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Beneficial effects elicited by the root endophyte Piriformospora indica are widely known, but the mechanism by which these are achieved is still unclear. It is proposed that phytohormones produced by the fungal symbiont play a crucial role in the interaction with the plant roots. Biochemical analyses of the underlying biosynthetic pathways for auxin production have shown that, on tryptophan feeding, P. indica can produce the phytohormones indole-3-acetic acid (IAA) and indole-3-lactate (ILA) through the intermediate indole-3-pyruvic acid (IPA). Time course transcriptional analyses after exposure to tryptophan designated the piTam1 gene as a key player. A green fluorescence protein (GFP) reporter study and transcriptional analysis of colonized barley roots showed that piTam1 is induced during the biotrophic phase. Piriformospora indica strains in which the piTam1 gene was silenced via an RNA interference (RNAi) approach were compromised in IAA and ILA production and displayed reduced colonization of barley (Hordeum vulgare) roots in the biotrophic phase, but the elicitation of growth promotion was not affected compared with the wild-type situation. Our results suggest that IAA is involved in the establishment of biotrophy in P. indica–barley symbiosis and might represent a compatibility factor in this system.

A new type of coronavirus has been identified as the causative agent underlying Middle East Respiratory Syndrome (MERS). The MERS coronavirus (MERS-CoV) has spread in the Middle East, but cases originating in the Middle East have also occurred in the European Union and the USA. Eight hundred and thirty-seven cases of MERS-CoV infection have been confirmed to date, including 291 deaths. MERS-CoV has infected dromedary camel populations in the Middle East at high rates, representing an immediate source of human infection. The MERS-CoV spike (S) protein, a characteristic structural component of the viral envelope, is considered as a key target of vaccines against coronavirus infection. In an initial attempt to develop a MERS-CoV vaccine to ultimately target dromedary camels, we constructed two recombinant adenoviral vectors encoding the full-length MERS-CoV S protein (Ad5.MERS-S) and the S1 extracellular domain of S protein (Ad5.MERS-S1). BALB/c mice were immunized with both candidate vaccines intramuscularly and boosted three weeks later intranasally. All the vaccinated animals had antibody responses against spike protein, which neutralized MERS-CoV in vitro. These results show that an adenoviral-based vaccine can induce MERS-CoV-specific immune responses in mice and hold promise for the development of a preventive vaccine that targets the animal reservoir, which might be an effective measure to eliminate transmission of MERS-CoV to humans.

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD 600 ) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform.

Lactate is a valuable compound used in food, chemical, and pharmaceutical industries. High-value, optically pure L- or D-lactate can be synthesized microbially via specific dehydrogenases. The non-conventional yeast Scheffersomyces stipitis, which is known for fermenting both hexoses and pentoses, is a promising host for biochemical production from lignocellulosic biomass but does not naturally produce lactate. In this study, we engineered S. stipitis to produce lactate by expressing two codon-optimized bacterial L-lactate dehydrogenase genes under the control of strong native promoters. The engineered strain produced 7.42 g/L (0.46 g/g yield) and 11.67 g/L (0.58 g/g yield) lactate from glucose and xylose, respectively. The highest titer, 19.27 g/L (0.52 g/g yield), was achieved from 50 g/L xylose after 74 h. Increasing the fermentation temperature from 28 °C to 32 °C improved yield by 30%, while a neutralizing agent further enhanced yield by 25% and prevented lactate degradation following carbon depletion. Although the wildtype strain produced a significant amount of ethanol on both glucose and xylose, the engineered strain produced ethanol as a side product exclusively on glucose and not on xylose. This phenomenon could be advantageous for biotechnological applications and may reflect shifts in gene expression depending on the carbon source or even on the presence of lactate.

Background Despite the extensive endeavours, developing an effective malaria vaccine remains as a great challenge. Apical membrane antigen 1 (AMA-1) located on the merozoite surface of parasites belonging to the genus Plasmodium is involved in red blood cell invasion. Methods Influenza virus-like particle (VLP) vaccines containing codon-optimized or native (non-codon optimized) AMA-1 from Plasmodium berghei were generated. VLP-induced protective immunity was evaluated in a mouse model. Results Mice immunized with VLP vaccine containing the codon-optimized AMA-1 elicited higher levels of P. berghei -specific IgG and IgG2a antibody responses compared to VLPs containing non-codon optimized AMA-1 before and after challenge infection. Codon-optimized AMA-1 VLP vaccination induced higher levels of CD4 + T cells, CD8 + T cells, B cells, and germinal centre cell responses compared to non-codon optimized AMA-1 VLPs. Importantly, the codon-optimized AMA-1 VLP vaccination showed lower body weight loss, longer survival and a significant decrease in parasitaemia compared to non-codon optimized VLP vaccination. Conclusion Overall, VLP vaccine expressing codon-optimized AMA-1 induced better protective efficacy than VLPs expressing the non-codon optimized AMA-1. Current findings highlight the importance of codon-optimization for vaccine use and its potential involvement in future malaria vaccine design strategies.

Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.

Ustilago scitaminea is the causal agent of sugar-cane smut disease. There is, however, no genetic transformation method for it. Here we report the development of an efficient mutagenesis method based on Agrobacterium tumefaciens-mediated transformation. To improve transformation efficiency, a range of conditions, including the codon-usage preference of the selection marker gene, promoters and the culture conditions for transformation were optimized. A strong promoter to drive marker gene expression, optimized codon usage of selection marker gene, controlled water content and pH of co-culture medium were critical factors affecting transformation efficiency. Our findings provide a useful tool for genetic analysis of this important plant pathogen.

A novel liver-targeting interferon (IFN-CSP) was successfully over-expressed in our previous work. The in vitro and in vivo investigation revealed that IFN-CSP has significant anti-hepatitis B virus (HBV) effect and liver-targeting capacity. However, due to the IFN-CSP tends to form inclusion bodies in recombinant Escherichia coli ( E. coli ), efficient production of the soluble liver-targeting interferon is a challenge. In view of biomedical application, novel strategies for efficiently expressing liver-targeting interferon and overcoming its poor solubility are necessary and important. In the present study, a modified mu-IFN-CSP was designed base on the amino acid mutant of the native IFN-CSP. Meanwhile, the coding sequence of mu-IFN-CSP was optimized for E. coli preferred codon and the induction conditions for expression were optimized by an orthogonal test. After amino acid mutant, codon optimization and induction conditions optimization, the solubility of Mu-IFN-CSP in E. coli was up to 98.4%. The structural comparison and molecular dynamic simulation showed that the Mu-IFN-CSP formed three structure changes and were more stable than the native IFN-CSP. Tissue sections binding assays revealed that Mu-IFN-CSP was also able to specific binding to liver. In vitro anti-HBV activity assays showed that the soluble Mu-IFN-CSP has improved anti-HBV effect in HepG2.2.15 cells compared to the native IFN-CSP. The present study reports for the first time that liver-targeting interferon Mu-IFN-CSP can be expressed as soluble form, and also contributes to further support its application as liver-targeting anti-HBV medicine.

Yersinia pestis PsaA is an adhesin that is synthesized inside macrophages. Here, we evaluated the immune profile of codon-optimized Y. pestis PsaA synthesized in a live recombinant attenuated Salmonella vaccine (RASV) strain χ9558. Oral immunization of BALB/c mice with χ9558(pYA3705) delivering a secreted form of PsaA, elicited a systemic PsaA-specific immunoglobulin G (IgG) response but offered limited protection against lethal challenge with the intranasally introduced Y. pestis CO92 strain. Our results suggest that appropriate fine-tuning of Y. pestis PsaA delivery by RASV could improve its protective role in curtailing plague colonization and infection.

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.