budR表达水平对克雷伯氏菌甘油代谢的影响

通过过量表达和反义RNA抑制改变budR表达水平,考察budR表达水平对克雷伯氏菌甘油代谢的影响。过表达budR使得Bud B和Bud C的酶活提高了48.7%和69.7%,1,3-丙二醇产量降低了12.6%,2,3-丁二醇含量增加了73.3%,同时乙酸含量显著降低。反义RNA抑制budR表达后,Bud B和Bud C酶活分别降低了56%和78%,重组菌的生物量、2,3-丁二醇(BDO)、乙酸、乳酸等均表现为略有降低,1,3-丙二醇含量达到23.4g/L,提高约10%。上述结果进一步丰富了budR基因在调控bud操纵子的表达水平、2,3-丁二醇合成及克雷伯氏菌甘油代谢中的作用,为后续代谢改造克雷伯氏菌合成1,3-丙二醇提供了新的思路。     

生物催化合成1,3-丙二醇工艺的研究

以甘油为底物,利用克雷伯氏菌发酵生产1,3-丙二醇的实验中,考察了初始甘油浓度、温度、pH、通气策略等发酵条件对1,3-PDO产量的影响。实验结果表明：积累1,3-PDO的适合条件为：甘油的初始浓度为40g/L、发酵温度37℃、pH7.0、0.5V/V·min的通气量,发酵30h,反应液中PDO的产量可达57.63g/L。     

磷酸转移酶系统关键基因敲除对Klebsiella pneumoniae产1,3-丙二醇的影响

克雷伯氏菌(Klebsiella pneumoniae)在以葡萄糖作辅底物发酵甘油生产1,3-丙二醇(1,3-propanediol,1,3-PDO)过程中,由于细胞存在碳分解代谢抑制现象,葡萄糖优先于甘油被菌体吸收代谢用于细胞生长及副产物的累积,影响1,3-PDO的合成。基于K.pneumoniae葡萄糖转运及碳代谢调控相关的磷酸转移酶系统(phosphotransferase system,PTS),利用Red重组技术对PTS系统中与葡萄糖特异性转运相关的基因ptsG(编码葡萄糖特异性转运膜透性酶EⅡBC~(Glc))、crr(编码胞浆可溶性葡萄糖特异性转运酶EⅡAGlc)分别进行敲除,并考察上述基因缺失对细胞生长、1,3-PDO合成和副产物代谢的影响。结果显示,敲除ptsG、crr基因后,甘油转化率较野生菌分别提高26.2%和42.7%,其中突变株K.pneumoniaeΔcrr的1,3-PDO的产量达到23.1 g/L,提高35.8%。上述结果表明,敲除ptsG、crr基因改造PTS系统能够有效提高底物甘油利用率,强化1,3-PDO合成。     

发酵后期补加2种氮源对克雷伯氏菌合成1,3-丙二醇的影响

为探寻解决重要平台化合物1,3-丙二醇发酵后期菌体生长和1,3-丙二醇合成受限的方法,通过从菌体量、产物和关键酶活性及基因转录水平等方面,较全面地考察了发酵后期补加酵母膏和硫酸铵对克雷伯氏菌(Klebsiella pneumoniae)合成1,3-丙二醇的影响,结果为:添加两种氮源均有利于菌体生长;补加10 g/L酵母膏和硫酸铵,1,3-丙二醇产量由58.6 g/L分别提高到70.6 g/L和77.2 g/L;相对于酵母膏,硫酸铵对关键酶活性的增强更为明显,并使甘油脱氢酶(glycerol dehydrogenase,Dha D)在发酵后期始终维持较高水平,促进细胞生长和产物合成。此外,与补加酵母膏相比,补加硫酸铵后关键酶基因转录水平上调并不显著。表明硫酸铵主要通过直接激活关键酶活性促进细胞生长和产物合成。综上所述,发酵后期补加硫酸铵更利于1,3-丙二醇的生物合成,是提高发酵合成1,3-丙二醇水平的有效方式之一。     

1,3-丙二醇氧化还原酶同功酶基因yqhD在肺炎克雷伯氏菌中的表达研究

以大肠杆菌(Escherichia coli)基因组为模板,PCR扩增出1,3-丙二醇氧化还原酶同功酶基因yqhD,经测序确认,将yqhD基因与四环素抗性基因TetR同时插入表达载体pUC18构建重组质粒pUC18-yqhD-TetR,重组质粒转化Klebsiella pneumoniae ME308;37℃,经1.0mmol/LIPTG诱导8h,重组肺炎克雷伯氏菌中1,3-丙二醇氧化还原酶同功酶的酶活力达到4.16U/mg,而对照菌株的酶活仅为0.62U/mg;将重组菌在摇瓶中进行微氧发酵培养,经IPTG诱导,可将60g/L甘油转化为33.8g/L1,3-丙二醇。     

克雷伯氏菌产1,3-丙二醇aldH基因缺失菌株的构建

该研究利用λRed重组技术对克雷伯氏菌（Klebsiella pneumonia）中的乙醛脱氢酶（aldH）基因进行敲除,成功构建缺失菌株K.pneumonia2-1ΔaldH,与原始菌株相比,缺失菌株K.pneumonia2-1ΔaldH发酵液中乙醇产量由原来的7.24 g/L降为0.47 g/L,降低了93.51%;1,3-丙二醇产量由原来的78.83 g/L增长至82.55 g/L,提高了4.72.%;甘油转化率由60.64%增长至63.50%,提高了2.86%。缺失菌株K.pneumonia2-1ΔaldH 50 L发酵罐小试试验中,1,3-丙二醇的产量为78.13 g/L,乙醇产量为0.50 g/L。     

固定化重组大肠杆菌产1,3-丙二醇发酵条件的研究

利用海藻酸钙将重组大肠杆菌JM109(pHsh-dhaB-dhaG-dhaF-yqhD)固定化,并对影响重组菌固定化细胞发酵的营养因子进行研究。实验结果表明,该重组菌固定化细胞发酵的适宜培养基组成为:甘油80 g/L、酵母膏5.0 g/L、VB120.05 g/L以及KH2PO47.5 g/L;在此培养条件下,1,3-丙二醇产量、转化率及生产能力分别可达61.5 g/L、76.8%和2.57 g(L.h),与游离细胞相比,重组菌固定化细胞对底物甘油和产物1,3-丙二醇的耐受力明显提高,且1,3-丙二醇产量明显提高。     

用克雷伯氏菌批式流加发酵法生产1,3-丙二醇

通过对克雷伯氏菌在 7L发酵罐中厌氧间歇发酵甘油生产 1,3 丙二醇的实验研究 ,建立了一种与 pH调节相偶联的批式流加甘油发酵策略。考察了不同甘油维持浓度条件下的流加方式及不同培养方式对 1,3 丙二醇产率的影响。结果表明 ,甘油质量分数维持在 2 %的流加方式有利于 1,3 丙二醇的发酵生产 ,其在 30 5h内消耗甘油 2 80 g ,得到 1,3 丙二醇152 6 g ,摩尔转化率 6 5 5% ,生产强度 0 91g/L·h     

产1,3-丙二醇菌株的筛选、改良及发酵

从自然界筛选得到了9株可发酵甘油生产1,3 丙二醇的肠道细菌,经鉴定均为肺炎克雷伯氏菌.经化学诱变,菌株的1,3 丙二醇产量有所提高,其最终产量达到2%左右.     

底物流加策略对发酵法生产1,3-丙二醇的影响

研究了以甘油为底物 ,由克雷伯氏菌 (Klebsiellapneumoniae)生产 1 ,3 丙二醇的流加发酵中底物的不同流加策略 (如甘油反馈连续流加、脉冲流加、恒速流加、甘油 碱耦合流加、改进的甘油 碱耦合流加 )对发酵的影响。研究表明 ,采用改进的甘油 碱耦合流加策略 ,即甘油 碱耦合流加同甘油恒速流加相结合的流加策略 ,能够有效地控制发酵体系中甘油浓度 ,当发酵进行至 5 0h时 ,1 ,3 丙二醇浓度可达 5 4g/L ,生产强度为 1 0 8g/(L·h) ,明显优于其他流加策略下 1 ,3 丙二醇的发酵结果。     

无机盐和碳氮源对噬夏孢欧文氏菌积累类胡萝卜素的影响

供试的8种无机盐中,MgSO4的单因子效应最好,可促进噬夏孢欧文氏菌（Erwinia uredovora）的生长和类胡萝卜素合成。在供试的4种碳源中,葡萄糖是最适碳源。噬夏孢欧文氏菌不能利用无机氮源,在供试的有机氮源中以酵母膏最有利于类胡萝卜素合成。培养基中的含碳量保持在2．45～4．90g/L,含氮量保持在1．60～2．40g/L时,对噬夏孢欧文氏菌生长和类胡萝卜素合成均为有利,培养基的最适C:N为2．5:1。类胡萝卜素稳定性的初步实验证明,所得到的类胡萝卜素丙酮溶液在常温下稳定性较差,容易分解。高效液相色谱法（HPLC）测定结果表明,从噬夏孢欧文氏菌细胞得到的天然色素成分比较复杂,在菌体中共检测到11种色素成分。     

C- and N-catabolic utilization of tricarboxylic acid cycle-related amino acids by Scheffersomyces stipitis and other yeasts

Scheffersomyces stipitis and the closely related yeast Candida shehatae assimilated the L-amino acids glutamate, aspartate and proline as both carbon and nitrogen sole sources. We also found this rarely investigated ability in ascomycetous species such as Candida glabrata, C. reukaufii, C. utilis, Debaryomyces hansenii, Kluyveromyces lactis, K. marxianus, Candida albicans, L. elongisporus, Meyerozyma guilliermondii, C. maltosa, Pichia capsulata and Yarrowia lipolytica and in basidiomycetous species such as Rhodotorula rubra and Trichosporon beigelii. Glutamate was a very efficient carbon source for Sc. stipitis, which enabled a high biomass yield/mole, although the growth rate was lower when compared to growth on glucose medium. The cells secreted waste ammonium during growth on glutamate alone. In Sc. stipitis cultures grown in glucose medium containing glutamate as the nitrogen source the biomass yield was maximal, and ethanol concentration and specific ethanol formation rate were significantly higher than in glucose medium containing ammonium as the nitrogen source. Mainly C-assimilation of glutamate but also N-assimilation in glucose-containing medium correlated with enhanced activity of the NAD-dependent glutamate dehydrogenase 2 (GDH2). A Δgdh2 disruptant was unable to utilize glutamate as either a carbon or a nitrogen source; moreover, this disruptant was also unable to utilize aspartate as a carbon source. The mutation was complemented by retransformation of the GDH2 ORF into the Δgdh2 strain. The results show that Gdh2p plays a dual role in Sc. stipitis as both C- and N-catabolic enzyme, which indicates its role as an interface between the carbon and nitrogen metabolism of this yeast.     

Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strain

Saccharomyces cerevisiae SU50 strain was cultivated with different concentrations of glucose and galactose with the aim of increasing cutinase activity, cutinase yield on the carbon source, and bioreactor productivity. Cultivations in shake flasks with galactose as the sole carbon source, with sugar concentrations between 10 and 40 g/l, exhibited growth-associated cutinase production and a constant specificity of cutinase secretion. Furthermore, as the galactose concentration increased to values higher than 15 g/l, a progressively higher maximum specific galactose consumption rate and a consequent higher alcoholic fermentation occurred, resulting in progressively lower biomass yields on the carbon source and cutinase yields on biomass. Using high glucose and galactose concentrations in a well-aerated bioreactor resulted in a high biomass productivity (0.5 g dcw/l/h), a high cutinase yield on biomass (21.5 U/mg dew), a final high cutinase secretion efficiency (97%), and plasmid stability (99%). Based on these studies, a two phase fed-batch cultivation strategy was developed. A batch phase with high glucose and galactose concentrations, followed by a fed-batch with a constant feed rate with galactose as the sole carbon source in order to minimize the repression of the GAL 7 promoter, were established. The feed rate was estimated to maintain a pre-determined concentration of galactose (20 g/l) on the culture medium in order to maximize the efficiency of cutinase secretion and minimize the galactose alcoholic fermentation. By this cultivation strategy, enhancements of 3.6-fold in cutinase activity, 1.2-fold in cutinase yield on the carbon source, and 8.7-fold culture productivity were obtained in relation to a batch cultivation performed in shake flasks with 20 g/l of galactose.     

高温厌氧菌CBZ-S-MEE2利用不同碳源发酵产氢的研究

以从长白山温泉中分离的一株高温厌氧产氢菌CBZ-S-MEE2为菌种,对该菌利用不同碳源发酵产氢进行研究。利用葡萄糖进行发酵产氢时,最大产气速率为180mL(/L.h),产氢效率为1.8mol H2/mol葡萄糖,表明该菌可以快速利用葡萄糖发酵产氢,对葡萄糖的转化效率高。对未经预处理纤维素和以纤维素为主要成分的碳源进行发酵时,该菌在硫元素缺失或pH偏碱性的状态下不能充分的生长,产氢速率低。在改进试验中,通过维持较好的硫元素平衡,以纤维素为唯一碳源发酵时,最大产气速率为77mL(/L.h),产氢效率为7.5mol H2/gVS。表明高温厌氧菌CBS-Z-MEE2可以利用未经预处理的纤维素原料为唯一碳源进行发酵产氢,这为以纤维素为主要成分的生物质发酵产氢奠定了技术基础。     

利用玉米秸秆产油脂木霉菌株的筛选

对52株木霉菌株进行尼罗红染色、菌丝油脂提取和纤维素酶、木质素降解酶活力测定,发现一株哈茨木霉(Trichoderma harzianum)菌株Q2-37为最优菌株.该菌株干菌丝中的油脂含量为(20.5±0.36)％.以玉米秸秆粉为唯一碳源的培养基质上培养Q2-37,每利用100g玉米秸秆粉可以产生(1.188±0.028)g油脂.对受试菌株提取得到的油脂含量进行分析发现,木霉干菌丝的油脂含量范围在(12.6±0.31)％～(20.5±0.36)％.该研究表明,木霉利用植物秸秆生产油脂为生物柴油提供廉价原料是具有潜力的.     

流加混合碳源的谷氨酸发酵工艺研究

采用糖蜜与葡萄糖的混合液作为流加碳源，对谷氨酸发酵工艺进行了探索。试验采用葡萄糖为基础碳源，以发酵初糖浓度为160g／L的培养基进行谷氨酸发酵，以糖蜜与葡萄糖的混合液为流加碳源，结果表明当糖蜜中还原糖占混合液总还原糖30％时，发酵产酸水平和糖酸转化率分别达到142.2g／L和64．88％，与葡萄糖作为唯一流加碳源的谷氨酸发酵工艺比较，其发酵水平十分相近。但生产谷氨酸的碳源成本可降低5％左右。     

不同碳源培养胶球藻C-169净化糖蜜酵母废水

本项目系统研究了三种碳源(CO_2、醋酸和甘油)添加到预处理后的糖蜜酵母废水,在藻菌共生条件下培养胶球藻C-169的生长情况及强化废水净化效果。结果表明,与通入空气相比,补充2%CO_2,能显著提高细胞的平均比生长速率、生物质产率,分别是通入空气的6.49、10.4倍,显著提高了对脱色废水的总氮、总磷和氨氮去除率(p0.05),但降低了废水COD(化学需氧量)去除率。研究发现胶球藻C-169能够利用甘油作为其生长的碳源,在0～20 g/L甘油浓度时,随着甘油浓度的提高,细胞生长速率增大,总氮、总磷去除率提高,并在20g/L时获得最高生物量干重达到8.80g/L,平均比生长速率是不添加甘油的2.07倍,生物量产率达0.70 g/(L·d)。甘油浓度在15 g/L以上时,总氮、总磷去除率分别高达85%、95%以上,极显著地提高了废水COD(p0.01)。添加1～4g/L醋酸,均会抑制胶球藻C-169的生长,降低废水净化效果。因此在使用C-169净化COD较高的废水时,可添加甘油作为其生长的碳源。     

杏鲍菇液态发酵培养的初步研究

杏鲍菇是一种珍贵的药食皆宜的真菌.为在较短时间内大量获得杏鲍菇菌体,该试验探讨了液态发酵方法制备杏鲍菇.试验以杏鲍菇菌丝体干重为指标,通过单因素试验和正交试验,对杏鲍菇液态发酵培养基和液态发酵条件进行了优化.试验结果表明,杏鲍菇液态发酵的最佳碳源为葡萄糖,最佳氮源为蛋白胨,当培养基配方为葡萄糖30g/L、蛋白胨5g/L、KH2PO40.8g/L、MgSO41.2g/L、VB110mg/L时杏鲍菇菌丝体生物量较高.在摇瓶培养条件中研究了种龄、装液量、初始pH值、接种量、温度、转速、培养时间等对液态发酵杏鲍菇菌体生物量的影响.得出最佳培养条件:种子培养6d,装液量20％ (v/v),接种量为10％ (v/v),培养基的初始pH值为自然,培养温度为25℃,摇瓶转速为140r/min,发酵8d,杏鲍菇菌丝产量可达8.23g/L.     

Transport of glycerol by Pediococcus pentosaceus isolated from wine

Pediococcus pentosaceus N(5)p is a strain isolated from wine that uses glycerol as its sole carbon source, mainly via the glycerol kinase pathway. The transport of glycerol was investigated in resting cells of this strain. Glycerol uptake followed a Michaelis-Menten relationship with an observed apparent K(m) of 33 microM and a V(max) of 2.5 nmol/min/mg of cell protein. The transport system was specific for glycerol, which was present in the cells grown either on glycerol or glucose suggesting its constitutive nature. The presence of uptake when resting cells were treated with HgCl(2) and the absence of counterflow indicate that facilitated diffusion is not involved in glycerol transport. On the other hand, glycerol uptake was inhibited by the metabolic poisons that affect ATP availability by acting on either electron transport or ATPase activity, and by the proton-conducting uncouplers without any effect on glycerol kinase activity. The restoration of glycerol uptake in de-energized cells by the addition of glucose and low concentration of cyanide-m-chlorophenyl hydrazone was achieved. These results, the first in the genus Pediococcus, provide evidence for an energy-dependent uptake of glycerol that involves the proton motive force directly or coupled with ATP synthesis.     

Growth and energy generation by Enterococcus faecium FAIR-E 198 during citrate metabolism

Citrate metabolism by Enterococcus faecium FAIR-E 198, isolated from Greek Feta cheese, was studied in various growth media containing citrate either in the presence of glucose, or as the sole carbon source, both under aerobic and anaerobic conditions. In de Man-Rogosa-Sharpe (MRS) broth with increasing citrate concentrations, cometabolism of citrate and glucose took place. Glucose was stoichiometrically converted into lactate, while citrate into acetate. Glucose consumption and biomass yield were enhanced with increasing initial citrate concentrations, even though maximum specific growth rate was not. When citrate was used as the sole carbon source in increasing initial concentrations, the main end product was acetate. Small amounts of lactate, formate, ethanol, and acetoin were also produced. In all cases, no significant differences were observed between aerobic and anaerobic conditions. However, when citrate was used as sole carbon source, formate production was favored in the absence of oxygen. The present work shows that E. faecium is able to utilize citrate in synthetic media, either in the presence of glucose or as the sole carbon source, resulting in energy production and the formation of aroma compounds.