High-throughput evaluation of hemolytic activity through precise measurement of colony and hemolytic zone sizes of engineered Bacillus subtilis on blood agar.

Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.

Engineering Escherichia coli for efficient glutathione production.

Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing gshA and gshB, which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δggt and ΔpepT); deleting gor (Δgor), which encodes glutathione oxide reductase; attenuating glutathione uptake (ΔyliABCD); and enhancing cysteine production (PompF-cysE). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of O-acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in E. coli. Our findings provide a great potential of E. coli fermentation process for the industrial production of glutathione.

High-level phenol bioproduction by engineered Pichia pastoris in glycerol fed-batch fermentation using an efficient pertraction system.

This study aimed to establish a high-level phenol bioproduction system from glycerol through metabolic engineering of the yeast Pichia pastoris (Komagataella phaffii). Introducing tyrosine phenol-lyase to P. pastoris led to a production of 59 mg/L of phenol in flask culture. By employing a strain of P. pastoris that overproduces tyrosine-a precursor to phenol-we achieved a phenol production of 1052 mg/L in glycerol fed-batch fermentation. However, phenol concentrations exceeding 1000 mg/L inhibited P. pastoris growth. A phenol pertraction system utilizing a hollow fiber membrane contactor and tributyrin as the organic solvent was developed to reduce phenol concentration in the culture medium. Integrating this system with glycerol fed-batch fermentation resulted in a 214 % increase in phenol titer (3304 mg/L) compared to glycerol fed-batch fermentation alone. These approaches offer a significant framework for the microbial production of chemicals and materials that are highly toxic to microorganisms.

Efficient production of long double-stranded RNAs applicable to agricultural pest control by Corynebacterium glutamicum equipped with coliphage T7-expression system.

RNA-based pesticides exert their function by suppressing the expression of an essential gene in the target pest through RNA interference caused by double-stranded RNA (dsRNA). Here, we selected target genes for growth suppression of the solanaceous crop pests ladybird beetle (Henosepilachna vigintioctopunctata) and Colorado potato beetle (Leptinotarsa decemlineata)-the death-associated inhibitor of apoptosis protein 1 gene (diap1), and an orthologous gene of the COPI coatomer protein complex (copI), respectively. We constructed a cost-competitive overproduction system for dsRNA using Corynebacterium glutamicum as a host bacterium. The dsRNA expression unit was equipped with two sets of promoters and terminators derived from coliphage T7, and the convergent expression system was designed to be selectively transcribed by T7 RNA polymerase. This expression system efficiently overproduced both target dsRNAs. On culture in a jar fermentor, the yield of diap1-targeting dsRNA (approximately 360 bp) was > 1 g per liter of culture. Long-chain diap1-targeting dsRNAs (up to around 1 kbp) could be produced without a substantial loss of efficiency. dsRNA accumulated in C. glutamicum significantly suppressed larval growth of H. vigintioctopunctata. The dsRNA expression technology developed here is expected to substantially reduce dsRNA production costs. Our method can be applied for a wide range of industrial uses, including agricultural pest control. KEY POINTS: • Overexpression of dsRNA was achieved in C. glutamicum using a coliphage T7 system. • The best strain produced > 1 g/L of the target dsRNA species, for use as an insecticide. • The developed system efficiently produced long dsRNA species, up to ~ 1 kbp.

Author Correction: A highly thermostable crude endoglucanase produced by a newly isolated Thermobifida fusca strain UPMC 901.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A highly thermostable crude endoglucanase produced by a newly isolated Thermobifida fusca strain UPMC 901.

A thermophilic Thermobifida fusca strain UPMC 901, harboring highly thermostable cellulolytic activity, was successfully isolated from oil palm empty fruit bunch compost. Its endoglucanase had the highest activity at 24 hours of incubation in carboxymethyl-cellulose (CMC) and filter paper. A maximum endoglucanase activity of 0.9 U/mL was achieved at pH 5 and 60 °C using CMC as a carbon source. The endoglucanase properties were further characterized using crude enzyme preparations from the culture supernatant. Thermal stability indicated that the endoglucanase activity was highly stable at 70 °C for 24 hours. Furthermore, the activity was found to be completely maintained without any loss at 50 °C and 60 °C for 144 hours, making it the most stable than other endoglucanases reported in the literature. The high stability of the endoglucanase at an elevated temperature for a prolonged period of time makes it a suitable candidate for the biorefinery application.

Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control.

Double-stranded RNA (dsRNA) inducing RNA interference (RNAi) is expected to be applicable to management of agricultural pests. In this study, we selected a ladybird beetle, Henosepilachna vigintioctopunctata, as a model target pest that devours vegetable leaves, and examined the effects of feeding the pest sterilized microbes highly accumulating a target dsRNA for RNAi induction. We constructed an efficient production system for diap1*-dsRNA, which suppresses expression of the essential gene diap1 (encoding death-associated inhibitor of apoptosis protein 1) in H. vigintioctopunctata, using an industrial strain of Corynebacterium glutamicum as the host microbe. The diap1*-dsRNA was overproduced in C. glutamicum by convergent transcription using a strong promoter derived from corynephage BFK20, and the amount of dsRNA accumulated in fermented cells reached about 75 mg per liter of culture. In addition, we developed a convenient method for stabilizing the dsRNA within the microbes by simply sterilizing the diap1*-dsRNA-expressing C. glutamicum cells with ethanol. When the sterilized microbes containing diap1*-dsRNA were fed to larvae of H. vigintioctopunctata, diap1 expression in the pest was suppressed, and the leaf-feeding activity of the larvae declined. These results suggest that this system is capable of producing stabilized dsRNA for RNAi at low cost, and it will open a way to practical application of dsRNA as an environmentally-friendly agricultural insecticide.

Overexpression system for recombinant RNA in Corynebacterium glutamicum using a strong promoter derived from corynephage BFK20.

In recent years, it has been shown that recombinant RNA molecules have a great potential in mRNA therapy and as novel agricultural pesticides. We developed a fundamental system for efficient production of target RNA molecules in Corynebacterium glutamicum, composed of a strong promoter named F1 and a terminator derived from corynephage BFK20 in a high-copy number plasmid vector. As a target model RNA for overexpression, we designed and used an RNA molecule [designated U1A*-RNA, ∼160 nucleotides (nt) long] containing a stem/loop II (SL-II, hairpin-II) structure from U1 small nuclear RNA (snRNA), which binds to U1A protein, forming a U1 sn-ribonucleoprotein, which is essential in the pre-mRNA splicing process. C. glutamicum strains harboring the U1A*-RNA expression plasmid were cultured and the total RNA was analyzed. We observed prominent expression of RNA corresponding to the U1A*-RNA transcript along with lower expression of a 3'-region-truncated form of the transcript (∼110 nt) in an rnc (encoding RNase III)-deficient strain. We also found that the produced U1A*-RNA bound to the U1A RNA-binding domain protein, which was separately prepared with C. glutamicum. In a batch cultivation using a fermentor, the total accumulated amount of the target RNA reached about 300 mg/L by 24 h. Thus, our results indicated that our system can serve as an efficient platform for large-scale preparation of an RNA of interest.

High copy number mutants derived from Corynebacterium glutamicum cryptic plasmid pAM330 and copy number control.

A high copy number mutant plasmid, designated pVC7H1, was isolated from an Escherichia coli-Corynebacterium glutamicum shuttle vector pVC7N derived from cryptic plasmid pAM330 that was originally found in Brevibacterium lactofermentum 2256 (formally C. glutamicum ATCC 13869). The copy number of pVC7N was estimated to be about 11 per chromosome, whereas pVC7H1 displayed a copy number of 112 per chromosome in C. glutamicum. The mutation (designated copA1) was in a region between long inverted repeats (designated the copA1 region) and was identified as a single base conversion of cytosine to adenine. By introduction of a cytosine to guanine mutation (designated copA2) at the same site as copA1, a further high copy number mutant (>300 copies of the plasmid per chromosome) was generated. Through genetic and RNA-Seq analyses of the copA1 region, it was determined that a small RNA (designated sRNA1) is produced from the upstream region of repA, a gene encoding a possible replication initiator protein, and sRNA1 is a possible regulator of the copy number of pAM330-replicon-contaning plasmids. Determination of the precise transcription start sites of sRNA1 and repA-mRNA suggested that sRNA1 could sequester a presumed ribosome binding site of repA-mRNA from ribosomes by an antisense RNA-mediated mechanism. Our data also indicate that the secondary-structure of sRNA1 is crucial for its function in plasmid copy number control.

Plasmid copy number mutation in repA gene encoding RepA replication initiator of cryptic plasmid pHM1519 in Corynebacterium glutamicum.

Cryptic plasmid pHM1519 is a rolling-circular replication mode plasmid of the pCG1 plasmid family in coryneform bacteria. The derived shuttle vector pPK4 is maintained at about 40-50 copies per chromosome in Corynebacterium glutamicum 2256 (ATCC 13869). We found that a mutation (designated copA1) within the repA gene encoding essential initiator protein RepA of the pHM1519-replicon increased the copy number of the mutant plasmid to about 800 copies per chromosome. The mutation was a single G to A base transition, which changed Gly to Glu at position 429 of the amino acid sequence of RepA. In silico secondary structure prediction of RepA suggested that Gly429 is situated in a disordered region in a helix-turn-helix motif, which is a typical DNA-binding domain. This study shows the first example of a high copy number of a C. glutamicum cryptic plasmid caused by an altered replication initiator protein.

Identification of novel long chain N-acylhomoserine lactones of chain length C20 from the marine phototrophic bacterium Rhodovulum sulfidophilum.

Gram-negative bacterial quorum sensing is mainly regulated by an extracellularly produced N-acylhomoserine lactone (AHL). AHL consists of a lactone ring and an acyl chain, which generally varies from C4 to C18 in length and affords species-specific variety. In this study, we developed an ultra-high performance liquid chromatography tandem mass spectrometry system and detected two kinds of long chain AHLs with chain length C20 from the reverse-phase thin layer chromatography-fractionated cultured supernatant of the marine photosynthetic bacterium Rhodovulum sulfidophilum. By fragmentation search analysis to detect compounds with a homoserine lactone ring moiety for data dependent acquisition, a minor AHL, presumed to be 3-OH-C18-homoserine lactone (HSL), was also found. Among the detected C20-HSLs, 3-OH-C20-HSL was structurally identified and 3-OH-C20:1-HSL was strongly suggested. To our knowledge, this is the first report to show a novel AHL with the longest C20 acyl side chain found to date. ABBREVIATIONS: AGC: automatic gain control; AHL: N-acylhomoserine lactone; CD: cyclodextrin; CID: collision induced dissociation; DDA: data dependent acquisition; EPI: enhanced product ion; FISh: fragment ion search; HCD: high energy collisional dissociation; HSL: homoserine lactone; IT: injection time; LC: liquid chromatography; MS: mass spectrometry; PRM: parallel reaction monitoring; RP: reverse phase; SRM: selected reaction monitoring; TLC: thin layer chromatography; UHPLC: ultra high performance liquid chromatography.

A novel galactolipase from a green microalga Chlorella kessleri: purification, characterization, molecular cloning, and heterologous expression.

We have identified an enzyme, galactolipase (ckGL), which hydrolyzes the acyl ester bond of galactolipids such as digalactosyldiacylglycerol (DGDG), in the microalga Chlorella kessleri. Following purification of the enzyme to electrophoretic homogeneity from cell-free extract, the maximum activity toward DGDG was observed at pH 6.5 and 37 °C. ckGL was Ca2+-dependent enzyme and displayed an apparent molecular mass of approx. 53 kDa on SDS-PAGE. The substrate specificity was in the order: DGDG (100%) > monogalactosyldiacylglycerol ≈ phosphatidylglycerol (~ 40%) > sulfoquinovosyldiacylglycerol (~ 20%); the enzyme exhibited almost no activity toward glycerides and other phospholipids. Gas chromatography analysis demonstrated that ckGL preferably hydrolyzed the sn-1 acyl ester bond in the substrates. The genomic DNA sequence (5.6 kb) containing the ckGL gene (designated glp1) was determined and the cDNA was cloned. glp1 was composed of 10 introns and 11 exons, and the 1608-bp full-length cDNA encoded a mature ckGL containing 475 amino acids (aa), with a presequence (60 aa) containing a potential chloroplast transit peptide. Recombinant functional ckGL was produced in Escherichia coli. Although the deduced aa sequence of ckGL contained the typical GXSXG motif of serine hydrolases together with conserved histidine and aspartate residues which would form part of the catalytic triad of α/ß-hydrolases, ckGL showed no significant overall similarity with known lipases including GLs from Chlamydomonas reinhardtii and Aspergillus japonicus, indicating that ckGL is a novel GL. ckGL, with high specificity for DGDG, could be applicable to food processing as an enzyme capable of improving material textures.

Biotemplated Synthesis of TiO2-Coated Gold Nanowire for Perovskite Solar Cells.

Fibrous nanomaterials have been widely employed toward the improvement of photovoltaic devices. Their light-trapping capabilities, owing to their unique structure, provide a direct pathway for carrier transport. This paper reports the improvement of perovskite solar cell (PSC) performance by a well-dispersed TiO2-coated gold nanowire (GNW) in a TiO2 cell layer. We used an artificially designed cage-shaped protein to synthesize a TiO2-coated GNW in aqueous solution under atmospheric pressure. The artificially cage-shaped protein with gold-binding peptides and titanium-compound-biomineralizing peptides can bind GNWs and selectively deposit a thin TiO2 layer on the gold surface. The TiO2-coated GNW incorporated in the photoelectrodes of PSCs increased the external quantum efficiency within the range of 350-750 nm and decreased the internal resistance by 12%. The efficient collection of photogenerated electrons by the nanowires boosted the power conversion efficiency by 33% compared to a typical mesoporous-TiO2-nanoparticle-only electrode.

Selection of a novel peptide aptamer with high affinity for TiO2-nanoparticle through a direct electroporation with TiO2-binding phage complexes.

We have developed an easy and rapid screening method of peptide aptamers with high affinity for a target material TiO2 using M13 phage-display and panning procedure. In a selection step, the phage-substrate complexes and Escherichia coli cells were directly applied by electric pulse for electroporation, without separating the objective phages from the TiO2 nanoparticles. Using this simple and rapid method, we obtained a novel peptide aptamer (named ST-1 with the sequence AYPQKFNNNFMS) with highly strong binding activity for TiO2. A cage-shaped protein fused with both ST-1 and an available carbon nanotube-affinity peptide was designed and produced in E. coli. The multi-functional supraprotein could efficiently mineralize a titanium-compound around the surface of single-wall carbon nanotubes (SWNTs), indicating that the ST-1 is valuable in the fabrication of nano-composite materials with titanium-compounds. The structural analysis of ST-1 variants indicated the importance of the N-terminal region (as a motif of AXPQKX6S) of the aptamer in the TiO2-binding activity.

Thermo-stable carbon nanotube-TiO2 nanocompsite as electron highways in dye-sensitized solar cell produced by bio-nano-process.

We produced a thermostable TiO2-(anatase)-coated multi-walled-carbon-nanotube (MWNT) nanocomposite for use in dye-sensitized solar cells (DSSCs) using biological supuramolecules as catalysts. We synthesized two different sizes of iron oxide nanoparticles (NPs) and arrayed the NPs on a silicon substrate utilizing two kinds of genetically modified cage-shaped proteins with silicon-binding peptide aptamers on their outer surfaces. Chemical vapor deposition (CVD) with the vapor-liquid-solid phase (VLS) method was applied to the substrate, and thermostable MWNTs with a diameter of 6 ± 1 nm were produced. Using a genetically modified cage-shaped protein with carbon-nanomaterials binding and Ti-mineralizing peptides as a catalyst, we were able to mineralize a titanium compound around the surface of the MWNT. The products were sintered, and thin TiO2-layer-coated MWNTs nanocomoposites were successfully produced. Addition of a 0.2 wt% TiO2-coated MWNT nanocomposite to a DSSC photoelectrode improved current density by 11% and decreased electric resistance by 20% compared to MWNT-free reference DSSCs. These results indicate that a nanoscale TiO2-layer-coated thermostable MWNT structure produced by our mutant proteins works as a superior electron transfer highway within TiO2 photoelectrodes.

Biological construction of single-walled carbon nanotube electron transfer pathways in dye-sensitized solar cells.

We designed and mass-produced a versatile protein supramolecule that can be used to manufacture a highly efficient dye-sensitized solar cell (DSSC). Twelve single-walled carbon-nanotube (SWNT)-binding and titanium-mineralizing peptides were genetically integrated on a cage-shaped dodecamer protein (CDT1). A process involving simple mixing of highly conductive SWNTs with CDT1 followed by TiO2 biomineralization produces a high surface-area/weight TiO2 -(anatase)-coated intact SWNT nanocomposite under environmentally friendly conditions. A DSSC with a TiO2 photoelectrode containing 0.2 wt % of the SWNT-TiO2 nanocomposite shows a current density improvement by 80% and a doubling of the photoelectric conversion efficiency. The SWNT-TiO2 nanocomposite transfers photon-generated electrons from dye molecules adsorbed on the TiO2 to the anode electrode swiftly.

A novel bifunctional protein supramolecule for construction of carbon nanotube-titanium hybrid material.

Carbon nanotube-TiO(2) hybrid materials with many nano-scale cavities were realized using a bifunctional protein supramolecule possessing carbonaceous material-binding peptides and Ti-binding peptides. The obtained structure was confirmed to consist of a central nanotube, surrounding proteins, and a swathing titanium-layer. All processes were carried out at room temperature, using an environmentally friendly method.

Improvement of L-lysine production by Methylophilus methylotrophus from methanol via the Entner-Doudoroff pathway, originating in Escherichia coli.

To improve the amino acid production by metabolic engineering, eliminating the pathway bottleneck is known to be very effective. The metabolic response of Methylophilus methylotrophus upon the addition of glucose and of pyruvate was investigated in batch cultivation. We found that the supply of pyruvate is a bottleneck in L-lysine production in M. methylotrophus from methanol as carbon source. M. methylotrophus has a ribulose monophosphate (RuMP) pathway for methanol assimilation, and consequently synthesized fructose-6-phosphate is metabolized to pyruvate via the Entner-Doudoroff (ED) pathway, and the ED pathway is thought to be the main pathway for pyruvate supply. An L-lysine producer of M. methylotrophus with an enhanced ED pathway was constructed by the introduction of the E. coli edd-eda operon encoding the enzyme involving the ED pathway. In this strain, the overall enzymatic activity of ED pathway, which is estimated by measuring the activities of 6-phosphogluconate dehydrogenase plus 2-keto-3-deoxy-6-phosphogluconate aldolase, was about 20 times higher than in the parent. This strain produced 1.2 times more L-lysine than the parent producer. Perhaps, then, the supply of pyruvate was a bottleneck in L-lysine production in the L-lysine producer of M. methylotrophus.

Disruption of metF increased L-lysine production by Methylophilus methylotrophus from methanol.

Methionine auxotrophic mutants of Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine, and mutated lysE (lysE24) encoding the L-lysine exporter from Corynebacterium glutamicum 2256, produced higher amounts of L-lysine from methanol as sole carbon source than did other amino acid auxotrophic mutants. Especially, the M. methylotrophus 102 strain, carrying both dapA24 and lysE24, produced L-lysine in more than 1.5 times amounts higher than the parent. A single-base substitution was identified in this auxotroph in codon-329 of the open reading frame of metF, encoding 5,10-methylene-tetra-hydrofolate reductase. We constructed a metF disruptant mutant carrying both dapA24 and lysE24, and confirmed increases in L-lysine production. This is the first report to the effect that metF deficient increased L-lysine production in methylotroph.

Innovative metabolic pathway design for efficient l-glutamate production by suppressing CO2 emission.

In the pathway of L-glutamic acid (L-Glu) biosynthesis in Corynebacterium glutamicum, 1 mol of L-Glu is synthesized from 1 mol of glucose at a cost of 1 mol of carbon dioxide (CO(2)), with a maximum theoretical yield of 81.7% by weight. We have designed an innovative pathway for efficient L-Glu production employing phosphoketolase (PKT) to bypass the CO(2)-releasing pyruvate dehydrogenase reaction, thereby increasing the maximum theoretical yield of L-Glu from glucose to up to 98.0% by weight (120% mol/mol L-Glu produced/glucose consumed). The xfp gene encoding PKT was cloned from Bifidobacterium animalis and overexpressed under the strong cspB promoter in C. glutamicum. A functional enzyme was detected in an L-Glu-producing strain of C. glutamicum (odhA). When cells of this producer strain with the xfp gene and those without the xfp gene were cultivated in a controlled fermentation system, the L-Glu production yield of the strain expressing the xfp gene was much higher than that of the original strain, coupled with the suppression of CO(2) emission. Consequently, we could successfully enhance L-glutamate production by installing the PKT pathway of B. animalis into C. glutamicuml-Glu metabolism, and this novel metabolic design will be able to increase L-Glu production yield beyond the maximum theoretical yield obtained from the conventional metabolic pathway of biosynthesis from glucose.