•CRISPR-Cas9 assisted gene disruption was demonstrated for the first time in higher fungi.•The ura3 gene of Ganoderma lucidum was disrupted by the codon-optimized Cas9 and in vitro transcribed gRNA.•This work may help to provide a widely applicable approach of gene disruption in higher fungi.Higher fungi are regarded as promising cell factories for production of bioactive secondary metabolites, but there is a lack of methods of genetic manipulation, such as gene disruption, which hinders the studies on biosynthesis and its regulation of those useful natural products. In this study, the CRISPR-Cas9 assisted gene disruption was established for the first time in higher fungi by taking Ganoderma species as typical examples. With double strand break (DSB) introduced by CRISPR-Cas9, non-homologous end joining (NHEJ) was induced and further assisted the gene disruption. As proof of concept, the ura3 gene of G. lucidum 260125 and G. lingzhi was successfully disrupted by the codon-optimized Cas9 and in vitro transcribed gRNA. This work may help to provide a widely applicable approach of gene disruption in higher fungi.Download high-res image (109KB)Download full-size image

•An aerobic-anaerobic shift was applied to increase glutarate production by recombinant E. coli.•An anaerobically-inducible nar promoter was used for oxygen-sensitive hgdABC expression.•Enhanced production of glutarate was reached by combining fermentation and genetic strategies.Synthetic biology provides a significant platform in creating novel pathways/organisms for producing useful compounds, while it remains a challenge to enhance the production efficiency. Recently we constructed a recombinant Escherichia coli for glutarate production using a synthetic α-ketoacid reduction pathway, in which α-ketoglutarate is reduced to 2-hydroxyglutarate then converted to glutarate. However, the production titer was low, which may be due to 1) oxygen-sensitive nature of 2-hydroxyglutaryl-CoA dehydratase (HgdABC) and 2) limited cell growth in anaerobic cultivation. Therefore, we developed an aerobic-anaerobic two-stage strategy by growing more cells aerobically, then shifting to anaerobic cultivation to ensure the functional HgdABC for glutarate biosynthesis. The two-stage cultivation resulted in higher production of glutarate and other two C5 dicarboxylic acids – glutaconate and 2-hydroxylglutarate than the original anaerobic process. Furthermore, we used an anaerobically-inducible nar promoter to improve the hgdABC expression responding to aerobic-anaerobic shift. Finally, the glutarate, glutaconate and 2-hydroxyglutarate titer was increased about 2, 5 and 3 times, reaching 11.6, 108.8 and 399.5 mg/L, respectively. The work demonstrated an effective strategy for ameliorating α-ketoacid reduction pathway to produce C5 dicarboxylic acids, as well as the potential of integration of bioprocess and metabolic engineering for enhancing chemicals production by an engineered microorganism.Download high-res image (146KB)Download full-size image

Antibody–drug conjugates (ADCs), which combine the exquisite specificity of antibodies with the cell-killing ability of cytotoxic drug payloads, have emerged as an attractive means for treating cancers. All (pre)clinical ADCs employ biosynthesized cytotoxins as their ADC payload precursors (APPs). The cost-effective bioproduction of APPs is receiving great interest from both academia and industry. Given the lack of systematic overviews of the topic, we provide the current status of APPs and focus on their state-of-the-art bioproduction strategies, illustrated with typical examples and critical analyses. Challenges in further enhancing the bioproduction efficiency of APPs and other cytotoxins are also discussed. This research has implications for bioprocess and metabolic engineering, systems and synthetic biology, and biopharmaceutical drug discovery, development, and industrialization.

•A novel synthetic pathway for glutarate production was constructed in Escherichia coli.•A biochemical gap from trans-glutaconyl-CoA to glutarate was filled with trans-enoyl-CoA reductases (Ter).•Glutarate production was enhanced by employing a Ter mutant (I287V) from Treponema denticola.Glutarate is an important C5 linear chain dicarboxylic acid having wide applications in chemical industry. In this work we report glutarate production by Escherichia coli with a newly constructed biosynthetic pathway containing part of known glutaconate biosynthetic pathway and a gap-filling module employing trans-enoyl-CoA reductase (Ter). Overall the artificial pathway comprises reduction of the central carbon metabolite α-ketoglutarate to 2-hydroxyglutarate, activation to 2-hydroxyglutaryl-CoA, dehydration to trans-glutaconyl-CoA, hydrogenation to glutaryl-CoA by Ter and thioester hydrolysis to finally yield glutarate. The pathway introduced into E. coli resulted in a recombinant strain that produced 3.8 mg/L of glutarate together with 27.7 mg/L of glutaconate in anaerobic culture mode. The glutarate production increased by approximately 50% through the mutation of Ter from Treponema denticola. The results demonstrated biosynthesis of glutarate via a non-natural synthetic pathway, which may enable its biobased production from renewable resources.Download high-res image (130KB)Download full-size image

Lavendamycin methyl ester (LME) is a derivative of a highly functionalized aminoquinone alkaloid lavendamycin and could be used as a scaffold for novel anticancer agent development. This work demonstrated LME production by cultivation of an engineered strain of Streptomyces flocculus CGMCC4.1223 ΔstnB1, while the wild-type strain did not produce. To enhance its production, the effect of shear stress and oxygen supply on ΔstnB1 strain cultivation was investigated in detail. In flask culture, when the shaking speed increased from 150 to 220 rpm, the mycelium was altered from a large pellet to a filamentous hypha, and the LME production was almost doubled, while no significant differences were observed among varied filling volumes, which implied a crucial role of shear stress in the morphology and LME production. To confirm this suggestion, experiments with agitation speed ranging from 400 to 1,000 rpm at a fixed aeration rate of 1.0 vvm were conducted in a stirred tank bioreactor. It was found that the morphology became more hairy with reduced pellet size, and the LME production was enhanced threefolds when the agitation speed increased from 400 to 800 rpm. Further experiments by varying initial kLa value at the same agitation speed indicated that oxygen supply only slightly affected the physiological status of ΔstnB1 strain. Altogether, shear stress was identified as a major factor affecting the cell morphology and LME production. The work would be helpful to the production of LME and other secondary metabolites by filamentous microorganism cultivation.

The effects of oxygen limitation on the production of ganoderic acid (GA), a secondary metabolite with antitumor activity, and on transcription levels of triterpene biosynthesis genes were investigated in liquid cultures of Ganoderma lucidum. A low oxygen supply level was beneficial to total GA biosynthesis, but negative to the cell growth. The higher GA production was obtained under micro-aerobic conditions (i.e. initial overall kLa values at 0.02 and 0.13/h). The maximum GA production of 272.3 ± 11.5 mg/L was obtained at an initial overall kLa of 0.13/h, which was 1.7-fold that at a normal cultivation condition (an initial kLa of 5.51/h). For four major individual GAs, the production level of GA-Mk, -T, -S, and -Me in the hypoxia-induced cells was increased by 50, 87, 62, and 111%, compared with that of the control, respectively. Meanwhile, the transcriptions of four key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate-5-pyrophosphate decarboxylase, squalene synthase and squalene epoxidase in the triterpene biosynthetic pathway were up-regulated under the hypoxia condition (at an initial kLa of 0.13/h). Reactive oxygen species was generated in response to hypoxia, which seemed to be involved in the regulation of GA biosynthesis. The information obtained provides an insight into the role of oxygen limitation in the GA biosynthesis, and will be helpful for optimizing the fermentation process on a large scale.

In this work, the performance and adsorption characteristics of macroporous resins for the recovery and enrichment of ganoderic acid (GA)-Mk and GA-T from Ganoderma lucidum mycelia were systematically evaluated. ADS-8 resin displayed the best adsorption and desorption capacities among the tested resins based on batch experiments. The interaction between solute and ADS-8 resin at different temperatures was described in terms of Langmuir and Freundlich isotherms, and the equilibrium experimental data were well fitted to the two isotherms. Thermodynamic analysis indicated the exothermic and spontaneous nature of the adsorption process. The adsorption capacity of ADS-8 resin was found to depend strongly on the pH value of the initial solution. Dynamic adsorption and desorption tests were performed on an ADS-8 resin-packed column to obtain optimal parameters for recovering GA-Mk and GAT from G. lucidum extract. Under optimized conditions, a laboratory scale-up preparation of GA-Mk and GA-T was carried out. The contents of GA-Mk and GA-T were increased from 45 to 22 mg/g in the crude extract to 352 and 141 mg/g in the final product with recovery yields of 90.1 and 72.2%, respectively. These results demonstrated that ADS-8 resin chromatography could act as a useful approach for obtaining ganoderic acids from G. lucidum mycelia.

Validamycin A (VAL-A) is an important and widely used agricultural antibiotic. In this study, statistical screening designs were applied to identify significant medium variables for VAL-A production and to find their optimal levels. The optimized medium caused 70% enhancement of VAL-A production. The difference between optimized medium and original medium suggested that low nitrogen source level might attribute to the enhancement of VAL-A production. The addition of different nitrogen sources to the optimized medium inhibited VAL-A production, which confirmed the importance of nitrogen concentration for VAL-A production. Furthermore, differences in structural gene transcription and enzyme activity between the two media were assayed. The results showed that lower nitrogen level in the optimized medium could regulate VAL-A production in gene transcriptional level. Our previous study indicated that the transcription of VAL-A structural genes could be enhanced at elevated temperature. In this work, the increased fermentation temperature from 37 to 42 °C with the optimized medium enhanced VAL-A production by 39%, which testified to the importance of structural gene transcription in VAL-A production. The information is useful for further VAL-A production enhancement.

In order to enhance the production of validamycin A (VAL-A), a widely used agricultural antibiotic, a temperature shift strategy was developed in the fermentation of Streptomyces hygroscopicus 5008. VAL-A production and the transcriptional levels of its structural genes were enhanced in the optimal temperature shift condition. The addition of diphenyleneiodonium [DPI, reactive oxygen species (ROS) inhibitor] inhibited intracellular ROS level and VAL-A production, which indicated that ROS signal might contribute to the enhancement of VAL-A production in the temperature shift process. The transcriptional levels of stress response sigma factors SigmaB and SigmaH as well as global regulator PhoRP were enhanced, which suggested that these regulators might participate in the signal pathway. This study developed a useful strategy for VAL-A production. It will help to further understand the regulation mechanism of ROS on VAL-A synthesis. The involvement of ROS in this process will encourage researchers to develop new ROS induction strategies to enhance VAL-A production.

Thermophilic enzymes are in high demand for various applications due to their prolonged lifetimes and high reaction rates at elevated temperatures. In this work, an open reading frame TM0295, which encodes a putative transaldolase (TAL) from a hyper-thermophilic microorganism, Thermotoga maritima, was cloned and expressed in Escherichia coli. The enzyme activity of transaldolase at high temperatures (e.g., at 80 °C) was reported here for the first time. The recombinant T. maritima transaldolase was extremely thermostable, with a half-life time of 198 and 13.0 h at 60 °C and 80 °C, respectively. The estimated total turn-over number was 1.5 × 106 mol of product per mol of enzyme at 80 °C. This enzyme also exhibited high activities within a broad pH range of 6.0–9.0. This ultra-thermostable TAL with high activity shows great potential for use in such applications as the production of enzymatic biofuels production and the synthesis of high-value carbohydrates by cell-free synthetic pathway biotransformation.

Suppression subtractive hybridization (SSH) was preformed to investigate the differences of gene expression between the shaking culture mode and the liquid static culture mode which favors ganoderic acids production in Ganoderma lucidum. One novel gene preferentially expressed in liquid static culture was identified and analyzed. Its full length cDNA sequence and the 5′-flanking region were then obtained by rapid amplification of cDNA ends (RACE) and self-formed adaptor PCR (SEFA-PCR), respectively. Nucleotide sequence of the gene is not homologous to any of the known Ganoderma genes. The sequence analysis revealed that the open reading frame of this gene encodes a protein of 371 amino acids that has high homology with the mitogen-activated protein kinase (MAPK) of other five species-Postia (97%), Coprinopsis (91%), Neurospora (86%), Aspergillus (83%), and Saccharomyces (80%)-so that it can be defined as a G. lucidum MAPK gene (GenBank accession number: JF781125). Computer assisted analysis revealed that this new G. lucidum MAPK gene contains thirteen exons and twelve introns. The quantitative real-time RT-PCR (qRT-PCR) analysis showed that this new gene had a much higher expression level in liquid static culture than in traditional shaking culture. Results of this research established a good foundation for further study on the functions of the G. lucidum MAPK at the molecular level.

A new sesquiterpene (Z)-7-acetoxy-methyl-11-methyl-3-methylenedodeca-1,6,10-triene (AMDT) was isolated and identified from the methanol extract of the hairy root culture of Artemisia annua. The structure of AMDT was determined based on the analysis of spectroscopic data, notably of the 2D NMR spectra. This new compound showed cytotoxicity against human tumor cell lines 95-D and HeLa with IC50 values of 27.08 and 20.12 μmol/L, respectively.

Ansamitocin P-3 (AP-3) is an antitumor agent produced by Actinosynnema pretiosum with a market demand for large and cheap supply, but its productivity is yet low. This work investigated the effects of ammonium in medium on the productivity, enzyme activities, and gene transcription for AP-3 biosynthesis. As observed, AP-3 production was depressed by medium ammonium, although the dry cell weight and the consumption of total sugar and isobutanol were not influenced obviously. From the onset of AP-3 accumulation, isobutyrate accumulation showed a different behavior in medium with or without ammonium. Isobutanol dehydrogenase activity was enhanced during production phase in medium with ammonium, but valine dehydrogenase activity was not substantially changed. qRT-PCR analysis revealed that transcriptional levels of structure gene asm14, asm24, asm43, and asm19 were down-regulated by medium ammonium. The transcription of regulatory gene asm2, asm29, and asm31 were slightly up-regulated while that of asm39 was down-regulated by ammonium. The results indicated that inhibition of AP-3 production by ammonium might be related to the AP-3 ester side chain supply and the repression of gene transcription responsible for 3-amino-5-hydroxybenzoic acid and methoxymalonyl-ACP biosynthesis. The information is useful for future AP-3 productivity enhancement.

A bioreactor is the core of biological processes. To design an appropriate bioreactor system for a particular bioprocess, intensive studies on the biological system, such as cell growth, metabolism, genetic manipulation, and protein or other product expression are needed to understand the cells’ requirement on their physical and chemical environments. It is also necessary to control and optimize the bioreactor environment via operating variables in order to favor the desired functions of cells and achieve cost-effective large-scale manufacture. This article briefly describes fundamental design principles and new types of bioreactors such as centrifugal impeller and wave bioreactors. Bioreactor operation factors and modes including mixing, oxygen supply, shear force, fed-batch, and perfusion cultures are discussed. The trends in bioreactor engineering are also briefly shown.

Liquid static cultivation of Ganoderma lucidum was previously found to be very efficient for improving the production of its valuable antitumor compound ganoderic acid (GA) (Fang and Zhong in Biotechnol Prog 18:51–54, 2002). In this work, effects of oxygen concentration within the range of 21–100% (v/v) in the gaseous phase on the mycelia growth, GA production, and gene transcription of key enzymes for GA biosynthesis in liquid static cultures of G. lucidum were investigated. A high cell density of 29.8 ± 1.7 g/l DW and total GA production of 1427.2 ± 74.2 mg/l were obtained under an optimal gaseous O2 level of 80%. The expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, squalene synthase and lanosterol synthase genes of GA biosynthetic pathway as detected by quantitative real-time PCR was also affected by the gaseous oxygen concentration in the liquid static culture. H2O2 was generated as reactive oxygen species in response to high oxygen concentrations in the gas phase, and it seemed to be involved in the regulation of GA biosynthesis. The information obtained in this study provided an insight into the role of gaseous O2 in the GA production and it will be helpful for further enhancing its productivity.

Ganoderic acids (GAs), a kind of highly oxygenated lanostane-type triterpenoids, are important bioactive constituents of the famous medicinal mushroom Ganoderma lucidum. They have received wide attention in recent years due to extraordinarily pharmacological functions. Submerged fermentation of G. lucidum is viewed as a promising technology for production of GAs, and substantial efforts have been devoted to process development for enhancing GA production in the last decade. This article reviews recent publication about fermentative production of GAs and their potential applications, especially the progresses toward manipulation of fermentation conditions and bioprocessing strategies are summarized. The biosynthetic pathway of GAs is also outlined.

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 μM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 ± 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.

Two-stage culture was efficient in enhancing total ganoderic acid (GA) production by Ganoderma lucidum (Fang and Zhong, Biotechnol Prog 18:51–54, 2002). As different GAs have different bioactivities, it is critical to understand the kinetics of individual GA production during fermentation, but no related information is yet available. To understand the regulation of GA biosynthesis, investigation of the accumulation of intermediate (lanosterol) and by-product (ergosterol) and of the expression of three important biosynthetic genes was also conducted in liquid shaking and static cultures of G. lucidum. The results showed that the content of individual GAs increased rapidly in the liquid static culture, and their maximum value was 6- to 25-fold that of shaking culture while lanosterol content in the former was lower than the latter. The transcript of squalene synthase (SQS), lanosterol synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase in liquid static culture was 4.3-, 2.1-, and 1.9-fold that of the shaking culture, respectively. Higher GA content in liquid static culture was related to increased transcription of those genes especially SQS. The work is helpful to the production of individual GAs and provided an insight into why the liquid static culture was superior to the shaking culture in view of biosynthetic gene expression.

N-Acyl homoserine lactone (AHL) is a widely conserved quorum sensing (QS) signal of Gram-negative bacteria and has received attention in fighting against human diseases and environmental pollution. However, a method for quantifying AHL is lacking although it is urgently required for diagnosis and bioprocess manipulation. This work screened out an aromatics degrader Pseudomonas aeruginosa for biosensing system development, which produced a blue–green pigment regulated by the RhlI–RhlR QS system. By taking advantage of the recognition of N-butyryl homoserine lactone (BHL, the signal molecule of RhlI–RhlR QS system and an AHL) by the product of rhlR, a new whole-cell biosensor P. aeruginosa ΔrhlIR/pYC-rhlR (rhlI−rhlR++) was developed. It was constructed through abolishing its BHL production by in-frame deletion of rhlIR and over-expressing rhlR by introducing a multi-copy plasmid pYC-rhlR into ΔrhlIR. By using the pigment production which responded to exogenous BHL as biosensor output, BHL quantification in samples was simply done spectrophotometrically. Under optimum conditions, the calibration curve had the limit of detection (LOD), the 50% activation/effect concentration, the limit of quantification (LOQ), and the quantitative detection range of 1.3 nM, 2.77 ± 0.45 μM, 5.7 nM and 0.11–49.7 μM, respectively. The biosensor output was stable, culture samples could be stored 10 days under −20 °C, and this sensing system was resistant to interferences by toxic aromatic pollutants. It was successfully applied to environmental samples even without extraction. The new whole-cell biosensing system provided a simple, stable, toxic pollutants-tolerant, and cost-effective tool for quantitative investigation of the QS signals’ role in environmental processes.

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg1 + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg1 + Re in the ALR was increased from 42.5 ± 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 ± 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg1 + Re in the ALR reached 5.66 ± 0.38 mg L−1 d−1, which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb1) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.

Higher fungi with greater than 70 000 species are regarded as a rich source of various natural compounds including terpenoids, the production of which represents a wide range of interest in pharmaceutical and healthcare industries. This review summarizes the current knowledge of terpenoids synthesized by higher fungi, and highlights the current state-of-the-art regarding genetic manipulation of higher fungi. As the focus, this article will discuss the most recent approaches enabling native hosts and heterologous microbes to efficiently produce various terpenoids, especially with regard to the construction of ‘smart’ higher-fungus cell factories. The merits and demerits of heterologous versus native hosts as cell factories will also be debated.

The traditional Chinese medicinal mushroom, Ganoderma lucidum, has been used in Asia for several thousand years for the prevention and treatment of a variety of diseases, including cancer. In previous work, we purified ganoderic acid T (GA-T) from G. lucidum [28]. In the present study, we investigate the functions of GA-T in terms of its effects on invasion in vitro and metastasis in vivo. A trypan blue dye exclusion assay indicates that GA-T inhibits proliferation of HCT-116 cells, a human colon carcinoma cell line. Cell aggregation and adhesion assays show that GA-T promotes homotypic aggregation and simultaneously inhibits the adhesion of HCT-116 cells to the extracellular matrix (ECM) in a dose-dependent manner. Wound healing assays indicate that GA-T also inhibits the migration of HCT-116 cells in a dose-dependent manner, and it suppresses the migration of 95-D cells, a highly metastatic human lung tumor cell line, in a dose- and time-dependent manner. In addition, GA-T inhibits the nuclear translocation of nuclear factor-κB (NF-κB) and the degradation of inhibitor of κB-α (IκBα), which leads to down-regulated expression of matrix metalloproteinase-9 (MMP-9), inducible nitric oxide synthase (iNOS), and urokinase-type plasminogen activator (uPA). Animal and Lewis Lung Carcinoma (LLC) model experiments demonstrate that GA-T suppresses tumor growth and LLC metastasis and down-regulates MMP-2 and MMP-9 mRNA expression in vivo. Taken together, these results demonstrate that GA-T effectively inhibits cancer cell invasion in vitro and metastasis in vivo, and thus it may act as a potential drug for treating cancer.

•Large silk proteins of MaSp2 were designed and produced in E. coli.•Low induction temperature was useful in enhancing the protein production titer.•The production titer of MaSp2 obtained was the highest as reported.•The work may be helpful to cost-effective large-scale production of silk proteins.Spider dragline silk exhibits excellent mechanical properties that make it a promising protein polymer for industrial and biomedical applications. Since farming spiders is not feasible due to their highly territorial nature, recombinant production of dragline silk proteins in a foreign host has received great attention. However, their production titer remains low, because efficient expression of very large, highly repetitive, glycine-rich silk proteins is a challenge. This work demonstrates the design and high-level production of large dragline silk proteins of major ampullate spidroin 2 (MaSp2) in Escherichia coli by synthetic biology approach. The expression levels of MaSp2 with molecular weight of 28.3–256.5 kDa were significantly elevated by down-shifting the induction temperature. The beneficial effect was found to be at least partially attributed to the improved plasmid maintenance in the recombinant cells. Combination of induction temperature downshift with the glycyl-tRNA pool increase in E. coli led to enhanced biosynthesis of glycine-rich silk proteins. A high production titer of about 3.6 g l−1 of a 201.6-kDa MaSp2 protein was achieved in a 3-L fed-batch bioreactor, which was the highest as reported. The developed approach may be useful to cost-effective large-scale production of silk proteins.Download high-res image (180KB)Download full-size image

The function of p53 induced by ganoderic acids (GAs) in anti-invasion was unknown, although our previous work reported the inhibition of tumor invasion and metastais by Ganoderic acid T (GA-T). This work indicated that GA-T promoted cell aggregation, inhibited cell adhesion and surpressed cell migration with a dose-dependent manner in human colon tumor cell lines of HCT-116 p53+/+ and p53−/−. Furthermore, comparing the ratios of HCT-116 p53+/+ and p53−/− cells, p53 modified GA-T inhibition of migration and adhesion and GA-T promotion of cell aggregation, and p53 also modified GA-T inhibition of NF-κB nuclear translocation, IκBα degradation, and down-regulation of urokinase-type plaminogen activator (uPA), matrix metalloproteinase-2/9 (MMP-2/9), inducible nitric oxide synthase (iNOS/NOS2) protein expression and inducible nitric oxide (NO) production. The results indicated that p53 played an important role in anti-invasion of GA-T in human carcinoma cells. p53 may be an important target for GA-T inhibiting human carcinoma cells anti-invasion.

•Hyaluronic acid (HA) could promote the proliferation of hAMSCs.•HA treatment dose not affect the pluripotency of hAMSCs.•HA increases hAMSCs proliferation through activation of Wnt/β-catenin signaling.This study investigated the pro-proliferative effect of hyaluronic acid (HA) on human amniotic mesenchymal stem cells (hAMSCs) and the underlying mechanisms. Treatment with HA increased cell population growth in a dose- and time-dependent manner. Analyses by flow cytometry and immunocytochemistry revealed that HA did not change the cytophenotypes of hAMSCs. Additionally, the osteogenic, chondrogenic, and adipogenic differentiation capabilities of these hAMSCs were retained after HA treatment. Moreover, HA increased the mRNA expressions of wnt1, wnt3a, wnt8a, cyclin D1, Ki-67, and β-catenin as well as the protein level of β-catenin and cyclin D1 in hAMSCs; and the nuclear localization of β-catenin was also enhanced. Furthermore, the pro-proliferative effect of HA and up-regulated expression of Wnt/β-catenin pathway-associated proteins - wnt3a, β-catenin and cyclin D1 in hAMSCs were significantly inhibited upon pre-treatment with Wnt-C59, an inhibitor of the Wnt/β-catenin pathway. These results suggest that HA may positively regulate hAMSCs proliferation through regulation of the Wnt/β-catenin signaling pathway.

Validamycin A (VAL-A), produced by Streptomyces hygroscopicus, is an important anti-fungal agro-antibiotic. In this work, the effect of fermentation temperature on VAL-A biosynthesis by S. hygroscopicus 5008 was investigated between 28 °C and 42 °C, and an interesting threshold of temperature for VAL-A biosynthesis was found between 35 °C and 37 °C. At a relatively higher temperature, a much higher VAL-A productivity was obtained together with faster protein synthesis and sugar consumption. Transcriptional analysis of samples from early, middle and late stages of fermentation at various temperatures demonstrated that three operons, valABC, valKLMN and valG, for all eight necessary structure genes, were dramatically promoted when temperature reached the threshold. Activities of both glucose-6-phosphate dehydrogenase (G6PDH) of pentose-phosphate pathway and ValG of VAL-A biosynthesis were also enhanced at a higher cultivation temperature. The interesting temperature effect with a 2 °C threshold shift from 35 °C to 37 °C on the antibiotic biosynthesis was understood to be related to the gene transcriptional levels and key enzyme activities.

The effect of ferrous sulfate addition on production of cordycepin (3′-deoxyadenosine), a useful bioactive product with some pharmacological activities, was investigated in submerged cultures of Cordyceps militaris in shake flasks. The results showed that the optimal addition condition was on day 0 with 1 g/L of ferrous sulfate, and the maximal amount of cordycepin reached 596.59 ± 85.5 mg/L, about 70% higher than the control without ferrous sulfate addition. Meanwhile, the consumption of inosine 5′-monophosphate (IMP), a potential precursor of cordycepin, was decreased rapidly. Transcription levels of important genes encoding adenylosuccinate synthetase (purA), IMP cyclohydrolase (purH) and IMP dehydrogenase (guaB) in the purine nucleotide biosynthetic pathway were also studied. Compared to the control, the transcription level of purA was significantly up-regulated in ferrous sulfate supplemented cultures, while purH and guaB were slightly down-regulated. This work indicated that ferrous sulfate addition was a simple and useful strategy for improving cordycepin production, and the related information might be helpful for further manipulation and understanding of the cordycepin biosynthesis.Highlights► Ferrous sulfate addition was found useful to enhance the cordycepin production. ► The related gene expression was studied for better manipulation of the cordycepin biosynthesis. ► The relationship between IMP content and cordycepin production was studied. ► purA gene expression was found important in cordycepin synthesis.

In this work, the effect of N,N′-dicyclohexylcarbodiimide (DCCD) on ginsenoside biosynthesis in suspension cultures of Panax ginseng cells was investigated. The optimal concentration and timing of DCCD addition were found to be 10 μM and on day 4 of cultivation. Under this condition, the maximal content of total ginsenosides increased to 3.0-fold that of untreated control, and the contents of Rg-group (Rg1 and Re) ginsenosides and Rb1 were 2.5- and 8.9-fold higher, respectively, which coincided with elevated activities of protopanaxatriol biosynthetic enzyme protopanaxadiol 6-hydroxylase and UDPG-ginsenoside Rd glucosyltransferase that converts Rd to Rb1. In addition, DCCD treatment induced the activity of defense response enzyme, phenylalanine ammonia lyase. To gain a better understanding of the molecular processes underlying the elicitation, we examined nitric oxide (NO) content and expression levels of the triterpene biosynthetic genes encoding squalene synthase (sqs), squalene epoxidase (se), and dammarenediol-II synthase (ds). It was found that DCCD up-regulated NO generation and transcription levels of sqs, se and ds. Interestingly, these effects of DCCD were compromised by an NO biosynthetic inhibitor, while an NO donor alone recapitulated the elicitation effect of DCCD on ginsenoside biosynthesis. These results suggest that DCCD may induce the ginsenoside biosynthesis via NO signaling in the P. ginseng cells. The information obtained might also be helpful to hyperproduction of valuable secondary metabolites in other plant cell cultures.Highlights► N,N′-Dicyclohexylcarbodiimide induced ginsenoside biosynthesis in P. ginseng. ► Elicitor increased the enzyme activities and transcription levels of biosynthetic genes. ► The signaling molecule, nitric oxide, plays an important role in induction. ► This approach may be helpful in other plant cell culture systems.

Polyhexamethylene guanidine hydrochloride (PHMG) possesses great potential in the development of covalently bound permanent sterile-surface materials for hospital infection control. This study aimed at evaluating the extensive activity of PHMG and its three novel analogs, including polybutamethylene guanidine hydrochloride, polyoctamethylene guanidine hydrochloride (POMG) and poly(m-xylylene guanidine hydrochloride), against 370 clinical strains, especially 96 isolates of which were antibiotics-resistant. Their in vitro antimicrobial activities were determined by testing the minimal inhibitory concentration (MIC) and time-killing curves. POMG, the novel oligoguanidine had significantly lower MIC values (0.5–16 mg/L) against 370 antibiotics-susceptible and -resistant clinical strains compared to PHMG (1–64 mg/L) and chlorhexidine digluconate (2–64 mg/L). Interestingly, POMG displayed excellent activity against meticillin resistant-Staphylococcus aureus (1–8 mg/L) and -coagulase-negative staphylococci (1–2 mg/L), vancomycin resistant Enterococcus faecium (2–4 mg/L), multidrug resistant Pseudomonas aeruginosa (8–16 mg/L), ceftazidime resistant-Citrobacter spp. (1–4 mg/L) and -Enterobacter spp. (2–4 mg/L). PHMG was especially efficient against methicillin resistant-S. aureus and -coagulase-negative staphylococci (1–8 mg/L). The presented extensive activity of POMG and PHMG against antibiotic-resistant bacteria provides encouraging reference information for the using and further development of cationic guanidine-based polymers in the biomedical material field.Download full-size imageHighlights► Three novel analogs of polyhexamethylene guanidine hydrochloride were synthesized. ► One new analog with evidently improved antimicrobial activity was demonstrated. ► Guanidine polymer analogs exhibited extensive activity against resistant bacteria. ► Cationic guanidine polymers were promising as materials against resistant bacteria.