常压室温等离子体诱变选育高产油脂皮状丝孢酵母的研究
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摘要
研究了常压室温等离子体(ARTP)诱变时间对皮状丝孢酵母(Trichosporon cutaneum)致死率的影响,考察了不同质量浓度芝麻酚对皮状丝孢酵母生长的抑制程度,建立了一种快速高效选育高产油脂菌株的方法。结果表明:经过ARTP的40 s诱变后,接种到质量浓度为0. 15 mg/m L的芝麻酚培养基中28℃培养2 d,筛选出生长旺盛的菌株,然后接种到48孔液体培养基培养,并利用96孔板尼罗红荧光染色对培养菌株进行高通量筛选,选育出一株高产油脂菌株Trichosporon cutaneum A1。该菌株较原始菌株的生物量、油脂产量、油脂含量分别提高了41. 6%、123. 1%、57. 4%,且具有较好的遗传稳定性;通过气相色谱分析,其油脂脂肪酸组成与原始菌株相同,均为十六烷酸、硬脂酸、油酸、亚油酸。说明建立的芝麻酚抗性筛选和荧光标记高通量筛选相结合的ARTP选育方法是可行的,为其他产油脂菌株的筛选提供重要参考。
The effect of atmospheric room temperature plasmas( ARTP) mutagenesis time on the lethality rate of Trichosporon cutaneum was studied. The inhibition of the different mass concentration sesamol on the growth of Trichosporon cutaneum was investigated. The method of rapid and high breeding on the high-producing oil strain was established. The results showed that the Trichosporon cutaneum strain A1 with high-producing oil was obtained through the procedure including screening the vigorous growth strain in the 0. 15 mg/mL sesamol medium after culturing at 28 ℃ for 2 d using the strains mutagenized for 40 s through ARTP,then was inoculated to 48 pore liquid medium and high throughput screening of the cultured strains was conducted using fluorescence detection after Nile red staining in 96 pore plates. The biomass,oil production and oil content of the strain increased by 41. 6%,123. 1% and 57. 4% respectively,and the strain had good genetic stability. The fatty acids of the microbiological oil mainly contained palmitic acid,stearic acid,oleic acid and linoleic acid,which was the same as the original strain. The method of ARTP breeding was effective combined resistant screening to the sesamol with fluorescent-labeled high throughput screening, and it provided important reference for screening other oil producing strains.
The effect of atmospheric room temperature plasmas( ARTP) mutagenesis time on the lethality rate of Trichosporon cutaneum was studied. The inhibition of the different mass concentration sesamol on the growth of Trichosporon cutaneum was investigated. The method of rapid and high breeding on the high-producing oil strain was established. The results showed that the Trichosporon cutaneum strain A1 with high-producing oil was obtained through the procedure including screening the vigorous growth strain in the 0. 15 mg/mL sesamol medium after culturing at 28 ℃ for 2 d using the strains mutagenized for 40 s through ARTP,then was inoculated to 48 pore liquid medium and high throughput screening of the cultured strains was conducted using fluorescence detection after Nile red staining in 96 pore plates. The biomass,oil production and oil content of the strain increased by 41. 6%,123. 1% and 57. 4% respectively,and the strain had good genetic stability. The fatty acids of the microbiological oil mainly contained palmitic acid,stearic acid,oleic acid and linoleic acid,which was the same as the original strain. The method of ARTP breeding was effective combined resistant screening to the sesamol with fluorescent-labeled high throughput screening, and it provided important reference for screening other oil producing strains.
引文
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[3]柳杰,刘文慧,王晚晴,等.产油微生物及其发酵原料的研究进展[J].环境工程,2017(3):132-136.
[4]吴亦楠,邢新会,张翀,等.ARTP生物育种技术与装备研发及其产业化发展[J].生物产业技术,2017(1):37-45.
[5]刘雅婷,刘宏娟,王艳萍,等.常压室温等离子体诱变粘红酵母筛选高产油脂菌株及发酵条件优化[J].中国油脂,2015,40(1):83-87.
[6]金丽华,方明月,张翀,等.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[J].生物工程学报,2011,27(3):461-467.
[7]王美珠.产油皮状丝孢酵母耐高温菌株的诱变筛选及发酵调控[D].上海:华东理工大学,2013.
[8]程万,林辉,赵宇华,等.苹果酸酶调控微生物油脂积累的研究进展[J].科技通报,2010,26(6):853-857.
[9]孟鑫,尚宏丽,郑益.过量表达苹果酸酶对E.coli脂肪酸合成能力的影响[J].食品工业科技,2013,34(12):207-209.
[10]KIMURA K,YAMAOKA M.KAMISAKA Y.Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence[J].J Microbiol Meth,2003,56(3):331-338.
[11]林义,钟添华,骆祝华,等.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[J].微生物学通报,2012,39(1):125-137.
[12]朱启思,老国丹,关则恳,等.油茶籽油脂肪酸组成及掺混大豆油的检测[J].粮食储藏,2015,44(2):37-41.
[13]FAN X G,WU H Y,LI G L,et al.Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening[J].Plos One,2017,12(5):1-13.