Characterization of Amycolatopsis 75iv2 dye-decolorizing peroxidase on O-glycosides.

Dye-decolorizing peroxidases are heme peroxidases with a broad range of substrate specificity. Their physiological function is still largely unknown, but a role in the depolymerization of plant cell wall polymers has been widely proposed. Here, a new expression system for bacterial dye-decolorizing peroxidases as well as the activity with previously unexplored plant molecules are reported. The dye-decolorizing peroxidase from Amycolatopsis 75iv2 (DyP2) was heterologously produced in the Gram-positive bacterium Streptomyces lividans TK24 in both intracellular and extracellular forms without external heme supplementation. The enzyme was tested on a series of O-glycosides, which are plant secondary metabolites with a phenyl glycosidic linkage. O-glycosides are of great interest, both for studying the compounds themselves and as potential models for studying specific lignin-carbohydrate complexes. The primary DyP reaction products of salicin, arbutin, fraxin, naringin, rutin, and gossypin were oxidatively coupled oligomers. A cleavage of the glycone moiety upon radical polymerization was observed when using arbutin, fraxin, rutin, and gossypin as substrates. The amount of released glucose from arbutin and fraxin reached 23% and 3% of the total substrate, respectively. The proposed mechanism suggests a destabilization of the ether linkage due to the localization of the radical in the para position. In addition, DyP2 was tested on complex lignocellulosic materials such as wheat straw, spruce, willow, and purified water-soluble lignin fractions, but no remarkable changes in the carbohydrate profile were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive. IMPORTANCE: Peroxidases require correct incorporation of the heme cofactor for activity. Heterologous overproduction of peroxidases often results in an inactive enzyme due to insufficient heme synthesis by the host organism. Therefore, peroxidases are incubated with excess heme during or after purification to reconstitute activity. S. lividans as a production host can produce fully active peroxidases both intracellularly and extracellularly without the need for heme supplementation. This reduces the number of downstream processing steps and is beneficial for more sustainable production of industrially relevant enzymes. Moreover, this research has extended the scope of dye-decolorizing peroxidase applications by studying naturally relevant plant secondary metabolites and analyzing the formed products. A previously overlooked artifact of radical polymerization leading to the release of the glycosyl moiety was revealed, shedding light on the mechanism of DyP peroxidases. The key aspect is the continuous addition, rather than the more common approach of a single addition, of the cosubstrate, hydrogen peroxide. This continuous addition allows the peroxidase to complete a high number of turnovers without self-oxidation.

Biotransformation of maize bran-derived ferulic acid to vanillin using an adapted strain of Amycolatopsis sp. ATCC 39116.

Maize bran, an agro-processing waste residue, is a good source of ferulic acid that can be further valorized for vanillin production. However, extraction of ferulic acid from natural sources has been challenging due to low concentrations and intensive extraction procedures. In the present work, ferulic acid streams (purities ranging from 5% to 75%) extracted from maize bran using thermochemical methods were evaluated for biotransformation to vanillin, employing Amycolatopsis sp. as a whole-cell biocatalyst. Initial adaptation studies were critical in improving ferulic acid assimilation and its conversion to vanillin by 65% and 56%, respectively by the fourth adaptation cycle. The effect of cell's physiological states and vanillic acid supplementation on vanillin production was studied using standard ferulic acid as a substrate in an effort to achieve further improvement in vanillin yield. In the presence of vanillic acid, 18 h cultured cells using 2 g/L of standard and isolated ferulic acid produced vanillin concentrations of up to 0.71 and 0.48 g/L, respectively. Furthermore, intermediates involved in the ferulic acid catabolic pathway and their interrelations were studied using GC-MS analysis. Results indicated that two different routes were involved in the catabolism of standard ferulic acid, and similar metabolic routes were observed for an isolated ferulic acid stream. These findings effectively evaluated isolated ferulic acid for sustainable vanillin production while reducing agro-industrial waste pollution.

Metabolic engineering of the shikimate pathway in Amycolatopsis strains for optimized glycopeptide antibiotic production.

Glycopeptide antibiotics (GPA) consist of a glycosylated heptapeptide backbone enriched in aromatic residues originating from the shikimate pathway. Since the enzymatic reactions within the shikimate pathway are highly feedback-regulated, this raises the question as to how GPA producers control the delivery of precursors for GPA assembly. We chose Amycolatopsis balhimycina, the producer of balhimycin, as a model strain for analyzing the key enzymes of the shikimate pathway. A. balhimycina contains two copies each of the key enzymes of the shikimate pathway, deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh), with one pair (Dahpsec and Pdhsec) encoded within the balhimycin biosynthetic gene cluster and one pair (Dahpprim and Pdhprim) in the core genome. While overexpression of the dahpsec gene resulted in a significant (>4-fold) increase in balhimycin yield, no positive effects were observed after overexpression of the pdhprim or pdhsec genes. Investigation of allosteric enzyme inhibition revealed that cross-regulation between the tyrosine and phenylalanine pathways plays an important role. Tyrosine, a key precursor of GPAs, was found to be a putative activator of prephenate dehydratase (Pdt), which catalyzes the first step reaction from prephenate to phenylalanine in the shikimate pathway. Surprisingly, overexpression of pdt in A. balhimycina led to an increase in antibiotic production in this modified strain. In order to demonstrate that this metabolic engineering approach is generally applicable to GPA producers, we subsequently applied this strategy to Amycolatopsis japonicum and improved the production of ristomycin A, which is used in diagnosis of genetic disorders. Comparison of "cluster-specific" enzymes with the isoenzymes from the primary metabolism's pathway provided insights into the adaptive mechanisms used by producers to ensure adequate precursor supply and GPA yields. These insights further demonstrate the importance of a holistic approach in bioengineering efforts that takes into account not only peptide assembly but also adequate precursor supply.

Phosphatidylglycerol-specific phospholipase C from Amycolatopsis sp. NT115 strain: purification, characterization, and gene cloning.

Recently, phosphatidylglycerol (PG) focused on its important role in chloroplast photosynthesis, mitochondrial function of the sperm, an inhibitory effect on SARS-CoV-2 ability to infect naïve cells, and reducing lung inflammation caused by coronavirus disease 2019. To develop an enzymatic PG determination method as the high-throughput analysis of PG, a PG-specific phospholipase C (PG-PLC) was found in the culture supernatant of Amycolatopsis sp. NT115. PG-PLC (54 kDa by SDS-PAGE) achieved the maximal activity at pH 6.0 and 55 °C and was inhibited by detergents, such as Briji35, Tween 80, and sodium cholate, but not by EDTA and metal ions, except for Zn2+. The open reading frame of the PG-PLC gene consisted of 1620 bp encoding 515-amino-acid residues containing the preceding 25-amino-acid residues (Tat signal peptide sequence). The putative amino acid sequence of PG-PLC was highly similar to those of metallophosphoesterases; however, its substrate specificity was completely different from those of known PLCs.

Amycolachromones A-F, Isolated from a Streptomycin-Resistant Strain of the Deep-Sea Marine Actinomycete Amycolatopsis sp. WP1.

In this study, a detailed chemical investigation of a streptomycin-resistant strain of the deep-sea marine, actinomycete Amycolatopsis sp. WP1, yielded six novel amycolachromones A-F (1-6), together with five known analogues (7-11). Amycolachromones A-B (1-2) possessed unique dimer skeletons. The structures and relative configurations of compounds 1-11 were elucidated by extensive spectroscopic data analyses combined with X-ray crystal diffraction analysis. Plausible biogenetic pathways of amycolachromones A-F were also proposed.

Biological evaluation and spectral characterization of a novel tetracenomycin X congener.

The aromatic polyketide tetracenomycin X (TcmX) was recently found to be a potent inhibitor of protein synthesis; its binding site is located in a unique locus within the tunnel of the large ribosomal subunit. The distinct mode of action makes this relatively narrow class of aromatic polyketides promising for drug development in the quest to prevent the spread of drug-resistant pathogens. Here we report the isolation and structure elucidation of a novel natural tetracenomycin X congener - 6-hydroxytetraceonomycin X (6-OH-TcmX). In contrast to TcmX, 6-OH-TcmX exhibited lower antimicrobial and cytotoxic activity, but comparable in vitro protein synthesis inhibition ability. A survey on spectral properties of tetracenomycins revealed profound differences in both UV-absorption and fluorescence spectra between TcmX and 6-OH-TcmX, suggesting a significant influence of 6-hydroxylation on the tetracenomycin X chromophore. Nonetheless, characteristic spectral properties of tetracenomycins make them suitable candidates for semi-synthetic drug development (e.g., for targeted delivery, chemical biology, or cell imaging).

Strategy for Producing the High-Quality Glycopeptide Antibiotic A82846B in Amycolatopsis orientalis Based on the CRISPR-Cas12a System.

Oritavancin is a new-generation semisynthetic lipoglycopeptide antibiotic used to prevent the spread of vancomycin-resistant Gram-positive bacteria. The glycopeptide A82846B is the direct precursor of oritavancin. Considering the structural similarity between A82846B and vancomycin, the vancomycin producer Amycolatopsis orientalis was used as a chassis for the construction of a strain producing high-quality A82846B. To construct the A82846B synthetic pathway, we established a highly efficient CRISPR-Cas12a system by optimizing the conditions of conjugation and by screening the regulatory elements in the A. orientalis, which is difficult to be genetically manipulated. The efficiency of gene knockout was almost 100%. The glycosyltransferases module (gtfDE) and glycosyl synthesis module (vcaAEBD) in the vancomycin gene cluster were replaced with the corresponding glycosyltransferases module (gtfABC) and glycosyl synthesis module (evaAEBD) in the A82846B cluster, respectively. A82846B was successfully produced by the artificially constructed synthetic pathway. Moreover, the titer of A82846B was increased 80% by expressing the pathway-specific regulatory strR. This strategy has excellent potential for remodification of natural products to solve antibiotic resistance.

Enhancing Ristomycin A Production by Overexpression of ParB-Like StrR Family Regulators Controlling the Biosynthesis Genes.

Amycolatopsis sp. strain TNS106 harbors a ristomycin-biosynthetic gene cluster (asr) in its genome and produces ristomycin A. Deletion of the sole cluster-situated StrR family regulatory gene, asrR, abolished ristomycin A production and the transcription of the asr genes orf5 to orf39. The ristomycin A fermentation titer in Amycolatopsis sp. strain TNS106 was dramatically improved by overexpression of asrR and a heterologous StrR family regulatory gene, bbr, from the balhimycin-biosynthetic gene cluster (BGC) utilizing strong promoters and multiple gene copies. Ristomycin A production was improved by approximately 60-fold, resulting in a fermentation titer of 4.01 g/liter in flask culture, in one of the engineered strains. Overexpression of AsrR and Bbr upregulated transcription of tested asr biosynthetic genes, indicating that these asr genes were positively regulated by AsrR and Bbr. However, only the promoter region of the asrR operon and the intergenic region upstream of orf12 were bound by AsrR and Bbr in gel retardation assays, suggesting that AsrR and Bbr directly regulated the asrR operon and probably orf12 to orf14 but no other asr biosynthetic genes. Further assays with synthetic short probes showed that AsrR and Bbr specifically bound not only probes containing the canonical inverted repeats but also a probe with only one 7-bp element of the inverted repeats in its native context. AsrR and Bbr have an N-terminal ParB-like domain and a central winged helix-turn-helix DNA-binding domain. Site-directed mutations indicated that the N-terminal ParB-like domain was involved in activation of ristomycin A biosynthesis and did not affect the DNA-binding activity of AsrR and Bbr. IMPORTANCE This study showed that overexpression of either a native StrR family regulator (AsrR) or a heterologous StrR family regulator (Bbr) dramatically improved ristomycin A production by increasing the transcription of biosynthetic genes directly or indirectly. The conserved ParB-like domain of AsrR and Bbr was demonstrated to be involved in the regulation of asr BGC expression. These findings provide new insights into the mechanism of StrR family regulators in the regulation of glycopeptide antibiotic biosynthesis. Furthermore, the regulator overexpression plasmids constructed in this study could serve as valuable tools for strain improvement and genome mining for new glycopeptide antibiotics. In addition, ristomycin A is a type III glycopeptide antibiotic clinically used as a diagnostic reagent due to its side effects. The overproduction strains engineered in this study are ideal materials for industrial production of ristomycin A.

Rifamycin W Analogues from Amycolatopsis mediterranei S699 Δrif-orf5 Strain.

Rifamycin W, the most predominant intermediate in the biosynthesis of rifamycin, needs to undergo polyketide backbone rearrangement to produce rifamycin B via an oxidative cleavage of the C-12/C-29 double bond. However, the mechanism of this putative oxidative cleavage has not been characterized yet. Rif-Orf5 (a putative cytochrome P450 monooxygenase) was proposed to be involved in the cleavage of this olefinic moiety of rifamycin W. In this study, the mutant strain Amycolatopsis mediterranei S699 Δrif-orf5 was constructed by in-frame deleting the rif-orf5 gene to afford thirteen rifamycin W congeners (1-13) including seven new ones (1-7). Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Presumably, compounds 1-4 were derivatized from rifamycin W via C-5/C-11 retro-Claisen cleavage, and compounds 1-3, 9 and 10 featured a hemiacetal. Compounds 5-7 and 11 showed oxygenations at various sites of the ansa chain. In addition, compounds 1-3 exhibited antibacterial activity against Staphylococcus aureus with minimal inhibitory concentration (MIC) values of 5, 40 and 0.5 µg/mL, respectively. Compounds 1 and 3 showed modest antiproliferative activity against HeLa and Caco-2 cells with half maximal inhibitory concentration (IC50) values of about 50 µM.

Secondary Metabolites of the Genus Amycolatopsis: Structures, Bioactivities and Biosynthesis.

Actinomycetes are regarded as important sources for the generation of various bioactive secondary metabolites with rich chemical and bioactive diversities. Amycolatopsis falls under the rare actinomycete genus with the potential to produce antibiotics. In this review, all literatures were searched in the Web of Science, Google Scholar and PubMed up to March 2021. The keywords used in the search strategy were "Amycolatopsis", "secondary metabolite", "new or novel compound", "bioactivity", "biosynthetic pathway" and "derivatives". The objective in this review is to summarize the chemical structures and biological activities of secondary metabolites from the genus Amycolatopsis. A total of 159 compounds derived from 8 known and 18 unidentified species are summarized in this paper. These secondary metabolites are mainly categorized into polyphenols, linear polyketides, macrolides, macrolactams, thiazolyl peptides, cyclic peptides, glycopeptides, amide and amino derivatives, glycoside derivatives, enediyne derivatives and sesquiterpenes. Meanwhile, they mainly showed unique antimicrobial, anti-cancer, antioxidant, anti-hyperglycemic, and enzyme inhibition activities. In addition, the biosynthetic pathways of several potent bioactive compounds and derivatives are included and the prospect of the chemical substances obtained from Amycolatopsis is also discussed to provide ideas for their implementation in the field of therapeutics and drug discovery.

Identification of a conserved N-terminal domain in the first module of ACV synthetases.

The l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine synthetase (ACVS) is a trimodular nonribosomal peptide synthetase (NRPS) that provides the peptide precursor for the synthesis of ß-lactams. The enzyme has been extensively characterized in terms of tripeptide formation and substrate specificity. The first module is highly specific and is the only NRPS unit known to recruit and activate the substrate l-α-aminoadipic acid, which is coupled to the α-amino group of l-cysteine through an unusual peptide bond, involving its δ-carboxyl group. Here we carried out an in-depth investigation on the architecture of the first module of the ACVS enzymes from the fungus Penicillium rubens and the bacterium Nocardia lactamdurans. Bioinformatic analyses revealed the presence of a previously unidentified domain at the N-terminus which is structurally related to condensation domains, but smaller in size. Deletion variants of both enzymes were generated to investigate the potential impact on penicillin biosynthesis in vivo and in vitro. The data indicate that the N-terminal domain is important for catalysis.

Feeding strategies to optimize vanillin production by Amycolatopsis sp. ATCC 39116.

The growing consumer demand for natural products led to an increasing interest in vanillin production by biotechnological routes. In this work, the biotechnological vanillin production by Amycolatopsis sp. ATCC 39116 is studied using ferulic acid as precursor, aiming to achieve maximized vanillin productivities. During biotech-vanillin production, the effects of glucose, vanillin and ferulic acid concentrations in the broth proved to be relevant for vanillin productivity. Concerning glucose, its presence in the broth during the production phase avoids vanillin conversion to vanillic acid and, consequently, increases vanillin production. To avoid the accumulation of vanillin up to a toxic concentration level, a multiple-pulse-feeding strategy is implemented, with intercalated vanillin removal from the broth and biomass recovery. This strategy turned out fruitful, leading to 0.46 g L-1 h-1 volumetric productivity of vanillin of and a production yield of 0.69 gvanillin gferulic acid-1, which are among the highest values reported in the literature for non-modified bacteria.

Anti-hyperglycemic and genotoxic studies of 1-O-methyl chrysophanol, a new anthraquinone isolated from Amycolatopsis thermoflava strain SFMA-103.

The compound 1-O-methyl chrysophanol (OMC) which belongs to a class of hydroxyanthraquinones was isolated from Amycolatopsis thermoflava strain SFMA-103 and studied for their anti-diabetic properties. OMC was evaluated as an anti-diabetic agent based on in silico studies which initially predicted the binding energy with α-amylase (-188.81 KJ mol-1) and with α-glucosidase (70.53 KJ mol-1). Further, these results were validated based on enzyme inhibition assays where OMC demonstrated enzyme inhibitory activity towards α-amylase (IC50 3.4 mg mL-1) and α-glucosidase (IC50 38.49 µg mL-1). To confirm the anti-diabetic activity, in vivo studies (oral dose in Wistar rats) revealed that OMC inhibited significantly the increase in glucose concentration at 100 mg/kg as compared to starch control (p < 0.05). Further, to understand the safety of OMC as a therapeutic agent, the genotoxic analysis was performed in both in vitro Chinese Hamster Ovary cells (250, 500, and 1000 µM/mL) and in vivo Swiss albino mice (250, 500, and 1000 mg/kg). In vitro results showed that OMC concentration of up to 250 µM/mL did not elicit significant changes in CAs, MI, and MN counts in CHO cells. Similarly, in mice experiments (i.p. injection), no significant changes in CAs, MI, and MN induction were observed till 500 mg/kg of OMC when compared with chrysophanic acid (Cy) (200 mg/kg). In addition, mice that received the lowest dose of OMC (250 mg/kg) did not show any histological changes in liver, kidney, and heart. The study concluded that five times higher therapeutic dose (100 mg/kg) of OMC can be utilized against hyperglycemia with no genotoxic effects.

Heterologous expression of the atypical tetracycline chelocardin reveals the full set of genes required for its biosynthesis.

BACKGROUND: Chelocardin (CHD) exhibits a broad-spectrum antibiotic activity and showed promising results in a small phase II clinical study conducted on patients with urinary tract infections. Importantly, CHD was shown to be active also against tetracycline-resistant Gram-negative pathogens, which is gaining even more importance in today's antibiotic crisis. We have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete Amycolatopsis sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CD-CHD), which is currently in preclinical evaluation. A. sulphurea is difficult to genetically manipulate and therefore manipulation of the chd biosynthetic gene cluster in a genetically amenable heterologous host would be of high importance for further drug-discovery efforts. RESULTS: We report heterologous expression of the CHD biosynthetic gene cluster in the model organism Streptomyces albus del14 strain. Unexpectedly, we found that the originally defined CHD gene cluster fails to provide all genes required for CHD formation, including an additional cyclase and two regulatory genes. Overexpression of the putative pathway-specific streptomyces antibiotic regulatory protein chdB in A. sulphurea resulted in an increase of both, CHD and CD-CHD production. Applying a metabolic-engineering approach, it was also possible to generate the potent CHD analogue, CD-CHD in S. albus. Finally, an additional yield increase was achieved in S. albus del14 by in-trans overexpression of the chdR exporter gene, which provides resistance to CHD and CDCHD. CONCLUSIONS: We identified previously unknown genes in the CHD cluster, which were shown to be essential for chelocardin biosynthesis by expression of the full biosynthetic gene cluster in S. albus as heterologous host. When comparing to oxytetracycline biosynthesis, we observed that the CHD gene cluster contains additional enzymes not found in gene clusters encoding the biosynthesis of typical tetracyclines (such as oxytetracycline). This finding probably explains the different chemistries and modes of action, which make CHD/CD-CHD valuable lead structures for clinical candidates. Even though the CHD genes are derived from a rare actinomycete A. sulphurea, the yield of CHD in the heterologous host was very good. The corrected nucleotide sequence of the CHD gene cluster now contains all gene products required for the production of CHD in a genetically amenable heterologous host, thus opening new possibilities towards production of novel and potent tetracycline analogues with a new mode of action.

Amycolasporins and Dibenzoyls from Lichen-Associated Amycolatopsis hippodromi and Their Antibacterial and Anti-inflammatory Activities.

Eleven metabolites, six echinosporins (1-6), four dibenzoyls (7-10), and an aromatic compound (11), were isolated from the fermentation broth of lichen-associated Amycolatopsis hippodromi. The structures of the new compounds (1-5, 8-11) were elucidated by comprehensive spectroscopic analysis including data from experimental and calculated ECD spectra. Amycolasporins A-C (1-3) demonstrated antibacterial activities against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli with MIC values of 25 or 100 µg/mL. Amycolasporin C (3) and the known dibenzoyl (7) attenuated the production of NO due to the suppression of the expression of nitric oxide synthase (iNOS) in LPS-induced RAW 264.7 cells in a dose-dependent manner.

8-Deoxy-Rifamycin Derivatives from Amycolatopsis mediterranei S699 ΔrifT Strain.

Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain Amycolatopsis mediterranei S699 ΔrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1-11) and seven known analogues (12-18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4-11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus (MIC (minimal inhibitory concentration) values of 10, 20, 20, 20, 40 and 20 µg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50 (half maximal inhibitory concentration) values of 14.91, 44.78, 2.16, 18.67 and 8.07 µM, respectively.

Recent Advances in Microbial Production of cis,cis-Muconic Acid.

cis,cis-Muconic acid (MA) is a valuable C6 dicarboxylic acid platform chemical that is used as a starting material for the production of various valuable polymers and drugs, including adipic acid and terephthalic acid. As an alternative to traditional chemical processes, bio-based MA production has progressed to the establishment of de novo MA pathways in several microorganisms, such as Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida, and Saccharomyces cerevisiae. Redesign of the metabolic pathway, intermediate flux control, and culture process optimization were all pursued to maximize the microbial MA production yield. Recently, MA production from biomass, such as the aromatic polymer lignin, has also attracted attention from researchers focusing on microbes that are tolerant to aromatic compounds. This paper summarizes recent microbial MA production strategies that involve engineering the metabolic pathway genes as well as the heterologous expression of some foreign genes involved in MA biosynthesis. Microbial MA production will continue to play a vital role in the field of bio-refineries and a feasible way to complement various petrochemical-based chemical processes.

Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.

Differential Activation of Ferulic Acid Catabolic Pathways of Amycolatopsis sp. ATCC 39116 in Submerged and Surface Cultures.

Amycolatopsis sp. ATCC 39116 catabolizes ferulic acid by the non-oxidative deacetylation and ß-oxidation pathways to produce vanillin and vanillic acid, respectively. In submerged culture, vanillin productivity decreased more than 8-fold, when ferulic, p-coumaric, and caffeic acids were employed in pre-cultures of the microorganism in order to activate the ferulic acid catabolic pathways, resulting in a carbon redistribution since vanillic acid and guaiacol productivities increased more than 5-fold compared with control. In contrast, in surface culture, the effects of ferulic and sinapic acids in pre-cultures were totally opposite to those of the submerged culture, directing the carbon distribution into vanillin formation. In surface culture, more than 30% of ferulic acid can be used as carbon source for other metabolic processes, such as ATP regeneration. In this way, the intracellular ATP concentration remained constant during the biotransformation process by surface culture (100 µg ATP/mg protein), demonstrating a high energetic state, which can maintain active the non-oxidative deacetylation pathway. In contrast, in submerged culture, it decreased 3.15-fold at the end of the biotransformation compared with the initial content, showing a low energetic state, while the NAD+/NADH ratio (23.15) increased 1.81-fold. It seems that in submerged culture, low energetic and high oxidative states are the physiological conditions that can redirect the ferulic catabolism into ß-oxidative pathway and/or vanillin oxidation to produce vanillic acid.

Metabolic engineering of Amycolatopsis japonicum for optimized production of [S,S]-EDDS, a biodegradable chelator.

The actinomycete Amycolatopsis japonicum is the producer of the chelating compound [S,S]-ethylenediamine-disuccinc acid (EDDS). [S,S]-EDDS is an isomer of ethylenediamine-tetraacetic acid (EDTA), an economically important chelating compound that suffers from an extremely poor degradability. Frequent use of the persistent EDTA in various industrial and domestic applications has caused an accumulation of EDTA in soil as well as in aqueous environments. As a consequence, EDTA is the highest concentrated anthropogenic compound present in water reservoirs. The [S,S]-form of EDDS has chelating properties similar to EDTA, however, in contrast to EDTA it is readily biodegradable. In order to compete with the cost-effective chemical synthesis of EDTA, we aimed to optimize the biotechnological production of [S,S]-EDDS in A. japonicum by using metabolic engineering approaches. Firstly, we integrated several copies of the [S,S]-EDDS biosynthetic genes into the chromosome of A. japonicum and replaced the native zinc responsive promoter with the strong synthetic constitutive promoter SP44*. Secondly, we increased the supply of O-phospho-serine, the direct precursor of [S,S]-EDDS. The combination of these approaches together with the optimized fermentation process led to a significant improvement in [S,S]-EDDS up to 9.8 g/L with a production rate of 4.3 mg/h/g DCW.