Microbial production of 2-pyrone-4,6-dicarboxylic acid from lignin derivatives in an engineered Pseudomonas putida and its application for the synthesis of bio-based polyester.

Lignin valorization depends on microbial upcycling of various aromatic compounds in the form of a complex mixture, including p-coumaric acid and ferulic acid. In this study, an engineered Pseudomonas putida strain utilizing lignin-derived monomeric compounds via biological funneling was developed to produce 2-pyrone-4,6-dicarboxylic acid (PDC), which has been considered a promising building block for bioplastics. The biosynthetic pathway for PDC production was established by introducing the heterologous ligABC genes under the promoter Ptac in a strain lacking pcaGH genes to accumulate a precursor of PDC, i.e., protocatechuic acid. Based on the culture optimization, fed-batch fermentation of the final strain resulted in 22.7 g/L PDC with a molar yield of 1.0 mol/mol and productivity of 0.21 g/L/h. Subsequent purification of PDC at high purity was successfully implemented, which was consequently applied for the novel polyester.

Effectiveness and Safety of Panax ginseng Extract on Hepatic Dysfunction: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

BACKGROUND: The purpose of this study was to evaluate the efficacy and safety of Panax ginseng extract (GS-KG9) in the treatment of hepatic dysfunction. METHODS: A randomized, double-blind, placebo-controlled clinical trial was conducted from December 2017 to January 2019. The trial included 60 subjects between the ages of 19 and 70 who had higher alanine transaminase (ALT) levels than the normal upper limit. The subjects were randomly divided into two groups: GS-KG9 (n = 30) and placebo (n = 30). The former was administered three GS-KG9 capsules (3 g/day) and the latter three placebo capsules (3 g/day) twice each day orally after meals in the morning and evening for 12 weeks. The primary goal was to observe the changes in ALT and gamma-glutamyl transferase (GGT) levels. The safety of the treatment was assessed and adverse events (AEs) were recorded. RESULTS: Out of 60 subjects, nine were excluded from the efficacy analysis because they met the exclusion criteria. Therefore, a total of 51 subjects were evaluated for the effectiveness of the treatment (26 in the GS-KG9 group and 25 in the placebo group). After 12 weeks of treatment, the ALT levels were significantly reduced in the GS-KG9 group compared to the placebo group (p=0.009). The GGT level of the GS-KG9 group was significantly lower than that of the placebo group (p=0.036). Mild AEs, such as diarrhea, occurred during the study. There were no significant differences between the two groups. CONCLUSION: The results of this trial suggest that GS-KG9 might be an effective and safe option for mild hepatic dysfunction. This trial is registered with KCT0004080.

26-Week Repeated Dose Oral Toxicity Study of KCHO-1 in Sprague-Dawley Rats.

OBJECTIVES: KCHO-1(Mecasin), also called Gamijakyakgamchobuja-tang originally, is a combination of some traditional herbal medicines in East Asia. This medicine has been used mainly for alleviating neuropathic pains for centuries in Korean traditional medicine. KCHO-1 was developed to treat pain, joint contracture and muscular weakness in patients with amyotrophic lateral sclerosis. This study was carried out to investigate the chronic toxicity of KCHO-1 oral administration in rats for 26 weeks. METHODS: Sprague-Dawely rats were divided into four groups and 10 rats were placed in the control group and the high-dose group, respectively. Group 1 was the control group and the remaining groups were the experimental groups. In the oral toxicity study, 500 mg/kg, 1,000 mg/kg, and 2,000 mg/kg of KCHO-1 were administered to the experimental group, and 10 ml/kg of sterile distilled water was administered to the control group. Survival rate, body weight, feed intake, clinical signs, and visual findings were examined. Urinalysis, ophthalmologic examination, necropsy, organ weight, hematologic examination, blood chemical examination and histopathologic examination were performed. RESULTS: Mortality and toxicological lesions associated with the administration of test substance were not observed in all groups. CONCLUSION: NOAEL(No observed adverse effect level) of KCHO-1 is higher than 2000 mg/kg/day. And, the above findings suggest that treatment with KCHO-1 is relatively safe.

High-Level Conversion of l-lysine into Cadaverine by Escherichia coli Whole Cell Biocatalyst Expressing Hafnia alvei l-lysine Decarboxylase.

Cadaverine is a C5 diamine monomer used for the production of bio-based polyamide 510. Cadaverine is produced by the decarboxylation of l-lysine using a lysine decarboxylase (LDC). In this study, we developed recombinant Escherichia coli strains for the expression of LDC from Hafnia alvei. The resulting recombinant XBHaLDC strain was used as a whole cell biocatalyst for the high-level bioconversion of l-lysine into cadaverine without the supplementation of isopropyl ß-d-1-thiogalactopyranoside (IPTG) for the induction of protein expression and pyridoxal phosphate (PLP), a key cofactor for an LDC reaction. The comparison of results from enzyme characterization of E. coli and H. alvei LDC revealed that H. alvei LDC exhibited greater bioconversion ability than E. coli LDC due to higher levels of protein expression in all cellular fractions and a higher specific activity at 37 °C (1825 U/mg protein > 1003 U/mg protein). The recombinant XBHaLDC and XBEcLDC strains were constructed for the high-level production of cadaverine. Recombinant XBHaLDC produced a 1.3-fold higher titer of cadaverine (6.1 g/L) than the XBEcLDC strain (4.8 g/L) from 10 g/L of l-lysine. Furthermore, XBHaLDC, concentrated to an optical density (OD600) of 50, efficiently produced 136 g/L of cadaverine from 200 g/L of l-lysine (97% molar yield) via an IPTG- and PLP-free whole cell bioconversion reaction. Cadaverine synthesized via a whole cell biocatalyst reaction using XBHaLDC was purified to polymer grade, and purified cadaverine was successfully used for the synthesis of polyamide 510. In conclusion, an IPTG- and PLP-free whole cell bioconversion process of l-lysine into cadaverine, using recombinant XBHaLDC, was successfully utilized for the production of bio-based polyamide 510, which has physical and thermal properties similar to polyamide 510 synthesized from chemical-grade cadaverine.

Metabolic engineering of Corynebacterium glutamicum for the production of glutaric acid, a C5 dicarboxylic acid platform chemical.

Corynebacterium glutamicum was metabolically engineered for the production of glutaric acid, a C5 dicarboxylic acid that can be used as platform building block chemical for nylons and plasticizers. C. glutamicum gabT and gabD genes and Pseudomonas putida davT and davD genes encoding 5-aminovalerate transaminase and glutarate semialdehyde dehydrogenase, respectively, were examined in C. glutamicum for the construction of a glutaric acid biosynthesis pathway along with P. putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. The glutaric acid biosynthesis pathway constructed in recombinant C. glutamicum was engineered by examining strong synthetic promoters PH30 and PH36, C. glutamicum codon-optimized davTDBA genes, and modification of davB gene with an N-terminal His6-tag to improve the production of glutaric acid. It was found that use of N-terminal His6-tagged DavB was most suitable for the production of glutaric acid from glucose. Fed-batch fermentation using the final engineered C. glutamicum H30_GAHis strain, expressing davTDA genes along with davB fused with His6-tag at N-terminus could produce 24.5 g/L of glutaric acid with low accumulation of l-lysine (1.7 g/L), wherein 5-AVA accumulation was not observed during fermentation.

Hwanglyunhaedok Pharmacopuncture versus Saline Pharmacopuncture on Chronic Nonbacterial Prostatitis/Chronic Pelvic Pain Syndrome.

PURPOSE: Chronic nonbacterial prostatitis (CP)/chronic pelvic pain syndrome (CPPS) is known not to have a successful therapy as yet. More recently, acupuncture, some herbal compounds, and trigger point injection have been reported to be beneficial. Pharmacopuncture is an herbal acupuncture having these benefits. The aim of this study was to compare the effect of HP (Hwanglyunhaedok Pharmacopuncture) versus normal saline injection (Saline Pharmacopuncture, SP) on CP/CPPS. METHODS: A retrospective follow up study of 63 patients who were diagnosed with CP/CPPS was performed. All patients were treated with electroacupuncture and injected with either 1 ml of HP or SP at CV1 as a standard treatment for 4 weeks. Thus, the patients were classified in two groups: HP (n = 32) and SP group (n = 31). Treatment was applied twice a week every third day for 4 weeks. After 4 weeks, the effect of pharmacopunture in both groups was compared using NIH-CPSI (National Institutes of Health-Chronic Prostatitis Symptom Index) and IPSS (International Prostate Symptom Score) before and after treatment. RESULTS: After treatment, the total NIH-CPSI scores were significantly reduced in both groups (p < 0.01). Pain domain scores in both groups showed significant decrease (p < 0.01). In HP group, urination (p < 0.05) and quality of life (p < 0.01) scores reduced significantly. In SP group, impact score showed significant decrease (p < 0.05). However, impact score in HP group and urination and quality of life scores in SP group didn't show any significance. IPSS score was reduced significantly after treatment in both groups (p < 0.05). CONCLUSIONS: These results suggest that pharmacopuncture and electroacupuncture treatment were effective on CP/CPPS. HP and SP didn't show any significant difference. However, it was also confirmed that HP is more favorable than SP to improve the symptoms of CP/CPPS.

Production of 5-aminovaleric acid in recombinant Corynebacterium glutamicum strains from a Miscanthus hydrolysate solution prepared by a newly developed Miscanthus hydrolysis process.

This study examined nine expired industrial Corynebacterium glutamicum strains with high lysine producing capability for enhanced production of 5-AVA. C. glutamicum KCTC 1857 exhibiting the highest lysine production was transformed with either original Pseudomonas putida davBA genes, encoding the 5-AVA biosynthesis pathway, or C. glutamicum codon-optimized davBA genes. C. glutamicum KCTC 1857 expressing the original genes had superior cell viability and 5-AVA production capability compared to the other strain. This strain produced 39.93g/L of 5-AVA, which is the highest titer reported to date in fed-batch fermentation from glucose. Indeed, Miscanthus hydrolysate solution prepared from a novel process, comprising pretreatment, hydrolysis, purification, and concentration, was used as feedstock for 5-AVA production. A total of 12.51g/L 5-AVA was produced from the Miscanthus hydrolysate; this value is 34.7% higher than that obtained from glucose in batch fermentation.

KCHO-1, a novel herbal anti-inflammatory compound, attenuates oxidative stress in an animal model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective death of motor neurons in the central nervous system. The main cause of the disease remains elusive, but several mutations have been associated with the disease process. In particular, mutant superoxide dismutase 1 (SOD1) protein causes oxidative stress by activating glia cells and contributes to motor neuron degeneration. KCHO-1, a novel herbal combination compound, contains 30% ethanol and the extracts of nine herbs that have been commonly used in traditional medicine to prevent fatigue or inflammation. In this study, we investigated whether KCHO-1 administration could reduce oxidative stress in an ALS model. KCHO-1 administered to ALS model mice improved motor function and delayed disease onset. Furthermore, KCHO-1 administration reduced oxidative stress through gp91phox and the MAPK pathway in both classically activated microglia and the spinal cord of hSOD1G93A transgenic mice. The results suggest that KCHO-1 can function as an effective therapeutic agent for ALS by reducing oxidative stress.

Historical Medical Value of Donguibogam.

Oriental medicine, since its origin in China, has had a long history extending over 2000 years. Today, it comprises several types of medicine predominately practiced in East Asia, including traditional Chinese, traditional Korean, and Kampo medicine. The distinctive medical system of traditional Korean medicine was established shortly after the publication of Donguibogam by Dr. Heo Jun in 1613. Donguibogam is highly acclaimed across East Asia; in 2009, in light of its historical medical value, the United Nations Educational, Scientific, and Cultural Organization registered the book on its cultural heritage list. Here, we review the historical medical value of Donguibogam. The findings confirm that Donguibogam developed a unique and independent form of traditional Korean medicine and innovatively reformed the disease classification system. Moreover, Donguibogam emphasized the importance of disease prevention and medical pragmatism. This book also accelerated the development of folk medicine. Owing to its historical medical value, Donguibogam is now considered the 'bible' of Oriental medicine. Its wide acceptance has contributed to the expansion of Korean medicine utilization among the general public. Donguibogam has also played an important role in the establishment of traditional Korean medicine as a universally valid and original form of medicine, independent of traditional Chinese medicine.

Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution.

BACKGROUND: Lignocellulosic raw materials have extensively been examined for the production of bio-based fuels, chemicals, and polymers using microbial platforms. Since xylose is one of the major components of the hydrolyzed lignocelluloses, it is being considered a promising substrate in lignocelluloses based fermentation process. Ralstonia eutropha, one of the most powerful and natural producers of polyhydroxyalkanoates (PHAs), has extensively been examined for the production of bio-based chemicals, fuels, and polymers. However, to the best of our knowledge, lignocellulosic feedstock has not been employed for R. eutropha probably due to its narrow spectrum of substrate utilization. Thus, R. eutropha engineered to utilize xylose should be useful in the development of microbial process for bio-based products from lignocellulosic feedstock. RESULTS: Recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes encoding xylose isomerase and xylulokinase respectively, was constructed and examined for the synthesis of poly(3-hydroxybutyrate) [P(3HB)] using xylose as a sole carbon source. It could produce 2.31 g/L of P(3HB) with a P(3HB) content of 30.95 wt% when it was cultured in a nitrogen limited chemically defined medium containing 20.18 g/L of xylose in a batch fermentation. Also, recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes produced 5.71 g/L of P(3HB) with a P(3HB) content of 78.11 wt% from a mixture of 10.05 g/L of glucose and 10.91 g/L of xylose in the same culture condition. The P(3HB) concentration and content could be increased to 8.79 g/L and 88.69 wt%, respectively, when it was cultured in the medium containing 16.74 g/L of glucose and 6.15 g/L of xylose. Further examination of recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes by fed-batch fermentation resulted in the production of 33.70 g/L of P(3HB) in 108 h with a P(3HB) content of 79.02 wt%. The concentration of xylose could be maintained as high as 6 g/L, which is similar to the initial concentration of xylose during the fed-batch fermentation suggesting that xylose consumption is not inhibited during fermentation. Finally, recombinant R. eutorpha NCIMB11599 expressing the E. coli xylAB gene was examined for the production of P(3HB) from the hydrolysate solution of sunflower stalk. The hydrolysate solution of sunflower stalk was prepared as a model lignocellulosic biomass, which contains 78.8 g/L of glucose, 26.9 g/L of xylose, and small amount of 4.8 g/L of galactose and mannose. When recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes was cultured in a nitrogen limited chemically defined medium containing 23.1 g/L of hydrolysate solution of sunflower stalk, which corresponds to 16.8 g/L of glucose and 5.9 g/L of xylose, it completely consumed glucose and xylose in the sunflower stalk based medium resulting in the production of 7.86 g/L of P(3HB) with a P(3HB) content of 72.53 wt%. CONCLUSIONS: Ralstonia eutropha was successfully engineered to utilize xylose as a sole carbon source as well as to co-utilize it in the presence of glucose for the synthesis of P(3HB). In addition, R. eutropha engineered to utilized xylose could synthesize P(3HB) from the sunflower stalk hydrolysate solution containing glucose and xylose as major sugars, which suggests that xylose utilizing R. eutropha developed in this study should be useful for development of lignocellulose based microbial processes.

Improving the synthesis of phenolic polymer using Coprinus cinereus peroxidase mutant Phe230Ala.

The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer.

The herbal extract KCHO-1 exerts a neuroprotective effect by ameliorating oxidative stress via heme oxygenase-1 upregulation.

KCHO-1 is a novel product comprised of 30% ethanol extracts obtained from nine medical herbs, which are commonly used in traditional Korean and Chinese medicine. The nine herbs include Curcuma longa, Salvia miltiorrhiza, Gastrodia elata, Chaenomeles sinensis, Polygala tenuifolia, Paeonia japonica, Glycyrrhiza uralensis, Atractylodes japonica and processed Aconitum carmichaeli. Recent studies have reported the beneficial effects of these herbs. The present study aimed to investigate the direct neuroprotective effects of KCHO­1 on HT22 mouse hippocampal cells, and to determine the possible underlying mechanisms. KCHO­1 significantly suppressed glutamate­ and hydrogen peroxide (H2O2)­induced cell damage, and reactive oxygen species generation. In addition, KCHO­1 increased the mRNA and protein expression levels of heme oxygenase (HO)­1. Tin protoporphyrin, which is an inhibitor of HO activity, partially suppressed the effects of KCHO­1. Furthermore, KCHO­1 significantly upregulated nuclear factor erythroid­derived 2­related factor­2 (Nrf2) nuclear translocation. Extracellular signal­regulated kinase (ERK) activation also appeared to be associated with KCHO­1­induced HO­1 expression, since the ERK inhibitor PD98059 suppressed HO­1 expression and prevented KCHO­1­induced cytoprotection. The results of the present study suggested that KCHO­1 may effectively prevent glutamate­ or H2O2­induced oxidative damage via Nrf2/ERK mitogen­activated protein kinase­dependent HO­1 expression. These data suggest that KCHO­1 may be useful for the treatment of neurodegenerative diseases.

Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD.

An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein.

Construction of heterologous gene expression cassettes for the development of recombinant Clostridium beijerinckii.

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.

Development of engineered Escherichia coli whole-cell biocatalysts for high-level conversion of L-lysine into cadaverine.

A whole-cell biocatalytic system for the production of cadaverine from L-lysine has been developed. Among the investigated lysine decarboxylases from different microorganisms, Escherichia coli LdcC showed the best performance on cadaverine synthesis when E. coli XL1-Blue was used as the host strain. Six different strains of E. coli expressing E. coli LdcC were investigated and recombinant E. coli XL1-Blue, BL21(DE3) and W were chosen for further investigation since they showed higher conversion yield of lysine into cadaverine. The effects of substrate pH, substrate concentrations, buffering conditions, and biocatalyst concentrations have been investigated. Finally, recombinant E. coli XL1-Blue concentrated to an OD(600) of 50, converted 192.6 g/L (1317 mM) of crude lysine solution, obtained from an actual lysine manufacturing process, to 133.7 g/L (1308 mM) of cadaverine with a molar yield of 99.90 %. The whole-cell biocatalytic system described herein is expected to be applicable to the development of industrial bionylon production process.

Optimized Transformation of Newly Constructed Escherichia coli-Clostridia Shuttle Vectors into Clostridium beijerinckii.

Three Escherichia coli-Clostridia shuttle vectors, pKBA411-MCS, pKBE411-MCS, and pKBM411-MCS, which contain p15A, ColE1, and pMB1 origins for replication in E. coli, respectively, along with the pAMB origin for replication in C. beijerinckii, were constructed and examined for their transformation efficiencies into Clostridium beijerinckii NCIMB8052. The transformation condition of pKBM411-MCS, which was optimized by varying resistance, buffer composition, and DNA concentration, was further employed for the transformation of the other plasmids, pKBA411-MCS and pKBE411-MCS into C. beijerinckii. It was found out that transformation efficiency is highly dependent on the origin of replication. The highest transformation efficiency of 7.44 × 10(3) colony-forming units per microgram of DNA was obtained at 5.0 kV cm(-1) field strength, 200 Ω resistance, 270 mM sucrose concentration, 150 ng µg(-1), and 3.0 µg DNA using pKBM411-MCS having pMB1 and pAMB origins of replication. The application of the newly constructed vector system was also investigated by introducing the putative alcohol dehydrogenase gene of C. beijerinckii.

Construction of Synthetic Promoter-Based Expression Cassettes for the Production of Cadaverine in Recombinant Corynebacterium glutamicum.

Corynebacterium glutamicum is an important microorganism in the biochemical industry for the production of various platform chemicals. However, despite its importance, a limited number of studies have been conducted on how to constitute gene expression cassettes in engineered C. glutamicum to obtain desired amounts of the target products. Therefore, in this study, six expression cassettes for the expression of the second lysine decarboxylase of Escherichia coli, LdcC, were constructed using six synthetic promoters with different strengths and were examined in C. glutamicum for the production of cadaverine. Among six expression cassettes, the expression of the E. coli ldcC gene under the PH30 promoter supported the highest production of cadaverine in flask and fed-batch cultivations. A fed-batch fermentation of recombinant C. glutamicum expressing E. coli ldcC gene under the PH30 promoter resulted in the production of 40.91 g/L of cadaverine in 64 h. This report is expected to contribute toward developing engineered C. glutamicum strains to have desired features.

Development of rice bran treatment process and its use for the synthesis of polyhydroxyalkanoates from rice bran hydrolysate solution.

Rice bran treatment process for the production of 43.7 kg of hydrolysate solution containing 24.41 g/L of glucose and small amount of fructose from 5 kg of rice bran was developed and employed to produce polyhydroxyalkanoates in recombinant Escherichia coli and Ralstonia eutropha strains. Recombinant E. coli XL1-Blue expressing R. eutropha phaCAB genes and R. eutropha NCIMB11599 could produce poly(3-hydroxybutyrate) with the polymer contents of 90.1 wt% and 97.2 wt%, respectively, when they were cultured in chemically defined MR medium and chemically defined nitrogen free MR medium containing 10 mL/L of rice bran hydrolysate solution, respectively. Also, recombinant E. coli XL1-Blue and recombinant R. eutropha 437-540, both of which express the Pseudomonas sp. phaC1437 gene and the Clostridium propionicum pct540 gene could produce poly(3-hydroxybutyrate-co-lactate) from rice bran hydrolysate solution. These results suggest that rice bran may be a good renewable resource for the production of biomass-based polymers by recombinant microorganisms.

A 4-week Repeated dose Oral Toxicity Study of Mecasin in Sprague-Dawley Rats to Determine the Appropriate Doses for a 13-week, Repeated Toxicity Test.

OBJECTIVES: In this study, we investigated the 4-week repeated-dose oral toxicity of gami-jakyak gamcho buja decoction (Mecasin) to develop safe treatments. METHODS: In order to investigate the 4-week oral toxicity of Mecasin, we administered Mecasin orally to rats. Sprague-Dawley (SD) rats were divided into four groups of five male and five female animals per group: group 1 being the control group and groups 2, 3, and 4 being the experimental groups. Doses of Mecasin of 500, 1,000, and 2,000 mg/kg of body weight were administered to the experimental groups, and a dose of normal saline solution of 10 mL/kg was administered to the control group. We examined the survival rate, weight, clinical signs, and gross findings for four weeks. This study was conducted under the approval of the Institutional Animal Ethics Committee. RESULTS: No deaths occurred in any of the four groups. No significant changes in weights or food consumption between the control group and the experimental groups were observed. Serum biochemistry revealed that some groups showed significant decrease in inorganic phosphorus (IP) (P < 0.05). During necropsy on the rats, one abnormal macroscopic feature, a slight loss of fur, was observed in the mid dosage (1,000 mg/ kg) male group. No abnormalities were observed in any other rats. In histopathological findings, the tubular basophilia and cast of the kidney and extramedullary hematopoiesis of the spleen were found. However, those changes were minimal and had occurred naturally or sporadically. No other organ abnormalities were observed. CONCLUSION: During this 4-week, repeated, oral toxicity test of Mecasin in SD rats, no toxicity changes due to Mecasin were observed in any of the male or the female rats in the high dosage group. Thus, we suggest that the doses in a 13-week, repeated test should be 0, 500, 1,000, and 2,000 mg/kg respectively.

Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack.