Molecular Characterization of Novel Cucurbit Aphid Borne Yellows Virus Strains Infecting Squash and Watermelon in India

The pathogen responsible for yellowing and downward rolling of leaves of squash and watermelon plants from Uttar Pradesh state, India, was identified as probably strains of Cucurbit aphid-borne yellows virus (CABYV) through RT-PCR using universal Polerovirus primers followed by sequencing. The full-length genome sequences of an isolate from squash (POL-SQ - 5650 nt) and one from watermelon (POL-WM - 5647nt) were determined by sequencing the products from RT-PCR with six sets of primers with overlapping products. Sequence comparison and phylogenetic analysis showed that these isolates had closest identity with a recombinant strain obtained between CABYV and Melon aphid-borne yellows virus (MABYV) reported from Taiwan infecting Luffa aegyptiaca (CABYV-R-TW82) rather than other Asian, American, or European isolates. The deduced amino acid sequences of the P0, P1 and P1-P2 proteins showed >10% variation, whereas the P3, P4 and P3-P5 proteins showed <10% variation when compared to the corresponding proteins of other strains of CABYV worldwide. Thus, according to the Polerovirus species demarcation threshold, these new sequences should be regarded as representing strains of a novel previously undescribed Polerovirus species. However, based on their sequence similarity and phylogenetic grouping with the recombinant strain from Taiwan we suggest these sequences represent recombinant strains of CABYV. These are the first full-length genome sequences for CABYV strains from India and this study adds watermelon as host for CABYV in India.

The recombinant pseudorabies virus expressing porcine deltacoronavirus spike protein is safe and effective for mice

Background Porcine deltacoronavirus (PDCoV) is a new pathogenic porcine intestinal coronavirus, which has appeared in many countries since 2012. PDCoV disease caused acute diarrhea, vomiting, dehydration and death in piglets, resulted in significant economic loss to the pig industry. However, there is no commercially available vaccine for PDCoV. In this study, we constructed recombinant pseudorabies virus (rPRVXJ-delgE/gI/TK-S) expressing PDCoV spike (S) protein and evaluated its safety and immunogenicity in mice. Results The recombinant strain rPRVXJ-delgE/gI/TK-S obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster syrian kidney-21 (BHK-21) cells and is safe to mice. After immunizing mice with rPRVXJ-delgE/gI/TK-S, the expression levels of IFN-γ and IL-4 in peripheral blood of mice were up-regulated, the proliferation of spleen-specific T lymphocytes and the percentage of CD4+ and CD8+ lymphocytes in mice spleen was increased. rPRVXJ-delgE/gI/TK-S showed good immunogenicity for mice. On the seventh day after booster immunity, PRV gB and PDCoV S specific antibodies were detected in mice, and the antibody level continued to increase, and the neutralizing antibody level reached the maximum at 28 days post- immunization (dpi). The recombinant strain can protect mice with 100% from the challenge of virulent strain (PRV XJ) and accelerate the detoxification of PDCoV in mice. Conclusion The recombinant rPRVXJ-delgE/gI/TK-S strain is safe and effective with strong immunogenicity and is expected to be a candidate vaccine against PDCoV and PRV.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

Background Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. Results Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. Conclusions This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Norovirus GII.2[P16] strain in Shenzhen, China: a retrospective study

Background Norovirus (NoV) is the main cause of non-bacterial acute gastroenteritis (AGE) outbreaks worldwide. From September 2015 through August 2018, 203 NoV outbreaks involving 2500 cases were reported to the Shenzhen Center for Disease Control and Prevention. Methods Faecal specimens for 203 outbreaks were collected and epidemiological data were obtained through the AGE outbreak surveillance system in Shenzhen. Genotypes were determined by sequencing analysis. To gain a better understanding of the evolutionary characteristics of NoV in Shenzhen, molecular evolution and mutations were evaluated based on time-scale evolutionary phylogeny and amino acid mutations. Results A total of nine districts reported NoV outbreaks and the reported NoV outbreaks peaked from November to March. Among the 203 NoV outbreaks, 150 were sequenced successfully. Most of these outbreaks were associated with the NoV GII.2[P16] strain (45.3%, 92/203) and occurred in school settings (91.6%, 186/203). The evolutionary rates of the RdRp region and the VP1 sequence were 2.1 × 10–3 (95% HPD interval, 1.7 × 10–3–2.5 × 10–3) substitutions/site/year and 2.7 × 10–3 (95% HPD interval, 2.4 × 10–3–3.1 × 10–3) substitutions/site/year, respectively. The common ancestors of the GII.2[P16] strain from Shenzhen and GII.4 Sydney 2012[P16] diverged from 2011 to 2012. The common ancestors of the GII.2[P16] strain from Shenzhen and previous GII.2[P16] (2010–2012) diverged from 2003 to 2004. The results of amino acid mutations showed 6 amino acid substitutions (*77E, R750K, P845Q, H1310Y, K1546Q, T1549A) were found only in GII.4 Sydney 2012[P16] and the GII.2[P16] recombinant strain. Conclusions This study illustrates the molecular epidemiological patterns in Shenzhen, China, from September 2015 to August 2018 and provides evidence that the epidemic trend of GII.2[P16] recombinant strain had weakened and the non-structural proteins of the recombinant strain might have played a more significant role than VP1.

In vivo production of pederin by labrenzin pathway expansion

SummaryPederin is a potent polyketide toxin that causes severe skin lesions in humans after contact with insects of genus Paederus. Due to its potent anticancer activities, pederin family compounds have raised the interest of pharmaceutical industry. Despite extensive studies on the cluster of biosynthetic genes responsible for the production of pederin, it has not yet been possible to isolate and cultivate its bacterial endosymbiont producer. However, the marine bacterium Labrenzia sp. PHM005 was recently reported to produce labrenzin, the closest pederin analog. By cloning a synthetic pedO gene encoding one of the three O-methyltraferase of the pederin cluster into Labrenzia sp. PHM005 we have been able to produce pederin for the first time by fermentation in the new recombinant strain.

Construction of model strain of yeast Saccharomyces cerevisiae with regulated expression of recombinant human alpha-synuclein

Background. Improper folding and accumulation of a-synuclein aggregates are among the causes of Parkinson’s disease. The most important factor influencing the process of α-synuclein aggregation is the level of this protein in neurons which depends on the balance between its synthesis, degradation and secretion. Under certain conditions, when α-synuclein is synthesized at a high level, monomers of this protein can aggregate on the lipid membrane, which leads to the formation of amyloids, fibrils and protofibrils unable to perform their physiological functions. Since it is virtually impossible to study the properties of α-synuclein in vivo, researchers are actively using model biological systems (single-celled microorganisms, human cell lines, animal models etc.). The aim of this study was to construct a recombinant strain of Saccharomyces cerevisiae with controlled expression of human α-synuclein to study the regulation and properties of this protein and for screening for new low molecular weight chemi­cal compounds which can induce α-synuclein aggregation and/or degradation. Materials and methods. A recombinant strain of S. cerevisiae with controlled expression of α-synuclein conjugated to a green fluorescent protein was isolated. Western blotting with specific anti-α-synuclein antibodies was used to detect recombinant α-synuclein in yeast cells. Intracellular localization of heterologous chimeric green fluorescent protein conjugated to α-synuclein was also examined by fluorescence microscopy. Results. To construct a recombinant strain of S. cerevisiae, the coding sequence of the human wild-type α-synuclein gene was expressed under the regulated promoter of the ScMET25 gene. Analysis of the effect of different concentrations of exogenous methionine as a factor regulating the expression of the ScMET25 promoter on the content of recombinant protein showed that the expression of the human α-synuclein gene in S. cerevisiae is repressed in the presence of methionine at a concentration of 10 mg/L and higher. During long-term cultivation of yeast cells, this effect decreased due to the depletion of methionine in the growth medium. As a result, recombinant protein synthesis was restored, and α-synuclein content in such cells approached that of cells grown in a medium with a low concentration of (5 mg/L), or without methionine. It was also found that overproduction of recombinant α-synuclein in S. cerevisiae cells had virtually no effect on culture growth, indicating the absence or a very weak toxic effect of human α-synuclein on yeast physiology. Conclusions. The obtained data indicate a concentration-dependent effect of methionine on the level of recombinant α-synuclein synthesis in S. cerevisiae yeast cells. Such controlled expression of the studied protein can be used to screen for compounds capable of promoting dose-dependent aggregation or degradation of α-synuclein in yeast cells and potentially in human cells as well.

Obtaining a fluorescently labeled endophytic strain of bacteria Herbaspirillum lusitanum P6-12 for their detection in vivo and in vitro

The Herbaspirillum lusitanum P6-12 strain containing the vector plasmid pJN105TurboGFP, which encodes the synthesis of the green fluorescent protein GFP, and which has resistance to the antibiotic gentamicin, was obtained by electroporation. The constructed strain of H. lusitanum P6-12 in cultural, morphological and biochemical properties did not differ from the original typical natural strain of H. lusitanum P6-12. On solid growth media, the recombinant strain formed yellow-green colonies, fluorescent under UV irradiation. Upon inoculation with the resulting culture of plant objects, a green glow of the marked H. lusitanum P6-12 cells, actively colonizing the internal tissues of the host plant, was observed. The created strain can be used as a model strain for studying the patterns and characteristics of the behaviour of organisms in integrated systems, including for tracking bacterial cells during interaction with plants, assessing their survival, competitiveness, etc.

Klebsiella Pneumoniae Metabolic Regulation and its Application in 1,3-Propanediol Production

Klebsiella pneumoniae is a well-known model organism for glycerol metabolism to produce 1,3-propanediol (1,3-PD), a valuable chemical intermediate for materials, such as polyesters. However, the relatively low conversion rate and productivity, as well as the accumulation of by-products such as lactic acid, ethanol and acetic acid, inhibit the production of 1,3-PD. Hereby, the 1,3-PD metabolism in K. pneumoniae was regulated through pathway engineering by using CRISPR-Cas9 technology for the first time to knock out the ldhA gene of lactate dehydrogenase,the adhE gene of alcohol dehydrogenase and the ack gene of acetate kinase respectively as needed and constructed recombinant bacteria ldhA(−), ldhA(−)-ack(−), ldhA(−)-adhE(−) and ldhA(−)-adhE(−)-ack(−), all of which showed a decrease in by-product production, leading to a higher NADH availability, and 1,3-BD production was significantly increased. In the shake flask fermentation, the 1,3-PD yield and conversion rate of the recombinant strain ldhA(−), ldhA(−)-ack(−), ldhA(−)-adhE(−), ldhA(−)-adhE(−)-ack(−) were higher than those of the parent strain. In the fed-batch fermentation, the 1,3-PD yield and conversion rate of the recombinant strain ldhA(−) were higher than those of the parent strain. The biomass of the recombinant strain ldhA(−)-adhE(−)-ack(−) was reduced due to the accumulation of acetic acid, but its 1,3-PD conversion rate was still higher than that of the parent strain. The higher productivity and fewer by-products concluded that the four Klebsiella pneumoniae recombinant strains could be promising industrial strain for economical production of 1,3-PD.