
- 批准号:10607101
- 负责人:
- 金额:$ 6.91万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-05-01 至 2024-08-31
- 项目状态:已结题
- 来源:
- 关键词:AcidsAlkenesAnabolismArchitectureAreaBinding ProteinsBinding SitesBioinformaticsBiological ModelsBiomedical EngineeringCarrier ProteinsChimera organismChimeric ProteinsComplexDevelopmentDirected Molecular EvolutionDockingElementsEngineeringEnzymesFatty AcidsFutureGatekeepingGene ClusterGene ExpressionGenerationsGoalsHealthHomologous GeneHumanHybridsIndividualKnowledgeLengthLibrariesLigaseLinkLogicMass FragmentographyMass Spectrum AnalysisMetabolicMetabolic PathwayMetabolismMethodsModelingNatural ProductsNuclear Magnetic ResonancePathway interactionsProcessProductionPropertyProteinsPseudomonas putidaResearch ProposalsRibosomesStructureStructure-Activity RelationshipSystemTemperatureTertiary Protein StructureTherapeuticTranslationsType I Polyketide SynthaseWorkanalogbioactive natural productsexperimental studyimprovedin vivoliquid chromatography mass spectrometrymetabolic engineeringmetabolomicsnatural product derivativenovelpeptide synthasepharmacologicpolyketide synthasepreventprotein protein interactionscaffoldscreeningsecondary metaboliteyeast two hybrid system
项目摘要
SUMMARY ABSTRACT
Natural products are potently active, privileged scaffolds that form the basis of our therapeutic arsenal across
all areas of human health. The continued development of natural products and their analogs will provide access
to compounds with improved activity and pharmacological properties while decreasing off-target effects. The
bioengineering of individual biosynthetic enzymes is one method of generating such novel secondary
metabolites. However, bioengineering efforts are often stymied due to a lack of fundamental understanding of
the discrete enzymatic transformations responsible for natural product biosynthesis. Likewise, whole pathway
metabolic engineering focused on generating novel secondary metabolites with targeted structural alterations
requires detailed knowledge of individual biosynthetic steps.
Fatty acyl-AMP ligases (FAALs) are pivotal biosynthetic domains that draw fatty acids from primary
metabolism for incorporation into more complex natural product scaffolds. The FAAL domains are often linked
with multidomain polyketide synthases either in cis or trans via structural linker regions or docking domains,
respectively. We hypothesize that these linker regions and docking domains are crucial to the transfer of fatty
acid chains of specific lengths to the downstream polyketide synthase domains and that we can modulate this
transfer by maintaining the appropriate key elements. This proposal seeks to identify the key residues that control
the activation and transfer of fatty acid chains in a model system for application to more complex pathways. As
well, we seek to develop a robust heterologous host capable of producing these fatty acid-containing metabolites.
In Aim I, we will use our model system olefin (Ols) synthase to identify and modulate the gate-keeping linker
regions and docking domains that govern fatty acid integration into secondary metabolites. Directed evolution
experiments using the bacterial two-hybrid system will allow us to dissect the key docking domains found in Ols
homologs that contain a trans enzymatic structure. In a complementary system, we will perform directed
evolution experiments targeted towards the linker regions of cis Ols synthases and directly assess metabolite
production via a temperature selection screening. Aim I will uncover the key structural elements in the model Ols
synthase for future bioengineering of more complex natural product enzymes with similar biosynthetic logic.
In Aim II, we propose to develop Pseudomonas putida for the heterologous expression of fatty acid-
containing natural products. The pathways for our model Ols synthase as well as the biosynthetic gene cluster
encoding for micacocidin production will be expressed in P. putida. The production and bioengineering of the
FAAL-ACP domains to integrate acyl chains of varying length will be encoded in this heterologous host to
facilitate engineering efforts. The bioengineering of the FAAL-ACP loading modules in Ols synthase and the
micacocidin pathway will be guided by our work from Aim I.
总结抽象
项目成果
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