Increasing isobutanol yield by double-gene deletion of PDC6 and LPD1 in Saccharomyces cerevisiae

As a new biofuel, isobutanol has received more attentions in recent years. Because of its high tolerance to higher alcohols, Saccharomyces cerevisiae has potential advantages as a platform microbe to produce isobutanol. In this study, we investigated integration effects of enhancing valine biosynthesis by overexpression of ILV2 and BAT2 with eliminating ethanol formation by deletion of PDC6 and decreasing acetyl-Co A biosynthesis by deletion of LPD1 on isobutanol titers. Our results showed that deletion of LPD1 in strains overexpressing BAT2 and ILV2 increased isobutanol titer by 5.3-fold compared with control strain. Additional deletion of PDC6 in lpd1Δ strains carrying overexpressed BAT2 and ILV2 further increased isobutanol titer by 1.5 fold. Overexpression of BAT2 and ILV2 in lpd1Δ strains and pdc6Δ strains decreased ethanol titers. Glycerol titers of the engineered strains did not have greater changes than that of control strain, while their acetic acid titers were higher, perhaps due to the imbalance of cofactors in isobutanol synthesis. Our researches suggest that double-gene deletion of PDC6 and LPD1 in strains overexpressing BAT2 and ILV2 could increase isobutanol production dramatically than single-gene deletion of PDC6 or LPD1. This study reveals the integration effects of overexpression of ILV2/BAT2 and double-gene deletion of LPD1 and PDC6 on isobutanol production, and helps understanding future developments of engineered strains for producing isobutanol.

Phase Equilibrium of Isobutanol in Supercritical CO2

Vapor-liquid phase equilibrium data including composition,densities,molar volume and equilibrium constant of isobutanol in supercritical carbon dioxide from 313.2K to 353.2K were measured in a variable-volume visual cell.The properties of critical point were obtained by extrapolation.The results showed that critical temperature,critical pressure and critical compressibility factor of CO2-isobutanol system decreased with the increase of critical CO2 content.The phase equilibrium model was established by Peng-Robinson equation of state and van der Waals-2 mixing regulation,and model parameters were determined by optimization calculation of nonlinear least square method.The correlation between calculated values and the experimental data showed good agreement.

Kinetic Study of Esterification of Lactic Acid with Isobutanol and n-Butanol Catalyzed by Ion-exchange Resins

The esterification reactions of lactic acid with isobutanol and n-butanol have been studied in the presence of acid ion-exchange resin Weblyst D009.The influences of catalyst loading,stirrer speed,catalyst particle size,initial reactant molar ratio and temperature on the reaction rate have been examined.Experimental kinetic data were correlated by using the Pseudo-homogeneous,Langmuir-Hinshelwood and Eley-Rideal models.Nonideality of the liquid phase was taken into account by using activities instead of molar fractions.The activity coefficients were calculated according to the group contribution method UNIFAC.Provided that the nonideality of the liquid is taken into account,the esterification kinetics of lactic acid with isobutanol and n-butanol catalyzed by the acid ion-exchange resin can be described using all three models with reasonable errors.

Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol,a next-generation biofuel,in Saccharomyces cerevisiae.Here,we explore how two of these strategies,pathway re-localization and redox cofactor-balancing,affect the performance and physiology of isobutanol producing strains.We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced(NADPH-dependent)or redox-balanced(NADH-dependent)ketol-acid reductoisomerase enzyme.We then conducted transcriptomic,proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains.Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes.Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version,albeit at low overall pathway flux.Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters,which are required cofactors for the dihydroxyacid dehydratase enzyme.We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation,thereby increasing cellular iron levels.The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold,demonstrating that both localizations can support flux to isobutanol.

Separation of n-heptane/isobutanol via eco-efficient vapor recompression-assisted distillation:process optimization and control strategy

In this study,vapor recompression and heat integration assisted distillation arrangements with either the low or high pressure in the reflux drum are proposed to reduce and/or eliminate the application of the costly refrigerant for the separation of n-heptane and isobutanol mixture.The high-pressure arrangement with vapor recompression and heat integration is the most attractive among these four intensified configurations since it can reduce total annual cost by 18.10%,CO_(2) emissions by 75.01%based on natural gas(78.78%based on heavy oil fuel),and second-law efficiency by 61.20%compared to a conventional refrigerated distillation system.Furthermore,exergy destruction in each component is calculated for the heat integration configurations and is shown in pie diagrams.The results demonstrate that the high-pressure configuration presents unique advantages in terms of thermodynamic efficiency compared to the low-pressure case.In addition,dynamic control investigation is performed for the economically efficient arrangement and good product compositions are well controlled through a dual-point temperature control strategy with almost negligible product offsets and quick process responses when addressing 20%step changes in production rate and feed composition.Note that there are no composition measurement loops in our developed control schemes.

Waste Cashew Apple (Anacardium occidentale) as Feedstock for Simultaneous Production of Two Main Ecofriendly Fuels

The present work investigated an effective low-cost production of bioethanol by the use of rejected cashew apples (CAs) in Ivorian plantations. Fresh CAs were cut into 8-10 mm slices and submitted to a drying cycle of two periods (for the sake of easing their rehydration) in an oven with forced air convection. Temperature was first set at 30˚C for 3 hours, and then raised at 50˚C until constant weight. Drying brought about 82% weight loss, and the dried slices were rehydrated in a ratio of 1:4 (w/v) in warm distilled water to reconstitute a 10 Brix degree (˚B) juice with 1.042 g/cm3 density. The sugar content of the juice was increased to 20˚B (syrup) by thermal evaporation. The process was optimized using a response surface methodology (RSM) by applying a central composite plan in order to minimize heat-sensitive compound degradation. The optimal operating conditions for temperature and time of heating were precisely 68,239˚C and 83,314 min, respectively. The commercial baker’s yeast Saccharomyces cerevisiae was used to seed the 20˚B cashew apple syrup following a batch fermentation at 30˚C. The total alcohol content recorded after 24 hours was 8.24% ± 0.11% made up of almost 97% of ethanol and isobutanol (a higher alcohol). Analysis of alcoholic profiles by flam ionization detector-gas chromatography (GC-FID) showed an ethanol content of 3.92% and an almost similar but higher quantity of isobutanol (4.05%) with the latter being a by-product. As bio-based isobutanol attracts more and more attention due to its wide application and excellent fuel performance as compared to ethanol, it emerged from this study that neglected cashew apples can be successfully employed as valuable raw material for the simultaneous production of both biofuels currently used as sustainable sources of renewable energy.

酿酒酵母异丁醇合成途径调控的研究进展

酿酒酵母是工业化生产中常用的微生物,具有耐低pH值、抗逆性强等优点。利用微生物进行生物基产品的生产已成为绿色生物制造的重大发展方向。异丁醇是一种支链高级醇,在化工、能源等领域有较好的应用前景。利用工程酿酒酵母合成的异丁醇是一种可持续、可再生、经济环保的生物能源,有望成为下一代生物燃料。构建高效合成异丁醇的酿酒酵母细胞工厂可以实现生物燃料异丁醇的绿色制造,但目前酿酒酵母合成异丁醇的能力不足,限制了异丁醇的工业化发展,而与合成生物学相结合的理性代谢调控策略是提高微生物异丁醇合成能力的有效方法之一。本文围绕异丁醇合成途径构建、竞争途径阻断、辅因子平衡、合成途径重定位、转录调控因子调节以及耐受性强化几方面,综述了酿酒酵母中异丁醇合成调控策略的研究进展。这些策略显著提高了工程酵母异丁醇的转化率和产率,但在异丁醇工业化生产上仍存在许多不足。为了推动异丁醇的工业化生产,本文讨论了目前酿酒酵母生产异丁醇的瓶颈及解决方法,以期对异丁醇的绿色制造提供有价值的参考。

低产高级醇酵母的筛选及其发酵特性研究

为了得到低产高级醇的酵母菌株,采用传统筛选方式从发酵酒醅中筛选菌株,采用分子生物学技术对其进行鉴定并研究其发酵特性。结果表明,从样品中得到1株低产高级醇菌株F11,其异丁醇和异戊醇的总产量为199.09 mg/L,比对照菌株低22.50%;该菌株耐受性强,在温度42℃、糖度25°Bx、乙醇体积分数12%,乳酸体积分数5%,乙酸体积分数5‰,己酸体积分数1‰的条件下仍能正常生长,鉴定结果表明该菌株为库德里阿兹威毕赤酵母(Pichia kudriavzevii)。该菌株产高级醇的阶段主要在主发酵期,其挥发性代谢物中酯、醇、酸、醛类化合物占挥发物总量的84.16%,适合在发酵酒和蒸馏酒中应用。

异丁醇改性六羟甲基三聚氰胺的合成及其消抑泡性能

采用六羟甲基三聚氰胺(HMM)和异丁醇进行醚化反应,合成一种多支链结构化合物异丁醇改性六羟甲基三聚氰胺消抑泡材料(HMIB),通过傅里叶变换红外光谱仪(FT-IR)对产物进行结构表征。将HMIB与STK系列表面活性剂STK332、STK542及STK416复配,测试并对比HMIB及复配产物分别在阴离子表面活性剂十二烷基苯磺酸钠(SDBS)、阳离子表面活性剂十二烷基三甲基溴化铵(DTAB)、非离子表面活性剂Lutensol A9N(AEO-9)、工业品白猫洗涤剂4种起泡液中的消泡时间以及消抑泡率。结果表明:HMIB复配产物的消抑泡率均大于HMIB的消抑泡率,HMIB与表面活性剂STK416复配在白猫洗涤剂起泡液中的消泡率最高,为95.29%,HMIB和STK332复配后在白猫洗涤剂中的抑泡率最高,可达76.47%。

碱金属助剂对MnO_x/ZrO_2催化合成甲醇及异丁醇反应性能的影响

研究了MnOx/ZrO2 催化剂中添加的碱金属助剂对以合成气合成甲醇及异丁醇反应的影响 ,并采用BET ,XRD和NH3 TPD等表征技术 ,考察了碱金属助剂的添加对催化剂结构和表面性质的影响 .结果表明 ,碱金属助剂对MnOx/ZrO2 催化剂的催化性能有明显影响 .在实验条件下 ,添加 1 0 %K(K2 CO3作为前驱物 )的催化剂 ,其催化性能最好 ,醇的选择性高达 74 8% ,异丁醇选择性达 16 3 % .添加碱金属助剂也使MnOx/ZrO2 催化剂的表面性质发生了很大变化 。

焙烧温度对K-Cu/Zn/La/ZrO_2催化剂上异丁醇合成的影响

采用共沉淀法制备了Cu/Zn/La/ZrO2催化剂,并浸渍K对其进行改性。以合成气合成异丁醇为探针反应,考察了焙烧温度对K-Cu/Zn/La/ZrO2催化剂合成反应的影响。采用XRD、TG、BET、NH3-TPD、H2-TPR、Raman及XPS等手段表征了催化剂的结构和表面性质。结果表明,催化剂体相结构、比表面积与其催化活性没有直接关系;催化剂表面强酸中心的存在不利于异丁醇的生成;Cu-Zn-Zr活性组分间协同作用对催化剂活性影响很大,450℃焙烧温度下,Cu-Zn-Zr活性组分间协同作用最强,CuO最容易还原,催化活性最高,此时CO的转化率(物质的量)达到69.47%,醇中异丁醇的选择性(质量分数)为19.09%,甲醇+异丁醇的选择性(质量分数)为95.02%。

锆系催化剂上合成气合成甲醇、异丁醇的研究

沉淀法制备Ｚｒ－Ｋ催化剂，由合成气合成甲醇和异丁醇，异丁醇的重量百分组成在１０ＭＰａ时达１５．１３％、时空产率在１４ＭＰａ时达６．９２ｍｌ／ｈ·Ｌｃａｔ。催化剂的制备条件、制备方法、助剂Ｋ_２Ｏ含量以及反应的工艺条件对催化剂的活性、选择性有着明显的影响。

超临界相CO加氢合成甲醇、异丁醇的研究

以正十一～十三烷的混合物为超临界介质，在反应温度３６０～４１０℃、合成气压力７５ＭＰａ、进气空速１７００ｈ－１、介质压力１７８ＭＰａ、总压９３ＭＰａ的实验条件下，研究了固定床反应器中Ｚｎ－Ｃｒ、Ｃｕ－Ｚｎ－Ｃｒ催化剂在超临界相和气相条件下合成甲醇、异丁醇的性能。结果表明，超临界相反应的ＣＯ转化率高于气相反应。在超临界条件下反应，醇类选择性随着温度升高下降较慢，而气相反应醇类选择性随着温度升高下降较快。气相反应产物以甲醇、异丁醇为主，含少量乙醇和正丙醇，超临界相反应的产物分布与气相反应的明显不同，甲醇含量减少，乙醇、正丙醇和异丁醇都有不同程度增加。超临界流体的存在对合成醇链增长有影响。

酿造条件对苹果白兰地中异丁醇、异戊醇及苯乙醇含量的影响

采用顶空固相微萃取-气相色谱质谱联用(HS-SPME-GC-MS)分析了酿酒酵母菌种、发酵温度、酵母接种浓度、可同化氮和碳源对苹果白兰地酿造过程中异丁醇、异戊醇及苯乙醇含量的影响。结果表明,采用酿酒酵母(CICC 32130)、发酵温度20℃,酵母接种浓度1×104 cfu/m L的发酵条件苹果白兰地酒中异丁醇、异戊醇及苯乙醇含量相对较低;可同化氮与苹果白兰地酿造中高级醇的形成密切相关,在发酵液中添加适宜的铵态氮有利于降低异丁醇、异戊醇及苯乙醇的含量,但是不宜大量添加一种氨基酸,以防相应的高级醇大量产生;而为提高酒精度在苹果白兰地酿造中过多添加碳源不仅会阻碍酵母的正常代谢,还会因其它营养物质的相对缺乏导致酵母异常发酵,进一步导致苹果白兰地酒中异丁醇、异戊醇及苯乙醇含量的增加。

异丁醇萃取三种酶解产物苦味肽的研究

以牡蛎、鸡肉和南极磷虾为原料,选用胰蛋白酶和风味蛋白酶组成复合酶对其进行酶解,以感官评分和肽分子量分布为指标,考察异丁醇萃取3种酶解产物苦味肽过程中感官评分和肽分子量分布的变化。研究结果表明:异丁醇萃取牡蛎、鸡肉和南极磷虾乙醇相上清液感官评定都具有苦味,苦味评分值分别为6.8,4.4,5.0分。多肽分子量测定表明异丁醇萃取牡蛎、鸡肉和南极磷虾乙醇相上清液分别含有94.86%,97.61%和97.44%小于5ku的多肽片段。采用80%乙醇萃取酶解产物,再经过异丁醇萃取乙醇相,是一种快速萃取牡蛎、鸡肉和南极磷虾酶解产物中苦味肽的方法。

稀土助剂对Zr－Mn－K催化剂上甲醇、异丁醇合成的影响

在Ｚｒ－Ｍｎ－Ｋ催化剂中添加稀土氧化物助剂Ｐｒ６Ｏ_（１１）、Ｓｍ_２Ｏ_３可以显著提高合成气合成甲醇、异丁醇的活性，特别是异丁醇的时空产率在４００℃、１０ＭＰａ、５０００^（－１）的反应条件下，由原来的５．９分别增加到１１．５、９．８ｍｌ／ｈ·ｌｃａｔ．。催化剂的ＸＲＤ、ＬＲＳ、ＴＰＲ、ＴＥＭ图和比表面测定结果一致表明，稀土元素可使催化剂晶粒细化、高度分散，稳定了四方相。ＸＰＳ结果表明：ＲＥＯ的加入使表面Ｍｎ富集，且催化剂表面Ｍｎ和Ｋ的比例影响其合成甲醇、异丁醇的活性与选择性。

Pd对Zr-Mn-K催化CO加氢合成甲醇与异丁醇的影响

考察了添加Pd对Zr Mn K催化CO加氢合成甲醇与异丁醇的影响 .与Zr Mn K催化剂相比 ,Pd/Zr Mn K催化剂的催化活性及异丁醇选择性明显升高 .在SV =10 0 0 0h-1,p =8 0MPa和T =6 73K的反应条件下 ,1 5 %Pd/Zr Mn K催化剂上醇的时空收率由原来的 12 2 8升高到 2 32 6 g/ (kg·h) ,异丁醇的选择性由原来的 15 5 %升高到 2 1 7% .当Pd的添加量大于1 5 %时 ,催化剂的活性及异丁醇选择性开始下降 .H2 和CO吸附实验结果表明 ,Pd/Zr Mn K催化剂表面维持有较高的H2 和CO吸附量 ,有利于甲醇与异丁醇生成速率的提高 .当Pd的添加量超过 1 5 %时 ,Pd的分散度降低 ,粒径变大 ,吸附H2 和CO的量减少 。

产异丁醇大肠杆菌工程菌的构建

酮酸脱羧酶是异丁醇生物合成的关键酶,而大肠杆菌中没有此基因。将乳脂乳球菌NIZO B1157的2-酮酸脱羧酶基因kdcA克隆到非生产菌株大肠杆菌中,同时串联表达与前体物质酮酸相关的alsS、ilvC和ilvD基因,构建了串联表达质粒pSTV29-alsS-ilvC-ilvD-kdcA。大肠杆菌工程菌成功表达了4个基因,并能利用葡萄糖发酵产异丁醇,发酵24 h后最高产量为3 g/L。

异丁醇生物合成的研究进展

异丁醇一直以来作为重要的化工原料被用于诸多领域。近年来发现异丁醇也可以作为新一代的生物燃料,并且具有高热值、易混合、高辛烷值等特有的优势,体现出了巨大的发展潜力,越来越受到人们的关注。就异丁醇作为新型生物燃料的生产现状和生物合成异丁醇在国内外的研究进展进行介绍与论述,并对生物合成异丁醇的未来发展的趋势进行展望。

超细ZrO_2催化剂上合成甲醇和异丁醇

采用超临界干燥法制备ＺｒＯ２超细催化剂，用于合成气高选择性合成甲醇、异丁醇。考察了碱金属助剂、焙烧温度、焙烧时间、助剂Ｋ２Ｏ含量、成型压力及反应温度等对合成甲醇、异丁醇的影响。结果是浸渍２％Ｋ２Ｏ、经６００℃焙烧４ｈ后得到的催化剂可高选择性合成甲醇、异丁醇，在４２０℃、１０ＭＰａ、５０００ｈ－１条件下生成异丁醇的组成为１６．３１％（ｍａｓｓ），时空产率为８．９０ｍｌ／（ｈ·Ｌ）。