Directed biosynthesis of fluorinated polyketides

Rittner, Alexander; Joppe, Mirko; Schmidt, Jennifer J.; Mayer, Lara Maria; Heid, Elia; Sherman, David H.; Grininger, Martin

doi:10.1101/2021.07.30.454469

Cited by 3 publications

(24 citation statements)

References 31 publications

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“…Finally, in the third mechanism, the two steps are swapped in sequence and the extender substrate is added to the acyl-KS followed by decarboxylation. Figure is adapted from Blaquiere et al 43 and was published by Rittner and Joppe et al 44 The substrate specificity of the KS plays a crucial role for the biosynthesis of fatty acids and polyketides. In mammalian FASs, the KS is specific for fully saturated acyl moieties and the turnover rates increase with chain length until C12 and decrease then again for longer chains.…”

Section: Ketosynthasesmentioning

confidence: 99%

“…The order of assembled building blocks during the biosynthesis is illustrated by grey dots with increasing grey scale starting with the first building block in light grey. The figure was published by Rittner and Joppe et al 44…”

Section: Pikromycin Synthasementioning

confidence: 99%

“…74 (B) The DEBS/FAS hybrids were created by exchanging AT6 (H1) or LD-AT6 (H2) with MAT or LD-MAT, respectively. The figure was adapted from Rittner and Joppe et al 44 We expressed the DEBS M6 WT and both hybrids in E. coli and purified them using Ni-NTA resin (figure 48 A) followed by SEC (figure 48 B-C). The hybrids were received in similar amounts as the WT, but the overall yields of the dimeric species decreased to 50% for H1 and 25% for H2, because of different oligomerization ratios (figure 48 C-D).…”

Section: Figure 47⎮mentioning

confidence: 99%

“…Final substrate concentrations in the assay were 0.3 µM enzyme, 2 mM trans-1-decalone and 60 µM NADPH. The figure was published by Rittner and Joppe et al 44…”

Section: Figure 47⎮mentioning

confidence: 99%

“…Furthermore, H1 was not able to produce the reduced fluorinated TKL (7), demonstrating a strict substrate specificity of the KR as reported before. 30 44 Figure includes data received from the master thesis of Elia Heid. 183 Finally, we aimed at employing H1 to produce DEB macrolactones, derivatized at position C2.…”

Section: Characterization Of Debs/fas Hybridsmentioning

confidence: 99%

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Structure and Function of Megasynthases

Joppe¹

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In this thesis, we characterized megasynthases such as fatty acid synthases (FASs) and polyketide synthases. The obtained insights into structure and function were used to engineer such systems to produce new-to-nature compounds. The in vitro characterization of megasynthases requires reproducible access to these enzymes in high quality. Therefore, we established purification strategies for the yeast FAS and the methylsalicylic acid synthase (MSAS) from Saccharopolyspora erythraea (SerMSAS) and applied the latter one on MSAS from Penicillium patulum (PenPaMSAS) and on 6-deoxyerythronolide B synthase (DEBS) module 6. With the purified samples, we were able to obtain initial structural data for SerMSAS and solve the complete structure of the yeast FAS (PDB: 6TA1). On the example of the yeast FAS, we could show that the sample can suffer from adsorption to the water-air interface during the grid preparation for electron microscopy and presented how the use of graphene-based grids can overcome this problem. The combined structural and functional analysis of the yeast FAS showed that the structural domains trimerization module and dimerization module 2 are not essential for the assembly of the whole system. Therefore, they can potentially be used for domain exchange approaches. The in-depth functional analysis of SerMSAS revealed that not SerMSAS itself releases the product, but a 3-oxoacyl-(acyl-carrier protein) synthase like enzyme within the gene cluster transfers 6-methyl salicylic acid from SerMSAS to another carrier protein for subsequent modifications. In contrast, we showed that PenPaMSAS can release its product by hydrolysis and that non-native substrates can be incorporated although at significantly slower turnover rates compared to the native starter substrate. Our further investigation demonstrated that the substrate specificity of the acyltransferase (AT) is a critical factor for the incorporation of non-native substrates. With the insight from the functional and structural characterization, we engineered megasynthases for the biosynthesis of natural product derivatives. We targeted the AT of PenPaMSAS for active site mutagenesis and discovered a mutant which can transfer non-native substrates significantly faster (~200-300%). Additionally, the malonyl/acetyl transferase (MAT) of the mammalian FAS was used as a promising target for protein engineering because of its previously reported properties including polyspecificity, fast transfer kinetics, robustness, and plasticity. We showed that the MAT can transfer fluorinated substrates and accept the acyl carrier protein of DEBS module 6. By exchanging the substrate specific AT of DEBS with the polyspecific MAT of the mammalian FAS, we demonstrated an efficient DEBS/FAS hybrid and an optimal truncation site for the applied ATs. In contrast to the wild type system, the DEBS/FAS enzyme was able to synthesize demethylated and fluorinated derivatives. The production and purification of a fluoro-methyl-disubstituted polyketide was of particular interest, as it has a high potential for the generation of new drugs and shows the potential of protein engineering. Furthermore, the incorporation of the disubstituted substrate had important implication in the mechanistic details of the ketosynthase-mediated C-C bond formation.

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Section: Ketosynthasesmentioning

confidence: 99%

Section: Pikromycin Synthasementioning

confidence: 99%

Section: Figure 47⎮mentioning

confidence: 99%

“…Final substrate concentrations in the assay were 0.3 µM enzyme, 2 mM trans-1-decalone and 60 µM NADPH. The figure was published by Rittner and Joppe et al 44…”

Section: Figure 47⎮mentioning

confidence: 99%

Section: Characterization Of Debs/fas Hybridsmentioning

confidence: 99%

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Structure and Function of Megasynthases

Joppe¹

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Enzyme Engineering Renders Chlorinase the Activity of Fluorinase

Jiang,

Yao,

Niu

et al. 2024

J. Agric. Food Chem.

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Organofluorine compounds have attracted substantial attention owing to their wide application in agrochemistry. Fluorinase (FlA) is a unique enzyme in nature that can incorporate fluorine into an organic molecule. Chlorinase (SalL) has a similar mechanism as fluorinase and can use chloride but not fluoride as a substrate to generate 5′-chloro-deoxyadenosine (5′-ClDA) from S-adenosyl-L-methionine (SAM). Therefore, identifying the features that lead to this selectivity for halide ions is highly important. Here, we engineered SalL to gain the function of FlA. We found that residue Tyr70 plays a key role in this conversion through alanine scanning. Site-saturation mutagenesis experiments demonstrated that Y70A/C/S/T/G all exhibited obvious fluorinase activity. The G131S mutant of SalL, in which the previously thought crucial residue Ser158 for fluoride binding in FlA was introduced, did not exhibit fluorination activity. Compared with the Y70T single mutant, the double mutant Y70T/W129F increased 5′-fluoro-5-deoxyadenosine (5′-FDA) production by 76%. The quantum mechanics (QM)/molecular mechanics (MM) calculations suggested that the lower energy barriers and shorter nucleophilic distance from F − to SAM in the mutants than in the SalL wild-type may contribute to the activity. Therefore, our study not only renders SalL the activity of FlA but also sheds light on the enzyme selectivity between fluoride versus chloride.

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Striving for sustainable biosynthesis: discovery, diversification, and production of antimicrobial drugs in Escherichia coli

Iacovelli

Sokolova

Haslinger

2022

Biochemical Society Transactions

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New antimicrobials need to be discovered to fight the advance of multidrug-resistant pathogens. A promising approach is the screening for antimicrobial agents naturally produced by living organisms. As an alternative to studying the native producer, it is possible to use genetically tractable microbes as heterologous hosts to aid the discovery process, facilitate product diversification through genetic engineering, and ultimately enable environmentally friendly production. In this mini-review, we summarize the literature from 2017 to 2022 on the application of Escherichia coli and E. coli-based platforms as versatile and powerful systems for the discovery, characterization, and sustainable production of antimicrobials. We highlight recent developments in high-throughput screening methods and genetic engineering approaches that build on the strengths of E. coli as an expression host and that led to the production of antimicrobial compounds. In the last section, we briefly discuss new techniques that have not been applied to discover or engineer antimicrobials yet, but that may be useful for this application in the future.

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Directed biosynthesis of fluorinated polyketides

Cited by 3 publications

References 31 publications

Structure and Function of Megasynthases

Structure and Function of Megasynthases

Enzyme Engineering Renders Chlorinase the Activity of Fluorinase

Striving for sustainable biosynthesis: discovery, diversification, and production of antimicrobial drugs in Escherichia coli

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