摘要
燃料乙醇作为生物能源的一种,具有悠久的历史,但一直以来燃料乙醇生产成本较高,无法与传统燃料形成竞争优势。据分析,以淀粉质为原料生产燃料乙醇,原料和能耗成本可占总成本的90%,因此采用更廉价的原料并降低能耗是降低燃料乙醇生产成本的主要途径。菊芋(Helianthus tuberosus Linn)和木薯(Manihot esculenta Crantz)作为生产燃料乙醇的原料作物,具有很好的发展潜力。目前,工业上发酵生产乙醇的主要微生物为酿酒酵母(Saccharomyces cerevisiae),但是酿酒酵母不能直接利用菊粉和淀粉,构建可分泌菊粉酶或淀粉酶的基因工程菌株,可以解决弥补这个缺陷。
本论文首先利用基因敲除技术构建了二倍体高产酒精酿酒酵母WO菌株的尿嘧啶缺陷型菌株W12d,并构建了基于rDNA和6序列的并可以与W12d尿嘧啶缺陷互补的多位点整合型表达载体pMIRSC21、pMIRSC11和pMIDSC11。利用W12d和这些载体成功构建了可以直接利用菊粉或淀粉的酿酒酵母工程菌株,并成功用于糖化菊粉或木薯淀粉发酵生产酒精。
从海洋藻类藻表面分离得到的季也蒙毕赤酵母(Pichia guilliermondii)菌株1可以向培养基分泌大量的菊粉酶。利用pMIRSC21,将海洋季也蒙毕赤酵母strain1菊粉酶基因INUl整合到酿酒酵母W12d染色体上表达后,重组子PguINU14S-04在YPI液体培养基中,28℃条件下,以180rpm振荡培养72h,菊粉酶活力可达34.5+0.3U/mL,且PguINU14S-04产酶能力稳定。在5-L发酵罐中,利用PguINU14S-04同步糖化30%(w/v)菊粉发酵生产酒精,经120h发酵,酒精浓度最终可以达到14.7±0.5%(v/v,20),乙醇转化率为1g菊粉可生产0.386g乙醇;同样条件下,发酵50%菊芋干粉提取物,可以得到12.6±0.4%(v/v,20℃)的酒精,总糖利用率91.9±0.5%,乙醇转化率为1g糖可生产0.331g酒精。通过对表达载体和INUl基因进行改造,得到了产菊粉酶能力更强的转化子PguINU14SM-R27,在同样培养条件下,菊粉酶酶活可以达到42.3±0.4U/mL比PguINU14S-04提高了22.6%。利用PguINU14SM-R27同步糖化30%(w/v)菊粉发酵生产酒精,最终可以得到浓度为14.8±0.2%(v/v,20℃)的酒精,乙醇转化率为1g菊粉可生产0.389g乙醇,发酵时间比PguINU14S-04缩短了12h。
扣囊复膜孢酵母(Saccharomycopsis fibuligera) IFO0111糖化酶Glm可以催化水解生淀粉,而扣囊复膜孢酵母A11菌株α-淀粉酶Alp1可以加快淀粉的液化。本论文同时利用表达载体pMIRSC11、pMIDSC11实现了扣囊复膜孢酵母A11菌株α-淀粉酶基因ALP1和IFO0111菌株糖化酶基因GLM在酿酒酵母W12d中的共表达,筛选到一个淀粉酶活性较高的转化子AMY-39,经72h振荡培养,α-淀粉酶活性可达22.5±0.5U/mL,糖化酶活性可达14.3±0.4U/mL,淀粉酶总活性可达43.2±0.8U/mL。利用转化子AMY-39糖化20%木薯淀粉生产酒精,经120h发酵,酒精浓度可达10.4±0.2%(v/v,20℃),淀粉利用率89%,酒精转化效率为1g木薯淀粉可生产0.413g乙醇。
Fuel ethanol has become very important alternative fuel, however, due to thehigh production cost it could not compete with other available energy sources.Analyzing the cost of fuel ethanol production from starchy raw material, the materialcost and the energy consumption could represent90%. Therefore, adoption of cheaperraw materials and decrease of the energy consumption are the principal approaches inorder to reduce the cost of fuel ethanol production. Helianthus tuberosus Linn andManihot esculenta Crantz could be good candidates for ethanol production. Currently,Saccharomyces cerevisiae is the main producer of bioethanol as it is capable ofproducing high concentration of ethanol from glucose and sucrose and has highethanol tolerance. However, S. cerevisiae can not synthesize and secreteinulinase/amylase that hydrolyzes inulin/starch into fructose and/or glucose.Therefore, in order to overcome these drawbacks it is very important to clone andactively express the inulinase/amylase gene in S. cerevisiae.
For the purpose of obtaining strains yielding high concentration ofamylase/inulinase in controlled fermentation from inulin or cassava starch, a uracilauxotroph strain W12d was constructed by the disruption URA3gene of S. cerevisiaestrain W0, and three multiple integration expression vectors based on rDNA orδ-element were constructed successfully and designated as pMIRSC21, pMIRSC11,pMIDSC11, respectively.
Pichia guilliermondii strain1derived from the surface of marine alga can secrethigh concentration of inulinase into the medium. Inulinase gene INU1from P.guilliermondii strain1was expressed after integrating into chromosome of W12d withthe help of expression vector pMIRSC21. A transformant PguINU14S-04was obtained which could yield stable and high concentration of inulinase i.e.34.5±0.3U/mL in YPI medium after72hours culture at28°C. The transformant couldstably produce high activity of inulinase during long-term batch cultivations.Therefore, it could be used for simultaneous saccharification of inulin and ethanolfermentation. During5-L fermentation, ethanol concentration in the fermentationmedium containing30.0%inulin was14.7%(v/v) and the ethanol productivity wasover0.386g of ethanol per g of inulin. When the tuber meal of H. tuberosus Linn(50.0%) was fermented into ethanol by the transformant PguINU14S-04,12.6%(v/v)ethanol was produced within120h and the ethanol productivity was over0.331g ofethanol per g of sugar. By vector alteration and INU1gene modification, atransformant PguINU14SM-R27was obtained, which could yield22.6%(42.3±0.4U/mL) more inulinase than PguINU14S-04under the same cultureconditions. When PguINU14SM-R27was used for simultaneous saccharification ofinulin and ethanol fermentation, the ethanol concentration reached up to14.8±0.2%v/v20°C, the ethanol productivity was over0.389g of ethanol per g of inulin,and the fermentation time was108h,12h less compared with PguINU14S-04.
Saccharomycopsis fibuligera IFO0111glucoamylase (Glm) could catalyticallyhydroyze raw starch into glucose,while α-amylase (Alp1) from S. fibuligera A11could accelerate the liquation of starch. GLM and ALP1were co-expressed in W12dsuccessfully by using two expression vectors i.e. pMIRSC11and pMIDSC11together.A high amylase yielding transformant AMY-39was obtained, which yielded22.5+0.5U/ml α-amylase,14.3±0.4U/mL glucoamylase and43.2±0.8U/ml totalamylase in YPS medium after72hours culture at28°C. AMY-39was used to produceethanol from fermentation medium containing20%(w/v) cassava starch bysimultaneous saccharification. On completion of fermentation, the concentration ofethanol in the medium was10.4±0.2%(v/v,20°C), the utilization ratio of starch was89%, and the ethanol productivity was0.413g ethanol per g of cassava starch.
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