Synthetic Zippers as an Enabling Tool for Engineering of Non‐Ribosomal Peptide Synthetases**

Bozhüyük, Kenan A. J.; Watzel, Jonas; Abbood, Nadya; Bode, Helge B.

doi:10.1002/anie.202102859

Cited by 36 publications

(41 citation statements)

References 48 publications

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“…Very soon, this may already be necessary for synthetic NRPSs. 39 Such systems are currently on the rise due to the increased availability of synthetic biology methods and they could be included, for example, as type IIS or type IIIS, etc. 39 There will always be different opinions about the structure of such a nomenclature, and there is probably no perfect solution that will satisfy everyone.…”

Section: Discussionmentioning

confidence: 99%

“…39 Such systems are currently on the rise due to the increased availability of synthetic biology methods and they could be included, for example, as type IIS or type IIIS, etc. 39 There will always be different opinions about the structure of such a nomenclature, and there is probably no perfect solution that will satisfy everyone. For a critical reection on this issue, it is crucial to clarify whether it is still useful to base an updated nomenclature on the PKS/FAS types, i.e.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Ribosome-independent peptide biosynthesis: the challenge of a unifying nomenclature

2022

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Ribosome-independent peptide biosynthesis: the challenge of a unifying nomenclature

2022

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“…The hty BGC provides an important biosynthetic precursor L-homotyrosine which was recognized by the fourth A domain of the NRPS EcdA. Understanding the biosynthetic mechanism of echinocandin B will facilitate us to take advantage of synthetic biology techniques to bioengineer NRPSs to generate bioactive compounds [ 49 , 50 , 51 ].…”

Section: Fungal Non-ribosomal Peptide Biosynthesis Involving Two Sepa...mentioning

confidence: 99%

Biosynthesis of Fungal Natural Products Involving Two Separate Pathway Crosstalk

Dai

Shen

Zhang

et al. 2022

JoF

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Fungal natural products (NPs) usually possess complicated structures, exhibit satisfactory bioactivities, and are an outstanding source of drug leads, such as the cholesterol-lowering drug lovastatin and the immunosuppressive drug mycophenolic acid. The fungal NPs biosynthetic genes are always arranged within one single biosynthetic gene cluster (BGC). However, a rare but fascinating phenomenon that a crosstalk between two separate BGCs is indispensable to some fungal dimeric NPs biosynthesis has attracted increasing attention. The hybridization of two separate BGCs not only increases the structural complexity and chemical diversity of fungal NPs, but also expands the scope of bioactivities. More importantly, the underlying mechanism for this hybridization process is poorly understood and needs further exploration, especially the determination of BGCs for each building block construction and the identification of enzyme(s) catalyzing the two biosynthetic precursors coupling processes such as Diels–Alder cycloaddition and Michael addition. In this review, we summarized the fungal NPs produced by functional crosstalk of two discrete BGCs, and highlighted their biosynthetic processes, which might shed new light on genome mining for fungal NPs with unprecedented frameworks, and provide valuable insights into the investigation of mysterious biosynthetic mechanisms of fungal dimeric NPs which are constructed by collaboration of two separate BGCs.

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“…[146] Recently the possibilities of combining NRPS enzymes were enhanced with the introduction of synthetic zippers. [147] Synthetic zipper pairs were shown to efficiently link two separate polypeptide chains, even allowing the generation of peptide libraries. [147] Insertion / deletion of domains…”

Section: Generating "New" Natural Products By Nrps Pks Engineering Ap...mentioning

confidence: 99%

Biosynthesis of selected natural products from entomopathogenic bacteria

Westphalen¹

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This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis. The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively. PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF. The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering. The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.

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Synthetic Zippers as an Enabling Tool for Engineering of Non‐Ribosomal Peptide Synthetases**

Cited by 36 publications

References 48 publications

Ribosome-independent peptide biosynthesis: the challenge of a unifying nomenclature

Ribosome-independent peptide biosynthesis: the challenge of a unifying nomenclature

Biosynthesis of Fungal Natural Products Involving Two Separate Pathway Crosstalk

Biosynthesis of selected natural products from entomopathogenic bacteria

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