Enhanced production of D-(-)-3-hydroxybutyric acid by recombinant Escherichia coli

Wild-type bacteria including Escherichia coli normally do not produce extracellular D-(-)-3-hydroxybutyric acid (3HB). To produce extracellular chiral 3HB, a new pathway for synthesis of 3HB was constructed by simultaneous expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), phosphor-transbutyrylase (ptb) and butyrate kinase (buk) in E. coli strain DH5alpha. E. coli DH5alpha containing any one of the four plasmids pBHR69, pUCAB, p68CM or pKKAB that harbor the phbA and phbB genes produced small amounts of 3HB, ranging from 75 to 400 mg l(-1), while E. coli DH5alpha harboring p68CMPTK containing genes of phbA, phbB, ptb and buk increased the 3HB concentration to 1.4 g l(-1) in shake flasks supplemented with LB broth and 20 g l(-1) glucose. 3HB production was further improved to over 2 g l(-1) in shake flasks when E. coli DH5alpha hosted two plasmids simultaneously that separately contained phbA and phbB in one plasmid while ptb and buk in the other. A batch fermentation run in a 5-l fermenter produced approximately 5 g l(-1) 3HB after 24 h. A fed-batch process increased 3HB production to 12 g l(-1) after 48 h of fermentation.     

Biosynthesis of enantiopure (S)-3-hydroxybutyric acid in metabolically engineered Escherichia coli

A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the beta-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl beta-D: -thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the beta-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l(-1) from 20 g l(-1) of glucose in Luria-Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l(-1) (OD(600) = 30 and 60), respectively. When protein expression was induced at 60 of OD(600) and glucose was fed to the concentration of 15 g l(-1), 10.3 g l(-1) of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l(-1)h(-1). Lowering glucose concentration to 5 g l(-1) and induction of protein expression at 30 of OD(600) significantly reduced final S3HB concentration to 3.7 g l(-1), which also resulted in the decrease of the S3HB productivity to 0.05 g l(-1)h(-1).     

Purification and properties of D-(-)-3-hydroxybutyrate oligomer hydrolase of Paracoccus denitrificans

D-(-)-3-Hydroxybutyrate (3HB) oligomer hydrolase was purified from Paracoccus denitrificans. The enzyme was a monomeric protein with an approximate molecular mass of 31 kDa. The isoelectric point of the enzyme was 5.2. Optimum temperature and pH were 35-40 degrees C and 8.0, respectively. The enzyme activity was not affected by sulfhydryl reagents but strongly inhibited by serine proteinase inhibitors. Both 3HB trimer and 3HB dimer were hydrolyzed by the enzyme, indicating that the enzyme is not 3HB dimer hydrolase but 3HB oligomer hydrolase. para-Nitrophenyl esters of short-chain fatty acids were also hydrolyzed by the enzyme. 3HB dimer was hydrolyzed somewhat faster than 3HB trimer. The level of the enzyme activity was almost constant, irrespective of carbon sources for the bacterial growth and of the cultivation conditions.     

Microbial production of R-3-hydroxybutyric acid by recombinant E. coli harboring genes of phbA, phbB, and tesB

Production of R-3-hydroxybutyric acid (3HB) was observed when genes of β-ketothiolase (PhbA), acetoacetyl CoA reductase (PhbB), and thioesterase II (TesB) were jointly expressed in Escherichia coli. TesB, generally regarded as a medium chain length acyl CoA thioesterase, was found, for the first time, to play an important role for transforming short chain length 3-hydroxybutyrate-CoA to its free fatty acid, namely, 3HB. E. coli BW25113 (pSPB01) harboring phbA, phbB, and tesB genes produced approximately 4 g/l 3HB in shake flask culture within 24 h with glucose used as a carbon source. Under anaerobic growth conditions, 3HB production was found to be more effective, achieving 0.47 g 3HB/g glucose compared with only 0.32 g 3HB/g glucose obtained from aerobic process. When growth was conducted on sodium gluconate, 6 g/l 3HB was obtained. In a 24-h fed-batch growth process conducted in a 6-l fermentor containing 3 l glucose mineral medium, 12 g/l 3HB was produced from 17 g/l cell dry weight (CDW). This was the highest 3HB productivity achieved by a one-stage fermentation process for 3HB production. Liu and Ouyang contributed equally to the paper.     

Stereoselective effects of 3-hydroxybutyrate on glucose utilization of rat cardiomyocytes

In researches of ketone bodies, D-3-hydroxybutyrate (D-3HB) is usually the major one which has been investigated; in contrast, little attention has been paid to L-3-hydroxybutyrate (L-3HB), because of its presence in trace amounts, its dubious metabolism, and a lack of knowledge about its sources. In the present study we determined the distributions of enantiomers of 3-hydroxybutyrate (3HB) in rat brain, liver, heart, and kidney homogenates, and we found the heart homogenate contained an enriched amount of L-3HB (37.67 microM/mg protein) which generated a significant ratio of 66/34 (D/L). The ratio was altered to be 87/13 in the diabetic rat heart homogenate. We subsequently found this changed ratio of D/L-3HB may contribute to reduce glucose utilization in cardiomyocytes. Glucose utilization by cardiomyocytes with 5 mM of D-3HB was decreased to 61% of the control, but no interference was observed when D-3HB was replaced with L-3HB, suggesting L-3HB is not utilized for the energy fuel as other ketone bodies are. In addition, the reduced glucose utilization caused by D-3HB gradually recovered in a dose-dependent manner with administration of additional L-3HB. The results gave the necessity of taking L-3HB together with D-3HB into account with regard to glucose utilization, and L-3HB may be a helpful substrate for improving inhibited cardiac pyruvate oxidation caused by hyperketonemia.     

Synthesis of poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) by a Sinorhizobium fredii strain

AIMS: The potential of a Sinorhizobium fredii strain to produce a copolymer from glucose and sodium dodecanoate substrates was investigated. METHODS AND RESULTS: Using an orthogonal design in a flask-shaker culture system, the vital regulation conditions for copolymer synthesis were optimized. These optimal results were applied to further studies in a two-stage fed-batch fermentation with a 10-l fermentor. When the biomass approached 33.5 g l(-1) dry cells at 35 h, 7 mmol l(-1) sodium dodecanoate was added into the broth to trigger the copolymer synthesis. After further culturing for 3 h, the copolymer product could be 17.14 g l(-1). The molecular structure of the copolymer was determined to be a poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) [P (HB-HO)] by nuclear magnetic resonance. The content of HB and HO in P (HB-HO) was 79.2% (w/w) and 20.8% (w/w) respectively. The molecular weight of the P (HB-HO) was measured as 1.85 x 10(5) Da by a viscosity method. CONCLUSION: The results demonstrated that the S. fredii strain used could be a potential candidate for the industrial production of the copolymer. SIGNIFICANCE AND IMPACT OF THE STUDY: Some basic fermentation parameters were acquired through the fed-batch culturing experiments and they should be applicable in developing large-scale fermentation technologies for producing the P (HB-HO) copolymers.     

Quisqualic Acid Modulates Kainate Responses in Cultured Cerebellar Granule Cells

The activation of kainic acid and quisqualic acid receptors in cultured cerebellar granule cells stimulated the release of preaccumulated D-[3H]aspartate. The effect of kainate could be distinguished from that of quisqualate by its sensitivity to the antagonists kynurenic acid and 2,3-cis-piperidine dicarboxylic acid. At a concentration of kainic acid (50 microM) close to its half-maximal releasing effect, simultaneous addition of quisqualic acid (10-50 microM) resulted in a significant dose-dependent inhibition of the kainate-induced component of D-[3H]aspartate release, which was monitored by the progressive decrease in sensitivity of the evoked release to kynurenic acid. In contrast, when kainic acid was used at a subeffective concentration (10 microM), addition of low doses of quisqualate (2-5 microM) resulted in a synergistic effect on D-[3H]aspartate release. Under these conditions, the effect of the two agonists was sensitive to kynurenic acid. Kainic acid (50-100 microM) also caused a dose-dependent, kynurenic acid-sensitive accumulation of cyclic GMP (cGMP) in granule cell cultures. Quisqualic acid was, by itself, ineffective and prevented, in a dose-dependent manner, the kainate-induced cGMP formation (IC50 = 5 microM). Finally, the guanylate cyclase activator sodium nitroprusside greatly enhanced cGMP formation but had no effect on D-[3H]aspartate release. Together, these results demonstrate the existence of complex interactions between quisqualic and kainic acids and indicate that the effects of the two glutamate agonists on D-[3H]aspartate release and on cGMP accumulation are independent.     

Production of poly(4-hydroxybutyric acid) by fed-batch cultures of recombinant strains of Escherichia coli

A recombinant strain of Escherichia coli was used to produce poly(4-hydroxybutyric acid), P(4HB), homopolyester by fed-batch culture in M9 mineral salts medium containing glucose and 4-hydroxybutyric acid as carbon sources. The final cell dry weight, P(4HB) concentration and P(4HB) content were 12.6 g/l, 4.4 g/l, and 36% of cell dry weight, respectively, in a 27-l stirred and aerated fermenter after 60 h of fed-batch fermentation at constant pH.     

Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid

AIMS: To understand the impact of assimilable nitrogen and pantothenic acid on fermentation rate and synthesis of volatile compounds by Saccharomyces under fermentative conditions. METHODS AND RESULTS: A 2 x 3 factorial experimental design was employed with the concentrations of yeast assimilable nitrogen (YAN) (60 and 250 mg l(-1)) and pantothenic acid (10, 50 and 250 microg l(-1)) as variables. In media containing 250 microg l(-1) pantothenic acid, H2S production by two different species of Saccharomyces decreased when YAN was increased from 60 to 250 mg l(-1). Conversely, H2S production was significantly higher when the concentration of assimilable nitrogen was increased if pantothenic acid was deficient (10 or 50 microg l(-1)). Yeast synthesis of other volatile compounds were impacted by both assimilable nitrogen and pantothenic acid. CONCLUSIONS: While growth and fermentative rate of Saccharomyces was more influenced by nitrogen than by pantothenic acid, complicated interactions exist between these nutrients that affect the synthesis of volatile compounds including H2S. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has important implications for the winemaking industry where a better understanding of the nutritional requirements of Saccharomyces is necessary to reduce fermentation problems and to improve final product quality.     

营养条件对兽疫链球菌发酵生产透明质酸的影响

透明质酸 (Ｈｙａｌｕｒｏｎｉｃａｃｉｄ ,简称ＨＡ)是Ｎ 乙酰氨基葡萄糖胺和葡萄糖醛酸以 β 1 3糖苷键和 β 1 4糖苷键连接而成的二糖单体重复构建而成的杂多糖 ,广泛存在于高等动物的结缔组织内。由于结构上的特点 ,ＨＡ具有很高的粘弹性和极强的保水性等特征 ,已被大量用于医学医药、化妆品工业[1,2 ] 。1937年Ｋｅｎｄａｌｌ[3 ] 等发现用溶血性链球菌 (Ｓｔｒｅｐｔｏｃｏｃｃｕｓｈａｅｍｏｌｙｔｉｃｕｓ)可以产生ＨＡ。其后 ,陆续发现许多能产生ＨＡ的微生物菌种 ,逐渐开发出一条可替代传统的动物组织提取法[4 ] 生产ＨＡ的新途径…     

Microbial production of medium-chain-length 3-hydroxyalkanoic acids by recombinant Pseudomonas putida KT2442 harboring genes fadL, fadD and phaZ

Monomers of microbial polyhydroxyalkanoates, mainly 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO), were produced by overexpressing polyhydroxyalkanoates depolymerase gene phaZ, together with putative long-chain fatty acid transport protein fadL of Pseudomonas putida KT2442 and acyl-CoA synthetase (fadD) of Escherichia coli MG1655 in P. putida KT2442. FadL(Pp), which is responsible for free fatty acid transportation from the extracellular environment to the cytoplasm, and FadD(Ec), which activates fatty acid to acyl-CoA, jointly reinforce the fatty acid beta-oxidation pathway. Pseudomonas putida KT2442 (pYZPst01) harboring polyhydroxyalkanoates depolymerase gene phaZ of Pseudomonas stutzeri 1317 produced 1.37 g L(-1) extracellular 3HHx and 3HO in shake flask studies after 48 h in the presence of sodium octanoate as a sole carbon source, while P. putida KT2442 (pYZPst06) harboring phaZ(Pst), fadD(Ec) and fadL(Pp) achieved 2.32 g L(-1) extracellular 3HHx and 3HO monomer production under the same conditions. In a 48-h fed-batch fermentation process conducted in a 6-L fermentor with 3 L sodium octanoate mineral medium, 5.8 g L(-1) extracellular 3HHx and 3HO were obtained in the fermentation broth. This is the first time that medium-chain-length 3-hydroxyalkanoic acids (mcl-3HA) were produced using fadL(Pp) and fadD(Ec) genes combined with the polyhydroxyalkanoates depolymerase gene phaZ.     

Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata

Feasibility of producing (R)-3-hydroxybutyric acid ((R)-3-HB) using wild type Azohydromonas lata and its mutants (derived by UV mutation) was investigated. A. lata mutant (M5) produced 780 mg/l in the culture broth when sucrose was used as the carbon source. M5 was further studied in terms of its specificity with various bioconversion substrates for production of (R)-3-HB. (R)-3-HB concentration produced in the culture broth by M5 mutant was 2.7-fold higher than that of the wild type strain when sucrose (3% w/v) and (R,S)-1,3-butanediol (3% v/v) were used as carbon source and bioconversion substrate, respectively. Bioconversion of resting cells (M5) with glucose (1% v/w), ethylacetoacetate (2% v/v), and (R,S)-1,3-butanediol (3% v/v), resulted in (R)-3-HB concentrations of 6.5 g/l, 7.3 g/l and 8.7 g/l, respectively.     

Effect of mixed organic substrate on α-tocopherol production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in photoheterotrophic culture

Effects of organic carbon sources on cell growth and alpha-tocopherol productivity in wild and chloroplast-deficient W14ZUL strains of Euglena gracilis under photoheterotrophic culture were investigated. In both strains, the increase in cell growth was particularly high when glucose was added as the sole organic carbon source. On the other hand, alpha-tocopherol production per dry cell weight was enhanced by adding ethanol. Ethanol addition also increased the chlorophyll concentration in wild strain and mitochondria activity in W14ZUL strain. For effective alpha-tocopherol production, the effects of mixture of glucose and ethanol were investigated. The results showed that, when a mixture of glucose (6 g/l) and ethanol (4 g/l) was used, alpha-tocopherol productivity per culture broth was 3.89 x 10(-2) mg l(-1) h(-1), which was higher than the value obtained without addition of organic carbon source (0.92 x 10(-2) mg l(-1) h(-1)). In addition, under fed-batch cultivation using an internally illuminated photobioreactor, the alpha-tocopherol production per culture broth was 23.43 mg/l, giving a productivity of 16.27 x 10(-2) mg l(-1) h(-1).     

Production of poly(D-3-hydroxybutyrate) from CO(2), H(2), and O(2) by high cell density autotrophic cultivation of Alcaligenes eutrophus

Hydrogen-oxidizing bacterium, Alcaligenes eutrophus autotrophically produces biodegradable plastic material, poly(D-3-hydroxybutyrate), P(3HB), from carbon dioxide, hydrogen, and oxygen. In autotrophic cultivation of the microorganism, it is essential to eliminate possible occurrence of gas explosions from the fermentation process. We developed a bench-plant scale, recycled-gas, closed-circuit culture system equipped with several safety features to perform autotrophic cultivation of A. eutrophus by maintaining the oxygen concentration in the substrate gas phase below the lower limit for a gas explosion (6.9%). The culture vessel utilized a baskettype agitator, resulting in a K(L) a value of 2970 h(-1). Oxygen gas was also directly fed to the fermentor separately from the other gases. As a result, 91.3 g . dm(-3) of the cells and 61.9 g . dm(-3) of P(3HB) were obtained after 40 h of cultivation under this oxygen-limited condition. The results compared favorably with those reported for mass production of P(3HB) by heterotrophic fermentation. (c) 1995 John Wiley & Sons, Inc.     

High-cell-density cultivation of Schizochytrium sp. in an ammonium/pH-auxostat fed-batch system

Thraustochytrids, in particular Schizochytrium spp., are used for the production of the valuable polyunsaturated fatty acid, docosahexaenoic acid (DHA; 22:6 n-3). Growth of Schizochytrium sp. G13/2S in a defined medium was initially made in shake-flask cultures to determine the optimum concentrations of glucose (100-200 g l(-1)) and ammonia ( approximately 300 mg l(-1)) that could be used by this microorganism. In subsequent fermenter cultures, a pH-auxostat method was used to maintain NH(3) from 200-300 mg l(-1). During the first 49 h of fermentation, 150 g glucose l(-1) produced 63 g cell dry wt l(-1). Although growth was not limited by the supply of nitrogen, total fatty acids were at 25% cell dry wt which is more than half the final lipid content of commercially-grown Schizochytrium biomass which uses N-limited medium in the final stages for maximum lipid accumulation. This strategy is therefore useful for the cultivation of Schizochytrium to a high cell density up to the point when lipid accumulation can be triggered by N exhaustion.     

Side-chain effect of second monomer units on crystalline morphology, thermal properties, and enzymatic degradability for random copolyesters of (R)-3-hydroxybutyric acid with (R)-3-hydroxyalkanoic acids

Three types of random copolymers with 94 mol % (R)-3-hydroxybutyric acid (3HB) and 6 mol % (R)-3-hydroxyalkanoic acids with different side-chain lengths, (R)-3-hydroxypentanoic acid (3HV), (R)-3-hydroxyhexanoic acid (3HHx), and medium-chain-length (R)-3-hydroxyalkanoic acids (mcl-3HA, C8-C12), were prepared by biological synthetic techniques. The solid-state structure and thermal properties of melt-crystallized films for copolymers were characterized by means of wide-angle X-ray diffraction, small-angle X-ray scattering, differential scanning calorimetry, and optical microscopy. The randomly distributed second monomer units, except for 3HV in copolyesters, act as defects of the P(3HB) crystal and are excluded from the P(3HB) crystalline lamellae. The lamellar thickness of copolymers decreased with an increase in the side-chain length of second monomer units. In addition, the growth rate of spherulites decreased with an increase in the carbon numbers of second monomer units at an identical crystallization temperature. These results indicate that a steric bulkiness of the second monomer unit affects the crystallization of (R)-3HB segments in random copolyesters. An enzymatic degradation test of melt-crystallized copolymer films was carried out in the presence of PHB depolymerase from Alcaligenes faecalis T1. Erosion rate of copolyesters was dependent on both the crystallinity and the lamellar thickness of samples. As the result, the rate of enzymatic degradation for copolymer films increased with an increase in the carbon numbers of second monomer units.     

Biotechnological production of lactic acid integrated with fishmeal wastewater treatment by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

Fishmeal wastewater, a seafood processing waste, was utilized for production of lactic acid and fungal biomass by Rhizopus oryzae AS 3.254 with the addition of sugars. The 30 g/l exogenous glucose in fishmeal wastewater was superior to starch in view of productivities of lactic acid and fungal biomass, and COD reduction. Fishmeal wastewater can be a replacement for peptone which was the most suitable nitrogen source for lactic acid production among the tested organic or inorganic nitrogen sources. Exogenous NaCl (12 g/l) completely inhibited the production of lactic acid and fungal growth. In the medium of COD 5,000 mg/l fishmeal wastewater with the addition of 30 g/l glucose, the maximum productivity of lactic acid was 0.723 g/l h corresponding to productivity of fungal biomass 0.0925 g/l h, COD reduction 84.9% and total nitrogen removal 50.3% at a fermentation time of 30 h.     

l-trans-Pyrrolidine-2,4-Dicarboxylic Acid-Evoked Striatal Glutamate Levels Are Attenuated by Calcium Reduction, Tetrodotoxin, and Glutamate Receptor Blockade

Abstract: l - trans -Pyrrolidine-2,4-dicarboxylic acid ( l - trans -PDC) reverses plasma membrane glutamate transporters and elevates extracellular glutamate levels in vivo. We investigated the possibility that l - trans -PDC-stimulated glutamate levels are mediated partially by increases in transsynaptic activity. Therefore, the degree to which l - trans -PDC-evoked glutamate levels depend on calcium, sodium-channel activation, and glutamate-receptor activation was investigated by infusing via reverse microdialysis (a) 0.1 m M calcium, (b) 1 µ M tetrodotoxin, a selective blocker of voltage-dependent sodium channels, (c) R (−)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a selective NMDA-receptor antagonist, or (d) LY293558, a selective α-amino-3-hydroxy-5-methylisoxazole-4-propionate antagonist. In separate experimental groups, l - trans -PDC-evoked glutamate levels were reduced significantly by 55% in the presence of 0.1 m M calcium and by 46% in the presence of tetrodotoxin. Additionally, CPP and LY293558 significantly attenuated l - trans -PDC-evoked glutamate levels without altering basal glutamate levels. These data suggest that glutamate transporter reversal by l - trans -PDC initially elevates extracellular glutamate levels enough to stimulate postsynaptic glutamate receptors within the striatum. It is proposed that glutamate-receptor stimulation activates a positive feedback loop within the basal ganglia, leading to further glutamate release from corticostriatal and thalamostriatal afferents. Therefore, either extracellular striatal calcium reduction or tetrodotoxin perfusion leads to decreased action potential-dependent glutamate release from these terminals. In addition, blocking glutamate receptors directly reduces medium spiny neuronal firing and indirectly attenuates corticostriatal and thalamostriatal activity, resulting in an overall depression of l - trans -PDC-stimulated glutamate levels.