Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae

Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD(+)-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Delta null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Deltafdh2Delta double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.     

重组大肠杆菌合成光学纯L-苯基乳酸

以巨大芽孢杆菌Z2013513基因组DNA为模板,分别PCR扩增得到L-乳酸脱氢酶基因(ldh L)和葡萄糖脱氢酶基因(gdh),将gdh与ldh L分别连接至表达载体p ETDuet,获得共表达质粒p ETDuet-ldh L-gdh。经转化和验证获得重组菌大肠杆菌BL21(DE3)/p ETDuet-ldh L-gdh。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和比酶活力分析表明重组蛋白L-乳酸脱氢酶和葡萄糖脱氢酶均成功表达且具有酶活力。在37℃、200 r/min条件下,经60 min反应,催化底物苯丙酮酸合成24.26 mmol/L L-苯基乳酸。产物L-苯基乳酸光学纯度(99%),底物摩尔转化率(59.55%),结果表明此重组体系可用于高效合成高光学纯L-苯基乳酸。     

Effect of dimorphic regulation on heterologous glucose oxidase production by Mucor circinelloides

The production of Aspergillus niger glucose oxidase (GOX) and native amylase by the recombinant M. circinelloides KFA199 strain under conditions of dimorphic growth was investigated. The recombinant KFA199 strain was compared to its parental ATCC 1216b strain and a wild‐type CBS 232.29 strain under similar morphology‐controlled conditions. Cultivation in Vogel's medium supplemented with ergosterol/Tween‐80 and sparged with nitrogen gas was most suitable for yeast‐like biomass production under anaerobic conditions. Anaerobic growth was characterized by high levels of ethanol formation and linear growth rates of 0.24–0.05/h, indicating metabolic stress. Subsequent to anaerobic growth, cultures were shifted to aerobic conditions to induce aerobic mycelial growth. GOX produced by the recombinant KFA199 after the shift to aerobic conditions was poorly secreted and accumulated intracellularly to 0.56 U/ml_culture. Amylase production by the KFA199, ATCC12b and CBS 232.29 strains was determined during growth on starch after the shift to aerobic culture. Growth‐associated amylase production by the ATCC 1216b (0.63 U/ml_culture) and wild‐type CBS 232.29 (0.33 U/ml_culture) strains was substantially higher than by the recombinant KFA199 strain (0.07 U/ml_culture), which may be related to the leucine auxotrophy of the transformation host, or genetic changes induced during transformation of the KFA199 strain. Copyright © 2010 John Wiley & Sons, Ltd.     

无外源脯氨酸发酵产反式-4-羟脯氨酸重组大肠杆菌的构建及发酵优化

为实现无外源脯氨酸的条件下直接从葡萄糖转化生成反式-4-羟脯氨酸,本实验采用定点突变和基因共表达的方法构建重组大肠杆菌:首先对L-脯氨酸生物合成途径中的关键酶谷氨酸激酶进行定点突变E143A、K145A,以增强L-脯氨酸生物合成能力;然后引入脯氨酸4-羟化酶基因,通过两个基因的共表达可以实现从葡萄糖到反式-4-羟脯氨酸的连续转化,而不再需要添加外源L-脯氨酸。得到的L-脯氨酸生物合成能力增强的菌株在摇瓶阶段L-脯氨酸的产量可达到1.4 g/L;pro BA2与hyp双基因共表达菌株的反式-4-羟脯氨酸产量为98.9 mg/L,比原始菌株提高了一倍,经发酵优化后得到培养基为:葡萄糖10 g/L,胰蛋白胨15 g/L,硫酸亚铁3 mmol/L,硫酸镁1 g/L,磷酸氢二钾3 g/L,氯化钙0.015 g/L,在这个培养条件下反式-4-羟脯氨酸的产量为220.0 mg/L,比优化前提高1.2倍。      

克雷伯氏菌乙醛脱氢酶基因过表达及产乙醇发酵工艺优化

为获得高效产乙醇的基因重组菌,该研究从克雷伯氏菌（Klebsiella sp.）WL1316中克隆乙醛脱氢酶aldh基因,进行同源过表达,考察其利用木质纤维素水解液中葡萄糖和木糖的能力,并以乙醇含量为响应值,通过单因素试验及响应面试验对其发酵稻草水解液产乙醇的工艺条件进行优化。结果表明,成功构建了同源过表达aldh基因的重组菌株aldh-pET-28a-Klebsiella sp. WL1316,其保持了野生菌株Klebsiella sp. WL1316利用木质纤维素水解液中葡萄糖和木糖的特性,且发酵稻草水解液产乙醇的最优发酵工艺为发酵时间60 h,异丙基硫代半乳糖苷（IPTG）诱导浓度1.5 mmol/L,发酵培养基初始pH值7.3,初始还原糖含量59 g/L。在此最优工艺条件下,重组菌株的乙醇含量为（5.39±0.51）g/L,是野生菌株[（3.67±0.32）g/L]的1.47倍,表明aldh基因的过表达促进了乙醇产量的提高。     

抗氯嘧磺隆黑曲霉乙酰乳酸合成酶（ALS）酶学特性研究

本文比较了不同黑曲霉（Aspergillus niger）菌株对大豆田除草剂氯嘧磺隆的耐药性,并且对抗性菌株中靶标酶乙酰乳酸合成酶（ALS）的酶学特性进行了研究。结果发现TR-H为抗性菌株,其ALS酶活性显著高于其它菌株。酶学特性分析显示,TR-H菌株的最佳培养时间为84h,最佳反应温度为35℃,最适反应pH为7.3,酶反应的初速度范围为0～60min。以丙酮酸钠为反应底物时,其米氏常数（Km）为39.41mmol/L,最大反应速度（Vmax）为232.2μg/mL·h。     

酿酒酵母代谢木糖工程菌的构建

通过PCR方法从休哈塔假丝酵母基因组DNA中克隆得到木糖还原酶(XR)基因XYL1和木糖醇脱氢酶(XDH)基因XYL2,将其分别连接到酵母表达载体pYES2上,得到重组表达载体pYES2-XYL1和pYES2-XYL2,从pYES2-XYL1上克隆得到含半乳糖启动子的XYL1,将其连接到pYES2-XYL2序列的下游,得到重组表达载体pYES2-XYL1-XYL2,通过电转化方法将pYES2-XYL1-XYL2转入酿酒酵母宿主菌INVSc1。在初始pH为5.5,温度为33℃,前5h转速为150r/min,后变为50r/min的条件下,乙醇产量为33.45g/L。     

米根霉AS 3.819基因启动子片段的克隆及功能鉴定

从L-乳酸生产菌米根霉（Rhizopus oryzae）菌株AS 3.819基因组DNA中分别扩增得到了乳酸脱氢酶基因（ldhA）、丙酮酸脱氢酶基因（pdcA）、淀粉糖化酶基因（amyA）以及磷酸甘油酸酯激酶基因（pgk1）的启动子片段,并构建启动子探针载体pUKMR,以β-内酰胺酶基因（bla）为报告基因在大肠杆菌JM109中对这些启动子片段进行筛选及启动活性检测。结果表明：4 种启动子片段成功启动报告基因表达;在非底物诱导情况下,ldhA和pgk1启动子启动活性较强;在有合适碳源底物诱导情况下pdcA和amyA启动子拥有更高的启动活性;ldhA基因的启动子启动活性随着启动子片段长度的增加有一定提高,而在长度为500 bp以上时,其启动活性变化不明显。本研究为Rhizopus oryzae提供了一种快速简便的启动子捕获分离及启动活性检测方法。     

Effect of Heating Temperature and Muscle Type on Porcine Muscle Extracts as Determined by Reverse Phase High Performance Liquid Chromatography

Samples from porcine longissimus muscles were heated to temperatures ranging from 40–80°C to reach endpoint temperatures (0 min) or held at endpoint temperature for 30 min. Proteins in water-soluble extracts of these samples were separated and quantified by reverse phase high performance liquid chromatography (RP-HPLC). After heating to 60°C for 0 min or to 55°C and holding for 30 min, lactate dehydrogenase (LDH), pyruvate kinase (PK) and myoglobin appeared to comprise the bulk of the remaining soluble protein. RP-HPLC analysis of water soluble extracts from longissimus dorsi, serratus ventralis and psoas major muscles from barrows and sows indicated differences in proportions of LDH and myoglobin.     

Disruption of genes encoding pyruvate dehydrogenase kinases leads to retarded growth on acetate and ethanol in Saccharomyces cerevisiae

Two open reading frames, YIL042c (PKP1) and YGL059w, with 25% sequence similarity to human pyruvate dehydrogenase kinases, were shown to have protein kinase activity. Using GFP fusions, it was demonstrated that the proteins localize in discrete submitochondrial regions. Strains with a null mutation in these loci grew poorly on acetate and ethanol as carbon sources. Doubling times increased from ca. 4 h in the wild-type to > 6 h for the mutants. Growth rates of the mutants could be restored to wild-type levels by simultaneous disruption of the PDA1 gene, encoding the E1alpha subunit of the pyruvate dehydrogenase complex. This observation and the pyruvate dehydrogenase activities measured in the mutant strains and the wild-type grown on glucose or acetate suggest that the slow growth phenotype on C2 carbon sources is caused by a futile cycle in which phosphoenolpyruvate is converted back to acetyl coenzyme A.     

甜玉米芯多糖对胰岛素抵抗HepG2细胞糖代谢功能的影响

目的：探讨甜玉米芯多糖（sweet corncob polysaccharide，SCP）组分SCP-80-I对胰岛素抵抗HepG2（insulin resistant HepG2，IR-HepG2）细胞糖代谢功能的影响。方法：确立IR-HepG2细胞模型建立的最佳条件，并由此建立IR-HepG2细胞模型；分别以50、100、200、400 μg/mL剂量的SCP-80-I孵育细胞24 h，评价其对细胞葡萄糖消耗的影响，同时利用噻唑蓝法评价其细胞毒性；检测氧化应激标志物超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）及活性氧（reactive oxygen species，ROS）的水平，测定细胞内糖原积累水平及糖酵解关键限速酶己糖激酶（hexokinase，HK）、丙酮酸激酶（pyruvate kinase，PK）的活力。结果：确立以1×10－6 mol/L胰岛素处理24 h作为诱导IR-HepG2细胞形成的最佳条件，并成功建立IR-HepG2细胞模型；在孵育实验中，SCP-80-I能极显著增加IR-HepG2细胞的葡萄糖摄取量（P＜0.01），细胞活力随SCP-80-I质量浓度的增加呈先上升后下降的趋势；200 μg/mL SCP-80-I处理组与模型对照组相比，胞内MDA含量极显著下降，SOD活力极显著提高，ROS水平极显著下降（P＜0.01）；胞内糖原含量极显著增加（P＜0.01），HK与PK活力极显著增加（P＜0.01）。结论：推测SCP-80-I的降血糖功效与其缓解氧化应激所造成的肝脏细胞损伤，改善胰岛素抵抗肝脏细胞的糖代谢功能有关。     

Redirection of carbon flux to lysine in a recombinant of Corynebacterium lactofermentum ATCC 21799 by limited supply of pantothenate

To increase carbon flux to lysine, minimized production of amino acids that are biosynthetically related to lysine, for example, isoleucine and valine, is required. By limiting the supply of pantothenate, the precursor of coenzyme A, the carbon flux was redirected from isoleucine and valine to lysine in the recombinant of Corynebacterium lactofermentum ATCC 21799 containing the plasmid pGC77. The pGC77 contains hom(dr), thrB, and ilvA encoding feedback-deregulated homoserine dehydrogenase, homoserine kinase, and threonine dehydratase, respectively. At 250 microM of isopropyl-beta-d-thiogalactopyranoside, the recombinant (pGC77) produced lysine, valine, and isoleucine. Limiting the supply of pantothenate from 300 microg/l to 30 microg/l resulted in an increase in lysine (from 4.5 to 6.4 g/l) and decreases in valine (from 3.1 to 1.6 g/l) and isoleucine (from 0.9 to 0.3 g/l) production. The concentration of pyruvate was higher and that of acetate lower in the pantothenate-limited culture than in the control, suggesting that the limited supply of pantothenate delayed the conversion of pyruvate to acetyl-CoA. Increased availability of pyruvate by limiting the supply of pantothenate might favor the integration of pyruvate into the lysine branch. The results of this study are useful for the production of lysine with decreased concentrations of byproducts.     

重组谷氨酸棒杆菌全细胞催化一步法合成高果糖浆

高果糖浆是一种可替代蔗糖的甜味剂,在食品和饮料行业应用广泛.该研究实现了经密码子优化的密苏里游动放线菌(Actinoplanes missouriensis)CICIM B0118(A)来源的葡萄糖异构酶在食品安全菌株谷氨酸棒杆菌(Corynebacterium glutamicum)13032中的异源表达,构建了重组...     

乳酸代谢通量调控规律的研究

选取了8株嗜酸乳杆菌进行分批发酵培养,分别测定了它们对数生长期乳酸发酵途径中10种相关酶的活性,分析了乳酸的代谢通量,并首次引入数量遗传学方法利用通径分析研究了酶活性对乳酸通量的直接和间接影响,建立了类似于代谢控制系数的决定系数R2(i)。该系数实际上反映了酶对乳酸通量的控制程度,研究结果显示,丙酮酸激酶(R2(PK)=0.3705)、己糖激酶(R2(HK)=0.3053)、磷酸丙糖异构酶(R2(TPI)=0.2733)和乳酸脱氢酶(R2(LDH)=0.2601)对乳酸通量具有相对较高的决定系数,对乳酸通量起主要控制作用;3-磷酸甘油醛脱氢酶(R2(GAPDH)= -0.1320)的决定系数为负值,对乳酸通量具有较为明显的负控制作用。本文首次应用数量遗传学方法研究相关酶对乳酸代谢通量的控制作用,这将为代谢控制分析提供新的思路。     

Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol.     

枸杞果汁发酵过程复合乳酸菌的实时定量检测

利用植物乳植杆菌（Lactiplantibacillus plantarum）、发酵粘液乳杆菌（Limosilactobacillus fermentum）和瑞士乳杆菌（Lactobacillus helveticus）混合菌种进行枸杞果汁发酵。该研究对乳酸菌亚致死修复液的组成和修复温度进行优化,分别针对3株乳酸菌设计特异性引物,利用叠氮溴化丙锭（PMA）-荧光定量聚合酶链式反应（fqPCR）的方法,实时荧光定量检测枸杞果汁发酵过程中乳酸菌活菌数。结果表明,优化修复液的组成为蛋白胨1 g/L、牛肉浸出粉0.3 g/L、氯化钠0.5 g/L、吐温80 0.10 g/L、丙酮酸钠0.09 g/L、过氧化氢酶0.04 g/L、MgCl2 3 mmol/L、Na2HPO4 1 mmol/L、MnCl2 2 mmol/L和FeCl2 2 mmol/L。在27 ℃条件下培养15 min,乳酸菌亚致死细胞修复率达到97%。利用该方法实现了枸杞果汁发酵过程不同乳酸菌的实时定量检测,为今后枸杞果汁发酵生产过程中的微生物动态监测提供了方法。     

Effects of phosphoglycerate kinase overproduction in Saccharomyces cerevisiae on the physiology and plasmid stability.

In this report the effects of phosphoglycerate kinase (PGK) overproduction on the physiology and plasmid stability in baker's yeast Saccharomyces cerevisiae containing the PGK1 gene on an episomal plasmid are described. This examination reveals that there is a preferred intracellular level for this enzyme, amounting to 10-15% of the total soluble protein. Strains containing the plasmid and the host strain were grown in non-selective batch cultures and continuous culture, under different growth conditions. Plasmid-containing yeast strains stabilize the copy number of the episomal plasmid at a level at which the PGK concentration is about 12%. This stabilization is due to an equilibrium between normal plasmid loss and selective pressure because of advantages resulting from the increased amount of PGK under glucose-limited conditions. During respiro-fermentative growth, PGK-overproducing cells showed an increased respiration rate and decreased fermentative activity, compared to the host strain. The PGK1 gene can be applied as a direct positive selection marker to obtain a high episomal plasmid stability during growth on glucose. The results are consistent with previously reported data on the physiology and gene stability of PGK-overproducing yeast cells that contain multiple copies of the PGK1 gene integrated into the genome.     

镁、钼对野油菜黄单胞菌代谢关键酶的影响

野油菜黄单胞菌(Xanthomonas campestris)是一种利用碳水化合物为主要底物发酵产生酸性胞外杂多糖黄原胶的革兰氏阴性菌。本实验研究了通过添加不同浓度的Mg2+、MoO2-对野油菜黄单胞菌代谢过程中关键酶:葡萄糖-6-磷酸脱氢酶,丙酮酸激酶在不同发酵时期的活性变化。结果表明,Mg2+添加量为1ml即在发酵液中为0.5g/50ml时葡萄糖-6-磷酸脱氢酶活性明显提高,MoO2-在发酵液中浓度为0.20g/50ml时丙酮酸激酶活性提高,表明适量的镁、钼对上述酶活性有调控作用。     

The influence of oxygen on the differentiation of temperature-sensitive and temperature-insensitive Lactococcus lactis subsp. cremoris starter strains

The differentiation of temperature-insensitive and temperature-sensitive strains of Lactococcus lactis subsp. cremoris by using a modified sodium-β-glycerophosphate/milk medium is described (temperature-insensitive strains are defined as those that continue to grow at 38°C and temperature-sensitive strains as those that do not grow, or grow poorly, at 38°C). The physiological basis for the differentiation assay was examined by using L. lactis subsp. cremoris strains 2146 (temperature-insensitive) and 2182 (temperature-sensitive) as test strains. After aerobic incubation on the medium, strain 2146 formed uniform colonies, 0·5 mm in diameter, while strain 2182 formed larger colonies, 1·0–1·5 mm in diameter. The differential was dependent on the medium constituents, on an aerobic gas phase, and on the effects of H₂O₂ generated within the colonies. The addition of 0·5% (w/v) pyruvate to the medium facilitated the growth of colonies of strain 2146 to 3-mm diameter, while the colony size of strain 2182 remained at 1-mm diameter, and thus the colony-size differential between strains was reversed. The growth of both strains was inhibited at 0·4–0·8% air saturation during suspension culture in sodium-β-glycerophosphate/milk medium. The inclusion of catalase in the cultures overcame the growth inhibition. There was no observable difference between the two strains in their oxygen sensitivity or NADH oxidase/peroxidase enzymology.