Construction,Expression, and Characterization of Recombinant Pfu DNA Polymerase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His₆-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni²⁺ chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Molecular evidence for hybridization between invasive <Emphasis Type="Italic">Solidago canadensis</Emphasis> and native <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">virgaurea</Emphasis>

Hybridization between alien and native species is biologically very important and could lead to genetic erosion of native taxa. Solidago × niederederi was discovered over a century ago in Austria and described by Khek as a natural hybrid between the alien (nowadays regarded also as invasive) S. canadensis and native S. virgaurea. Although interspecific hybridization in the genus Solidago is considered to be relatively common, hybrid nature of S. × niederederi has not been independently proven using molecular tools, to date. Because proper identification of the parentage for the hybrid Solidago individuals solely based on morphological features can be misleading, in this paper we report an additive polymorphism pattern expressed in the ITS sequences obtained from individuals representing S. × niederederi, and confirm the previous hypothesis that the parental species of this hybrid are S. canadensis and S. virgaurea. Additionally, based on variability at the cpDNA rpl32-trnL locus, we showed that in natural populations hybridization occurs in both directions.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.     

<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

Detection of pea wilt pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis> using DNA-based markers

Identification of the fungus Fusarium oxysporum f. sp. pisi (Fop), the causal organism of wilt disease of pea, is a time consuming and arduous task. Diagnosis of Fop by traditional means requires more than 2 months and involves two steps, identification of species using morphological characters and formae specialis ‘pisi’ using pathogenicity assays. The ambiguous morphological differences between F. solani and F. oxysporum further complicate the diagnosis of F. oxysporum. A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) based method was developed to detect Fop from India. A PCR–RFLP marker, HPACAPS1₃₈₀, generated after restriction of 28S rDNA region with enzyme MvaI, detected accurately the Fop among several other fungi with detection sensitivity of 5 fg of Fop genomic DNA. In a mixture of Fop and pea DNA, the sensitivity was 500 pg of Fop DNA in 50 ng of pea DNA. The assay was further refined to detect the Fop from infected tissues and infested soil. The current assay can detect Fop from culture, plant tissues and soil in a considerably shorter period of time compared to traditional methods.     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration,production, and storage of artificial seeds in <Emphasis Type="Italic">Ceropegia barnesii</Emphasis>, an endangered plant

Approaches for in vitro regeneration and fabrication of synthetic seeds were formulated to support restoration in the wild and genetic manipulation of Ceropegia barnesii (categorized as endemic and endangered). MS medium augmented with 4 mg L^?1 benzyl adenine was most advantageous for the production of multiple shoots from nodal explants. Fabrication of synthetic seeds was accomplished by sodium alginate encapsulation of nodes from microshoots. The most favorable medium combination for the induction of multiple shoots from synthetic seeds was MS medium complemented with 4 mg L^?1 benzyl adenine and 1 mg L^?1 gibberelic acid. Following root induction promoted by half strength MS basal medium augmented with indolebutyric acid, multiple shoots were subjected to hardening. Influence of vesicular-arbuscular mycorrhizal fungi on the hardening trials was investigated and it was observed that dual inoculation of Glomus aggregatum and G. intraradices enhanced the survival rate. The encapsulated nodes of C. barnesii were tested for their capability to endure different temperatures during storage and the optimal temperature for storage was found to be 4°C. A methodology for initiation of somatic embryogenesis from C. barnesii is also reported here, but embryos could not be induced to develop further. The micropropagated plants were reintroduced in to their natural habitat. This is the first report on micropropagation of C. barnesii.     

The <Emphasis Type="Italic">yajC</Emphasis> gene from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and its role in ethanol tolerance

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-d-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.     

Development and characterization of <Emphasis Type="Italic">japonica</Emphasis> rice lines carrying the brown planthopper-resistance genes <Emphasis Type="Italic">BPH12</Emphasis> and <Emphasis Type="Italic">BPH6</Emphasis>

The brown planthopper (Nilaparvata lugens Stål; BPH) has become a severe constraint on rice production. Identification and pyramiding BPH-resistance genes is an economical and effective solution to increase the resistance level of rice varieties. All the BPH-resistance genes identified to date have been from indica rice or wild species. The BPH12 gene in the indica rice accession B14 is derived from the wild species Oryza latifolia. Using an F₂ population from a cross between the indica cultivar 93-11 and B14, we mapped the BPH12 gene to a 1.9-cM region on chromosome 4, flanked by the markers RM16459 and RM1305. In this population, BPH12 appeared to be partially dominant and explained 73.8% of the phenotypic variance in BPH resistance. A near-isogenic line (NIL) containing the BPH12 locus in the background of the susceptible japonica variety Nipponbare was developed and crossed with a NIL carrying BPH6 to generate a pyramid line (PYL) with both genes. BPH insects showed significant differences in non-preference in comparisons between the lines harboring resistance genes (NILs and PYL) and Nipponbare. BPH growth and development were inhibited and survival rates were lower on the NIL-BPH12 and NIL-BPH6 plants compared to the recurrent parent Nipponbare. PYL-BPH6 + BPH12 exhibited 46.4, 26.8 and 72.1% reductions in population growth rates (PGR) compared to NIL-BPH12, NIL-BPH6 and Nipponbare, respectively. Furthermore, insect survival rates were the lowest on the PYL-BPH6 + BPH12 plants. These results demonstrated that pyramiding different BPH-resistance genes resulted in stronger antixenotic and antibiotic effects on the BPH insects. This gene pyramiding strategy should be of great benefit for the breeding of BPH-resistant japonica rice varieties.     

Distribution and habitats of <Emphasis Type="Italic">Phengaris</Emphasis> (<Emphasis Type="Italic">Maculinea</Emphasis>) butterflies and population ecology of <Emphasis Type="Italic">Phengaris</Emphasis><Emphasis Type="Italic">teleius</Emphasis> in China

Many species of the butterfly genus Phengaris are regarded as endangered in many parts of their distribution. Several species are also widely distributed across northern China. Due to land use change and overgrazing, their habitats are declining and many patches have been lost. This paper investigates the distribution and habitats of the Chinese Phengaris species (of the subgenus Maculinea). Shrub-grassland near forests seem the most frequent habitat for Phengaris, while flat open grasslands are mostly over-grazed and thus survival for Phengaris butterflies there seems difficult. Throughout Europe, P. teleius is an endangered species, while there is still no information on its status in China. To improve the knowledge on the population ecology of P. teleius, its population structure, adult behaviour and movement were studied through mark–release–recapture methods in the Qinling Mountains of Taibai County. Eight grassland patches which were potentially suitable were found in the area in 2013. In total, 480 individuals (274 females) were marked, resulting in an overall recapture rate of 16 %. The average daily population size was 44 butterflies (±23 SD) during the adult flight period. Sixty-seven percent of the females and 38 % of the males moved less than 50 m, and 17 % of recaptured females and 38 % of males moved more than 200 m. The mean movement distance was 107 ± 177 m for males and 182 ± 122 m for females. The majority of the recaptures (86 %) were made within the patches, only a few individuals (14 %) moved between patches. Due to human disturbance and destruction, all of the eight potentially suitable patches are becoming smaller and increasingly isolated, thus these populations of P. teleius may face an increasing risk of extinction, which may well be a tip of the iceberg of habitat loss and fragmentation of P. teleius in Taibai County and possibly beyond. Hence we hope our initial study of P. teleius could have positive impacts on the conservation of Phengaris butterflies in China.     

Activation of stilbene synthesis in cell cultures of <Emphasis Type="Italic">Vitis amurensis</Emphasis> by calcium-dependent protein kinases <Emphasis Type="Italic">VaCPK1</Emphasis> and <Emphasis Type="Italic">VaCPK26</Emphasis>

Stilbenes, including trans-resveratrol (3,4′,5-trihydroxy-trans-stilbene), are known to exert beneficial health effects and contribute to plant biotic stress resistance. Much remains to be discovered about the cell signaling pathways regulating stilbene biosynthesis. It has recently been shown that overexpression of the calcium-dependent protein kinase VaCPK20 gene considerably increased t-resveratrol accumulation in cell cultures of Vitis amurensis. In this study, we analyzed the involvement of other CDPK family members, VaCPK1 and VaCPK26, on stilbene synthesis and biomass production by cell cultures of V. amurensis. We showed that overexpression of the VaCPK1 and 26 genes induced production of stilbenes by 1.7–4.6-fold (for VaCPK1) and by 2.5–6.2-fold (for VaCPK26) in several independently established cell lines compared to the empty vector-transformed control. Using HPLC-UV-MS, we detected five stilbenes in the grape cells: t-resveratrol diglucoside, t-piceid, t-resveratrol, ε- and δ-viniferin. The VaCPK1- and VaCPK26-transformed calli were capable of producing 1.4–3.1 and 1.8–4.9 mg/l of t-resveratrol, respectively (up to 0.4 for and 0.6 mg/g of dry weight for VaCPK26 and VaCPK1, respectively), while the control line synthesized only 0.5 mg/l of t-resveratrol (0.07 mg/g DW). The up-regulation of t-resveratrol production in the VaCPK1- and VaCPK26-overexpressing grape calli correlated with a significant up-regulation of stilbene synthase (STS) gene expression, especially VaSTS7. The data indicate that VaCPK1 and 26 genes, which are close homologues of VaCPK20, are positive regulators of stilbene biosynthesis in grapevine.