维生素C二步发酵中离子注入诱变巨大芽孢杆菌的生物学效应

巨大芽孢杆菌BM279是经低能N 离子注入诱变原始菌株BM80而得到的维生素C高转化率伴生菌株。通过对离子注入前后出发菌和突变株的生理、生化等生物学特点比较,探讨了离子注入巨大芽孢杆菌对2-酮基-L-古龙酸(2KGA)高转化率的促进机理。离子注入对巨大芽孢杆菌自身的生长无明显影响,BM279呈现出与BM80基本一致的生长曲线;但BM279对混菌发酵体系中产酸菌GO29的细胞增殖有显著促进作用。BM279在混菌发酵过程中分泌较多的碱性物,有利于维持GO29生长、代谢的pH环境。BM279培养42 h,其胞外活性物质对GO29的糖酸转化活力较BM80有显著提高,且分泌时间较BM80推迟6 h。利用层析技术分别从BM80、BM279胞外液中纯化了L-山梨糖脱氢酶(L-sorbose dehydrogenase,SDH)激活蛋白(SSPBM80和SSPBM279),后者比活较前者高出50%,对GO29中的SDH酶活有更强促进作用。     

玻璃化法对离子注入拟南芥幼苗过程中的冻害防护效应

离子注入过程中活体材料往往由于真空造成的冻害而不能存活.文章以拟南芥幼苗为对象,尝试了玻璃化法对注入过程中冻害的防护效应.结果显示,玻璃化处理的2 d龄幼苗经液氮冻融处理后存活率最高,达80%以上.2 d龄幼苗经玻璃化处理后进行离子注入,在一定剂量范围内成活率在90%以上;而未经玻璃化处理的幼苗离子注入后均不能成活.玻璃化后注入的幼苗成活率随注入剂量的增加而呈下降趋势,但受注入的离子能量的影响不大.显示玻璃化法对离子注入过程中活体材料遭受的冻害有较好的防护效果,在应用上有一定的意义.     

D-葡萄糖串联发酵生产2-酮基-L-古龙酸的工艺条件

研究了在10L发酵罐中D-葡萄糖串联发酵生产维生素C前体——2-酮基-L-古龙酸的发酵工艺条件。第一步发酵采用欧文氏菌(Erwinia sp.)的突变株SCB247,培养36小时,可将D-葡萄糖转化成中间体2,5-二酮基-D-葡萄糖酸,在发酵液中约累积180mg/ml。第二步发酵采用棒状杆菌(Corynebacterium sp.)SCB3058,可将2,5-二酮基-D-葡萄糖酸专一性地还原生成2-酮基-L-古龙酸。在细胞生长进入对数生长期后期时,加入经十二烷基硫酸钠处理的第一     

低能离子束介导的遗传转化研究进展

低能离子束注入对细胞的刻蚀作用提供了外源遗传物质进入细胞的途径,离子束介导的遗传转化技术已在水稻,小麦,烟草,棉花等多种植物上取得成功,对离子束介导遗传转化的原理和最新进展进行了评述。．     

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。     

高渗条件下利用蔗糖提升2-酮基-L-古龙酸生产效率

旨在进一步提升维生素C前体2-酮基-L-古龙酸(2-KLG)的生产效率。在详细考察了2-KLG工业化生产过程中渗透压变化规律的基础上,研究了高渗对混合菌系细胞生长和2-KLG合成的影响,提出蔗糖促进伴生菌巨大芽胞杆菌Bacillus megaterium生长,进而促进普通生酮古龙酸菌Ketogulonigenium vulgare生长和产酸的策略。结果表明,2-KLG的积累和碱性物质的流加使渗透压上升了832mOsmol/kg;高渗抑制了巨大芽胞杆菌的生长(15.4%),从而抑制普通生酮古龙酸菌(31.7%)的生长,导致2-KLG产量和生产强度分别下降67.5%和69.3%(以1250mOsmol/kg为例);蔗糖的添加则显著促进巨大芽胞杆菌的生长,使高渗条件下(摇瓶,1250 mOsmol/kg)2-KLG产量(40.6g/L)提高87%;在3L发酵罐中,补加10mmol/L蔗糖使2-KLG发酵周期缩短10.8%,2-KLG生产强度提高10.4%。研究成果为在环境胁迫下提高混菌生产目标代谢产物的产量提供了潜在的策略。     

微生物混合培养从D山梨醇产生维生素C前体——2-酮基-L-古龙酸

氧化葡萄糖酸杆菌（Gluconobacter oxydans)SCB329以D－山梨醇为底物培养时可产生微量2－酮基－L－古龙酸;而葡萄糖酸杆菌（Gluconobacter sp.)SCB110能将D－山梨醇以较高效率转化为L－山梨糖,但不产2－酮基－L－古龙酸。将两种微生物在以山梨醇为底物的培养基中混合培养,其代谢产物经分离提纯后进行熔点测定、元素分析、红外吸收光谱测定等,确定其主要的代谢产物是2－酮基－L－古龙酸。     

低能离子束在生物技术中的应用研究

自从发现离子注入生物效应后,低能离子与生物体系相互作用研究在我国率先兴起,并很快投入应用。简要介绍低能离子注入生物效应的机理研究和应用研究的进展状况,并展望未来 。     

Gelatin enhances 2-keto-L-gulonic acid production based on Ketogulonigenium vulgare genome annotation

In the two-step fermentative production of vitamin C, its precursor 2-keto-l-gulonic acid (2-KLG) was synthesized by Ketogulonicigenium vulgare through co-culture with Bacillus megaterium. The reconstruction of the amino acid metabolic pathway through completed genome sequence annotation demonstrated that K. vulgare was deficient in one or more key enzymes in the de novo biosynthesis pathways of eight different amino acids (l-histidine, l-glycine, l-lysine, l-proline, l-threonine, l-methionine, l-leucine, and l-isoleucine). Among them, l-glycine, l-proline, l-threonine, and l-isoleucine play vital roles in K. vulgare growth and 2-KLG production. The addition of those amino acids increased the 2-KLG productivity by 20.4%, 17.2%, 17.2%, and 11.8%, respectively. Furthermore, food grade gelatin was developed as a substitute for the amino acids to increase the cell concentration, 2-KLG productivity, and l-sorbose consumption rate by 10.2%, 23.4%, and 20.9%, respectively. As a result, the fermentation period decreased to 43 h in a 7-L fermentor.     

Manipulation of B. megaterium growth for efficient 2-KLG production by K. vulgare

In the two-step Vitamin C fermentative production, its precursor 2-keto-l-gulonic acid (2-KLG) was synthesized by Ketogulonicigenium vulgare through co-culture with Bacillus megaterium. The rates of K. vulgare cell growth and 2-KLG production were closely related with B. megaterium concentration in the co-culture system. To enhance the 2-KLG production efficiency, a strategy of manipulating B. megaterium growth in the co-culture system and properly releasing its intracellular components was introduced. Lysozyme was used specifically to damage B. megaterium cell wall structure and subsequently inhibit its cell growth. When 10,000 U mL⁻¹ lysozyme was fed to the co-culture system at 12 h, the growth rate of K. vulgare, sorbose consumption rate, and 2-KLG productivity could increase 27.4%, 37.1%, and 28.2%, respectively.     

建立低能离子束介导小麦转基因方法并获得转GUS基因植株

研究了注入离子种类、能量、剂量等参数对于低能离子束介导的遗传转化的影响,建立了适于小麦成熟胚转化的组培条件和筛选程序。以携带ＧＵＳ基因的质粒为供体,进行了报告基因转化研究。分子生物学证据表明ＧＵＳ基因已整合到小麦基因组中。３个小麦品种的抗性愈伤转化率分别为９．５％、１０．８％、１１．２％,再生植株转化率分别为１．４％、３．４％、１．７％,首次证明了离子束介导小麦遗传转化是可行的,为离子束介导小麦遗     

Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-l-gulonic acid from d-sorbitol

2-Keto-l-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step fermentation route from d-sorbitol. However, this route involves three bacteria, making the mix-culture system complicated and redundant. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. In this study, different combinations of five l-sorbose dehydrogenases (SDH) and two l-sorbosone dehydrogenases (SNDH) from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an industrial strain used for the conversion of d-sorbitol to l-sorbose. The optimum combination produced 4.9 g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4 g/L of 2-KLG after 168 h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 39.2 g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C.     

高浓度2-KLG对氧化葡萄糖酸杆菌WSH-003中2-KLG合成途径关键基因表达的影响

【目的】研究高浓度的2-KLG对其生产菌株氧化葡萄糖酸杆菌生产过程中关键的脱氢酶合成基因、辅因子合成基因及其转运蛋白编码基因的影响。【方法】测定高浓度梯度2-KLG下氧化葡萄糖酸杆菌的生长情况,确定合适的添加浓度对氧化葡萄糖酸杆菌进行胁迫。使用实时定量PCR技术检测2-KLG合成中关键山梨醇脱氢酶基因sld AB、关键辅因子PQQ合成基因pqq ABCDE及5个潜在转运蛋白合成基因的变化。【结果】根据氧化葡萄糖酸杆菌在2-KLG高浓度梯度下生长测定实验结果,选定40、80和120 g/L 2-KLG作为添加浓度。实时定量PCR结果显示,在高浓度的2-KLG压力下,PQQ合成基因pqq ABCDE未受到显著影响,山梨醇脱氢酶基因sld AB以及部分PQQ潜在转运蛋白编码基因的表达均显著下调。【结论】高浓度2-KLG会抑制氧化葡萄糖酸杆菌中山梨醇脱氢酶基因的表达,有可能会影响辅酶PQQ的转运,但不会显著影响辅酶PQQ的合成。     

离子束介导玉米DNA影响水稻幼苗根系蛋白水解酶的表达

利用离子束介导法把玉米DNA导入水稻豫粳6号种子胚中,通过复性电泳技术,分析水稻幼苗根系中蛋白水解酶在pH4.5、pH7.0和pH8.5的表达情况.结果表明:(1)在不同pH条件下处理,各蛋白水解酶种类与活性存在差异,酸性、中性、碱性条件下,检到的蛋白水解酶酶带依次增多,酸性条件下酶活性较弱,中性和碱性条件下酶活性较强;(2)在3种pH条件下,离子束辐照处理均有部分蛋白水解酶带缺失,同时,在中性和碱性条件下检出50kD新酶带,说明离子束辐照可影响水稻蛋白水解酶表达;(3)离子束介导玉米DNA水稻幼苗根系中,pH4.5时无新蛋白水解酶酶带检出,pH7.0和pH8.5时均检到多条新酶带且活性较强.说明离子束介导玉米DNA引起水稻幼根蛋白水解酶表达发生变化.     

Mathematical modeling of in vitro enzymatic production of 2-Keto-L-gulonic acid using NAD(H) or NADP(H) as cofactors

A 2-Keto-L-gulonic acid (2-KLG) production process using stationary Pantoea citrea cells and a Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme has been developed which may represent an improved method of vitamin C biosynthesis. Experimental data was collected using the F22Y/A272G 2,5-DKG reductase mutant and NADP(H) as a cofactor. An extensive kinetic analysis was performed and a kinetic rate equation model for this process was developed. A recent protein engineering effort has resulted in several 2,5-DKG reductase mutants exhibiting improved activity with NADH as a cofactor. The use of NAD(H) in the bioreactor may be preferable due to its increased stability and lower cost. The kinetic parameters in the rate equation model have been replaced in order to predict 2-KLG production with NAD(H) as a cofactor. The model was also extended to predict 2-KLG production in the presence of a range of combined cofactor concentrations. This analysis suggests that the use of the F22Y/K232G/R238H/A272G 2,5-DKG reductase mutant with NAD(H) combined with a small amount of NADP(H) could provide a significant cost benefit for in vitro enzymatic 2-KLG production.     

分阶段pH调控提高2-酮基-L-古龙酸生产

为了提高酮古龙酸菌Ketogulonicigenium vulgare和巨大芽胞杆菌Bacillus megaterium生产2-酮基-L-古龙酸(2-KLG)的生产效率,分析了pH对K.vulgare和B.megaterium生长和产酸的影响,发现K.vulgare和B.megaterium的最适生长pH值分别为6.0和8.0,但是K.vulgare的糖酸转化活力在pH7.0时达到最大值,因此提出了三阶段pH控制策略(第一阶段:0～8h,pH8.0;第二阶段:8～20h,pH6.0;第三阶段:20h至发酵结束,pH7.0)以促进K.vulgare生长和2-KLG生产。结果表明,三阶段pH控制策略的实施进一步提高了2-KLG的产量(77.3g/L)、生产强度(1.38g/(L·h))和L-山梨糖消耗速率(1.42g/(L·h)),分别比恒定pH7.0时提高了9.7%、33.2%和25.7%。     

A subset of asialo GM1+ cells play a protective role in the occurrence of graft-versus-host disease in mice

In three different murine models of bone marrow (BM) transplantation the capacity of asialo GM1+ cells to suppress graft-vs-host disease (GVHD) was investigated. In a first model, total lymphoid irradiation (TLI)-treated BALB/C mice were given 1 mg of anti-asialo GM1 antibody. This led to the disappearance of functional suppressor cells after TLI. Injections of anti-asialo GM1 into TLI-treated BALB/C mice before infusion of 30 x 10(6) fully allogeneic (C3H) BM cells, led to a significantly decreased survival rate as compared to TLI-treated mice injected with control serum before BM transplantation (survival 29 and 83%, respectively, at 120 days after transplantation, p = 0.0032 log rank). The mortality of the former group was due to GVHD as 1 degree all dying animals showed clinical and histologic signs of GVHD, 2 degrees all animals were chimeric and 3 degrees mice receiving no or syngeneic BALB/C BM had excellent survival rates excluding BM aplasia or increased susceptibility for infections as reason for the mortality of the allogeneic BM recipients. In a second model, asialo GM1+ cells were removed in vitro from the C3H BM inoculum before injection into lethally irradiated (9 Gy) BALB/C recipients. In mice kept in specific pathogen-free conditions, this procedure resulted into a significant mortality (12/12) as compared to mice receiving BM pretreated with control serum (1/12, p = 0.0001 log rank). When kept in conventional housing, GVHD occurred in both groups but much earlier in the group receiving anti-asialo GM1-treated BM (median survival time 6 vs 46 days for the control mice, p = 0.001 log rank). No animal receiving anti-asialo GM1 and treated with syngeneic BM died, thus excluding toxicity, increased susceptibility to infections, or decreased graft take as a cause of mortality. In a last model, asialo GM1 cells were removed from syngeneic BM in a BM transplantation model in which T cell-depleted syngeneic (BALB/C) and non-T cell-depleted allogeneic (C3H) BM was administered to lethally irradiated (9 Gy) BALB/C mice. Also in this model GVHD-related mortality only occurred in the group of mice receiving syngeneic BM from which asialo GM+ cells were depleted before infusion (3/12). Our experiments thus clearly show that asialo GM1+ cells from both recipient (the TLI model) as well as donor origin (the TBI experiments) can suppress the occurrence of GVHD.     

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.     

A high throughput method to screen companion bacterium for 2-keto-l-gulonic acid biosynthesis by co-culturing Ketogulonicigenium vulgare

Bacillus megaterium is widely used as companion bacterium in the two-step biosynthesis of 2-keto-l-gulonic acid (2-KLG) by Ketogulonicigenium vulgare. To screen efficiently target companion strains from large numbers of random mutants, a screen method based on spectrophotometry and 24-well microtiter plates was developed and validated on an integrated library of 450 transposon random insertional mutants and two sporulation-defective mutants. The co-culture processes were classified into three groups (low, intermediate and high performance) by K-mean clustering analysis. In addition, mutant m71 was successfully screened out from the library. The substrate conversion ratio of m71 and K. vulgare co-culture process after 72 h was decreased by about 38% compared with that of the wild-type co-culture process in 750 ml flasks. These results indicated that the proposed high throughput method is feasible for screening target companions for the co-culture process of 2-KLG biosynthesis.     

Development of chemically defined media supporting high cell density growth of Ketogulonicigenium vulgare and Bacillus megaterium

The immediate precursor of L-ascorbic acid, or vitamin C, is 2-keto-l-gulonic acid (2-KLG). This is commonly produced commercially by Ketogulonicigenium vulgare and Bacillus megaterium, using corn steep liquor powder (CSLP) as an organic nitrogen source. In this study, the effects of the individual CSLP components (amino acids, vitamins, and metal elements) on 2-KLG production were evaluated, with the aim of developing a complete, chemically defined medium for 2-KLG production. Forty components of CSLP were analyzed, and key components were correlated to biomass, 2-KLG productivity, and consumption rate of L-sorbose. Glycine had the greatest effect, followed by serine, biotin, proline, nicotinic acid, and threonine. The combination of 0.28 g L⁻¹ serine, 0.36 g L⁻¹glycine, 0.18 g L⁻¹ threonine, 0.28 g L⁻¹proline, 0.19 g L⁻¹nicotinic acid, and 0.62 mg L⁻¹biotin in a chemically defined medium produced the highest maximum biomass concentration (4.2 × 10⁹ cfu mL⁻¹), 2-KLG concentration (58 g L⁻¹), and yield (0.76 g g⁻¹) after culturing for 28 h.