Presentation Information
[1MENP-07]Precise- and multistep-engineering of type I polyketide synthases
○Kei Kudo1, Kazuo Shin-ya1 (1. National Institute of Advanced Industrial Science and Technology (AIST) (Japan))
Keywords:
Polyketide,Genetic engineering,Skeletal editing,Actinomycete,Heterologous expression
[Purpose]
Type I polyketide synthases (PKSs) are large, multifunctional enzymes responsible for the biosynthesis of structurally diverse polyketides. Their modular architecture makes PKSs attractive targets for engineering carbon skeletons; however, precise and multistep manipulation of highly repetitive PKS genes remains challenging. Here, we aimed to establish a reliable PKS engineering methodology by combining in vitro genome editing with heterologous expression in Streptomyces host strains.
[Method]
PKS gene clusters for rapamycin and mediomycin biosynthesis were cloned into bacterial artificial chromosomes (BACs). The BACs were edited in vitro using Cas9-mediated digestion guided by pairs of designed sgRNAs, followed by insertion of engineered fragments via Gibson assembly. All modifications were verified by Sanger sequencing. The engineered BACs were introduced into Streptomyces avermitilis and Streptomyces lividans, respectively, for heterologous expression and metabolite analysis.
[Results]
The rapamycin PKS was first used to establish a precise editing workflow suitable for highly similar PKS sequences. In vitro Cas9 digestion combined with Gibson assembly enabled stepwise and reliable modification of PKS genes, resulting in the production of over 20 rapamycin derivatives through domain-swapping experiments.The mediomycin PKS was then employed for multistep engineering to reconstruct tetrafibricin biosynthesis, a pathway no longer active in its native producer. A five-step modular engineering achieved skeletal editing of mediomycin while retaining 26% of the original production titer (76% geometric mean across steps). Comparative analysis showed that cleavage at the post-acyltransferase (AT) linker preserved productivity more effectively than cleavage at the ketosynthase (KS)-AT linker.
[Consideration]
Off-target effects are a concern in Cas9-based manipulation of high-GC and repetitive PKS genes, especially in vivo. Editing BACs (up to 250 kbp) in vitro substantially reduces off-target risks compared with in vivo editing of entire Streptomyces chromosomes (8–10 Mbp), making this approach well suited for PKS engineering. Although rapamycin derivatization highlights the tolerance of PKS assembly lines to modification, productivity losses indicate the need for strategies beyond simple domain swapping. Use of the post-AT cut site preserves the KS-AT didomain architecture. In the tetrafibricin reconstruction, repeated use of this site enabled robust production of expected products, suggesting minimal mismatch between upstream and downstream modules. It’s noteworthy that, as all engineered modules employed malonyl-CoA–specific ATs in our case, no unnatural α-substituents were introduced to KS domains.
[Conclusion]
Successful skeletal remodeling of complex polyketides demonstrates that PKS engineering can serve as a synthetic strategy for skeletal editing. This work set a stage to leverage modular PKSs’ potential for rational construction of complex carbon architectures.
Type I polyketide synthases (PKSs) are large, multifunctional enzymes responsible for the biosynthesis of structurally diverse polyketides. Their modular architecture makes PKSs attractive targets for engineering carbon skeletons; however, precise and multistep manipulation of highly repetitive PKS genes remains challenging. Here, we aimed to establish a reliable PKS engineering methodology by combining in vitro genome editing with heterologous expression in Streptomyces host strains.
[Method]
PKS gene clusters for rapamycin and mediomycin biosynthesis were cloned into bacterial artificial chromosomes (BACs). The BACs were edited in vitro using Cas9-mediated digestion guided by pairs of designed sgRNAs, followed by insertion of engineered fragments via Gibson assembly. All modifications were verified by Sanger sequencing. The engineered BACs were introduced into Streptomyces avermitilis and Streptomyces lividans, respectively, for heterologous expression and metabolite analysis.
[Results]
The rapamycin PKS was first used to establish a precise editing workflow suitable for highly similar PKS sequences. In vitro Cas9 digestion combined with Gibson assembly enabled stepwise and reliable modification of PKS genes, resulting in the production of over 20 rapamycin derivatives through domain-swapping experiments.The mediomycin PKS was then employed for multistep engineering to reconstruct tetrafibricin biosynthesis, a pathway no longer active in its native producer. A five-step modular engineering achieved skeletal editing of mediomycin while retaining 26% of the original production titer (76% geometric mean across steps). Comparative analysis showed that cleavage at the post-acyltransferase (AT) linker preserved productivity more effectively than cleavage at the ketosynthase (KS)-AT linker.
[Consideration]
Off-target effects are a concern in Cas9-based manipulation of high-GC and repetitive PKS genes, especially in vivo. Editing BACs (up to 250 kbp) in vitro substantially reduces off-target risks compared with in vivo editing of entire Streptomyces chromosomes (8–10 Mbp), making this approach well suited for PKS engineering. Although rapamycin derivatization highlights the tolerance of PKS assembly lines to modification, productivity losses indicate the need for strategies beyond simple domain swapping. Use of the post-AT cut site preserves the KS-AT didomain architecture. In the tetrafibricin reconstruction, repeated use of this site enabled robust production of expected products, suggesting minimal mismatch between upstream and downstream modules. It’s noteworthy that, as all engineered modules employed malonyl-CoA–specific ATs in our case, no unnatural α-substituents were introduced to KS domains.
[Conclusion]
Successful skeletal remodeling of complex polyketides demonstrates that PKS engineering can serve as a synthetic strategy for skeletal editing. This work set a stage to leverage modular PKSs’ potential for rational construction of complex carbon architectures.
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