Engineered Biosynthesis of Alkyne-Tagged Polyketides by Type I PKSs

Porterfield, William; Poenateetai, Nannalin; Zhang, Wenjun

doi:10.1016/j.isci.2020.100938

Cited by 11 publications

(6 citation statements)

References 73 publications

(77 reference statements)

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“…3a ). In addition, an analogous strategy of optimizing the ACP n /KS n+1 chain transfer interface was shown recently to substantially improve interaction between an ACP (JamC) derived from the jamaicamide B biosynthetic pathway, and the first chain extension module of the lipomycin PKS (LipPKS1) 51 .…”

Section: Resultsmentioning

confidence: 99%

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

et al. 2022

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The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites − a reduction in chain length of 14 carbons relative to the 51-membered parental compounds − but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.

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Section: Resultsmentioning

confidence: 99%

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

et al. 2022

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“…12B ). 89 These findings have shed light on the discovery and development of synthetic tools for the de novo biosynthesis of various acetylenic products.…”

Section: Engineered Biosynthesis Of Alkyne-tagged Productsmentioning

confidence: 98%

“…They further tried to generalize this strategy to other kinds of PKSs by employing two well studied type I PKSs, LipPKS1 and DEBSM6, to produce the target alkyne-tagged polyketides. 89 LipPKS1 is the first module in lipomycin biosynthesis and DEBSM6 is the last PKS module in the erythromycin biosynthetic pathway. 90,91 Engineered LipPKS1 and DEBSM6 have been constructed to utilize malonyl-CoA instead of methylmalonyl-CoA as the extender unit, with the promiscuous DEBS thioesterase, to promote the release of a carboxylic acid product.…”

Section: Engineered Biosynthesis Of Alkyne-tagged Productsmentioning

confidence: 99%

“… 92 Zhang and co-workers further removed the AT and ACP loading domains of the engineered LipPKS1, to create a truncated version that facilitates the alternative starter unit incorporation. The two engineered modules (LipPKS1* and DEBSM6*), in combination with JamABC working as a trans loading system, were employed to produce the alkyne-tagged polyketide 3-hydroxydec-9-ynoic acid ( 44 ) (compound 1 in ref. 89 ) ( Fig.…”

Section: Engineered Biosynthesis Of Alkyne-tagged Productsmentioning

confidence: 99%

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Biosynthesis of alkyne-containing natural products

et al. 2021

RSC Chem. Biol.

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“…[150] Obviously, the different strategies can also be combined as was shown in the example of an alkyne polyketide product where docking domains as well as site directed mutagenesis of the carrier protein were employed. [151] In the pursue of de novo assembly of specialised metabolites, bioinformatic tools such as ClusterCAD for PKS are employed and will in the future allow computer-aided re-engineering of NRPS and PKS assembly lines. [143] In conclusion, the knowledge from these studies can help elucidate the biosynthesis of novel natural product and in turn every discovery of a novel mechanism increases the possibilities of the engineering toolbox.…”

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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Engineered Biosynthesis of Alkyne-Tagged Polyketides by Type I PKSs

Cited by 11 publications

References 73 publications

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

Biosynthesis of alkyne-containing natural products

Biosynthesis of selected natural products from entomopathogenic bacteria

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