Precultivation of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> DSM2314 in the presence of furfural decreases inhibitory effects of lignocellulosic by-products during <Emphasis Type="SmallCaps">l</Emphasis>(+)-lactic acid fermentation

By-products resulting from thermo-chemical pretreatment of lignocellulose can inhibit fermentation of lignocellulosic sugars to lactic acid. Furfural is such a by-product, which is formed during acid pretreatment of lignocellulose. pH-controlled fermentations with 1 L starting volume, containing YP medium and a mixture of lignocellulosic by-products, were inoculated with precultures of Bacillus coagulans DSM2314 to which 1 g/L furfural was added. The addition of furfural to precultures resulted in an increase in l(+)-lactic acid productivity by a factor 2 to 1.39 g/L/h, an increase in lactic acid production from 54 to 71 g and an increase in conversion yields of sugar to lactic acid from 68 to 88 % W/W in subsequent fermentations. The improved performance was not caused by furfural consumption or conversion, indicating that the cells acquired a higher tolerance towards this by-product. The improvement coincided with a significant elongation of B. coagulans cells. Via RNA-Seq analysis, an upregulation of pathways involved in the synthesis of cell wall components such as bacillosamine, peptidoglycan and spermidine was observed in elongated cells. Furthermore, the gene SigB and genes promoted by SigB, such as NhaX and YsnF, were upregulated in the presence of furfural. These genes are involved in stress responses in bacilli.     

Sucrose dependent mineral phosphate solubilization in <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> PSI3 by heterologous overexpression of periplasmic invertases

Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.     

Enzymatic production of (R)-phenylacetylcarbinol by pyruvate decarboxylase from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Herein, we synthesized (R)-phenylacetylcarbinol (PAC), a pharmaceutical intermediate for ephedrine and pseudoephedrine, from benzaldehyde and pyruvate using a recombinant pyruvate decarboxylase (PDC) from Zymomonas mobilis. A whole cell reaction consisting of 30 mM benzaldehyde, 60 mM pyruvate, and a mutant PDC enzyme (PDC W329M; 1.6 mg DCW/mL) produced 12.4 mM (R)-PAC and less than 0.3 mM benzyl alchohol in 15 h at 20°C, outperforming the crude enzyme extract reaction (1.2 mM (R)-PAC) and minimizing formation of benzyl alchohol, the major by-product of S. cerevisiae whole cell reaction. These observations suggested that recombinant E. coli whole cell reactions are more efficient than crude enzyme extract or yeast-based reactions. We also demonstrated that the E. coli whole cell reaction performed effectively without expensive thiamin diphosphate cofactor. Finally, whole cell reaction (8 mg DCW/mL) was carried out with 200 mM benzaldehyde, 400 mM pyruvate in 10 mL of 500 mM phosphate buffer (pH 6.5), and 72 mM (R)-PAC was produced with 36% conversion for 15 h. © KSBB     

The effects of condensed tannins derived from senescing <Emphasis Type="Italic">Rhizophora mangle</Emphasis> leaves on carbon,nitrogen and phosphorus mineralization in a <Emphasis Type="Italic">Distichlis spicata</Emphasis> salt marsh soil

Background and aims

Low nitrogen negatively affects soil fertility and plant productivity. Glucose-6-phosphate dehydrogenase (G6PDH) and Epichloë gansuensis endophytes are two factors that are associated with tolerance of Achnatherum inebrians to abiotic stress. However, the possibility that E. gansuensis interacts with G6PDH in enhancing low nitrogen tolerance of host grasses has not been examined.

Methods

A. inebrians plants with (E+) and without E. gansuensis (E?) were subjected to different nitrogen concentration treatments (0.1, 1, and 7.5 mM). After 90 days, physiological studies were carried out to investigate the participation of G6PDH in the adaption of host plants to low nitrogen availability.

Results

Low nitrogen retarded the growth of A. inebrians. E+ plants had higher total dry weight, chlorophyll a and b contents, net photosynthesis rate, G6PDH activity, and GSH content, while having lower plasma membrane (PM) NADPH oxidase activity, NADPH/NADP⁺ ratios, and MDA and H₂O₂ than in E? A. inebrians plants under low nitrogen concentration.

Conclusions

The presence of E. gansuensis played a key role in maintaining the growth of the A. inebrians plants under low nitrogen concentration by regulating G6PDH activity and the NADPH/NADP⁺ ratio and improving net photosynthesis rate.

     

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (<Emphasis Type="Italic">R</Emphasis>)-phenyl-1,2-ethanediol

Objectives

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee _p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis.

Results

The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2^A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2^A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h^?1 g^?1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee _p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2^A250I-expressing E. coli/Aueh2 ^A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee _p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee _p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h.

Conclusions

Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2^A250I is an effective method for preparing (R)-PED with high ee _p and yield.

     

A new source of resistance to 2-furaldehyde from <Emphasis Type="Italic">Scheffersomyces</Emphasis> (<Emphasis Type="Italic">Pichia</Emphasis>) <Emphasis Type="Italic">stipitis</Emphasis> for sustainable lignocellulose-to-biofuel conversion

Aldehyde inhibitory compounds derived from lignocellulosic biomass pretreatment have been identified as a major class of toxic chemicals that interfere with microbial growth and subsequent fermentation for advanced biofuel production. Development of robust next-generation biocatalyst is a key for a low-cost biofuel production industry. Scheffersomyces (Pichia) stipitis is a naturally occurring C-5 sugar utilization yeast; however, little is known about the genetic background underlying its potential tolerance to biomass conversion inhibitors. We investigated and identified five uncharacterized putative aryl-alcohol dehydrogenase genes (SsAADs) from this yeast as a new source of resistance against biomass fermentation inhibitor 2-furaldehyde (furfural) by gene expression, gene cloning, and direct enzyme assay analysis using partially purified proteins. All five proteins from S. stipitis showed furfural reduction using cofactor NADH. An optimum active temperature was observed at 40 °C for SsAad1p; 30 °C for SsAad3p, SsAad4p, and SsAad5p; and 20 °C for SsAad2p. SsAad2p, SsAad3p, and SsAad4p showed tolerance to a wide range of pH from 4.5 to 8, but SsAad1p and SsAad5p were sensitive to pH changes beyond 7. Genes SsAAD2, SsAAD3, and SsAAD4 displayed significantly enhanced higher levels of expression in response to the challenge of furfural. Their encoding proteins also showed higher levels of specific activity toward furfural and were suggested as core functional enzymes contributing aldehyde resistance in S. stipitis.     

<Emphasis Type="Italic">Enterococcus faecalis</Emphasis> promotes osteoclast differentiation within an osteoblast/osteoclast co-culture system

Objective

To investigate the effects of heat-killed Enterococcus faecalis ATCC 29212 and P25RC clinical strain (derived from an obturated root canal with apical periodontitis) on osteoclast differentiation within an osteoblast/osteoclast co-culture system.

Results

Heat-killed E. faecalis significantly increased the proportion of multinucleated osteoclastic cells (MNCs) within the co-culture system. The IL-6 level was significantly increased upon exposure to heat-killed E. faecalis. Gene expression levels of NFATc1 and cathepsin K were significantly up-regulated compared to the untreated control. EphrinB2 and EphB4 expressions at both the mRNA and protein levels were also significantly upregulated compared to the untreated control.

Conclusions

Heat-killed E. faecalis can induce osteoclast differentiation within the osteoblast/osteoclast co-culture system in vitro, possibly through ephrinB2-EphB4 bidirectional signaling.

     

Discovery of novel feruloyl esterase activity of BioH in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)

Objectives

To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3.

Results

The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K _m values of 0.48, 6.3, and 1.9 mM, respectively, and k _cat /K _m values of 9.3, 3.8, and 3.8 mM^?1 s^?1, respectively.

Conclusions

Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.

     

Effect of ATP and Bax on the apoptosis of <Emphasis Type="Italic">Eimeria tenella</Emphasis> host cells

Background

Eimeria tenella (E. tenella) is a species of Eimeria that causes haemorrhagic caecal coccidiosis, resulting in major economic losses in the global poultry industry. After E. tenella infection, the amount of ATP and Bax in host cells showed highly significant changes. Therefore, it is necessary to investigate the effects of ATP and Bax on the apoptosis of E. tenella host cells.

Results

The ATP-treated group and the V5-treated group had higher E. tenella infection rates than the untreated group at 24, 48, 72, 96, and 120 h after infection with E. tenella. The results of flow cytometry showed that compared with the control group, the mitochondrial permeability transition pore (MPTP) opening in the untreated group was highly significantly increased (P?<?0.01) at 4, 24, 48, 72, 96, and 120 h. Moreover, results from Hoechst-Annexin V-PI staining and flow cytometry showed that the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were significantly lower (P?<?0.05) or highly significantly lower (P?<?0.01) than those of the control group at 4 h, while the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were higher at varying degrees than those in the control group at 24–120 h (P?<?0.05 or P?<?0.01). After treatment with ATP and Bax inhibitors, the rates of early apoptosis, late apoptosis, and necrosis, in addition to the MPTP opening in both the ATP-treated and V5-treated groups, were significantly lower (P?<?0.05) or highly significantly lower (P?<?0.01) than those in the untreated group.

Conclusions

ATP and Bax play important roles in regulating the apoptosis of E. tenella host cells.

     

Transmission and characterization of <Emphasis Type="Italic">bla</Emphasis><Subscript>NDM-1</Subscript> in <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> at a teaching hospital in Yunnan,China

Background

In recent years, New Delhi metallo-beta-lactamases 1 (bla _NDM-1) has been reported with increasing frequency and become prevalent. The present study was undertaken to investigate the epidemiological dissemination of the bla _NDM-1 gene in Enterobacter cloacae isolates at a teaching hospital in Yunnan, China.

Methods

Antimicrobial susceptibility testing was performed using VITEK 2 system and E test gradient strips. The presence of integrons and insertion sequence common region 1 were examined by PCR and sequencing. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Conjugation experiments and Southern blot hybridization were performed to determine the transferability of plasmids.

Results

Ten E. cloacae isolates and their Escherichia coli transconjugants were exhibited similar resistant patterns to carbapenems, cephalosporins and penicillins. 8 (80%) of E. cloacae isolates carried class 1 integron and 1 (12.5%) carried class 2 integron. Integron variable regions harbored the genes which encoded resistance to aminoglycosides (aadA1, aadA2, aadA5, aadB, aac(6′)-Ib-cr), sulfamethoxazole/trimethoprim (dfrA17, dfrA12, dfrA15) and Streptozotocin (sat2). Six E. cloacae isolates belonged to ST74 and exhibited highly similar PFGE patterns. Each isolate shared an identical plasmid with ~33.3 kb size that carried the bla _NDM-1 gene, except T3 strain, of which the bla _NDM-1 gene was located on a ~50 kb plasmid.

Conclusions

Our findings suggested that plasmid was able to contribute to the dissemination of bla _NDM-1. Hence, more attention should be devoted to monitor the dissemination of the bla _NDM-1 gene due to its horizontal transfer via plasmid. In addition, nosocomial surveillance system should actively monitor the potential endemic clone of ST74 to prevent their further spread.

     

NtRING1, putative RING-finger E3 ligase protein,is a positive regulator of the early stages of elicitin-induced HR in tobacco

Key message

NtRING1 is a RING-finger protein with a putative E3 ligase activity. NtRING1 regulates HR establishment against different pathogens. Loss-/gain-of-function of NtRING1 altered early stages of HR phenotype establishment.

Abstract

Plant defence responses against pathogens often involve the restriction of pathogens by inducing a hypersensitive response (HR). cDNA clones DD11-39, DD38-11 and DD34-26 were previously obtained from a differential screen aimed at characterising tobacco genes with an elicitin-induced HR-specific pattern of expression. Our precedent observations suggested that DD11-39, DD38-11 and DD34-26 might play roles in the HR establishment. Only for DD11-39 a full-length cDNA sequence was obtained and the corresponding protein encoded for a type-HC RING-finger/putative E3 ligase protein which we termed NtRING1. The expression of NtRING1 was upregulated upon HR induction by elicitin, Ralstonia solanacearum, or tobacco mosaic virus (TMV) in tobacco. Silencing of NtRING1 remarkably delayed the establishment of elicitin-induced HR in tobacco as well as the expression of different early induction genes in tissues undergoing HR. Accordingly, transient overexpression of NtRING1 accelerated the HR launching upon elicitin treatment. Taking together, our data suggests that NtRING1 plays a functional role in the early establishment of HR.

     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

High yield expression in a recombinant <Emphasis Type="Italic">E. coli</Emphasis> of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Background

Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

Results

Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.

Conclusions

Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

     

An engineered non-oxidative glycolysis pathway for acetone production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli.

Results

Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain.

Conclusions

Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.

     

Contributions of <Emphasis Type="Italic">TaSUTs</Emphasis> to grain weight in wheat under drought

Key message

The homologous genes to OsSUT1-5 in wheat were identified and detailed analysed. TaSUT1 was the predominant sucrose transporter group and it illustrated the genotypic variations towards drought during grain filling.

Abstract

Sucrose transporters (SUT) play crucial roles in wheat stem water soluble carbohydrate (WSC) remobilization to grain. To determine the major functional SUT gene groups in shoot parts of wheat during grain development, drought tolerant varieties, Westonia and Kauz, were investigated in field drought experiments. Fourteen homologous genes to OsSUT1-5 were identified on five homeologous groups, namely TaSUT1_4A, TaSUT1_4B, TaSUT1_4D; TaSUT2_5A, TaSUT2_5B, TaSUT2_5D; TaSUT3_1A, TaSUT3_1D; TaSUT4_6A, TaSUT4_6B, TaSUT4_6D; TaSUT5_2A, TaSUT5_2B, and TaSUT5_2D, and their gene structures were analysed. Wheat plants above the ground were harvested from pre-anthesis to grain maturity and the stem, leaf sheath, rachis, lemma and developing grain were used for analysing TaSUT gene expression. Grain weight, thousand grain weight, kernel number per spike, biomass and stem WSC were characterized. The study showed that among the five TaSUT groups, TaSUT1 was the predominant sucrose transporting group in all organs sampled, and the expression was particularly high in the developing grain. In contrast to TaSUT1, the gene expression levels of TaSUT2, TaSUT3 and TaSUT4 were lower, except for TaSUT3 which showed preferential expression in the lemma before anthesis. The TaSUT5 gene group was very weakly expressed in all tissues. The upregulated gene expression of TaSUT1 Westonia type in stem and grain reveal a crucial role in stem WSC remobilization to grain under drought. The high TaSUT1 gene expression and the significant correlations with thousand grain weight (TGW) and kernel number per spike demonstrated the contribution in Kauz’s high grain yield in an irrigated environment and high TGW in Westonia under drought stress. Further molecular level identification is required for gene marker development.