代谢改造重组谷氨酸棒杆菌C4途径高效合成5-氨基乙酰丙酸

5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)在医药和农业等领域有着广泛作用,目前主要采用大肠杆菌或谷氨酸棒杆菌以微生物发酵法合成.为了进一步提高谷氨酸棒杆菌合成5-ALA的能力,对其C4代谢途径进行了系统代谢改造.首先分别在谷氨酸棒杆菌中异源表达荚膜红杆菌和沼泽红假单胞菌的5-氨基乙酰丙酸...     

δ-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA

Abstract Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of δ-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis . The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.     

5-Aminolevulinic acid formation from glutamate via the C5 pathway in Clostridium thermoaceticum

A cell-free extract of the anaerobic eubacterium, Clostridium thermoaceticum, catalyzes the synthesis of 5-aminolevulinic acid (ALA) from glutamate via the C5 pathway. The enzyme reaction resembles that of higher plants and algae in cofactor requirements and sensitivity to ribonuclease. From the phylogenetic distribution it is proposed that the C5 pathway evolved earlier than the ALA synthase pathway.     

Enhancement of Growth and Fruit Maturity in 2-year-old Grapevines cv. Delaware by 5-Aminolevulinic Acid

The influence of 5-aminolevulinic acid (ALA) on the growth, photosynthesis, and fruit quality of gibberellin-induced seedless 2-year-old “Delaware“ grapevine was examined. The result of soil treatment (0.1–10 mg l⁻¹) and foliar spray treatment (30–300 mg l⁻¹) with ALA after flowering was significant growth improvement, up to 55% in the lateral shoot, and up to 38% in leaf area. Optimal doses were 1 mg l⁻¹ soil treatment and 100 mg l⁻¹ foliar treatment. The photosynthetic rate of plants treated with ALA increased by a significant 9.2%–22.5%. Relative to the control, based on measurements of the 5th leaf of each shoot in the ripening period, the optimum concentrations for growth and photosynthesis enhancement in grapevines were 100 mg l⁻¹ (foliar treatment) and 1 mg l⁻¹ (soil treatment). Total plant weight at harvest increased a significant 13% at the optimal treatment doses. In terms of fruit quality, the cluster fresh weight increased a significant 44.9%–53% and fruit colour showed a tendency to become darker in all plants treated with ALA. The °Brix value in the plant treated with 100 mg l⁻¹ ALA was a significant 2.7% higher than that of the control. We consider that leaf area and photosynthetic rates during cultivation are important for the acquisition of photoassimilates and that these are likely to be causally associated with improvement of total dry weight and advance of fruit quality. In addition, a possibility of advancing the harvest time of grapes by ALA treatment was shown.     

Carboxylic acid consumption and production by Corynebacterium glutamicum

Corynebacterium glutamicum is well-known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Previous studies of the effect of gradients in scale-down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation. This study, therefore, addresses the impact of carboxylic acids, for example, acetate and l -lactate, on the cultivation of the cadaverine producing strain C. glutamicum DM1945Δact3:P_tuf-ldcC^opt and their potential role in scale up related performance losses. A fluctuating power input in shake flask and stirred tank cultivations with mineral salt was applied to mimic discontinuous oxygen availability. Results demonstrate, whenever sufficient oxygen was available, C. glutamicum recovered from previously occurring stressful conditions like an oxygen limiting phase. Reassimilation of acids was detected simultaneously. In cultures, which were supplemented with either acetate or l -lactate, a rapid cometabolization of both acids in presence of glucose was observed, showing conversion rates of 7.8 and 3.8 mmol g_{cell dry weight}⁻¹ hr⁻¹, respectively. Uptake of these acids was accompanied by increased oxygen consumption. Proteins related to oxidative stress response, glycogen synthesis, and the main carbon metabolism were found in altered concentrations under oscillatory cultivation conditions. (Proteomics data are available via ProteomeXchange with identifier PXD012760). Virtually no impact on growth or product formation was observed. We conclude that the reduced growth and product formation in scale-down cultivations when complex media was used is not caused by the accumulation of carboxylic acids.     

Determination of 5-aminolevulinic acid in blood plasma, tissues and cell cultures by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method for determining 5-aminolevulinic acid (ALA) in biological samples is described. ALA is derivatized with o-phthaldehyde to give a compound with favorable properties for high-performance liquid chromatography with electrochemical detection. The method does not require extensive pretreatment of the samples and its detection limit is in the range of 1 pmol/20 μl injection. This method was applied to the determination of plasma ALA from normal and lead-exposed subjects, where 0.26±0.08 μM (n=30) and 2.6±0.75 μM (n=30), respectively were found. We also determined ALA in rat tissues, namely liver and brain, and the uptake of ALA by cultured fibroblasts and hepatocytes to illustrate the diversified applicability of the method.     

5-氨基酮戊酸声动力疗法诱导人骨肉瘤细胞凋亡的研究

目的:探讨5-氨基酮戊酸声动力疗法(5-ALA-SDT)对人骨肉瘤细胞株U2-OS细胞凋亡的影响。方法:对数生长期的U2-OS细胞随机分为空白对照组(control)、声敏剂组(5-ALA)、超声组(ultrasound),声动力组(5-ALA-SDT)。采用流式细胞术检测细胞凋亡率,荧光探针及共聚焦激光扫描显微镜检测细胞内荧光定位,用DCFH-DA检测活性氧(ROS)的产生情况。结果:与单纯超声组,单纯5-氨基酮戊酸组和对照组相比较,5-氨基酮戊酸介导的声动力组细胞存活率(40.18%±1.35%)均明显下降(P0.01),细胞内荧光定位表明5-ALA扩散在细胞质和主要累积在U2-OS细胞线粒体内,并且5-氨基酮戊酸介导的声动力组ROS产生的百分率(34.3±2.4)%较单纯超声组、单纯5-氨基酮戊酸组和对照组明显升高,差异有统计学意义(P0.01)。结论:5-氨基酮戊酸介导的声动力作用可能通过线粒体途径对诱导U2-OS细胞凋亡。     

Dimethyl Sulfoxide and Ebselen Prevent Convulsions Induced by 5-Aminolevulinic Acid

We investigated whether intrastriatal (i.s.) administration of 5-aminolevulinic acid (ALA) induces oxidative damage and whether behavioral alterations induced by i.s. administration of ALA could be affected by antioxidants. Unilateral injection of ALA (6 micromol/striatum) increased (approximately 30%) thiobarbituric acid-reactive substances (TBARS), but did not affect striatal content of total thiol groups. ALA-induced body asymmetry was not prevented by pretreatment with ascorbic acid (100 mg/kg, s.c.), dimethyl sulfoxide (DMSO, 0.5 microl/striatum, i.s.) or ebselen (10 nmol/striatum, i.s.). ALA-induced convulsions were not prevented by ascorbic acid, but were partially prevented by DMSO and completely prevented by ebselen. Ebselen completely prevented the increase of striatal TBARS induced by ALA. Results obtained suggest the involvement of reactive species in ALA-induced convulsions and may be of value in understanding the physiopathology of neurological dysfunctions associated to ALA overload.     

5-Aminolevulinic acid inhibits [3H]muscimol binding to human and rat brain synaptic membranes

The interaction of 5-aminolevulinic acid (ALA) with GABA_A receptors has been proposed to underlie the neurological dysfunctions of ALA-accumulating disorders, such as acute intermittent porphyria. The effects of ALA on [³H]muscimol binding to human and rat cerebral cortical membranes were compared. ALA (0.1–10 mM) significantly inhibited the binding of [³H]muscimol (12 nM), with a similar potency in rat and human membranes (IC₅₀ = 199 vs. 228 M, respectively). Kinetical analysis revealed that ALA (1 mM) significantly increased the Kd and decreased the Bmax of [³H]muscimol to both rat (100 and 50%, respectively) and human (200 and 40%, respectively) membranes, indicating a mixed-type inhibition. The similarity in the potency and mechanism of the ALA-induced inhibition of muscimol binding in rat and human membranes indicate that rat studies are useful to evaluate the neurotoxic properties of ALA towards the human GABAergic system, and may help to understand the pathophysiology of porphyria.     

Enhancement of 5-Aminolevulinic acid-induced oxidative stress on two cancer cell lines by gold nanoparticles

Abstract

5-Aminolevulinic acid (5-ALA) and its methyl ester (5-ALA-Me) at mM concentration levels induce oxidative stress via the production of reactive oxygen species (ROS). Human cancer cell lines (MCF-7 and HepG2) incubated in the dark in the simultaneous presence of 5.0 mM or more 5-ALA or 5-ALA-Me (for MCF-7) and 7 µg/mL of 15 nm citrate capped gold nanoparticles (AuNPs) were damaged more seriously compared to those in the presence of the levulinic acid alone. Damage is visible in electron micrographs which reveal similar morphology both in the presence or absence of AuNPs. Cytotoxicity was observed irrespective of the presence of serum and medium. Production of ROS in cell free samples containing 5-ALA-Me was monitored by EPR as the DMPO-OH spin adduct and also showed a catalytic effect of AuNPs. Both SOD and CAT inhibited the production of ROS and also reduced cytotoxicity in the cell samples. These observations can be explained by initial attack on the cell membrane by ROS produced in the medium outside the cell and provide insight into possible uses of 5-ALA in cancer chemotherapy.     

High‐titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum

Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. The wild microbial producer of HA, streptococcus, was restricted by its potential pathogens, hence different recombinant hosts are being explored. In this work, we engineered Corynebacterium glutamicum, a GRAS (Generally Recognized as Safe) organism free of exotoxins and endotoxins to produce HA with high titer and satisfied M_w. The ssehasA gene encoding hyaluronan synthase (HasA) was artificially synthesized with codon preference of C. glutamicum. Other genes involved in the HA synthetic pathway were directly cloned from the C. glutamicum genome. The operon structures and constitutive or inducible promoters were particularly compared and the preferred environmental conditions were also optimized. Using glucose and corn syrup powder as carbon and nitrogen sources, batch cultures of the engineered C.glutamicum with operon ssehasA‐hasB driven by Ptac promoter were performed in a 5 L fermentor. The maximal HA titer, productivity and yield reached 8.3 g/L, 0.24 g/L/h and 0.22 gHA/gGlucose, respectively; meanwhile the maximal M_w was 1.30 MDa. This work provides a safe and efficient novel producer of HA with huge industrial prospects.     

5-Aminolevulinic acid production by Chlorella sp. during heterotrophic cultivation in the dark

5-Aminolevulinic acid (ALA) was produced aerobically in the dark during growth on glucose by a newly isolated Chlorella sp. When levulinic acid (20 mM), a competitive inhibitor of ALA dehydratase, was added repeatedly to the medium, about 1.5 mM of ALA was produced extracellularly. Glutamate (30 mM) added with levulinic acid (20 mM, given repeatedly) enhanced ALA production up to 1.9 mM, indicating that ALA might be synthesized via the C-5 pathway.K. Sasaki was with the Hiroshima-Denki Institute of Technology, 6-20-1, Nakano, Akiku, Hiroshima, 739-03, Japan; and is now with the Department of Biotechnology. The University of New South Wales, Sydney, NSW, 2052, Australia; K. Watanabe, T. Tanaka and Y. Hotta are with the Cosmo Research Institute, 1134-2, Gongendo, Satte, Saitama, 340-01, Japan. S. Nagai is with the Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 4-1, Kagamiyama 1 chome, Higashi-Hiroshima, 724. Japan.     

Characterization of a secondary uptake system for l-glutamate in Corynebacterium glutamicum

Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K _m of 0.6 mM and a V _max of 15 nmol min⁻¹ (mg dw)⁻¹. For the co-transported sodium ions, a relatively low K _m of 3.3 mM was determined.     

Modification of histidine biosynthesis pathway genes and the impact on production of l-histidine in Corynebacterium glutamicum

Histidine biosynthesis in Corynebacterium glutamicum is regulated not only by feedback inhibition by the first enzyme in the pathway, but also by repression control of the synthesis of the histidine enzymes. C. glutamicum histidine genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. We constructed plasmid pK18hisDPtac to replace the native hisD promoter with the tac promoter, and overexpressed phosphoribosyl-ATP-pyrophosphohydrolase, encoded by hisE, and ATP-phosphoribosyltransferase, encoded by hisG. The l-histidine titer at 0.85 g l^?1 was 80 % greater in the transformed bacterium and production of byproducts, l-alanine and l-tryptophan, was significantly decreased. However, accumulation of glutamic acid increased by 58 % (2.8 g l^?1). This study represents the first attempt to substitute the histidine biosynthesis pathway promoter in the chromosome with a stronger promoter to increase histidine production.     

Increasing available NADH supply during succinic acid production by Corynebacterium glutamicum

A critical factor in the biotechnological production of succinic acid with Corynebacterium glutamicum is the sufficient supply of NADH. It is conceivable that cofactor availability and the proportion of cofactor in the active form may play an important role in dictating the succinic acid yield. PntAB genes from Escherichia coli can directly catalyze the reversible hydride transfer and adjust the dynamic balance between NADP(H) and NAD(H). Hence, we studied the physiological effect of coenzyme systems by expressing the membrane‐bound transhydrogenase pntAB genes. We have shown experimentally that the pntAB genes could function as an alternative source of NADH. In an anaerobic fermentation with C. glutamicum NC‐3‐pntAB, a 16% higher succinic acid yield and a 57% higher production from glucose were obtained by pntAB expression. Moreover, the formation of by‐products was significantly decreased. The concomitant increase in the consumption of intracellular NADPH from 0.6 to 1.2 mmol/g CDW and the increased NADH/NAD⁺ ratio resulted from introduction of pntAB, suggesting that the membrane‐bound transhydrogenase converted excess NADPH to NADH for succinic acid production. Finally, we explored whether the transhydrogenase had different effects on the succinic acid formation on different carbon sources. The succinic acid yield was increased in the presence of pntAB by 16% on glucose, 7% on sucrose, and without large influence on fructose and xylose. The results of this study demonstrated that the effectiveness of cofactor manipulation could be a promising strategy applied in metabolic engineering. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:12–19, 2015     

Purification and characterization of 5-aminolevulinic acid dehydratase from Methanosarcina barken

Abstract 5-Aminolevulinic acid dehydratase from the archaebacterium Methanosarcina barken resembles the mammalian and yeast enzymes in its activation by Zn²⁺, whereas its activation by K⁺ resembles the characteristic of bacterial enzymes. This enzyme is activated with Ni²⁺ which is a component of F₄₃₀, a cofactor present mainly in methanogens. The M _r of 280000 for the native enzyme and 30 000 ± 2000 for the individual subunit suggest that the enzyme is composed of eight apparently indentical subunits similar to mammalian and yeast enzymes. The enzyme has two pH optima, at 8.5 and 9.4. Higher levels of 5-aminolevulinic acid dehydratase in acetate-grown cells suggest the possibility that regulation and control of this enzyme could be different on various growth substrates.     

Use of wood vinegar to enhance 5-aminolevulinic acid production by selected Rhodopseudomonas palustris in rubber sheet wastewater for agricultural use

This study aimed to produce inexpensive 5-aminolevulinic acid (ALA) in a non-sterile latex rubber sheet wastewater (RSW) by Rhodopseudomonas palustris TN114 and PP803 for the possibility to use in agricultural purposes by investigating the optimum conditions, and applying of wood vinegar (WV) as an economical source of levulinic acid to enhance ALA content. The Box–Behnken Design experiment was conducted under microaerobic-light conditions for 96 h with TN114, PP803 and their mixed culture (1:1) by varying initial pH, inoculum size (% v/v) and initial chemical oxygen demand (COD, mg/L). Results showed that the optimal condition (pH, % inoculum size, COD) of each set to produce extracellular ALA was found at 7.50, 6.00, 2000 for TN114; 7.50, 7.00, 3000 for PP803; and 7.50, 6.00, 4000 for a mixed culture; and each set achieved COD reduction as high as 63%, 71% and 75%, respectively. Addition of the optimal concentration of WV at mid log phase at 0.63% for TN114, and 1.25% for PP803 and the mixed culture significantly increased the ALA content by 3.7–4.2 times (128, 90 and 131 μM, respectively) compared to their controls. ALA production cost could be reduced approximately 31 times with WV on the basis of the amount of levulinic acid used. Effluent containing ALA for using in agriculture could be achieved by treating the RSW with the selected ALA producer R. palustris strains under the optimized condition with a little WV additive.