• Journal of Microbiology and Biotechnology
• /
• 제8권6호
• /
• pp.714-718
• /
• 1998

Various mutants either lacking or having decreased levels of light-harvesting complexes and reaction center complex were obtained with a high frequency by an increased formation of poly-3-hydroxybutyrate (PHB) in Rhodobacter sphaeroides. One of the photosynthesis-defective mutants, PY-17, which was devoid of any of the light-harvesting complexes (B800-850, B875) as well as the reaction center complex, was analyzed further. The mutant showed substantial transcription of the puhA, pufKBALMX, and pucBAC operons coding for the structural proteins of the photosynthetic complexes although each of the activities was lower than that of the wild type. Translation of the pufKBALMX and pucBAC operons were also active in the mutant although with activities different from the corresponding one of the wild type. From these results the mutation appears to exert its effect at the post-translational level of the photosynthetic complex assembly. Complementation of the photosynthesis-defective phenotype of the mutant was achieved with an about 12-kb DNA region containing the puhA gene. The relationship between the formation of PHB and photosynthetic complexes is discussed.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제9권3호
• /
• pp.196-200
• /
• 2004

The supra molecular weight poly(〔R〕-3-hydroxybutyrate) (PH B), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineered Escherichia coli strain and its fermentation for high level production of supra molecular weight PHB. Recombinant E. coli strains, harboring plasm ids of different copy numbers containing the Alcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinant E. coli XL1-Blue, harboring a medium-copy-number pJC2 containing the A. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced in Ralstonia eutropha or recombinant E. coli.

• 생명과학회지
• /
• 제18권12호
• /
• pp.1625-1630
• /
• 2008

본 연구는 생분해성 플라스틱인 poly-${\beta}$-hydroxybutyrate (PHB)의 생산단가를 낮추기 위한 노력으로 저가의 기질로부터 PHB 대량생산을 위한 기초자료를 얻는데 그 목적을 두었다. Hemicellulose hydrolysate는 지구상에 풍부하게 존재하는 저가의 waste by-product로서 xylose가 많이 포함되어 있다. 본 연구에서는 xylose로부터 PHB를 생산할 수 있는 균주를 토양에서 분리하여, 분류학적 위치를 밝히고, 균체 생육 최적 조건, PHB 생산을 위한 최적 발효 배양 조건, PHB의 구조 확인 등을 검토 하였으며, 그 결과는 다음과 같다. 토양으로부터 분리한 균주 J-65는 형태학적, 배양적, 생화학적 및 partial 16S rRNA sequence에 근거하여 Bacillus megaterium로 동정하였다. B. megaterium J-65의 균체 생육 최적 조건은 온도 $37^{\circ}C$, 초발 pH 8.0이었으며 2% xylose, 0.25% $(NH_4)_2SO_4$, 0.3% $Na_2HPO_4{\cdot}12H_2O$, 0.1% $KH_2PO_4$였다. PHB 축적에 영향을 미치는 요인을 검토하기 위해 균체생육 최적배지에서 $37^{\circ}C$, 24시간 1차 배양한 후, 균체를 회수하여 각종 영양분이 결핍된 배지에 2차 배양을 실시한 결과 B. megaterium J-65는 균형생육조건(balanced-growth condition)에서 PHB를 합성하는 균주로 나타났다. PHB보다 물성이 향상된 PHB/HV 공중 합체를 생산하기 위하여 보조기질로 propionic acid를 첨가하였을 때, 0.1% propionic acid 농도에서 HV mol%가 14%인 PHB/HV 공중합체가 합성되었다. 5 l 용량의 발효조에 B. megaterium J-65를 회분배양하였을 때 배양 21시간에 건조균체량 10 g/l, PHB 3.5 g/l를 얻을 수 있었고, 유가배양을 실시한 결과 배양 48시간에 건조균체량 26.52g/l, PHB 9.28 g/l를 얻을 수 있었다. 생산된 PHB를 alkaline solution 처리와 chloroform을 이용한 유기용매 추출법을 이용하여 추출.정제한 후 Gas Chromatography로 정제를 확인하고 300MHz 1H-NMR을 실시한 결과 3-hydroxybutyrate의 homopolymer임을 확인하였다.

• Journal of Microbiology and Biotechnology
• /
• 제16권6호
• /
• pp.901-910
• /
• 2006

Poly(3-hydroxybutyrate) [P(3HB)] is a microbial polyester intracellularly accumulated as distinct granules in numerous microorganisms as an energy and carbon storage material. Recombinant Escherichia coli harboring the heterologous P(3HB) biosynthesis genes accumulates large amounts of P(3HB) granules, yet the granule-associated proteins have not been identified. Therefore, this study reports on an analysis of the P(3HB) granule-associated proteome in recombinant E. coli. Fiye proteins out of 7 spots identified were found to be involved in functions of translation, heat-stress responses, and P(3HB) biosynthesis. Two of the major granule-associated proteins, IbpA/B, which are already known to bind to recombinant proteins forming inclusion bodies in E. coli, were further analyzed. Immunoblotting and immunoelectron microscopic studies with IbpA/B antibodies clearly demonstrated the binding and localization of IbpA/B to P(3HB) granules. IbpA/B seemed to play an important role in recombinant E. coli producing P(3HB) by stabilizing the interface between the hydrophobic P(3HB) granules and the hydrophilic cytoplasm. Thus, IbpA/B were found to act like phasins in recombinant E. coli, as they are the major proteins bound to the P(3HB) granules, affect the morphology of the granules, and reduce the amount of cytosolic proteins bound to the P(3HB) granules.

• Journal of Microbiology and Biotechnology
• /
• 제15권5호
• /
• pp.1115-1119
• /
• 2005

The photoheterotrophic $H_2$ production of Rhodobacter sphaeroides was significantly increased through disruption of the genes coding for uptake hydrogenase and poly-${\beta}$-hydroxybutyrate (PHB) synthase (Lee et al., Appl. Microbiol. Biotechnol. 60: 147-153, 2002). In this work, we further removed the B800-850 light-harvesting (LH) complex from the strain and found an increase in $H_2$ production at the light-saturating cell growth (${\ge}10$ Watts $[W]/m^2$). Neither the mutant nor the wild-type produced more $H_2$ at the brighter light. Accordingly, light does not appear to be limited for the $H_2$ production by the presence of B800-850. However, increase in the level of the spectral complexes resulted in decrease of $H_2$ production. Thus, although the B875 is essential for light harvesting, the consumption of cellular energy for the synthesis of B800-850 and the surplus LH complexes may reduce the energy flow into the $H_2$ production of R. sphaeroides.

• Asian-Australasian Journal of Animal Sciences
• /
• 제23권10호
• /
• pp.1299-1307
• /
• 2010

This study was conducted to evaluate the effect of dietary antioxidant and energy density on performance and antioxidative status in transition cows. Forty cows were randomly allocated to 4 dietary treatments in a $2{\times}2$ factorial design. High or low energy density diets (1.43 or 1.28 Mcal $NE_L$/kg DM, respectively) were formulated with or without antioxidant (AOX, a dry granular blend of ethoxyquin and tertiary-butylhydroquinone; 0 or 5 g/cow per d). These diets were fed to cows for 21 days pre-partum. During the post-partum period, all cows were fed the same lactation diets, and AOX treatment followed as for the pre-partum period. Feeding a high energy diet depressed the DMI, milk yield, and 4% fat-corrected milk (FCM) of cows. However, AOX inclusion in the diet improved the milk and 4% FCM yields. There was an interaction of energy density by AOX on milk protein, milk fat and total solids contents. Feeding a high energy diet pre-partum increased plasma glucose and ${\beta}$-hydroxybutyrate, whereas dietary AOX decreased plasma ${\beta}$-hydroxybutyrate value during the transition period. There were also interactions between time and treatment for plasma glutathione peroxidase activity and malondialdehyde content during the study. Cows fed high energy diets pre-partum had higher plasma glutathione peroxidase activity 3 days prior to parturition, compared with those on low energy diets. Inclusion of AOX in diets decreased plasma glutathione peroxidase activity in cows 3 and 10 days pre-partum. Addition of AOX significantly decreased malondialdehyde values at calving. Energy density induced marginal changes in fatty acid composition in the erythrocyte membrane 3 days post-partum, while AOX only significantly increased cis-9, trans-11 conjugated linoleic acid composition. The increase in fluidity of the erythrocyte membrane was only observed in the high energy treatment. It is suggested that a diet containing high energy density pre-partum may negatively affect the anti-oxidative status, DMI and subsequent performance. Addition of AOX may improve the anti-oxidative status and reduce plasma ${\beta}$-hydroxybutyrate, eventually resulting in improved lactation performance; the response to AOX addition was more pronounced on the high energy diet.

• Asian-Australasian Journal of Animal Sciences
• /
• 제19권3호
• /
• pp.356-362
• /
• 2006

This study aimed to assess the effects of neutral detergent fiber (NDF) from rice straw on blood metabolites and productivity of dairy cows in the tropics. Eight Holstein ${\times}$ indigenous (75-87.5% Holstein) were randomly allocated to dietary treatments according to a double $4{\times}4$ Latin square design. Contents of roughage NDF from rice straw were 12.8, 14.8, 16.8 and 18.8% dry matter (DM) and concentrate NDF content was 10.2% DM, so that levels of dietary NDF were 23, 25, 27 and 29% DM. Dietary treatments were isonitrogenous and isocaloric diets. The average maximum and minimum temperature humidity index during the experimental period were 85.6 and 76.9, respectively. Plasma glucose (p<0.05), dry matter intake and 4% fat corrected milk (p<0.01) decreased with increasing contents of roughage NDF from rice straw, while rectal temperature, serum urea, cortisol, low density lipoprotein-cholesterol (p<0.05), serum total protein and ${\beta}$-hydroxybutyrate (p<0.01) increased. Serum triglyceride, total cholesterol and high density lipoprotein-cholesterol were not affected (p>0.05). Decreased rectal temperature and concentrations of serum cortisol were associated with decreasing levels of ${\beta}$-hydroxybutyrate and serum urea, but with increased concentrations of plasma glucose, DMI and 4% fat corrected milk indicating that lactating cows fed lower fiber diets were in lower oxidative stress. Therefore higher intake and metabolic efficiency in dairy cows fed diets lower in roughage NDF from rice straw would be of an advantage in productive systems under tropical conditions.

• 한국미생물·생명공학회지
• /
• 제51권3호
• /
• pp.257-267
• /
• 2023

The potential polyhydroxyalkanoates (PHA)-producing bacteria, Bacillus megaterium PP-10, was successfully isolated and studied its feasibility for utilization of pineapple peel waste (PPW) as a cheap carbon substrate. The PPW was pretreated with 1% (v/v) H₂SO₄ under steam sterilization and about 26.4 g/l of total reducing sugar (TRS) in pineapple peel hydrolysate (PPH) was generated and main fermentable sugars were glucose and fructose. A maximum cell growth and PHA concentration of 3.63 ± 0.07 g/l and 1.98 ± 0.09 g/l (about 54.58 ± 2.39%DCW) were received in only 12 h when grown in PPH. Interestingly, PHA productivity and biomass yield (Y_x/s) in PPH was about 4 times and 1.5 times higher than in glucose. To achieve the highest DCW and PHA production, the optimal culture conditions e.g. carbon to nitrogen ratios of 40 mole/mole, incubation temperature at 35℃ and shaking speed of 200 rpm were performed and a maximum DCW up to 4.24 ± 0.04 g/l and PHA concentration of 2.68 ± 0.02 g/l (61% DCW) were obtained. The produced PHA was further examined its monomer composition and found to contain only 3-hydroxybutyrate (3HB). This finding corresponded with the presence of class IV PHA synthase gene. Finally, certain thermal properties of the produced PHA i.e. the melting temperature (T_m) and the glass transition temperature (T_g) were about 176℃ and -4℃, respectively whereas the M_w was about 1.07 KDa ; therefore, the newly isolated B. megaterium PP-10 is a promising bacterial candidate for the efficient conversion of low-cost PPH to PHA.

• Asian-Australasian Journal of Animal Sciences
• /
• 제16권1호
• /
• pp.88-95
• /
• 2003

The objective of the present study was to determine the effect of exogenous bovine somatotropin on the mammary function in late lactating crossbred Holstein cows. Twelve 87.5% late lactating Holstein cows, approximately 30 weeks postpartum, were divided into two groups of 6 animals each. Animals in the control group were given sodium bicarbonate buffer by subcutaneous injection, while animals in the treated group were given recombinant bovine somatotropin (bST) by subcutaneous injection with 500 mg of bST (14 day prolonged-release bST). After bST injection, milk yield significantly increased from the control level on day 8 to day 20 (p<0.05) with a concomitant increase in mammary blood flow (p<0.01). An increase in mammary blood flow in response to bST treatment was greater than an increase in milk production. An increased plasma concentration of IGF-I coincided with an increase in mammary blood flow in animals treated with bST. There were no significant changes in the concentration of arterial plasma glucose concentration, the arteriovenous concentration difference (A-V difference) and mammary extraction ratio while the mammary glucose uptake increased when compared to the control group. The concentration of arterial plasma triglyceride decreased throughout the experimental period in animals give bST. The plasma concentration of acetate, and the mammary uptake for acetate significantly increased (p<0.05) after bST treatment. The action of bST did not affect the plasma concentration, A-V difference and extraction ratio across the mammary gland for $\beta$-hydroxybutyrate. The concentrations of milk fat and lactose tended to increase during bST treatment. Milk protein concentration initially increased in the first few days and decreased after bST injection when compared to the pretreated period. The present results indicated that bST could affect the mammary function in late lactating cows by increase in milk yield involving changes in both extra-mammary and intra-mammary mechanisms. The exogenous bST exerted its galactopoietic action through an increase in circulating IGF-I of the late lactating Crossbred Holstein cattle.

• Journal of Microbiology and Biotechnology
• /
• 제25권2호
• /
• pp.152-161
• /
• 2015

CodY is a highly conserved protein in low G+C gram-positive bacteria that regulates genes involved in sporulation and stationary-phase adaptation. Bacillus thuringiensis is a grampositive bacterium that forms spores and parasporal crystals during the stationary phase. To our knowledge, the regulatory mechanism of CodY in B. thuringiensis is unknown. To study the function of CodY protein in B. thuringiensis, BMB171codY^- was constructed in a BMB171 strain. A shuttle vector containing the ORF of cry1Ac10 was transformed into BMB171 and BMB171codY^-, named BMB171cry1Ac and BMB171codY^-cry1Ac, respectively. Some morphological and physiological changes of codY mutant BMB171codY^-cry1Ac were observed. A comparative proteomic analysis was conducted for both BMB171codY^-cry1Ac and BMB171cry1Ac through two-dimensional gel electrophoresis and MALDI-TOF-MS/MS analysis. The results showed that the proteins regulated by CodY are involved in microbial metabolism, including branched-chain amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, and energy metabolism. Furthermore, we found CodY to be involved in sporulation, biosynthesis of poly-β-hydroxybutyrate, growth, genetic competence, and translation. According to the analysis of differentially expressed proteins, and physiological characterization of the codY mutant, we performed bacterial one-hybrid and electrophoretic mobility shift assay experiments and confirmed the direct regulation of genes by CodY, specifically those involved in metabolism of branched-chain amino acids, ribosomal recycling factor FRR, and the late competence protein ComER. Our data establish the foundation for in-depth study of the regulation of CodY in B. thuringiensis, and also offer a potential biocatalyst for functions of CodY in other bacteria.