Heterologous expression of pediocin/papA in Bacillus subtilis.

Listeria monocytogenes is a food-borne pathogen. Pediocin is a group IIα bacteriocin with anti-listeria activity that is naturally produced by Pediococcus acidilactic and Lactobacillus plantarum. The pedA/papA gene encodes pediocin/plantaricin. In native hosts, the expression and secretion of active PedA/PapA protein rely on the accessory protein PedC/PapC and ABC transporter PedD/PapD on the same operon. The excretion machines were also necessary for pediocin protein expression in heterologous hosts of E. coli, Lactobacillus lactis, and Corynebacterium glutamicum. In this study, two vectors carrying the codon sequence of the mature PapA peptide were constructed, one with and one without a His tag. Both fragments were inserted into the plasmid pHT43 and transformed into Bacillus subtilis WB800N. The strains were induced with IPTG to secrete the fused proteins PA1 and PA2. Supernatants from both recombinant strains can inhibit Listeria monocytogenes ATCC54003 directly. The fused protein possesses inhibition activity as a whole dispense with removal of the leading peptide. This is the first report of active pediocin/PapA expression without the assistance of PedCD/PapCD in heterogeneous hosts. In addition, the PA1 protein can be purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography.

Improvement of butanol production by the development and co-culture of C. acetobutylicum TSH1 and B. cereus TSH2.

Butanol fermentation comprises two successive and distinct stages, namely acidogenesis and solventogenesis. The current lack of clarity regarding the underlying metabolic regulation of fermentation impedes improvements in biobutanol production. Here, a proteomics study was performed in the acidogenesis phase, the lowest pH point (transition point), and the solventogenesis phase in the butanol-producing symbiotic system TSH06. Forty-two Clostridium acetobutylicum proteins demonstrated differential expression levels at different stages. The protein level of butanol dehydrogenase increased in the solventogenesis phase, which was in accordance with the trend of butanol concentration. Stress proteins were upregulated either at the transition point or in the solventogenesis phase. The cell division-related protein Maf was upregulated at the transition point. We disrupted the maf gene in C. acetobutylicum TSH1, and Bacillus cereus TSH2 was added to form a new symbiotic system. TSH06△maf produced 13.9 ± 1.0 g/L butanol, which was higher than that of TSH06 (12.3 ± 0.9 g/L). Butanol was furtherly improved in fermentation at variable temperature with neutral red addition for both TSH06 and TSH06△maf. The butanol titer of the maf deletion strain was higher than that of the wild type, although the exact mechanism remains to be determined.

ARTP mutation and genome shuffling of ABE fermentation symbiotic system for improvement of butanol production.

Butanol is an ideal renewable biofuel which possesses superior fuel properties. Previously, butanol-producing symbiotic system TSH06 was isolated in our lab, with microoxygen tolerance ability. To boost butanol yield for large-scale industrial production, TSH06 was used as parental strain and subjected to atmospheric and room temperature plasma (ARTP) and four rounds of genome shuffling (GS). ARTP mutant and GS strain were co-cultured with facultative anaerobic Bacillus cereus TSH2 to form a symbiotic system with microoxygen tolerance, which was then subjected to fermentation. Relative messenger RNA (mRNA) level of key enzyme gene was measured by real-time PCR. The highest butanol titer of TS4-30 reached 15.63 g/L, which was 34% higher than TSH06 (12.19 g/L). Compared with parental strain, mRNA of acid-forming gene in TS4-30 decreased in acidogenesis phase, while solvent-forming gene increased in solventogenesis phase. This gene expression pattern was consistent with high butanol yield and low acid level in TS4-30. In summary, symbiotic system TS4-30 was obtained with butanol titer improvement and microoxygen tolerance.

Interactions between Bacillus cereus CGMCC 1.895 and Clostridium beijerinckii NCIMB 8052 in coculture for butanol production under nonanaerobic conditions.

Low oxygen tolerance and substrate restriction continues to hamper the process of biobutanol industrialization. In this work, butanol fermentation with cocultures of Bacillus cereus China General Microbiological Culture Collection Center (CGMCC) 1.895 and Clostridium beijerinckii NCIMB 8052 under nonanaerobic conditions was investigated, and the interactions between these two strains were examined. The addition of B. cereus CGMCC 1.895 resulted in higher oxygen tolerance and a wider range of substrate utilization, compared with the pure culture of C. beijerinckii NCIMB 8052. Butanol concentration reached 10.49 g/L with an optimized inoculation size of 90% under nonanaerobic conditions, and this concentration was close to that of pure C. beijerinckii NCIMB 8052 culture under anaerobic conditions. Dynamic relative abundance analysis demonstrated that the ratio of C. beijerinckii NCIMB 8052 accounted for nearly 99% of the cocultured cells. Furthermore, the substrate utilization range was expanded, allowing the use of corn mash for butanol production. The final concentration of butanol and total solvents was 6.78 and 10.52 g/L, respectively. Coculture also was performed successfully in a 5-L fermenter and 8.75 g/L butanol was obtained. Dynamic dissolved oxygen analysis demonstrated that B. cereus consumed the dissolved oxygen in the broth and resulted in the anaerobic condition for C. beijerinckii.

Butanol production under microaerobic conditions with a symbiotic system of Clostridium acetobutylicum and Bacillus cereus.

BACKGROUND: One major problem of ABE (acetone, butanol and ethanol) fermentation is high oxygen sensitivity of Clostridium acetobutylicum. Currently, no single strain has been isolated or genetically engineered to produce butanol effectively under aerobic conditions. In our previous work, a symbiotic system TSH06 has been developed successfully by our group, and two strains, C. acetobutylicum TSH1 and Bacillus cereus TSH2, were isolated from TSH06. RESULTS: Compared with single culture, TSH06 showed promotion on cell growth and solvent accumulation under microaerobic conditions. To simulate TSH06, a new symbiotic system was successfully re-constructed by adding living cells of B. cereus TSH2 into C. acetobutylicum TSH1 cultures. During the fermentation process, the function of B. cereus TSH2 was found to deplete oxygen and provide anaerobic environment for C. acetobutylicum TSH1. Furthermore, inoculation ratio of C. acetobutylicum TSH1 and B. cereus TSH2 affected butanol production. In a batch fermentation with optimized inoculation ratio of 5 % C. acetobutylicum TSH1 and 0.5 % B. cereus TSH2, 11.0 g/L butanol and 18.1 g/L ABE were produced under microaerobic static condition. In contrast to the single culture of C. acetobutylicum TSH1, the symbiotic system became more aerotolerant and was able to produce 11.2 g/L butanol in a 5 L bioreactor even with continuous 0.15 L/min air sparging. In addition, qPCR assay demonstrated that the abundance of B. cereus TSH2 increased quickly at first and then decreased sharply to lower than 1 %, whereas C. acetobutylicum TSH1 accounted for more than 99 % of the whole population in solventogenic phase. CONCLUSIONS: The characterization of a novel symbiotic system on butanol fermentation was studied. The new symbiotic system re-constructed by co-culture of C. acetobutylicum TSH1 and B. cereus TSH2 showed excellent performance on butanol production under microaerobic conditions. B. cereus TSH2 was a good partner for C. acetobutylicum TSH1 by providing an anaerobic environment. During fermentation process, the high ratio of Clostridium and low ratio of Bacillus composition indicated that this symbiotic system was an effective and easily controlled cultivation model for ABE fermentation under microaerobic conditions.

Isolation and characterisation of non-anaerobic butanol-producing symbiotic system TSH06.

Butanol-producing microorganisms are all obligate anaerobes. In this study, a unique symbiotic system TSH06 was isolated to be capable of producing butanol under non-anaerobic condition. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) revealed that two strains coexist in TSH06. The two strains were identical to Clostridium acetobutylicum and Bacillus cereus, respectively. They were isolated individually and named as C. acetobutylicum TSH1 and B. cereus TSH2. C. acetobutylicum TSH1 is a butanol-producing, obligate anaerobic strain. Facultative anaerobic B. cereus TSH2 did not possess the ability of butanol production; however, it offered C. acetobutylicum TSH1 the viability under non-anaerobic condition. Moreover, B. cereus TSH2 enhanced butanol yield and speed of fermentation. TSH06 produced 12.97 g/L butanol and 15.39 g/L total solvent under non-anaerobic condition, which is 25 and 24 %, respectively, higher than those of C. acetobutylicum TSH1. In addition, TSH06 produced butanol faster under non-anaerobic condition than under anaerobic condition. Butanol accounted for more than 80 % of total solvent, which is higher than the known report. TSH06 was stable during passage. In all, TSH06 is a promising candidate for industrialisation of biobutanol with high yield, high butanol proportion, easy-handling and time-saving system. These results demonstrated the potential advantage of symbiosis. This study also provides a promising strategy for butanol fermentation.

Vestigial like 4 regulates the adipogenesis of classical brown adipose tissue.

Brown adipose tissue (BAT) is mammals' primary non-shivering thermogenesis organ, and the molecular mechanisms regulating BAT growth and adipogenesis are largely unknown. The Hippo-YAP pathway has been well-known for controlling organ size, and Vestigial like 4 (VGLL4) is a transcriptional regulator that modulates the Hippo-YAP pathway by competing against YAP for binding to TEAD proteins. In this study, we dissected the function of VGLL4 in regulating BAT development. We generated a conventional Vgll4 mutant mouse line, in which the two Tondu (TDU) domains of VGLL4 were disrupted. We found that deletion of the TDU domains of VGLL4 resulted in perinatal lethality and paucity of the interscapular BAT. Histological and magnetic resonance imaging studies confirmed that the adipogenesis of BAT was impaired in Vgll4 mutants. Adeno-associated virus (AAV) mediated, brown adipocyte-specific overexpression of VGLL4 increased BAT volume and protected the adult male mice from acute cold stress. Genomic studies suggest that VGLL4/TEAD1 complex directly regulates the myogenic and adipogenic gene expression programs of BAT. In conclusion, our data identify VGLL4 as a previously unrecognized adipogenesis factor that regulates classical BAT development.

Increased Sulfiredoxin Expression in Gastric Cancer Cells May Be a Molecular Target of the Anticancer Component Diallyl Trisulfide.

Sulfiredoxin (Srx) is a newly discovered antioxidant enzyme playing a role in the catalytic reduction of oxidative modifications. Srx is overexpressed in a variety of cancers. It may promote carcinogenesis as well as tumor progression. In this study, we report for the first time that Srx expression might be positively associated with the development of gastric cancer and tumor malignancy. Immunohistochemistry showed that, compared to normal tissues (42%, 20/47), Srx expression in gastric tumors (85%, 40/47) was much more common (chi-square test, p<0.01). In addition, the staining of Srx was stronger in poorly differentiated gastric cancer than in well-differentiated gastric cancer. Western blotting showed that, in the gastric tumor cell line BGC823, the Srx protein was upregulated in response to H2O2 treatment, although it was inadequate to counteract the increased oxidative stress, as indicated by the gradually increasing level of malondialdehyde (MDA). In addition, Srx expression, MDA levels, and ROS levels in BGC823 cells were markedly inhibited upon treatment with diallyl trisulfide (DATS), a major constituent of garlic oil with proven anticancer effects. These results suggest that Srx may be an oxidative stress marker. Antioxidation may account for the anticancer potential of garlic.

Soluble expression of active human beta-defensin-3 in Escherichia coli and its effects on the growth of host cells.

BACKGROUND: Human beta-defensin-3 (HBD(3)) is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coli (E. coli) is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD(3) protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways. METHODS: We first cloned the complementary DNA coding for the mature chain of HBD(3), inserted it into the vector PGEX-KG then transformed E. coli BL21 (DE3) with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-beta-D-galactopyranoside (IPTG) the transformed E. coli produced a recombinant glutathione S-transferase and HBD(3) (GST-HBD(3)) fusion protein. The fusion protein was treated with thrombin to produce pure HBD(3) protein then the antimicrobial activity of HBD(3) was evaluated in a liquid microdilution assay. RESULTS: The fusion protein GST-HBD(3) was efficiently cleaved by thrombin and yielded HBD(3) that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 microg/ml. The E. coli strain expressing the recombinant protein did not grow slower than the empty vector strain. CONCLUSION: Active HBD(3) in E. coli by expressing the recombinant protein GST-HBD(3) could be produced, and suicide did not occur in the E. coli strain expressing the recombinant protein.

Insulin-like growth factor 2 as a candidate gene influencing growth and carcass traits and its bialleleic expression in chicken.

We have identified DNA polymorphisms in the gene of insulin-like growth factor 2 by PCR-SSCP in a resource population, which was generated by Silky reciprocally crossing to Broilers. A C --> G mutation was detected in the exon 2 (at position 71) by sequencing. This single nucleotide polymorphism (SNP) was found to be associated with production traits. Chicken with BB genotype showed more chest angle width but less 3 week body weight and glandular stomach weight than chicken with AA genotype (P < 0.05); while the heterozygote (AB genotype) chicken had more abdominal fat weight, eviscerated yield with giblet than AA homozygote chicken. Further analysis showed that there were different genetic effects on some traits between heterozygote AB (paternal allele given first) and heterozygote BA: chickens with genotype BA had more birth weight and breast weight but less abdominal fat weight than chickens with genotype AB (P < 0.05), which could be hypothetically contributed by genome imprinting. Therefore, Silky chickens were selected for production of heterozygotes to confirm whether IGF2 locus was imprinting. Progeny from heterozygote x homozygote reciprocal cross was assayed for expression after the genotype was determined. The transcription of IGF2 was detected by RT-PCR-SSCP. IGF2 gene was expressed bialleleically in 1-day-old neonatal liver and 90-day-old liver, kidney, heart, and muscle of both heterozygote AB and BA chickens. Therefore, IGF2 was not an imprinting gene in chicken. The different genetic effects between the heterozygote AB and BA remain to be elucidated.

[Key enzymes in butanol fermentation by a facultative anaerobe Bacillus sp. TSH1].

Bacillus sp. TSH1 is a butanol-producing microorganism newly isolated in our laboratory; it can grow and ferment under facultative anaerobic conditions, while sharing similar fermentation pathways and products with Clostridium acetobutylicum. To illustrate the relationships between the products and the enzyme activities in Bacillus sp. TSH1, key butanol- and ethanol-forming enzymes were studied, including butyraldehyde dehydrogenase, butanol dehydrogenase and alcohol dehydrogenase. The activities of the three enzymes increased rapidly after the initiation of fermentation. Activities of three enzymes peaked before 21 h, and simultaneously, product concentrations also began to increase gradually. The maximum activity of alcohol dehydrogenase was 0.054 U/mg at 12 h, butyraldehyde dehydrogenase 0.035 U/mg at 21 h and butanol dehydrogenase 0.055 U/mg at 15 h. The enzyme activities then decreased, but remained constant at a low level after 24 h, while the concentrations of butanol, acetone, and ethanol continued increasing until the end of the fermentation. The results will attribute to the understanding of the butanol metabolic mechanism, and provide a reference for further study of a facultative Bacillus metabolic pathway.

Improved succinate production by metabolic engineering.

Succinate is a promising chemical which has wide applications and can be produced by biological route. The history of the biosuccinate production shows that the joint effort of different metabolic engineering approaches brings successful results. In order to enhance the succinate production, multiple metabolical strategies have been sought. In this review, different overproducers for succinate production, including natural succinate overproducers and metabolic engineered overproducers, are examined and the metabolic engineering strategies and performances are discussed. Modification of the mechanism of substrate transportation, knocking-out genes responsible for by-products accumulation, overexpression of the genes directly involved in the pathway, and improvement of internal NADH and ATP formation are some of the strategies applied. Combination of the appropriate genes from homologous and heterologous hosts, extension of substrate, integrated production of succinate, and other high-value-added products are expected to bring a desired objective of producing succinate from renewable resources economically and efficiently.

Effects of pH and dissolved CO2 level on simultaneous production of 2,3-butanediol and succinic acid using Klebsiella pneumoniae.

The influences of pH and dissolved CO2 level on the regulation of growth and formation of catabolic end products have been investigated in Klebsiella pneumoniae. With increasing CO2 levels, there were no apparent changes in 2,3-butanediol production but succinic acid productions were enhanced significantly. A novel strategy for co-production of 2,3-butanediol and succinic acid using K. pneumoniae was developed by controlling pH and dissolved CO2 concentration in fermentation medium. Under the optimum condition, maximal 77.1 g l(-1) 2,3-butanediol and 28.7 g l(-1) succinic acid were obtained after 60 h of fed-batch fermentation, giving a 2,3-butanediol+succinic acid yield of 1.03 mol mol(-1) glucose. This type of fermentation producing two commercial interests at the same fermentation process might be considered for a promising biological production process which will decrease the production cost by sharing the operation and recovery cost.