利用辅因子工程策略提高酿酒酵母中S-腺苷蛋氨酸的生物合成

通过代谢工程策略改造酿酒酵母胞内辅因子的形式和浓度,分析辅因子NADPH对于产物S-腺苷蛋氨酸(SAM)合成的作用并总结能量代谢和其他物质代谢的规律,为高产SAM菌株的代谢工程改造提供理论基础。由于酿酒酵母中的NADPH在线粒体和细胞质中的代谢相对独立,因此以酿酒酵母BY4741单倍体模式菌株为研究对象,研究了不同亚细胞结构内NADPH对于产物合成的影响。通过激光共聚焦显微镜证实了NADH激酶在酿酒酵母线粒体和细胞质中的表达。实验结果表明NADPH的提高有利于酿酒酵母胞内SAM的合成。发酵24 h,菌株NBYSM-1胞内SAM浓度较对照菌提高3.28倍,菌株NBYSM-2胞内SAM浓度提高1.79倍。其中重组菌株NBYSM-1合成SAM的能力和胞内NADPH/NADP~+比率均明显高于重组菌株NBYSM-2。因此,NADPH调控策略有望成为提高SAM产量的有力工具并应用于其他辅因子依赖化合物的合成。     

丙酮酸和乙酰-CoA协同促进L-亮氨酸的合成

【目的】通过理性改造柠檬酸合酶(citrate synthase,CS)、丙酮酸脱氢酶系E1p (pyruvate dehydrogenase complex,PDHC,编码基因aceE)和ATP-柠檬酸裂解酶(ATP-Citrate lyase,ACL),有效供应胞内丙酮酸和乙酰-CoA,以提高L-亮氨酸产量。【方法】以谷氨酸棒杆菌(Corynebacterium glutamicum)为底盘细胞,分析不同CS和PDHC酶活水平对L-亮氨酸合成的影响。随后,考查协同改造CS和PDHC或引入绿硫菌(Chlorobium tepidum)中ACL对L-亮氨酸合成的影响。【结果】低强度的CS酶活(即重组菌XL-3 P_(dapA-R2)gltA)有利于L-亮氨酸的合成,L-亮氨酸产量达到17.5±0.6 g/L。而改变PDHC酶活水平不利于L-亮氨酸的合成。此外,以启动子P_(dapA-R2)控制CS表达,而以启动子P_(gapA)控制PDHC表达时(即重组菌XL-4),可实现胞内丙酮酸和乙酰-CoA的有效供给,L-亮氨酸产量达到20.2±1.7 g/L,且显著降低副产物产量。若在重组菌XL-4中引入C.tepidum,ACL会显著抑制菌体生长而不利于L-亮氨酸合成,而引入到出发菌XL-3中因胞内丙酮酸和乙酰-CoA得到有效供给,目标重组菌XL-5L-亮氨酸产量达到18.5±1.2 g/L,比出发菌株XL-3增加了14.2%。【结论】重组菌XL-4中因协同控制CS和PDHC酶活,从而实现胞内丙酮酸和乙酰-CoA有效供给,促进L-亮氨酸的合成。该研究结果对后续利用代谢工程技术强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。     

利用五碳糖产高纯度L-乳酸的大肠杆菌基因工程菌的构建

[目的]本研究以已敲除多个产杂酸酶基因的大肠杆菌(Escherichia coli)乙醇工程菌SZ470(△frdBC △ldhA △ackA △focA-pflB △pdhR::pflBp6-pflBrbs-aceEF-lpd)为起始菌株,进一步敲除其乙醇脱氢酶(alcohol dehydrogenase,ADH)基因,同时插入带有自身启动子的乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢酶(L-lactate dehydrogenase,LLDH)基因,构建可利用五碳糖同型发酵L-乳酸重组大肠杆菌.[方法]利用λ噬菌体Red重组系统构建乙醇脱氢酶基因(adhE)缺失菌株Escherichia coli JH01,并克隆P.acidilactici的ldhL基因,利用染色体插入技术将其整合到JH01基因组,构建产L-乳酸大肠杆菌基因工程菌Escherichia coli JH12,利用无氧发酵15 L发酵罐测定重组菌株L-乳酸产量.[结果]工程菌JH12在15 L发酵罐中以6％的葡萄糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为1.46 g/(L·h),乳酸生产强度为1.14 g/(L·h),乳酸的产量达到41.13 g/L.发酵产物中未检测到琥珀酸、甲酸的生成,仅有少量乙酸生成,L-乳酸纯度达95.69％(L-乳酸在总发酵产物的比率).工程菌JH12以6％的木糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为0.88 g/(L·h),乳酸生产强度为0.60 g/(L·h),乳酸的产量达到34.73 g/L.发酵产物中杂酸少,乳酸的纯度高达98％.[结论]本研究通过基因敲除、染色体插入及无氧进化筛选获得一株产L-乳酸的大肠杆菌工程菌JH12,该菌株不需利用外源质粒,稳定性好,可利用五碳糖进行发酵,发酵产物中杂酸少,L-乳酸的纯度高.本研究为L-乳酸大肠杆菌工程菌的构建提供一定的技术支持,同时也为大肠杆菌L-乳酸的工业化生产提供了参考依据.     

酿酒酵母POS5基因过表达对L-异亮氨酸合成的促进作用

L-异亮氨酸是人体必需氨基酸之一,具有很大的商业价值。在谷氨酸棒杆菌中,合成一分子L-异亮氨酸需要消耗四分子NADPH。因此,提高胞内NADPH浓度是提高L-异亮氨酸产量的重要手段之一。在乳糖发酵短杆菌JHI3-156中过量表达酿酒酵母的NADH激酶编码基因POS5△MTS,发酵48 h表达菌株JHI3-156/pDXW-10-POS5△MTS的胞内NADP~+浓度增加了27μmol/L(17%),NADPH浓度增加了36μmol/L(96%);发酵72 h后L-异亮氨酸产量是(3.02±0.52)g/L,比对照菌(1.96±0.04)g/L提高了54%。本研究表明,过表达POS5△MTS基因能提高NADPH的供应,促进了L-异亮氨酸的生物合成。     

强化类球红细菌辅因子NADPH再生以提高法尼醇的产量

法尼醇(Farnesol,FOH)是由焦磷酸异戊烯基(IPP)和焦磷酸二甲基烯丙基(DMAPP)合成的法尼酰基焦磷酸盐(FPP)去焦磷酸化作用生成的。在类球红细菌中IPP和DMAPP是由MEP途径生成,而完整的MEP途径需要消耗大量的辅因子NADPH,增加胞内NADPH的量有可能强化FOH的合成。文中从增加NADPH的生成和降低NADPH的消耗这两个策略出发,分别干扰了编码6-磷酸葡萄糖异构酶基因(pgi)和谷氨酸脱氢酶基因(gdhA)的表达,同时强化了磷酸戊糖途径中6-葡萄糖磷酸脱氢酶基因(zwf)和6-葡萄糖酸磷酸脱氢酶基因(gnd)的表达。实验结果表明,经改造的菌株NADPH含量显著增加,干扰菌株中菌株RSpgii的产量较高,为3.91 mg/g,在过表达的菌株中同时过表达zwf和gnd基因的重组菌株(RSzg)的FOH产量提高到了3.43 mg/g。为了获得FOH产量更高的菌株,以RSpgii为出发菌株,分别与zwf和gnd组合调控,获得的菌株RSzgpi的产量达到了最高量为4.48 mg/g,是出发菌株RS-GY2产率的2.24倍。     

溶氧量与谷氨酸棒杆菌代谢

正自从1957年Kinoshita等首次描述谷氨酸棒杆菌(Corynebacterium glutamicum)为谷氨酸产生菌[1]以来,其已成为用于氨基酸生产的主要菌株。目前,全世界每年利用谷氨酸棒杆菌生产约100万t L-谷氨酸用于食品调味剂和约45万t L-赖氨酸用作食品添加剂[2]。通过谷氨酸棒状杆菌发酵获得谷氨酸的发酵水平已较高,通过进一步优化工艺来提高产量具有较大困难[3]。     

黑曲霉糖化酶基因启动子功能鉴定

从糖化酶工业生产菌株Aspergillus nigerCICIM F0410基因组DNA中扩增糖化酶基因启动子(PglaA),并将该启动子替换质粒pRS303K上KmR基因启动子,构建成糖化酶基因启动子功能检测质粒pRS-PglaA-KmR。将pRS-PglaA-KmR转入E.coliJM109中,得到重组菌E.coli(pRS-PglaA-KmR)。通过对重组菌的氨基糖苷磷酸转移酶基因活性检测,表明PglaA在E.coli中具有驱动KmR基因表达的活性。采用不同诱导物进行培养发现,葡萄糖、蔗糖、乳糖、麦芽糖或玉米淀粉,可以不同程度增强PglaA的强度。     

代谢工程大肠杆菌利用甘油高效合成L-乳酸

以甘油为碳源高效合成L-乳酸有助于推进油脂水解产业和生物可降解材料制造业的共同发展。为此,首先分别从凝结芽胞杆菌Bacillus coagulans CICIM B1821和大肠杆菌Escherichia coli CICIM B0013中克隆了L-乳酸脱氢酶基因BcoaLDH和D-乳酸脱氢酶 (LdhA) 的启动子片段PldhA。将两条DNA片段连接组成了表达盒PldhA-BcoaLDH。然后将上述表达盒通过同源重组删除FMN为辅酶的L-乳酸脱氢酶编码基因lldD的同时克隆入ldhA基因缺失菌株E. coli CICIM B0013-080C (ack-pta pps pflB dld poxB adhE frdA ldhA)的染色体上,获得了L-乳酸高产菌株E. coli CICIM B0013-090B (B0013-080C,lldD::PldhA-BcoaLDH)。考察了菌株CICIM B0013-090B不同培养温度下代谢利用甘油和合成L-乳酸的特征后,建立并优化了一种新型L-乳酸变温发酵工艺。在7 L发酵罐上,发酵27 h,积累L-乳酸132.4 g/L,产酸强度4.90 g/(L·h),甘油到L-乳酸的得率为93.7％,L-乳酸的光学纯度达到99.95％。     

基于时间序列转录组筛选谷氨酸棒杆菌内源高效组成型启动子

启动子是重要的转录调控元件,广泛用于工业菌株的代谢工程改造。谷氨酸棒杆菌Corynebacterium glutamicum是重要的氨基酸生产菌株,但已报道的组成型强启动子较少。对谷氨酸高产菌Corynebacterium glutamicum SL4发酵过程的10个时间点样品进行转录组测序,筛选在发酵过程中稳定转录并且转录水平最高的10个基因;分别克隆其启动子序列至红色荧光蛋白(RFP)报告系统,通过荧光强度表征启动子在SL4菌株中的强度,再在野生型C. glutamicum ATCC 13869和ATCC 13032中验证部分启动子的通用性;并采用LacZ蛋白进一步评价强启动子的表达效果。结果显示,成功筛选到3个可以通用的组成型启动子P_(cysK)、P_(gapA)和P_(fumC)。其中P_(cysK)的表达强度最高,与诱导型强启动子P_(tac)对比,在SL4和13869菌株中均达到其2倍(RFP)和4倍(LacZ)以上;在ATCC 13032菌株中,P_(cysK)的表达强度为P_(tac)的0.3-0.4倍。Pcys K首次被报道为强启动子,可用于谷氨酸棒杆菌强化合成途径的代谢工程改造。     

氨基脱氧分支酸合成酶对Corynebacterium glutamicum SYPS-062积累L-丝氨酸的影响

为阐明氨基脱氧分支酸合成酶(ADC合成酶)在Corynebacterium glutamicum SYPS-062体内积累L-丝氨酸过程中的作用,通过交叉PCR以及同源重组的方法敲除叶酸途径关键酶ADC合成酶的编码基因pabAB,构建了叶酸缺陷型菌株Corynebacterium glutamicum SYPS-062△pabAB,同时构建pabAB基因增强表达重组菌C.glutamicum SYPS-062(pJC Ⅰ-pabAB).分别考察了ADC合成酶对菌株生长的影响、对L-丝氨酸降解途径关键酶丝氨酸羟甲基转移酶(SHMT)的影响以及其对L-丝氨酸积累的影响.结果表明,与出发菌株相比,增强表达基因pabAB重组菌的ADC合成酶的酶活力提高了33%.SHMT酶的酶活力提高了30%,其最大比生长速率(μm)提高了48%,单位细胞产酸率(Yp/x)降低了36.2%;而敲除基因pabAB重组菌的ADC合成酶的酶活力降低了61%.SHMT酶的酶活力降低了20%,最大比生长速率降低了32%,单位细胞产酸率提高了12%.     

Role of glucose in the bioconversion of fructose into mannitol by Candida magnoliae

The most efficient substrate for mannitol production by Candida magnoliae HH-01 is fructose; glucose and sucrose can also be converted into mannitol but with lower conversion yields. Mannitol dehydrogenase was purified and characterized; it had the highest activity with fructose as the substrate and used only NADPH. In fed-batch fermentation with glucose, the production of mannitol from fructose ceased when the glucose was exhausted but it was reinitiated with the addition of glucose, implying that glucose plays an important role in NADPH regeneration.     

Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in <Emphasis Type="Italic">Schizochytrium</Emphasis> sp. HX-308

Docosahexaenoic acid (DHA) production in Schizochytrium sp. HX-308 was evaluated by detecting enzymatic activities of ATP:citrate lyase (EC 4.1.3.8), malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) at different fermentation stages. According to the analysis, a regulation strategy was proposed which reinforced acetyl-CoA and NADPH supply at a specific fermentation stage. DHA content of total fatty acids was increased from 35 to 60% by the addition of 4 g/L malic acid at the rapid lipid accumulation stage. Total lipid content also showed an apparent increase of 35% and reached 19 g/L when 40 mL ethanol/L was added at the late lipid accumulation stage.     

酿酒酵母中NAD激酶同功酶对偶数位不饱和脂肪酸β-氧化的影响

ATP-NAD激酶利用ATP,催化NAD磷酸化,生成NADP,而ATP-NADH激酶则催化NAD和NADH发生磷酸化。酿酒酵母细胞内存在三种NAD激酶同功酶Utr1p、Pos5p和Yef1p,它们都是ATP-NADH激酶,对细胞内NADP(H)的供应起到重要作用。酵母偶数位双键不饱和脂肪酸的β-氧化依赖于过氧化物酶体基质内的NADPH。通过构建NAD激酶基因的单、双基因缺失株,并验证它们和对照菌株对不饱和脂肪酸的氧化、利用能力,证实NAD激酶同功酶,尤其是Pos5p,对过氧化物酶体基质内NADP(H)的供应起着重要作用,并推测NADP可以从过氧化物酶体膜外转运至过氧化物酶体基质内。     

Iron Mediated Nitrate Reductase Activity in Different Parts of Young Maize Seedlings

Incubation of 5-d-old maize seedlings in the half-strength Hoagland's nutrient solution containing 10 mM KNO₃ with FeCl₃ or FeSO₄ (0.5 or 2.0 mM) caused a significant increase in nitrate reductase (NR) activity and slightly increased total protein content in root, shoot and scutellum. In case of root, NADPH:NR activity was inhibited contrary to the NADH:NR activity. In spite of NR activity, nitrate uptake was inhibited from 13 to 37 % by the iron. The results presented demonstrate an isoform specific, organ specific, and to some extent salt specific responses of NR to iron.     

Neutrophil priming mechanisms of sulfolipid-I and n-formyl-methionyl-leucyl-phenylalanine

The principal sulfatide of virulentMycobacterium tuberculosis, sulfolipid-I (SL-I), both directly stimulates neutrophil superoxide (O ₂ ^– ) release and, at substimulatory concentrations, primes these cells for markedly enhanced oxidative responsiveness to other stimuli. The present study was undertaken to clarify the priming mechanisms by comparing cellular events following priming doses of SL-I with those following priming with N-formyl-methionyl-leucyl-phenylalanine (FMLP). We compared the involvement of the calcium cation (Ca²⁺), as well as membrane protein kinase C (PKC) activity and the translocation of NADPH oxidase-cytosolic cofactor effected by priming levels of the two agonists. The investigation led to two important conclusions. First, we clearly demonstrate that priming by both SL-I and FMLP results from activation of cellular processes that are not involved in direct oxidative activation. For example, whereas direct induction of O ₂ ^– generation by FMLP and SL-I required increases in intracellular Ca²⁺, an increase in intracellular calcium concentration ([Ca²⁺]_i) above basal levels was not required for priming. Second, we identified key differences in the cellular responses to priming doses of SL-I and FMLP. Whereas increased membrane PKC activity caused by priming doses of FMLP was only partially blocked by chelation of intracellular Ca²⁺, Ca²⁺ chelation completely inhibited the increase in membrane PKC activity caused by SL-I. NADPH oxidase-cytosolic factor translocation to plasma membranes was completely blocked by pertussis toxin when priming doses of SL-I were used. This guanine-nucleotide-binding protein inhibitor had no effect on FMLP-dependent translocation of the oxidase cofactors. The comparative approach introduced in this report provides a valuable and novel method to discern the complex interactions of various cellular processes that regulate the state of activation of stimulated cells.     

Enzymatic synthesis of 2-keto- -gluconate and 2-keto- -galactonate from -glucose and -galactose with cell culture of Pseudomonas fluorescens and 2-keto-galactonate from -galactono 1,4-lactone with partially purified 2-ketogalactonate reductase

2-Keto- -gluconate and 2-keto- -galactonate were prepared from -glucose (with a yield of 40%) and -galactose (with a yield of 25%), respectively, with cell culture of Pseudomonas fluorescens. However, 2-keto- -mannoate was not prepared in this method. The time courses of the reactions showed that 2-keto- -gluconic acid and 2-keto- -galactonic acid were produced from -glucose and -galactose through -gluconate and -galactonate, respectively. When using -galactono 1,4-lactone as a starting material, 2-keto- -galactonate was produced with partially purified NADP-dependent 2-ketogalactonate reductase from P. fluorescens. Some fundamental properties of the 2-ketogalactonate reductase were compared with those of 2-ketogluconate reductase from Acetobacter and Gluconobacter.     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.     

Transgenic rice plants conferring increased tolerance to rice blast and multiple environmental stresses

We isolated a rice gene (denoted YK1), which showed78 percent amino acid sequence homology to the maize HM1gene. A chimeric gene consisting of a promoter and first intron of maizeubiquitin gene and the cDNA of YK1 was introduced intorice via Agrobacterium mediated transformation. Transgenic riceplants overexpressing this chimeric gene were resistant to rice blast(Magnaporthe grisea) disease, which is one of the mostserious pathogens in rice. Furthermore, the same transgenic plants conferredhigh tolerance to several abiotic stresses such as NaCl, UV-C, submergence, andhydrogen peroxide.     

Chloroplast biogenesis 76. Regulation of 4-vinyl reduction during conversion of divinyl Mg-protoporphyrin IX to monovinyl protochlorophyllide a is controlled by plastid membrane and stromal factors

Most of the chlorophyll (Chl) a of green plants is formed via two biosynthetic routes, namely the carboxylic divinyl and monovinyl chlorophyll biosynthetic routes. These two routes are linked by (4-vinyl) reductases that convert divinyl tetrapyrroles to monovinyl tetrapyrroles by reduction of the vinyl group at position four of the macrocycle to ethyl. The activities of these two routes are very sensitive to cell disruption. For example in barley leaves, cell disruption, a mandatory step during plastid isolation, results in partial inactivation of the carboxylic divinyl route. Investigations with subplastidic fractions revealed that the carboxylic divinyl and monovinyl biosynthetic routes were regulated by a delicate interaction that involved plastid membranes, stroma, and reduced pyridine nucleotides. While the monovinyl biosynthetic route was very active in isolated plastid membranes, activation of the divinyl biosynthetic route required the joint presence of plastid membranes and stroma. Contrary to expectation, activity of the carboxylic divinyl biosynthetic route was greatly enhanced by addition of NADPH to the lysing buffer used during plastid membranes and stroma preparation. NADPH in cooperation with the plastid stroma may play an important regulatory role during the biosynthesis of divinyl and monovinyl protochlorophyllide a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glucose-6-phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition

The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies,NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP⁺ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903–904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP⁺] ratio.