Genome-scale metabolic network models for industrial microorganisms metabolic engineering: Current advances and future prospects.

The construction of high-performance microbial cell factories (MCFs) is the centerpiece of biomanufacturing. However, the complex metabolic regulatory network of microorganisms poses great challenges for the efficient design and construction of MCFs. The genome-scale metabolic network models (GSMs) can systematically simulate the metabolic regulation process of microorganisms in silico, providing effective guidance for the rapid design and construction of MCFs. In this review, we summarized the development status of 16 important industrial microbial GSMs, and further outline the technologies or methods that continuously promote high-quality GSMs construction from five aspects: I) Databases and modeling tools facilitate GSMs reconstruction; II) evolving gap-filling technologies; III) constraint-based model reconstruction; IV) advances in algorithms; and V) developed visualization tools. In addition, we also summarized the applications of GSMs in guiding metabolic engineering from four aspects: I) exploring and explaining metabolic features; II) predicting the effects of genetic perturbations on metabolism; III) predicting the optimal phenotype; IV) guiding cell factories construction in practical experiment. Finally, we discussed the development of GSMs, aiming to provide a reference for efficiently reconstructing GSMs and guiding metabolic engineering.

Identification of Emerging Industrial Biotechnology Chassis Vibrio natriegens as a Novel High Salt-Tolerant and Feedstock Flexibility Electroactive Microorganism for Microbial Fuel Cell.

The development of MFC using electroactive industrial microorganisms has seen a surge of interest because of the co-generation for bioproduct and electricity production. Vibrio natriegens as a promising next-generation industrial microorganism chassis and its application for microbial fuel cells (MFC) was first studied. Mediated electron transfer was found in V. natriegens MFC (VMFC), but V. natriegens cannot secrete sufficient electron mediators to transfer electrons to the anode. All seven electron mediators supplemented are capable of improving the electronic transfer efficiency of VMFC. The media and carbon sources switching study reveals that VMFCs have excellent bioelectricity generation performance with feedstock flexibility and high salt-tolerance. Among them, 1% glycerol as the sole carbon source produced the highest power density of 111.9 ± 6.7 mW/cm2. The insight of the endogenous electronic mediators found that phenazine-1-carboxamide, phenazine-1-carboxylic acid, and 1-hydroxyphenazine are synthesized by V. natriegens via the shikimate pathway and the phenazine synthesis and modification pathways. This work provides the first proof for emerging industrial biotechnology chassis V. natriegens as a novel high salt-tolerant and feedstock flexibility electroactive microorganism for MFC, and giving insight into the endogenous electron mediator biosynthesis of VMFC, paving the way for the application of V. natriegens in MFC and even microbial electrofermentation (EF).

Achieving "Non-Foaming" Rhamnolipid Production and Productivity Rebounds of Pseudomonas aeruginosa under Weakly Acidic Fermentation.

The rhamnolipid production of Pseudomonas aeruginosa has been impeded by its severe foaming; overcoming the bottleneck of foaming has become the most urgent requirement for rhamnolipid production in recent decades. In this study, we performed rhamnolipid fermentation under weakly acidic conditions to address this bottleneck. The results showed that the foaming behavior of rhamnolipid fermentation broths was pH-dependent with the foaming ability decreasing from 162.8% to 28.6% from pH 8 to 4. The "non-foaming" rhamnolipid fermentation can be realized at pH 5.5, but the biosynthesis of rhamnolipids was significantly inhibited. Further, rhamnolipid yield rebounded from 8.1 g/L to 15.4 g/L after ultraviolet and ethyl methanesulfonate compound mutagenesis. The mechanism study showed that the species changes of rhamnolipid homologs did not affect the foaming behavior of the fermentation but had a slight effect on the bioactivity of rhamnolipids. At pH 8.0 to 5.0, increased surface tension, decreased viscosity and zeta potential, and aggregation of rhamnolipid molecules contributed to the "non-foaming" rhamnolipid fermentation. This study provides a promising avenue for the "non-foaming" rhamnolipid fermentation and elucidates the mechanisms involved, facilitating the understanding of pH-associated foaming behavior and developing a more efficient strategy for achieving rhamnolipid production.

Effects of shining pondweed (Potamogeton lucens) on bacterial communities in water and rhizosphere sediments in Nansi Lake, China.

Submerged macrophytes and microbial communities are important parts of lake ecosystems. In this study, the bacterial community composition in rhizosphere sediments and water from areas cultivated with (PL) and without (CK) shining pondweed (Potamogeton lucens Linn.) was investigated to determine the effects of P. lucens Linn. on the structure of the bacterial communities in Nansi Lake, China. Molecular techniques, including Illumina MiSeq and qPCR targeting of the 16S rRNA gene, were used to analyze the composition and abundance of the bacterial community. We found that bacterial alpha diversity was higher in PL water than in CK water, and the opposite trend was observed in sediment. In addition, 16S rRNA gene copy number in sediment was lower in PL than in CK. We found 30 (e.g., Desulfatiglans) and 29 (e.g., Limnohabitans) significantly different genera in sediment and water, respectively. P. lucens Linn. can change chemical properties in sediment and water and thereby affect the bacterial community. At the genus level, members of bacterial community clustered according to source (water/sediment) and area (PL/CK). Our study demonstrated that submerged macrophytes can affect the bacterial community composition in both sediment and water, suggesting that submerged macrophytes affect the transportation and cycling of nutrients in lake ecosystems.

Construction of an exosome-functionalized graphene oxide based composite bionic smart drug delivery system and its anticancer activity.

Graphene oxide has covalently modified by chito oligosaccharides andγ-polyglutamic acid to form GO-CO-γ-PGA, which exhibits excellent performance as a drug delivery carrier, but this carrier did not have the ability to actively target. In this study, the targeting property of breast cancer tumor cell exosomes was exploited to give GO-CO-γ-PGA the ability to target breast tumor cells (MDA-MB-231), and the drug mitoxantrone (MIT) was loaded to finally form EXO-GO-CO-γ-PGA-MIT with an encapsulation efficiency of 73.02%. The pH response of EXO-GO-CO-γ-PGA showed a maximum cumulative release rate of 56.59% (pH 5.0, 120 h) and 6.73% (pH 7.4, 120 h) for MIT at different pH conditions.In vitrocellular assays showed that EXO-GO-CO-γ-PGA-MIT was more potent in killing MDA-MB-231 cells due to its targeting ability and had a significantly higher pro-apoptotic capacity compared to GO-CO-γ-PGA-MIT. The results showed that this bionic nano-intelligent drug delivery system has good drug slow release function and it can increase the local drug concentration of tumor and enhance the pro-apoptotic ability of MIT, so this newly synthesized bionic drug delivery carriers (EXO-GO-CO-γ-PGA-MIT) has potential application in breast cancer treatment.

Foaming of rhamnolipids fermentation: impact factors and fermentation strategies.

Rhamnolipids have recently attracted considerable attentions because of their excellent biosurfactant performance and potential applications in agriculture, environment, biomedicine, etc., but severe foaming causes the high cost of production, restraining their commercial production and applications. To reduce or eliminate the foaming, numerous explorations have been focused on foaming factors and fermentation strategies, but a systematic summary and discussion are still lacking. Additionally, although these studies have not broken through the bottleneck of foaming, they are conducive to understanding the foaming mechanism and developing more effective rhamnolipids production strategies. Therefore, this review focuses on the effects of fermentation components and control conditions on foaming behavior and fermentation strategies responded to the severe foaming in rhamnolipids fermentation and systematically summarizes 6 impact factors and 9 fermentation strategies. Furthermore, the potentialities of 9 fermentation strategies for large-scale production are discussed and some further strategies are suggested. We hope this review can further facilitate the understanding of foaming factors and fermentation strategies as well as conducive to developing the more effective large-scale production strategies to accelerate the commercial production process of rhamnolipids.

A Targeted Nano Drug Delivery System of AS1411 Functionalized Graphene Oxide Based Composites.

A novel method for the preparation of antitumor drug vehicles has been optimized. Biological materials of chitosan oligosaccharide (CO) and γ-polyglutamic acid (γ-PGA) have previously been employed as modifiers to covalently modify graphene oxide (GO), which in turn loaded doxorubicin (DOX) to obtain a nano drug delivery systems of graphene oxide based composites (GO-CO-γ-PGA-DOX). The system was not equipped with the ability of initiative targeting, thus resulting into toxicity and side effects on normal tissues or organs. In order to further improve the targeting property of the system, the nucleic acid aptamer NH2 -AS1411 (APT) of targeted nucleolin (C23) was used to conjugate on GO-CO-γ-PGA to yield the targeted nano drug delivery system APT-GO-CO-γ-PGA. The structure, composition, dispersion, particle size and morphology properties of the synthesized complex have been studied using multiple characterization methods. Drug loading and release profile data showed that APT-GO-CO-γ-PGA is provided with high drug loading capacity and is capable of controlled and sustained release of DOX. Cell experimental results indicated that since C23 was overexpressed on the surface of Hela cells but not on the surface of Beas-2B cells, APT-GO-CO-γ-PGA-DOX can target Hela cells and make increase toxicity to Hela cells than Beas-2B cells, and the IC50 value of APT-GO-CO-γ-PGA-DOX was 3.23±0.04 µg/mL. All results proved that APT-GO-CO-γ-PGA can deliver antitumor drugs in a targeted manner, and achieve the effect of reducing poison, which indicated that the targeted carrier exhibits a broad application prospect in the field of biomedicine.

Corynebacterium glutamicum Mycoredoxin 3 protects against multiple oxidative stresses and displays thioredoxin-like activity.

Glutaredoxins (Grxs) and thioredoxins (Trxs) play a critical role in resistance to oxidative conditions. However, physiological and biochemical roles of Mycoredoxin 3 (Mrx3) that shared a high amino acid sequence similarity to Grxs remain unknown in Corynebacterium glutamicum. Here we showed that mrx3 deletion strains of C. glutamicum was involved in the protection against oxidative stress. Recombinant Mrx3 not only catalytically reduced the disulfide bonds in ribonucleotide reductase (RNR), insulin and 5,5'-dithiobis-(2-nitro-benzoicacid) (DTNB), but also reduced the mixed disulphides between mycothiol (MSH) and substrate, which was exclusively linked to the thioredoxin reductase (TrxR) electron transfer pathway by a dithiol mechanism. Site-directed mutagenesis confirmed that the conserved Cys17 and Cys20 in Mrx3 were necessary to maintain its activity. The mrx3 deletion mutant showed decreased resistance to various stress, and these sensitive phenotypes were almost fully restored in the complementary strain. The physiological roles of Mrx3 in resistance to various stress were further supported by the induced expression of mrx3 under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. Thus, we presented the first evidence that Mrx3 protected against various oxidative stresses by acting as a disulfide oxidoreductase behaving like Trx.

Molecular mechanisms of Mycoredoxin-1 in resistance to oxidative stress in Corynebacterium glutamicum.

Glutaredoxins (Grxs) with Cys-Pro-Phe (Tyr)-Cys motif and a thioredoxin fold structure play an important role in the anti-oxidant system of bacteria by catalyzing a variety of thiol-disulfide exchange reactions with a 2-Cys mechanism or a 1-Cys mechanism. However, the catalytic and physiological mechanism of Corynebacterium glutamicum Mycoredoxin 1 (Mrx1) that shares a high amino acid sequence similarity to Grxs has not been fully elucidated. Here, we report that Mrx1 has a protective function against various adverse conditions, and the decrease of cell viability to various stress conditions by deletion of the Mrx1 in C. glutamicum was confirmed in the mrx1 mutant. The physiological roles of Mrx1 in defence to oxidative stress were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. As well as reducing mycothiol (MSH) mixed disulfide bonds via a 1-Cys mechanism, C. glutamicum Mrx1 catalytically reduced the disulfides in the Ib RNR, insulin and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by exclusively linking the MSH/Mtr (mycothiol disulfide reductase)/NADPH electron pathway via a 2-Cys mechanism. Thus, we present the first evidence that the Mrx1 is able to protect against the damaging effects of various exogenous stresses by acting as a disulfide oxidoreductase, thereby giving a new insight in how C. glutamicum survives oxidative stressful conditions.

Optimization and scale-up of the production of rhamnolipid by Pseudomonas aeruginosa in solid-state fermentation using high-density polyurethane foam as an inert support.

To be competitive with common synthetic surfactants, the cost of production of rhamnolipid must be minimized by the fermentation process of non-foaming and low impurities. Herein, a novel solid-state fermentation process was developed for production of rhamnolipid by Pseudomonas aeruginosa SKY. The results were shown that high-density polyurethane foam is a satisfactory alternative to agro-industrial by-products for SSF of rhamnolipid. Palm oil and NaNO3 were promising carbon source and nitrogen source, respectively. Response surface methodology was employed to enhance the production of rhamnolipid. Palm oil, NaNO3 and liquid-to-solid ratios were significant factors. The optimal medium was developed as: 73.6 g/l palm oil; 3.0 g/l g NaNO3; 1.1 g NaCl; 1.1 g KCl; 3.4 g KH2PO4; 4.4 g K2HPO4; 0.5 g MgSO4·7H2O and 37.2 liquid-to-solid ratios. An overall 1.39-fold increase in rhamnolipid production was achieved in the optimized medium as compared with the unoptimized basal medium. Air pressure pulsation solid-state fermentation (APP-SSF) was applied to the experiment of scale-up for improving transfer efficiency of heat and mass. The yield of rhamnolipid reached 39.8 g/l in a 30 l APP-SSF fermenter. The crude extract of rhamnolipid lowered the surface tension of water to 28 mN/m and kept the critical micelle concentration at 50 mg/l. The work revealed the SSF with HPUF as an inert support was a promising fermentation system that could effectively produce rhamnolipid with low impurities, high productivity and low cost of production at a large scale.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance.

MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to ß-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Characterization of Xi-class mycothiol S-transferase from Corynebacterium glutamicum and its protective effects in oxidative stress.

BACKGROUND: Oxidative stress caused by inevitable hostile conditions during fermentative process was the most serious threat to the survival of the well-known industrial microorganism Corynebacterium glutamicum. To survive, C. glutamicum developed several antioxidant defenses including millimolar concentrations of mycothiol (MSH) and protective enzymes. Glutathione (GSH) S-transferases (GSTs) with essentially defensive role in oxidative stress have been well defined in numerous microorganisms, while their physiological and biochemical functions remained elusive in C. glutamicum thus far. RESULTS: In the present study, we described protein NCgl1216 belonging to a novel MSH S-transferase Xi class (MstX), considered as the equivalent of GST Xi class (GSTX). MstX had a characteristic conserved catalytic motif (Cys-Pro-Trp-Ala, C-P-W-A). MstX was active as thiol transferase, dehydroascorbate reductase, mycothiolyl-hydroquinone reductase and MSH peroxidase, while it showed null activity toward canonical GSTs substrate as 1-chloro-2,4-dinitrobenzene (CDNB) and GST Omega's specific substance glutathionyl-acetophenones, indicating MstX had some biochemical characteristics related with mycoredoxin (Mrx). Site-directed mutagenesis showed that, among the two cysteine residues of the molecule, only the residue at position 67 was required for the activity. Moreover, the residues adjacent to the active Cys67 were also important for activity. These results indicated that the thiol transferase of MstX operated through a monothiol mechanism. In addition, we found MstX played important role in various stress resistance. The lack of C. glutamicum mstX gene resulted in significant growth inhibition and increased sensitivity under adverse stress condition. The mstX expression was induced by stress. CONCLUSION: Corynebacterium glutamicum MstX might be critically involved in response to oxidative conditions, thereby giving new insight in how C. glutamicum survived oxidative stressful conditions.

OsmC in Corynebacterium glutamicum was a thiol-dependent organic hydroperoxide reductase.

Bacterial antioxidants play a vital role in the detoxification of exogenous peroxides. Several antioxidant defenses including low-molecular-weight thiols (LMWTs) and protective enzymes were developed to help the bacterium withstand the adverse stress. Although osmotically induced bacterial protein C (OsmC), classified as the organic hydroperoxide reductase (Ohr)/OsmC superfamily, has been demonstrated in some mycobacterial species, including M. tuberculosis and M. smegmatis, its physiological and biochemical functions in C. glutamicum remained elusive. Here we found the lack of C. glutamicum osmC gene resulted in decreased cell viability and increased intracellular reactive oxygen species accumulation under organic hydroperoxides (OHPs) stress conditions. The osmC expression was induced in the multiple antibiotic resistance regulator MarR-dependent manner by OHPs, and not by other oxidants or osmotic stress. Peroxide reductase activity showed that OsmC had a narrow range of substrates-only degrading OHPs, and detoxified OHPs mainly by linking the alkyl hydroperoxide reductase (AhpD) system (AhpD/dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide acyltransferase (SucB)). Site-directed mutagenesis confirmed Cys48 was the peroxidatic cysteine, while Cys114 was the resolving Cys residue that formed an intramolecular disulfide bond with oxidized Cys48. Therefore, C. glutamicum OsmC was a thiol-dependent OHP reductase and played important role of protection against OHPs together with Ohr.

Structural characterization and immunomodulating activities of polysaccharides from a newly collected wild Morchella sextelata.

A purified polysaccharide was acquired from a newly collected wild Morchella. The strain identification initially showed that the strain was Morchella sextelata. This study aimed to investigate the structural features and immunomodulating activities of the polysaccharide. Polysaccharide extracted from mycelia was purified by DEAE-cellulose chromatography and Sephadex G-25 size-exclusion chromatography in sequence. The main fraction of polysaccharide (MSP-II) was obtained during purification process. High Performance Liquid Chromatography (HPLC) analysis revealed that MSP-II was composed of Glc, Ara, Gal, Man, Rha, Fuc, GalUA and GluUA in ratio of 34.95:8.7:9.55:4.55:5.0:1.45:12.7:7.65. The structure of MSP-II was furtherly analyzed by FT-IR spectrum and 1H and 13C NMR spectroscopy, the results showed that MSP contained ß-glycosidic bonds as well as α-glycosidic linkages. In vitro tests proved that MSP-II could not only promote the proliferation and phagocytosis of RAW264.7 cells, but also induce the section of nitric oxide (NO) of macrophages. Consequently, the polysaccharide has a potent immunomodulatory activity by stimulating macrophages and can be considered as a novel potential immunopotentiator in medical and food industries.

Enhanced stability of manganese superoxide dismutase by amino acid replacement designed via molecular dynamics simulation.

In order to improve manganese-SOD stability, three mutations were constructed via site-directed mutagenesis, and the root mean square fluctuation (RMSF) and root mean square deviation (RMSD) were used as stability assessment indexes. The amino acids of V140, E155 and E215 from wild-type mouse Mn-SOD was replaced to L140, W155 and W215, and a recombinant plasmid containing DNA segment coding wild-type and mutant Mn-SOD protein was transformed into Escherichia coli BL21 for expression. The highest enzyme activity of the mutations-MnSOD was 2050 U/mg. In addition, the recombinant protein, TM-MnSODV140L, E155W, E215W exhibited higher working temperature and improved stability compared with the wild-type Mn-SOD. Furthermore, CD spectrum analysis of the improved mutants and wild-type enzyme showed that there was no significant change in their secondary structures. This study not only expands the scope of the application of enzymes, but also helps us understand the relationship between protein structure and function.

OhsR acts as an organic peroxide-sensing transcriptional activator using an S-mycothiolation mechanism in Corynebacterium glutamicum.

BACKGROUND: Corynebacterium glutamicum is a well-known producer of various L-amino acids in industry. During the fermenting process, C. glutamicum unavoidably encounters oxidative stress due to a specific reactive oxygen species (ROS) produced by consistent adverse conditions. To combat the ROS, C. glutamicum has developed many common disulfide bond-based regulatory devices to control a specific set of antioxidant genes. However, nothing is known about the mixed disulfide between the protein thiol groups and the mycothiol (MSH) (S-mycothiolation)-based sensor. In addition, no OhrR (organic hydroperoxide resistance regulator) homologs and none of the organic hydroperoxide reductase (Ohr) sensors have been described in the alkyl hydroperoxide reductase CF-missing C. glutamicum, while organic hydroperoxides (OHPs)-specific Ohr was a core detoxification system. RESULTS: In this study, we showed that the C. glutamicum OhsR acted as an OHPs sensor that activated ohr expression. OhsR conferred resistance to cumene hydroperoxide (CHP) and t-butyl hydroperoxide but not H2O2, hypochlorous acid, and diamide; this outcome was substantiated by the fact that the ohsR-deficient mutant was sensitive to OHPs but not inorganic peroxides. The DNA binding activity of OhsR was specifically activated by CHP. Mutational analysis of the two cysteines (Cys125 and Cys261) showed that Cys125 was primarily responsible for the activation of DNA binding. The oxidation of Cys125 produced a sulfenic acid (C125-SOH) that subsequently reacted with MSH to generate S-mycothiolation that was required to activate the ohr expression. Therefore, OhsR regulated the ohr expression using an S-mycothiolation mechanism in vivo. CONCLUSION: This is the first report demonstrating that the regulatory OhsR specifically sensed OHPs stress and responded to it by activating a specific ohr gene under its control using an S-mycothiolated mechanism.

[Construction and optimization of Escherichia coli for producing rhamnolipid biosurfactant].

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.

Rhamnolipid production, characterization and fermentation scale-up by Pseudomonas aeruginosa with plant oils.

OBJECTIVES: To be competitive with common chemical surfactants, the cost of rhamnolipid production must be minimized by selecting suitable substrates and optimizing the fermentation process. RESULTS: With different plant oils as substrates, Pseudomonas aeruginosa TIB-R02 can produce rhamnolipids with different structural characteristics that were confirmed by HPLC/MS analysis. Different rhamnolipids had different performances in interfacial tension. The production of rhamnolipid was greatly enhanced by fermentation optimization with palm oil as substrate. A fermentation-defoaming tandem system was developed to resolve the problems of foaming and medium overflow during scale-up. Finally, the titer of rhamnolipid reached 60 g/l and the yield reached 80% in a 300 l fermentation-defoaming tandem system. CONCLUSIONS: The work reveals the potential for producing high-performance rhamnolipids from renewable resources on a large scale.

[Expression of proteins in p53 (p14ARF-mdm2-p53-p21WAF/CIP1) pathway and their significance in exocrine pancreatic carcinoma].

OBJECTIVE: To investigate the expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins and their relationship in exocrine pancreatic carcinoma. METHODS: Specimens of pancreatic carcinoma, adjacent non-neoplastic pancreatic tissue and pancreatic benign lesions were examined for p14(ARF), p53, mdm2 and p21(WAF/CIP1) protein expression by tissue microarray technique and immunohistochemistry. RESULTS: The expression of p14(ARF), p53, mdm2 and p21(WAF/CIP1) proteins in pancreatic carcinoma were 35.3% (59/167), 57.5% (96/101), 64.1% (107/167) and 39.5% (66/167) respectively. The expression of p53 proteins was increased in pancreatic carcinoma (P < 0.01), while the expression of p14(ARF) and p21(WAF/CIP1) proteins was reduced (P < 0.05), as compared with that in non-neoplastic pancreatic tissue. p21(WAF/CIP1) protein expression in pancreatic carcinoma significantly correlated with the age of patients and perineural invasion (P < 0.05). p53 protein expression correlated significantly with tumor differentiation, lymph node metastasis and perineural invasion (P < 0.05). Mdm2 protein expression correlated significantly with tumor differentiation (P < 0.05), while p14(ARF) protein expression correlated significantly with the age of patients and metastasis (P < 0.05). There was also statistic correlation between the expression of these four genes (P < 0.05). CONCLUSIONS: Overexpression of p53 and mdm2 and loss of p14(ARF) and p21(WAF/CIP1) expression may contribute to the pathogenesis of pancreatic carcinoma. These proteins play a critical role in cell cycle arrest and apoptosis after DNA damage through p14(ARF)-mdm2-p53-p21(WAF/CIP1) pathway. Detection of p53 and Mdm2 protein overexpression may be useful in evaluation of the aggressiveness of pancreatic carcinoma.

[Fine needle aspiration cytology of eyelid sebaceous gland carcinoma and its differential diagnosis].

OBJECTIVE: To investigate the fine needle aspiration cytology (FNAC) features and differential diagnosis of eyelid sebaceous gland carcinoma. METHODS: Four cases of eyelid sebaceous gland carcinoma diagnosed by FNAC were reported and confirmed by biopsy. Three of the cases were in early stages with tumor sizes smaller than 10 mm in diameter and without metastasis. The smears were stained by routine H & E and SudanIII methods. The cytologic findings were described and compared to corresponding histological features, and moreover, compared to chalazion, pilomatrixoma and eyelid basal cell carcinoma. RESULTS: Neither hemorrhage nor infection were found after the examination. Abundant cells were observed in the sebaceous carcinoma FNAC smears. Two types of tumor cells were found: one showed tumor cells differentiating toward sebaceous gland, with large pale cells and vacuolated cytoplasm, the other demonstrated poorly-differentiated cell with dark and irregular nuclei. Numerous vacuoles with inequality of size were found in cytoplasm or in background in all four cases, and the SudanIII stain showed that these vacuoles contained lipid. Some smears demonstrated cells with basaloid, fusiform or squamous features, corresponding to various histopathological types. In contrast, smears of chalazion displayed inflammatory granuloma, containing several types of inflammatory cells without malignant cells. Smears of pilomatrixoma were cellular with three cell populations, which included bland sheets of basaloid cells, nucleated basophilic cells and anucleated keratinized "ghost cells", along with calcific debris. The smears of basal cell carcinoma were typically less cellular, more tightly cohesive and had smaller clusters of uniform hyperchromatic basaloid cells without vacuolization in cytoplasm or background. Overall, the cytological features of eyelid sebaceous carcinoma were distinct from those of chalazion, pilomatricoma and basal cell carcinoma. CONCLUSIONS: FNAC is a safe and effective approach for the diagnosis of eyelid sebaceous carcinoma and lipid stain is useful in differential diagnosis. The application of FNAC may be important in reaching an early diagnosis and initial treatment of eyelid nodule.