重组大肠杆菌发酵生产树莓酮

树莓酮是一种在工业上有多种用途的重要香料物质。将来源于不同植物的4-香豆酰辅酶A连接酶(4-coumarate:Co A ligase,4CL1)、苄基丙酮合酶(benzalacetone synthase,BAS)和苄基丙酮还原酶(raspberry ketone/zingerone synthase,RZS1)基因分别克隆到载体p CDF-DUET-1和p ET-DUET-1中,构建重组质粒并转入大肠杆菌W3110(DE3)中,成功获得了以对香豆酸为前体发酵生产覆盆子酮的重组菌株WCC-1。该菌摇瓶发酵培养2 h后,添加0. 8 mmol/L的异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside,IPTG),30℃诱导,发酵72 h条件下,产覆盆子酮的质量浓度达到125. 86 mg/L;在3 L发酵罐中进行补料分批发酵68 h,覆盆子酮的质量浓度提高到178. 13 mg/L。该研究展现了微生物发酵生产树莓酮的前景。     

链格孢菌侵染采后伽师瓜抗病关键酶活性变化规律

该文将链格孢菌无损接种于采后伽师瓜大、小、无网纹处,通过研究苯丙烷代谢相关酶及细胞壁降解酶活性变化规律,比较伽师瓜不同网纹处、不同组织抗病性差异。结果表明：大网纹组发病率显著高于其他组,在侵染18 d时病害发生率为大网纹组89.00% 、小网纹组83.33% 、无网纹组78.00% 。苯丙氨酸解氨酶（phenylalanine ammonia lyase,PAL）、4-香豆酰辅酶 A 连接酶（4-coumaroyl-Coa ligase,4CL）、几丁质酶（chitinase,CHT）、β-1,3 葡聚糖酶（β-1,3-glucanase,GLU）均呈先上升后下降的趋势,接种组均高于对照组,差异显著（p＜0.05）;无网纹组外、内果皮的PAL活性峰值均高于大网纹组和小网纹组,活性为135.16、221.81 U/g,小网纹组果肉处PAL峰值高于大网纹组和无网纹组,活性为44.42 U/g;无网纹组外、内果皮及果肉的4CL活性峰值均高于小网纹组和大网纹组,活性为44.42、131.28、221.13 U/g;无网纹组外、内果皮及果肉的CHT及GLU活性峰值均高于大网纹组和小网纹组,CHT活性分别为170.30、153.60、121.48 U/g,GLU 活性分别为 29.84、19.27、15.71 U/g。 PAL、4CL、CHT、GLU 活性可以在一定程度上反映伽师瓜对于链格孢菌侵染的抵抗能力,无网纹最强,小网纹次之,大网纹最弱;外果皮最强,内果皮次之,果肉最弱。     

茉莉酸甲酯处理对鲜切菠萝品质及抗氧化活性的影响

研究茉莉酸甲酯处理对鲜切菠萝贮藏期间品质和抗氧化活性的影响。先将完整的菠萝果实在20 ℃条件 下分别用浓度为0、1、10、100 μmol/L的茉莉酸甲酯熏蒸12 h,然后进行鲜切加工并于15 ℃条件下贮藏48 h。结 果表明,茉莉酸甲酯处理可促进鲜切菠萝总酚含量和1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl, DPPH）自由基清除能力上升,抑制可溶性固形物及可滴定酸含量下降,而对色泽及菌落总数无显著影响 （P＞0.05）。其中,10 μmol/L茉莉酸甲酯处理效果最好,能显著地诱导鲜切菠萝贮藏期间苯丙氨酸解氨酶和肉桂 酸-4-羟化酶活力的上升（P＜0.05）,延缓4-香豆酸辅酶A连接酶活力的下降（P＜0.05）,从而促进总酚和总黄酮的 积累,提高DPPH自由基清除能力。这些结果表明,茉莉酸甲酯处理可保持鲜切菠萝的品质并提高其抗氧化活性。     

酿酒酵母表面展示表达的β-1,3-1,4-葡聚糖酶酶学性质研究

固定化酶是酵母表面展示技术的1个重要应用方向。本文应用食品级酵母展示表达系统进行表达,成功获得具有生物活性且固定在酿酒酵母细胞表面的β-1,3-1,4-葡聚糖酶,并测定其酶学性质。结果表明,与分泌表达的自由酶相比,展示表达的β-1,3-1,4-葡聚糖酶的酶学性质发生了改变。其最适温度为60℃,热稳定性增强。50℃保温3 h,对酶活几乎没有影响。60℃保温1 h后的酶活为初始酶活的129.2%。随着该温度下保温时间的延长,酶活迅速下降,保温3h后的酶活为初始酶活的64.6%。70℃保温1 h,酶活增加到初始酶活的109.2%;1 h后酶活开始下降;70℃保温3 h后残留酶活仅为初始酶活的35.8%。展示表达的β-1,3-1,4-葡聚糖酶最适pH为6.0,在pH 4~7范围内酶的稳定性较好。     

纳豆激酶基因在酿酒酵母中的表达

目的:从纳豆芽孢杆菌基因组DNA中扩增纳豆激酶基因,以实现该基因在酿酒酵母中的表达.方法:通过PCR方法扩增纳豆激酶基因,利用DNA重组技术构建重组质粒pYES2-NK,转化酿酒酵母H158感受态细胞;经β-半乳糖诱导表达后,用纤维蛋白平板法检测纤溶酶活性.结果:克隆到大小为1192bp的纳豆激酶基因,编码397个氨基酸;活性检测表明酿酒酵母发酵液的上清液具有纤溶酶活性.结论:纳豆激酶基因在重组酿酒酵母中实现了分泌表达.     

人β-防御素3基因的克隆及其在酿酒酵母中的表达

以质粒pPIC9K．hBD3为模板,扩增得到一段上游带信号肽因子（a-factor）的人3-防御素-3（hBD-3）基因,克隆至酿酒酵母穿梭质粒pYES2中,构建了酿酒酵母表达载体pYES2．a-factor-hBD3（pYa-hBD3）。将重组质粒转化至SaccharomycescerevisiaeINVSCl中,鉴定阳性重组子,经2％半乳糖诱导表达。实验发现表达所得hBD-3对金黄色葡萄球菌（ATCC6538）和大肠杆菌（ATCC10231）具有明显的抑菌活性。hBD-3基因片段在酿酒酵母中的表达,为进一步探讨hBD-3的生物活性及防御素应用安全性打下基础。     

产5-氨基乙酰丙酸酿酒酵母工程菌株的构建

5-氨基乙酰丙酸(5-aminolevulinic acid,ALA)是一种天然存在的非蛋白质类氨基酸,在医学和农业领域应用广泛。为构建生产ALA的酿酒酵母细胞工厂,作者首先在酿酒酵母中分别表达了来源于酿酒酵母和类球红细菌中编码ALA生物合成C4途径ALA合酶的hem1和hemA基因,同时又表达了来源于大肠杆菌编码C5途径关键酶谷氨酰-tRNA还原酶和谷氨醛氨基转移酶的基因hemA和hemL。结果表明,表达酿酒酵母自身来源的hem1基因更利于ALA的合成,ALA产量为327.6 mg/L。在此基础上将C4和C5途径的基因hem1、hemA和hemL在酿酒酵母中过量表达,在添加前体物质甘氨酸和琥珀酸的条件下,ALA产量为525.8 mg/L,实现了在酿酒酵母中合成ALA。     

瑞氏木霉内切葡聚糖酶基因在酿酒酵母中的表达研究

将PCR合成的瑞氏木霉 (Trichodermareesei) β 内切葡聚糖酶I(EGI)cDNA基因片断分别插入酵母met1 0和 pgk1启动子和终止子序列之间 ,构建了在不同启动子控制下 ,具有不同拷贝数的eg1表达分泌质粒pRS41 5ME、pRS41 5PE和 pRS42 5PE。通过电转化使重组质粒转移至实验室酿酒酵母H1 5 8菌株中 ,分别得到了 3株酵母转化子H1 p、H2p和H1m。在 3株酵母转化子中 ,重组 β 内切葡聚糖酶I都能在酶自身信号肽序列引导下进行分泌型表达。在YPD培养基中 3株重组酵母生长速率大致相同 ,H1m ,H1 p与H2 p的内切葡聚糖酶活力分别为 70 .4,1 2 6.7和 1 2 5 .0U/mL。     

酿酒酵母胱硫醚β-裂解酶的异源表达及催化生成糠硫醇

为明晰糠硫醇生物转化机制,将来源于酿酒酵母（Saccharomyces cerevisiae）G20的胱硫醚β-裂解酶（cystathionine β-lyase,Str3p）在大肠杆菌（Escherichia coli）中实现异源表达,并验证其催化性质。正交设计试验优化大肠杆菌基因工程菌蛋白表达的诱导条件,其最适诱导表达条件为诱导温度20 ℃、异丙基硫代半乳糖苷浓度0.5 mmol/L、诱导时间13 h。该条件下获得的菌体经超声破碎和镍柱亲和层析,纯化得到的可溶性Str3p分子质量约为52 kDa,其表达量达1.26 mg/mL,较优化前的表达量和酶活力分别提高41.7%和38.6%。实验发现Str3p能够催化裂解半胱氨酸-糠醛加合物生成糠硫醇,且催化过程受pH值影响较大,在pH 8.0时糠硫醇产量达到47 μmol/L。本研究确定Str3p在E.coli BL21中的异源表达及催化特性,为生物转化合成天然糠硫醇提供新的参考途径。     

硬脂酰辅酶A去饱和酶1基因表达对草原安格斯牛血液脂肪酸组成的影响

研究硬脂酰辅酶A去饱和酶1(stearoyl-coenzyme A desaturase 1,SCD1)基因表达对草原安格斯牛血液脂肪酸组成的影响,采集38头平均体质量(698±34)?kg、48月龄草原安格斯牛血液样品,测定其脂肪酸组成与含量,采用实时荧光定量聚合酶链式反应测定SCD1基因表达量,一代测序技术检测SC...     

Energy‐dependent effects of resveratrol in Saccharomyces cerevisiae

The metabolic effects induced by resveratrol have been associated mainly with the consumption of high‐calorie diets; however, its effects with standard or low‐calorie diets remain unclear. To better understand the interactions between resveratrol and cellular energy levels, we used Saccharomyces cerevisiae as a model. Herein it is shown that resveratrol: (a) decreased cell viability in an energy‐dependent manner; (b) lessening of cell viability occurred specifically when cells were under cellular respiration; and (c) inhibition of oxygen consumption in state 4 occurred at low and standard energy levels, whereas at high energy levels oxygen consumption was promoted. These findings indicate that the effects of resveratrol are dependent on the cellular energy status and linked to metabolic respiration. Importantly, our study also revealed that S. cerevisiae is a suitable and useful model to elucidate the molecular targets of resveratrol under different nutritional statuses. Copyright © 2016 John Wiley & Sons, Ltd.     

D-lactic acid production by metabolically engineered Saccharomyces cerevisiae

Poly D-lactic acid is an important polymer because it improves the thermostability of poly L-lactic acid by the stereo complex formation. We constructed a metabolically engineered Saccharomyces cerevisiae that produces D-lactic acid efficiently. In this recombinant, the coding region of pyruvate decarboxylase 1 (PDC1) was completely deleted, and two copies of the D-lactate dehydrogenase (D-LDH) gene from Leuconostoc mesenteroides subsp. mesenteroides strain NBRC3426 were introduced into the genome. The D-lactate production reached 61.5 g/l, the amount of glucose being transformed into D-lactic acid being 61.2% under neutralizing conditions. Additionally, the yield of free D-lactic acid was also shown to be 53.0% under non-neutralizing conditions. It was confirmed that D-lactic acid of extremely high optical purity of 99.9% or higher. Our finding obtained the possibility of a new approach for pure d-lactic acid production without a neutralizing process compared with other techniques involving lactic acid bacteria and transgenic Escherichia coli.     

Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe

The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology. The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The heterologous protein is expressed at a high level in cell extracts of a S. cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of l-lactate during fermentation on glucose-rich medium in the presence of malic acid. Isotopic filiation was used to demonstrate that 75% of the l-lactate produced originates from endogenous l-malate and 25% from exogenous l-malate. Moreover, although a small amount of exogenous l-malate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded l-malate was converted into l-lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways. These results indicate that the sole limiting step for S. cerevisiae in achieving malolactic fermentation is in malate transport. This was confirmed using a different model, S. pombe, which efficiently degrades l-malate. Total malolactic fermentation was obtained in this strain, with most of the l-malate converted into l-lactate and CO₂. Moreover, l-malate was used preferentially by the malolactic enzyme in this strain also.     

酿酒酵母工程菌产青蒿酸的发酵动力学研究

以青蒿酸产量为考察指标,在50 L发酵罐中对酿酒酵母（Saccharomyces cerevisiae）工程菌1211发酵产青蒿酸的溶氧参数进行优化。在此基础上,根据Logistic方程及Luedeking-Piret方程构建酿酒酵母工程菌1211分批发酵产青蒿酸的动力学模型。结果表明,当溶氧参数为25%～30%时,青蒿酸产量最高,为（6 269.6±100.3）mg/L。青蒿酸合成与菌体生长呈现部分生长偶联型。通过Origin 9.0软件对动力学模型进行非线性拟合,发现S. cerevisiae工程菌1211的菌体繁殖生长、青蒿酸合成以及基质消耗动力学模型的拟合度R2分别达到了0.995 85、0.979 04和0.995 48,该动力学模型能够很好的描述S. cerevisiae工程菌1211分批发酵过程。该研究为青蒿酸的低成本发酵及工业化大规模发酵生产提供了理论基础。     

重组酿酒酵母发酵生产瓦伦西亚烯及其衍生物

以酿酒酵母为宿主菌株,重构其体内代谢途径,生物合成瓦伦西亚烯及其衍生物。该研究在酿酒酵母菌株中引入来源于纯天仙子的细胞色素P450单加氧酶(cytochrome P450 monooxygenase,HPO)、来源于黄扁柏的瓦伦西亚烯氧化酶(valencene oxidase,Cn VO),并构建了它们对应的4个突变体HPOM、Cn VO-3、Cn VO-4、Cn VO-34,与来源于拟南芥的细胞色素还原酶(cytochrome P450 reductase,At CPR)组合表达,通过静息细胞试验,筛选得到表达HPO和HPOM的菌株催化瓦伦西亚烯的效果最优,选取HPOM作为后续试验的基础。进一步引入来自黄扁柏的瓦伦西亚烯合成酶(valencene synthase,Cn VS),过表达醇脱氢酶(alcohol dehydrogenase,ADH1),截短的3-羟基-3-甲基戊二酰辅酶A还原酶(truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase,t HMG1)。最终获得1株原位生产瓦伦西亚烯及其衍生物的重组酵母菌株PK2-24,进行3 L发酵罐的发酵试验,158 h后总萜产量达310. 94 mg/L,较原始菌株提高了111倍。该研究为利用酵母规模化生产瓦伦西亚烯及其衍生物奠定了重要基础。     

低产尿素黄酒酵母工程菌的酿造特性

为考察酵母工程菌在黄酒酿造过程中的发酵性能及其降低发酵液中尿素和氨基甲酸乙酯(ethyl carba-mate,EC)的能力,以前期构建的降低黄酒中尿素和EC效果最好的酵母工程菌N85^DUR1,2-c为研究对象,利用单因素试验考察黄酒发酵工艺对其降低发酵液中尿素和EC能力的影响,并对其在生产试验过程中的发酵性能进行研究。结果表明,酵母接种量、发酵温度以及麦曲添加量等工艺参数对工程菌N85^DUR1,2-c低产尿素和EC的性能没有明显的影响,且含量低于亲本菌株。50 kL生产试验表明,工程菌N85^DUR1,2-c所酿黄酒中理化指标含量正常,符合黄酒国标的要求。而N85^DUR1,2-c发酵液中尿素和EC的含量分别为(2.4±0.2)mg/L和(14.9±0.6)μg/L,较亲本菌株分别降低了90.7%和54.6%,且贮存过程中EC含量增加缓慢。说明酵母工程菌N85^DUR1,2-c在不改变黄酒优良品质的前提下,能够显著地降低发酵液中尿素的含量,可以从根源上减少黄酒中EC的积累,提高饮用...     

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac⁺ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac⁺ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac⁺ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.     

The determinants of heat-shock element-directed lacZ expression in Saccharomyces cerevisiae.

Heat-shock induction of heat-shock protein genes is due to a specific promoter element (the heat-shock element, HSE). This study used lacZ under HSE control (HSE-lacZ) to characterize HSE activity in Saccharomyces cerevisiae cells of different physiological states and differing genetic backgrounds. In batch fermentations HSE-lacZ induction by heat shock was maximal in exponential growth, and showed marked decline with the approach to stationary phase. Expression in the absence of heat shock was unaffected by growth phase, indicating that the growth-dependent expression of many yeast heat-shock genes uses promoter elements in addition to the HSE. Heat-induced expression was strongly influenced by the temperature at which cultures were grown. While basal, uninduced expression was constant during growth at different temperatures to 30 degrees C, induction by transfer to 39 degrees C was reduced by increases in growth temperature as low as 18-24 degrees C. Maximal HSE-lacZ induction (30- to 50-fold) was in cultures grown at low temperatures (18-24 degrees C), then heat shocked at 39 degrees C. Ethanol was a poor inducer. Mutations having little effect on HSE-lacZ expression included a respiratory petite; ubi4 (which inactivates the poly-ubiquitin gene); also ubc4 and ubc5 (which each inactivate one of the ubiquitin ligases involved in degradation of aberrant protein). pep4-3 increased both basal and induced beta-galactosidase about two-fold, probably because of slower turnover of this enzyme in pep4-3 strains.