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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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... supplementation or expression of the PQQ biosynthetic gene cluster using whole cell proteomic analysis. With this approach we looked for proteins that were up-regulated upon addition of 10 µM or 1 mM of PQQ, or upon integration of a PQQ biosynthetic gene cassette. In total, we identied 77 proteins that were upregulated under all three conditions (Fig. 4A), 44 of which were involved in metabolism. Figure 4B shows the pathways enriched after the integration of the PQQ biosynthetic cassette. We then ranked these proteins according to the ratio of enrichment. Among the top 10 pathways, six are involved in the biosynthesis of cofactors or maintenance of the intracellular redox state (fatty ...
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... total, we identied 77 proteins that were upregulated under all three conditions (Fig. 4A), 44 of which were involved in metabolism. Figure 4B shows the pathways enriched after the integration of the PQQ biosynthetic cassette. We then ranked these proteins according to the ratio of enrichment. ...
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... and citrate cycle). 22% of the enzymatic steps (7/32, guaB, ndk, ndh, gltBD, idh, korAB) upregulated are involved with generation of the cofactors NADH, NADPH or ATP, 9% of the enzymatic steps (3/32, acc, bkd, fabG) upregulated are involved with generation of CoA precursors, and the pathways related to removal of H 2 O 2 are also enhanced (Fig. 4C). This is in accordance with a previous report that PQQ stimulates respiration and relieves oxidative stress in mammalian cells 22 . To provide evidence to support this hypothesis, we determined the intracellular levels of ATP, NADP, NADPH, NAD and NADH. The intracellular level of ATP and the ratios of NADPH to NADP and NADH to NAD ...
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... report that PQQ stimulates respiration and relieves oxidative stress in mammalian cells 22 . To provide evidence to support this hypothesis, we determined the intracellular levels of ATP, NADP, NADPH, NAD and NADH. The intracellular level of ATP and the ratios of NADPH to NADP and NADH to NAD increased by 32%, 392% and 253%, respectively ( Fig. 4D), indicating that PQQ improved natural products biosynthesis by enhancing cofactor ...
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... conrmation of the identity of the molecule came with the identication of a BGC in S. rapamycinicus matching the previously reported BGC for mediomycins in Streptomyces mediocidicus ATCC23936 26 . The BGC identied in S. rapamycinicus is 88% homologous to Mediomycin A in S. rapamycinicus ( Figure S4), indicating this compound can also be produced by Streptomyces rapamycinicus. Besides S. rapamycinicus, a mediomycin BGC was also found in several other strains of clade 12 ( Figure S5). ...
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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.
