ENGINEERING A HIGH-YIELD GLUTATHIONE STRAIN OF Hansenula polymorpha USING ION BEAM IMPLANTATION

To generate an industrial strain of Hansenula polymorpha capable of yielding greater levels of glutathione (GSH), wild strain H. polymorpha DL-1 cells were mutated using a nitrogen ion beam, a novel mutagen. At an energy level of 20 keV and dose of 2.13 × 10¹⁶ ions/cm², H. polymorpha strain 28 (HP28) with a high-yield of GSH was screened. HP28 intracellular GSH levels reached 337.16 mg/L by ion beam implantation, 1.56 times greater than that of the wild type strain when the fermentation time was shortened from 48 hr to 42 hr, greatly improving efficiency and reducing the cost of industrial-scale production. The enhanced efficiency of HP28 is promising for GSH production from lignocellulosic materials. Therefore, the ion beam implantation would be a cost-effective alternative to the conventional mutation method for engineering yeast and improving its utility.     

Changes in DNA base sequences in the mutant of Arabidopsis thaliana induced by low-energy N+ implantation

To reveal the mutation effect of low-energy ion implantation on Arabidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N⁺ with the dose of 80×10¹⁵ ions/cm² was used for Random Amplified Polymorphic DNA (RAPD) and base sequence analysis. The results indicated that among total 397 RAPD bands observed, 52 bands in T80II were different from those of wild type showing a variation frequency 13.1%. In comparison with the sequences of A. thalianain GenBank, the RAPD fragments in T80II were changed greatly in base sequences with an average rate of one base change per 16.8 bases. The types of base changes included base transition, transversion, deletion and insertion. Among the 275 base changes detected, single base substitutions (97.09%) occurred more frequently than base deletions and insertions (2.91%). And the frequency of base transitions (66.55%) was higher than that of base transversions (30.55%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases in cloned DNA fragments in T80II. It seems that thymine was more sensitive to the irradiation than other bases. The flanking sequences of the base changes in RAPD fragments in T80II were analyzed and the mutational “hotspot” induced by low-energy ion implantation was discussed.     

N+离子注入选育高产氨基酰化酶菌株的研究

选用N~+离子注入的方法对米曲霉(Aspergillus oryzae)CICC 2339-1进行诱变育种,通过三角瓶发酵法筛选氨基酰化酶高产株。N~+离子注入选择能量为10 KeV,剂量在(1.30～4.94)×10~(15)ions/cm~2之间。根据剂量与存活率以及剂量与突变率曲线选择最佳的注入剂量。通过三角瓶发酵筛选得到突变菌株SN-110-15其酶活提高率为139.5%,诱变试验效果显著。     

Mutation of Gluconobacter oxydans and Bacillus megaterium in a two-step process of l-ascorbic acid manufacture by ion beam

AIM: To increase the transformation rate of l-sorbose to 2-keto-l-gulonic (2-KLG) acid in a two-step process of l-ascrobic acid manufacture by ion beam. METHODS AND RESULTS: Gluconobacter oxydans (GO29) and Bacillus megaterium (BM80) were used in the present study. Ion implantation was carried out with the heavy ion implantation facility at the institute of Plasma Physics in China. 2-KLG in whole culture broth was determined by iodometry. Mutants were screened by single-colony isolation and 2-KLG accumulation in broth. GO29 and BM80 were implanted by either hydrogen ions (H(+)) or nitrogen ions (N(+)) with various doses, respectively. The average transformation rate of GM112-302 bred by ion beam in Gram-molecule was increased from 79.3 to 94.5% after eight passages in shaking flasks. Furthermore, in 180-ton fermentors in Jiangsu Jiangshan Pharmaceutical Co. Ltd, the transformation rate was stable at 92.0%, indicating a producer could get 0.99 kg of gulonic acid from 1.0 kg of sorbose. CONCLUSION: Ion beam as a new mutation source had potential advantages in breeding. Comparing with original mixture GO29 and BM80, GM112-302 is more efficient in accumulating 2-KLG, especially at the later phase. SIGNIFICANCE AND IMPACT OF THE STUDY: GM112-302 bred by ion beam implantation dramatically increased the transformation rate by 19.2%, which greatly increased efficiency and reduced the cost of l-ascorbic acid manufacture in a two-step process.     

Effect of Temperature on Growth of Bacillus sp. T-4, a Thermophilic Acidophilic Bacterium

The lipid distribution and function in the thylakoid membranes from a thermophilic cyanobacterium, Mastigocladus laminosus, were investigated. The thylakoid membranes were treated with digitonin and separated on a DEAE-cellulose column into fractions enriched in photosystem I or II complex. Lipid analyses showed a specific distribution of anionic lipids among the fractions. A mild delipidation of the membranes with cholate indicates that monogalactosyl diacylglycerol (MGDG) and sulfoquinovosyl diacylglycerol (SQDG) are released rapidly, while the major parts of digalactosyl diacylglycerol (DGDG) and phosphatidylglycerol (PG) are tightly associated with membranes, suggesting a different distribution between the two groups of lipids. Measurements of fluorescence of delipidated and reconstituted thylakoids showed the contribution of lipids to energy transfer. MGDG enhanced all the original fluorescence of thylakoids, while acidic PG and SQDG stimulated fluorescence of photosystem I and antena chlorophyll-protein complexes. DGDG was less effective under the conditions tested.     

Biological effects of low-energy C ion implantation on sesame ( Sesamum indicum L.)

Field cultivation experiments on white sesame (Sesamum indicum L.) seeds implanted with low-energy C ion showed that different dosages of C ion implantation produce different biological effects. Sesame plants in 6 different dosage groups with C ion density respectively at 1 × 10¹¹, 1 × 10¹², 1 × 10¹⁵, 5 × 10¹⁵, 1 × 10¹⁶, 5 × 10¹⁶ ion/cm² were superior to the control group in plant height, leaf number, stalk diameter and leaf size. Further, sesame plants in these groups flower and seed earlier than those in the control group, and single plant yield also increased. Of all the groups, the 5 × 10¹⁵ ion/cm² dosage group yielded the best effect, whereas the 1 × 10¹⁷/cm² dosage group showed an evident inhibitory effect of ion implantation on the germination and growth of the sesame seeds. __________ Translated from Journal of Beijing Normal University (Natural Science), 2006, 42 (1): 95–97 [译自: 北京师范大学学报 (自然科学版)]     

离子注入选育霉酚酸高产菌株及其发酵条件研究

离子注入技术是20世纪80年代兴起的一种综合诱变技术,其应用于生物工程已取得了丰硕成果,但在霉酚酸产生菌的诱变育种中的应用还未见报道。短密青霉菌(Penicillium brevicompactum)M_51是从土壤中分离得到的MPA产生菌F_663经过紫外线、微波等诱变处理得到的。为获得霉酚酸的高产工业菌株,进一步对该菌株进行了离子注入诱变处理。用15keV氮离子分5个剂量进行处理,结果显示,随离子注入剂量增加,存活率呈现较明显的下降_上升_下降的“马鞍型”变化趋势。在剂量为140×2.6×1013ions/cm2时,菌株变异率及正变率均最高,分别达到88.9%和63.4%。用HPLC定量测定发酵液中霉酚酸的含量,筛选到产霉酚酸能力提高30.1%的突变株M_163。经过连续传代试验,其遗传性状稳定。对发酵条件的优化结果显示最佳种龄为24h;用正交试验方法对发酵培养基中的碳、氮源进行优化,得到较优配方。突变株M_163在最优发酵条件下,霉酚酸摇瓶发酵单位可达2819μg/mL。野生菌株F_663的MPA产量为133μg/mL,经过5代诱变育种及发酵条件优化,产量提高了20.2倍。     

Apoptosis. Signaling pathways and cell ion and water balance

Current data on the alteration of the monovalent ion balance, pH and the membrane potential during apoptosis are summarized and considered with respect to ionic mechanisms of the apoptotic cell shrinkage. A brief survey of the main signaling pathways involved in apoptosis, such as receptor-and mitochondria-mediated pathways of the caspase-dependent and caspase-independent apoptosis is given. The data on the alteration of the distinct ion transporters and channels of the plasma membrane during apoptosis are considered.     

Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation

Aims: Isolation, characterization and identification of Phaffia sp. ZJB 00010, and improvement of astaxanthin production with low‐energy ion beam implantation. Methods and Results: A strain of ZJB 00010, capable of producing astaxanthin, was isolated and identified as Phaffia rhodozyma, based on its physiological and biochemical characteristics as well as its internal transcribed spacer (ITS) rDNA gene sequence analysis. With low‐energy ion beam implantation, this wild‐type strain was bred for improving the yield of astaxanthin. After ion beam implantation, the best mutant, E5042, was obtained. The production of astaxanthin in E5042 was 2512 μg g^?1 (dry cell weight, DCW), while the wild‐type strain was about 1114 μg g^?1 (DCW), an increase of 125·5%. Moreover, the fermentation conditions of mutant E5042 for producing astaxanthin were optimized. The astaxanthin production under the optimized conditions was upscaled and studied in a 50‐l fermentor. Conclusions: A genetically stable mutant strain with high yield of astaxanthin was obtained using low‐energy ion beam implantation. This mutant may be a suitable candidate for the industrial‐scale production of astaxanthin. Significance and Impact of the Study: Astaxanthin production in Phaffia rhodozyma could be fficiently improved by low‐energy ion beam implantation, which is a new technology in the mutant breeding of micro‐organisms. The mutant obtained in this work could potentially be utilized in industrial production of astaxanthin.     

Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms

The x-ray structure of LeuT, a bacterial homologue of Na⁺/Cl⁻-dependent neurotransmitter transporters, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion binding sites, NA1 and NA2, which are highly selective for Na⁺. Extensive all-atom free-energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na⁺ over K⁺ or Li⁺, the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In NA1, selectivity for Na⁺ over K⁺ arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In NA2, which comprises only neutral ligands, selectivity for Na⁺ is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the polypeptide chain surrounding the ion according to a “snug-fit” mechanism.     

低能氮离子注入大肠杆菌诱发的生物学效应研究

研究离子注入的诱变和导入外源DNA的生物学效应,用低能氮离子注入处理大肠杆菌野生型菌株MC4100A,将含水母绿色荧光蛋白基因的质粒导入细胞中。实验结果表明,在一些转化子中绿色荧光蛋白的表达或折叠受到了影响,一些转化子丧失了分裂后分离能力,且细胞膜有一定程度的损伤,为进一步研究微生物细胞分裂和蛋白质折叠机理提供了菌株。故低能氮离子注入对微生物细胞的诱变效应和导入外源DNA效应的有机结合将使离子注入技术在生物学基础研究中有着广泛的应用前景。     

Desulfurization of 2,4,6,8-tetraethyl dibenzothiophene by recombinant Mycobacterium sp. strain MR65

Recombinant Mycobacterium sp. strain MR65 harboring dszABCD genes was used to desulfurize alkyl dibenzothiophenes (C_x-DBTs) in n-hexadecane. The specific desulfurization activity for 2,4,6,8-tetraethyl DBT (C₈-DBT) by DszC enzyme was about twice that for 4,6-dipropyl DBT (C₆-DBT). However, the degradation rate of 2,4,6,8-tetraethyl DBT in n-hexadecane by resting cells of strain MR65 was only about 40% of that of 4,6-dipropyl DBT. These results indicated that the desulfurization ability for Cx-DBTs by resting cells depends on carbon number substituted at positions 4 and 6 and that the rate-limiting step in the desulfurization reaction of highly alkylated C_x-DBTs is the transfer process from the oil phase into the cell.     

Aerobic reduction of manganese oxide by Salmonella sp. strain MR4

A new isolate of Salmonella, strain MR4, reduced Mn(IV)O₂ at 2.3 mM under aerobic conditions by about 83% over 24 h. Direct contact of cells to MnO₂ was not necessary as the cell-free spent medium produced a similar amount of Mn(II). Pyruvate (1.6 mM) and oxalate (0.8 mM) were identified in the culture medium and presumed to have a role in Mn(II) production in this microorganism.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was immobilized in various matrices namely, polyurethane foam (PUF), polyacrylamide, alginate and agar. The degradation rates of 20 and 40 mM p-cresol by the freely suspended cells and immobilized cells in batches and semi-continuous with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 20 and 40 mM p-cresol than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- immobilized cells could be reused for more than 35 cycles, without losing any degradation capacity and showed more tolerance to pH and temperature changes than free cells. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for degradation of p-cresol.     

Regulation of neuronal ion channels via P2Y receptors

Within the last 15 years, at least 8 different G protein-coupled P2Y receptors have been characterized. These mediate slow metabotropic effects of nucleotides in neurons as well as non-neural cells, as opposed to the fast ionotropic effects which are mediated by P2X receptors. One class of effector systems regulated by various G protein-coupled receptors are voltage-gated and ligand-gated ion channels. This review summarizes the current knowledge about the modulation of such neuronal ion channels via P2Y receptors. The regulated proteins include voltage-gated Ca²⁺ and K⁺ channels, as well as N-methyl-d-aspartate, vanilloid, and P2X receptors, and the regulating entities include most of the known P2Y receptor subtypes. The functional consequences of the modulation of ion channels by nucleotides acting at pre- or postsynaptic P2Y receptors are changes in the strength of synaptic transmission. Accordingly, ATP and related nucleotides may act not only as fast transmitters (via P2X receptors) in the nervous system, but also as neuromodulators (via P2Y receptors). Hence, nucleotides are as universal transmitters as, for instance, acetylcholine, glutamate, or -aminobutyric acid.