过表达MRP8提高酿酒酵母乙酸耐性及乙醇发酵效率

乙酸是木质纤维素水解液中含量较多的抑制物,因此提高酿酒酵母菌株对乙酸的耐受性有助于提高纤维素乙醇生产效率。本文中,笔者利用基于CRISPR/Cas9系统的基因组编辑技术过表达了酿酒酵母(Saccharomyces cerevisiae)S288c线粒体核糖体蛋白编码基因MRP8,并比较了过表达MRP8的菌株与对照菌株的生长和发酵特性。平板耐性检测发现,MRP8过表达明显提高了菌株的乙酸胁迫耐受性;乙醇发酵结果表明,在4.8 g/L乙酸胁迫条件下,过表达菌株MRP8-3在51 h消耗全部的葡萄糖,发酵时间缩短了25 h,显著优于相同时间的对照菌株。本研究结果为构建高效纤维素乙醇发酵的酿酒酵母菌株提供了新思路。     

紫外诱变结合驯化提高酿酒酵母对抑制物耐受性

[目的]提高工业酿酒酵母Sc4126对木质纤维素预处理过程中产生的发酵抑制物的耐受性。[方法]采用紫外诱变结合驯化对工业酿酒酵母Sc4126进行选育,对筛选出的三株菌株Sc4126-1、Sc4126-2、Sc4126-3在复合抑制剂和单一抑制剂存在下对比考查其发酵性能。[结果]相比原始菌株,3株菌株对抑制剂的耐受性均不同程度地提高。在复合抑制剂存在下,优势菌株Sc4126-1发酵时间为48 h,乙醇平均产率0.19 g/L·h,而原始菌株Sc4126发酵时间138 h,乙醇平均产率0.06 g/L·h,相比原始菌株,优势菌株发酵时间缩短65.22%,乙醇平均产率提高2.17倍。对于单一抑制剂,改造后的菌株对糠醛、香草醛的耐受性明显提高,而对乙酸的耐受性提高较少。[结论]通过紫外诱变结合驯化的方法,有效提高了工业酿酒酵母Sc4126对抑制剂的耐受性。     

过表达tRNA基因tL(CAA)K提高酿酒酵母乙酸耐受性

乙酸是木质纤维素类生物质水解液中的常见毒性抑制物,选育乙酸耐受性好的酿酒酵母菌株,有利于高效利用木质纤维素类生物质,发酵生产生物燃料和生物基化学品。目前对酿酒酵母抗逆性的研究多集中在转录水平,但对转运RNA (Transfer RNA,tRNA) 在耐受性中的作用研究较少。在对酿酒酵母抗逆性研究过程中发现,一些转运RNA基因在耐受性好的酿酒酵母菌株中转录明显上调。本文深入分析了精氨酸tRNA基因tR(ACG)D和亮氨酸tRNA基因tL(CAA)K过表达对酿酒酵母耐受木质纤维素水解液的影响。结果表明,在4.2 g/L乙酸胁迫条件下进行乙醇发酵时,过表达tL(CAA)K的菌株生长和发酵性能均优于对照酵母菌株,乙醇生产强度比对照菌株提高了29.41%,但过表达tR(ACG)D基因的菌株生长和代谢能力较对照菌株明显降低,体现了不同tRNA的不同调控作用。进一步分析发现,过表达tL(CAA)K的重组酵母菌株乙酸耐受性调控相关基因HAA1、MSN2和MSN4等胁迫耐受性相关转录因子编码基因的转录水平上调。本文的研究为选育高效利用木质纤维素资源进行生物炼制的酵母菌株提供了新的改造策略,也为进一步揭示酿酒酵母tRNA基因表达调控对抗逆性的影响提供了基础。     

选育耐受复合抑制剂酿酒酵母提高乙醇产量

能够耐受纤维素预处理中抑制剂的酿酒酵母对高效、经济生产纤维素乙醇至关重要。利用诱变结合驯化工程选育了一株可耐受复合抑制剂(1.3g/L糠醛、5.3g/L乙酸及1.0g/L苯酚)的工业酿酒酵母YYJ003。在pH 4.0的含有抑制剂的培养基中,耐受菌株乙醇产率是原始菌株的7.8倍,糠醛转化速率提高了5倍。在pH 5.5的复合抑制剂条件下,YYJ003发酵时间(16h)比野生菌株发酵时间(22h)缩短6h。在pH 4.0的未脱毒的玉米秸秆水热法预处理水解液中YYJ003的乙醇产率达到0.50g/g(乙醇/葡萄糖),乙醇产速达到4.16g/(L·h),而对照菌株无乙醇产出。     

紫外诱变驯化提高酿酒酵母木糖发酵的抑制物耐受性

利用驯化和紫外处理结合驯化的手段对一株木糖发酵工业菌株的抑制物耐受性进行提升。在反复批次培养过程中不断提高含9种抑制物的混合抑制物浓度,使细胞的生长和发酵逐渐适应高浓度抑制物环境,分离突变菌株并进行评价。紫外处理结合驯化比直接驯化能更有效的提升细胞对高浓度抑制物的耐受能力;通过突变菌株分离和筛选,获得3株抑制物耐受能力高于出发菌株的突变菌株,它们在抑制物浓度100%MI(甲酸1 g/L,乙酸3.5 g/L,乙酰丙酸1.5 g/L,糠醛1.5 g/L,5-羟甲基糠醛1.5g/L,丁香醛0.1 g/L,香草醛0.1 g/L,松柏醛0.025 g/L,肉桂酸0.025 g/L)条件下的木糖消耗率比出发菌株高出11.3%-23.2%。紫外诱变处理结合驯化过程可以有效提高酿酒酵母对混合抑制物的耐受性。     

絮凝基因FLO1及FLO1c高表达提高工业酿酒酵母乙酸耐受性及发酵性能

乙酸是生物质乙醇发酵过程中酵母细胞面临的重要抑制剂之一,对细胞生长及发酵性能有强烈的抑制作用。增强酵母菌对乙酸胁迫的耐受性对提高乙醇产率具有重要意义。用分别带有完整絮凝基因FLO1及其重复序列单元C发生缺失的衍生基因FLO1c的重组表达质粒分别转化非絮凝型工业酿酒酵母CE6,获得絮凝型重组酵母菌株6-AF1和6-AF1c。同时以空载体p YCPGA1转化CE6的菌株CE6-V为对照菌株。与CE6-V相比,絮凝酵母明显提高了对乙酸胁迫的耐受性。在0.6%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.56倍和1.62倍;在1.0%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.21倍和1.78倍。可见絮凝能力改造能明显提高工业酿酒酵母的乙酸胁迫耐受性及发酵性能,而且FLO1内重复序列单元C缺失具有更加明显的效果。     

定向进化Rho1提高酿酒酵母的乙醇耐受性和超高浓度乙醇发酵性能

燃料乙醇发酵过程中酿酒酵母细胞活性被高浓度乙醇严重抑制而导致发酵提前终止，生产强度严重降低，因此构建同时具有高耐受性、高发酵性能的菌株一直是发酵工业追求的目标。选取酿酒酵母细胞形态调节关键基因小GTP酶家族成员Rho1,构建易错PCR产物文库，以酿酒酵母S288c为出发菌株采取“富集-自然生长-复筛”的筛选策略，成功筛选得到两株乙醇胁迫耐受性与发酵性能均提高的突变株M2和M5。测序发现突变株过表达的Rho1序列出现了3～5个氨基酸的突变和大片段的缺失突变。以300 g/L起始葡萄糖进行乙醇发酵，72 h时，M2和M5的乙醇滴度比对照菌株分别提高了19.4%和22.3%,超高浓度乙醇发酵能力显著提高。本研究为利用蛋白定向进化方法改良酵母菌复杂表型提供了新的作用靶点。     

常压甘油自催化预处理麦草浓醪发酵纤维素乙醇

目前纤维素乙醇成本偏高的根本原因在于没有达到淀粉质乙醇发酵水平的"三高"(高浓度、高转化率和高效率)指标,提高水解糖液浓度和避免发酵抑制物来实现浓醪发酵,是解决问题的关键。文中以常压甘油自催化预处理麦草为底物,尝试采用不同发酵策略,探讨其浓醪发酵产纤维素乙醇的可行性。在优化培养条件(15%底物浓度,加酶量30 FPU/g干底物,温度37℃,接种量10%)下同步糖化发酵72 h,纤维素乙醇产量为31.2 g/L,转化率为73%,发酵效率0.43 g/(L·h);采用半同步(预酶解24 h)糖化发酵72 h,纤维素乙醇浓度达到33.7 g/L,转化率为79%,发酵效率为0.47 g/(L·h),其中(半)同步糖化发酵中90%以上纤维素已被糖化水解用于发酵;采用分批补料式半同步糖化发酵,补料到基质浓度相当于30%,发酵72 h时纤维素乙醇产量达到51.2 g/L,转化率为62%,发酵效率为0.71 g/(L·h)。在所有浓醪发酵中乙酸不足3 g/L,无糠醛和羟甲基糠醛等发酵抑制物。以上结果表明,常压甘油自催化预处理木质纤维素基质适用于纤维素乙醇发酵;分批补料式半同步糖化发酵策略可用来进行浓醪纤维素乙醇发酵;未来工作中提高基质纯度和强化酶解产糖是浓醪纤维素乙醇达到"三高"指标的关键。     

玉米秸秆酸解副产物对重组酿酒酵母6508-127发酵的影响

将木质纤维素类生物质如玉米秸秆等用稀酸水解预处理,在半纤维素水解为单糖的同时,水解液中还会产生一些可能对后续发酵有影响的副产物。本实验分别考查了在玉米秸秆稀酸水解液中检测出的乙酸、甲酸、香草醛、糠醛和羟甲基糠醛对重组木糖发酵菌株S. cerevisiae 6508-127生长和发酵的影响。结果表明,甲酸和乙酸对菌体生长的抑制强于乙醇生成,且甲酸的抑制程度远大于乙酸;2g/L香草醛可使菌体生长延滞期明显延长,而在较低浓度（≤1.2g/L）此现象不明显。糠醛在0.5-1.5g/L范围内对菌体生长有抑制作用,但使乙醇得率提高;羟甲基糠醛在0.2g/L浓度存在就使乙醇得率有明显降低,但使生物量得率提高;研究中还发现,糠醛、羟甲基糠醛和香草醛可被S. cerevisiae 6508-127代谢。     

分别利用产甘油假丝酵母抗逆转录因子CgSTB5和CgSEF1对酿酒酵母菌株糠醛耐受改造

【背景】纤维素是生物转化解决能源问题的主要原料之一,其水解物中存在严重影响抑制菌株生长的糠醛,需脱毒才可应用于发酵,提高菌株耐受性是解决纤维素水解液实际生产应用的关键。【目的】酿酒酵母(Saccharomyces cerevisiae)是主要的纤维素水解液发酵工业菌株,但糠醛耐受性较低,通过分子改造获得具有高糠醛耐受性的菌株。【方法】利用新获得的产甘油假丝酵母(Candidaglycerinogenes)的相关抗逆转录因子CgSTB5、CgSEF1和CgCAS5,通过分子技术进行S.cerevisiae改造,考察其对酿酒酵母糠醛耐受性的影响,并尝试应用于未脱毒纤维素乙醇发酵。【结果】单个表达CgSTB5和CgSEF1的酿酒酵母,通过菌株点板实验表明菌株的糠醛耐受性提高25%以上,并且摇瓶发酵结果显示糠醛降解性能明显提高,生长延滞期明显缩短,S.cerevisiae W303/p414-CgSTB5的未脱毒纤维素乙醇发酵生产效率提高12.5%左右。【结论】转录因子CgSTB5和CgSEF1均能对提高酿酒酵母糠醛耐受性起到重要作用,并且有助于提高酿酒酵母菌株未脱毒纤维素乙醇发酵性能。     

Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498

Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosics ethanol. In this study, we used acid pretreatment, delignification and enzymatic hydrolysis to evaluate the pretreatment to produce more sugar, to be fermented to ethanol. Dilute H(2)SO(4) (3.0%,v/v) treatment resulted in hydrolysis of hemicelluloses from lignocellulosic complex to pentose sugars along with other byproducts such as furfural, hydroxymethyl furfural (HMF), phenolics and acetic acid. The acid pretreated substrate was delignified to the extent of 93.2% by the combined action of sodium sulphite (5.0%,w/v) and sodium chlorite (3.0%,w/v). The remaining cellulosic residue was enzymatically hydrolyzed in 0.05 M citrate phosphate buffer (pH 5.0) using 3.0 U of filter paper cellulase (FPase) and 9.0 U of beta-glucosidase per mL of citrate phosphate buffer. The maximum enzymatic saccharification of cellulosic material (82.8%) was achieved after 28 h incubation at 50 degrees C. The fermentation of both acid and enzymatic hydrolysates, containing 18.24 g/L and 37.47 g/L sugars, with Pichia stipitis and Saccharomyces cerevisiae produced 7.13 g/L and 18.52 g/L of ethanol with corresponding yield of 0.39 g/g and 0.49 g/g, respectively.     

混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究

【目的】构建可用于纤维素乙醇高效生产的混合糖发酵重组酿酒酵母菌株,并利用菊芋秸秆为原料进行乙醇发酵。【方法】筛选在木糖中生长较好的酿酒酵母YB-2625作为宿主菌,构建木糖共代谢菌株YB-2625 CCX。进一步通过r DNA位点多拷贝整合的方式,以YB-2625 CCX为出发菌株构建木糖脱氢酶过表达菌株,并筛选得到优势菌株YB-73。采用同步糖化发酵策略研究YB-73的菊芋秸秆发酵性能。【结果】YB-73菌株以90 g/L葡萄糖和30 g/L木糖为碳源进行混合糖发酵,乙醇产量比出发菌株YB-2625 CCX提高了13.9%,副产物木糖醇产率由0.89 g/g降低至0.31 g/g,下降了64.6%。利用重组菌YB-73对菊芋秸秆进行同步糖化发酵,48 h最高乙醇浓度达到6.10%(体积比)。【结论】通过转入木糖代谢途径以及r DNA位点多拷贝整合过表达木糖脱氢酶基因可有效提高菌株木糖发酵性能,并用于菊芋秸秆的纤维素乙醇生产。这是首次报道利用重组酿酒酵母进行菊芋秸秆原料的纤维素乙醇发酵。     

纤维素降解产物对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响

研究纤维素酸水解产生的4种副产物乙酸、甲酸、糠醛、5-羟甲基糠醛及发酵产物乙醇对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响。结果表明:5.0 g/L乙酸和1.0 g/L甲酸对葡萄糖和木糖共发酵具有明显的抑制作用;1.0 g/L糠醛和5-羟甲基糠醛基本不影响K.marxianus 1727发酵葡萄糖,且能够被K.marxianus1727转化为毒性相对较低的物质。由于5-羟甲基糠醛的转化速率慢,对K.marxianus 1727发酵木糖的抑制程度大于糠醛。乙醇对K.marxianus 1727发酵木糖具有抑制作用,当乙醇质量浓度大于20 g/L时,生物量及木糖利用率约是对照的44%和70%。     

Improvement of robustness and ethanol production of ethanologenic Saccharomyces cerevisiae under co-stress of heat and inhibitors

Bioethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, yeast cells are challenged by various environmental stresses during the industrial process of ethanol production. The robustness under heat, acetic acid, and furfural stresses was improved for ethanologenic S. cerevisiae in this work using genome shuffling. Recombinant yeast strain R32 could grow at 45°C, and resist 0.55% (v/v) acetic acid and 0.3% (v/v) furfural at 40°C. When ethanol fermentation was conducted at temperatures ranging from 30 to 42°C, recombinant strain R32 always gave high ethanol production. After 42 h of fermentation at 42°C, 187.6 ± 1.4 g/l glucose was utilized by recombinant strain R32 to produce 81.4 ± 2.7 g/l ethanol, which were respectively 3.4 and 4.1 times those of CE25. After 36 h of fermentation at 40°C with 0.5% (v/v) acetic acid, 194.4 ± 1.2 g/l glucose in the medium was utilized by recombinant strain R32 to produce 84.2 ± 4.6 g/l of ethanol. The extent of glucose utilization and ethanol concentration of recombinant strain R32 were 6.3 and 7.9 times those of strain CE25. The ethanol concentration produced by recombinant strain R32 was 8.9 times that of strain CE25 after fermentation for 48 h under 0.2% (v/v) furfural stress at 40°C. The strong physiological robustness and fitness of yeast strain R32 support its potential application for industrial production of bioethanol from renewable resources such as lignocelluloses.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

The acid hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol sulfuric acid per liter pyrolysate using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol in 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol in 18 h. The results showed that the acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by S. cerevisiae (R) in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

自絮凝酵母高浓度重复批次乙醇发酵

利用发酵性能优良的自絮凝酵母Saccharomyces cerevisiaeflo,研究开发了重复批次高浓度乙醇发酵系统,以节省下游加工过程的能耗。在终点乙醇浓度达到120g/L左右的条件下,发酵系统的乙醇生产强度达到8.2g/(L·h)。然而实验中发现,随着发酵批次的增多,自絮凝酵母沉降性能逐渐下降,从发酵液中沉降分离所需时间相应延长,导致发酵液中高浓度乙醇对酵母的毒害作用加剧,影响其发酵活性和发酵系统运行的稳定性,发酵装置运行11个批次后无法继续运行。实验结果表明,絮凝能力下降导致的酵母絮凝颗粒尺度减小是其沉降性能下降的主要原因。进一步研究发现,酵母的絮凝能力通过再培养可以恢复。在此基础上对发酵系统操作进行改进,每批发酵结束后可控采出一定比例菌体,调节系统的酵母细胞密度和乙醇生产强度以刺激酵母增殖,保持其絮凝能力。在达到相同发酵终点乙醇浓度条件下,虽然发酵系统的乙醇生产强度降低到4.0g/(L·h),但运行10d后絮凝颗粒酵母尺度趋于稳定,继续运行14d,未发现絮凝颗粒酵母尺度继续下降的现象,系统可以稳定运行。     

Potential inhibitors from wet oxidation of wheat straw and their effect on ethanol production of Saccharomyces cerevisiae: wet oxidation and fermentation by yeast

Alkaline wet oxidation (WO) (using water, 6.5 g/L sodium carbonate and 12 bar oxygen at 195 degrees C) was used as pretreatment method for wheat straw (60 g/L), resulting in a hydrolysate and a cellulosic solid fraction. The hydrolysate consisted of soluble hemicellulose (8 g/L), low-molecular-weight carboxylic acids (3.9 g/L), phenols (0.27 g/L = 1.7 mM) and 2-furoic acid (0.007 g/L). The wet oxidized wheat straw hydrolysate caused no inhibition of ethanol production by Saccharomyces cerevisiae ATCC 96581. Nine phenols and 2-furoic acid, identified to be present in the hydrolysate, were each tested in concentrations of 50-100 times the concentration found in the hydrolysate for their effect on fermentation by yeast. At these high concentrations (10 mM), 4-hydroxybenzaldehyde, vanillin, 4-hydroxyacetophenone and acetovanillone caused a 53-67% decrease in the volumetric ethanol productivity in S. cerevisiae compared to controls with an ethanol productivity of 3.8 g/L. The phenol acids (4-hydroxy, vanillic and syringic acid), 2-furoic acid, syringaldehyde and acetosyringone were less inhibitory, causing a 5-16% decrease in ethanol productivity. By adding the same aromatic compounds to hydrolysate (10 mM), it was shown that syringaldehyde and acetovanillone interacted negatively with hydrolysate components on the ethanol productivity. Fermentation in WO hydrolysate, that had been concentrated 6 times by freeze-drying, lasted 4 hours longer than in regular hydrolysate; however, the ethanol yield was the same. The longer fermentation time could not be explained by an inhibitory action of phenols alone, but was more likely caused by inhibitory interactions of phenols with carboxylic acids, such as acetic and formic acid.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

Acid-hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol/l sulfuric acid using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol after 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol after 18 h. The results showed that acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by it in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.