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Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering paradigm for different Streptomyces strains is challenging. That some bacteria and fungi are more adept than others at synthesizing natural products implies the existence of key genes co-evolved with the BGCs for high...
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... PQQ gene cluster in the PKS procient family contains 5 genes, pqqA-E (Fig. 3A). The biosynthesis of PQQ starts with the ribosomal production of a short peptide encoded in pqqA, which then undergoes a series of modications to form the characteristic pyridine and pyrrole rings in PQQ 21 . To test the contribution of PQQ on natural product production, the full operon containing 5 biosynthetic genes (pqqABCDE) were ...
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... kasOp*, and introduced into the model strain Streptomyces coelicolor M145. The blue color of actinorhodin, a polyketide naturally produced by S. coelicolor, was followed as indicator of titer for this class of compounds. Between 60 to 72 hr of growth, introduction of the PQQ gene cluster brought about 1.8-2.2 fold increase in actinorhodin titer (Fig. ...
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... processes within the cell. This could be altered ux such as inhibition on fatty acid biosynthesis or improving respiration, leading to an increase in the accumulation of precursors, cofactors, or energy for the biosynthesis of natural products. Further supplementation of PQQ in the growth medium conrmed the positive effect on actinorhodin (Fig. ...
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... also investigated the impact of the PQQ gene cluster on enzymes directly involved in natural products biosynthesis with fractional proteome analysis. The expression of two actinorhodin PKSs (genes SCO5087 and SCO5088 in the S. coelicolor) were almost unchanged, indicating that the increase in actinorhodin was mainly through a change in metabolic ux and an increase in cofactors and precursors ( Figure S3). Unexpectedly, two enzymes of the coelimycin PKS (SCO6273 and SCO6275) -a 'so called' cryptic pathway which is normally silent in S. coelicolor -was signicantly up-regulated (1010% and 212%), indicating the metabolic change caused by expression of the PQQ gene cluster resulted in a regulatory change and thus activation of some natural product biosynthetic genes. ...
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... PQQ was shown to be effective in increasing actinorhodin production in S. coelicolor, it is possible that PQQ augmentation can be developed as a universal tool to enhance natural product biosynthesis and to activate silent biosynthetic gene clusters. To explore this idea, we introduced the PQQ gene cluster into 11 Streptomyces strains distributed across the Streptomyces genus ( Supplementary Fig. 3). The strains harboring an integrative vector carrying the PQQ gene cluster as well as strains with the empty vector were cultured in shake asks, and the changes of metabolites were measured using metabolomic analyses (Fig. 5A). ...
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Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we proposed that a set of genes co-evolved with BGCs to support biosynthetic profici...
Citations
... Moreover, we achieved tunable product profiles of RimPKS-TRs through substrate CoA pool engineering. PKS pathways are often tightly regulated in nature, as shown by wide presence of PKS pathway specific transcription regulators within the BGCs 56 , and more recent discovery of pyrroloquinoline quinone (PQQ) gene clusters that co-evolved with PKS BGCs and enhanced natural product production 57 . Another common PKS-related regulation approach prioritises (which was not certified by peer review) is the author/funder. ...
Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here, we present a novel approach utilising a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and NADPH-dependent terminal thioreductases (TRs), previously untapped in engineered PKSs. Reduction of the terminal aldehyde with specific alcohol dehydrogenases enables production of diols, oxidation enables production of hydroxy acids, and transamination with specific transaminases enables production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A (CoA)–specific acyltransferase (AT) in the extension module with methyl- or ethylmalonyl- CoA–specific ATs enables production of branched-chain diols and amino alcohols. In total, we demonstrated production of nine 1,3-diols (including the difficult-to-produce insect repellent and cosmetic ingredient 2-ethyl-1,3-hexanediol), six amino alcohols, and two carboxylic acids using our PKS platform in Streptomyces albus . Finally, tuning production of the PKS acyl-CoA substrates enabled production of high titers of specific diols and amino alcohols (1 g/L diol titer in shake flasks), demonstrating high tunability and efficiency of the platform.
