利用不同启动子控制木糖代谢关键酶基因构建可共代谢葡萄糖木糖重组酿酒酵母

利用不同强度的启动子调控木糖代谢关键酶活性,构建稳定代谢葡萄糖和木糖产乙醇的重组酿酒酵母。以本实验室专利菌株Saccharomyces cerevisiae Y5为宿主菌,将树干毕赤酵母Pichia stipitis CBS6054的木糖还原酶基因XYL1和木糖醇脱氢酶基因XYL2置于磷酸甘油酸激酶基因启动子(PGKp)控制下,酿酒酵母Y5内源的木酮糖激酶基因XKS1分别由己糖激酶基因启动子(HXK2p)及其内源启动子(XKS1p)控制。这3个基因连同各自表达元件导入宿主细胞中,打通其木糖上游代谢途径。酶活测定结果显示,HXK2p对木酮糖激酶表现出更强的启动效率。重组菌Y5-X3-1中木糖还原酶/木糖醇脱氢酶/木酮糖激酶(XR/XDH/XK)的酶活比值为1∶5∶4,其木糖消耗量是宿主菌的5倍,最高乙醇产量为24.35 g/L,达到理论值的73%。结果表明,通过调节XYL1、XYL2及XKS1启动子的强度,调控其表达水平,进而改变3种酶的活性水平,对于提高重组酿酒酵母利用木糖发酵产乙醇有明显效果。     

在酿酒酵母中共表达XYLA和XKS1基因后利用木糖的初步研究

为了使酿酒酵母较好地利用木糖产生乙醇,将来自Thermus thermophilus的木糖异构酶基因XYLA和酿酒酵母自身的木酮糖激酶基因XKS1,构建到酵母表达载体pESC-LEU中,导入酿酒酵母YPH499中,同时成功表达了两种酶基因。该菌以木糖为唯一碳源进行限氧发酵,木糖的利用率为9.64%,为宿主菌的4.17倍,产生2.22 mmol.L-1的乙醇。同时初步探讨了两种酶基因的表达量对酿酒酵母发酵木糖生成乙醇的影响。木糖异构酶对木糖的利用起关键性的作用,木酮糖激酶的过量表达不利于乙醇生成。     

酿酒酵母工业菌株中XI木糖代谢途径的建立

根据代谢工程原理,采取多拷贝整合策略,利用整合载体pYMIKP,将来自嗜热细菌Thermusthermophilus的木糖异构酶(XI)基因xylA和酿酒酵母(Saccharomycescerevisiae)自身的木酮糖激酶(XK)基因XKS1,插入酿酒酵母工业菌株NAN-27的染色体中,得到工程菌株NAN-114。酶活测定结果显示,NAN-114中XI和XK的活性均高于出发菌株NAN-27,表明外源蛋白在酿酒酵母工业菌株中得到活性表达。对木糖、葡萄糖共发酵摇瓶实验结果表明,工程菌NAN-114消耗木糖4.6g/L,产生乙醇6.9g/L,较出发菌株分别提高了43.8%和9.5%。首次在酿酒酵母工业菌株中建立了XI路径的木糖代谢途径。     

木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。     

系列过表达非氧化磷酸戊糖途径基因对重组酿酒酵母菌株木糖代谢的影响

【目的】通过系统研究一个、两个及多个非氧化磷酸戊糖(PP)途径基因组合过表达对酿酒酵母木糖代谢的影响,以优化重组菌株的构建过程,构建高效的木糖代谢酿酒酵母菌株。【方法】在酿酒酵母中双拷贝过表达上游代谢途径的关键酶(木糖还原酶XR,木糖醇脱氢酶XDH,木酮糖激酶XKS),在此基础上构建了一系列PP途径基因过表达菌株,并对其木糖发酵性能进行比较研究。【结果】木糖发酵结果显示,不同组合过表达PP途径基因能不同程度改善重组菌株的木糖发酵性能。其中,过表达PP途径全部基因(RKI1,RPE1,TAL1和TKL1)使菌株的发酵性能最优,其乙醇产率和产量较对照菌株分别提高了39.25%和12.57%,同时较其他基因组合过表达菌株也有不同程度的改善。【结论】通过构建PP途径基因不同组合过表达酿酒酵母菌株,首次对PP途径基因对酿酒酵母木糖代谢的影响进行了系统研究,结果表明,不同组合强化PP途径基因对重组菌株木糖代谢的影响存在差异,相对于其他基因过表达组合,同步过表达PP途径全部基因最有利于碳通量流向乙醇。     

木酮糖激酶表达水平对酿酒酵母木糖代谢产物流向的影响

在酿酒酵母中分别引入真菌和细菌的木糖代谢关键酶,木糖还原酶基因XYL1、木糖醇脱氢酶基因XYL2和木糖异构酶基因xylA. 并在此基础上以共转化策略超表达下游关键酶木酮糖激酶基因XKS1. 与亲本菌株相比,用pMA91和YEp24质粒表达XKS1的重组菌株,木酮糖激酶（xylulokinase,XK）活性分别提高了14和6.7倍. 在限氧条件下,重组菌株对木糖和葡萄糖的共发酵结果显示,表达XYL1,XYL2以及XKS1的重组菌株HSXY-251木糖消耗为12.4 g/L,提高了120.9%,乙醇产量达到9.4 g/L,提高了36%,副产物木糖醇产量为0.7 g/L,下降了84.9%.     

不同宿主来源的重组酿酒酵母混合糖代谢比较

【目的】研究不同工业酿酒酵母宿主背景对重组酵母木糖利用效率的影响。【方法】将木糖利用途径的木糖还原酶(XR)、木糖醇脱氢酶(XDH)和木酮糖激酶(XK)编码基因串联后分别转入3株不同的工业酿酒酵母中,得到重组酵母ZQ1、ZQ5和ZQ7。分别对3个木糖途径代谢基因的表达水平、酶活和重组菌株的木糖发酵效率进行比较。【结果】重组菌株在木糖代谢基因转录、酶活性和木糖利用性能方面有很大差异,其中ZQ5木糖代谢能力最强,ZQ7其次,ZQ1木糖利用能力最弱。ZQ7在初始木糖浓度为20 g/L时木糖利用速率快于ZQ5,表明木糖浓度对重组菌发酵性能评价具有影响。【结论】不同菌株的遗传背景和木糖浓度对重组菌木糖利用的影响很大,评价重组酵母的木糖利用需考虑宿主的遗传背景和底物浓度的影响。     

建立转酮酶酶活测定方法及应用

摘要：目的 转酮酶是非氧化磷酸化戊糖途径中的关键酶。5-磷酸木酮糖是分析测定转酮酶酶活性的底物,然而因该底物难以化学合成,生产成本高,从而导致厂家停止生产,目前市场上无法得到。因此有必要建立起新的转酮酶酶活测定方法。方法 （1）将酿酒酵母的木酮糖激酶基因（XKS1）克隆于pET30a载体。（2）纯化木酮糖激酶。（3）建立酶偶联反应,以木酮糖为底物,将其转化为5-磷酸木酮糖,用于转酮酶酶活的测定。结果 （1）成功地将XKS1基因克隆于pET 30a得到质粒pXZ-X004。（2）质粒pXZ-X004诱导表达产生C端His-tag标签的XK,用Ni柱分离得到纯化的木酮糖激酶(XK)。（3）纯化的XK活性为21.0 U/mg protein,当用12.5%的甘油保存于?80 ℃环境1个月,仍有74%的活性。（4）用纯化的XK建立了转酮酶（TK）酶活测定新方法,当TK酶活低至0.00875 U时仍能够利用新方法检测。结论 本实验通过克隆和表达XKS1基因,成功地建立了TK酶活性测定的新方法,并应用于木糖代谢工程菌株TK酶活的测定,为医学和生态学领域中TK酶活的分析奠定了基础。     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.     

The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism

Many yeast species have growth rates on D-xylulose of 25-130% of those on glucose, but for Saccharomyces cerevisiae this ratio is only about 6%. The xylulokinase reaction has been proposed to be the rate-limiting step in the D-xylulose fermentation with S. cerevisiae. Over-expression of xylulokinase encoding XKS1 stimulated growth on D-xylulose in a S. cerevisiae strain to about 20% of the growth rate on glucose and deletion of the gene prevented growth on D-xylulose and D-xylulose metabolism. We have partially purified the xylulokinase and characterised its kinetic properties. It is reversible and will also accept D-ribulose as a substrate.     

Optimal growth and ethanol production from xylose by recombinant Saccharomyces cerevisiae require moderate D-xylulokinase activity

D-Xylulokinase (XK) is essential for the metabolism of D-xylose in yeasts. However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S. cerevisiae on xylose. We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism in S. cerevisiae. The S. cerevisiae genetic background included single integrated copies of P. stipitis XYL1 and XYL2 driven by the S. cerevisiae TDH1 promoter. Multicopy and single-copy constructs with either XYL3 or XKS1, likewise under control of the TDH1 promoter, or with the native P. stipitis promoter were introduced into the recombinant S. cerevisiae. In vitro enzymatic activity of XK increased with copy number and promoter strength. Overexpression of XYL3 and XKS1 inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells. Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity. Uncontrolled XK expression in recombinant S. cerevisiae is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an S. cerevisiae tps1 mutant during glucose metabolism. To bypass this effect, we transformed cells with a tunable expression vector containing XYL3 under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose. The selected transformant had approximately four copies of XYL3 per haploid genome and had moderate XK activity. It converted xylose into ethanol efficiently.     

Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.     

Physiological and enzymatic comparison between Pichia stipitis and recombinant Saccharomyces cerevisiae on xylose fermentation

In order to better understand the differences in xylose metabolism between natural xylose-utilizing Pichia stipitis and metabolically engineered Saccharomyces cerevisiae, we constructed a series of recombinant S. cerevisiae strains with different xylose reductase/xylitol dehydrogenase/xylulokinase activity ratios by integrating xylitol dehydrogenase gene (XYL2) into the chromosome with variable copies and heterogeneously expressing xylose reductase gene (XYL1) and endogenous xylulokinase gene (XKS1). The strain with the highest specific xylose uptake rate and ethanol productivity on pure xylose fermentation was selected to compare to P. stipitis under oxygen-limited condition. Physiological and enzymatic comparison showed that they have different patterns of xylose metabolism and NADPH generation.     

Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae

To produce an industrial strain of Saccharomyces cerevisiae that metabolizes xylose, we constructed a rDNA integration vector and YIp integration vector, containing the xylose-utilizing genes, XYL1 and XYL2, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, and XKS1, which encodes xylulokinase (XK) from S. cerevisiae, with the G418 resistance gene KanMX as a dominant selectable marker. The rDNA results in integration of multiple copies of the target genes. The industrial stain of S. cerevisiae NAN-27 was transformed with the two integration vectors to produce two recombinant strains, S. cerevisiae NAN-127 and NAN-123. Upon transformation, multiple copies of the xylose-utilizing genes were integrated into the genome rDNA locus of S. cerevisiae. Strain NAN-127 consumed twice as much xylose and produced 39% more ethanol than the parent strain, while NAN-123 consumed 10% more xylose and produced 10% more ethanol than the parent strain over 94 h.     

Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability.

The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials. Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway. To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S. cerevisiae that also expresses the P. stipitis genes for XR and XDH. The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels. The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization. In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied. One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon. Under microaerobic conditions, 5.4 g L(-1) ethanol was produced from 47 g L(-1) xylose during 100 h. In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels. Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain. Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces. The ATP : ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4).