BackgroundCis, cis-muconic acid (MA) is a dicarboxylic acid of recognized industrial value. It provides direct access to adipic acid and terephthalic acid, prominent monomers of commercial plastics.ResultsIn the present work, we engineered the soil bacterium Corynebacterium glutamicum into a stable genome-based cell factory for high-level production of bio-based MA from aromatics and lignin hydrolysates. The elimination of muconate cycloisomerase (catB) in the catechol branch of the β-ketoadipate pathway provided a mutant, which accumulated MA at 100% molar yield from catechol, phenol, and benzoic acid, using glucose as additional growth substrate. The production of MA was optimized by constitutive overexpression of catA, which increased the activity of the encoded catechol 1,2-dioxygenase, forming MA from catechol, tenfold. Intracellular levels of catechol were more than 30-fold lower than extracellular levels, minimizing toxicity, but still saturating the high affinity CatA enzyme. In a fed-batch process, the created strain C. glutamicum MA-2 accumulated 85 g L−1 MA from catechol in 60 h and achieved a maximum volumetric productivity of 2.4 g L−1 h−1. The strain was furthermore used to demonstrate the production of MA from lignin in a cascade process. Following hydrothermal depolymerization of softwood lignin into small aromatics, the MA-2 strain accumulated 1.8 g L−1 MA from the obtained hydrolysate.ConclusionsOur findings open the door to valorize lignin, the second most abundant polymer on earth, by metabolically engineered C. glutamicum for industrial production of MA and potentially other chemicals.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0963-2) contains supplementary material, which is available to authorized users.

Streptomyces spp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline the performance of in these microbes. Here, we studied the production of pamamycins, macrodiolide homologs with a high activity against multiresistant pathogenic microbes, using recombinant Streptomyces albus J1074/R2. Talc particles (hydrous magnesium silicate, 3MgO•4SiO 2 •H 2 O) of micrometer size, added to submerged cultures of the recombinant strain, tripled pamamycin production up to 50 mg/L. Furthermore, they strongly affected morphology, reduced the size of cell pellets formed by the filamentous microbe during the process up to sixfold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and precursor (CoA thioester) supply of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3,341 genes (56% of all genes), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to 1,024-fold). Furthermore, the microparticles perturbed genes encoding for CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased fourfold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. The novel insights into the behavior

Background: Thioesters of coenzyme A participate in 5% of all enzymatic reactions. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5-10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance of these metabolites explains the high interest to trace and quantify them in microbial cells. Results: Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach assessed intracellular CoA thioesters down to the picomolar level and exhibited high precision and reproducibility for all microbes, as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA metabolism. A succinyl-CoA synthase defective mutant of C. glutamicum exhibited an unaffected level of succinyl-CoA that indicated a complete compensation by the l-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the actinomycete S. albus. The microbe revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. Conclusions: The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use one single protocol promises to facilitate automatized efforts, which rely on standardized workflows.

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO•4SiO 2 •H 2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L −1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L −1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of noncyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycinanalogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Background Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. Results Supplemented with l-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L−1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from l-valine catabolism. Unfavorably, l-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post l-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with l-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. Conclusions Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.