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1.
Chem Commun (Camb) ; 55(95): 14287-14290, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31712798

RESUMO

A novel bright near-infrared II (NIR-II, 1000-1700 nm) fluorescent probe with excellent water-solubility, superior photostability, and excellent in vitro and in vivo biocompatibility was facilely synthesized for in vivo biomedical imaging of xenograft breast tumor and chemically induced spontaneous breast carcinoma. To the best of our knowledge, it is the first time that the superior practical applications of this NIR-II probe in dimethylbenzanthracene (DMBA)-induced rat mammary carcinoma imaging and image-guided rat carcinoma surgery were demonstrated.

2.
Chembiochem ; 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31777147

RESUMO

A few acyltransferase (AT) domains of modular polyketide synthases (PKSs) recruit acyl carrier protein (ACP)-linked extender units with unusual C2 substituents to confer functionalities that are not available in coenzyme A (CoA)-linked ones. Here, an AT specific for methoxymalonyl (MOM)-ACP in the third module of the ansamitocin PKS was structurally and biochemically characterized. The AT uses a conserved tryptophan at the entrance of the substrate binding tunnel to discriminate between different carriers. A W275R mutation switches its carrier specificity from the ACP protein to the CoA molecule. The acyl-AT complex structures clearly show that the MOM-ACP accepted by the AT has the 2S instead of the opposite 2R stereochemistry that is predicted according to the biosynthetic derivation from a D-glycolytic intermediate. Together, these results reveal the structural basis of ATs recognizing ACP-linked extender units in polyketide biosynthesis.

3.
Chem Commun (Camb) ; 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31777862

RESUMO

Correction for 'A bright NIR-II fluorescent probe for breast carcinoma imaging and image-guided surgery' by Xiaodong Zeng et al., Chem. Commun., 2019, DOI: 10.1039/c9cc07694h.

4.
Magn Reson Med ; 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31765496

RESUMO

PURPOSE: To develop a simultaneous T1 , T2 , and ADC mapping method that provides co-registered, distortion-free images and enables multiparametric quantification of 3D brain coverage in a clinically feasible scan time with the MR Multitasking framework. METHODS: The T1 /T2 /diffusion weighting was generated by a series of T2 preparations and diffusion preparations. The underlying multidimensional image containing 3 spatial dimensions, 1 T1 weighting dimension, 1 T2 -preparation duration dimension, 1 b-value dimension, and 1 diffusion direction dimension was modeled as a 5-way low-rank tensor. A separate real-time low-rank model incorporating time-resolved phase correction was also used to compensate for both inter-shot and intra-shot phase inconsistency induced by physiological motion. The proposed method was validated on both phantom and 16 healthy subjects. The quantification of T1 /T2 /ADC was evaluated for each case. Three post-surgery brain tumor patients were scanned for demonstration of clinical feasibility. RESULTS: Multitasking T1 /T2 /ADC maps were perfectly co-registered and free from image distortion. Phantom studies showed substantial quantitative agreement ( R 2 = 0.999 ) with reference protocols for T1 /T2 /ADC. In vivo studies showed nonsignificant T1 (P = .248), T2 (P = .97), ADC (P = .328) differences among the frontal, parietal, and occipital regions. Although Multitasking showed significant differences of T1 (P = .03), T2 (P < .001), and ADC (P = .001) biases against the references, the mean bias estimates were small (ΔT1 % < 5%, ΔT2 % < 7%, ΔADC% < 5%), with all intraclass correlation coefficients greater than 0.82 indicating "excellent" agreement. Patient studies showed that Multitasking T1 /T2 /ADC maps were consistent with the clinical qualitative images. CONCLUSION: The Multitasking approach simultaneously quantifies T1 /T2 /ADC with substantial agreement with the references and is promising for clinical applications.

5.
Mol Microbiol ; 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31749226

RESUMO

Phosphorothioation (PT) involves the replacement of a nonbridging phosphate oxygen on the DNA backbone with sulfur. In bacteria, the procedure is both sequence- and stereo-specific. We reconstituted the PT reaction using purified DndCDE from Salmonella enterica and IscS from E. coli. We determined that the in vitro process of PT was oxygen-sensitive. Only one strand on a double-stranded (ds) DNA substrate was modified in the reaction. The modification was dominant between G and A in the GAAC/GTTC conserved sequence. The modification between G and T required the presence of PT between G and A on the opposite strand. Cysteine, S-adenosyl methionine (SAM), and the formation of an iron-sulfur cluster in DndCDE (DndCDE-FeS) were essential for the process. Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction. ATP promoted the reaction but was not essential. The data and conclusions presented suggest that the PT reaction in bacteria involves three steps. The first step is the binding of DndCDE-FeS to DNA and searching for the modification sequence, possibly with the help of ATP. Cysteine locks DndCDE-FeS to the modification site with an appropriate protein conformation. SAM triggers the radical SAM reaction to complete the oxygen-sulfur swapping.

6.
Artigo em Inglês | MEDLINE | ID: mdl-31704680

RESUMO

Hybrubins are "unnatural" alkaloids with the same 4'-methoxy-2,2'-bipyrrole-5'-methine moiety found in prodiginines and a different ring derived from tetramic acids. Here, we demonstrated that RedH, a homologue of prodigiosin synthetase PigC, was responsible for the biosynthesis of hybrubins A and B in Streptomyces lividans In vitro reactions indicated that RedH and PigC catalyzed the intermolecular condensation between 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde (MBC) and (Z)-5-ethylidenetetramic acid (ETA) to produce hybrubin B. Moreover, we demonstrated that RedH and PigC activated MBC via phosphorylation of the aldehyde group to form an intermediate Pi-MBC and that the subsequent condensation between Pi-MBC and (Z)-5-ethylidenetetramic acid occurs in a non-enzymatic way.IMPORTANCE Hybrubins is an emerging class of prodiginines possessing a new C-ring derived from 5'-substituted tetramic acids and the methylene bridge connecting the C-ring at a different position. We have supposed that condensation between 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde (MBC) and 5-ethylidenetetramic acid (ETA) yields the hybrid natural products hybrubins, which was proposed to be catalysed by the undecylprodigiosin synthetase RedH. However, it is doubted whether the RedH is able to catalyse another type of condensation between MBC and tetramic acids. In this study, we have demonstrated that the MBC-ETA condensation proceeds through RedH/PigC-catalysed enzymatic activation of MBC via phosphorylation and a non-enzymatic condensation of Pi-MBC with ETA. Since MBC analogues have been showed to be accepted by PigC, more hybrubin analogues might be produced by using combinations of MBC analogues and other tetramic acids in future studies.

7.
J Org Chem ; 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31729221

RESUMO

Tricyclic carbazole is an important scaffold in many naturally occurring metabolites, as well as valuable building blocks. Here we report the reconstitution of the ring A formation of the bacterial neocarazostatin A carbazole metabolite. We provide evidence of the involvement of two unusual aromatic polyketide proteins. This finding suggests how new enzymatic activities can be recruited to specific pathways to expand biosynthetic capacities. Finally, we leveraged our bioinformatics survey to identify the untapped capacity of carbazole biosynthesis.

8.
ACS Synth Biol ; 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31747253

RESUMO

Emerging antimicrobial resistant fungal pathogens are a growing threat, and fungicides with novel modes of action are urgently needed to prevent critical failures in global food security. Fenpicoxamid, the prodrug of UK-2A, is a member of a new class of antifungal agents that displays no cross-resistance to other fungicides. Rational engineering of its structure using a biosynthetic approach is a promising avenue for developing more potent fungicides. Herein, through in vitro enzymatic reconstitution, we elucidate the biosynthetic pathway of UK-2A. Its biosynthesis involves a flexible AMP-binding protein and dilactone formation assembly enzymes that are able to select and incorporate highly diverse substituted salicylic acids into the dilactone scaffold. By introducing diverse salicylic acids into the in vitro biosynthetic pathway, we successfully generate 14 novel deacyl UK-2A analogues. This study reveals the flexibility of the biosynthetic pathway of UK-2A and provides an effective solution to rationally engineer its crucial C3 moiety.

9.
Artigo em Inglês | MEDLINE | ID: mdl-31676476

RESUMO

Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside antibiotics, in which ribose and a pyrazole-derived base are linked by a C-glycosidic bond. However, the logic underlying the biosynthesis of these molecules has remained largely unexplored. Here, we report the discovery of the pathways for FOR-A and PRF-A biosynthesis from diverse actinobacteria, and propose that their biosynthesis is likely initiated by a lysine N6 -monooxygenase. Moreover, we show that the forT and prfT (involved in FOR-A and PRF-A biosynthesis, respectively) mutants are correspondingly capable of accumulating the unexpected pyrazole-related intermediates, 4-amino-3,5-dicarboxypyrazole and 3,5-dicarboxy-4-oxo-4,5-dihydropyrazole. We also decipher the enzymatic mechanism of ForT/PrfT for the C-glycosidic bond formation in FOR-A/PRF-A biosynthesis. To our knowledge, ForT/PrfT represents an example of ß-RFA-P (ß-ribofuranosyl-aminobenzene 5'-phosphate) synthase-like enzymes governing C-nucleoside scaffold construction in natural product biosynthesis. These data establish a foundation for combinatorial biosynthesis of related purine nucleoside antibiotics, and also open the way for target-directed genome mining of PRF-A/FOR-A related antibiotics.IMPORTANCE FOR-A and PRF-A are C-nucleoside antibiotics known for their unusual chemical structures and remarkable biological activities. Deciphering the enzymatic mechanism for the construction of C-nucleoside scaffold during FOR-A/PRF-A biosynthesis will not only expand biochemical repertoire for novel enzymatic reactions, but also permit the target-oriented genome mining of FOR-A/PRF-A related C-nucleoside antibiotics. Moreover, the availability of the FOR-A/PRF-A biosynthetic gene clusters will pave the way for the rational generation of designer FOR-A/PRF-A derivatives with enhanced/selective bioactivity via synthetic biology strategies.

10.
Artigo em Inglês | MEDLINE | ID: mdl-31565827

RESUMO

Sactionine-containing antibiotics (sactibiotics) are a growing class of peptide antibiotics belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. We report the characterization of thuricin Z, a novel sactibiotic from Bacillus thuringiensis. Unusually, the biosynthesis of thuricin Z involves two radical S-adenosylmethionine (SAM) enzymes, ThzC and ThzD. Although ThzC and ThzD are highly divergent from each other, these two enzymes produced the same sactionine ring in the precursor peptide ThzA in vitro. Thuricin Z exhibits narrow-spectrum antibacterial activity against Bacillus cereus. A series of analyses, including confocal laser scanning microscopy, ultrathin-sectioning transmission electron microscopy, scanning electron microscopy, and large-unilamellar-vesicle-based fluorescence analysis, suggested that thuricin Z binds to the bacterial cell membrane and leads to membrane permeabilization.

11.
ACS Synth Biol ; 8(9): 1991-1997, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31487454

RESUMO

Direct cloning of natural product pathways for efficient refactoring and heterologous expression has become an important strategy for microbial natural product research and discovery, especially for those kept silent or poorly expressed in the original strains. Accordingly, the development of convenient and efficient cloning approaches is becoming increasingly necessary. Here we presented an in vitro packaging mediated cloning approach that combines CRISPR/Cas9 system with in vitro λ packaging system, for targeted cloning of natural product pathways. In such a scheme, pathways of Tü3010 (27.4 kb) and sisomicin (40.7 kb) were respectively cloned, and stuR was further depicted to positively regulate Tü3010 production. In vitro packaging mediated approach not only enables to activate cryptic pathways, but also facilitates refactoring or interrogating the pathways in conjunction with various gene editing systems. This approach features an expedited, convenient, and generic manner, and it is conceivable that it may be widely adopted for targeted cloning of the natural product pathways.

12.
Org Lett ; 21(18): 7592-7596, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31490082

RESUMO

Pentalenolactone is a microbial sesquiterpenoid with antibiotic activity. Its biosynthetic pathway was elucidated by a combination of genetic and biochemical characterizations of all genes involved. For the related neopentalenoketolactone biosynthetic gene cluster from Streptomyces avermitilis, an α-ketoglutarate-dependent mononuclear nonheme iron enzyme, PtlD, was proposed to catalyze both desaturation and olefin epoxidation reactions. Yet, these activities remained to be validated by in vitro biochemical evidence. In this report, we demonstrated that PtlD has multiple activities, including hydroxylation, desaturation, and epoxidation, and confirmed the presence of the elusive epoxide intermediate in a neopentalenoketolactone pathway.

13.
Nat Commun ; 10(1): 4248, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31534134

RESUMO

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD-RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.

14.
Appl Microbiol Biotechnol ; 103(21-22): 8785-8797, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31515597

RESUMO

Ophiobolins (ophs) are characteristic 5-8-5 tricyclic sesterterpenes with potential pharmaceutical activities. Ophiobolin synthase is a bifunctional terpene synthase (BTS) that catalyzes both chain elongation and cyclization. In Aspergillus ustus 094102, ophiobolin accumulation was involved with not only ophiobolin synthase C25 (Au8003) but also other four gene clusters containing C15 (Au6298), C20 (Au13192 and Au11565), and C30 (Au3446) terpene synthases. In this report, overexpression of codon-optimized gene Au8003 resulted in a detectable production of oph F in E. coli. In subsequent modulation of culture conditions, pentose arabinose allowed a more than 10-fold improvement of production than that of glycerol. To achieve a higher titer, the whole mevalonate pathway and an additional copy of isopentenyl diphosphate isomerase gene were assembled, leading to approximately 24-fold and 60-fold yield increases, respectively. The above four terpene synthase genes related to ophiobolin production in strain 094102 were individually or combinatorially overexpressed with Au8003 to mimic the original fungal biosynthesis. The biosynthesis of oph scaffold was increased by short-chain terpene synthases (C15 and C20), among which the C15 synthase gene contributed the highest yield of 82.76 mg/L at 96 h; the multi-gene combinatorial results suggested that cyclization might be a rate-limiting step. Further protein engineering including fusion tags and phylogenetically based mutations on the rate-limiting cyclization part of Au8003 enabled a further yield improvement (> 150 mg/L at 96 h) in shake flasks. These multiple approaches for sesterterpene skeleton production using engineered E. coli may be applicable for cost-effective, high-yield productions of ophiobolins and other compounds synthesized by BTSs.

15.
Biotechnol J ; : e1900175, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31520458

RESUMO

Lipopeptides are produced by nonribosomal peptide synthetases (NRPSs) and contain diverse fatty acyl moieties that are major determinants of antibiotic potency. The lipid chains are incorporated into peptidyl backbones via lipoinitiation, a process comprising free fatty acid activation and the subsequent starter condensation domain (C1)-catalyzed conjugation of fatty acyl moieties onto the aminoacyl substrates. Thus, a thorough understanding of lipoinitiation biocatalysts would significantly expand their potential to produce novel antibiotics. Here, biochemical assays, in silico analysis, and mutagenesis studies are used to ultimately identify the specific amino acid residues that control the fatty acyl substrate selectivity of C1 in lipopeptide A54145. In silico docking study has identified four candidate amino acids, and subsequent in vitro assays confirmed their functional contribution to the channel that controls substrate selectivity. Two engineered variants with single point mutations in C1 are found to alter the substrate selectivity toward nonnatural fatty acyl substrates. The detailed mechanistic insights into the catalytic contribution of C1 obtained from the present study will facilitate future NPRS biocatalyst efforts.

16.
J Agric Food Chem ; 67(40): 11148-11157, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31532654

RESUMO

Lycopene is widely used in foods, cosmetics, nutritional supplements, and pharmaceuticals. Microbial production of lycopene has been intensively studied. However, there are few systematic engineering studies on Saccharomyces cerevisiae aimed at achieving high-yield lycopene production. In the current study, by employing a systematic optimization strategy, we screened the key lycopene biosynthetic genes, crtE, crtB, and crtI, from diverse organisms. By adjusting the copy number of these three key genes, knocking out endogenous bypass genes, increasing the supply of the precursor acetyl-CoA, balancing NADPH utilization, and regulating the GAL-inducible system, we constructed a high-yield lycopene-producing strain BS106, which can produce 310 mg/L lycopene in shake-flask fermentation, with gene expression controlled by glucose. In optimized two-stage fed-batch fermentation, BS106 produced 3.28 g/L lycopene in a 7 L fermenter, which is the highest concentration achieved in S. cerevisiae to date. It will decrease the consumption of tomatoes for lycopene extraction and increase the market supply of lycopene.


Assuntos
Licopeno/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vias Biossintéticas , Fermentação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Mol Cell ; 76(1): 126-137.e7, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31444107

RESUMO

A surprising complexity of ubiquitin signaling has emerged with identification of different ubiquitin chain topologies. However, mechanisms of how the diverse ubiquitin codes control biological processes remain poorly understood. Here, we use quantitative whole-proteome mass spectrometry to identify yeast proteins that are regulated by lysine 11 (K11)-linked ubiquitin chains. The entire Met4 pathway, which links cell proliferation with sulfur amino acid metabolism, was significantly affected by K11 chains and selected for mechanistic studies. Previously, we demonstrated that a K48-linked ubiquitin chain represses the transcription factor Met4. Here, we show that efficient Met4 activation requires a K11-linked topology. Mechanistically, our results propose that the K48 chain binds to a topology-selective tandem ubiquitin binding region in Met4 and competes with binding of the basal transcription machinery to the same region. The change to K11-enriched chain architecture releases this competition and permits binding of the basal transcription complex to activate transcription.

18.
Biotechnol J ; : e1900212, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31469239

RESUMO

Fluorescence imaging, as a commonly used scientific tool, is widely applied in various biomedical and material structures through visualization technology. Highly selective and sensitive luminescent biological probes, as well as those with good water solubility, are urgently needed for biomedical research. In contrast to the traditional aggregation-caused quenching of fluorescence, in the unique phenomenon of aggregation-induced emission (AIE), the individual luminogens have extremely weak or no emissivity because they each have free intramolecular motion; however, when they form aggregates, these components immediately "light up". Since the discovery of "turn-on" mechanism, researchers have been studying and applying AIE in a variety of fields to develop more sensitive, selective, and efficient strategies for the AIE dyes. There are numerous advantages to the use of AIE-based methods, including low background interference, strong contrast, high performance in intracellular imaging, and the ability for long-term monitoring in vivo. In this review, two typical examples of AIEgens, TPE-Cy and TPE-Ph-In, are described, including their structure properties and applications. Recent progress in the biological applications is mainly focused on. Undoubtedly, in the near future, an increasing number of encouraging and practical ideas will promote the development of more AIEgens for broad use in biomedical applications.

19.
Biotechnol J ; 14(11): e1900114, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31294913

RESUMO

Xylose/glucose isomerases are important industrial enzymes that are most widely used in food industries; however, their previously reported expression levels do not meet the requirements for industrial application. Here, an antibiotic resistance marker (ARM)-free system driven by ribosomal RNA (rRNA) promoters is developed to obtain high-level xylose/glucose isomerase (XI/GI) expression in Streptomyces rubiginosus (S. rubiginosus). The rRNA promoter rrnD yields the highest glucose isomerase production titer of XIs/GIs, which is eight times higher than that of ermEp* and 2.6 times higher than that of kasOp*. The integrated ARM gene is removed by further introduction of the Cre plasmid with a temperature-sensitive replicon. The production titer of XIs/GIs is further improved by replacing the xylR gene with an additional expression glucose isomerase cassette at the xylR locus. Ultimately, the glucose isomerase activity reaches up to 79.7 ± 7.5 U mL-1 at 96 h. The results support the robustness and stability of XI/GI production with this ARM-free system using optimal ribosomal promoters in S. rubiginosus, demonstrating strong potential in large-scale industrial applications. Besides, the results imply that rRNA promoters are strong promoters that can be used for protein engineering or metabolic engineering.

20.
Nucleic Acids Res ; 47(14): 7690-7702, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31260525

RESUMO

Bacterial toxin-antitoxin pairs play important roles in bacterial multidrug tolerance. Gcn5-related N-acetyltransferase (GNAT) toxins inhibit translation by acetylation of aminoacyl-tRNAs and are counteracted by direct contacts with cognate ribbon-helix-helix (RHH) antitoxins. Our previous analysis showed that the GNAT toxin KacT and RHH antitoxin KacA of Klebsiella pneumoniae form a heterohexamer in solution and that the complex interacts with the cognate promoter DNA, resulting in negative autoregulation of kacAT transcription. Here, we present the crystal structure of DNA-bound KacAT complex at 2.2 Å resolution. The crystal structure revealed the formation of a unique heterohexamer, KacT-KacA2-KacA2-KacT. The direct interaction of KacA and KacT involves a unique W-shaped structure with the two KacT molecules at opposite ends. Inhibition of KacT is achieved by the binding of four KacA proteins that preclude the formation of an active KacT dimer. The kacAT operon is auto-regulated and we present an experimentally supported molecular model proposing that the KacT:KacA ratio controls kacAT transcription by conditional cooperativity. These results yield a profound understanding of how transcription GNAT-RHH pairs are regulated.

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