一株高产纤维素酶的柄篮状菌EMM的基因改造和培养条件优化

发布时间：2018-07-06 19:36

本文选题：纤维素酶 + 柄蓝状菌　；参考：《武汉工程大学》2016年硕士论文

【摘要】：柄蓝状菌EMM是本实验室从原始菌OPC4诱变而来的一株高产纤维素酶菌,由于其超高的蛋白分泌量和酶活稳定性,在产纤维素酶的生产方面表现出很好的工业化前景。但其β-木糖苷酶的酶活过低,严重影响降解木质纤维素的综合能力,所以本文通过基因工程的方法对其进行改造。由于采用PEG—CaCl2的转化方法,其重点在于原生质的制备与再生。从制备材料,菌龄,渗透压稳定剂,酶的种类,酶的浓度,酶解时间及再生方式对柄蓝状菌EMM原生质的制备与再生进行优化。其最适宜的原生质的制备与再生条件为:以菌龄为48小时的菌丝为材料,在渗透压稳定剂1.2MMgSO_4的条件下,以50mg/mL的裂解酶,温度30度,酶解3h,以先液体培养基孵化过夜再混合倒板的再生方式再生。原生质体的释放量可达8.73±0.8×10~8个/g,再生率可达17.7±1.9%。里氏木霉作为纤维素酶的商业生产菌株,其β-木糖苷酶活较高。从里氏木霉T.reesei Rut-C30和柄蓝状菌EMM里分别克隆出β-木糖苷酶基因,通过同源重组的方式,用添加了PCBH启动子和TCBH终止子的C30β-木糖苷酶基因替换掉原来EMM的β-木糖苷酶基因,来达到提高EMMβ-木糖苷酶活的目的。转化后在含有潮霉素B的PDB培养液连续传代3次后,涂布在潮霉素B平板上筛选得到37个转化子,经PCR验证10个为hph阳性转化子,用同源同组引物鉴定,其中2个为同源同组转化子,其他为随机插入转化子。10个阳性转化子与原始EMM同时发酵取第7天最大酶活检测,对比发现:转化子的滤纸酶活FPA均高于EMM,最高达60%,然而β-木糖苷酶活几乎几乎没有差距。由于以潮霉素B为标记基因选择强度和稳定性不高,导致需要通过含潮霉素B液体培养基传代来筛选阳性转化子,耗时过多效率不高。通过UV诱变的方法,在1.5g/mL 5-FOA的筛选压力下,从EMM诱变得到一株尿嘧啶营养缺陷型EMU6。从OPC4的基因组里克隆出2553bp的pyrF基因,通过NCBI比对发现,pyrF为编码乳清酸磷酸核糖基转移酶的基因,影响尿嘧啶的合成。通过测序对比发现,尿嘧啶营养缺陷型EMU6的pyrF基因在1160bp处有一个核苷酸缺失。为了确认pyrF可以作为一个标记基因,将带有pyrF的质粒pGEF2通过PEG介导的方法转入EMU6中。通过PCR分析,pyrF已经整合到EMU6的基因组当中,使EMM重新获得了合成尿嘧啶的能力。从以上结果可以的出结论,基于pyrF作为标记基因的转化系统已经构建成功。为了构建一个可以重复利用的pyrF标记基因,在pyrF基因的一侧添加了一段490bp的正向重复序列,这样在利用pyrF筛选出转化子后,在5-FOA的选择压力下,一侧的正向重复序列与pyrF中的重复部分发生同源重组而导致pyrF标记自动删除。通过这种策略可以达到pyrF标记基因重复使用的目的,使后面利用EMM作为细胞工厂表达外源基因的工作成为可能。
[Abstract]:Blue stalk bacteria EMM is a cellulase producing strain which was induced from the original strain OPC4 in our laboratory. Because of its high protein secretion and enzyme activity stability, EMM has a good prospect of industrialization in the production of cellulase. However, the enzyme activity of 尾 -xylosidase was too low, which seriously affected the comprehensive ability of degradation of lignocellulose. Therefore, the genetic engineering method was used to modify the 尾 -xylosidase. Due to PEG-CaCl _ 2 transformation, the emphasis is on the preparation and regeneration of protoplasm. The preparation and regeneration of EMM protoplasm were optimized from preparation materials, bacterial age, osmotic pressure stabilizer, enzyme type, enzyme concentration, enzymatic hydrolysis time and regeneration method. The optimum conditions for protoplast preparation and regeneration were as follows: the mycelium of 48 hours was used as the material, the osmotic pressure stabilizer was 1.2 MMgSO4, the lyase was 50 mg / mL, the temperature was 30 degrees. After 3 h enzymatic hydrolysis, regeneration was carried out in liquid medium incubated overnight and then mixed with inverted plate. The release of protoplasts was 8.73 卤0.8 脳 10 ~ 8 / g, and the regeneration rate was 17.7 卤1.9. Trichoderma rii, as a commercial cellulase producing strain, has high 尾-xylosidase activity. The 尾 -xylosidase gene was cloned from T. reesei Rut-C30 and E. solanum EMM, respectively. By homologous recombination, the 尾 -xylosidase gene was replaced by the C30 尾 -xylosidase gene which added PCBH promoter and TCBH Terminator. To increase the activity of EMM 尾-xylosidase. After transformation, 37 transformants were screened on hygromycin B plate after three successive passages in the culture medium containing hygromycin B. 10 transformants were identified as hph positive transformants by PCR, and identified by homologous primers. Two of them were homologous transformants, the other were randomly inserted transformants. Ten positive transformants were fermented with the original EMM at the same time to detect the maximum enzyme activity on the 7th day. The results showed that the filter paper enzyme activity of the transformants was higher than that of EMMs, and the highest was 60%. However, there was almost no difference in 尾 -xylosidase activity. Because the selection intensity and stability of hygromycin B as marker gene is not high, it is necessary to screen positive transformants by subculture of hygromycin B liquid medium, and the time consuming is not high. Under the screening pressure of 1.5 g / mL 5-FOA, a uracil nutrition deficient strain EMU6 was obtained from EMM by UV mutagenesis. The pyrF gene of 2553bp was cloned from the genome of OPC4. By NCBI comparison, it was found that pyrF was a gene encoding whey acid phosphate ribonyltransferase, which affected the synthesis of uracil. By sequencing comparison, it was found that the pyrF gene of EMU6 had a nucleotide deletion at 1160bp. In order to confirm that pyrF can be used as a marker gene, the plasmid pGEF2 containing pyrF was transferred into EMU6 by PEG mediated method. PCR analysis showed that pyrF had been integrated into EMU6 genome, and EMM was able to synthesize uracil. From the above results, we can conclude that the transformation system based on pyrF as a marker gene has been successfully constructed. In order to construct a reusable pyrF marker gene, a forward repeat sequence of 490bp was added to the side of the pyrF gene, so that after screening out the transformant using pyrF, the selection pressure of 5-FOA was applied. The homologous recombination of the forward repeats on one side with the repeats in pyrF leads to the automatic deletion of pyrF tags. This strategy can achieve the purpose of repeated use of pyrF marker genes and make it possible to express foreign genes by using EMM as a cell factory.
【学位授予单位】：武汉工程大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ925

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