Increased Thermostability of an Engineered Flavin-Containing Monooxygenase to Remediate Trimethylamine in Fish Protein Hydrolysates.

Protein hydrolysates made from marine by-products are very nutritious but frequently contain trimethylamine (TMA), which has an unattractive fish-like smell. Bacterial trimethylamine monooxygenases can oxidize TMA into the odorless trimethylamine N-oxide (TMAO) and have been shown to reduce TMA levels in a salmon protein hydrolysate. To make the flavin-containing monooxygenase (FMO) Methylophaga aminisulfidivorans trimethylamine monooxygenase (mFMO) more suitable for industrial application, we engineered it using the Protein Repair One-Stop Shop (PROSS) algorithm. All seven mutant variants, containing 8 to 28 mutations, displayed increases in melting temperature of between 4.7°C and 9.0°C. The crystal structure of the most thermostable variant, mFMO_20, revealed the presence of four new stabilizing interhelical salt bridges, each involving a mutated residue. Finally, mFMO_20 significantly outperformed native mFMO in its ability to reduce TMA levels in a salmon protein hydrolysate at industrially relevant temperatures. IMPORTANCE Marine by-products are a high-quality source for peptide ingredients, but the unpleasant fishy odor caused by TMA limits their access to the food market. This problem can be mitigated by enzymatic conversion of TMA into the odorless TMAO. However, enzymes isolated from nature must be adapted to industrial requirements, such as the ability to tolerate high temperatures. This study has demonstrated that mFMO can be engineered to become more thermostable. Moreover, unlike the native enzyme, the best thermostable variant efficiently oxidized TMA in a salmon protein hydrolysate at industrial temperatures. Our results present an important next step toward the application of this novel and highly promising enzyme technology in marine biorefineries.

Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1.

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to -25°C. P355's stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (k cat ) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

A ShK-like Domain from Steinernema carpocapsae with Bioinsecticidal Potential.

Entomopathogenic nematodes are used as biological control agents against a broad range of insect pests. We ascribed the pathogenicity of these organisms to the excretory/secretory products (ESP) released by the infective nematode. Our group characterized different virulence factors produced by Steinernema carpocapsae that underlie its success as an insect pathogen. A novel ShK-like peptide (ScK1) from this nematode that presents high sequence similarity with the ShK peptide from a sea anemone was successfully produced recombinantly in Escherichia coli. The secondary structure of ScK1 appeared redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with an alpha-helical secondary structure. Thermal denaturation of the ScK1 allowed estimating the melting temperature to 59.2 ± 0.1 °C. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 16.9 µM per adult within 24 h. Oral administration of the fusion protein significantly reduced the locomotor activity of insects after 48 h (p < 0.05, Tukey's test). These data show that this nematode expresses insecticidal peptides with potential as next-generation insecticides.

The bone-degrading enzyme machinery: From multi-component understanding to the treatment of residues from the meat industry.

Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm Osedax mucofloris, and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.6%) encoding such enzymes were retrieved. The taxonomic assignation and the median identity of 72.2% to homologous proteins reflect microbial and functional novelty associated to a specialized bone-degrading marine community. Binning suggests that only one generalist hosting all ten targeted activities, working in synergy with multiple specialists hosting a few or individual activities. Collagenases were the most abundant enzyme class, representing 48% of the total hits. A total of 47 diverse enzymes, representing 8 hydrolytic activities, were produced in Escherichia coli, whereof 13 were soluble and active. The biochemical analyses revealed a wide range of optimal pH (4.0-7.0), optimal temperature (5-65 °C), and of accepted substrates, specific to each microbial enzyme. This versatility may contribute to a high environmental plasticity of bone-degrading marine consortia that can be confronted to diverse habitats and bone materials. Through bone-meal degradation tests, we further demonstrated that some of these enzymes, particularly those from Flavobacteriaceae and Marinifilaceae, may be an asset for development of new value chains in the biorefinery industry.

Two-step functional screen on multiple proteinaceous substrates reveals temperature-robust proteases with a broad-substrate range.

To support the bio-based industry in development of environment-friendly processes and products, an optimal toolbox of biocatalysts is key. Although functional screen of (meta)genomic libraries may potentially contribute to identifying new enzymes, the discovery of new enzymes meeting industry compliance demands is still challenging. This is particularly noticeable in the case of proteases, for which the reports of metagenome-derived proteases with industrial applicability are surprisingly limited. Indeed, proteolytic clones have been typically assessed by its sole activity on casein or skim milk and limited to mild screening conditions. Here, we demonstrate the use of six industry-relevant animal and plant by-products, namely bone, feather, blood meals, gelatin, gluten, and zein, as complementary substrates in functional screens and show the utility of temperature as a screening parameter to potentially discover new broad-substrate range and robust proteases for the biorefinery industry. By targeting 340,000 clones from two libraries of pooled isolates of mesophilic and thermophilic marine bacteria and two libraries of microbial communities inhabiting marine environments, we identified proteases in four of eleven selected clones that showed activity against all substrates herein tested after prolonged incubation at 55 °C. Following sequencing, in silico analysis and recombinant expression in Escherichia coli, one functional protease, 58% identical at sequence level to previously reported homologs, was found to readily hydrolyze highly insoluble zein at temperatures up to 50 °C and pH 9-11. It is derived from a bacterial group whose ability to degrade zein was unknown. This study reports a two-step screen resulting in identification of a new marine metagenome-derived protease with zein-hydrolytic properties at common biomass processing temperatures that could be useful for the modern biorefinery industry. KEY POINTS: • A two-step multi-substrate strategy for discovery of robust proteases. • Feasible approach for shortening enzyme optimization to industrial demands. • A new temperature-tolerant protease efficiently hydrolyzes insoluble zein.

Deciphering a Marine Bone-Degrading Microbiome Reveals a Complex Community Effort.

The marine bone biome is a complex assemblage of macro- and microorganisms; however, the enzymatic repertoire to access bone-derived nutrients remains unknown. The bone matrix is a composite material made up mainly of organic collagen and inorganic hydroxyapatite. We conducted field experiments to study microbial assemblages that can use organic bone components as nutrient source. Bovine and turkey bones were deposited at 69 m depth in a Norwegian fjord (Byfjorden, Bergen). Metagenomic sequence analysis was used to assess the functional potential of microbial assemblages from bone surface and the bone-eating worm Osedax mucofloris, which is a frequent colonizer of whale falls and known to degrade bone. The bone microbiome displayed a surprising taxonomic diversity revealed by the examination of 59 high-quality metagenome-assembled genomes from at least 23 bacterial families. Over 700 genes encoding enzymes from 12 relevant enzymatic families pertaining to collagenases, peptidases, and glycosidases putatively involved in bone degradation were identified. Metagenome-assembled genomes (MAGs) of the class Bacteroidia contained the most diverse gene repertoires. We postulate that demineralization of inorganic bone components is achieved by a timely succession of a closed sulfur biogeochemical cycle between sulfur-oxidizing and sulfur-reducing bacteria, causing a drop in pH and subsequent enzymatic processing of organic components in the bone surface communities. An unusually large and novel collagen utilization gene cluster was retrieved from one genome belonging to the gammaproteobacterial genus Colwellia IMPORTANCE Bones are an underexploited, yet potentially profitable feedstock for biotechnological advances and value chains, due to the sheer amounts of residues produced by the modern meat and poultry processing industry. In this metagenomic study, we decipher the microbial pathways and enzymes that we postulate to be involved in bone degradation in the marine environment. We here demonstrate the interplay between different bacterial community members, each supplying different enzymatic functions with the potential to cover an array of reactions relating to the degradation of bone matrix components. We identify and describe a novel gene cluster for collagen utilization, which is a key function in this unique environment. We propose that the interplay between the different microbial taxa is necessary to achieve the complex task of bone degradation in the marine environment.

Fragment Exchange Plasmid Tools for CRISPR/Cas9-Mediated Gene Integration and Protease Production in Bacillus subtilis.

Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool for targeted genome editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial species. A collection of vectors compatible with high-throughput (HTP) fragment exchange (FX) cloning for heterologous expression in Escherichia coli and Bacillus was previously developed. This vector catalogue was through this work supplemented with editing plasmids for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to the stable integration of the gene into the amyE locus of a seven-protease-deficient KO7 strain. The newly engineered B. subtilis strain allowed the successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering, and contribute to a cloning platform for a quick transition from HTP enzyme expression to production through the fermentation of industrially relevant B. subtilis and related strains.IMPORTANCE We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host.

Use of Flavin-Containing Monooxygenases for Conversion of Trimethylamine in Salmon Protein Hydrolysates.

Enzymatic processing of fish by-products for recovery of peptides (hydrolysates) is a promising technology to reach food grade ingredients of high nutritional quality. Despite this, their bitter taste and "fish" odor block implementation in food products and limit their economic potential. Trimethylamine (TMA) is a known contributor to malodor in fish. Current strategies to mask or remove the odor either are not effective or give rise to undesirable side effects. As an alternative approach to remediate TMA, we propose a novel enzymatic strategy to convert TMA into the odorless trimethylamine N-oxide (TMAO) using TMA monooxygenases (Tmms). We identified a diverse set of bacterial Tmms using a sequence similarity network. Purified, recombinant enzymes were assessed for their biocatalytic capacity by monitoring NADPH consumption and TMAO generation. Selected Tmms were subjected to biochemical characterization and investigated for their ability to oxidize TMA in an industry-relevant substrate. From the 45 bacterial Tmm candidates investigated, eight enzymes from four different taxa were selected for their high activity toward TMA. The three most active enzymes were shown to vary in temperature optimum, with the highest being 45°C. Enzymatic activity dropped at high temperatures, likely due to structural unfolding. The enzymes were all active from pH 6.0 to 8.5, with functional stability being lowest around the optimal pH. All three Tmms, given sufficient NADPH cofactor, were found to generate TMAO in the TMA-rich salmon protein hydrolysate. The Tmms serve as unique starting points for engineering and should be useful for guiding process development for marine biorefineries.IMPORTANCE Enzyme-based conversion of marine biomass to high-quality peptide ingredients leaves a distinct smell of "fish" caused by the presence of trimethylamine, which limits their economic potential. We suggest an enzymatic solution for converting trimethylamine to the odorless trimethylamine N-oxide as a novel strategy to improve the smell quality of marine protein hydrolysates. Following a systematic investigation of 45 putative bacterial trimethylamine monooxygenases from several phyla, we expand the repertoire of known active trimethylamine monooxygenases. As a proof-of-concept, we demonstrate that three of these enzymes oxidized trimethylamine in an industry-relevant salmon protein hydrolysate. Our results add new oxidoreductases to the industrial biocatalytic toolbox and provide a new point of departure for enzyme process developments in marine biorefineries.

Development of Versatile Vectors for Heterologous Expression in Bacillus.

The discovery of new enzymes for industrial application relies on a robust discovery pipeline. Such a pipeline should facilitate efficient molecular cloning, recombinant expression and functional screening procedures. Previously, we have developed a vector set for heterologous expression in Escherichia coli. Here, we supplement the catalogue with vectors for expression in Bacillus. The vectors are made compatible with a versatile cloning procedure based on type IIS restriction enzymes and T4 DNA ligase, and encompass an effective counter-selection procedure and complement the set of vectors with options for secreted expression. We validate the system with expression of recombinant subtilisins, which are generally challenging to express in a heterologous system. The complementarity of the E. coli and Bacillus systems allows rapid switching between the two commonly used hosts without comprehensive intermediate cloning steps. The vectors described are not limited to the expression of certain enzymes, but could also be applied for the expression of other enzymes for more generalized enzyme discovery or development.

A rapid solubility-optimized screening procedure for recombinant subtilisins in E. coli.

Subtilisins and other serine proteases are extensively used in the detergent, leather and food industry, and frequently under non-physiological conditions. New proteases with improved performance at extreme temperatures and in the presence of chemical additives may have great economical potential. The increasing availability of genetic sequences from different environments makes homology-based screening an attractive strategy for discovery of new proteases. A prerequisite for large-scale screening of protease-encoding sequences is an efficient screening procedure. We have developed and implemented a screening procedure that encompasses cloning of candidate sequences into multiple expression vectors, cytoplasmic expression in E. coli, and a casein-based functional screen. The procedure is plate-format compatible and can be completed in only four days, starting from the gene of interest in a suitable cloning vector. The expression vector suite includes six vectors with combinations of maltose-binding protein (MBP) or the small ubiquitin-related modifier (SUMO) for increased solubility, and polyhistidine tags for downstream purification. We used enhanced green fluorescent protein and four Bacilli subtilisins to validate the screening procedure and our results show that proteins were expressed, soluble and active. Interestingly, the highest activities were consistently achieved with either MBP or SUMO fusions, thus demonstrating the merit of including solubility tags. In conclusion, the results demonstrate that our approach can be used to efficiently screen for new subtilisins, and suggest that the approach may also be used to screen for proteins with other activities.

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-ß-Lactamase GIM-1.

Metallo-ß-lactamases (MBLs) hydrolyze virtually all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative ß-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with ß-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the ß-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

Cold shock induction of recombinant Arctic environmental genes.

BACKGROUND: Heterologous expression of psychrophilic enzymes in E. coli is particularly challenging due to their intrinsic instability. The low stability is regarded as a consequence of adaptation that allow them to function at low temperatures. Recombinant production presents a significant barrier to their exploitation for commercial applications in industry. METHODS: As part of an enzyme discovery project we have investigated the utility of a cold-shock inducible promoter for low-temperature expression of five diverse genes derived from the metagenomes of marine Arctic sediments. After evaluation of their production, we further optimized for soluble production by building a vector suite from which the environmental genes could be expressed as fusions with solubility tags. RESULTS: We found that the low-temperature optimized system produced high expression levels for all putatively cold-active proteins, as well as reducing host toxicity for several candidates. As a proof of concept, activity assays with one of the candidates, a putative chitinase, showed that functional protein was obtained using the low-temperature optimized vector suite. CONCLUSIONS: We conclude that a cold-shock inducible system is advantageous for the heterologous expression of psychrophilic proteins, and may also be useful for expression of toxic mesophilic and thermophilic proteins where properties of the proteins are deleterious to the host cell growth.

Structural and biochemical characterization of VIM-26 shows that Leu224 has implications for the substrate specificity of VIM metallo-ß-lactamases.

During the last decades antimicrobial resistance has become a global health problem. Metallo-ß-lactamases (MBLs) which are broad-spectrum ß-lactamases that inactivate virtually all ß-lactams including carbapenems, are contributing to this health problem. In this study a novel MBL variant, termed VIM-26, identified in a Klebsiella pneumoniae isolate was studied. VIM-26 belongs to the Verona integron-encoded metallo-ß-lactamase (VIM) family of MBLs and is a His224Leu variant of the well-characterized VIM-1 variant. In this study, we report the kinetic parameters, minimum inhibitory concentrations and crystal structures of a recombinant VIM-26 protein, and compare them to previously published data on VIM-1, VIM-2 and VIM-7. The kinetic parameters and minimum inhibitory concentration determinations show that VIM-26, like VIM-7, has higher penicillinase activity but lower cephalosporinase activity than VIM-1 and VIM-2. The four determined VIM-26 crystal structures revealed mono- and di-zinc forms, where the Zn1 ion has distorted tetrahedral coordination geometry with an additional water molecule (W2) at a distance of 2.6-3.7 Å, which could be important during catalysis. The R2 drug binding site in VIM-26 is more open compared to VIM-2 and VIM-7 and neutrally charged due to Leu224 and Ser228. Thus, the VIM-26 drug binding properties are different from the VIM-2 (Tyr224/Arg228) and VIM-7 (His224/Arg228) structures, indicating a role of these residues in the substrate specificity.

High quality draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a sea squirt in Northern Norway.

Here we report the 8 Mb high quality draft genome of Streptomyces sp. strain AW19M42, together with specific properties of the organism and the generation, annotation and analysis of its genome sequence. The genome encodes 7,727 putative open reading frames, of which 6,400 could be assigned with COG categories. Also, 62 tRNA genes and 8 rRNA operons were identified. The genome harbors several gene clusters involved in the production of secondary metabolites. Functional screening of the isolate was positive for several enzymatic activities, and some candidate genes coding for those activities are listed in this report. We find that this isolate shows biotechnological potential and is an interesting target for bioprospecting.

His224 alters the R2 drug binding site and Phe218 influences the catalytic efficiency of the metallo-ß-lactamase VIM-7.

Metallo-ß-lactamases (MBLs) are the causative mechanism for resistance to ß-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T(m)) of 48.5°C, compared to 55.3 °C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higher Tm (57.1 °C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T(m), 62.9 °C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement.

BACKGROUND: In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening. RESULTS: In our case study, three homologous ß-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccdB-gene. CONCLUSION: We provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.