高产紫杉醇菌株的诱变选育及其差异表达基因消减cDNA文库的构建

[目的]选育高产紫杉醇菌株,并构建选育到的高产紫杉醇菌株与出发菌株HD1-3差异表达的cDNA消减文库.[方法]分别采用硫酸二乙酯和紫外线与硫酸二乙酯复合诱变处理菌株HD1-3孢子;以选育到的高产紫杉醇菌株为tester,菌株HD1-3为driver,应用抑制性消减杂交技术构建选育到的高产紫杉醇菌株与菌株HD1-3差异表达的cDNA消减文库.[结果]试验确定的Nodulisporium sylviforme紫杉醇产生菌HD1-3孢子复合诱变的适宜条件为:将106cfu/mL孢子悬液经过8%硫酸二乙酯处理15 min后,在电磁搅拌下,用紫外灯(30 w,距离30 cm)照射处理45 s,获得了1株遗传性状稳定、高产紫杉醇的突变株--UD14-11,其紫杉醇产量从出发菌株HD1-3的232.73±4.61μg/L提高至312.81±7.51μg/L;构建的文库滴度为1.2×107cfu/mL,阳性克隆率75.3%,片段大小主要集中在300 bp-1.0kb.[结论]选育到了1株遗传性状稳定、高产紫杉醇突变株;成功地构建了高产紫杉醇菌株UD14-11与菌株HD1-3差异表达的cDNA消减文库,为寻找、分离微生物生物合成紫杉醇相关基因和利用基因工程或代谢工程手段定向设计改造菌株奠定基础.     

产紫杉醇菌株原生质体诱变育种的研究

对紫杉醇产生菌NCEU-1的原生质体进行了紫外线和氯化锂复合诱变,筛选制霉菌素抗性突变株,共筛选出了4株正突变株。经发酵筛选试验,获得了一株遗传性状稳定、高产紫杉醇的原生质体诱变菌株——UL_04-5,其紫杉醇产量从出发菌株的314.07μg/L提高至418.24μg/L。     

提高展青霉(Penicillium patulum)产灰黄霉素效价的诱变育种研究

为进一步筛选高产灰黄霉素的工业生产菌株,分别对前期采用紫外线-氯化锂（UV-LiCl）、半导体激光（LD laser）及CO2激光（CO2laser）对展青霉FS80-1复合诱变获得三株高产菌株进行液体发酵和固体培养比较。结果表明,通过UV-LiCl复合诱变获得突变菌株GM120-43的液体发酵产灰黄霉素效价11 982μg/mL,比出发菌株提高37.52%,固体培养效价为89 496μg/g（干重）,比出发菌株提高80.04%。;半导体激光诱变获得突变株LD100-1的液体发酵效价9 440μg/mL,固体培养效价119 766μg/g干重,比出发菌株FS80-1提高了140%;两个突变株的生物学特性均发生不同程度的变化,突菌株GM120-43适合于液体发酵生产,突变株LD100-1适合于固体发酵培养。     

紫杉醇高产菌株的原生质体诱变选育及其遗传变异初探

以紫杉醇产生菌NCEU_1为出发菌株,分别采用紫外线和紫外线与氯化锂复合诱变方法对其原生质体进行诱变,获得了两株高产紫杉醇的突变株———UV4 0 - 1 9和UL50 - 6 ,其紫杉醇产量从出发菌株的314 0 7μg L分别提高至376 38μg L和392 6 3μg L ;同时,又采用RAPD和同工酶技术对出发菌株NCEU_1与两高产株UV4 0 - 1 9和UL50 - 6 间的遗传差异进行了研究。结果表明,出发菌株与诱变菌株之间以及两诱变菌株之间都存在明显差异,为进一步研究与紫杉醇合成相关基因及诱变株产量提高的分子机制奠定了基础     

常压室温等离子体(ARTP)诱变快速选育高产DHA的裂殖壶菌突变株

旨在建立一种能够快速便捷的诱变选育高产DHA菌株的方法。出发菌株Schizochytrium sp.ATCC 20888悬浮液经过常压室温等离子体(ARTP)处理后,涂布到2,2’-联吡啶平上板培养。将所得的突变菌株摇瓶发酵培养,通过磷酸香草醛油脂快速检测法和气相色谱分析从突变菌株中筛选得到DHA高产菌株。结果表明,裂殖壶菌诱变选育条件为ARTP为处理时间15 s,气量10 L/min,电功率100 W;2,2’-联吡啶浓度为100μmol/L。通过该方法可以获得高产DHA的菌株。其中D32菌株DHA生产能力提升显著,比初始菌株提升了29.8%,DHA产量达到7.31g/L。D32菌株与出发菌株相比,主要的饱和脂肪酸含量显著下降(P0.005),而不饱和脂肪酸含量显著增加(P0.005)。经5次传代后性状稳定,本方法快捷高效,同时也为其他多不饱和脂肪的诱变选育方法提供参考。     

激光与抗性突变筛选技术相结合选育抗肿瘤抗生素博安霉素高产菌株

目的:获得博安霉素高产菌株,同时比较了铜蒸汽激光与妥布霉素抗性及二者复合诱变的选育效果。方法:采用铜蒸汽激光辐照30 min与妥布霉素100 r/m L抗性处理及其复合诱变选育博安霉素产生菌轮枝链霉菌(S.verticillus)B-31。结果:在复合诱变组中,获得一株高产突变株GB-160,经发酵罐应用后,发酵单位较出发菌株提高1.5倍,并且遗传性能稳定。结论:该方法能有效获得抗生素高产优质菌株。在医药生物工程中,具有较高的实用价值,为其它药物微生物选育提供借鉴。     

阿魏菇多糖高产菌的离子束和激光复合诱变育种

目的:诱变筛选阿魏菇多糖高产菌,探索食用菌的离子束和激光复合诱变育种方法。方法:尝试用阿魏菇菌丝单细胞为靶材,以离子束注入和激光辐照为复合诱变手段,采用营养缺陷型筛选办法定性初筛,摇瓶发酵定量复筛。结果:通过离子束注入诱变,获得了2株菌丝体多糖产量分别达到551.80mg/L和659.46mg/L、较1号出发菌株提高了46.5%和75.2%的多糖高产菌PFPH-1和PFPH-2;在此基础上,以激光辐照为复合诱变手段,获得了1株菌丝体多糖产量达到762.50 mg/L、较出发菌株PFPH-2提高了15.63%的多糖高产菌PFPH-3。结论:离子束和激光复合诱变育种方法在改良阿魏菇多糖高产性状方面诱变功效显著。     

L-异亮氨酸高产菌选育及其培养基优化

旨在诱变选育L-异亮氨酸高产菌,并探索突变株最佳发酵条件。利用传统化学诱变结合常压室温等离子体生物诱变体系对实验室保藏的Brevibacterium flavum I-12进行逐级诱变,选育2-噻唑丙氨酸(2-TA)和磺胺胍(SG)高抗性和在琥珀酸平板上能快速生长的突变菌株。随后,在单因素实验的基础上,利用响应面设计优化出目的突变株摇瓶发酵培养基组分的最佳参数水平。结果显示,经过一系列诱变和筛选,成功选育出一株在40 g/L的2-TA和5 g/L的SG,且以琥珀酸为唯一碳源的培养基上快速生长突变株,命名为B. flavum TA-6,该菌株产酸达26.2±0.5 g/L,比出发菌株提高了44.75%,而副产物L-缬氨酸和L-亮氨酸积累量明显降低。经响应面法优化发酵条件后,突变株产酸可达27.8±0.5 g/L,比优化前提高了6.1%。通过传统化学诱变结合ARTP生物诱变体系,成功选育出一株杂酸降低的L-异亮氨酸高产菌TA-6,该菌株具有潜在生产应用价值。     

高产DHA破囊壶菌Aurantiochytrium sp. PKU#SW7诱变株的筛选

【目的】对野生菌株Aurantiochytrium sp.PKU#SW7诱变育种,筛选高产DHA突变株。【方法】采用UV诱变和化学药物胁迫筛选方式,以菌株的生物量、油脂产量、DHA产量作为筛选指标,获得高产DHA突变株。【结果】经鉴定获得一株DHA高产突变株PKU#PM003,该菌株传代4次后仍保持较好的遗传稳定性。摇瓶发酵后,PKU#PM003生物量产量高达6.62 g/L,比原始菌株5.95 g/L提高了11.26%,脂肪酸含量高达4.01 g/L,比原始菌株3.18 g/L提高了26.1%,DHA在脂肪酸中所占比例由29.97%增加到33.43%,产量提高了41.01%,油脂突变效果显著。【结论】突变株PKU#PM003可作为性状优良的工业化发酵生产菌种,并在DHA产量提升上仍具有巨大的空间。     

具有双重抗生素抗性的ε-聚赖氨酸高产菌株选育及生理特性

以ε-聚赖氨酸产量为1.60g/L的Streptomyces albulus M-Z18为出发菌株,利用核糖体工程技术选育具有双重抗生素抗性的ε-聚赖氨酸高产菌株,并对高产菌株和出发菌株的生理生化性能进行比较。通过链霉素诱变成功选育出了1株遗传稳定的ε-聚赖氨酸产生菌S.albulus S-7,ε-聚赖氨酸产量为2.03g/L;对S.albulus S-7叠加巴龙霉素,获得1株遗传稳定的具有双重抗性的ε-聚赖氨酸产生菌S.albulus SP-14,ε-聚赖氨酸产量为2.37g/L,比出发菌株S.albulus M-Z18的ε-聚赖氨酸产量增加了48.10%。使用链霉素和巴龙霉素选育具有双重抗生素抗性的ε-聚赖氨酸高产菌株是一种有效的手段。     

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%。     

Screening the Fusarium graminearum inhibitory mutant strain from Bacillus subtilis by atmospheric-pressure plasma jet

Aims:  The aim of this study was to improve the antagonistic activity of Bacillus subtilis JA towards Fusarium graminearum by screening high-yielding mutant using the atmospheric-pressure plasma jet (APPJ).
Methods and Results:  Atmospheric-pressure plasma jet was applied as mutagenic source for breeding high-yielding mutant strain. Helium was used as APPJ operating gas. The mutation effects of different treatment times of APPJ were studied. The mutant strain designated as B. subtilis B06 was successfully screened out, which showed higher antagonistic activity against F. graminearum in vitro . Its inhibition zone against the indicator fungus increased by 23% compared to the original one. HPLC and ESI (electrospray ionization) mass spectrometry analysis indicated that antifungal compounds produced by the mutant and original strain belonged to the lipopeptide, surfactin and iturin families. The mutant strain showed favourable properties of faster growth in the fermentation process and higher production of antibiotics. The lipopeptide production of the mutant was 2·3-fold as that of the original strain.
Conclusions:  A mutant strain with strong antagonistic activity and high yielding of antibiotics was obtained by APPJ in this study. The mutant could be used as a promising biocontrol agent in agriculture.
Significance and Impact of Study:  This study provides a novel mutagenic source for breeding high-yielding microbial mutant, which would be very useful in the application of some valuable metabolites from micro-organism.     

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 %).     

培养条件对产油微生物生长的影响

为了筛选出高产油菌株, 首先采用细胞形态学方法与细胞化学方法(苏丹III染色法)对4株高产油脂菌株进行初筛, 并通过索氏提取法对初筛菌株油脂含量进行分析, 确定M2菌株为实验菌株, 其油脂含量达53.09%。为了增加产油微生物油脂产量, 本试验考察了不同发酵条件对其细胞生长和油脂积累的影响。优化工艺参数为: 10° Bx玉米皮渣水解液为培养基质, 0.2% NaNO3为氮源, pH 6.0、28oC下发酵培养6 d, 微生物油脂含量75.21%, 菌体生物量30.40 g/L, 油脂产量22.86 g/L。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 主要含有16碳和18碳系脂肪酸, 可作为生物柴油的原料, 不饱和脂肪酸含量达68%, 可应用于医药化工领域。     

Identification and characterization of a Na(+)-independent neutral amino acid transporter that associates with the 4F2 heavy chain and exhibits substrate selectivity for small neutral D- and L-amino acids

A cDNA was isolated from the mouse brain that encodes a novel Na(+)-independent neutral amino acid transporter. The encoded protein, designated as Asc-1 (asc-type amino acid transporter 1), was found to be structurally related to recently identified mammalian amino acid transporters for the transport systems L, y(+)L, x(C)(-), and b(0,+), which are linked, via a disulfide bond, to the type II membrane glycoproteins, 4F2 heavy chain (4F2hc), or rBAT (related to b(0,+) amino acid transporter). Asc-1 required 4F2hc for its functional expression. In Western blot analysis in the nonreducing condition, a 118-kDa band, which seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc, was detected in the mouse brain. The band shifted to 33 kDa in the reducing condition, confirming that Asc-1 and 4F2hc are linked via a disulfide bond. Asc-1-mediated transport was not dependent on the presence of Na(+) or Cl(-). Although Asc-1 showed a high sequence homology (66% identity at the amino acid level) to the Na(+)-independent broad scope neutral amino acid transporter LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y. (1999) J. Biol. Chem. 274, 19745-19751), Asc-1 also exhibited distinctive substrate selectivity and transport properties. Asc-1 preferred small neutral amino acids such as Gly, L-Ala, L-Ser, L-Thr, and L-Cys, and alpha-aminoisobutyric acid as substrates. Asc-1 also transported D-isomers of the small neutral amino acids, in particular D-Ser, a putative endogenous modulator of N-methyl-D-aspartate-type glutamate receptors, with high affinity. Asc-1 operated preferentially, although not exclusively, in an exchange mode. Asc-1 mRNA was detected in the brain, lung, small intestine, and placenta. The functional properties of Asc-1 seem to be consistent with those of a transporter subserving the Na(+)-independent small neutral amino acid transport system asc.     

复合诱变被孢霉原生质体获高产γ-亚麻酸的研究

以拉曼被孢霉（Mortierella remanniana）为出发菌株M₅,通过原生质体制备,经亚硝酸和激光等进行复合诱变;进行初筛,经摇瓶发酵法复筛,并测定其相关性能指标,获得一生产性能比出发菌株显著提高的突变株M_?;其干菌体收率为46.8g/L、油脂产率达34.6g/L、γ-亚麻酸的产率达12.5g/L,分别是出发菌株的1.73倍、2.02倍和2.6倍。通过基因传代实验,说明突变株的基因可稳定遗传。     

常压室温等离子体快速诱变产油酵母的条件及其突变株的特性

采用新型常压室温等离子体射流诱变产油酵母,结合快速突变产油酵母操作条件及基于96孔板的高通量筛选手段,获得了一系列增殖速度和产油量发生变化的突变株。在等离子体对菌株致死率为99％的条件下获得的以突变株增殖速度为指标的正突变率达到27.2％。用含酵母粉 (10 g/L)、蛋白胨 (10 g/L) 及葡萄糖 (20 g/L) 的酵母膏胨葡萄糖培养基进行发酵实验表明,筛选得到的高产突变体产油量从对照株的1.87％ (W/W) 增加到4.07％ (W/W)。     

高浓度丁醇浸泡及N＋束注入诱变双重作用筛选丁醇高产菌

通过高浓度丁醇浸泡处理丙酮丁醇梭菌（Clostridiumacetobutylicum）CL-2,筛选得到一株丁醇耐受能力提高并溶剂产量增加的菌株BR30—2,丁醇产量达11．77g/L,比CL-2提高了16．65％。以BR30—2作为出发菌株,进行N＋束注入诱变,筛选得到高产菌株BH．9,丁醇产量达14．5g/L,总溶剂为23．14g／L。在BH-9发酵过程中添加0．1％丁酸钠,丁醇产量达到16．59g/L,丁醇比例提高至67．38％。     

高产γ-氨基丁酸低产桔霉素红曲霉(Monascus ruber)突变子1047筛选与发酵特性研究

以红曲米中筛选到的红色红曲霉菌株G为原始菌株,通过农杆菌介导T-DNA插入突变技术,成功构建了含有1 483株红曲霉突变子的T-DNA插入突变库。用HPLC等方法从突变库中筛选出10株γ-氨基丁酸(GABA)产量稳定高于原始菌株G的突变子,薄层层析结合HPLC技术分析了10株突变子发酵液中桔霉素的含量;其中突变子1047的GABA产量较高,为1.169g/L,是原始菌株G(GABA产量0.472g/L)的247.67%,桔霉素含量稳定低于原始菌株G。以红曲霉菌株G和突变子1047为实验材料,通过5 L发酵罐发酵并定时取样,HPLC等方法精确分析发酵液各种活性物质的含量;结果显示,突变子1047生长速度稍快于原始菌株G;GABA、莫纳可林K(Monacolin K)、红曲色素色价分别为2.201 g/L、83.892 mg/L、21.984 U/mL,是原始菌株G的279.67%、108.01%和182.35%;而桔霉素产量为1.976 mg/L,是原始菌株G的41.71%。因此,利用TDNA插入的方法对红曲霉进行育种,能产生稳定的遗传变化,在红曲霉资源的保护和利用上有一定潜力。     

小克银汉霉γ-亚麻酸高产菌株的快速筛选和发酵条件研究

以小克银汉霉C0为出发菌株,经过5-氟尿嘧啶和紫外线复合诱变,采用抗失水苹果酰肼与抗低温(15℃)相结合的筛选方法,获得一株生产性能比出发菌株显著提高的突变株C23。采用5L全自动发酵罐对小克银汉霉C23发酵生产γ-亚麻酸的pH值控制和补料工艺进行研究,发现将发酵液pH值维持在5.5,当发酵进行到60h、84h、108h时,分别补糖15g/L,发酵192h后收获,结果生物量、油脂产量和γ-亚麻酸产量分别达到49.88g/L、21.93g/L、2.69g/L。