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ARTP酵母突变株U1-58提高转化玉米芯产生物乙醇的能力

作者:清华研究院生物育种中心 发布时间:2017/09/07 浏览量:293


本期推荐天津科技大学陈叶福教授科研团队,利用现代ARTP育种技术,诱变选育木糖发酵酵母(Spathaspora passalidarum),获得高效利用木糖产乙醇的突变株S. passalidarum U1-58。突变株S. passalidarum U1-58,不但利用木糖产乙醇的能力提高了72.1%,而且还具耐高温发酵性能力,在41℃条件下,乙醇产量提高了166.31%。与文献报道的纤维素生产乙醇菌株相比,显著提高了乙醇产量和耐高温能力。该研究成果,2017年发表在国际主流学术top期刊《生物资源技术》上(SCI IF 5.65)(Efficient utilization of hemicellulose and cellulose in alkali liquor-pretreated corncob for bioethanol production at high solid loading by Spathaspora passalidarum U1-58. Bioresource Technology 232(2017)168-175 )。

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木质纤维素是自然界中最丰富的可再生资源,其中纤维素、半纤维素、木质素所占比例约为4:3:3。纤维素的水解产物为葡萄糖,酿酒酵母能够高效地将其转化为生物乙醇;而半纤维素主要水解产物为木糖,但在葡萄糖存在的情况下,木糖的有效利用受到巴斯德效应的影响,其在生物乙醇发酵过程中利用率很低,提高其转化乙醇的应用仍然具挑战性。利用基因工程手段、原生质体融合等遗传操作和进化适应手段,酵母转化木糖的能力有了一定的提高。酵母利用木质纤维素转化为生物乙醇,已经取得较大的进步,但降低生成成本,提高经济效益,仍然是研究人员的追求。


天津科技大学陈叶福教授科研团队利用ARTP技术从S. passalidarum NRRL Y-27907(U1)诱变筛选到突变株U1-58,该突变株相比原始菌株U1,其木糖消耗率和乙醇生产率分别达到1.18g/L/h(提高了42.17%)、0.49g/L/h(提高了68.97%),乙醇的产量从20.58g/L增加到35.41g/L,增长率达到72.1%。同时该突变体U1-58在30℃至41℃下表现出良好的高温发酵性能,33℃乙醇产量达到最高0.45g/g,为理论产率的97.61%;在41℃下乙醇产量较原始菌株U1提高166.31%。


为了进一步深入研究发酵性能,陈叶福教授将突变株U1-58利用玉米芯进行分离的水解和共发酵(SHCF)和同步糖化和共发酵(SSCF),SHCF中最高乙醇产量达到42.46 g/L,产率达到72.12 %;SSCF中最高乙醇产量达到53.24 g/L,产率达到75.35 %。对SSCF后的培养基进行分析(发酵96小时后),SSCF中消耗了86.20%的纤维素和82.99%的半纤维素,根据产量折算分析,即使培养基中的纤维素按照理论产率全部转化为乙醇,剩余的乙醇至少10.49 g/L由半纤维素转化(相当于理论产量37.59%)。


令人欣喜的是ARTP突变株U1-58具有反巴士德效应。原始菌株U1在葡萄糖耗尽前,木糖利用是非常缓慢,而突变株U1-58在葡萄糖浓度低于60 g/L时,能够快速消耗木糖,推断可能是由于ARTP技术产生基因突变导致用于木糖和葡萄糖的共同转运蛋白更高效以及参加乙醇发酵的酶活性更强,类似的结果以前未见报道。


所以,ARTP诱变S. passalidarum不仅十分有效,而且得到令人欣喜的反巴斯德效应菌株,充分说明了ARTP育种技术,在菌株改良方面具有独特的优越性。


文章精选内容如下:


1、突变菌株U1-58利用葡萄糖和木糖共转化生产乙醇


原始菌株U1与突变菌株U1-58发酵性能比较

Table 2 Fermentation performance evaluation of U1 and U1-58 with the YPX medium.

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耐高温验证

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Fig. 1. Ethanol production (a) and xylose consumption (b) of U1-58 and U1 at different temperatures. The fermentation medium contained 100 g/L xylose, 22 g/L yeast extract, 2.5 g/L K2HPO4, 0.1 g/L MgSO4, pH was adjusted to 5.5. The fermentation was conducted in 250 mL shake flasks with a working volume of 100 mL at 120 rpm for 120 h.


葡萄糖和木糖的混合糖培养的发酵性能

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Fig. 2. Comparison of the fermentation performance of U1-58 (a) and U1 (b) with the mixed sugar of glucose and xylose. The mimicked hydrolysate containing 70 g/L of  xylose and 50 g/L of glucose, nutrient sources (22 g/L yeast extract, 2.5 g/L K2HPO4, 0.1 of MgSO4) were also added, and pH was adjusted to 5.5. The fermentation was conducted in 250 mL shake flasks with a working volume of 100 mL at 120 rpm for 120 h.


2、突变菌株U1-58预处理玉米芯的SHCF


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Fig. 3. Optimization of the solid-to-liquid ratio (a), the dosage of cellulase A1 (b) and xylanse B1 (c) for SHCF. The initial solid-to-liquid ratio of 1:5 (w/v), cellulase A1 at 20 FPU/g DM, and xylanase B1 at 10,000 IU/g DM was adopted, nutrient sources (22 g/L yeast extract, 2.5 g/L K2HPO4, 0.1 of MgSO4) were also added. The parameters were optimized one after another, and the fermentation was conducted in 250 mL shake flasks with a working volume of 100 mL at 120 rpm for 120 h.


3、突变菌株U1-58预处理玉米芯的SSCF

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Fig. 6. The effect of rotation rate on SSCF performance. 4 g pretreated corncob, 20 mL nutrient substances (2.67 g/L NH4Cl, 3 g/L K2HPO4, 0.2 of MgSO4), and cellulase A1 at 20 FPU/g DM and xylanase B1 at 6000 IU/g DM were added and mixed thoroughly. The fermentation was conducted in 150 mL shake flasks with a working volume of 50 mL at 150 rpm for 120 h.


4、U1-58生物乙醇潜力的评价


Table 3  Comparison of high ethanol titer production by simultaneous glucose and xylose utilization.

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