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。     

酿酒酵母Mbp1缺陷型菌株的构建及其乙醇发酵特性

【目的】研究酿酒酵母(Saccharomyces cerevisiae)工业菌株Mbp1基因的功能,探讨Mbp1基因对酿酒酵母乙醇发酵性能的影响。【方法】以酿酒酵母MF1015为出发菌株,用PCR方法构建Mbp1基因敲除组件Loxp-KanMX-Loxp,将敲除组件转化两种配型的酿酒酵母单倍体,通过单倍体复倍获得敲除Mbp1基因的二倍体突变菌株,研究突变菌株形态变化及乙醇发酵特性。【结果】敲除Mbp1基因后突变菌株生长曲线无显著变化,出芽率降低,细胞体积增大19.2%,对饥饿更敏感,较早出现假菌丝。甘蔗糖蜜在静置条件下发酵,突变菌株的乙醇产量明显低于野生型;在130 r/min的条件下发酵,突变菌株和野生型发酵液中的乙醇产量基本相同。【结论】Mbp1基因缺失使酿酒酵母的乙醇发酵能力下降并影响细胞的形态分化。     

酿酒酵母adh2和ald6双基因缺失突变株的构建

酿酒酵母乙醇合成代谢过程中, 阻断或削弱乙醛至乙酸代谢流不但能增强乙醇合成流, 同时还能降低发酵乙酸含量。本研究以乙醇脱氢酶Ⅱ(adh2)基因缺陷型酿酒酵母YS2-Dadh2为出发菌株, 应用长侧翼同源两步PCR(LFH-PCR)策略构建乙醛脱氢酶Ⅵ(ald6)基因敲除组件, 转化酿酒酵母YS2-Dadh2敲除ald6基因, 之后转入表达质粒pSH65到阳性克隆中, 半乳糖诱导表达Cre重组酶切除Kanr基因筛选标记, 最后, 传代丢失质粒pSH65获得单倍体ald6基因缺失突变株。利用同样的敲除组件和技术再次敲除其等位基因, 最终获得双基因缺失突变株YS2-△adh2-Dald6。发酵实验表明与出发菌株YS2相比, 突变株乙酸合成量降低18%, 乙醇最高产量提高12.5%。     

代谢工程与全基因组重组构建酿酒酵母抗逆高产乙醇菌株

将酿酒酵母海藻糖代谢工程与全基因组重组技术相结合,改良工业酿酒酵母菌株的抗逆性和乙醇发酵性能。对来源于二倍体出发菌株Zd4的两株优良单倍体Z1和Z2菌株进行杂交获得基因组重组菌株Z12,并对Z1和Z2先进行(1)过表达海藻糖-6-磷酸合成酶基因 (TPS1) ,(2)敲除海藻糖水解酶基因 (ATH1), (3)同时过表达 TPS1和敲除ATH1, 经此三种基因工程操作后再进行杂交获得代谢工程菌株的全基因组重组菌株Z12ptps1、Z12 Δath1和Z12pTΔA。与亲株Zd4相比,Z12及结合代谢工程获得的菌株在高糖、高乙醇浓度与高温条件下生长与乙醇发酵性能都有不同程度的改进。对比研究结果表明:在高糖发酵条件下,同时过表达 TPS1和敲除ATH1 的双基因操作工程菌株胞内海藻糖积累、乙醇主发酵速率和乙醇产量相对于亲株的提高幅度要大于只过表达 TPS1,或敲除ATH1 的工程菌。结合了全基因组重组后获得的二倍体工程菌株Z12pTΔA,与原始出发菌株Zd4及重组子Z12相比,主发酵速率分别提高11.4%和6.3%,乙醇产量提高7.0%和4.1%,与其胞内海藻糖含量高于其它菌株、在胁迫条件下具有更强耐逆境能力相一致。结果证明,海藻糖代谢工程与杂交介导的全基因组重组相结合,是提高酿酒酵母抗逆生长与乙醇发酵性能的有效策略与技术途径。     

利用SPT3的定向进化提高工业酿酒酵母乙醇耐受性

利用对转录因子的定向进化可对多基因控制的性状进行有效的代谢工程改造。本研究对酿酒酵母负责胁迫相关基因转录的SAGA复合体成分SPT3编码基因进行易错PCR随机突变,并研究了SPT3的定向进化对酿酒酵母乙醇耐性的影响。将SPT3的易错PCR产物连接改造的pYES2.0表达载体并转化酿酒酵母Saccharomyces cerevisiae4126,构建了突变体文库。通过筛选在高浓度乙醇中耐受性提高的突变株,获得了一株在10%(V/V)乙醇中生长较好的突变株M25。该突变株利用125g/L的葡萄糖进行乙醇发酵时,终点乙醇产量比对照菌株提高了11.7%。由此表明,SPT3是对酿酒酵母乙醇耐性进行代谢工程改造的一个重要的转录因子。     

黄脂菌素生物合成基因簇中调控基因xanR3的功能

【目的】研究黄脂菌素产生菌灰黄链霉菌中编码ArsR家族转录调控蛋白(Arsenical resistance regulator)的xanR3基因的功能。【方法】利用大肠杆菌和链霉菌双亲本接合转移的方法,构建xanR3基因缺失突变株及回补突变株。利用cDNA在相邻同方向的基因间隔区进行PCR确定黄脂菌素生物合成基因簇中的转录单元。利用荧光定量RT-PCR方法进行突变株中黄脂菌素生物合成基因簇转录水平的检测。【结果】对得到的xanR3基因缺失突变株及回补突变株进行发酵,发现xanR3基因缺失突变株产黄脂菌素能力下降,回补菌株中黄脂菌素产量相比缺失突变株有一定程度的恢复,但仍未达到野生型水平。经鉴定,黄脂菌素生物合成基因簇中共有18个共转录单元,其中4个共转录单元在?xanR3突变株中转录水平明显下降。【结论】ArsR家族转录调控基因xanR3是黄脂菌素生物合成的正调控基因。     

slnN基因在盐霉素生物合成中的调控功能分析

【目的】研究盐霉素生物合成基因簇上游潜在调控基因slnN的功能。【方法】本实验利用遗传操作技术,分别对白色链霉菌出发菌株Streptomyces albus BK3-25中的slnN基因进行敲除和过表达,然后利用抑菌圈实验和发酵实验,分别检测不同衍生菌株中盐霉素生物合成产量的变化。同时利用qRT-PCR分析衍生菌株与原始出发菌株之间的结构基因表达差异。【结果】结果表明在slnN基因缺失株(slnNDM)中,盐霉素的表达水平提高了35%左右;而在slnN基因过表达株(slnNOE)中,盐霉素产量下降达43%左右。qRT-PCR分析进一步发现slnN基因缺失,会引起slnO和slnA1基因的上调;而slnN基因过表达后,一方面会下调slnO与slnA1基因的表达,另一方面引起slnT1、slnF基因上调。【结论】本研究证实slnN基因对盐霉素的生物合成具有明显的负调控作用,其机制有待进一步研究。     

酿酒酵母蔗糖关键代谢途径suc2基因的敲除及其蔗糖代谢特性的变化分析

蔗糖基生物质是热带和亚热带地区重要的生物质材料,因而在微生物发酵和微生物代谢原料中具有重要的地位。酿酒酵母(Saccharomyces cerevisiae)具有以蔗糖为原料进行代谢的能力,在酿酒酵母的基因组中蔗糖水解酶基因共有6个结构基因。本研究以酿酒酵母INVSC1为出发菌株,首先利用基因敲除技术构建suc2基因缺失菌株,然后将suc2基因回补,从而研究suc2基因对酿酒酵母蔗糖关键代谢途径及蔗糖代谢特性的影响。以蔗糖为碳源的发酵培养基中,在静置条件下发酵,suc2基因缺失菌株失去了利用蔗糖代谢的能力,回补菌株则恢复了对蔗糖的代谢;而且回补菌株对蔗糖的利用率及乙醇产量均比出发菌株有所提高。suc2基因是酿酒酵母蔗糖代谢的关键基因,对蔗糖的代谢具有决定性作用,可以作为蔗糖代谢途径改造的一个关键点。     

酿酒酵母SNF4基因敲除缺失菌株的构建

构建一株酿酒酵母SNF4基因缺失菌株并研究其对乙醇产量的影响。扩增带有SNF4基因上下游同源序列和Kanr筛选标记的SNF4基因敲除组件,转化到酿酒酵母YS2获得阳性克隆子,然后将质粒pSH65转到阳性克隆子中,半乳糖诱导pSH65表达Cre酶切除Kanr筛选标记,获得SNF4等位基因完全缺失菌株YS2-△SNF4。发酵实验结果表明,缺失菌株YS2-△SNF4乙醇产量较出发菌株提高了7.57％。利用Cre-LoxP系统,成功构建了SNF4等位基因完全缺失菌株并提高乙醇产生量。     

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。     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60-3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8-13% vs. 33-47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Selection and study of potent lactose-fermenting yeasts

Whey-fermenting Kluyveromyces cultures were revealed among 105 yeast strains assimilating lactose. Eighteen most potent strains isolated from milk products fermented galactose, sucrose, and raffinose, in addition to lactose. Many yeast strains fermented inulin. Most strains were resistant to cycloheximide and grew in medium containing glucose, NaCl, and ethanol at concentrations of up to 50, 11-12, and 10-12%, respectively (4 degrees C). Three strains had mycocinogenic activity. After fermentation of whey with selected yeast strains at 30 degrees C for 2-3 days, ethanol concentration was 4-5%.     

Ethanol fermentation by nystatin-resistant strains of Saccharomyces cerevisiae

Nystatin-resistant mutants of haploid and polyploid strains of Saccharomyces cerevisiae were isolated by plating on gradient plates with increasing nystatin concentrations (60–3000 U/ml). Some of the mutants were defective in ergosterol biosynthesis, and produced zymosterol and cholestatetraenol-like sterols. Those mutants which do not form ergosterol produce less ethanol than the parent strains. They also had lower viability during fermentation of glucose solutions (8–13% vs. 33–47%). This became more pronounced in fermentations of higher concentrations of glucose. A nystatin-resistant but ergosterol-forming mutant had a similar fermentation capacity to the parent strain.     

Optimization of batch alcoholic fermentation of glucose syrup substrate

The quantitative effects of substrate concentration, yeast concentration, and nutrient supplementation on ethanol content, fermentation time, and ethanol productivity were investigated in a Box–Wilson central composite design experiment, consisting of five levels of each variable, High substrate concentration, up to 30° Brix, resulted in higher ethanol content (i.e., up to 15.7% w/v or 19.6% v/v) but longer fermentation time and hence lower ethanol productivity. Increasing yeast concentration, on the other hand, resulted in shorter fermentation time and higher productivity. The highest ethanol productivity of about 21 g EiOH/L h was obtained at low substrate concentration (i.e., 12° Brix), low alcohol content (i.e., 6% by weight), high yeast concentration (i.e., 4.4%), and high supplementation of yeast extract (i.e., 2.8). Productivity of this magnitude is substantially higher that that of the traditional batch fermentation of fed-batch fermentation. It is comparable to the results of continuous fermentation but lower than those of vacuum fermentation but lower than those of vacuum fermentation. Optimal conditions for maximal ethanol productivity can be established by a multiple regression analysis technique and by plotting the contours of constant response to conform to the constraints of individual operations.     

Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions

Taking continuous ethanol fermentation with the self‐flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300 µm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300 µm grown in presence or absence of 0.05 g L^?1 zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300 µm exhibited highest ethanol production (110.0 g L^?1) and glucose uptake rate (286.69 C mmol L^?1 h^?1), which are in accordance with the increased flux from pyruvate to ethanol and decreased flux to glycerol. And in the meantime, zinc supplementation further increased ethanol production and cell viability comparing with the control. Zinc addition enhanced the carbon fluxes to the biosynthesis of ergosterol (28.6%) and trehalose (43.3%), whereas the fluxes towards glycerol, protein biosynthesis, and tricarboxylic acid cycle significantly decreased by 37.7%, 19.5%, and 27.8%, respectively. This work presents the first report on the regulation of metabolic flux by the size of yeast flocs and zinc supplementation, which provides the potential for developing engineering strategy to optimize the fermentation system. Biotechnol. Bioeng. 2010;105: 935–944. © 2009 Wiley Periodicals, Inc.     

Mechanisms of enhanced cellulosic bioethanol fermentation by co-cultivation of Clostridium and Thermoanaerobacter spp

Engineering microbial consortia capable of efficient ethanolic fermentation of cellulose is a strategy for the development of consolidated bioprocessing for bioethanol production. Co-cultures of cellulolytic Clostridium thermocellum with non-cellulolytic Thermoanaerobacter strains (X514 and 39E) significantly improved ethanol production by 194-440%. Strain X514 enhanced ethanolic fermentation much more effectively than strain 39E in co-cultivation, with ethanol production in X514 co-cultures at least 62% higher than that of 39E co-cultures. Comparative genome sequence analysis revealed that the higher ethanolic fermentation efficiency in strain X514 was associated with the presence of a complete vitamin B(12) biosynthesis pathway, which is incomplete in strain 39E. The significance of the vitamin B(12)de novo biosynthesis capacity was further supported by the observation of improved ethanol production in strain 39E by 203% following the addition of exogenous vitamin B(12). The vitamin B(12) biosynthesis pathway provides a valuable biomarker for selecting metabolically robust strains for bioethanol production.     

Ethanol fermentation of HTST extruded rye grain by bacteria and yeasts

High temperature extrusion cooking of rye was used as a pretreatment for ethanol fermentation, and yeasts and bacteria were compared for their fermentation rates. Extrusion cooking caused, on average, a 7.5% increase in ethanol yield in comparison to autoclaved samples. The best results were achieved for grain with a moisture of 21-23% which was extruded at temperatures of 160-180 °C. Extrusion decreased the relative viscosity of rye grain water extracts, so it was possible to mash it without α-amylase. The efficiency of fermentation of extruded rye without Termamyl was equal to that of autoclaved and traditionally mashed rye (using α-amylase). The rate of fermentation of extruded rye grain by Zymomonas was higher during the first stage, but the final ethanol yield was similar for the bacterium and the yeast. Though both microorganisms gave good quality distillates, the concentration of compounds other than ethanol achieved from extruded rye mashes, which were fermented by Z. mobilis, was five times lower than for yeasts.     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.