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.

Positive effect of phasin in biohydrogen production of non polyhydroxybutyrate-producing Clostridium acetobutylicum ATCC 824.

In this study, the goal was to enhance the tolerance of Clostridium acetobutylicum ATCC 824 to biomass-based inhibitory compounds for biohydrogen production and evaluate various known genes that enhance the production of biochemicals in various hosts. The introduction of phaP, the major polyhydroxyalkanoate granule-associated protein that has been reported as a chaperone-like protein resulted in increased tolerance to inhibitors and leads to higher levels of hydrogen production, cell growth, and glucose consumption in the presence of these inhibitors. It was observed that the introduction of phaP led to an increase in the transcription of the hydrogenase gene, whereas transcription of the chaperone functional genes decreased compared to the wild type. Finally, the introduction of phaP could significantly enhance biohydrogen production by 2.6-fold from lignocellulosic hydrolysates compared to that of wild type. These findings suggested that the introduction of phaP could enhance growth and biohydrogen production, even in non-polyhydroxyalkanoate-producing strains.

Validating a Xylose Regulator to Increase Polyhydroxybutyrate Production for Utilizing Mixed Sugars from Lignocellulosic Biomass Using Escherichia coli.

Polyhydroxybutyrate (PHB) production from lignocellulosic biomass is economically beneficial. Because lignocellulosic biomass is a mixture rich in glucose and xylose, Escherichia coli, which prefers glucose, needs to overcome glucose repression for efficient biosugar use. To avoid glucose repression, here, we overexpressed a xylose regulator (xylR) in an E. coli strain expressing bktB, phaB, and phaC from Cupriavidus necator and evaluated the effect of xylR on PHB production. XylR overexpression increased xylose consumption from 0% to 46.53% and produced 4.45-fold more PHB than the control strain without xylR in a 1% sugar mixture of glucose and xylose (1:1). When the xylR-overexpressed strain was applied to sugars from lignocellulosic biomass, cell growth and PHB production of the strain showed a 4.7-fold increase from the control strain, yielding 2.58 ± 0.02 g/l PHB and 4.43 ± 0.28 g/l dry cell weight in a 1% hydrolysate mixture. XylR overexpression increased the expression of xylose operon genes by up to 1.7-fold. Moreover, the effect of xylR was substantially different in various E. coli strains. Overall, the results showed the effect of xylR overexpression on PHB production in a non-native PHB producer and the possible application of xylR for xylose utilization in E. coli.

Maximization of 3-hydroxyhexanoate fraction in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) using lauric acid with engineered Cupriavidus necator H16.

As polyhydroxybutyrate (P(3HB)) was struggling with mechanical properties, efforts have been directed towards increasing mole fraction of 3-hydroxyhexanoate (3HHx) in P(3HB-co-3HHx) to improve the properties of polyhydroxyalkanoates (PHAs). Although genetic modification had significant results, there were several issues related to cell growth and PHA production by deletion of PHA synthetic genes. To find out easier strategy for high 3HHx mole fraction without gene deletion, Cupriavidus necator H16 containing phaC2Ra-phaACn-phaJ1Pa was examined with various oils resulting that coconut oil gave the highest 3HHx mole fraction. When fatty acid composition analysis with GC-MS was applied, coconut oil was found to have very different composition from other vegetable oil containing very high lauric acid (C12) content. To find out specific fatty acid affecting 3HHx fraction, different fatty acids from caproic acid (C6) to stearic acid (C18) was evaluated and the 3HHx mole fraction was increased to 26.5 ± 1.6 % using lauric acid. Moreover, the 3HHx mole fraction could be controlled from 9 % to 31.1 % by mixing bean oil and lauric acid with different ratios. Produced P(3HB-co-3HHx) exhibited higher molecular than P(3HB-co-3HHx) from phaB-deletion mutant. This study proposes another strategy to increase 3HHx mole fraction with easier way by modifying substrate composition without applying deletion tools.

A strategy to promote the convenient storage and direct use of polyhydroxybutyrate-degrading Bacillus sp. JY14 by lyophilization with protective reagents.

BACKGROUND: Bioplastics are attracting considerable attention, owing to the increase in non-degradable waste. Using microorganisms to degrade bioplastics is a promising strategy for reducing non-degradable plastic waste. However, maintaining bacterial viability and activity during culture and storage remains challenging. With the use of conventional methods, cell viability and activity was lost; therefore, these conditions need to be optimized for the practical application of microorganisms in bioplastic degradation. Therefore, we aimed to optimize the feasibility of the lyophilization method for convenient storage and direct use. In addition, we incoporated protective reagents to increase the viability and activity of lyophilized microorganisms. By selecting and applying the best protective reagents for the lyophilization process and the effects of additives on the growth and PHB-degrading activity of strains were analyzed after lyophilization. For developing the lyophilization method for protecting degradation activity, it may promote practical applications of bioplastic-degrading bacteria. RESULTS: In this study, the polyhydroxybutyrate (PHB)-degrading strain, Bacillus sp. JY14 was lyophilized with the use of various sugars as protective reagents. Among the carbon sources tested, raffinose was associated with the highest cell survival rate (12.1%). Moreover, 7% of raffionose showed the highest PHB degradation yield (92.1%). Therefore, raffinose was selected as the most effective protective reagent. Also, bacterial activity was successfully maintained, with raffinose, under different storage temperatures and period. CONCLUSIONS: This study highlights lyophilization as an efficient microorganism storage method to enhance the applicability of bioplastic-degrading bacterial strains. The approach developed herein can be further studied and used to promote the application of microorganisms in bioplastic degradation.

Complementation of reducing power for 5-hydroxyvaleric acid and 1,5-pentanediol production via glucose dehydrogenase in Escherichia coli whole-cell system.

One of the key intermediates, 5-hydroxyvaleric acid (5-HV), is used in the synthesis of polyhydroxyalkanoate monomer, δ-valerolactone, 1,5-pentanediol (1,5-PDO), and many other substances. Due to global environmental problems, eco-friendly bio-based synthesis of various platform chemicals and key intermediates are socially required, but few previous studies on 5-HV biosynthesis have been conducted. To establish a sustainable bioprocess for 5-HV production, we introduced gabT encoding 4-aminobutyrate aminotransferase and yqhD encoding alcohol dehydrogenase to produce 5-HV from 5-aminovaleric acid (5-AVA), through glutarate semialdehyde in Escherichia coli whole-cell reaction. As, high reducing power is required to produce high concentrations of 5-HV, we newly introduced glucose dehydrogenase (GDH) for NADPH regeneration system from Bacillus subtilis 168. By applying GDH with D-glucose and optimizing the parameters, 5-HV conversion rate from 5-AVA increased from 47% (w/o GDH) to 82% when using 200 mM (23.4 g/L) of 5-AVA. Also, it reached 56% conversion in 2 h, showing 56 mM/h (6.547 g/L/h) productivity from 200 mM 5-AVA, finally reaching 350 mM (41 g/L) and 14.6 mM/h (1.708 g/L/h) productivity at 24 h when 1 M (117.15 g/L) 5-AVA was used. When the whole-cell system with GDH was expanded to produce 1,5-PDO, its production was also increased 5-fold. Considering that 5-HV and 1,5-PDO production depends heavily on the reducing power of the cells, we successfully achieved a significant increase in 5-HV and 1,5-PDO production using GDH.

Polyhydroxybutyrate (PHB) production from sugar cane molasses and tap water without sterilization using novel strain, Priestia sp. YH4.

The production cost of biodegradable polymer like polyhydroxybutyrate (PHB) is still higher than that of petroleum-based plastics. A potential solution for reducing its production cost is using a cheap carbon source and avoiding a process of sterilization. In this study, a novel PHB-producing microbial strain, Priestia sp. YH4 was screened from the marine environment using sugarcane molasses as the carbon source without sterilization. Culture conditions, such as carbon, NaCl, temperature, pH, inoculum size, and cultivation time, were optimized for obtaining the highest PHB production by YH4 resulting in 5.94 g/L of dry cell weight (DCW) and 61.7 % of PHB content in the 5 mL culture. In addition, it showed similar PHB production between the cultures with or without sterilization in Marine Broth media. When cultured using only tap water, sugarcane molasses, and NaCl in a 5 L fermenter, 24.8 g/L DCW was produced at 41 h yielding 13.9 g/L PHB. Finally, DSC (Differential Scanning Calorimetry) and GPC (Gel Permeation Chromatography) were used to analyze thermal properties and molecular weights resulting in Tm = 167.2 °C, Tc = 67.3 °C, Mw = 2.85 × 105, Mn = 1.05 × 105, and PDI = 2.7, respectively. Therefore, we showed the feasibility of more economical process for PHB production by finding novel strain, utilizing molasses with minimal media components and avoiding sterilization.

Macroalgae as a source of sugar and detoxifier biochar for polyhydroxyalkanoates production by Halomonas sp. YLGW01 under the unsterile condition.

Macroalgae (seaweed) is considered a favorable feedstock for polyhydroxyalkanoates (PHAs) production owing to its high productivity, low land and freshwater requirement, and renewable nature. Among different microbes Halomonas sp. YLGW01 can utilize algal biomass-derived sugars (galactose and glucose) for growth and PHAs production. Biomass-derived byproducts furfural, hydroxymethylfurfural (HMF), and acetate affects Halomonas sp. YLGW01 growth and poly(3-hydroxybutyrate) (PHB) production i.e., furfural > HMF > acetate. Eucheuma spinosum biomass-derived biochar was able to remove 87.9 % of phenolic compounds from its hydrolysate without affecting sugar concentration. Halomonas sp. YLGW01 grows and accumulates a high amount of PHB at 4 % NaCl. The use of detoxified unsterilized media resulted in high biomass (6.32 ± 0.16 g cdm/L) and PHB production (3.88 ± 0.04 g/L) compared to undetoxified media (3.97 ± 0.24 g cdm/L, 2.58 ± 0.1 g/L). The finding suggests that Halomonas sp. YLGW01 has the potential to valorize macroalgal biomass into PHAs and open a new avenue for renewable bioplastic production.

Revealing the key gene involved in bioplastic degradation from superior bioplastic degrader Bacillus sp. JY35.

The use of bioplastics, which can alleviate environmental pollution caused by non-degradable bioplastics, has received attention. As there are many types of bioplastics, method that can treat them simultaneously is important. Therefore, Bacillus sp. JY35 which can degrade different types of bioplastics, was screened in previous study. Most types of bioplastics, such as polyhydroxybutyrate (PHB), (P(3HB-co-4HB)), poly(butylene adipate-co-terephthalate) (PBAT), polybutylene succinate (PBS), and polycaprolactone (PCL), can be degraded by esterase family enzymes. To identify the genes that are involved in bioplastic degradation, analysis with whole-genome sequencing was performed. Among the many esterase enzymes, three carboxylesterase and one triacylglycerol lipase were identified and selected based on previous studies. Esterase activity using p-nitrophenyl substrates was measured, and the supernatant of JY35_02679 showed strong emulsion clarification activity compared with others. In addition, when recombinant E. coli was applied to the clear zone test, only the JY35_02679 gene showed activity in the clear zone test with bioplastic containing solid cultures. Further quantitative analysis showed 100 % PCL degradation at 7 days and 45.7 % PBS degradation at 10 days. We identified a gene encoding a bioplastic-degrading enzyme in Bacillus sp. JY35 and successfully expressed the gene in heterologous E. coli, which secreted esterases with broad specificity.

Enhancement of biohydrogen production in Clostridium acetobutylicum ATCC 824 by overexpression of glyceraldehyde-3-phosphate dehydrogenase gene.

In the dark fermentation of hydrogen, development of production host is crucial as bacteria act on substrates and produce hydrogen. The present study aimed to improve hydrogen production through the development of Clostridium acetobutylicum as a superior biohydrogen producer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which produces NADH/NADPH for metabolites and energy in primary pathways, was introduced to enhance hydrogen production. The strain CAC824-G containing gapC that encodes GAPDH showed a 66.3 % higher hydrogen production than the wild-type strain, with increased NADH and NADPH pools. Glucose consumption and other byproducts, such as acetone, butanol, and ethanol, were also high in CAC824-G. Overexpression of gapC resulted in increased hydrogen production with sugars obtained from different biomass, even in the presence of inhibitors such as vanillin, 5-hydroxymethylfufural, acetic acid, and formic acid. Our results imply that overexpression of gapC in Clostridium is possible to expand the production of the reported biochemicals to produce hydrogen.

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.

Strategy for efficiently utilizing Escherichia coli cells producing isobutanol by combining isobutanol and indigo production systems.

Isobutanol is a potential biofuel, and its microbial production systems have demonstrated promising results. In a microbial system, the isobutanol produced is secreted into the media; however, the cells remaining after fermentation cannot be used efficiently during the isobutanol recovery process and are discarded as waste. To address this, we aimed to investigate the strategy of utilizing these remaining cells by combining the isobutanol production system with the indigo production system, wherein the product accumulates intracellularly. Accordingly, we constructed E. coli systems with genes, such as acetolactate synthase gene (alsS), ketol-acid reductoisomerase gene (ilvC), dihydroxyl-acid dehydratase (ilvD), and alpha-ketoisovalerate decarboxylase gene (kivD), for isobutanol production and genes, such as tryptophanase gene (tnaA) and flavin-containing monooxygenase gene (FMO), for indigo production. This system produced isobutanol and indigo simultaneously while accumulating indigo within cells. The production of isobutanol and indigo exhibited a strong linear correlation up to 72 h of production time; however, the pattern of isobutanol and indigo production varied. To our knowledge, this study is the first to simultaneously produce isobutanol and indigo and can potentially enhance the economy of biochemical production.

Direct Monitoring of Membrane Fatty Acid Changes and Effects on the Isoleucine/Valine Pathways in an ndgR Deletion Mutant of Streptomyces coelicolor.

NdgR, a global regulator in soil-dwelling and antibiotic-producing Streptomyces, is known to regulate branched-chain amino acid metabolism by binding to the upstream region of synthetic genes. However, its numerous and complex roles are not yet fully understood. To more fully reveal the function of NdgR, phospholipid fatty acid (PLFA) analysis with gas chromatography-mass spectrometry (GC-MS) was used to assess the effects of an ndgR deletion mutant of Streptomyces coelicolor. The deletion of ndgR was found to decrease the levels of isoleucine- and leucine-related fatty acids but increase those of valine-related fatty acids. Furthermore, the defects in leucine and isoleucine metabolism caused by the deletion impaired the growth of Streptomyces at low temperatures. Supplementation of leucine and isoleucine, however, could complement this defect under cold shock condition. NdgR was thus shown to be involved in the control of branched-chain amino acids and consequently affected the membrane fatty acid composition in Streptomyces. While isoleucine and valine could be synthesized by the same enzymes (IlvB/N, IlvC, IlvD, and IlvE), ndgR deletion did not affect them in the same way. This suggests that NdgR is involved in the upper isoleucine and valine pathways, or that its control over them differs in some respect.

Polyhydroxybutyrate production from crude glycerol using a highly robust bacterial strain Halomonas sp. YLGW01.

Petrochemical-based plastics are hardly biodegradable and a major cause of environmental pollution, and polyhydroxybutyrate (PHB) is attracting attention as an alternative due to its similar properties. However, the cost of PHB production is high and is considered the greatest challenge for its industrialization. Here, crude glycerol was used as a carbon source for more efficient PHB production. Among the 18 strains investigated, Halomonas taeanenisis YLGW01 was selected for PHB production due to its salt tolerance and high glycerol consumption rate. Furthermore, this strain can produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3 HV)) with 17 % 3 HV mol fraction when a precursor is added. PHB production was maximized through medium optimization and activated carbon treatment of crude glycerol, resulting in 10.5 g/L of PHB with 60 % PHB content in fed-batch fermentation. Physical properties of the produced PHB were analyzed, i.e., weight average molecular weight (6.8 × 105), number average molecular weight (4.4 × 105), and the polydispersity index (1.53). In the universal testing machine analysis, the extracted intracellular PHB showed a decrease in Young's modulus, an increase in Elongation at break, greater flexibility than authentic film, and decreased brittleness. This study confirmed that YLGW01 is a promising strain for industrial PHB production using crude glycerol.

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.

Evaluation of Non-Thermal Decontamination Processes to Have the Equivalence of Thermal Process in Raw Ground Chicken.

The present study was aimed at examining the antibacterial effects of non-thermal decontamination processes, which are equivalent to thermal treatment, to ensure microbiological safety of raw ground chicken. Escherichia coli or Salmonella were inoculated into 25 g of raw ground chicken samples. The raw ground chicken samples were non-treated or treated with high hydrostatic pressure (HHP) at 500 MPa (1-7 min), light-emitting diode (LED) irradiation at 405 nm wavelength (30-120 min), and heat at 70°C, 90°C (1-60 min), and 121°C (1-15 min). E. coli and Salmonella cell counts were enumerated after treatments. Moreover, the color parameters of treated raw ground chicken were analyzed. HHP treatment reduced E. coli and Salmonella cell counts by more than 5 Log CFU/g and more than 6 Log CFU/g after 7 min and 1 min, respectively; these effects were equivalent to those of thermal treatment. However, LED irradiation reduced Salmonella cell counts by only 0.9 Log CFU/g after 90 min of treatment, and it did not reduce E. coli cell counts for 90 min. Compared with those of the non-treated samples, the ΔE (total color difference) values of the samples treated with HHP were high, whereas the ΔE values of the samples treated with LED irradiation were low (1.93-2.98). These results indicate that despite color change by HHP treatment, HHP treatment at 500 MPa could be used as a non-thermal decontamination process equivalent to thermal treatment.

Evaluation of infrared assisted freeze drying for strawberry snacks: drying kinetics, energy efficiency and quality attributes.

Feasibility of infrared assisted freeze drying (IRAFD) was evaluated for production of the strawberry snacks. Infrared (IR) radiation provided the driving force of ice sublimation during freeze drying (FD). Different IRAFD conditions were tested, including the continuous IRAFD-1.6 kW/m2 and IRAFD-1.6 kW/m2 at different weight reductions (20%, 40%, and 60%). Conventional FD had a total drying time of 691 ± 19 min, whereas continuous IRAFD significantly reduced the drying time to 309 ± 32 min. Continuous IRAFD also reduced the amount of consumed electrical energy by 42% compared to that of FD. A long duration of IR radiation produced a soft texture in the snacks. Drying kinetics were analyzed using various models, including the Page model, exponential model, and Henderson and Pabis model. The Page model provided the best fit to the experimental drying curve. This study showed the potential of IRAFD in producing value-added fruit snacks with good textural quality and efficient use of energy.