原生质体融合技术选育高产乙醇酵母
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摘要
发酵生产燃料乙醇越来越受到人们的重视。目前,发酵生产乙醇的菌种主要以酵母菌为主。本课题主要的研究内容是对发酵生产乙醇的菌种诱变、筛选、驯化以及原生质体融合,目的是筛选出耐高温、耐乙醇且高效产乙醇的酵母菌株。
本文从实验室保藏的30℃米酒酵母M和市售安琪酵母A出发,分别进行紫外(UV)和硫酸二乙酯(DES)复合诱变以及2,3,5-氯化三苯基四氮唑(TTC)显色筛选。经过UV+DES诱变后,米酒酵母M-UV-DES3(以下简称M-3)在37℃下发酵糖醇转化率为0.475 g·g~(-1),提高了10.9%。TTC法活化筛选安琪酵母A的结果表明活化后的安琪酵母纯度高,产乙醇能力均一,糖醇转化率普遍达到0.41g·g~(-1)以上。其中A-8 37℃发酵的乙醇产量为72 g·l~(-1),糖醇转化率达到0.481g·g~(-1)。
考查了葡萄糖浓度对M-3和A-8的影响。当初始葡萄糖浓度达到200g·l~(-1)以上时,酵母受到乙醇和葡萄糖的双重抑制,发酵最大乙醇浓度不再随着葡萄糖浓度升高而升高。故对其进行乙醇浓度梯度驯化培养,以提高其耐乙醇性能。经过多批次的驯化,M-3和A-8的耐乙醇浓度都达到120 g·1~(-1)。
对驯化后的M-3和A-8进行单倍体分离和筛选,从中选出生物量较高的单倍体SM-4(15.74 g·1~(-1))和乙醇含量较高的单倍体SA-5(127.5 g·l~(-1))作为原生质体融合的亲株。
对亲株的原生质体制备和再生条件进行了试验,其最佳条件为:采用25ml 0.05mol/L EDTA和1 m1 0.5mol/1β-巯基乙醇混合液预处理1h后,用4%的蜗牛酶酶解90min。亲株SM-4的原生质体形成率为89.3%,再生率为22.6%。亲株SA-5的原生质体形成率为91.45%,再生率为17.9%。
采用紫外灭活的方法对SM-4的进行彻底灭活,条件为:20W紫外灯下距离20cm垂直照射4min。采用高温热灭活的方法对SA-5进行彻底灭活,条件为:65℃下水浴处理30min。
用PEG诱导灭活的双亲原生质体融合,经TTC平板初筛,发酵复筛以及多批次遗传稳定性实验,获得一株生物量高的高产乙醇融合株F-13,其生物量为26 g·1~(-1),乙醇产量为135 g·l~(-1),比亲株SA-5和SM-4分别提高了7.3%和17%,耐乙醇能力达到160 g·1~(-1)。
沿用米酒酵母和安琪酵母原生质体制备融合的方法,以管囊酵母单倍体菌株1770 S-5和米酒酵母单倍体菌株SM-4为亲株,对其原生质体分别进行热灭活和紫外灭活后融合。
对融合子TTC平板筛选,发酵筛选和5个批次遗传稳定性实验,我们筛选到了一株能发酵100 g·l~(-1)混合糖(葡萄糖:木糖=3:1),乙醇产量和总糖转化率都较高的融合株F-9,其发酵4个批次的乙醇产量为25.55 g·l~(-1),总糖转化率为0.25 g·g~(-1)。
Fermentation production of fuel ethanol is attracting increasing attention.Currently,yeast strains were mostly used in the production of industrial ethanol fermentation.The main goal of this research was to find a Saccharomyces cerevisiae strain which showed high ethanol production with heat and ethanol resistance by means of mutation,domestication and protoplast fusion.
Firstly,rice wine yeast M cultured at 30℃and Angel yeast A were chosen as the initial strains.Strain M-UV-DES 3(abbreviation M-3) growing at 37℃was screened through combination mutation of UV and DES.The ethanol yield was 0.475 g·g~(-1),which increased 10.9%in comparison with the original strain M.
TTC method was used in screening of Angel yeast A.The ethanol yield of most strains has achieved over 0.41g·g~(-1).Ethanol production from A-8 was obviously improved to 72.0 g·l~(-1) in 37℃fermentation and its ethanol was 0.481g·g~(-1).
Secondly,the effects of glucose concentration to M-3 and A-8 fermentation were studied.The fermentation performances of yeasts were simultaneously inhibited by both ethanol and glucose,where initial glucose concentration above 200g.l~(-1).The maximum of ethanol maintained at a similar level.In this case,ethanol gradient domestication was applied to improve their alcohol-resistant properties.Through repeated batch domestications,the ethanol tolerance capacity of both M-3 and A-8 reached 120 g.l~(-1).
Then,haploid strain SM-4 from M-3 with high biomass and SA-5 from A-8 with high ethanol concentration were slected as parental strains for protoplast fusion.
The optimum conditions of formation and regeneration of both parental strains were as below:the cells were pretreated with a mixture of 25ml 0.05mol/L EDTA and 1ml 0.5mol/lβ-ME for 1h,then cell walls were broke with 4%snailase for 1.5h.The protoplast formation rate and regeneration rate of SM-4 were 89.3%and 22.6%respectively,and for SA-5,they were 91.45%and 17.9%.
The protoplast of SM-4 was inactivated with UV,the optimum conditions were:treating for 4min below the UV lamp(20W).The protoplast of SA-5 was inactivated with heating,the optimum conditions were:treating for 30min in electric-heated thermostatic water bath(65℃).
The inactivated protoplast fusion was induced by PEG.We obtained a fusant F-13 which can produce ethanol at a high concentration after TTC screening,fermentation and 5-batches inheritance stability study.The biomass reached 26g.1~(-1) with its ethanol concentration in fermentation broth 135 g.1~(-1),which 7.3%and 17%higher than SA-5 and SM-4 respectively,the ethanol tolerance capacity achieved 160 g.1~(-1).
Utilizing methods above,protoplast fusion was accomplished between haploid strain 1770 S-5 of Pachysolen tannophilus and SM-4 after heat and UV inactivation,respectively.
A fusant F-9 was abtained after TTC screening,fermentation and 5-batches inheritance stability study.Its ethanol concentration in fermentation broth was 25.55 g.1~(-1) with ethanol yield 0.25 g.g~(-1).
本文从实验室保藏的30℃米酒酵母M和市售安琪酵母A出发,分别进行紫外(UV)和硫酸二乙酯(DES)复合诱变以及2,3,5-氯化三苯基四氮唑(TTC)显色筛选。经过UV+DES诱变后,米酒酵母M-UV-DES3(以下简称M-3)在37℃下发酵糖醇转化率为0.475 g·g~(-1),提高了10.9%。TTC法活化筛选安琪酵母A的结果表明活化后的安琪酵母纯度高,产乙醇能力均一,糖醇转化率普遍达到0.41g·g~(-1)以上。其中A-8 37℃发酵的乙醇产量为72 g·l~(-1),糖醇转化率达到0.481g·g~(-1)。
考查了葡萄糖浓度对M-3和A-8的影响。当初始葡萄糖浓度达到200g·l~(-1)以上时,酵母受到乙醇和葡萄糖的双重抑制,发酵最大乙醇浓度不再随着葡萄糖浓度升高而升高。故对其进行乙醇浓度梯度驯化培养,以提高其耐乙醇性能。经过多批次的驯化,M-3和A-8的耐乙醇浓度都达到120 g·1~(-1)。
对驯化后的M-3和A-8进行单倍体分离和筛选,从中选出生物量较高的单倍体SM-4(15.74 g·1~(-1))和乙醇含量较高的单倍体SA-5(127.5 g·l~(-1))作为原生质体融合的亲株。
对亲株的原生质体制备和再生条件进行了试验,其最佳条件为:采用25ml 0.05mol/L EDTA和1 m1 0.5mol/1β-巯基乙醇混合液预处理1h后,用4%的蜗牛酶酶解90min。亲株SM-4的原生质体形成率为89.3%,再生率为22.6%。亲株SA-5的原生质体形成率为91.45%,再生率为17.9%。
采用紫外灭活的方法对SM-4的进行彻底灭活,条件为:20W紫外灯下距离20cm垂直照射4min。采用高温热灭活的方法对SA-5进行彻底灭活,条件为:65℃下水浴处理30min。
用PEG诱导灭活的双亲原生质体融合,经TTC平板初筛,发酵复筛以及多批次遗传稳定性实验,获得一株生物量高的高产乙醇融合株F-13,其生物量为26 g·1~(-1),乙醇产量为135 g·l~(-1),比亲株SA-5和SM-4分别提高了7.3%和17%,耐乙醇能力达到160 g·1~(-1)。
沿用米酒酵母和安琪酵母原生质体制备融合的方法,以管囊酵母单倍体菌株1770 S-5和米酒酵母单倍体菌株SM-4为亲株,对其原生质体分别进行热灭活和紫外灭活后融合。
对融合子TTC平板筛选,发酵筛选和5个批次遗传稳定性实验,我们筛选到了一株能发酵100 g·l~(-1)混合糖(葡萄糖:木糖=3:1),乙醇产量和总糖转化率都较高的融合株F-9,其发酵4个批次的乙醇产量为25.55 g·l~(-1),总糖转化率为0.25 g·g~(-1)。
Fermentation production of fuel ethanol is attracting increasing attention.Currently,yeast strains were mostly used in the production of industrial ethanol fermentation.The main goal of this research was to find a Saccharomyces cerevisiae strain which showed high ethanol production with heat and ethanol resistance by means of mutation,domestication and protoplast fusion.
Firstly,rice wine yeast M cultured at 30℃and Angel yeast A were chosen as the initial strains.Strain M-UV-DES 3(abbreviation M-3) growing at 37℃was screened through combination mutation of UV and DES.The ethanol yield was 0.475 g·g~(-1),which increased 10.9%in comparison with the original strain M.
TTC method was used in screening of Angel yeast A.The ethanol yield of most strains has achieved over 0.41g·g~(-1).Ethanol production from A-8 was obviously improved to 72.0 g·l~(-1) in 37℃fermentation and its ethanol was 0.481g·g~(-1).
Secondly,the effects of glucose concentration to M-3 and A-8 fermentation were studied.The fermentation performances of yeasts were simultaneously inhibited by both ethanol and glucose,where initial glucose concentration above 200g.l~(-1).The maximum of ethanol maintained at a similar level.In this case,ethanol gradient domestication was applied to improve their alcohol-resistant properties.Through repeated batch domestications,the ethanol tolerance capacity of both M-3 and A-8 reached 120 g.l~(-1).
Then,haploid strain SM-4 from M-3 with high biomass and SA-5 from A-8 with high ethanol concentration were slected as parental strains for protoplast fusion.
The optimum conditions of formation and regeneration of both parental strains were as below:the cells were pretreated with a mixture of 25ml 0.05mol/L EDTA and 1ml 0.5mol/lβ-ME for 1h,then cell walls were broke with 4%snailase for 1.5h.The protoplast formation rate and regeneration rate of SM-4 were 89.3%and 22.6%respectively,and for SA-5,they were 91.45%and 17.9%.
The protoplast of SM-4 was inactivated with UV,the optimum conditions were:treating for 4min below the UV lamp(20W).The protoplast of SA-5 was inactivated with heating,the optimum conditions were:treating for 30min in electric-heated thermostatic water bath(65℃).
The inactivated protoplast fusion was induced by PEG.We obtained a fusant F-13 which can produce ethanol at a high concentration after TTC screening,fermentation and 5-batches inheritance stability study.The biomass reached 26g.1~(-1) with its ethanol concentration in fermentation broth 135 g.1~(-1),which 7.3%and 17%higher than SA-5 and SM-4 respectively,the ethanol tolerance capacity achieved 160 g.1~(-1).
Utilizing methods above,protoplast fusion was accomplished between haploid strain 1770 S-5 of Pachysolen tannophilus and SM-4 after heat and UV inactivation,respectively.
A fusant F-9 was abtained after TTC screening,fermentation and 5-batches inheritance stability study.Its ethanol concentration in fermentation broth was 25.55 g.1~(-1) with ethanol yield 0.25 g.g~(-1).
引文
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