Updated component analysis method for naturally occurring sophorolipids from Starmerella bombicola.

Sophorolipids (SLs) are promising glycolipid biosurfactants as they are easily produced and functional. SLs from microorganisms are comprised of mixtures of multiple derivatives that have different structures and properties, including well-known acidic and lactonic SL (ASLs and LSLs, respectively). In this study, we established a method for analyzing all SL derivatives in the products of Starmerella bombicola, a typical SL-producing yeast. Detailed component analyses of S. bombicola products were carried out using reversed-phase high-performance liquid chromatography and mass spectrometry. Methanol was used as the eluent as it is a good solvent for all SL derivatives. With this approach, it was possible to not only quantify the ratio of the main components of ASL, LSL, and SL glycerides but also confirm trace components such as SL mono-glyceride and bola-form SL (sophorose at both ends); notably, this is the first time these components have been isolated and identified successfully in naturally occurring SLs. In addition, our results revealed a novel SL derivative in which a fatty acid is bonded in series to the ASL, which had not been reported previously. Using the present analysis method, it was possible to easily track compositional changes in the SL components during culture. Our results showed that LSL and ASL are produced initially and that SL glycerides accumulate from the middle stage during the fermentation process. KEY POINTS: • An easy and detailed component analysis method for sophorolipids (SLs) is introduced. • Multiple SL derivatives were identified different from known SLs. • A novel hydrophobic acidic SL was isolated and characterized.

Biosynthetic ability of diverse basidiomycetous yeast strains to produce the natural antioxidant ergothioneine.

Sixteen strains of basidiomycetous yeasts were evaluated for their capability to produce ergothioneine (EGT), an amino acid derivative with strong antioxidant activity. The cells were cultured in either two synthetic media or yeast mold (YM) medium for 72 h, after which cytosolic constituents were extracted from the cells with hot water. After analyzing the extracts via liquid chromatography-mass spectrometry (LC-MS), we found that all strains produced varying amounts of EGT. The EGT-producing strains, including Ustilago siamensis, Anthracocystis floculossa, Tridiomyces crassus, Ustilago shanxiensis, and Moesziomyces antarcticus, were subjected to flask cultivation in YM medium. U. siamensis CBS9960 produced the highest amount of EGT at 49.5 ± 7.0 mg/L after 120 h, followed by T. crassus at 30.9 ± 1.8 mg/L. U. siamensis was also cultured in a jar fermenter and produced slightly higher amounts of EGT than under flask cultivation. The effects of culture conditions, particularly the addition of precursor amino acids, on EGT production by the selected strains were also evaluated. U. siamensis showed a 1.5-fold increase in EGT production with the addition of histidine, while U. shanxiensis experienced a 1.8-fold increase in EGT production with the addition of methionine. These results suggest that basidiomycetous yeasts could serve an abundant source for natural EGT producers.

First direct evidence for direct cell-membrane penetrations of polycationic homopoly(amino acid)s produced by bacteria.

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. Although the biological significance of polycationic homopoly(amino acid)s remains unclear, increasing attention has recently been focused on their potential use to achieve cellular internalization. Here, for the first time, we provide direct evidence that two representative bacterial polycationic isopeptides, ε-poly-L-α-lysine (ε-PαL) and ε-oligo-L-ß-lysine (ε-OßL), were internalized into mammalian cells by direct cell-membrane penetration and then diffused throughout the cytosol. In this study, we used clickable ε-PαL and ε-OßL derivatives carrying a C-terminal azide group, which were enzymatically produced and then conjugated with a fluorescent dye to analyze subcellular localization. Interestingly, fluorescent proteins conjugated with the clickable ε-PαL or ε-OßL were also internalized into cells and diffused throughout the cytosol. Notably, a Cre recombinase conjugate with ε-PαL entered cells and mediated the Cre/loxP recombination, and ε-PαL was found to deliver a full-length IgG antibody to the cytosol and nucleus.

Synthesis of Mannosylerythritol Lipid Analogues and their Self-Assembling Properties, Recovery Effects on Damaged Skin Cells, and Antibacterial Activity.

Synthesis of three types of purpose-designed mannosylerythritol lipid (MEL)-D analogues with decanoyl groups, ß-GlcEL-D, α-GlcEL-D, and α-MEL-D, was accomplished utilizing our boron-mediated aglycon delivery (BMAD) methods. Their self-assembling properties, recovery effects on damaged skin cells, and antibacterial activity were evaluated. It was revealed, for the first time, that α-GlcEL-D and α-MEL-D only generated giant vesicles, indicating that slight differences in the steric configuration of an erythritol moiety and fatty acyl chains affect the ability to form vesicles. Analogue α-MEL-D exhibited significant recovery effects on damaged skin cells. Furthermore, α-MEL-D exhibited antibacterial activity as high as that for MEL-D, indicating that α-MEL-D is a promising artificial sugar-based material candidate for enhancing the barrier function of the stratum corneum, superior to a known cosmetic ingredient, and possesses antibacterial activity.

Metabolomic Evaluation of the Central Metabolic Pathways of Mannosylerythritol Lipid Biosynthesis in Moesziomyces antarcticus T-34.

Moesziomyces antarcticus is a basidiomycetous yeast that produces mannosylerythritol lipids (MELs), which have potential applications as bio-based functional materials in various oleochemical industries, the cosmetics, toiletry, agriculture, and pharmaceutical industries. To better understand the MEL producer, we characterized the central metabolic pathways of M. antarcticus strain T-34 grown on glucose or olive oil via metabolomics. The relative fatty acid content was higher in the cells cultured in olive oil compared to glucose, while the acetyl-CoA content was lower in cells cultured in olive oil. The levels of the tricarboxylic acid cycle metabolites citrate/isocitrate, α-ketoglutarate, and succinate were lower in olive oil compared to glucose, while fumarate and malate levels exhibited the opposite pattern. Pyruvate was not detected in olive oil compared to glucose culture. The levels of glycerol, as well as trehalose, myo-inositol, threitol/erythritol, and mannitol/sorbitol, were higher in olive oil compared to glucose cultures. The ATP level was lower in olive oil compared to glucose culture, although the assimilation of fatty acids produced by digestion of olive oil should promote large amounts of ATP production. The possibility that ATP regeneration by respiratory chain complex promote oil utilization and MEL production in M. antarcticus T-34 was found based on the results of this metabolomic analysis.

Self-Assembling Properties and Recovery Effects on Damaged Skin Cells of Chemically Synthesized Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs), which are one of the representative sugar-based biosurfactants (BSs) produced by microorganisms, have attracted much attention in various fields in the sustainable development goals (SDGs) era. However, they are inseparable mixtures with respect to the chain length of the fatty acids. In this study, self-assembling properties and structure-activity relationship (SAR) studies of recovery effects on damaged skin cells using chemically synthesized MELs were investigated. It was revealed, for the first time, that synthetic and homogeneous MELs exhibited significant self-assembling properties to form droplets or giant vesicles. In addition, a small difference in the length of the fatty acid chains of the MELs significantly affected their recovery effects on the damaged skin cells. MELs with medium or longer length alkyl chains exhibited much higher recovery effects than that of C18-ceramide NP.

Evaluating haloarchaeal culture media for ultrahigh-molecular-weight polyhydroxyalkanoate biosynthesis by Haloferax mediterranei.

A series of culture media for haloarchaea were evaluated to optimize the production of ultrahigh-molecular-weight (UHMW) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei. Cells of H. mediterranei grew (> 1 g/L of dry cell weight) and accumulated PHBV upon flask cultivation in 10 medium types with neutral pH and NaCl concentration > 100 g/L. Molecular weight and compositional analysis revealed that the number-average molecular weight (Mn) of PHBV produced with six selected types of media ranged from 0.8 to 3.5 × 106 g/mol and the 3-hydroxyvalerate (3HV) composition ranged from 8 to 36 mol%. Cultivation in two NBRC media, 1214 and 1380, resulted in the production of PHBV with an Mn of more than 3.0 × 106 g/mol and a weight-average molecular weight of more than 5.0 × 106 g/mol, indicating the production of UHMW-PHBV. These culture media contained small amount of complex nutrients like yeast extract and casamino acids, suggesting that H. mediterranei likely produced UHMW-PHBV on poor nutrient condition. Haloferax mediterranei grown in NBRC medium 1380 produced PHBV with the highest 3HV composition. A solvent-cast film of UHMW-PHBV with 26.4 mol% 3HV produced from 1-L flask cultivation with NBRC medium 1380 was found to be flexible and semi-transparent. Thermal analysis of the UHMW-PHBV cast film revealed melting and glass-transition temperatures of 90.5 °C and - 2.7 °C, respectively. KEY POINTS: • Haloarchaeal culture media were evaluated to produce UHMW-PHBV by H. mediterranei. • UHMW-PHBV with varied molecular weight was produced dependent on culture media. • Semi-transparent film could be made from UHMW-PHBV with 26.4 mol% 3HV.

Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme.

The yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) secretes a xylose-induced biodegradable plastic-degrading enzyme (PaE). To suppress degradation of PaE during production and storage, we targeted the inhibition of proteolytic enzyme activity in P. antarctica. Proteases A and B act as upper regulators in the proteolytic network of the model yeast, Saccharomyces cerevisiae. We searched for orthologous genes encoding proteases A and B in the genome of P. antarctica GB-4(0) based on the predicted amino acid sequences. We found two gene candidates, PaPRO1 and PaPRO2, with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPaPRO1 and ΔPaPRO2, and evaluated PaE stability in culture by immunoblotting analysis. Both mutants exhibited sufficient production of PaE without degradation fragments, while the parent strain exhibited the degradation fragments. Therefore, we concluded that the protease A and B orthologous genes are related to the degradation of PaE. To produce a large quantity of PaE, we made a PaPRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPaPRO2 mutant of XG8 was able to produce PaE without the degradation fragments during large-scale cultivation in a 3-L jar fermenter for 3 days at 30°C. After terminating the agitation, the PaE activity in the XG8 ΔPaPRO2 mutant culture was maintained for the subsequent 48 h incubation at 25°C regardless of remaining cells, while activity in the XG8 control was reduced to 55.1%. The gene-deleted mutants will be useful for the development of industrial processes of PaE production and storage.

Bio-Based, Flexible, and Tough Material Derived from ε-Poly-l-lysine and Fructose via the Maillard Reaction.

We report a bio-based, soft, elastic, and tough material prepared from a mixture of ε-poly-l-lysine (ε-PL) and d-fructose. The obtained complex was insoluble in water, whereas its ingredients had high water solubility. This complex was likely formed via Schiff base formation and subsequent rearrangement reactions, that is, the Maillard reaction, because the reaction occurred between reducing sugars and cationic polyelectrolytes having primary and secondary amino groups. The progress of the Maillard reaction was investigated by proton nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. Mechanical properties of the complexes were evaluated by tensile testing, and the properties of the optimized complex [ε-PL/fructose = 60:40 (w/w), maximum stress = 27.9 MPa, strain at break = 46%, Young's modulus = 741.6 MPa] resembled those of some petroleum-based plastics. Additionally, the ε-PL/fructose complex displayed antimicrobial activity against Bacillus subtilis. These ε-PL/fructose complexes have biological properties such as antimicrobial activity, low toxicity toward mammals, and biodegradability, which are attributable to the intrinsic nature of ε-PL, as well as enhanced mechanical properties and water resistance compared with pure ε-PL.

Moldable Material from ε-Poly-l-lysine and Lignosulfonate: Mechanical and Self-Healing Properties of a Bio-Based Polyelectrolyte Complex.

A moldable material from a natural cationic polyelectrolyte, ε-poly-l-lysine (ε-PL), was prepared by mixing with two lignosulfonates a reagent for research (L-SO3Na) and a commercially available purified lignosulfonate (Pearllex NP). The obtained ε-PL/lignosulfonate complexes demonstrated the ability to be tuned from a rigid form, such as polystyrene or poly(methyl methacrylate), to a soft elastomer form such as silicone by varying the lignosulfonate species and composition. The maximum toughness of the complex (8.4 MJ/m3) was superior to that of ε-PL or lignosulfonate-derived polyelectrolyte complexes. In addition, the ε-PL/lignosulfonate complex showed self-healing properties due to the many reversible ionic bonds in the complex. The preparation process for the novel complex was simple, involving the mixing and drying of an aqueous solution of the polyelectrolyte without any extra reagents (organic solvents, condensation reagents, and cross-linker). Thus, given these many advantages and the excellent biodegradability of the components, the ε-PL/lignosulfonate complex is expected to be useful as a sustainable structural material.

Antimicrobial Activity of ε-Poly-l-lysine after Forming a Water-Insoluble Complex with an Anionic Surfactant.

ε-Poly-l-lysine (ε-PL) is one of the few homopoly(amino-acid)s occurring in nature. ε-PL, which possesses multiple amino groups, is highly soluble in water, where it forms the antimicrobial polycationic chain (PLn+). Although the high water-solubility is advantageous for the use of ε-PL as a food preservative, it has limited the applicability of ε-PL as a biopolymer plastic. Here, we report on the preparation and availability of a water-insoluble complex formed with PLn+ and an anionic surfactant, bis(2-ethylhexyl) sulfosuccinate (BEHS-, is also commercialized as AOT) anion. The PLn+/BEHS--complex, which is soluble in organic solvents, was successfully used as a coating material for a cellulose acetate membrane to create a water-resistant antimicrobial membrane. In addition, the thermoplastic PLn+/BEHS--complex was able to be uniformly mixed with polypropylene by heating, resulting in materials exhibiting antimicrobial activities.

Real-Time Observation of Enzymatic Polyhydroxyalkanoate Polymerization Using High-Speed Scanning Atomic Force Microscopy.

The initial stage of in vitro polyhydroxyalkanoate (PHA) polymerization by PHA synthase from Ralstonia eutropha (PhaCRe) on a mica substrate in water was observed using high-speed scanning atomic force microscopy (HS-AFM). Before PHA polymerization, the adsorption-desorption cycle of the PhaCRe molecule on mica was observed in real time. For approximately 30 s after the addition of the PHA monomer, no significant change was observed on the mica substrate, but PhaCRe could be transformed into an active enzyme in water upon contact with the monomer during this period. Subsequently, linearly elongating rod-shaped objects were observed on the mica substrate, plausibly as a result of the polymerization reaction. The height of these elongating objects was considerably larger than the expected height for a single PHA chain. This observation suggests that PHA chains generated during the reported experiments might form some kind of a semiregular structure.

Poly(hydroxyalkanoate) Generation from Nonchiral Substrates Using Multiple Enzyme Immobilizations on Peptide Nanofibers.

We developed a method for the immobilization of multiple active enzymes, allowing the production of chiral products from nonchiral substrates with recycling of expensive cofactors. Using a rapid, two-step process under nondenaturing conditions, we could preserve enzyme activity by separating the production of an immobilization scaffold from the attachment of the enzymes. The technique is applicable to a wide range of enzymes and will facilitate simple, cost-effective enzyme immobilization for research and industrial purposes. An (R)-specific poly(hydroxyalkanoate) synthase (PhaCRe from Ralstonia eutropha), an (S)-specific dehydrogenase (FadB from Pseudomonas putida), and an (R)-specific hydratase (PhaJ4Pa from P. aeruginosa) were immobilized by affinity tag-assisted binding to self-assembled antiparallel type ß-sheets with a coiled fiber structure formed from a decapeptide (P-K-F-K-I-I-E-F-E-P). The functionalized scaffolds were capable of producing poly(3-hydroxybutyrate) from ß-butyrolactone with the recycling of coenzyme A. Enzyme immobilization was confirmed by fluorescence microscopy using fusion proteins of the enzymes with fluorescent marker proteins, and activity was confirmed by spectroscopic activity assays.

Colorimetric method to detect ε-poly-l-lysine using glucose oxidase.

We describe a new colorimetric assay method using glucose oxidase (GOx) to detect ε-poly-l-lysine (εPL). This method uses εPL's remarkable effect of promoting the enzymatic reaction of GOx with ferricyanide ion. This reaction reduces ferricyanide ion to ferrocyanide ion, accompanied by a color change from yellow to colorless. In this colorimetric assay, the detection limit of εPL was estimated to be approximately 0.5 mg/L when purified εPL samples were used. εPL has usually been produced by a fermentation process using Streptomyces albulus species. The components of the culture broth showed interference effects against the assay method. However, due to the high sensitivity of the assay method for εPL, εPL could be detected in the culture broth without any pretreatment. The detectable concentration of εPL in the culture broth, cPL,ac, was estimated to be approximately 20 mg/L. By combining the Berlin blue reaction with this method, the cPL,ac was reduced to 10 mg/L. In light of the proposed method's simplicity and sensitivity, it could be useful for screening εPL synthetic enzymes and microorganisms.

Characterization of binding preference of polyhydroxyalkanoate biosynthesis-related multifunctional protein PhaM from Ralstonia eutropha.

The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaMRe) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaCRe) was activated by the presence of PhaMRe but PHA synthase from Aeromonas caviae (PhaCAc) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaMRe interacted with PhaCRe directly, but did not interact with PhaCAc. These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaMRe. Further analyses indicated that PhaMRe has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaA Re, phaB Re, and phaC Re/phaC Ac) and the phaM Re gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaMRe decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaCRe-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaMRe expression was observed in PhaCAc-expressing E. coli. These observations suggest that PhaMRe affects cellular physiology through the PhaM-PhaC interaction.

Separation and Purification of ε-Poly-L-lysine with Its Colorimetric Determination Using Dipicrylamine.

A separation method for a biopolymer, ε-poly-L-lysine (εPL), with its colorimetric determination using a yellow anionic dye, dipicrylamine anion (DPA(-)), is presented. The εPL-producing culture broth was mixed with a NaDPA solution to precipitate the εPL in polycationic form with the DPA(-) anion. The precipitate was dissolved into acetonitrile (AN). The AN solution was yellowish, and gave an absorption maximum at around 420 nm. Thus, εPL in the culture broth can be assayed colorimetrically a with multiwell microtiter plate. By the addition of bis(triphenylphosphoranylidene)ammonium chloride, the polycationic εPL was precipitated from the AN solution as the hydrochloride salt. On the other hand, the DPA(-) anion remained in the AN solution as the quaternary ammonium salt. Thus, εPL larger than tetramer can be separated and purified after the high-throughput screening of the synthetic enzyme.

Genome-based analysis and gene dosage studies provide new insight into 3-hydroxy-4-methylvalerate biosynthesis in Ralstonia eutropha.

Recombinant Ralstonia eutropha strain PHB(-)4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB(-)4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB(-)4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.

Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase.

In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc.

Efficient production of active polyhydroxyalkanoate synthase in Escherichia coli by coexpression of molecular chaperones.

The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter(-1)), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter(-1)) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.