Improved Production of Recombinant Myrosinase in Pichia pastoris

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

Background The production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation, and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements, is the impact of target protein sequon accessibility during glycosylation. Results Here, we explore a series of genetic and process engineering strategies to increase recombinant N-linked glycosylation, mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductase or disulphide-bond isomerase activity. These approaches achieve up to twofold improvement in glycosylation efficiency. Furthermore, we also demonstrate that supplementation with the chemical oxidant cystine enhances the titre of glycoprotein in an oxidoreductase knockout strain by improving total protein production and cell fitness, while at the same time maintaining higher levels of glycosylation efficiency. Conclusions In this study, we demonstrate that improved protein glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native host such as Campylobacter jejuni and mammalian cells. Furthermore, it provides insight into strain engineering and bioprocess strategies, to improve glycoprotein yield and titre, and to avoid physiological burden of unfolded protein stress upon cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production.

Intermolecular latency regulates the essential C-terminal signal peptidase and sortase of the Porphyromonas gingivalis type-IX secretion system

Porphyromonas gingivalis is a keystone pathogen of the human dysbiotic oral microbiome that causes severe periodontitis. It employs a type-IX secretion system (T9SS) to shuttle proteins across the outer membrane (OM) for virulence. Uniquely, T9SS cargoes carry a C-terminal domain (CTD) as a secretion signal, which is cleaved and replaced with anionic lipopolysaccharide by transpeptidation for extracellular anchorage to the OM. Both reactions are carried out by PorU, the only known dual-function, C-terminal signal peptidase and sortase. PorU is itself secreted by the T9SS, but its CTD is not removed; instead, intact PorU combines with PorQ, PorV, and PorZ in the OM-inserted “attachment complex.” Herein, we revealed that PorU transits between active monomers and latent dimers and solved the crystal structure of the ∼260-kDa dimer. PorU has an elongated shape ∼130 Å in length and consists of seven domains. The first three form an intertwined N-terminal cluster likely engaged in substrate binding. They are followed by a gingipain-type catalytic domain (CD), two immunoglobulin-like domains (IGL), and the CTD. In the first IGL, a long “latency β-hairpin” protrudes ∼30 Å from the surface to form an intermolecular β-barrel with β-strands from the symmetric CD, which is in a latent conformation. Homology modeling of the competent CD followed by in vivo validation through a cohort of mutant strains revealed that PorU is transported and functions as a monomer through a C690/H657 catalytic dyad. Thus, dimerization is an intermolecular mechanism for PorU regulation to prevent untimely activity until joining the attachment complex.

Optimisation of Neuraminidase Expression for Use in Drug Discovery by Using HEK293-6E Cells

Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (>3000 EU/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding.

A Jacalin-like lectin-domain-containing protein of Sclerospora graminicola act as an apoplastic virulence effector in plant–oomycete interactions

SUMMARYThe plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant– pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the Jacalin-related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from S. graminicola (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs via protein mass spectrometry analysis of extracellular fluid from S. graminicola-inoculated foxtail millet leaves. SgJRLs consist of a Jacalin-like lectin domain and an N-terminal putative secretion signal, and SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N-terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with GFP localized to the apoplastic space in N. benthamiana leaves. INF1-mediated induction of defense-related genes was suppressed by co-expression of SG06536-GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defense responses.

Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system

Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system named Out is necessary for bacterial virulence. Its study in D. dadantii showed that it secretes a wide range of pectinolytic enzymes. However, the full repertoire of exoproteins it can secrete has not been identified. No secretion signal present on the protein allows the identification of substrates of a type 2 secretion system. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced encoding a protein with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and showed that Svf proteins are present with a variable number of copies, up to three in D. fanghzongdai, in other Pectobacteriaceae. This work opens the way to the study of the role in virulence of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

Comparative expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide

Currently, the Russian market of phospholipase A2 enzyme preparations is represented by commercial preparations of foreign manufacturers: Nagase (Japan) and Maxapal (the Netherlands). However, the growing demand and the need to reduce the cost of production of phospholipase A2 require the development of new super-producers of phospholipase A2. In this connection, the aim of the work is to compare the expression of recombinant phospholipase A2 in Komagataella phaffii depending on the modification of the alpha-factor signaling peptide. The object of the study is the recipient yeast strain Komagataella phaffii X-33. The studies were conducted in accordance with generally accepted norms and approaches. Phospholipase A2 genes from Streptomyces violaceoruber were used for this worK. The target sequences were synthesized in the company "Eurogen" (Russia) and cloned as part of the TE vector pUC57. In the course of the work, the genetic constructs pPICZaA-Pla2 and PPICZmf4iA-Pla2 containing the Streptomyces violaceoruber phospholipase A2 gene were assembled under the native signal a-MF and its modification mf4i. The transformation of the yeast Komagataella phaffii X-33 with the obtained genetic constructs was also carried out. As a result of the conducted studies, it was shown that on average, there were no significant differences in the level of expression and specific activity of recombinant phospholipase A2 in methylotrophic yeast K. Phaffii X-33 when using the native a-MF secretion signal and its modified version mf4i. However, the use of the secretion factor mf4i allows for higher production of phospholipase A2 in individual clones. The obtained data indicate the prospects of using the secretion factor mf4i to create super-producers of enzymes based on yeast K. Phaffii X-33.

The Arabidopsis WRR4A and WRR4B paralogous NLR proteins both confer recognition of multiple Albugo candida effectors

The oomycete Albugo candida causes white blister rust, an important disease of Brassica crops. Distinct races of A. candida are defined by their specificity for infecting different host species. The White Rust Resistance 4 (WRR4) locus in Col-0 accession of Arabidopsis thaliana contains three genes that encode TIR-NLR resistance proteins. The Col-0 alleles of WRR4A and WRR4B confer resistance to at least four A. candida races (2, 7 and 9 from B. juncea, B. rapa and B. oleracea, respectively, and Race 4 from Capsella bursa-pastoris). Resistance mediated by both paralogs can be overcome by Col-0-virulent isolates of Race 4. After comparing repertoires of candidate effectors in resisted and resistance-breaking strains, we used transient co-expression in tobacco or Arabidopsis to identify effectors recognized by WRR4A and WRR4B. A library of CCG effectors from four A. candida races was screened for WRR4A- or WRR4B- dependent elicitation of hypersensitive response (HR). These CCG genes were validated for WRR-dependent HR by bombardment assays in wild type Col-0, wrr4A or wrr4B mutants. Our analysis revealed eight WRR4A-recognized CCGs and four WRR4B-recognized CCGs. Remarkably, the N-terminal region of 100 amino acids after the secretion signal is sufficient for WRR4A recognition of these eight recognized effectors. This multiple recognition capacity potentially explains the broad-spectrum resistance to many A. candida races conferred by WRR4 paralogs.

Phenolic acid-degrading Paraburkholderia prime decomposition in forest soil

Plant-derived phenolic acids are catabolized by soil microorganisms whose activity may enhance the decomposition of soil organic carbon (SOC). We characterized whether phenolic acid-degrading bacteria enhance SOC mineralization in forest soils when primed with 13C-labeled p-hydroxybenzoic acid (pHB). We further tested whether pHB-induced priming could explain differences in SOC content among mono-specific tree plantations in a 70-year-old common garden experiment. pHB addition primed significant losses of SOC (3–13 µmols C g−1 dry wt soil over 7 days) compared to glucose, which reduced mineralization (-3 to -8 µmols C g−1 dry wt soil over 7 days). The principal degraders of pHB were Paraburkholderia and Caballeronia in all plantations regardless of tree species or soil type, with one predominant phylotype (RP11ASV) enriched 23-fold following peak pHB respiration. We isolated and confirmed the phenolic degrading activity of a strain matching this phylotype (RP11T), which encoded numerous oxidative enzymes, including secretion signal-bearing laccase, Dyp-type peroxidase and aryl-alcohol oxidase. Increased relative abundance of RP11ASV corresponded with higher pHB respiration and expression of pHB monooxygenase (pobA), which was inversely proportional to SOC content among plantations. pobA expression proved a responsive measure of priming activity. We found that stimulating phenolic-acid degrading bacteria can prime decomposition and that this activity, corresponding with differences in tree species, is a potential mechanism in SOC cycling in forests. Overall, this study highlights the ecology and function of Paraburkholderia whose associations with plant roots and capacity to degrade phenolics suggest a role for specialized bacteria in the priming effect.