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The use of the pJB658-based expression system to produce granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-[alpha]2b (IFN-[alpha]2b), and single-chain antibody variable fragment (scFv-phOx) both intracellularly lacking signal sequences and through secretion with signal sequences during high-cell-density cultivation is described.

The unique properties of plasmid pJB658 are utilized to express a host-toxic antibody fragment, scFv-phOx, in high-cell-density cultivation of Escherichia coli. The findings indicate that soluble hapten 2-phenyloxazol-5-one could be produced to a high volumetric yield in high-cell-density cultures of Escherichia coli.

Abstract Background During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples. Results Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis. Conclusions Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.

Interfibrillar phases and bonding in cellulose nanofibril (CNF)-based composites are crucial for materials performances. In this study, we investigated the influence of CNF surface characteristics, the guluronic acid/mannuronic acid ratio, and the molecular weight of alginates on the structure, mechanical, and barrier properties of CNF/alginate composite films. Three types of CNFs with varying surface charges and nanofibril dimensions were prepared from wood pulp fibers. The interfacial bonding through calcium ion cross-linking between alginate and carboxylated CNFs (TCNFs) led to significantly enhanced stiffness and strength due to the formation of an interpenetrating double network, compared to composites from alginates and CNFs with native negative or cationic surface charges. Various alginates extracted from Alaria esculenta (AE) and Laminaria hyperborea (LH) were also examined. The TCNF/AE composite, prepared from alginate with a high mannuronic acid proportion and high molecular weight, exhibited a Young's modulus of 20.3 GPa and a tensile strength of 331 MPa under dry conditions and a Young's modulus of 430 MPa and a tensile strength of 9.3 MPa at the wet state. Additionally, the TCNF/AE composite demonstrated protective properties as a barrier coating for fruit, significantly reducing browning of banana peels and weight loss of bananas stored under ambient conditions.

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α-l-guluronate oligosaccharides with a lower content of β-d-mannuronate residues compared to what can be achieved using chemical depolymerization alone., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024. Published by Elsevier Ltd.)

Alginates are valued in many industries, due to their versatile properties. These polysaccharides originate from brown algae (Phaeophyceae) and some bacteria of the Azotobacter and Pseudomonas genera, consisting of 1 → 4 linked β-d-mannuronic acid (M), and its C5-epimer α-l-guluronic acid (G). Several applications rely on a high G-content, which confers good gelling properties. Because of its high natural G-content (F G  = 0.60-0.75), the alginate from Laminaria hyperborea (LH) has sustained a thriving industry in Norway. Alginates from other sources can be upgraded with mannuronan C-5 epimerases that convert M to G, and this has been demonstrated in many studies, but not applied in the seaweed industry. The present study demonstrates epimerisation directly in the process of alginate extraction from cultivated Saccharina latissima (SL) and Alaria esculenta (AE), and the lamina of LH. Unlike conventional epimerisation, which comprises multiple steps, this in-process protocol can decrease the time and costs necessary for alginate upgrading. In-process epimerisation with AlgE1 enzyme enhanced G-content and hydrogel strength in all examined species, with the greatest effect on SL (F G from 0.44 to 0.76, hydrogel Young's modulus from 22 to 34 kPa). As proof of concept, an upscaled in-process epimerisation of alginate from fresh SL was successfully demonstrated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa . Previous studies suggested that the disruption of calcium (Ca 2+ )-DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca 2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca 2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca 2+ -dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures.

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. KEY POINTS: • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase., (© 2023. The Author(s).)

Alginate is a polysaccharide consisting of β-D-mannuronate (M) and α-L-guluronate (G) produced by brown algae and some bacterial species. Alginate has a wide range of industrial and pharmaceutical applications, owing mainly to its gelling and viscosifying properties. Alginates with high G content are considered more valuable since the G residues can form hydrogels with divalent cations. Alginates are modified by lyases, acetylases, and epimerases. Alginate lyases are produced by alginate-producing organisms and by organisms that use alginate as a carbon source. Acetylation protects alginate from lyases and epimerases. Following biosynthesis, alginate C-5 epimerases convert M to G residues at the polymer level. Alginate epimerases have been found in brown algae and alginate-producing bacteria, predominantly Azotobacter and Pseudomonas species. The best characterised epimerases are the extracellular family of AlgE1-7 from Azotobacter vinelandii(Av). AlgE1-7 all consist of combinations of one or two catalytic A-modules and one to seven regulatory R-modules, but even though they are sequentially and structurally similar, they create different epimerisation patterns. This makes the AlgE enzymes promising for tailoring of alginates to have the desired properties. The present review describes the current state of knowledge regarding alginate-active enzymes with focus on epimerases, characterisation of the epimerase reaction, and how alginate epimerases can be used in alginate production., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of β-lactoglobulin A (β-LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. β-LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on β-LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to β-LgA, as concluded from saturation transfer difference and 1 H-ligand-targeted NMR analyses. Molecular docking using Glide within the Schrödinger suite 2016-1 revealed the orientation of AOSs to only vary slightly at the preferred β-LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the β-LgA complexes may help explain the first steps toward disordered β-LgA alginate coacervate structures., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Plant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54-1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50-55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer. KEY POINTS: • Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity. • Overexpression of the ORFs produced a novel P. rubens strain with improved activity. • The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This chapter describes methods for cultivation and characterization of the growth of Mycolicibacterium spp. mutants in a microbioreactor system in the presence of steroids and/or phytosterols followed by high-throughput mass spectrometry analysis to describe their ability to convert phytosterols into the target steroid androstenedione (AD). We focus on Mycolicibacterium neoaurum NRRL B-3805 ΔkstD which can convert phytosterol into androstenedione (AD) as one of its major steroid products, and mutants thereof with increased tolerance towards this end-product. By using BioLector 48-well plates with optodes at the bottom of each well, bacterial growth can be monitored online despite the turbidity of the growth medium resulting from non-dissolved phytosterol and steroid particles. To cope with the large number of samples that accumulate during growth experiments in microbioreactors and similar formats (e.g., microtiter plates), protocols for extraction and subsequent RapidFire-MS analysis are presented. This reduces the analysis time per sample to 10 s from 10 min required for regular LC-MS analysis., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The chapter describes the bioconversion of phytosterols into androstenedione (AD) by Mycolicibacterium spp. in shake flasks and fermenters, as well as LC-MS-based methods for analysis of phytosterols and steroid products. Phytosterols are derived as by-products of vegetable oil refining and manufacture of wood pulp. They contain the same four-ring nucleus as steroids and may be converted to high-value steroids by removing the sidechain at C17 and minor changes at other sites in the ring structure. Many bacteria, including Mycolicibacterium spp., can degrade phytosterols. Mutants of Mycolicibacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and androstadienedione (ADD). The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic for the cells. Two main strategies have been employed to overcome this challenge: the use of two-phase systems and the addition of chemically modified cyclodextrins. The latter method is used here. Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as hints how to minimize the practical problems due to the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: The aim of this study was to develop a high-throughput robotic microtiter plate-based screening assay for Candida albicans, optimizing growth conditions to replicate the filamentous biofilm growth found in vivo, and subsequently, to demonstrate the assay by evaluating the effect of nutritional drinks alone and in combination with the antifungal amphotericin B (AmB)., Methods and Results: Candida albicans cultured in a defined growth medium showed filamentous growth in microcolonies, mimicking the morphology of oral mucosal disease (oral candidiasis). Addition of nutrient drinks containing fruit juices, fish oil and whey protein to the medium resulted in changed morphology and promoted growth as free yeast cells and with weak biofilm structures. Minimum inhibitory concentration of AmB on the biofilms was 0.25 μg ml -1 , and this was eightfold reduced (0.0038 μg ml -1 ) in the presence of the nutritional drinks., Conclusions: The established assay demonstrated applicability for screening of antifungal and anti-biofilm effects of bioactive substances on C. albicans biofilm with clinically relevant morphology., Significance and Impact of the Study: Candida albicans is the causative agent of the majority of fungal infections globally. The filamentous morphology of C. albicans and the ability to form biofilm are traits known to increase virulence and resistance towards antifungals. This study describes the development of a plate-based in vitro screening method mimicking the filamentous morphology of C. albicans found in vivo. The assay established can thus facilitate efficient antifungal drug discovery and development., (© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Rotting wood is inhabited by a large diversity of bacteria, fungi, and insects with complex environmental relationships. The aim of this work was to study the composition of the microbiota (bacteria and fungi) in decaying wood from a northwest Spanish forest as a source of industrially relevant microorganisms. The analyzed forest is situated in a well-defined biogeographic area combining Mediterranean and temperate macrobioclimates. Bacterial diversity, determined by metagenome analyses, was higher than fungal heterogeneity. However, a total of 194 different cultivable bacterial isolates (mainly Bacillaceae , Streptomycetaceae , Paenibacillaceae , and Microbacteriaceae ) were obtained, in contrast to 343 fungal strains (mainly Aspergillaceae , Hypocreaceae , and Coniochaetaceae ). Isolates traditionally known as secondary metabolite producers, such as Actinobacteria and members of the Penicillium genus, were screened for their antimicrobial activity by the detection of antibiotic biosynthetic clusters and competitive bioassays against fungi involved in wood decay. In addition, the ability of Penicillium isolates to degrade cellulose and release ferulic acid from wood was also examined. These results present decaying wood as an ecologically rich niche and a promising source of biotechnologically interesting microorganisms.

Alginates are linear polysaccharides produced by brown algae and some bacteria and are composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). Alginate has numerous present and potential future applications within industrial, medical and pharmaceutical areas and G rich alginates are traditionally most valuable and frequently used due to their gelling and viscosifying properties. Mannuronan C-5 epimerases are enzymes converting M to G at the polymer level during the biosynthesis of alginate. The Azotobacter vinelandii epimerases AlgE1-AlgE7 share a common structure, containing one or two catalytic A-modules (A), and one to seven regulatory R-modules (R). Despite the structural similarity of the epimerases, they create different M-G patterns in the alginate; AlgE4 (AR) creates strictly alternating MG structures whereas AlgE1 (ARRRAR) and AlgE6 (ARRR) create predominantly G-blocks. These enzymes are therefore promising tools for producing in vitro tailor-made alginates. Efficient in vitro epimerization of alginates requires availability of recombinantly produced alginate epimerases, and for this purpose the methylotrophic yeast Hansenula polymorpha is an attractive host organism. The present study investigates whether H. polymorpha is a suitable expression system for future large-scale production of AlgE1, AlgE4, and AlgE6. H. polymorpha expression strains were constructed using synthetic genes with reduced repetitive sequences as well as optimized codon usage. High cell density cultivations revealed that the largest epimerases AlgE1 (147 kDa) and AlgE6 (90 kDa) are subject to proteolytic degradation by proteases secreted by the yeast cells. However, degradation could be controlled to a large extent either by co-expression of chaperones or by adjusting cultivation conditions. The smaller AlgE4 (58 kDa) was stable under all tested conditions. The results obtained thus point toward a future potential for using H. polymorpha in industrial production of mannuronan C-5 epimerases for in vitro tailoring of alginates., Competing Interests: AD and MP were employed by the ARTES Biotechnology GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tøndervik, Aune, Degelmann, Piontek, Ertesvåg, Skjåk-Bræk and Sletta.)

The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5-epimerases determine the monomer composition by catalyzing the epimerization of β-d-mannuronic acid (M) residues into α-l-guluronic acid (G) residues. The molecular weight is affected by alginate lyases, which catalyze a β-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar, and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5-epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by nuclear magnetic resonance (NMR) spectroscopy. Our results show that calcium promotes lyase activity, whereas NaCl reduces the lyase activity of AlgE7. By using defined polymannuronan (polyM) and polyalternating alginate (polyMG) substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study, we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 functions as the catalytic acid. IMPORTANCE Postharvest valorization and upgrading of algal constituents are promising strategies in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens further applications of bacterial epimerases and lyases in the enzymatic tailoring of alginate polymers.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to polysaccharide lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics data set. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly-β-d-mannuronate, and poly-α-l-guluronate within a broad range of pHs, temperatures, and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as a catalytic acid, indeed is essential for catalysis, whereas mutation of tyrosine Y446, previously proposed to act as a catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l- erythro -5-hexulosuronate), which are formed after ring opening, and, finally, two epimers of a 5-member hemiketal called 4-deoxy-d- manno -hexulofuranosidonate (DHF), formed through intramolecular cyclization of hydrated DEH. The detection and nuclear magnetic resonance (NMR) assignment of these hemiketals refine our current understanding of alginate degradation. IMPORTANCE The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases, and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here, we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at a high industrially relevant temperature (≥60°C), this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation revealed previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases., (Copyright © 2021 American Society for Microbiology.)

Background: Butanol (n-butanol) has been gaining attention as a renewable energy carrier and an alternative biofuel with superior properties to the most widely used ethanol. We performed 48 anaerobic fermentations simultaneously with glucose and xylose as representative lignocellulosic sugars by Clostridium beijerinckii NCIMB 8052 in BioLector® microbioreactors to understand the effect of different sugar mixtures on fermentation and to demonstrate the applicability of the micro-cultivation system for high-throughput anaerobic cultivation studies. We then compared the results to those of similar cultures in serum flasks to provide insight into different setups and measurement methods., Results: ANOVA results showed that the glucose-to-xylose ratio affects both growth and production due to Carbon Catabolite Repression. The study demonstrated successful use of BioLector® system for the first time for screening several media and sugar compositions under anaerobic conditions by using online monitoring of cell mass and pH in real-time and at unprecedented time-resolution. Fermentation products possibly interfered with dissolved oxygen (DO) measurements, which require a careful interpretation of DO monitoring results., Conclusions: The statistical approach to evaluate the microbioreactor setup, and information obtained in this study will support further research in bioreactor and bioprocess design, which are very important aspects of industrial fermentations of lignocellulosic biomass.

Francisellosis in fish is caused by the facultative intracellular Gram-negative bacterial pathogens Francisella noatunensis ssp. noatunensis and Francisella orientalis . The disease is affecting both farmed and wild fish worldwide and no commercial vaccines are currently available. In this study, we tested isolated membrane vesicles (MVs) as possible vaccine candidates based on previous trials in zebrafish ( Danio rerio ) indicating promising vaccine efficacy. Here, the MV vaccine-candidates were tested in their natural hosts, Atlantic cod ( Gadus morhua L.) and Nile tilapia ( Oreochromis niloticus ). Injection of MVs did not display any toxicity or other negative influence on the fish and gene expression analysis indicated an influence on the host immune response. However, unlike in other tested fish species, a protective immunity following vaccine application and immunization period could not be detected in the Atlantic cod or tilapia. Further in vivo studies are required to achieve a better understanding of the development of immunological memory in different fish species.

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae ( Ascophyllum nodosum, Durvillea potatorum , Laminaria hyperborea and Lessonia nigrescens ) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance ( 1 H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

Alginate is a linear copolymer composed of 1→4 linked β-d-mannuronic acid (M) and its epimer α-l-guluronic acid (G). The polysaccharide is first produced as homopolymeric mannuronan and subsequently, at the polymer level, C-5 epimerases convert M residues to G residues. The bacterium Azotobacter vinelandii encodes a family of seven secreted and calcium ion-dependent mannuronan C-5 epimerases (AlgE1-AlgE7). These epimerases consist of two types of structural modules: the A-modules, which contain the catalytic site, and the R-modules, which influence activity through substrate and calcium binding. In this study, we rationally designed new hybrid mannuronan C-5 epimerases constituting the A-module from AlgE6 and the R-module from AlgE4. This led to a better understanding of the molecular mechanism determining differences in MG- and GG-block-forming properties of the enzymes. A long loop with either tyrosine or phenylalanine extruding from the β-helix of the enzyme proved essential in defining the final alginate block structure, probably by affecting substrate binding. Normal mode analysis of the A-module from AlgE6 supports the results., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii , similarly to Pseudomonas aeruginosa . An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction., (Copyright © 2020 Mærk, Jakobsen, Sletta, Klinkenberg, Tøndervik, Ellingsen, Valla and Ertesvåg.)

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) ( Sc LPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface., (© 2019 Jensen et al.)

Fighting bacterial resistance is one of the concerns in modern days, as antibiotics remain the main resource of bacterial control. Data shows that for every antibiotic developed, there is a microorganism that becomes resistant to it. Natural polymers, as the source of antibacterial agents, offer a new way to fight bacterial infection. The advantage over conventional synthetic antibiotics is that natural antimicrobial agents are biocompatible, non-toxic, and inexpensive. Chitosan is one of the natural polymers that represent a very promising source for the development of antimicrobial agents. In addition, chitosan is biodegradable, non-toxic, and most importantly, promotes wound healing, features that makes it suitable as a starting material for wound dressings. This paper reviews the antimicrobial properties of chitosan and describes the mechanisms of action toward microbial cells as well as the interactions with mammalian cells in terms of wound healing process. Finally, the applications of chitosan as a wound-dressing material are discussed along with the current status of chitosan-based wound dressings existing on the market., Competing Interests: The authors declare no conflict of interest.

Chitin deacetylases are a group of enzymes catalysing the conversion of chitin to chitosan. Obtaining chitosan with established deacetylation degree and pattern is important for biomedical and biotechnological applications. Understandings of the structural properties of chitin deacetylases and the specificity of their interactions with chitin may conduct to the control of the pattern of deacetylation of chitosan. Our study is focused on the characterization and comparison of the structural and physicochemical properties of chitin deacetylases from fungi and marine bacteria. Despite the low sequences identity for the investigated chitin deacetylases, there are amino acids belonging to their active sites that are strongly conserved. Moreover, they reveal an increased structural similarity of their catalytic domains, reflecting the common biological function of these enzymes. The studied enzymes present dissimilar local physicochemical properties of their catalytic cavities that could be responsible of their distinct deacetylation patterns. Molecular docking studies reflect that deacetylation efficiency is also distinct for the chitin and partially deacetylated chitin oligomers and that N-acetylglucosamine units and some partially deacetylated chitin oligomers could have inhibitory effect against chitin deacetylases belonging to fungi and marine bacteria., (Copyright © 2019 Elsevier Inc. All rights reserved.)

With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown that alginate from algal sources all contain a fraction of very long G-blocks (VLG), that is, consecutive sequences of guluronic acid (G) residues of more than 100 residues. By comparing the gelling properties of these materials with in vitro epimerized polymannuronic acid (poly-M) with shorter G-blocks, but comparable with the G-content, we could demonstrate that VLG have a large influence on gelling properties. Hypothesized to function as reinforcement bars, VLG prevents the contraction of the gels during formation (syneresis) and increases the Young's modulus (strength of the gel). Here we report that these VLG structures are also present in alginates from Azotobacter vinelandii and that these polymers consequently form stable, low syneretic gels with calcium, comparable in mechanical strength to algal alginates with the similar monomeric composition. The bacterium expresses seven different extracellular mannuronan epimerases (AlgE1-AlgE7), of which only the bifunctional epimerase AlgE1 seems to be able to generate the long G-blocks when acting on poly-M. The data implies evidence for a processive mode of action and the necessity of two catalytic sites to obtain the observed epimerization pattern. Furthermore, poly-M epimerized with AlgE1 in vitro form gels with comparable or higher rigidity and gel strength than gels made from brown seaweed alginate with matching G-content. These findings strengthen the viability of commercial alginate production from microbial sources.

A family of seven mannuronan C5-epimerases (AlgE1-AlgE7) produced by Azotobacter vinelandii is able to convert β-d-mannuronate (M) to its epimer α-l-guluronate (G) in alginates. Even sharing high sequence homology at the amino acid level, they produce distinctive epimerization patterns. The introduction of new G-blocks into the polymer by in vitro epimerization is a strategy to improve the mechanical properties of alginates as biomaterial. However, epimerization is hampered when the substrate is modified or in the gelled state. Here it is presented how native and engineered epimerases of varying size perform on steric hindered alginate substrates (modified or as hydrogels). Reducing the size of the epimerases enables the epimerization of otherwise inaccessible regions in the alginate polymer. Even though the reduction of the size affects the productive binding of epimerases to the substrate, and hence their activity, the smaller epimerases could more freely diffuse into calcium-alginate hydrogel and epimerize it., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although nanotoxicology has become a large research field, assessment of cytotoxicity is often reduced to analysis of one cell line only. Cytotoxicity of nanoparticles is complex and should, preferentially, be evaluated in several cell lines with different methods and on multiple nanoparticle batches. Here we report the toxicity of poly(alkyl cyanoacrylate) nanoparticles in 12 different cell lines after synthesizing and analyzing 19 different nanoparticle batches and report that large variations were obtained when using different cell lines or various toxicity assays. Surprisingly, we found that nanoparticles with intermediate degradation rates were less toxic than particles that were degraded faster or more slowly in a cell-free system. The toxicity did not vary significantly with either the three different combinations of polyethylene glycol surfactants or with particle size (range 100-200 nm). No acute pro- or anti-inflammatory activity on cells in whole blood was observed., Competing Interests: The authors declare no conflict of interest.

Background: Polysaccharides often are necessary components of bacterial biofilms and capsules. Production of these biopolymers constitutes a drain on key components in the central carbon metabolism, but so far little is known concerning if and how the cells divide their resources between cell growth and production of exopolysaccharides. Alginate is an industrially important linear polysaccharide synthesized from fructose 6-phosphate by several bacterial species. The aim of this study was to identify genes that are necessary for obtaining a normal level of alginate production in alginate-producing Pseudomonas fluorescens., Results: Polysaccharide biosynthesis is costly, since it utilizes nucleotide sugars and sequesters carbon. Consequently, transcription of the genes necessary for polysaccharide biosynthesis is usually tightly regulated. In this study we used an engineered P. fluorescens SBW25 derivative where all genes encoding the proteins needed for biosynthesis of alginate from fructose 6-phosphate and export of the polymer are expressed from inducible Pm promoters. In this way we would avoid identification of genes merely involved in regulating the expression of the alginate biosynthetic genes. The engineered strain was subjected to random transposon mutagenesis and a library of about 11500 mutants was screened for strains with altered alginate production. Identified inactivated genes were mainly found to encode proteins involved in metabolic pathways related to uptake and utilization of carbon, nitrogen and phosphor sources, biosynthesis of purine and tryptophan and peptidoglycan recycling., Conclusions: The majority of the identified mutants resulted in diminished alginate biosynthesis while cell yield in most cases were less affected. In some cases, however, a higher final cell yield were measured. The data indicate that when the supplies of fructose 6-phosphate or GTP are diminished, less alginate is produced. This should be taken into account when bacterial strains are designed for industrial polysaccharide production.

The chapter describes the bioconversion of phytosterols to androstenedione (AD) with Mycobacterium spp. in shake flasks and fermenters, as well as LC-MS based methods for analysis of phytosterols and steroid products.Phytosterols are derived as a by-product of vegetable oil refining and of manufacture of wood pulp. Phytosterols contain the same four-ring nucleus as steroids, and may be converted to high-value steroids by removing the side chain at C17 and minor changes at other sites in the ring structure.Many bacteria, including Mycobacterium spp., are able to degrade phytosterols. Mutants of Mycobacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and/or androstadienedione (ADD).The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic to cells.Two main strategies have been employed to overcome this challenge: the use of two-phase systems, and the addition of chemically modified cyclodextrins. The latter method is used here.Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as suggestions to minimize the practical problems caused by the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described.

Activation of silent biosynthetic gene clusters in Streptomyces bacteria via overexpression of cluster-specific regulatory genes is a promising strategy for the discovery of novel bioactive secondary metabolites. This approach was used in an attempt to activate a cryptic gene cluster in a marine sponge-derived Streptomyces albus PVA94-07 presumably governing the biosynthesis of peptide-based secondary metabolites. While no new peptide-based metabolites were detected in the recombinant strain, it was shown to produce at least four new analogues of deferoxamine with additional acyl and sugar moieties, for which chemical structures were fully elucidated. Biological activity tests of two of the new deferoxamine analogues revealed weak activity against Escherichia coli. The gene knockout experiment in the gene cluster targeted for activation, as well as overexpression of certain genes from this cluster did not have an effect on the production of these compounds by the strain overexpressing the regulator. It seems plausible that the production of such compounds is a response to stress imposed by the production of an as-yet unidentified metabolite specified by the cryptic cluster.

Human chitotriosidase (HCHT) is one of two active glycoside hydrolase family 18 chitinases produced by humans. The enzyme is associated with several diseases and is thought to play a role in the anti-parasite responses of the innate immune system. HCHT occurs in two isoforms, one 50 kDa (HCHT50) and one 39 kDa variant (HCHT39). Common for both isoforms is a catalytic domain with the (β/α)8 TIM barrel fold. HCHT50 has an additional linker-region, followed by a C-terminal carbohydrate-binding module (CBM) classified as CBM family 14 in the CAZy database. To gain further insight into enzyme functionality and especially the effect of the CBM, we expressed both isoforms and compared their catalytic properties on chitin and high molecular weight chitosans. HCHT50 degrades chitin faster than HCHT39 and much more efficiently. Interestingly, both HCHT50 and HCHT39 show biphasic kinetics on chitosan degradation where HCHT50 is faster initially and HCHT39 is faster in the second phase. Moreover, HCHT50 produces distinctly different oligomer distributions than HCHT39. This is likely due to increased transglycosylation activity for HCHT50 due the CBM extending the positive subsites binding surface and therefore promoting transglycosylation. Finally, studies with both chitin and chitosan showed that both isoforms have a similarly low degree of processivity. Combining functional and structural features of the two isoforms, it seems that HCHT combines features of exo-processive and endo-nonprocessive chitinases with the somewhat unusual CBM14 to reach a high degree of efficiency, in line with its alleged physiological task of being a "complete" chitinolytic machinery by itself., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The bacterium Pseudomonas fluorescens switches to an alginate-producing phenotype when the pleiotropic anti-sigma factor MucA is inactivated. The inactivation is accompanied by an increased biomass yield on carbon sources when grown under nitrogen-limited chemostat conditions. A previous metabolome study showed significant changes in the intracellular metabolite concentrations, especially of the nucleotides, in mucA deletion mutants compared to the wild-type. In this study, the P. fluorescens SBW25 wild-type and an alginate non-producing mucA- ΔalgC double-knockout mutant are investigated through model-based (13)C-metabolic flux analysis ((13)C-MFA) to explore the physiological consequences of MucA inactivation at the metabolic flux level. Intracellular metabolite extracts from three carbon labelling experiments using fructose as the sole carbon source are analysed for (13)C-label incorporation in primary metabolites by gas and liquid chromatography tandem mass spectrometry., Results: From mass isotopomer distribution datasets, absolute intracellular metabolic reaction rates for the wild type and the mutant are determined, revealing extensive reorganisation of carbon flux through central metabolic pathways in response to MucA inactivation. The carbon flux through the Entner-Doudoroff pathway was reduced in the mucA- ΔalgC mutant, while flux through the pentose phosphate pathway was increased. Our findings also indicated flexibility of the anaplerotic reactions through down-regulation of the pyruvate shunt in the mucA- ΔalgC mutant and up-regulation of the glyoxylate shunt., Conclusions: Absolute metabolic fluxes and metabolite levels give detailed, integrated insight into the physiology of this industrially, medically and agriculturally important bacterial species and suggest that the most efficient way of using a mucA- mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation.

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.

Phenazine natural products/compounds possess a range of biological activities, including anti-microbial and cytotoxic, making them valuable starting materials for drug development in several therapeutic areas. These compounds are biosynthesized almost exclusively by eubacteria of both terrestrial and marine origins from erythrose 4-phosphate and phosphoenol pyruvate via the shikimate pathway. In this paper, we report isolation of actinomycete bacteria from marine sediment collected in the Trondheimfjord, Norway. Screening of the isolates for biological activity produced several "hits", one of which was followed up by identification and purification of the active compound from the actinomycete bacterium Streptosporangium sp. The purified compound, identified as 1,6-dihydroxyphenazine-5,10-dioxide (iodinin), was subjected to extended tests for biological activity against bacteria, fungi and mammalian cells. In these tests, the iodinin demonstrated high anti-microbial and cytotoxic activity, and was particularly potent against leukaemia cell lines. This is the first report on the isolation of iodinin from a marine-derived Streptosporangium.

The polysaccharide alginate is produced by brown algae and some bacteria and is composed of the two monomers, β-D-mannuronic acid (M) and α-L-guluronic acid (G). The distribution and composition of M/G are important for the chemical-physical properties of alginate and result from the activity of a family of mannuronan C-5 epimerases that converts M to G in the initially synthesized polyM. Traditionally, G-rich alginates are commercially most interesting due to gelling and viscosifying properties. From a library of mutant epimerases we have isolated enzymes that introduce a high level of G-blocks in polyM more efficiently than the wild-type enzymes from Azotobacter vinelandii when employed for in vitro epimerization reactions. This was achieved by developing a high-throughput screening method to discriminate between different alginate structures. Furthermore, genetic and biochemical analyses of the mutant enzymes have revealed structural features that are important for the differences in epimerization pattern found for the various epimerases.

Background: Alginate is an industrially important polysaccharide, currently produced commercially by harvesting of marine brown sea-weeds. The polymer is also synthesized as an exo-polysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter, and these organisms may represent an alternative alginate source in the future. The current work describes an attempt to rationally develop a biological system tuned for very high levels of alginate production, based on a fundamental understanding of the system through metabolic modeling supported by transcriptomics studies and carefully controlled fermentations., Results: Alginate biosynthesis in Pseudomonas fluorescens was studied in a genomics perspective, using an alginate over-producing strain carrying a mutation in the anti-sigma factor gene mucA. Cells were cultivated in chemostats under nitrogen limitation on fructose or glycerol as carbon sources, and cell mass, growth rate, sugar uptake, alginate and CO(2) production were monitored. In addition a genome scale metabolic model was constructed and samples were collected for transcriptome analyses. The analyses show that polymer production operates in a close to optimal way with respect to stoichiometric utilization of the carbon source and that the cells increase the uptake of carbon source to compensate for the additional needs following from alginate synthesis. The transcriptome studies show that in the presence of the mucA mutation, the alg operon is upregulated together with genes involved in energy generation, genes on both sides of the succinate node of the TCA cycle and genes encoding ribosomal and other translation-related proteins. Strains expressing a functional MucA protein (no alginate production) synthesize cellular biomass in an inefficient way, apparently due to a cycle that involves oxidation of NADPH without ATP production. The results of this study indicate that the most efficient way of using a mucA mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation., Conclusions: The insights gained in this study should be very useful for a future efficient production of microbial alginates.

Despite recent improvement in therapy, acute myeloid leukemia (AML) is still associated with high lethality. In the presented study, we analyzed the bioactive compound iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from a marine actinomycetes bacterium for the ability to induce cell death in a range of cell types. Iodinin showed selective toxicity to AML and acute promyelocytic (APL) leukemia cells, with EC50 values for cell death up to 40 times lower for leukemia cells when compared with normal cells. Iodinin also successfully induced cell death in patient-derived leukemia cells or cell lines with features associated with poor prognostic such as FLT3 internal tandem duplications or mutated/deficient p53. The cell death had typical apoptotic morphology, and activation of apoptotic signaling proteins like caspase-3. Molecular modeling suggested that iodinin could intercalate between bases in the DNA in a way similar to the anti-cancer drug daunorubicin (DNR), causing DNA-strand breaks. Iodinin induced apoptosis in several therapy-resistant AML-patient blasts, but to a low degree in peripheral blood leukocytes, and in contrast to DNR, not in rat cardiomyoblasts. The low activity towards normal cell types that are usually affected by anti-leukemia therapy suggests that iodinin and related compounds represent promising structures in the development of anti-cancer therapy.

We previously designed the consensus signal peptide (CSP) and demonstrated that it can be used to strongly stimulate heterologous protein production in Escherichia coli. A comparative study using CSP and two bacterial signal sequences, pelB and ompA, showed that the effect of signal sequences on both expression level and translocation efficiency can be highly protein specific. We report here the generation of CSP mutant libraries by a combinatorial mutagenesis approach. Degenerated CSP oligonucleotides were cloned in frame with the 5' end of the bla gene, encoding the mature periplasmic β-lactamase released from its native signal sequence. This novel design allows for a direct selection of improved signal sequences that positively affect the expression level and/or translocation efficiency of β-lactamase, based on the ampicillin tolerance level of the E. coli host cells. By using this strategy, 61 different CSP mutants with up to 8-fold-increased ampicillin tolerance level and up to 5.5-fold-increased β-lactamase expression level were isolated and characterized genetically. A subset of the CSP mutants was then tested with the alternative reporter gene phoA, encoding periplasmic alkaline phosphatase (AP), resulting in an up to 8-fold-increased production level of active AP protein in E. coli. Moreover, it was demonstrated that the CSP mutants can improve the production of the medically important human interferon α2b under high-cell-density cultivations. Our results show that there is a clear potential for improving bacterial signal sequences by using combinatorial mutagenesis, and bioinformatics analyses indicated that the beneficial mutations could not be rationally predicted.

The uncontrolled, often inappropriate use of antibiotics has resulted in the increasing prevalence of antibiotic-resistant pathogens, with major cost implications for both United States and European health care systems. We describe the utilization of a low-molecular-weight oligosaccharide nanomedicine (OligoG), based on the biopolymer alginate, which is able to perturb multidrug-resistant (MDR) bacteria by modulating biofilm formation and persistence and reducing resistance to antibiotic treatment, as evident using conventional and robotic MIC screening and microscopic analyses of biofilm structure. OligoG increased (up to 512-fold) the efficacy of conventional antibiotics against important MDR pathogens, including Pseudomonas, Acinetobacter, and Burkholderia spp., appearing to be effective with several classes of antibiotic (i.e., macrolides, β-lactams, and tetracyclines). Using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), increasing concentrations (2%, 6%, and 10%) of alginate oligomer were shown to have a direct effect on the quality of the biofilms produced and on the health of the cells within that biofilm. Biofilm growth was visibly weakened in the presence of 10% OligoG, as seen by decreased biomass and increased intercellular spaces, with the bacterial cells themselves becoming distorted and uneven due to apparently damaged cell membranes. This report demonstrates the feasibility of reducing the tolerance of wound biofilms to antibiotics with the use of specific alginate preparations.