产对香豆酸酿酒酵母工程菌株的构建与优化

对香豆酸是黄酮类、芪类等天然活性化合物的重要前体,在生物医药、食品等行业应用广泛。与传统植物提取和化学合成相比,微生物合成对香豆酸因其具有生产周期短、转化效率高等优势而得到广泛关注。为构建高产对香豆酸酵母工程菌株,以酿酒酵母为出发菌,通过敲除酪氨酸合成竞争路径基因ARO10和PDC5,突变芳香族氨基酸合成调控基因ARO4~(K229L)与ARO7~(G141S)、解除酪氨酸负反馈抑制、并整合酪氨酸解氨酶FjTAL,获得的工程菌C001对香豆酸产量为296.73 mg/L。为进一步提高对香豆酸合成前体积累,分别敲除8个与氨基酸、糖类等转运相关基因并强化糖异生途径,分析其对对香豆酸积累的影响。结果表明,敲除GAL2及过表达EcppsA,对香豆酸产量提高至475.11 mg/L。最后,分析了FjTAL蛋白锚定至酵母液泡对产物积累的影响,结果表明其定位液泡后对香豆酸产量明显提升,达到593.04mg/L。通过强化前体物供应,阻断竞争旁路途径,利用亚细胞定位等策略有效提高对香豆酸产量,为后续黄酮类及芪类化合物的合成提供高效平台菌株,具有重要的应用前景。     

产对香豆酸酿酒酵母菌株的构建及优化

对香豆酸(p-coumaric acid)作为苯丙素类物质、芪类物质及黄酮类物质的重要前体化合物,在生物医药、化妆品及食品工业中均有广泛的应用价值。以酿酒酵母作为底盘菌株,利用合成生物学原理构建一株高产对香豆酸的人工酵母细胞。通过对比不同拷贝数的酪氨酸解氨酶(tyrosine ammonia lyase)合成的对香豆酸产量,发现随着基因拷贝数的增加对香豆酸的产量也相应提高;同时对酪氨酸的负反馈调控相关的蛋白质进行氨基酸定点突变得到Aro4pK229L和Aro7pG141S,利用delta位点将突变后的基因整合至酵母基因组,并挑取24株构建成功的酵母细胞进行发酵验证,发现菌株最高产量与最低产量相差28.87mg/L;为了进一步增加对香豆酸的代谢通量,对生成芳香醇类物质的旁路基因ARO10和PDC5进行敲除,发现同时敲除两个基因的菌株对香豆酸的产量最高,是敲除前产量的2.05倍(从42.71mg/L到87.56mg/L)。此外,通过设计前体酪氨酸的梯度添加实验,发现当添加1mmol/L的酪氨酸时,对香豆酸产量达到峰值(174.57±0.30)mg/L,相较于未添加时提高了将近1倍。通过运用合成生物学原理在酿酒酵母中实现了对香豆酸的高产,为后续的芪类化合物和黄酮类化合物生物合成奠定了基础。     

高活性酪氨酸解氨酶的表征及其在对香豆酸生物合成中应用

对香豆酸(p-coumaric acid)具有抗菌、抗氧化和预防心血管疾病的作用,也是许多重要化合物的前体或中间体,被广泛应用于食品、化妆品和医药等领域。酪氨酸解氨酶(tyrosine ammonia-lyase,TAL)能直接催化酪氨酸脱氨生成对香豆酸。然而,缺少高活性和高底物耐受性的酪氨酸解氨酶限制了对香豆酸的高效生物合成。为了提高对香豆酸的合成能力,本研究挖掘了2个黄杆菌来源的酪氨酸解氨酶,分别是柱状黄杆菌(Flavobacterium columnare)来源的Fc-TAL2和顺天黄杆菌(Flavobacterium suncheonense)来源的Fs-TAL。异源表达纯化表征分析显示,Fc-TAL2和Fs-TAL的最适温度和最适pH相同,分别为55℃、pH 9.5。在最适条件下,Fs-TAL的比酶活为82.47 U/mg,而Fc-TAL2的比酶活为13.27 U/mg。结构模拟和比对分析显示,内盖环上保守的Y50残基酚羟基朝向和到底物的距离是造成Fs-TAL活性高于Fc-TAL2的主要原因。全细胞催化研究进一步证实Fs-TAL具有较高活性和特异性,能够催化10 g/L酪氨酸...     

大肠杆菌中从头合成白藜芦醇途径的设计及优化

白藜芦醇是一种极具药用价值的植物源芪类化合物。为了在E.coli实现白藜芦醇的从头合成,构建了由酪氨酸解氨酶(TAL),香豆酸-CoA合成酶(4CL)和白藜芦醇合成酶(STS)组成的非天然合成途径。经3天发酵后,白藜芦醇产量仅为2.67 mg/L,而其中间体香豆酸的积累达到了95.64 mg/L。为了进一步改善异源途径的效率,对4CL和STS模块采取融合表达、高拷贝表达及启动子工程改造的策略,最终使白藜芦醇产量提高到了9.6倍,达到了25.76 mg/L,同时香豆酸的积累减少到了20.38 mg/L。这些研究结果为更高效白藜芦醇从头合成工程菌的构建及最终实现白藜芦醇的微生物大规模生产奠定了基础。     

苯丙氨酸代谢途径关键酶:PAL、C4H、4CL研究新进展

主要阐述了苯丙氨酸代谢途径中三种关键酶:苯丙氨酸解氨酶(PAL)、肉桂酸4-羟基化酶(C4H)、4-香豆酸辅酶A连接酶(4CL)的研究进展,希望能为研究植物次生代谢途径的研究工作者提供一些帮助。     

大肠杆菌中从头合成白藜芦醇途径的设计及优化

白藜芦醇是一种极具药用价值的植物源芪类化合物。为了在E. coli实现白藜芦醇的从头合成,构建了由酪氨酸解氨酶（TAL）,香豆酸-CoA合成酶（4CL）和白藜芦醇合成酶（STS）组成的非天然合成途径。经3天发酵后,白藜芦醇产量仅为2.67 mg/L,而其中间体香豆酸的积累达到了95.64 mg/L。为了进一步改善异源途径的效率,对4CL和STS模块采取融合表达、高拷贝表达及启动子工程改造的策略,最终使白藜芦醇产量提高到了9.6倍,达到了25.76 mg/L,同时香豆酸的积累减少到了20.38 mg/L。这些研究结果为更高效白藜芦醇从头合成工程菌的构建及最终实现白藜芦醇的微生物大规模生产奠定了基础。     

反应条件下苯丙氨酸解氨酶的活力稳定性

在苯丙氨酸解氨酶(PAL)的作用下由肉桂酸和氨合成L-苯丙氨酸(L-Phe)是酶法合成该氨基酸的重要途径,国外已利用该途径进行L-苯丙氨酸的工业生产,但是该过程仍存在着转化率低和酶活力稳定性差的问题。为解决这些问题,有必要在现有基础上开展提高酶活力稳定性的研究。     

构建酿酒酵母工程菌合成香紫苏醇

香紫苏醇是一种来源于植物的双环二萜醇,常用于香味成分且具有重要生物学活性。为实现香紫苏醇的微生物生产,以酿酒酵母为宿主,表达焦磷酸赖百当烯二醇酯合酶和香紫苏醇合酶,构建香紫苏醇的人工生物合成途径。发现过表达前体代谢关键酶、蛋白质融合增强底物通道效应及去除异源蛋白信号肽等,有利于香紫苏醇合成。在摇瓶培养条件下,组合优化得到的工程菌株S6的香紫苏醇产量达到8.96 mg/L。研究结果对其他萜类化合物的异源生物合成具有参考价值。     

大肠杆菌高效合成L-苯甘氨酸的研究进展

自然界存在着多种氨基酸,除用于蛋白质合成的20种外,大量用于合成具有生物活性的物质,广泛应用于食品、医药等多个领域。其中,非天然芳香族氨基酸L-苯甘氨酸作为一种重要的组成单元广泛的应用于盘尼西林、维吉霉素S、原始霉素I等β-内酰胺类抗生素的生物合成当中。目前L-苯甘氨酸主要通过化学法合成,但该方法合成收率低、污染大,且不易得到单一手性的化合物。由于生物合成L-苯甘氨酸具有反应条件温和、产物立体选择性好的优势,因此受到了广泛的关注。通过对L-苯甘氨酸两条生物合成途径的解析,合成所需相关酶的筛选及辅因子平衡再生等,逐步形成了以苯乙酮酸、扁桃酸和L-苯丙氨酸为底物的合成线路。主要对L-苯甘氨酸的生物合成途径及生物合成策略展开综述,为研究者提供优化方向,以期为高效工业化生物合成L-苯甘氨酸提供理论参考。     

粘红酵母产L-苯丙氨酸解氨酶发酵培养基的优化

通过单因子和正交试验 ,对粘红酵母产 L -苯丙氨酸解氨酶 ( PAL )培养基进行优化 ,L-苯丙氨酸的积累浓度可以从 2 .0 g/1 0 0 ml提高到 3 .3 g/1 0 0 ml,最终得到了 L-苯丙氨酸解氨酶发酵的最适条件     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

Compartmentation of phenylacetic and cinnamic acid synthesis in spinach

Applying labelled phenylalanine or tyrosine to purified intact spinach chloroplasts, only the corresponding phenylacetic acids but not the cinnamic acids could be detected. The addition of mercaptoethanol or dl -dithiothreitol and the variation of light conditions had only a slight effect. However, cinnamic acids could be found together with phenylacetic acids in leaf homogenates indicating the presence of phenylalanine and/or tyrosine ammonia lyase outside the spinach chloroplasts. Similar results were obtained with barley leaf homogenates, where cinnamic acids were the main products. Reviewing recent findings on amino acid synthesis in spinach leaves, it may be concluded that the synthesis of aromatic amino acids is restricted to the chloroplast, whereas the metabolism of secondary aromatic compounds is predominantly localized outside the chloroplasts.     

细胞色素P450还原酶与CYP17的共表达及其功能分析*

17α-羟基黄体酮(17α-OH-PROG)是甾体激素类药物的关键中间体,其生物合成主要由细胞色素单加氧酶(CYP17)催化生成。在此过程中,细胞色素 P450还原酶(cytochrome P450 reductase,CPR)作为细胞色素P450 酶电子传递链的重要组成部分,直接影响CYP17的催化效率。为研究不同来源CPR与17α-羟化酶的适配性,首先以人源17α-羟化酶作为研究对象,构建了表达质粒pPIC3.5k-hCYP17,获得了重组毕赤酵母菌株。其次筛选获得3种不同来源CPR,构建了表达质粒 pPICZX-CPR,获得17α-羟化酶与CPR共表达菌株,并在毕赤酵母中进行转化实验,对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析。结果显示,重组菌株具有17α-羟化酶活性,能够催化黄体酮生成目标产物17α-OH-PROG 以及副产物16α-羟基黄体酮(16α-OH-PROG)。不同来源的CPR与17α-羟化酶共表达与仅表达17α-羟化酶的产率相比均有所提高,其中hCPR-CYP17共表达菌株表现出最高的转化水平,17α-OH-PROG产率提高42%。上述结果表明:17α-羟化酶基因与CPR共表达能够提高其黄体酮17α-羟基化水平。为甾体黄体酮17α-羟基化的生物催化研究提供思路,对甾体药物的工业生产具有重要意义。     

Stearidonic acid (18:4ω3) in Primula florindae

Stearidonic acid (18:4ω3), which is reported to be of rare occurrence in the plant kingdom and which is of considerable dietary and pharmaceutical interest has been found in three closely related Primula species. It occurs, together with γ-linolenic acid (3–4% of the seed oil total fatty acids), in significant percentages in Primula florindae (11%), P. sikkimensis (14%) and P. alpicola (14%). 18:4(ω3 may also be of chemotaxonomic interest in the genus Primula, as high levels may be typical for section Sikkimensis. The only commercial plant source of stearidonic acid known so far is the seed oil of Ribes nigrum.     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

基于重组毕赤酵母的fusaruside生物合成

目的:构建产fusaruside的毕赤酵母菌株,解决天然小分子免疫抑制剂fusaruside的来源问题。方法:从禾谷镰刀菌Fusarium graminearum PH-1中扩增获得合成fusaruside的相关基因-3位去饱和酶[Δ3(E)-SD]和10位去饱和酶[Δ10(E)-SD]基因;并通过2A肽策略构建两种基因的共表达载体,转化到毕赤酵母GS115中进行双酶的诱导表达;对诱导后的毕赤酵母菌体进行甲醇和二氯甲烷的处理后,经高效液相色谱质谱联用仪(HPLC-MS)检测其中产物变化。结果:3位去饱和酶和10位去饱和酶在毕赤酵母中成功共表达,SDS-PAGE显示3位去饱和酶分子量约为48kDa,10位去饱和酶分子量约为65kDa; HPLC-MS显示重组酵母可以产生fusaruside。结论:与fusaruside原产菌株镰刀菌相比,该酵母菌的发酵时间更短、产量更高,为fusaruside的进一步开发与应用奠定基础。     

Changes of shikimate pathway in glyphosate tolerant alfalfa cell lines with reduced embryogenic ability

Glyphosate tolerant cell lines were selected from highly embryogenic cell suspension culture ofMedicago sativa L. Resistant cell lines showed significant reduction of embryogenic ability and during long-term culture in the presence of glyphosate gradual loss of this ability was observed. After glyphosate treatment the increased activity of 5-enolpyruvylshikimate-3-phosphate synthase in tolerant cell lines overcame the block in aromatic amino acid synthesis which was observed in control cell lines. Glyphosate caused marked increase in the content of shikimic acid in both control and tolerant cell lines but the accumulation of shikimic acid was considerably lower in tolerant calli. Significant qualitative and quantitative differences were found in the content of individual phenolic acids. The considerable decrease in the amount of cinnamic acid derivates and broader spectrum of hydroxybenzoic acids suggest in tolerant cell lines the activation of alternative pathway not regulated by phenylalanine ammonia lyase. The possible role of altered pool of phenolic acids on the embryogenic ability is discussed.     

Cinnamic acid production using <Emphasis Type="Italic">Streptomyces lividans</Emphasis> expressing phenylalanine ammonia lyase

Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans, and its expression was confirmed by Western blot analysis. After 4 days cultivation using glucose as carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose or xylan as carbon source, the maximal level of cinnamic acid reached 460, 300, and 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds.     

Production of resveratrol from tyrosine in metabolically engineered Saccharomyces cerevisiae

Resveratrol, a polyphenol compound found in grape skins, has been proposed to account for the beneficial effects of red wine against heart disease. To produce resveratrol in Saccharomyces cerevisiae, four heterologous genes were introduced: the phenylalanine ammonia lyase gene from Rhodosporidium toruloides, the cinnamic acid 4-hydroxylase and 4-coumarate:coenzyme A ligase genes both from Arabidopsis thaliana, and the stilbene synthase gene from Arachis hypogaea. When this recombinant yeast was cultivated by batch fermentation in YP medium containing 2% galactose, it produced 2.6mg/L p-coumaric acid and 3.3mg/L resveratrol. In order to increase the pool of malonyl-CoA, a key precursor in resveratrol biosynthesis, the acetyl-CoA carboxylase (ACC1) gene was additionally overexpressed in the yeast by replacing the native promoter of the ACC1 gene with the stronger GAL1 promoter and this resulted in enhanced production of resveratrol (4.3mg/L). Furthermore, when tyrosine was supplemented in the medium, the concentration of resveratrol increased up to 5.8mg/L. This result illustrates a possible strategy for developing metabolically engineered yeast strain for the economical production of resveratrol from cheap amino acids.     

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35 mg/L naringenin and 37 mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158 mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.