Identification of crucial bottlenecks in engineered polyketide biosynthesis

Grote, Marius; Kushnir, Susanna; Pryk, Niclas; Möller, David R.; Erver, Julian; Ismail-Ali, A.; Schulz, Frank

doi:10.1039/c9ob00831d

Cited by 7 publications

(10 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…32 In that light, the increase as well as the decrease in turnover rates was expected, provided that it is the substrate specificity of the acceptor KS that limits the rate of a chimeric PKS. 26,41 In addition, we systematically varied the protein-protein interactions within the chimeric KSmutated PKSs; i.e., the protein:protein interface emerging from the upstream ACP docking to the downstream KS for translocation and the structure of the substrate presented to the downstream KS for uptake and processing. Overall, our approach yielded a large body of data giving insight into mechanistic details of PKS-mediated product synthesis outlined in the following on three examples:…”

Section: Resultsmentioning

confidence: 99%

The Ketosynthase Domain Constrains the Design of Polyketide Synthases

Klaus

Buyachuihan

Grininger

2020

Preprint

View full text Add to dashboard Cite

Modular polyketide synthases (PKSs) produce complex, bioactive secondary metabolites in assembly line-like multistep reactions. Longstanding efforts to produce novel, biologically active compounds by recombining intact modules to new modular PKSs have mostly resulted in poorly active chimeras and decreased product yields. Recent findings demonstrate that the low efficiencies of modular chimeric PKSs also result from rate limitations in the transfer of the growing polyketide chain across the non-cognate module:module interface and further processing of the non-native polyketide substrate by the ketosynthase (KS) domain. In this study, we aim at disclosing and understanding the low efficiency of chimeric modular PKSs and at establishing guidelines for modular PKSs engineering. To do so, we work with a bimodular PKS testbed and systematically vary substrate specificity, substrate identity, and domain:domain interfaces of the KS involved reactions. We observe that KS domains employed in our chimeric bimodular PKSs are bottlenecks with regards to both substrate specificity as well as interaction with the ACP. Overall, our systematic study can explain in quantitative terms why early oversimplified engineering strategies based on the plain shuffling of modules mostly failed and why more recent approaches show improved success rates. We moreover identify two mutations of the KS domain that significantly increased turnover rates in chimeric systems and interpret this finding in mechanistic detail.

show abstract

Section: Resultsmentioning

confidence: 99%

The Ketosynthase Domain Constrains the Design of Polyketide Synthases

Klaus

Buyachuihan

Grininger

2020

Preprint

View full text Add to dashboard Cite

show abstract

“…4,11 The inactivation of processing enzymes within a synthase usually results in large decreases in polyketide production and shunt products indicative of a stall in the assembly line. 12 The gatekeeping activities of KSs could explain these phenomena (Figure 1). Studies of processing enzyme knockouts in the monensin PKS showed KR knockouts are among the most severely affected.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Studies of processing enzyme knockouts in the monensin PKS showed KR knockouts are among the most severely affected. 12 This could be due to a general exclusion of the β-keto group by KSs that accept more reduced intermediates. The products of ER knockouts contain a new double bond rather than a new hydroxy group, as might be expected from the DH-mediated equilibrium between βhydroxy and α/β-unsaturated intermediates.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The authors of the study of the monensin processing enzyme knockouts considered KSs as potential gatekeepers; however, since only weak fingerprints at the dimer interface loop were observed for the KSs that accept α/β-unsaturated substrates, other enzymes were investigated for this role. 12 We looked beyond the four-residue stretch at the dimer interface loop and beyond the previously analyzed PKSs to more completely characterize KSs that accept intermediates of differing chemistries. Based on the chemistries at the αand βcarbons anticipated for their substrates, 739 KSs from 92 PKSs were divided into 16 families, which were subdivided into 115 groups based on the chemistry at the γ-carbon.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The inactivation of processing enzymes within a synthase usually results in large decreases in polyketide production and shunt products indicative of a stall in the assembly line . The gatekeeping activities of KSs could explain these phenomena (Figure ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

How cis-Acyltransferase Assembly-Line Ketosynthases Gatekeep for Processed Polyketide Intermediates

2021

View full text Add to dashboard Cite

With the redefinition of polyketide synthase (PKS) modules, a new appreciation of their most downstream domain, the ketosynthase (KS), is emerging. In addition to performing its well-established role of generating a carbon–carbon bond between an acyl-CoA building block and a growing polyketide, it may gatekeep against incompletely processed intermediates. Here, we investigate 739 KSs from 92 primarily actinomycete, cis-acyltransferase assembly lines. When KSs were separated into 16 families based on the chemistries at the α- and β-carbons of their polyketide substrates, a comparison of 32 substrate tunnel residues revealed unique sequence fingerprints. Surprisingly, additional fingerprints were detected when the chemistry at the γ-carbon was considered. Representative KSs were modeled bound to their natural polyketide substrates to better understand observed patterns, such as the substitution of a tryptophan by a smaller residue to accommodate an l-α-methyl group or the substitution of four smaller residues by larger ones to make better contact with a primer unit or diketide. Mutagenesis of a conserved glutamine in a KS within a model triketide synthase indicates that the substrate tunnel is sensitive to alteration and that engineering this KS to accept unnatural substrates may require several mutations.

show abstract

Polyether Cyclization Cascade Alterations in Response to Monensin Polyketide Synthase Mutations

et al. 2021

Self Cite

View full text Add to dashboard Cite

The targeted manipulation of polyketide synthases has in recent years led to numerous new-to-nature polyketides. For type I polyketide synthases the response of post-polyketide synthases (PKS) processing enzymes onto the most frequently polyketide backbone manipulations is so far insufficiently studied. In particular, complex processes such as the polyether cyclisation in the biosynthesis of ionophores such as monensin pose interesting objects of research. We present here a study of the substrate promiscuity of the polyether cyclisation cascade enzymes in monensin biosynthesis in the conversion of redox derivatives of the nascent polyketide chain. LC-HRMS/MS 2based studies revealed a remarkable flexibility of the post-PKS enzymes. They acted on derivatized polyketide backbones based on the three possible polyketide redox states within two different modules and gave rise to an altered polyether structure. One of these monensin derivatives was isolated and characterized by 2D-NMR spectroscopy, crystallography, and bioactivity studies. Results and DiscussionFor the interrogation of post-PKS processivity, it was first decided to investigate the effect of all three feasible different [a

show abstract

Identification of crucial bottlenecks in engineered polyketide biosynthesis

Abstract: Quo vadis combinatorial biosynthesis: STOP signs through substrate scope limitations lower the yields in engineered polyketide biosynthesis using cis-AT polyketide synthases.

Cited by 7 publications

References 64 publications

The Ketosynthase Domain Constrains the Design of Polyketide Synthases

The Ketosynthase Domain Constrains the Design of Polyketide Synthases

How cis-Acyltransferase Assembly-Line Ketosynthases Gatekeep for Processed Polyketide Intermediates

Polyether Cyclization Cascade Alterations in Response to Monensin Polyketide Synthase Mutations

Contact Info

Product

Resources

About