原生质体紫外诱变选育可利用廉价碳源的高产油新菌株

【目的】研究并建立利用原生质体紫外诱变技术选育可利用廉价碳源发酵的高产油新菌株的方法。【方法】采用1.5%蜗牛酶和1.0%纤维素酶混合液水解去除细胞壁得到2A00015(近平滑假丝酵母,Candida parapsilosis)的原生质体,将其放于紫外灯下诱变及再生壁培养,筛选获得可利用廉价碳源发酵的高产油酵母,并采用气相色谱质谱联用法(GC-MS)测定其脂肪酸组成。【结果】突变效果最好的突变菌株2A00015/25用葡萄糖发酵培养7 d后,其生物量、油脂产率和产油量分别为17.77 g/L、58.12%和10.32 g/L,较原始菌株分别提高了12.45%、23.32%和38.68%;利用废糖蜜发酵培养,其生物量、油脂产率和产油量分别为18.54 g/L、49.44%和9.17 g/L,较原始菌株分别提高了9.09%、21.16%和32.18%。利用废糖蜜培养其产油效率虽低于利用葡萄糖培养,但从环境保护及原材料成本的角度考虑,用废糖蜜作为碳源发酵培养产生油脂更具优势。诱变菌株利用废糖蜜发酵后产生油脂经检测含有8种脂肪酸,其脂肪酸组成与植物油近似,其中不饱和脂肪酸含量占脂肪酸总量的82.4%。【结论】通过利用原生质体紫外诱变技术,成功选育出一株新的可利用廉价碳源的高产油海洋菌株,产油率达到49.4%,提高了21.2%。     

从芽胞杆菌dhs-330转座突变库筛选获得高产表面活性剂突变株

目的:利用本实验室构建的芽胞杆菌dhs-330的转座突变体库,筛选表面活性剂高产菌株,并获得最佳发酵条件。方法:通过排油圈法初筛和测定表面张力复筛,从500个突变株中筛选出5株表面活性较高的突变株,对获得的5株突变株进行遗传稳定性分析,通过单因素实验对高产表面活性剂的发酵条件进行优化。结果:获得了5株表面活性较高的突变株,传代培养10代后突变株dhs-330-021的遗传稳定性和表面活性均较好,dhs-330-021的最佳培养基为葡萄糖32 g/L、酵母提取物1.2 g/L、尿素5.0 g/L、KH_2PO_4 0.8 g/L、Na_2HPO_4·12H_2O 3 g/L、MgSO_4·7H_2O 0.15g/L、微量元素溶液1 m L/L,发酵液稀释至1/10后的表面张力降低到30.392 mN/m。结论:运用转座突变技术进行高产表面活性剂菌株的选育,可获得遗传稳定的高产菌株。     

利用转座标签mTn-lacZ/leu2插入突变发酵性丝孢酵母2.1368-Leu?筛选高效产油突变株

为提高微生物油脂产率,降低其生产成本,以转座标签mTn-lacZ/leu2插入突变发酵性丝孢酵母2.1368-Leu?筛选高效产油突变株。利用LacZ显色反应、脂肪酸合成酶抑制剂Cerulenin和磷酸香草醛反应,最终在玉米秸秆糖化液中筛选出一株高效产油突变株2.1368-Leu?-7。结果表明其油脂含量为38.30％,比对照的29.33％高了8.97％,而其产油率为8.35％,比对照的6.92％提高了20.63％;在玉米秸秆糖化液中的糖利用率为77％,每100 g玉米秸秆可转化油脂8.32 g。可为未来生物柴油产业提供了廉价原料。     

戊糖乳杆菌发酵产乳酸及高产菌株诱变选育

戊糖乳杆菌(Lactobacillus pentosus)是能利用木质纤维素水解液发酵产乳酸的潜力菌株,发酵条件优化与高产菌株的选育是提高乳酸产量的重要手段。通过单因素试验、Plackett-Burman设计与响应面试验,对戊糖乳杆菌ATCC 8041产乳酸的发酵培养基及发酵条件进行了优化。结果表明,该菌株发酵培养基的最佳组合为葡萄糖93.11 g/L、酵母浸粉5.19 g/L、碳酸钙29.43 g/L、蛋白胨10.00 g/L、Na2HPO4·12H2O 5.00 g/L、Mg SO4 0.20 g/L、Mn SO4 50 mg/L;最佳发酵条件为37℃、p H6.5、接种量6%、装液量80%。在此优化条件下,该菌株发酵产乳酸为54.12 g/L。进一步以戊糖乳杆菌ATCC 8041为出发菌株,通过原生质体进行紫外诱变,经多重筛选,最终获得一株遗传稳定性好的高产乳酸突变株,命名为戊糖乳杆菌Lactic UVC-02,由中国典型培养物保藏中心保存,注册号为CCTCC M 2013209。该突变株Lactic UVC-02经葡萄糖发酵,乳酸产量达64.17 g/L,比出发菌株ATCC 8041(54.12g/L)提高18.6%。     

红酵母类胡萝卜素高产菌株的筛选及其发酵生理学条件研究

从数株红酵母中选出了1株产类胡萝卜素能力较强的红酵母RY－98（生物量,类胡萝卜素含量和产量分别为19．9g/L,334.8ug/g和6．7mg/L);研究了该菌株产类胡萝卜素的最适营养与环境条件,获得了最佳的发酵生理学条件;葡萄糖40g/L,(NH4)2SO4 10g/L,酵母膏3g/L,蕃茄汁2mL/L,花生油0.5mL/L,接种量30mLL初始pH6.0和通气量（培养基装置040ml/250mL:,在此初步优化的培养条件下,红酵母RY－98经72小时摇瓶发酵其生物量,类胡萝卜素含量和产量分别可达26．8g/L,386.9ug/g和10．4mg/L,依次比初筛中提高了34．7％,15．6％,和55．2％。     

汉逊德巴利酵母发酵葡萄糖生产D-阿拉伯糖醇

从378株耐高渗酵母中,筛选到1株由葡萄糖发酵高产D-阿拉伯糖醇的酵母。通过生理生化和分子生物学的鉴定,证实该菌株为Debaryomyces hansenii,保藏编号CICIM Y 0504。研究该酵母摇瓶发酵的主要影响因素,确定其摇瓶发酵条件为:葡萄糖200 g/L,酵母膏10 g/L,初始pH值3,装液量20 mL/250 mL,温度30℃。在此条件下发酵120 h,D-阿拉伯糖醇浓度达90.37 g/L,转化率45.18%。在15 L发酵罐对该酵母进行扩大培养,结果表明,初始葡萄糖浓度200 g/L的分批发酵产D-阿拉伯糖醇64.07 g/L,转化率33.94%;葡萄糖浓度控制在30～50 g/L的分批补料发酵产D-阿拉伯糖醇125 g/L,转化率37.5%。研究结果对葡萄糖发酵生产D-阿拉伯糖醇工业化的实现具有重要启示。     

高效利用木糖产油的氮离子注入Mortierella alpina诱变选育研究

木糖的有效利用是木质纤维素全利用的基础。为了获得高效利用木糖产油的高山被孢霉菌株,通过多轮氮离子束诱变,筛选出一株有效利用木糖的产油高山被孢霉1502．8（MortiereUaalpina1502—8,MAIS02．8）,并研究了以葡萄糖／木糖（W／W,5／3）组成的混合糖为碳源,突变株生长和油脂积累的特性。采用单因子和正交实验对培养基组成和发酵条件进行了优化,结果表明,诱变菌在温度28oC,pH8．0,接种量8％,装液量30％,蛋白胨0．76％和豆粕1％,分批补糖的最优发酵条件下发酵9d,生物量和菌体油脂积累量分别达29．8g／L和11．7g／L,较出发菌提高了2．59倍和2．05倍,同时原料糖利用率达到99．4％。     

叶绿素合成缺陷型小球藻突变株的选育及其蛋白产能和品质评价

本研究旨在利用常压室温等离子体(atmospheric pressure room temperature plasma, ARTP)诱变技术构建叶绿素合成缺陷型凯式小球藻突变株,筛选出极低叶绿素、适用于发酵生产蛋白质的新藻种。首先经优化诱变处理时间后建立了野生型兼养细胞的致死率曲线,在高于95%致死率条件下处理对数早期兼养细胞,基于可视化藻落颜色变化初筛获得4株突变株。随后在摇瓶中异养培养突变株,系统评价了蛋白生产性能,发现在含有30 g/L葡萄糖和5 g/L NaNO₃的Basal培养基中,突变株P. ks4表现最优,蛋白含量及产率分别为39.25%干重及1.15g/(L·d),氨基酸评分达101.34,叶绿素a含量下降98.78%且不含叶绿素b,含有叶黄素0.62 mg/g而使藻体呈金黄色。本研究为微藻替代蛋白的发酵生产提供了高性能、高品质的新种质P. ks 4。     

尼罗红染色法筛选产油酵母及定量检测胞内油脂含量的研究

【目的】建立产油酵母筛选以及胞内油脂含量测定的简便方法。【方法】利用尼罗红与胞内油脂成分结合后在紫外光照射下发出荧光且荧光强弱与油脂含量相关的原理。通过在添加尼罗红的培养基中培养酵母,并观察菌落荧光的方法对385株深海酵母进行产油脂菌株筛选,利用26S rDNA D1/D2区序列分析方法对筛选获得的产油酵母菌株进行鉴定,并以其中的一株高产油脂酵母(2A00015)为试验菌株,建立了一套尼罗红染色快速测定油脂含量的方法。【结果】获得22株产油酵母,其中油脂含量最高可达62.9%,经分子鉴定后显示这22株酵母分别属于(Candida viswanathii)、近平滑假丝酵母(Candidaparapsilosis)、粘质红酵母(Rhodotorula mucilaginosa)、汉逊德巴利酵母(Debaryomyceshansenii)、季也蒙毕赤酵母(Pichia guilliermondii)以及Rhodosporidium paludigenum酵母。尼罗红染色快速测定油脂含量方法的最佳检测条件为:菌悬液OD600小于1.2,尼罗红浓度0.5 mg/L,染色时间5 min,激发波长488 nm,发射波长570 nm。该测定方法得到相对荧光强度与称重法得到油脂含量呈正相关性,R2=0.9637。     

红酵母类胡萝卜素形成的生理学研究

从数株红酵母中选出 1株产类胡萝卜素能力较强的红酵母RY 98(生物量、类胡萝卜素含量和产量分别为19.9g/L ,334 .8μg/ g和 6 .7mg/L) ;研究了该菌株产类胡萝卜素的最适营养与环境条件 ,获得了最佳的发酵生理学条件 :葡萄糖 40 g/L ,(NH4 ) 2 SO4 10 g/L ,酵母膏 3g/L ,蕃茄汁 2mL/L ,花生油 0 .5mL/L ,接种量 30mL/L ,初始pH 6 .0和通气量 (培养基装量 ) 4 0mL/ 2 5 0mL。在此初步优化的培养条件下 ,红酵母RY 98经 72h摇瓶发酵其生物量、类胡萝卜素含量和产量分别可达 2 6 .8g/L ,386 .9μg/ g和 10 .4mg/L ,依次比初筛中提高了 34 .7% ,15 .6 %和 5 5 .2 %。     

裂殖壶菌高产油突变体的高通量筛选

二十二碳六烯酸(DHA)具有促进婴幼儿大脑和视网膜发育等多种生理功能,被广泛应用于食品、医药和养殖等行业。为了获得适合于工业化生产的高产油、高产DHA的裂殖壶菌工程株,文中建立了一套操作简单、快速准确的基于尼罗红染色的高通量筛选方案。首先利用紫外线(UVC)诱变的方式快速构建裂殖壶菌的随机突变体库。然后采用优化后的筛选条件如裂殖壶菌的最佳尼罗红染色条件(二甲基亚砜浓度为20%,尼罗红终浓度为2.0μg/mL,孵育时间为10 min,孵育温度为40℃)和更合理的筛选依据(多功能酶标仪实现高通量测量的单位细胞密度油脂量)等,对3 648株突变体进行筛选,得到了3株高产油突变体(D03432、D05106和D01521)。摇瓶发酵实验表明,这3株突变体在生物量、油脂含量和DHA产量上均高于野生型菌株,其中突变体D03432和D05106的油脂量分别达到了干重的64.74%和63.13%,远高于野生型菌株的43.19%。而且这两株突变体的DHA产量分别是野生型菌株的2.26倍和2.37倍。最后,对突变体D03432和D05106进行了5 L发酵罐发酵培养,相较于野生型菌株,这两株突变体不仅生物量和油脂含量有所增加,而且DHA产量更是分别增加了45.5 1%和66.46%,展现出较好的工业应用潜力。此外,本筛选方案对其他产油微生物高产油突变体的高通量筛选具有借鉴作用。     

Citrus residues isolates improve astaxanthin production by Xanthophyllomyces dendrorhous

The wild strain and two astaxanthin-overproducing mutant strains, W618 and GNG274, of Xanthophyllomyces dendrorhous were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium containing (per liter): 2 g KH2PO4, 0.5 g MgSO4, 2 g KNO3, and 1 g yeast extract, and supplemented with citrus residues isolates as a carbon source (citrus medium). The selected strain W618 was evaluated under various contents of citrus juice. At the content of 20% (v/v), the highest astaxanthin production reached 22.63 mg L(-1), which was two-fold more than that observed in yeast malt medium. Addition of 8% (v/v) n-hexadecane to the citrus medium was found to be optimal, increasing the astaxanthin yield by 21.7%.     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

Isolation of a high-specific-growth-rate mutant of Cellulomonas flavigena on sugar cane bagasse

By treatment of a wild-type strain of Cellulomonas flavigena with N-methyl-N'-nitro-N-nitrosoguanidine at 150 g/ml, mutants PN-7 and PN-10 were obtained, which produce 1.38 and 1.5 times more carboxymethylcellulase than the wild strain when cultured in a batch system with sugar cane bagasse as the sole carbon source. These mutants also exhibited higher specific growth rates compared to the wild strain. From a second mutagenesis of mutant PN-10, mutant PN-120 was obtained in continuous culture. This mutant was able to use a larger portion of sugar cane bagasse than did the wild-type and therefore its biomass yield was also higher. The mutant showed a specific growth rate on sugar cane bagasse threefold higher than the wild strain.     

The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.

The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.     

Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single-stage continuous culture.

Lipid accumulation of Candida 107, grown at dilution rates from 0.03 to the maximum of 0.21/h, with carbon, nitrogen, phosphate, and magnesium limitations in a chemostat, was maximal at about 40% (wt/wt) with nitrogen-limited medium at a dilution rate of 0.06/h, giving an efficiency of substrate conversion of 22 g of lipid per g of glucose consumed. At higher dilution rates the lipid content decreased. With carbon-limited growth, the highest lipid content (14%, wt/wt) was at the maximum dilution rate. High lipid contents also occurred with phosphate + nitrogen as double limitations of growth, with the lipid content of the yeast (about 35%, wt/wt) continuing to be near maximum at dilution rates also near maximum (0.17/h), thus giving the highest specific rate of lipid formation of any growth conditions (0.59 g of lipid/g of yeast per h). However, the efficiency of substrate utilization was only 5.2 g of lipid formed per 100 g of glucose consumed. The composition of the fatty acyl residues within the lipid remained constant over many weeks if the steady-state conditions remained unchanged. With carbon-limited growth, the degree of unsaturation of the fatty acids markedly decreased as the dilution rate was increased, but with nitrogen limitation the reverse trend was seen. In all cases, linoleic and oleic acids were the principal fatty acyl residues affected, and their relative proportions always varied in opposite directions. When magnesium was a limiting nutrient, there was a considerable increase in the proportion of myristic acid produced within the lipid. Neutral lipids (predominantly triglycerides) varied from 66 to 92% of the total lipid from carbon- and nitrogen-limited growth; phospholipids (varying from 2 to 25%) were highest in nitrogen-limited growth. The fatty acyl residues within each lipid fraction showed the same variations with changing growth rates.     

联合固氮菌Enterobactergergoviae57—7泌铵突变株的分离和特性

经 Tn5转座子诱变从野生型菌株 ( Enterobacter gergoviae 5 7- 7)筛选到抗甲胺 ( 0 .3mol/L )的泌铵突变株 MG61 ,该突变株在固氮生长时能分泌铵 2 .0 mmol/L。由于泌铵 MG61的生长比野生型菌株慢 ,在培养基中含铵 ( 5 mmol/L或 30 mmol/L)条件下 ,MG61的生长速率与对照相同 ,而远比野生型菌株慢 ,表明 MG 61不能很好地利用铵。在含 2 0 mmol/L铵的培养基中 MG 61仍表达 86%的固氮活性 ,而野生型菌株完全丧失了固氮活性。在谷氨酸存在下 MG61的生长速率及固氮酶活性都比对照高。在硝酸盐存在下 MG61的生长速率与对照相同 ,但泌铵量达 7.8mmol/L。     

The feasibility of growing cells of Saccharomyces cerevisiae for citronellol production in a continuous-closed-gas-loop bioreactor (CCGLB)

The present work aims to address the gas-phase biotransformation of geraniol into citronellol using growing cells of Saccharomyces cerevisiae (baker's yeast) in a continuous-closed-gas-loop bioreactor (CCGLB). This study revealed that the gaseous geraniol had a severe effect on the production of biomass during the growing cell biotransformation resulting in the decrease in the specific growth rate from 0.07 to 0.05 h?1. The rate of reaction of the growing cell biotransformation was strongly affected by agitation and substrate flow rates. The highest citronellol concentration of 1.18 g/L and initial rate of reaction of 7.06 × 10?? g/min g(cell) were obtained at 500 rpm and 8 L/min, respectively.     

Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changes

A stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid (PHA) biosynthesis genes was constructed. This plasmid was transferred to seven Escherichia coli strains (K12, B, W, XL1-Blue, JM109, DH5alpha, and HB101), which were subsequently compared for their ability to synthesize and accumulate ploy- (3-hydroxybutyric acid) (PHB). Growth of recombinant cells and PHB synthesis were investigated in detail in Luria-Bertani (LB) medium containing 20 g/L glucose. Cell growth, the rate of PHB synthesis, the extent of PHB accumulation, the amount of glucose utilized, and the amount of acetate formed varied from one strain to another. XL1-Blue (pSYL105) and B (pSYL105) synthesized PHB at the fastest rate, which was ca. 0.2 g PHB/g true cell mass-h, and produced PHB up to 6-7 g/L. The yields of cell mass, true cell mass, and PHB varied considerably among the strains. The PHB yield of XL1-Blue (pSYL105) in LB plus 20 g/L glucose was as high as 0.369 g PHB/g glucose. Strains W (pSYL105) and K12 (pSYL105) accumulated the least amount of PHB with the lowest PHB yield at the lowest synthesis rate. JM109 (pSYL105) accumulated PHB to the highest extent (85.6%) with relatively low true cell mass (0.77 g/L). Considerable filamentation of cells accumulating PHB was observed for all strains except for K12 and W, which seemed to be due either to the overexpression of the foreign PHA biosynthesis enzymes or to the accumulation of PHB. (c) 1994 John Wiley & Sons, Inc.     

Screening of lipid high producing mutant from rhodotorula glutinis by low ion implantation and study on optimization of fermentation medium

In order to obtain lipid producing strain with high-yield, the wild type stain Rhodotorula glutinis was treated by low ion implantation, and optimization of fermentation medium for higher lipid yield was carried out using mutant strain. It was found that the strain had a higher positive mutation rate when the output power was 10 keV and the dose of N⁺ implantation was 80 × 2.6 × 10¹³ ions/cm². Then a high-yield mutant strain D30 was obtained through cid-heating coupling ultrasonic method and lipid yield was 3.10 g/L. Additionally, the surface response method was used to optimize fermentation medium. The three significant factors (glucose, peptone, KH₂PO₄) were optimized using response surface methodology (RSM), and the optimized parameters of fermentation medium were as follows: glucose 73.40 g/L, peptone 1.06 g/L and KH₂PO₄ 3.56 g/L. Finally the fermentation characteristic of high-yield mutation strain D30 was studied, when fermentation time was 10 days, which lipid yield increased to 7.81 g/L. Fatty acid composition of the lipid was determined by GC, and the most represented fatty acids of mutant D30 were C16:0 (11.4 %), C16:1 (5.66 %), C18:1 (49.3 %), and C18:2 (27.0 %).