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Plant molecular farming (PMF) has been promoted since the 1990s as a rapid, cost-effective and (most of all) safe alternative to the cultivation of bacteria or animal cells for the production of biopharmaceutical proteins. Numerous plant species have been investigated for the production of a broad range of protein-based drug candidates. The inherent safety of these products is frequently highlighted as an advantage of PMF because plant viruses do not replicate in humans and vice versa. However, a more nuanced analysis of this principle is required when considering other pathogens because toxic compounds pose a risk even in the absence of replication. Similarly, it is necessary to assess the risks associated with the host system (e.g., the presence of toxic secondary metabolites) and the production approach (e.g., transient expression based on bacterial infiltration substantially increases the endotoxin load). This review considers the most relevant host systems in terms of their toxicity profile, including the presence of secondary metabolites, and the risks arising from the persistence of these substances after downstream processing and product purification. Similarly, we discuss a range of plant pathogens and disease vectors that can influence product safety, for example, due to the release of toxins. The ability of downstream unit operations to remove contaminants and process-related toxic impurities such as endotoxins is also addressed. This overview of plant-based production, focusing on product safety aspects, provides recommendations that will allow stakeholders to choose the most appropriate strategies for process development.

Spider silk is a natural fiber known as "biosteel" with the strongest composite performance, such as high tensile strength and toughness. It is also equipped with excellent biocompatibility and shape memory ability, thus shows great potential in many fields such as biomedicine and tissue engineering. Spider silk is composed of macromolecular spidroin with rich structural diversity. The characteristics of the primary structure of natural spidroin, such as the high repeatability of amino acids in the core repetitive region, the high content of specific amino acids, the large molecular weight, and the high GC content of the spidroin gene, have brought great difficulties in heterologous expression. This review discusses focuses on the relationship between the featured motifs of the microcrystalline region in the repetitive unit of spidroin and its structure, as well as the spinning performance and the heterologous expression. The optimization design for the sequence of spidroin combined with heterologous expression strategy has greatly promoted the development of the biosynthesis of spider silk proteins. This review may facilitate the rational design and efficient synthesis of recombinant spidroin.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus betacoronavirus within the family coronaviridae, which invades the central nervous system (CNS) via peripheral nervous system and causes encephalomyelitis or vomiting and wasting disease (VWD) in sucking piglets. Up to now, although few complete nucleotide sequences of PHEV have been reported, they are not annotated. This study aimed to illuminate genome characterization, phylogenesis and pathogenicity of the PHEV/2008 strain. The full length of the PHEV/2008 strain genome was 30,684 bp, with a G + C content of 37.27%. The genome included at a minimum of 11 predicted open reading frames (ORFs) flanked by 5′ and 3′ untranslated regions (UTR) of 211 and 289 nucleotides. The replicase polyproteins pp1a and pp1ab, which had 4382 and 7094 amino acid residues, respectively, were predicted to be cleaved into 16 subunits by two viral proteinases. Phylogenetic analysis based on the complete genome sequence revealed that PHEV/2008 strain was genetically different from other known PHEV types, which represented a novel genotype (GI-1). In addition, we found that PHEV/2008 was neurotropic and highly pathogenic to 4-week-old BALB/c mice. Taken together, this is the first detailed annotated, complete genomic sequence of a new genotype PHEV strain in China. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli , Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. [ABSTRACT FROM AUTHOR]

Autonomous retrotransposons, in which replication and transcription are coupled, encode the essential gag and pol genes as a fusion or separate overlapping form(s) that are expressed in single transcripts regulated by a common upstream promoter. The element-specific expression strategies have driven development of relevant translational recoding mechanisms including ribosomal frameshifting to satisfy the protein stoichiometry critical for the assembly of infectious virus-like particles. Retrotransposons with different recoding strategies exhibit a mosaic distribution pattern across the diverse families of reverse transcribing elements, even though their respective distributions are substantially skewed towards certain family groups. However, only a few investigations to date have focused on the emergence of retrotransposons evolving novel expression strategy and causal genetic drivers of the structural variants. In this study, the bulk of genomic and transcribed sequences of a Ty3/gypsy -like CsRn1 retrotransposon in Clonorchis sinensis were analyzed for the comprehensive examination of its expression strategy. Our results demonstrated that structural variants with single open reading frame (ORF) have recurrently emerged from precedential CsRn1 copies encoding overlapping gag - pol ORFs by a single-nucleotide insertion in an upstream region of gag stop codon. In the parasite genome, some of the newly evolved variants appeared to undergo proliferative burst as active master lineages together with their ancestral copies. The genetic event was similarly observed in Opisthorchis viverrini , the closest neighbor of C. sinensis , whereas the resulting structural variants might have failed to overcome purifying selection and comprised minor remnant copies in the Opisthorchis genome. [ABSTRACT FROM AUTHOR]

The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. [ABSTRACT FROM AUTHOR]

Densoviruses (DVs) are a group of viruses that contain a linear single-stranded DNA genome between 4–6 kb in length. Herein, we report a DV with a 5,480-nt genome, isolated from tobacco aphid (Myzus persicae nicotianae Blackman), named MpnDV. Unlike the genome of M. persicae densovirus (MpDV), which possesses five open reading frames (ORFs), the genome of MpnDV contains four putative ORFs—the nonstructural protein 1 (NS1) and NS2 from MpnDV are 98 - and 52-amino acids longer than those of MpDV, respectively, at the N-terminus, and the capsid proteins (VP) are 102 amino acids longer at the C-terminus than those of MpDV. Mapping of the MpnDV transcripts by RACE method indicated that the ORF of NS2 started at nt 340 and the right two putative ORFs were combined together by deleting two introns, one of 95 bp located at nt 2,932–3,026 and the other of 145 bp located at nt 4,715–4,859, suggesting transcript mapping was necessary for analyzing of genome organization. Alignment analysis indicated that MpnDV shows 97% sequence identity with MpDV, and that the shortened ORFs resulted from nucleotide indels, suggesting MpnDV and MpDV were two isolates of the same virus. Thus, MpnDV and MpDV clustered together in a tree-based analysis. The prevalence of MpnDV infection in wild populations of tobacco aphids differed among 29 locations; 34% of the 622 individuals sampled were positive. The genome organization, transcript strategy, and widespread distribution in wild populations suggest that MpnDV might possess a biological function different from that of MpDV. [ABSTRACT FROM AUTHOR]

Highlights: [•] Three transcripts of JcDNV, an unspliced 2.5kb VP mRNA encoding capsid proteins and two NS mRNAs, were identified. [•] The VP mRNA has a very short (3nt) 5′ untranslated region whereas the NS mRNAs have 83nt (unspliced) and 86nt (Spliced) 5′ UTR. [•] The VP and NS transcripts co-terminate in the middle of their respective strand and possess an overlapping sequence of 61nt at their 3′ termini. [•] 4 capsid proteins are generated by a leaky scanning mechanism. [Copyright &y& Elsevier]

Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi's L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO of 0.5 mM to the medium, and shaking off while cell mass reached to OD of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2′-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains. [ABSTRACT FROM AUTHOR]

The genome of Mythimna loreyi densovirus (MlDNV) was cloned into the pEMBL(19)+ vector. This clone was infectious upon transfection, both in LD cells and larvae. The genome possessed ITRs of 543 nucleotides of which the distal 126 nucleotides could form a hairpin. The nonstructural (NS) and structural (VP) genes were located on the 5′-halves of the complementary strands and their transcripts started 27 nts downstream of the ITRs. These transcripts had an overlap of 57 nucleotides in middle of the genome. The NS cassette consisted of three genes with NS1 and the overlapping NS2 downstream of NS3. The NS3 gene was spliced out from a fraction of the NS transcripts to allow leaky scanning translation of the downstream bicistronic NS1 and NS2 genes. The four VPs were similarly generated by leaky scanning translation of unspliced mRNA. The 5′-untranslated region of the VP transcript was only seven nucleotides long. [Copyright &y& Elsevier]