Tryptophan-Based Hyperproduction of Bioindigo by Combinatorial Overexpression of Two Different Tryptophan Transporters.

Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction from plants and chemical synthesis. Studies are being conducted to develop methods for environment-friendly and sustainable production of indigo using genetically engineered microbes. Here, to enhance the yield of bioindigo from an E. coli whole-cell system containing tryptophanase (TnaA) and flavin-containing monooxygenase (FMO), we evaluated tryptophan transporters to improve the transport of aromatic compounds, such as indole and tryptophan, which are not easily soluble and passable through cell walls. Among the three transporters, Mtr, AroP, and TnaB, AroP enhanced indigo production the most. The combination of each transporter with AroP was also evaluated, and the combination of AroP and TnaB showed the best performance compared to the single transporters and two transporters. Bioindigo production was then optimized by examining the culture medium, temperature, isopropyl ß-D-1-thiogalactopyranoside concentration, shaking speed (rpm), and pH. The novel strain containing aroP and tnaB plasmid with tnaA and FMO produced 8.77 mM (2.3 g/l) of bioindigo after 66 h of culture. The produced bioindigo was further recovered using a simple method and used as a watercolor dye, showing good mixing with other colors and color retention for a relatively long time. This study presents an effective strategy for enhancing indigo production using a combination of transporters.

Continuous production of gamma aminobutyric acid by engineered and immobilized Escherichia coli whole-cells in a small-scale reactor system.

γ-Amino butyric acid (GABA) is a non-proteinogenic amino acid and a human neurotransmitter. Recently, increasing demand for food additives and biodegradable bioplastic monomers, such as nylon 4, has been reported. Consequently, considerable efforts have been made to produce GABA through fermentation and bioconversion. To realize bioconversion, wild-type or recombinant strains harboring glutamate decarboxylase were paired with the cheap starting material monosodium glutamate, resulting in less by-product formation and faster production compared to fermentation. To increase the reusability and stability of whole-cell production systems, this study used an immobilization and continuous production system with a small-scale continuous reactor for gram-scale production. The cation type, alginate concentration, barium concentration, and whole-cell concentration in the beads were optimized and this optimization resulted in more than 95 % conversion of 600 mM monosodium glutamate to GABA in 3 h and reuse of the immobilized cells 15 times, whereas free cells lost all activity after the ninth reaction. When a continuous production system was applied after optimizing the buffer concentration, substrate concentration, and flow rate, 165 g of GABA was produced after 96 h of continuous operation in a 14-mL scale reactor. Our work demonstrates the efficient and economical production of GABA by immobilization and continuous production in a small-scale reactor.

Enhanced production of bio-indigo in engineered Escherichia coli, reinforced by cyclopropane-fatty acid-acyl-phospholipid synthase from psychrophilic Pseudomonas sp. B14-6.

Indigo dye is an organic compound with a distinctive blue color. Most of the indigo currently used in industry is produced via chemical synthesis, which generates a large amount of wastewater. Therefore, several studies have recently been conducted to find ways to produce indigo eco-friendly using microorganisms. Here, we produced indigo using recombinant Escherichia coli with both an indigo-producing plasmid and a cyclopropane fatty acid (CFA)-regulating plasmid. The CFA-regulating plasmid contains the cfa gene, and its expression increases the CFA composition of the phospholipid fatty acids of the cell membrane. Overexpression of cfa showed cytotoxicity resistance of indole, an intermediate product formed during the indigo production process. This had a positive effect on indigo production and cfa originated from Pseudomonas sp. B 14-6 was used. Optimal conditions for indigo production were determined by adjusting the expression strain, culture temperature, shaking speed, and isopropyl ß-D-1-thiogalactopyranoside concentration. Treatment with Tween 80 at a particular concentration to increase the permeability of the cell membrane had a positive effect on indigo production. The strain with the CFA plasmid produced 4.1 mM of indigo after 24 h of culture and produced 1.5-fold higher indigo than the control strain without the CFA plasmid that produced 2.7 mM.

Adsorptive removal of synthetic plastic components bisphenol-A and solvent black-3 dye from single and binary solutions using pristine pinecone biochar.

The existing study deals with adsorptive removal of the endocrine-disrupting chemical bisphenol-A and toxic azo dye solvent black-3 from single and binary solutions. These two chemicals are commonly used as an additive in the synthetic plastic industries. Among the tested twenty pristine and modified biochars, the pristine pinecone biochar produced at 750 °C revealed greater bisphenol-A removal. Simulation of the experimental data obtained for bisphenol-A and dye removal from the single-component solution offered a best-fit to Elovich (R2 > 0.98) and pseudo-second-order (R2 > 0.99) kinetic models, respectively. Whereas for the bisphenol-A + dye removal from binary solution, the values for bisphenol-A adsorption were best suited to Elovich (R2 > 0.98), while pseudo-second-order (R2 > 0.99) for dye removal. Similarly, the two-compartment model also demonstrated better values (R2 > 0.92) for bisphenol-A and dye removal from single and binary solutions with greater Ffast values (except for bisphenol-A in binary solution). The Langmuir isotherm model demonstrated the highest regression coefficient values (R2 > 0.99) for bisphenol-A and dye removal with the highest adsorption capacity of 38.387 mg g-1 and 346.856 mg g-1, correspondingly. Besides, the co-existence of humic acid revealed a positive impact on bisphenol-A removal, while the dye removal rate was slightly hindered in presence of humic acid. The absorption process showed monolayer coverage of biochar surface with contaminants using a chemisorption mechanism with fast reactions between functional groups on the adsorbate and adsorbent. Whereas the adsorption mechanism was primarily controlled by hydrogen bonding, hydrophobic and π-π electron-donor-acceptor interactions as confirmed by FTIR, XPS, and pH investigations.

Gamma aminobutyric acid (GABA) production in Escherichia coli with pyridoxal kinase (pdxY) based regeneration system.

Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid act as a major neurotransmitter inhibitor in the nervous system of mammals. It also used as a precursor of bioplastics synthesis such as N-methylpyrolidone and polyamide 4. Chemical-based synthesis methods have many environmental-related issues, so efforts have been made to develop biosynthetic methods to produce GABA. Glutamate decarboxylase (GAD) transforms L-glutamate to GABA using pyridoxal 5'-phosphate (PLP) as a cofactor. Bioconversion of GABA with whole cells overexpressing the glutamate decarboxylase has advantages of fewer byproducts and rapid reaction. However, there is a bottleneck in the whole-cell bioconversion system i.e., higher GABA production require a large amount of cofactor PLP which make the process costly. Therefore, pyridoxal kinase (PdxY) able to regenerate PLP was introduced in the whole-cell system to construct a new GABA producing system. Culture and reaction conditions were optimized, and 100% conversion of 0.6 M MSG was obtained. This study reports that a competitive level of GABA production could be achieved without supplying additional PLPs.

Novel Polyhydroxybutyrate-Degrading Activity of the Microbulbifer Genus as Confirmed by Microbulbifer sp. SOL03 from the Marine Environment.

Ever since bioplastics were globally introduced to a wide range of industries, the disposal of used products made with bioplastics has become an issue inseparable from their application. Unlike petroleum-based plastics, bioplastics can be completely decomposed into water and carbon dioxide by microorganisms in a relatively short time, which is an advantage. However, there is little information on the specific degraders and accelerating factors for biodegradation. To elucidate a new strain for biodegrading poly-3-hydroxybutyrate (PHB), we screened out one PHB-degrading bacterium, Microbulbifer sp. SOL03, which is the first reported strain from the Microbulbifer genus to show PHB degradation activity, although Microbulbifer species are known to be complex carbohydrate degraders found in high-salt environments. In this study, we evaluated its biodegradability using solid- and liquid-based methods in addition to examining the changes in physical properties throughout the biodegradation process. Furthermore, we established the optimal conditions for biodegradation with respect to temperature, salt concentration, and additional carbon and nitrogen sources; accordingly, a temperature of 37°C with the addition of 3% NaCl without additional carbon sources, was determined to be optimal. In summary, we found that Microbulbifer sp. SOL03 showed a PHB degradation yield of almost 97% after 10 days. To the best of our knowledge, this is the first study to investigate the potent bioplastic degradation activity of Microbulbifer sp., and we believe that it can contribute to the development of bioplastics from application to disposal.

Novel phasins from the Arctic Pseudomonas sp. B14-6 enhance the production of polyhydroxybutyrate and increase inhibitor tolerance.

Phasin (PhaP), one of the polyhydroxyalkanoate granule-associated protein, enhances cell growth and polyhydroxybutyrate (PHB) biosynthesis by regulating the number and size of PHB granules. However, few studies have applied phasins to various PHB production conditions. In this study, we identified novel phasin genes from the genomic data of Arctic soil bacterium Pseudomonas sp. B14-6 and determined the role of phaP1Ps under different PHB production conditions. Transmission electron microscopy and gel permeation chromatography revealed small PHB granules with high-molecular weight, while differential scanning calorimetry showed that the extracted PHB films had similar thermal properties. The phasin protein derived from Pseudomonas sp. B14-6 revealed higher PHB production and exhibited higher tolerance to several lignocellulosic biosugar-based inhibitors than the phasin protein of Ralstonia eutropha H16 in a recombinant Escherichia coli strain. The increased tolerance to propionate, temperature, and other inhibitors was attributed to the introduction of phaP1Ps, which increased PHB production from lignocellulosic hydrolysate (2.39-fold) in the phaP1Ps strain. However, a combination of phasin proteins isolated from two different sources did not increase PHB production. These findings suggest that phasin could serve as a powerful means to increase robustness and PHB production in heterologous strains.

Comparative Study of the Difference in Behavior of the Accessory Gene Regulator (Agr) in USA300 and USA400 Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA).

Community-associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) is notorious as a leading cause of soft tissue infections. Despite several studies on the Agr regulator, the mechanisms of action of Agr on the virulence factors in different strains are still unknown. To reveal the role of Agr in different CA-MRSA, we investigated the LACΔagr mutant and the MW2Δagr mutant by comparing LAC (USA300), MW2 (USA400), and Δagr mutants. The changes of Δagr mutants in sensitivity to oxacillin and several virulence factors such as biofilm formation, pigmentation, motility, and membrane properties were monitored. LACΔagr and MW2Δagr mutants showed different oxacillin sensitivity and biofilm formation compared to the LAC and MW2 strains. Regardless of the strain, the motility was reduced in Δagr mutants. And there was an increase in the long chain fatty acid in phospholipid fatty acid composition of Δagr mutants. Other properties such as biofilm formation, pigmentation, motility, and membrane properties were different in both Δagr mutants. The Agr regulator may have a common role like the control of motility and straindependent roles such as antibiotic resistance, biofilm formation, change of membrane, and pigment production. It does not seem easy to control all MRSA by targeting the Agr regulator only as it showed strain-dependent behaviors.

Polyhydroxyalkanoates (PHAs) degradation by the newly isolated marine Bacillus sp. JY14.

Polyhydroxyalkanoates (PHAs) are bioplastic substitutes for petroleum-derived plastics that may help to reduce the increasing environmental impact of plastic pollution. Among them, polyhydroxybutyrate (PHB) is a promising biopolymer, incentivizing many researchers to search for PHB-producing and PHB-degrading bacteria for improved PHB utilization. Many novel PHB-producing microorganisms have been discovered; however, relatively few PHB-degrading bacteria have been identified. Six PHB-degrading bacteria were found in marine soil and investigated their PHB-degrading abilities under various temperature and salinity conditions using solid-media based culture. Finally, thermotolerant and halotolerant PHB-degrader Bacillus sp. JY14 was selected. PHB degradation was confirmed by monitoring changes in the physical and chemical properties of PHB films incubated with Bacillus sp. JY14 using scanning electron microscopy, Fourier-transform infrared spectroscopy, and gel permeation chromatography. Further, PHB degradation ability of Bacillus sp. JY14 was measured in liquid culture by gas chromatography. After 14 days of cultivation with PHB film, Bacillus sp. JY14 achieved approximately 98% PHB degradation. Applying various bioplastics to assess the bacteria's biodegradation capabilities, the result showed that Bacillus sp. JY14 could also degrade P(3HB-co-4HB) and P(3HB-co-3HV). Overall, this study identified a thermotolerant and halotolerant bacteria capable of PHB degradation under solid and liquid conditions. These results suggest that this bacteria could be utilized to degrade various PHAs.

Engineering of Shewanella marisflavi BBL25 for biomass-based polyhydroxybutyrate production and evaluation of its performance in electricity production.

Polyhydroxybutyrate (PHB) is a biodegradable plastic with physical properties similar to petrochemically derived plastics. Here, Shewanella marisflavi BBL25 was engineered by inserting the pLW487 vector containing polyhydroxyalkanoates synthesis genes from Ralstonia eutropha H16. Under optimal conditions, the engineered S. marisflavi BBL25 produced 1.99 ± 0.05 g/L PHB from galactose. The strain showed high tolerance to various inhibitors and could utilize lignocellulosic biomass for PHB production. When barley straw hydrolysates were used as a carbon source, PHB production was 3.27 ± 0.19 g/L. In addition, PHB production under the microbial fuel cell system was performed to confirm electricity coproduction. The maximum electricity current output density was 1.71 mA/cm2, and dry cell weight (DCW) and PHB production were 11.4 g/L and 6.31 g/L, respectively. Our results demonstrated PHB production using various lignocellulosic biomass and the feasibility of PHB and electricity production, simultaneously, and it is the first example of PHB production in engineered Shewanella.

Screening of the strictly xylose-utilizing Bacillus sp. SM01 for polyhydroxybutyrate and its co-culture with Cupriavidus necator NCIMB 11599 for enhanced production of PHB.

Polyhydroxybutyrate (PHB) is a biodegradable plastic that can be used as an alternative to petrochemical-based plastics. PHB is produced by various microorganisms such as Ralstonia, Halomonas, and Bacillus species. However, there are very few strains that produce PHB using xylose, an abundant and inexpensive carbon source. In this study, ten xylose-utilizing PHB producers isolated from South Korean marine environments were screened and characterized. Among these isolates, Bacillus sp. SM01, a newly identified strain, produced the highest amount of PHB using xylose. Under optimal conditions, the maximum dry cell weight (DCW) was 3.41 ± 0.09 g/L, with 62% PHB content, and Bacillus sp. SM01 showed Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production with propionate; however, the growth of Bacillus sp. SM01 was greatly inhibited by the presence of glucose. Co-culturing Bacillus sp. SM01 with Cupriavidus necator NCIMB 11599 resulted in increased DCW, PHB production, and utilization of glucose and xylose, the main sugar of lignocellulosic biomass, compared with the monoculture. Our results indicated that this co-culture system can be used to increase PHB production and overcome the limitation of sugar consumption associated with Bacillus sp. SM01 and C. necator.