Two Cytochrome P450 Enzymes Form the Tricyclic Nested Skeleton of Meroterpenoids by Sequential Oxidative Reactions.

Meroterpenoid clavilactones feature a unique benzo-fused ten-membered carbocyclic ring unit with an α,ß-epoxy-γ-lactone moiety, forming an intriguing 10/5/3 tricyclic nested skeleton. These compounds are good inhibitors of the tyrosine kinase, attracting a lot of chemical synthesis studies. However, the natural enzymes involved in the formation of the 10/5/3 tricyclic nested skeleton remain unexplored. Here, we identified a gene cluster responsible for the biosynthesis of clavilactone A in the basidiomycetous fungus Clitocybe clavipes. We showed that a key cytochrome P450 monooxygenase ClaR catalyzes the diradical coupling reaction between the intramolecular hydroquinone and allyl moieties to form the benzo-fused ten-membered carbocyclic ring unit, followed by the P450 ClaT that exquisitely and stereoselectively assembles the α,ß-epoxy-γ-lactone moiety in clavilactone biosynthesis. ClaR unprecedentedly acts as a macrocyclase to catalyze the oxidative cyclization of the isopentenyl to the nonterpenoid moieties to form the benzo-fused macrocycle, and a multifunctional P450 ClaT catalyzes a ten-electron oxidation to accomplish the biosynthesis of the 10/5/3 tricyclic nested skeleton in clavilactones. Our findings establish the foundation for the efficient production of clavilactones using synthetic biology approaches and provide the mechanistic insights into the macrocycle formation in the biosynthesis of fungal meroterpenoids.

Security enhanced routing and spectrum allocation against crosstalk attacks for confidential lightpath in elastic optical networks.

Elastic optical network (EON) is a critical transmission infrastructure for emerging new applications due to its spectral efficiency and flexibility. Nowadays, numerous confidential lightpaths (CLPs) are carried over EON to support security-sensitive users. However, they are vulnerable to crosstalk attacks at the optical layer, typically aimed at eavesdropping on the carried data or even disrupting connections. Due to the transparent nature of the optical signals, such attacks are difficult to detect and could last for a long time, resulting in data leakage even spreading throughout the network. This paper presents a novel routing and spectrum allocation (RSA) algorithm to protect CLPs from crosstalk attacks. We investigate intra-channel and inter-channel crosstalk attacks and develop a metric to quantify crosstalk leakage risks (CLRs). We first formulate an ILP model to plan CLPs with a minimum CLR. To solve the same problem for large-scale networks, we also propose a heuristic algorithm, i.e., crosstalk-attack-aware RSA. Results indicate that the proposed algorithm is capable of reducing CLR by 23%.

Insights into inactivation and response mechanisms of sublethal Listeria monocytogenes treated by cold plasma with joint transcriptomics and metabolomics.

AIM: The aim of the current study is to elucidate the inactivation and molecular response pattern of sublethal Listeria monocytogenes to cold plasma-mediated two-pronged oxidative microenvironments from a high-throughput multi-omics perspective. METHODS AND RESULTS: First joint transcriptomics and metabolomics analyses revealed that significantly expressed genes and metabolites were mainly involved in enhanced transmembrane transport and Fe2+/Cu+ efflux, amino acid limitation, cytoplasmic pH homeostasis, reconfiguration of central carbon metabolism flux, and energy conservation strategy, which triggered the surge of intracellular endogenous oxidative stress and finally mediated bacterial ferroptosis and pathogenicity attenuation. Typical antioxidant systems such as the TrxR-Trx system and common antioxidant genes (e.g. sodA, katA, ahpC, trxA, spxA) were inhibited, and the more prominent antioxidant pathways include methionine metabolism, the pentose phosphate pathway, and glutathione metabolism, as well as the DNA repair systems. CONCLUSIONS: Therefore, our work confirmed from the transcriptional and metabolic as well as physiological levels that cold plasma-mediated intracellular oxidative stress induced big perturbations in pathways as a driving force for the inactivation and pathogenicity attenuation of L. monocytogenes. SIGNIFICANCE AND IMPACT OF STUDY: This study provided new insights for the construction of multi-dimensional mechanisms of bacterial inactivation and pathogenicity attenuation for the precise control and inactivation of microorganisms in plasma non-thermal processing.

Mining an O-methyltransferase for de novo biosynthesis of physcion in Aspergillus nidulans.

Physcion is one of natural anthraquinones, registered as a novel plant-derived fungicide due to its excellent prevention of plant disease. However, the current production of physcion via plant extraction limits its yield promotion and application. Here, a pair of polyketide synthases (PKS) in emodin biosynthesis were used as probes to mining the potential O-methyltransferase (OMT) responsible for physcion biosynthesis. Further refinement using the phylogenetic analysis of the mined OMTs revealed a distinct OMT (AcOMT) with the ability of transferring a methyl group to C-6 hydroxyl of emodin to form physcion. Through introducing AcOMT, we successfully obtained the de novo production of physcion in Aspergillus nidulans. The physcion biosynthetic pathway was further rationally engineered by expressing the decarboxylase genes from different fungi. Finally, the titer of physcion reached to 64.6 mg/L in shake-flask fermentation through enhancing S-adenosylmethionine supply. Our work provides a native O-methyltransferase for physcion biosynthesis and lays the foundation for further improving the production of physcion via a sustainable route. KEY POINTS: • Genome mining of the native O-methyltransferase responsible for physcion biosynthesis • De novo biosynthesis of physcion in the engineered Aspergillus nidulans • Providing an alternative way to produce plant-derived fungicide physcion.

Isotropic Contact Properties in Monolayer GeAs Field-Effect Transistors.

Owing to the tunable bandgap and high thermodynamic stability, anisotropic monolayer (ML) GeAs have arisen as an attractive candidate for electronic and optoelectronic applications. The contact properties of ML GeAs with 2D metal (graphene, Ti2CF2, V2CF2, and Ti3C2O2) and Cu electrodes are explored along two principal axes in field-effect transistors (FET) by employing ab initio electronic structure calculations and quantum transport simulations. Weak van der Waals interactions are found between ML GeAs and the 2D metal electrodes with the band structure of ML GeAs kept the same, while there is a strong interaction between ML GeAs and the Cu metal electrode, resulting in the obvious hybridization of the band structure. Isotropic contact properties are seen along the two principal directions. P-type lateral Schottky contacts are established in ML GeAs FETs with Ti3C2O2, graphene, and Ti2CF2 metals, with a hole Schottky barrier height (SBH) of 0.12 (0.20), 0.15 (0.11), and 0.29 (0.21) eV along the armchair (zigzag) direction, respectively, and an n-type lateral Schottky contact is established with the Cu electrode with an electron SBH of 0.64 (0.57) eV. Surprisingly, ML GeAs forms ideal p-type Ohmic contacts with the V2CF2 electrode. The results provide a theoretical foundation for comprehending the interactions between ML GeAs and metals, as well as for designing high-performance ML GeAs FETs.

Decoration of defective graphene with MoS2 enabling enhanced anchoring and catalytic conversion of polysulfides for lithium-sulfur batteries: a first-principles study.

The potential of carbon materials for electrochemical processes could be largely activated by the delicate regulation of their intrinsic defects, and this prospect could be further enhanced after hybridizing with other functional components. Herein, we, for the first time, systematically combine graphene possessing different intrinsic defects with MoS2 as a host material for sulfur in lithium-sulfur batteries using first-principles calculations. After introducing the intrinsic defects in graphene, the heterostructures provide moderate binding affinity to lithium polysulfides (LiPSs) and facilitate their chemical reactions due to the unsaturated coordination of defective carbon and the charge rearrangement inside the heterostructures. Specifically, graphene with intrinsic defects increases the active sites and improves the conductivity, while MoS2 can not only improve the adsorption for LiPSs, but also provide smooth Li diffusion pathways and catalyze the rapid conversion of LiPSs. Among all the calculated heterostructures, the single vacancy graphene/MoS2 heterostructure is considered to be the most promising sulfur host due to the strongest binding strength to LiPSs (3.10-0.72 eV) and the lowest free energy barrier for the sulfur reduction reaction (1.36 eV), which is attributed to the spin polarization near the carbon defect. This work could afford fruitful insights into the rational design of defect engineering in heterostructures.

Oxidative lesions and post-treatment viability attenuation of listeria monocytogenes triggered by atmospheric non-thermal plasma.

AIMS: The aim of the current study was to investigate the effect of plasma-mediated oxidative stress on the post-treatment viability of Listeria monocytogenes at the physiological and molecular levels. METHODS AND RESULTS: 107  CFU/ml L. monocytogenes in 10 ml phosphate-buffered saline (PBS) was treated with atmospheric non-thermal plasma for 0, 30, 60, 90 and 120 s respectively. Optical diagnostics using optical emission spectroscopy (OES) confirmed that dielectric barrier discharge (DBD) plasma was a significant source of ample exogenous reactive oxygen and nitrogen species (RONS). The development of extracellular main long-lived species was associated with plasma exposure time, accompanied by a massive accumulation of intracellular ROS in L. monocytogenes (p < 0.01). With the exception of virulence genes (hly), most oxidation resistance genes (e.g. sigB, perR, lmo2344, lmo2770 and trxA) and DNA repair gene (recA) were upregulated significantly (p < 0.05). A visible fragmentation in genomic DNA and a decline in the secretion of extracellular proteins and haemolytic activity (p < 0.01) were noticed. The quantitate oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) confirmed the viability attenuation from the aspect of energy metabolism. Survival assay in a real food system (raw milk) further suggested not only the viability attenuation, but also the resuscitation potential and safety risk of mild plasma-treated cells during post-treatment storage. CONCLUSION: DBD plasma had the potential to inactivate and attenuate the virulence of L. monocytogenes, and it was recommended that plasma exposure time longer than 120 s was more suitable for attenuating viability and avoiding the recovery possibility of L. monocytogenes in raw milk within 7 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The current results presented a strategy to inactivate and attenuate the viability of L. monocytogenes, which could serve as a theoretical basis for better application of non-thermal plasma in food in an effort to effectively combat foodborne pathogens.

Exosomes derived from MSC pre-treated with oridonin alleviates myocardial IR injury by suppressing apoptosis via regulating autophagy activation.

This study aimed to investigate the molecular mechanisms underlying the role of bone marrow mesenchymal stem cells (BMMSCs)-derived exosomes in ischaemia/reperfusion (IR)-induced damage, and the role of oridonin in the treatment of IR. Exosomes were isolated from BMMSCs. Western blot analysis was done to examine the expression of proteins including CD63, CD8, apoptotic-linked gene product 2 interacting protein X (AliX), Beclin-1, ATG13, B-cell lymphoma-2 (Bcl-2), apoptotic peptidase activating factor 1 (Apaf1) and Bcl2-associated X (Bax) in different treatment groups. Accordingly, the expression of CD63, CD81 and AliX was higher in BMMSCs-EXOs and IR + BMMSCs-EXOs + ORI groups compared with that in the BMMSCs group. And BMMSCs-derived exosomes inhibited the progression of IR-induced myocardial damage, while this protective effect was boosted by the pre-treatment with oridonin. Moreover, Beclin-1, ATG13 and Bcl-2 were significantly down-regulated while Apaf1 and Bax were significantly up-regulated in IR rats. And the presence of BMMSCs-derived exosomes partly alleviated IR-induced dysregulation of these proteins, while the oridonin pre-treatment boosted the effect of these BMMSCs-derived exosomes. The inhibited proliferation and promoted apoptosis of H9c2 cells induced by hypoxia/reperfusion (HR) were mitigated by the administration of BMMSCs-derived exosomes. Meanwhile, HR also induced down-regulation of Beclin-1, ATG13 and Bcl-2 expression and up-regulation of Apaf1 and Bax, which were mitigated by the administration of BMMSCs-derived exosomes. And oridonin pre-treatment boosted the effect of BMMSCs-derived exosomes. In conclusion, our results validated that BMMSCs-derived exosomes suppressed the IR-induced damages by participating in the autophagy process, while the pre-treatment with oridonin could boost the protective effect of BMMSCs-derived exosomes.

Schottky barrier heights in two-dimensional field-effect transistors: from theory to experiment.

Over the past decade, two-dimensional semiconductors (2DSCs) have aroused wide interest due to their extraordinary electronic, magnetic, optical, mechanical, and thermal properties, which hold potential in electronic, optoelectronic, thermoelectric applications, and so forth. The field-effect transistor (FET), a semiconductor gated with at least three terminals, is pervasively exploited as the device geometry for these applications. For lack of effective and stable substitutional doping techniques, direct metal contact is often used in 2DSC FETs to inject carriers. A Schottky barrier (SB) generally exists in the metal-2DSC junction, which significantly affects and even dominates the performance of most 2DSC FETs. Therefore, low SB or Ohmic contact is highly preferred for approaching the intrinsic characteristics of the 2DSC channel. In this review, we systematically introduce the recent progress made in theoretical prediction of the SB height (SBH) in the 2DSC FETs and the efforts made both in theory and experiments to achieve low SB contacts. From the comparison between the theoretical and experimentally observed SBHs, the emerging first-principles quantum transport simulation turns out to be the most powerful theoretical tool to calculate the SBH of a 2DSC FET. Finally, we conclude this review from the viewpoints of state-of-the-art electrode designs for 2DSC FETs.

Metabolomic analyses on microbial primary and secondary oxidative stress responses.

Food safety is veryimportant in our daily life. In food processing or disinfection, microorganisms are commonly exposed to oxidative stress perturbations. However, microorganisms can adapt and respond to physicochemical interventions, leading to difficulty and complexity for food safety assurance. Therefore, understanding the response mechanisms of microbes and providing an overview of the responses under oxidative stress conditions are beneficial for ensuring food safety for the industry. The current review takes the metabolomics approach to reveal small metabolite signatures and key pathway alterations during oxidative stress at the molecular and technical levels. These alterations are involved in primary oxidative stress responses due to inactivation treatments such as using hypochlorite (HOCl), hydrogen peroxide (H2 O2 ), electrolyzed water (EW), irradiation, pulsed light (PL), electron beam (EB), and secondary oxidative stress responses due to exposures to excessive conditions such as heat, pressure, acid, and alkaline. Details on the putative origin of exogenous or endogenous reactive oxygen species (ROS) are discussed, with particular attention paid to their effects on lipid, amino acid, nucleotide, and carbohydrate metabolism. In addition, mechanisms on counteracting oxidative stresses, stabilization of cell osmolality as well as energy provision for microbes to survive are also discussed.

Dietary methionine restriction attenuates renal ischaemia/reperfusion-induced myocardial injury by activating the CSE/H2S/ERS pathway in diabetic mice.

Methionine restrictive diet may alleviate ischaemia/reperfusion (I/R)-induced myocardial injury, but its underlying mechanism remains unclear. HE staining was performed to evaluate the myocardial injury caused by I/R and the effect of methionine-restricted diet (MRD) in I/R mice. IHC and Western blot were carried out to analyse the expression of CSE, CHOP and active caspase3 in I/R mice and hypoxia/reoxygenation (H/R) cells. TUNEL assay and flow cytometry were used to assess the apoptotic status of I/R mice and H/R cells. MTT was performed to analyse the proliferation of H/R cells. H2S assay was used to evaluate the concentration of H2S in the myocardial tissues and peripheral blood of I/R mice. I/R-induced mediated myocardial injury and apoptosis were partially reversed by methionine-restricted diet (MRD) via the down-regulation of CSE expression and up-regulation of CHOP and active caspase3 expression. The decreased H2S concentration in myocardial tissues and peripheral blood of I/R mice was increased by MRD. Accordingly, in a cellular model of I/R injury established with H9C2 cells, cell proliferation was inhibited, cell apoptosis was increased, and the expressions of CSE, CHOP and active caspase3 were dysregulated, whereas NaHS treatment alleviated the effect of I/R injury in H9C2 cells in a dose-dependent manner. This study provided a deep insight into the mechanism underlying the role of MRD in I/R-induced myocardial injury.

Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum.

Acremonium chrysogenum has been employed in the industrial production of cephalosporin C (CPC). However, there are still some impediments to understanding the regulation of CPC biosynthesis and improving strains due to the difficulty of genetic manipulation in A. chrysogenum, especially in the CPC high-producing strain C10. Here, an improved CRISPR-Cas9 system was constructed based on an U6/tRNA chimeric promoter. Using this system, high efficiency for single gene disruption was achieved in C10. In addition, double loci were simultaneously targeted when supplying with the homology-directed repair templates (donor DNAs). Based on this system, large DNA fragments up to 31.5 kb for the yellow compound sorbicillinoid biosynthesis were successfully deleted with high efficiency. Furthermore, CPC production was significantly enhanced when the sorbicillinoid biosynthetic genes were knocked out. This study provides a powerful tool for gene editing and strain improvement in A. chrysogenum.

Serum miR-195-5p is upregulated in gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is defined as varying degrees of glucose intolerance with an onset or first recognition during pregnancy in women without previously diagnosed diabetes. Accumulating evidence indicates that miRNAs exert crucial roles in the pathogenesis and development of diabetes, including GDM. In the present study, we aimed to determine the clinical performance of miR-195-5p in GDM. METHODS: First, the miR-195-5p expressions in serum samples from healthy pregnant women and women with GDM at 25 weeks pregnancy were detected using real-time polymerase chain reaction (RT-qPCR). Then, receive characteristic (ROC) curve was used to determine the diagnostic value of miR-195-5p in GDM. Finally, the correlation analysis of miR-195-5p expression with related clinicopathological factors was carried out to determine the clinical value of miR-195-5p in GDM. RESULTS: In this study, we found that miR-195-5p expression was significantly increased in serum samples from GDM patients as compared with that in healthy pregnancies. Furthermore, miR-195-5p might be a putative biomarker for GDM diagnosis with an area under the curve (AUC) of 0.8451; the cutoff value was 1.598, sensitivity was 73.69%, specificity was 96.85%, accuracy was 81.26%, and Youden index was 70.54%. Expression of miR-195-5p was positively associated with fasting plasma glucose, one-hour plasma glucose, and two-hour plasma glucose. CONCLUSION: miR-195-5p might function as a putative diagnostic biomarker for GDM and contribute to identifying at-risk mothers in pregnancy.

Bencycloquidium bromide nasal spray is effective and safe for persistent allergic rhinitis: a phase III, multicenter, randomized, double-blinded, placebo-controlled clinical trial.

PURPOSE: To investigate the efficacy and safety of bencycloquidium bromide nasal spray (BCQB) in patients with persistent allergic rhinitis (PAR). METHODS: We enrolled 720 patients from 15 hospitals across China and randomly assigned them into BCQB group or placebo group (90 µg per nostril qid) to receive a 4-week treatment. Visual analog scale (VAS) for rhinorrhea, sneezing, nasal congestion, itching and overall symptoms were recorded by patients every day. Anterior rhinoscopy scoring was completed by doctors on every visit. Adverse events were recorded in detail. RESULTS: A total of 354 and 351 patients were included in BCQB group and in placebo group. Baseline information was comparable. At the end of the trial, the decrease of VAS for rhinorrhea from baseline was 4.83 ± 2.35 and 2.46 ± 2.34 in BCQB group and placebo group, respectively (P < 0.001). The change ratio from baseline of VAS for rhinorrhea in BCQB group was 72.32%, higher than 31.03% in placebo group (P < 0.001). VAS for other symptoms and overall symptoms also improved significantly in the BCQB group, while no inter-group difference was found in anterior rhinoscopy scoring. The incidence of adverse reaction was similar between the two groups. Most reactions were mild and no severe reactions happened. CONCLUSION: 90 µg BCQB per nostril four times daily is effective and safe in the treatment of rhinorrhea as well as sneezing, nasal congestion and itching for patients with PAR. RETROSPECTIVELY REGISTERED: ChiCTR2000030924, 2020/3/17.

The disruption of verM activates the production of gliocladiosin A and B in Clonostachys rogersoniana.

Gliocladiosin A (1) and B (2), two dipeptides conjugated with macrolides, were identified from a verM disruption mutant of the Cordycep-colonizing fungus Clonostachys rogersoniana. The structures and absolute configurations of 1 and 2 were determined on the basis of spectroscopic data analysis, including MS, NMR, CD and X-ray diffraction. A biogenetic pathway for 1 and 2 was proposed. These two compounds showed moderate antibacterial effects.

Functional response of sediment bacterial community to iron-reducing bioaugmentation with Shewanella decolorationis S12.

Bioaugmentation with exogenously functional microbes is a widely used technology in bioengineering and environmental remediation. Generally, the colonization of inoculated bacteria is considered to be the determining factor in technical success. However, increasing reports have shown that bioaugmentation was still effective when the colonization of inoculated bacteria was unsuccessful. Here, an augmentation study with iron-reducing bacteria (IRB, Shewanella decolorationis S12) was conducted in Fe(II)-poor sediments to elucidate the role of exogenously inoculated bacteria for bioaugmentation performance. The results showed that a sufficient amount of IRB inputs enhanced the iron reduction in bioaugmented sediments, even though the exogenous IRB did not colonize after the beginning of the experiment (less than 1% at day 3). The iron reduction function responded to stimulation of the indigenous IRB community such as Clostridium, Cupriavidus, Fervidicella, and Acinetobacter, which comprised less than 1% in the initial sediments. Moreover, compared with microbial community in control sediment, more positive correlations between OTUs were observed for that in S12-added sediments upon network analysis. The pH and oxidation-reduction potential of sediment were found to be the predominant factors shaping the iron-reducing microbial communities. It meant that exogenous IRB successfully trigged functional community via altering microenvironment by the inoculated bacteria. Overall, this study provides a new insight into the understanding of the role of single strain addition in iron-reducing bioaugmentation.

Cold Plasma-Mediated Treatments for Shelf Life Extension of Fresh Produce: A Review of Recent Research Developments.

Fresh produce, like fruits and vegetables, are important sources of nutrients and health-promoting compounds. However, incidences of foodborne outbreaks associated with fresh produce often occur; it is thus important to develop and expand decay-control technologies that can not only maintain the quality but can also control the biological hazards in postharvest, processing, and storage to extend their shelf life. It is under such a situation that plasma-mediated treatments have been developed as a novel nonthermal processing tool, offering many advantages and attracting much interest from researchers and the food industry. This review summarizes recent developments of cold plasma technology and associated activated water for shelf life extension of fresh produce. An overview of plasma generation and its physical-chemical properties as well as methods for improving plasma efficiency are first presented. Details of using the technology as a nonthermal agent in inhibiting spoilage and pathogenic microorganisms, inactivating enzymes, and modifying the barrier properties or imparting specific functionalities of packaging materials to extend shelf life of food produce are then reviewed, and the effects of cold plasma-mediated treatment on microstructure and quality attributes of fresh produce are discussed. Future prospects and research gaps of cold plasma are finally elucidated. The review shows that atmospheric plasma-mediated treatments in various gas mixtures can significantly inhibit microorganisms, inactive enzyme, and modify packaging materials, leading to shelf life extension of fresh produce. The quality attributes of treated produce are not compromised but improved. Therefore, plasma-mediated treatment has great potential and values for its application in the food industry.

A Myb transcription factor represses conidiation and cephalosporin C production in Acremonium chrysogenum.

Acremonium chrysogenum is the industrial producer of cephalosporin C (CPC). We isolated a mutant (AC554) from a T-DNA inserted mutant library of A. chrysogenum. AC554 exhibited a reduced conidiation and lack of CPC production. In consistent with it, the transcription of cephalosporin biosynthetic genes pcbC and cefEF was significantly decreased in AC554. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed and sequence analysis indicated that a T-DNA was inserted upstream of an open reading frame (ORF) which was designated AcmybA. On the basis of sequence analysis, AcmybA encodes a Myb domain containing transcriptional factor. Observation of red fluorescent protein (RFP) tagged AcMybA showed that AcMybA is naturally located in the nucleus of A. chrysogenum. Transcriptional analysis demonstrated that the AcmybA transcription was increased in AC554. In contrast, the AcmybA deleted mutant (ΔAcmybA) overproduced conidia and CPC. To screen the targets of AcmybA, we sequenced and compared the transcriptome of ΔAcmybA, AC554 and the wild-type strain at different developmental stages. Twelve differentially expressed regulatory genes were identified. Taken together, our results indicate that AcMybA negatively regulates conidiation and CPC production in A. chrysogenum.

Enhancing the production of cephalosporin C through modulating the autophagic process of Acremonium chrysogenum.

BACKGROUND: Autophagy is used for degradation of cellular components and nutrient recycling. Atg8 is one of the core proteins in autophagy and used as a marker for autophagic detection. However, the autophagy of filamentous fungi is poorly understood compared with that of Saccharomyces cerevisiae. Our previous study revealed that disruption of the autophagy related gene Acatg1 significantly enhanced cephalosporin C yield through reducing degradation of cephalosporin biosynthetic proteins in Acremonium chrysogenum, suggesting that modulation of autophagic process is one promising way to increase antibiotic production in A. chrysogenum. RESULTS: In this study, a S. cerevisiae ATG8 homologue gene Acatg8 was identified from A. chrysogenum. Acatg8 could complement the ATG8 mutation in S. cerevisiae, indicating that Acatg8 is a functional homologue of ATG8. Microscope observation demonstrated the fluorescently labeled AcAtg8 was localized in the cytoplasm and autophagosome of A. chrysogenum, and the expression of Acatg8 was induced by nutrient starvation. Gene disruption and genetic complementation revealed that Acatg8 is essential for autophagosome formation. Disruption of Acatg8 significantly reduced fungal conidiation and delayed conidial germination. Localization of GFP-AcAtg8 implied that autophagy is involved in the early phase of conidial germination. Similar to Acatg1, disruption of Acatg8 remarkably enhanced cephalosporin C yield. The cephalosporin C biosynthetic enzymes (isopenicillin N synthase PcbC and isopenicillin N epimerase CefD2) and peroxisomes were accumulated in the Acatg8 disruption mutant (∆Acatg8), which might be the main reasons for the enhancement of cephalosporin C production. However, the biomass of ΔAcatg8 decreased drastically at the late stage of fermentation, suggesting that autophagy is critical for A. chrysogenum cell survival under nutrition deprived condition. Disruption of Acatg8 also resulted in accumulation of mitochondria, which might produce more reactive oxygen species (ROS) which promotes fungal death. However, the premature death is unfavorable for cephalosporin C production. To solve this problem, a plasmid containing Acatg8 under control of the xylose/xylan-inducible promoter was introduced into ∆Acatg8. Conidiation and growth of the recombinant strain restored to the wild-type level in the medium supplemented with xylose, while the cephalosporin C production maintained at a high level even prolonged fermentation. CONCLUSIONS: Our results demonstrated inducible expression of Acatg8 and disruption of Acatg8 remarkably increased cephalosporin C production. This study provides a promising approach for yield improvement of cephalosporin C in A. chrysogenum.

Metabolic engineering of Acremonium chrysogenum for improving cephalosporin C production independent of methionine stimulation.

BACKGROUND: Cephalosporin C (CPC) produced by Acremonium chrysogenum is one of the most important drugs for treatment of bacterial infectious diseases. As the major stimulant, methionine is widely used in the industrial production of CPC. In this study, we found methionine stimulated CPC production through enhancing the accumulation of endogenous S-adenosylmethionine (SAM). To overcome the methionine dependent stimulation of CPC production, the methionine cycle of A. chrysogenum was reconstructed by metabolic engineering. RESULTS: Three engineered strains were obtained by overexpressing the SAM synthetase gene AcsamS and the cystathionine-γ-lyase gene mecB, and disrupting a SAM dependent methyltransferase gene Acppm1, respectively. Overexpression of AcsamS resulted in fourfold increase of CPC production which reached to 129.7 µg/mL. Disruption of Acppm1 also increased CPC production (up to 135.5 µg/mL) through enhancing the accumulation of intracellular SAM. Finally, an optimum recombinant strain (Acppm1DM-mecBOE) was constructed through overexpressing mecB in the Acppm1 disruption mutant. In this strain, CPC production reached to the maximum value (142.7 µg/mL) which was 5.5-fold of the wild-type level and its improvement was totally independent of methionine stimulation. CONCLUSIONS: In this study, we constructed a recombinant strain in which the improvement of CPC production was totally independent of methionine stimulation. This work provides an economic route for improving CPC production in A. chrysogenum through metabolic engineering.