Itaconate Production from Crude Substrates with U. maydis: Scale-up of an Industrially Relevant Bioprocess.

BACKGROUND: Industrial by-products accrue in most agricultural or food-related production processes, but additional value chains have already been established for many of them. Crude glycerol has a 60% lower market value than commercial glucose, as large quantities are produced in the biodiesel industry, but its valorisation is still underutilized. Due to its high carbon content and the natural ability of many microorganisms to metabolise it, microbial upcycling is a suitable option for this waste product. RESULTS: In this work, the use of crude glycerol for the production of the value-added compound itaconate is demonstrated using the smut fungus Ustilago maydis. Starting with a highly engineered strain, itaconate production from an industrial glycerol waste stream was quickly established on a small scale, and the resulting yields were already competitive with processes using commercial sugars. Adaptive laboratory evolution resulted in an evolved strain with a 72% increased growth rate on glycerol. In the subsequent development and optimisation of a fed-batch process on a 1.5-2 L scale, the use of molasses, a side stream of sugar beet processing, eliminated the need for other expensive media components such as nitrogen or vitamins for biomass growth. The optimised process was scaled up to 150 L, achieving an overall titre of 72 g L- 1, a yield of 0.34 g g- 1, and a productivity of 0.54 g L- 1 h- 1. CONCLUSIONS: Pilot-scale itaconate production from the complementary waste streams molasses and glycerol has been successfully established. In addition to achieving competitive performance indicators, the proposed dual feedstock strategy offers lower process costs and carbon footprint for the production of bio-based itaconate.

Microparticles enhance the formation of seven major classes of natural products in native and metabolically engineered actinobacteria through accelerated morphological development.

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·4SiO2 ·H2 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 non-cyclized 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 anthramycin-analogs 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 synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.

Genomic-Led Discovery of a Novel Glycopeptide Antibiotic by Nonomuraea coxensis DSM 45129.

Glycopeptide antibiotics (GPAs) are last defense line drugs against multidrug-resistant Gram-positive pathogens. Natural GPAs teicoplanin and vancomycin, as well as semisynthetic oritavancin, telavancin, and dalbavancin, are currently approved for clinical use. Although these antibiotics remain efficient, emergence of novel GPA-resistant pathogens is a question of time. Therefore, it is important to investigate the natural variety of GPAs coming from so-called "rare" actinobacteria. Herein we describe a novel GPA producer-Nonomuraea coxensis DSM 45129. Its de novo sequenced and completely assembled genome harbors a biosynthetic gene cluster (BGC) similar to the dbv BGC of A40926, the natural precursor to dalbavancin. The strain produces a novel GPA, which we propose is an A40926 analogue lacking the carboxyl group on the N-acylglucosamine moiety. This structural difference correlates with the absence of dbv29-coding for an enzyme responsible for the oxidation of the N-acylglucosamine moiety. Introduction of dbv29 into N. coxensis led to A40926 production in this strain. Finally, we successfully applied dbv3 and dbv4 heterologous transcriptional regulators to trigger and improve A50926 production in N. coxensis, making them prospective tools for screening other Nonomuraea spp. for GPA production. Our work highlights genus Nonomuraea as a still untapped source of novel GPAs.

Viennamycins: Lipopeptides Produced by a Streptomyces sp.

Extracts from Streptomyces sp. S4.7 isolated from the rhizosphere of edelweiss, an alpine medicinal plant, exhibited activity against Gram-positive bacteria. LC-HRMS analyses of the extracts resulted in the detection of two unknown, structurally related lipopeptides that were assumed to be responsible for the antibiotic activity. LC-MS guided isolation and structure elucidation of viennamycins A and B (1 and 2) by HR-MS/MS, 1D and 2D NMR, and Marfey's analyses revealed them to be novel compounds, with viennamycin A containing cysteic acid, a unique feature for lipopeptides. Tests for antibacterial, antifungal, and cytotoxic activities of purified viennamycins, both with and without divalent cations, did not reveal any bioactivity, suggesting that their biological function, which could not be determined in the tests used, is atypical for lipopeptides. The genome of Streptomyces sp. S4.7 was sequenced and analyzed, revealing the viennamycin biosynthetic gene cluster. Detailed bioinformatics-based analysis of the viennamycin gene cluster allowed elucidation of the biosynthetic pathway for these lipopeptides.

Staphylococcus aureus responds to allicin by global S-thioallylation - Role of the Brx/BSH/YpdA pathway and the disulfide reductase MerA to overcome allicin stress.

The prevalence of methicillin-resitant Staphylococcus aureus (MRSA) in hospitals and the community poses an increasing health burden, which requires the discovery of alternative antimicrobials. Allicin (diallyl thiosulfinate) from garlic exhibits broad-spectrum antimicrobial activity against many multidrug resistant bacteria. The thiol-reactive mode of action of allicin involves its S-thioallylations of low molecular weight (LMW) thiols and protein thiols. To investigate the mode of action and stress response caused by allicin in S. aureus, we analyzed the transcriptome signature, the targets for S-thioallylation in the proteome and the changes in the bacillithiol (BSH) redox potential (EBSH) under allicin stress. Allicin caused a strong thiol-specific oxidative and sulfur stress response and protein damage as revealed by the induction of the PerR, HypR, QsrR, MhqR, CstR, CtsR, HrcA and CymR regulons in the RNA-seq transcriptome. Allicin also interfered with metal and cell wall homeostasis and caused induction of the Zur, CsoR and GraRS regulons. Brx-roGFP2 biosensor measurements revealed a strongly increased EBSH under allicin stress. In the proteome, 57 proteins were identified with S-thioallylations under allicin treatment, including translation factors (EF-Tu, EF-Ts), metabolic and redox enzymes (AldA, GuaB, Tpx, KatA, BrxA, MsrB) as well as redox-sensitive MarR/SarA-family regulators (MgrA, SarA, SarH1, SarS). Phenotype and biochemical analyses revealed that BSH and the HypR-controlled disulfide reductase MerA are involved in allicin detoxification in S. aureus. The reversal of protein S-thioallylation was catalyzed by the Brx/BSH/YpdA pathway. Finally, the BSSB reductase YpdA was shown to use S-allylmercaptobacillithiol (BSSA) as substrate to regenerate BSH in S. aureus. In conclusion, allicin results in an oxidative shift of EBSH and protein S-thioallylation, which can be reversed by YpdA and the Brx/BSH/YpdA electron pathways in S. aureus to regenerate thiol homeostasis.

Metabolite profiling at the cellular and subcellular level reveals metabolites associated with salinity tolerance in sugar beet.

Sugar beet is among the most salt-tolerant crops. This study aimed to investigate the metabolic adaptation of sugar beet to salt stress at the cellular and subcellular levels. Seedlings were grown hydroponically and subjected to stepwise increases in salt stress up to 300 mM NaCl. Highly enriched fractions of chloroplasts were obtained by non-aqueous fractionation using organic solvents. Total leaf metabolites and metabolites in chloroplasts were profiled at 3 h and 14 d after reaching the maximum salinity stress of 300 mM NaCl. Metabolite profiling by gas chromatography-mass spectrometry (GC-MS) resulted in the identification of a total of 83 metabolites in leaves and chloroplasts under control and stress conditions. There was a lower abundance of Calvin cycle metabolites under salinity whereas there was a higher abundance of oxidative pentose phosphate cycle metabolites such as 6-phosphogluconate. Accumulation of ribose-5-phosphate and ribulose-5-phosphate coincided with limitation of carbon fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Increases in glycolate and serine levels indicated that photorespiratory metabolism was stimulated in salt-stressed sugar beet. Compatible solutes such as proline, mannitol, and putrescine accumulated mostly outside the chloroplasts. Within the chloroplast, putrescine had the highest relative level and probably assisted in the acclimation of sugar beet to high salinity stress. The results provide new information on the contribution of chloroplasts and the extra-chloroplast space to salinity tolerance via metabolic adjustment in sugar beet.

Linking secondary metabolites to biosynthesis genes in the fungal endophyte Cyanodermella asteris: The anti-cancer bisanthraquinone skyrin.

Fungal aromatic polyketides display a very diverse and widespread group of natural products. Due to their excellent light absorption properties and widely studied biological activities, they offer numerous application for food, textile and pharmaceutical industry. The biosynthetic pathways of fungal aromatic polyketides usually involve a set of successive enzymes, in which a non-reductive polyketide synthase iteratively catalyzes the essential assembly of simple building blocks into (often polycyclic) aromatic compounds. However, only a limited number of such pathways have been described so far and further elucidation of the individual biosynthetic steps is needed to fully exploit the biotechnological and medicinal potential of these compounds. Here, we identified the bisanthraquinone skyrin as the main pigment of the fungus Cyanodermella asteris, an endophyte that has recently been isolated from the traditional Chinese medicinal plant Aster tataricus. The genome of C. asteris was sequenced, assembled and annotated, which enables first insights into a genome from a non-lichenized member of the class Lecanoromycetes. Genetic and in silico analyses led to the identification of a gene cluster of five genes suggested to encode the enzymatic pathway for skyrin. Our study is a starting point for rational pathway engineering in order to drive the production towards higher yields or more active derivatives. Moreover, our investigations revealed a large potential of secondary metabolite production in C. asteris as well as in all Lecanoromycetes of which genomes were available. These findings convincingly emphasize that Lecanoromycetes are prolific producers of secondary metabolites.

Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway.

BACKGROUND: Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. RESULTS: A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. CONCLUSION: The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.

The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum.

In order to isolate transcriptional regulatory proteins involved in L-methionine-dependent repression in Corynebacterium glutamicum, proteins binding to the putative promoter region upstream of the metY gene were isolated by DNA affinity chromatography. One of the isolated proteins was identified as a putative transcriptional repressor of the TetR-family by a mass spectrometry fingerprint technique based on the complete C. glutamicum genome sequence. The respective gene, designated mcbR, was deleted in the mutant strain C. glutamicum DR1. Using 2D-PAGE, the protein contents of the C. glutamicum wild type and the mutant strain DR1 grown in media with or without L-methionine supplementation were compared and a set of six proteins was identified. Their abundance was drastically enhanced in the mutant strain and no longer influenced by L-methionine added to the growth medium. The corresponding genes were identified by mass spectrometry fingerprint analysis. They included metY encoding O-acetyl-L-homoserine sulfhydrylase, metK encoding S-adenosyl-methionine synthethase, hom encoding homoserine dehydrogenase, cysK encoding L-cysteine synthase, cysI encoding an NADPH dependant sulfite reductase, and ssuD encoding an alkanesulfonate monooxygenase. Evidently, the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis of L-methionine in C. glutamicum. The C. glutamicum mcbR mutant can be considered to represent a first step in the construction of an L-methionine production strain.