Exploring codon adjustment strategies towards Escherichia coli-based production of viral proteins encoded by HTH1, a novel prophage of the marine bacterium Hypnocyclicus thermotrophus

Marine viral sequence space is immense and presents a promising resource for the discovery of new enzymes interesting for research and biotechnology. However, bottlenecks in the functional annotation of viral genes and soluble heterologous production of proteins hinder access to downstream characterization, subsequently impeding the discovery process. While commonly utilized for the heterologous expression of prokaryotic genes, codon adjustment approaches have not been fully explored for viral genes. Herein, the sequence-based identification of a putative prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, moderately thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. A prophage-associated gene cluster, consisting of 46 protein coding genes, was identified and given the proposed name Hypnocyclicus thermotrophus phage H1 (HTH1). HTH1 was taxonomically assigned to the viral family Siphoviridae, by lowest common ancestor analysis of its genome and phylogeny analyses based on proteins predicted as holin and DNA polymerase. The gene neighbourhood around the HTH1 lytic cassette was found most similar to viruses infecting Gram-positive bacteria. In the HTH1 lytic cassette, an N-acetylmuramoyl-L-alanine amidase (Amidase_2) with a peptidoglycan binding motif (LysM) was identified. A total of nine genes coding for enzymes putatively related to lysis, nucleic acid modification and of unknown function were subjected to heterologous expression in Escherichia coli. Codon optimization and codon harmonization approaches were applied in parallel to compare their effects on produced proteins. Comparison of protein yields and thermostability demonstrated that codon optimization yielded higher levels of soluble protein, but codon harmonization led to proteins with higher thermostability, implying a higher folding quality. Altogether, our study suggests that both codon optimization and codon harmonization are valuable approaches for successful heterologous expression of viral genes in E. coli, but codon harmonization may be preferable in obtaining recombinant viral proteins of higher folding quality.publishedVersio

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared to protein structures of similar proteins. The functional characterisation of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity towards thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterised with a temperature optimum at 85 °C. The extraordinary high melting temperature Tm 102.6 °C confirms fold stability up to approximately 100 °C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology

Peptidazių iš Geobacillus thermoleovorans DSM 15325 kolagenolizinio aktyvumo analizė: M3 proteolizinių fermentų šeimos M3B pošeimio oligopeptidazės ir U32 proteolizinių fermentų šeimos peptidazės charakterizavimas

The full understanding of laws of nature cannot be accomplished without comprehensive characterisation of collagenolysis as collagen is a major structural protein in organisms. The diversity of collagenolytic peptidases from non-pathogenic bacteria remains only fragmentary characterized, whereas the characterization of the diversity of collagenolytic peptidases from eukaryotes and pathogenic bacteria are far more comprehensive. The determined constitutive production of collagenases by moderate thermophile Geobacillus thermoleovorans DSM 15325 confirms the ability of thermophiles to be ever ready for collagen uptake. The identification of constitutive collagenolytic peptidases variety produced by geobacilli lead to substantial development of collagen degradation model and confirms importance of collagenolytic potential for adaptive plasticity of bacteria. Determined M3 family M3B subfamily oligopeptidases, secreted by geobacilli, characteristics indicate oligopeptidases nutritional importance for bacteria during the adaption for environmental changes. Combination of oligopeptidases characteristics also outlines biotechnological applicability of this thermostable hydrolase. Determined U32 family peptidase from geobacilli characteristics ensure objective understanding of U32 family peptidases characteristics and functional importance. U32 family peptidases ability to specifically in vitro interact with dsRNA was never determined for collagenolytic peptidases previously

The collagenolytic activity analysis of Geobacillus thermoleovorans DSM 15325 peptidases: characterization of M3 proteolytic enzymes family M3B subfamily oligopeptidase and U32 proteolytic enzymes family peptidase

The full understanding of laws of nature cannot be accomplished without comprehensive characterisation of collagenolysis as collagen is a major structural protein in organisms. The diversity of collagenolytic peptidases from non-pathogenic bacteria remains only fragmentary characterized, whereas the characterization of the diversity of collagenolytic peptidases from eukaryotes and pathogenic bacteria are far more comprehensive. The determined constitutive production of collagenases by moderate thermophile Geobacillus thermoleovorans DSM 15325 confirms the ability of thermophiles to be ever ready for collagen uptake. The identification of constitutive collagenolytic peptidases variety produced by geobacilli lead to substantial development of collagen degradation model and confirms importance of collagenolytic potential for adaptive plasticity of bacteria. Determined M3 family M3B subfamily oligopeptidases, secreted by geobacilli, characteristics indicate oligopeptidases nutritional importance for bacteria during the adaption for environmental changes. Combination of oligopeptidases characteristics also outlines biotechnological applicability of this thermostable hydrolase. Determined U32 family peptidase from geobacilli characteristics ensure objective understanding of U32 family peptidases characteristics and functional importance. U32 family peptidases ability to specifically in vitro interact with dsRNA was never determined for collagenolytic peptidases previously

Transcription analysis and cloning of u32.002 collagenolytic peptidase from geobacillus lituanicus dsm 15325t

Collagens are one of the most important proteins including their functions and biotechnological application. The biotechnological application of collagens wouldn‘t be possible without the process of collagenolysis which is catalysed by collagenolytic peptidases. The aim of this work was to perform transcriptional analysis of U32.002 (Helicobacter-type) collagenolytic peptidase gene and its cloning as well as basic analysis. The analysis of U32.002 peptidase gene transcription was performed after the extraction of total RNA from G. lituanicus DSM 15325T cells that were in two different stages of growth. Reverse transcription assays were performed after total RNA extraction. After the process of reverse transcription samples of cDNA were used for the diagnostic PGR that was carried out by using five different primers. This gene was cloned with and without putative signal/pro sequence in order to produce the preparation of U32.002 peptidase. Full sequence of U32.002 peptidase gene was cloned into pTZ57R/T and then into the expression vector pET28c(+). U32.002 gene without putative signal/pro sequence was cloned into pJET1.2, and then into pET28c(+). SDS-PAGE and MALDI-TOF analyses were performed in order to determine the fact of expression in E. coli BL21 (DE3). Full scale expression optimisation was performed, as well. The influence of calcium and zinc ions to the structural stability of U32.002 peptidase with putative signal/pro sequence was analysed. The collagenolytic activity of U32.002 peptidase was analysed by using zymography of native collagen, type I. The U32.002 peptidase of G. lituanicus DSM 15325T is transcribed constitutively during the exponential phase of growth, whereas only fragments of U32.002 gene transcript are observed in the later growth phases. It is evident that zinc ions increase structural thermostability of U32.002. The homodimer of U32.002 peptidase with putative signal/pro sequence was able to digest native type I collagen at 50 °C; pH 7,4

Evaluation of the potential of free and immobilized thermophilic bacterial enzymes in the degradation of agro-industrial wastes

Agro-industrial wastes are potential starting materials for the production of useful value-added compounds, including prebiotic oligosaccharides. In this paper, we evaluated the potential of thermophilic bacterial pectin- and xylan-degrading recombinant enzymes for the degradation of the agro-industrial wastes: apple pomace, wheat straw, wheat bran and distillers grains. For the immobilization of pectate lyase and xylanase, three different supports were used. The effect of enzyme immobilization was analyzed in terms of enhanced thermostability and activity against these wastes. For xylanase, the highest thermostability was achieved by immobilization on Sepabeads EC-EP/M. The best activity against bran and grains was obtained by immobilization on Sepabeads EC-HA/M. For pectate lyase, the highest thermostability was achieved by immobilization on Sepabeads EC-EP/M, however, activity against apple pomace pectin was slightly reduced by this immobilization. The length of oligosaccharides produced by both free and immobilized enzymes was also determined

Gene expression and activity analysis of the first thermophilic U32 peptidase

Peptidase family U32 is one of the few whose catalytic type and structure has not yet been described. It is generally accepted that U32 peptidases represent putative collagenases and contribute to the pathogenicity of some bacteria. Meanwhile, U32 peptidases are also found in nonpathogenic bacteria including thermophiles and hyperthermophiles. Here we report cloning of the U32.002 peptidase gene from thermophilic Geobacillus thermoleovorans DSM 15325 and demonstrate expression and characterization of the recombinant protein. It has been determined that U32.002 peptidase is constitutively expressed in the cells of thermophilic G. thermoleovorans DSM 15325. The recombinant oligomeric enzyme showed its activity only against heat-treated collagen. It was unable to degrade albumin, casein, elastin, gelatine and keratin. In contrast to this, the monomeric recombinant protein showed no activity at all. This paper is the first report about the thermophilic U32 peptidase. As the thermophilic bacteria are non-pathogenic, the role of constitutively expressed extracellular collagenolytic U32 peptidase in these bacteria is unclear

HPAEC-PAD analysis for determination of the amino acid profiles in protein fractions from oat flour combined with correction of amino acid loss during hydrolysis

Current derivatization-dependent approaches for amino acid composition analysis of cereal proteins have significant variability due to lack of direct analysis opportunities and loss of amino acids during protein-hydrolysis. To tackle these drawbacks, a novel direct, derivatization-free approach was successfully introduced, using HPAEC-PAD, and applied for analysis of hydrolyzed defatted oat flour and extracted flour protein fractions. The approach ensured reliable detection of amino acids, including L-tryptophan, as well as oxidation products of L-cysteine and L-methionine. A time course study, analysed by nonlinear least-square regression to determine rates of hydrolysis and loss of each amino acid, allowed comparison of the original mass fraction (AA0) of the respective amino acid in the oat flour mixture with the mass fraction obtained after 24 h hydrolysis (AA24). The difference between (AA0) and (AA24) was less than 0.05%, except for L-arginine (0.61%), glycine (0.14%), L-isoleucine (0.27%), and L-tryptophan (0.17%). The (AA0)s obtained corresponded to literature-data, and fitted with the amino acid composition estimated from deduced proteins encoded in the oat genome, except for L-arginine (27%) and L-glutamic acid/L-glutamine (10%). The amino acid composition estimation from sequence data indirectly confirmed that the high presence of L-arginine observed was a result of co-elution with unknown flour components

Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6, displays activity on oat fibre xylan for prebiotic production

Carbohydrate active enzymes are valuable tools in cereal processing to valorise underutilized side streams. By solubilizing hemicellulose and modifying the fibre structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibres; arabinoxylo-oligosaccharides (AXOS). The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature Tm of 61 °C and optimum reaction conditions were determined to 55 °C and pH 6.5 on wheat arabinoxylan (WAX). HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, while the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat β-glucan. In contrast to the commercially available homologue CtXyn5A, HhXyn5A gave a more specific HPAEC–PAD oligosaccharide product profile when using WAX and alkali extracted oat bran fibres as substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modelling of HhXyn5A and docking simulations with ligands XXXA3, XXXA3XX, and X5, concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer AXOS ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to lack of important ligand interacting residues, is suggested to cause the observed differences in substrate specificity and product formation