L-异亮氨酸高产菌选育及其培养基优化
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摘要
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。
This work aims to breed a strain yielding high L-isoleucine by mutagenesis,and explore the optimal fermentation conditionsfor the mutant strain. The original strain Brevibacterium flavum I-12 was mutagenized by conventional chemical mutagenesis and atmosphericand room temperature plasma(ARTP)biological breeding system. A mutant strain having high resistance to 2-thiazolyl alanine(2-TA)andsulfaguanidine(SG)and capable of rapid growth on a succinic acid plate was selected. Subsequently,the response surface design based onthe single factor experiment was used to optimize the optimal parameter level of the shake flask fermentation medium components for the targetmutant strain. Results showed that a strain producing L-isoleucine was obtained after multiple rounds of random mutagenesis and screening,which was resistant to 40 g/L of 2-TA and 5 g/L of SG and rapidly grew on the medium with succinate as the sole carbon source,and designatedas B. flavum TA-6. The L-isoleucine production by this mutant strain reached 26.2±0.5 g/L,which was 44.75% higher than that by the originalstrain(18.1±0.5 g/L);while the accumulations of side product L-valine and L-leucine significantly reduced. The L-isoleucine productionincreased to 27.8±0.5 g/L after optimizing the fermentation conditions by response surface method,which was 6.1% higher than that beforeoptimization. In conclusion,a strain yielding high L-isoleucine but producing low other acids,TA-6,is obtained by conventional chemicalmutagenesis and ARTP biological breeding system,thus it has potential production and application value in L-isoleucine fermentation industry.
This work aims to breed a strain yielding high L-isoleucine by mutagenesis,and explore the optimal fermentation conditionsfor the mutant strain. The original strain Brevibacterium flavum I-12 was mutagenized by conventional chemical mutagenesis and atmosphericand room temperature plasma(ARTP)biological breeding system. A mutant strain having high resistance to 2-thiazolyl alanine(2-TA)andsulfaguanidine(SG)and capable of rapid growth on a succinic acid plate was selected. Subsequently,the response surface design based onthe single factor experiment was used to optimize the optimal parameter level of the shake flask fermentation medium components for the targetmutant strain. Results showed that a strain producing L-isoleucine was obtained after multiple rounds of random mutagenesis and screening,which was resistant to 40 g/L of 2-TA and 5 g/L of SG and rapidly grew on the medium with succinate as the sole carbon source,and designatedas B. flavum TA-6. The L-isoleucine production by this mutant strain reached 26.2±0.5 g/L,which was 44.75% higher than that by the originalstrain(18.1±0.5 g/L);while the accumulations of side product L-valine and L-leucine significantly reduced. The L-isoleucine productionincreased to 27.8±0.5 g/L after optimizing the fermentation conditions by response surface method,which was 6.1% higher than that beforeoptimization. In conclusion,a strain yielding high L-isoleucine but producing low other acids,TA-6,is obtained by conventional chemicalmutagenesis and ARTP biological breeding system,thus it has potential production and application value in L-isoleucine fermentation industry.
引文
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[6]陈宁.氨基酸工艺学[M].北京:中国轻工业出版社, 2011.
[7]张雪,张晓菲,王立言,等.常压室温等离子体生物诱变育种及其应用研究进展[J].化工学报, 2014, 65(7):2676-2684.
[8]诸葛健,李华钟,王正祥.微生物遗传育种学[M].北京:化学工业出版社, 2009.
[9]刘俊梅,王庆,等.紫外线和硫酸二乙酯复合诱变选育PHB高产菌株[J].微生物学通报, 2016, 43(10):2242-2248.
[10]秦海斌. L-谷氨酸产生菌选育、发酵条件研究及其代谢流分析[D].无锡:江南大学, 2009.
[11]程功,徐建中,郭燕风,等.常压室温等离子体诱变选育L-精氨酸生产菌及发酵条件优化[J].微生物学通报, 2016, 43(2):360-369.
[12]周扬,薛正莲,夏俊,等.常压室温等离子体(ARTP)诱变及高通量筛选那西肽高产菌株[J].工业微生物, 2015(2):7-12.
[13]袁军,赵犇,孙梦玉,等.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[J].生物技术通报,2015, 31(10):199-204.
[14]Henderson JW, Ricker RD, Bidlingmeyer BA, et al. Rapid,accurate, sensitive, and reproducible HPLC analysis of aminoacids[S]. Agilent Application Note 5980-1193EN:1-10, 2006.
[15]唐艳,李晶,吴涛. L-异亮氨酸高效液相色谱检测方法的研究[J].发酵科技通讯, 2017, 46(4):220-223.
[16]马雷,程立坤,等.柠檬酸钠对L-异亮氨酸发酵及代谢流量分布的影响[J].天津科技大学学报, 2010, 25(3):14-18.
[17]Guillouet S, Rodal AA, An GH, et al. Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum[J]. Appl Microbiol Biotechnol, 2001, 57(5-6):667-673.
[18]Sahm H, Eggeling L, Morbach S, et al. Construction of L-isoleucineoverproducing strains of Corynebacterium glutamicum[J].Naturwissenschaften, 1999, 86(1):33-38.
[19]Yin LH, Hu XQ, Xu DQ, et al. Co-expression of feedback-resistantthreonine dehydratase and acetohydroxy acid synthase increaseL-isoleucine production in Corynebacterium glutamicum[J].Metabolic Engineering, 2012, 14(5):542.
[20]徐庆阳,孙家凯,等.过表达hom基因对谷氨酸棒杆菌发酵L-异亮氨酸的影响[J].天津科技大学学报, 2012(5):1-6.
[21]程功,徐建中,张伟国. L-精氨酸生物合成机制及其代谢工程育种研究进展[J].微生物学通报, 2016, 43(6):1379-1387.
[2]Yamamoto K, Tsuchisaka A, Yukawa H. Branched-chain aminoacids[J]. Methods in Enzymology, 2016, 166(7):1.
[3]Yoshinaga F, Kubota K, Jujita I, et al. Method of producingL-isoleucine by fermentation:US, 3767529 A[P]. 1973.
[4]Ikeda S, Fujita I, Yoshinaga F. Screening of L-isoleucine producersamong ethionnine resistant mutants of L-threonine producingbacteria[J]. Agric Bid Chem, 1976(40):511-516.
[5]谢飞,崔国英,王翀,等. L-异亮氨酸高产菌种的选育研究[J].中国酿造, 2014, 33(11):98-100.
[6]陈宁.氨基酸工艺学[M].北京:中国轻工业出版社, 2011.
[7]张雪,张晓菲,王立言,等.常压室温等离子体生物诱变育种及其应用研究进展[J].化工学报, 2014, 65(7):2676-2684.
[8]诸葛健,李华钟,王正祥.微生物遗传育种学[M].北京:化学工业出版社, 2009.
[9]刘俊梅,王庆,等.紫外线和硫酸二乙酯复合诱变选育PHB高产菌株[J].微生物学通报, 2016, 43(10):2242-2248.
[10]秦海斌. L-谷氨酸产生菌选育、发酵条件研究及其代谢流分析[D].无锡:江南大学, 2009.
[11]程功,徐建中,郭燕风,等.常压室温等离子体诱变选育L-精氨酸生产菌及发酵条件优化[J].微生物学通报, 2016, 43(2):360-369.
[12]周扬,薛正莲,夏俊,等.常压室温等离子体(ARTP)诱变及高通量筛选那西肽高产菌株[J].工业微生物, 2015(2):7-12.
[13]袁军,赵犇,孙梦玉,等.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[J].生物技术通报,2015, 31(10):199-204.
[14]Henderson JW, Ricker RD, Bidlingmeyer BA, et al. Rapid,accurate, sensitive, and reproducible HPLC analysis of aminoacids[S]. Agilent Application Note 5980-1193EN:1-10, 2006.
[15]唐艳,李晶,吴涛. L-异亮氨酸高效液相色谱检测方法的研究[J].发酵科技通讯, 2017, 46(4):220-223.
[16]马雷,程立坤,等.柠檬酸钠对L-异亮氨酸发酵及代谢流量分布的影响[J].天津科技大学学报, 2010, 25(3):14-18.
[17]Guillouet S, Rodal AA, An GH, et al. Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum[J]. Appl Microbiol Biotechnol, 2001, 57(5-6):667-673.
[18]Sahm H, Eggeling L, Morbach S, et al. Construction of L-isoleucineoverproducing strains of Corynebacterium glutamicum[J].Naturwissenschaften, 1999, 86(1):33-38.
[19]Yin LH, Hu XQ, Xu DQ, et al. Co-expression of feedback-resistantthreonine dehydratase and acetohydroxy acid synthase increaseL-isoleucine production in Corynebacterium glutamicum[J].Metabolic Engineering, 2012, 14(5):542.
[20]徐庆阳,孙家凯,等.过表达hom基因对谷氨酸棒杆菌发酵L-异亮氨酸的影响[J].天津科技大学学报, 2012(5):1-6.
[21]程功,徐建中,张伟国. L-精氨酸生物合成机制及其代谢工程育种研究进展[J].微生物学通报, 2016, 43(6):1379-1387.
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