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Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria

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Science China(Life Sciences) 2023年第1期66卷 127-136页

作者： Yu Jiang Bingbing Sun Fenghui Qian Feng Dong Chongmao Xu Wuling Zhong Rui Huang Qiwei Zhai Yu Jiang Sheng Yang Shanghai Taoyusheng Biotechology Co. Ltd.Shanghai 201201China CAS Key Laboratory of Synthetic Biology CAS Center for Excellence in Molecular Plant SciencesChinese Academy of SciencesShanghai 200032China Shanghai Research and Development Center of Industrial Biotechnology Shanghai 201201China CAS Key Laboratory of Nutrition Metabolism and Food SafetyShanghai Institute of Nutrition and HealthChinese Academy of SciencesShanghai 200031China

Phenylketonuria(PKU),a disease resulting in the disability to degrade phenylalanine(Phe)is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to *** an potential alternative to a protein-restricted diet,oral intake of engineered probiotics degrading Phe inside the body is a promising treatment,currently at clinical stage II(Isabella,et al.,2018).However,limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic’s ***,we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase(PAL)as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917(EcN)which overcomes the transportation *** metabolic engineering strategy was also combined with strengthening of Phe transportation,transportation of PAL-catalyzed trans-cinnamic acid and fixation of released *** of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the Pah^(F263S)PKU mouse model reduced blood Phe concentration by 44.4%compared to the control Ec N,independent of dietary protein ***8500 shows great potential in future applications for PKU therapy.

关键词： phenylalanine ammonia lyase cell surface display phenylketonuria TYS8500 oral administration

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Enhancement of phenylalanine ammonia lyase,Polyphenoloxidase,and Peroxidase in Cucumber Seedlings by Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae) Infestation

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Agricultural Sciences in China 2008年第1期7卷 82-87页

作者： ZHANG Shi-ze ZHANG Fan HUA Bao-zhen College of Plant Protection Northwest A ＆ F University Yangling 712100 P.R.China Institute of Plant and Environmental Protection Beijing Academy of Agricultural and Forestry Sciences Beijing 100089 P.R.China

In this study, the activities of phenylalanine ammonia lyase （PAL）, polyphenoloxidase （PPO）, and peroxidase （POD） were assayed in cucumber seedlings （Cucumis sativus L.） at 0, 6, 12, 24, 48, 72, and 96 h after they were infested by Bemisia tabaci （Gennadius） using spectrophotometric analysis. The results indicated that herbivore infestation increased the activities of PAL, PPO, and POD. The enzymes showed different activity levels at different times after the infestation. The PAL activity reached the first high peak by 23.1% at 6 h and the highest peak by 29.1% at 48 h compared to the control. The PPO activity reached the first high peak by 22.7% at 6 h and the highest peak by 52.6% at 24 h, and the POD activity reached the highest peak by 213.2% at 6 h and another higher peak value by 135.2% at 96 h. The results suggest that the enhanced activities of the enzymes may contribute to bioprotection of cucumber plants against B. tabaci infestation.

关键词： phenylalanine ammonia lyase polyphenoloxidase peroxidase Bemisia tabaci induce response

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Cis- and Trans-Cinnamic Acids Have Different Effects on the Catalytic Properties of Arabidopsis phenylalanine ammonia lyases PAL1, PAL2, and PAL4

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Journal of Integrative Plant Biology 2005年第1期47卷 67-75页

作者： Ming-JieCHEN VeerappanVIJAYKUMAR Bing-WenLU BingXIA NingLI DepartmentofBiology TheHongKongUniversityofScienceandTechnologyHongKongSpecialAdministrationRegionChina InstituteofBotanyofJiangsuProvinceandtheChineseAcademyofSciences NanjingBotanicalGardenNanjing210014China

Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.

关键词： Arabidopsis thaliana(L.) Heynh. cis-cinnamic acid enzymatic kinetics phenylalanine ammonia lyase

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Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis

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Journal of Integrative Plant Biology 2022年第9期64卷 1739-1754页

作者： Lu Yao Huanyu Zhang Yirong Liu Qiushuang Ji Jing Xie Ru Zhang Luqi Huang Kunrong Mei Juan Wang Wenyuan Gao School of Pharmaceutical Science and Technology Tianjin UniversityTianjin300072 China Wenzhou Safety(Emergency)Institute of Tianjin University Wenzhou325000 China Key Laboratory of Systems Bioengineering Ministry of EducationTianjin UniversityTianjin300072 China National Resource Center for Chinese Meteria Medica China Academy of Chinese Medical SciencesBeijing100700 China

The ginsenoside Rgfound in Panax species has extensive pharmacological properties,in particular anti-cancer ***,its natural yield in Panax plants is ***,we report a multimodular strategy to improve yields of Rgin a Panax ginseng chassis,combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene,phenylalanine ammonia lyase(PAL).We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2,a glycosyltransferase that directly catalyzes the biosynthesis of Rgfrom ***,we used clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside *** of PAL accelerated the formation of the xylem structure,significantly improving ginsenoside Rgaccumulation(to 6.19-fold higher than in thecontrol).Wecombinedoverexpression of the ginsenoside aglycon synthetic genes squalene epoxidase,Pq3-O-UGT2,and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside ***,we produced ginsenoside Rgat a yield of 83.6 mg/L in a shake flask(7.0 mg/g dry weight,21.12-fold higher than with wild-type cultures).The highproduction system established in this study could be a potential platform to produce the ginsenoside Rgcommercially for pharmaceutical use.

关键词： catalysis ginsenoside Rg3 metabolism regulation phenylalanine ammonia lyase

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Alteration in Biochemical Response in Sesamum indicum upon Different Plant-Pathogen Interactions

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Journal of Agricultural Science and Technology(B) 2011年第1期1卷 68-75页

作者： A. Sharma S, Sharma N. Joshi P. Sharma Department of Bioscience ＆ Biotechnology Banasthali University Banasthali 304022 India

Sesamum, an important oil yielding crop suffers a huge loss in its yield due to attack of large number of fungal pathogens. In semi-arid regions Sesamum is mainly affected by two major plant-pathogenic fungus viz. Macrophomina phaseolina and Fusarium oxysporum. The aim of the study was to analyze the metabolic alterations in Sesamum after infection with both pathogens. This accomplished by individually by （the word estimating is not quantitative） the levels of total phenolic compounds and the activities of phenylalanine ammonia lyase （PAL） of one week old plants. The PAL showed high activity in infected plants, revealing the active phase in the synthesis of secondary metabolites in the Sesamum plant after infection. As a consequence, in infected plants the contents of polyphenols along with salicylic acid （SA） considerably exceeded when compared to control plants. This in vivo study of M. phaseolina and F. oxysporum infection reveals the differences of resistance levels in sesame against these two pathogens. These results provide important information regarding the plant-pathogen interactions and also forfor Sesamum improvement programs seeking the adaptation to diverse range of fungal attack along with adverse environmental factors.

关键词： Fusarium oxysporum Macrophomina phaseolina phenylalanine ammonia lyase polyphenols salicylic acid Seasamum

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Cloning, Expression and Analysis of pal, a Positive Gene Involved in Capsaicin Biosynthesis in Capsicum chinense Jacq

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Agricultural Science & Technology 2013年第12期14卷 1723-1728页

作者：刘艳梅杨帆段军涛刘羽王晶莹郭庆勋吉林大学植物科学学院吉林长春130062

The cDNA sequence of Capal gene was cloned from Capsicum chinense Jacq by RT-PCR and sequenced. Bioinformatics analysis showed that Capal en- codes a putative polypeptide of 683 amino acids with a calculated molecular mass of 74.2 kD and a theoretical pl of 6.9. Multiple sequence alignments and phyloge- netic tree analyses showed that Capal protein of C. chinense is similar to that of Capsicum annuum var. conoides, with an overall sequence similarity of 96%. The prokaryotic expression vector pET-32a-pal was constructed and induced to express in E. coil BL21. The SDS-PAGE analysis showed that the relative molecular mass of the induced new protein is about 74 kD, which was basically identical with that predicted by DNAMAN software （74.3 kD）, Real-time PCR analysis showed that ex- ogenous jasmonic acid （JA） promoted pal expression. The accumulation of capsaicin in pepper was analyzed by high performance liquid chromatography （HPLC）, and the results indicated that exogenous jasmonic acid （JA） can promote the synthesis of capsaicin. This study will provide valuable experimental basis for the research of transcription regulation and explaining the gene function of pal.

关键词： phenylalanine ammonia lyase Capsaicin Prokaryotic expression Jas- monic acid

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桑树花青素生物合成相关基因的克隆与表达分析（英文）

桑树花青素生物合成相关基因的克隆与表达分析（英文）

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全国蚕桑资源多元化利用学术研讨会

作者：李军赵爱春刘长英余茂德西南大学家蚕基因组学国家重点实验室/生物技术学院

内容介绍桑椹从授粉到成熟一般要经历30到40天。桑椹在发育过程中,颜色会从一开始的绿色变成红色,最后转变为黑紫色。桑椹的色素被用作食品着色剂,其果实也具有良好的药用价值。目前,桑椹被广泛利用,既可被直接食用,也可用于制作果酱、... 详细信息

内容介绍桑椹从授粉到成熟一般要经历30到40天。桑椹在发育过程中,颜色会从一开始的绿色变成红色,最后转变为黑紫色。桑椹的色素被用作食品着色剂,其果实也具有良好的药用价值。目前,桑椹被广泛利用,既可被直接食用,也可用于制作果酱、冷冻甜品、冰淇淋和果酒等产品。目前,在植物界中,一些着有红色的花卉、水果和蔬菜是由于花青素的积累和组分的多样化促成,花青素的合成机制也已经被解析。花青素功能多样,包括抗辐射和抗氧化等,甚至还可以可吸引动物进行授粉和种子传播。花青素在等多种植物被分离和提取,在葡萄和蓝莓等果实大量积累,特别在桑椹中的花青素含量高达64.70±0.45 mg g-1（d.m.）,几乎是蓝莓的二倍之多。在桑椹中主要的花青素是矢车菊-3-芸香糖苷和矢车菊素-3-葡萄糖苷,然而,对于桑树花青素途径在分子水平的研究还很少。花青素的合成途径可以分为两个部分:上游和下游。上游基因包括苯丙氨酸解氨酶（PAL）、肉桂酸-4-羟化酶（C4H）、4-香豆酸:辅酶A连接酶（4CL）、查尔酮合成酶（CHS）、查尔酮异构酶（CHI）和黄酮3’-羟化酶（F3’H）,这些基因负责合成二羟黄酮醇。下游基因包括二羟黄酮醇还原酶（DFR）、花青苷合成酶（ANS）和UDP-葡萄糖:类黄酮3-葡糖基转移酶（UFGT）,最终导致花青素的形成。目前,涉及花青素合成途径的基因已在多种物种中被克隆和鉴定,包括花青素合成基因和转录因子。桑椹大部分颜色呈现出紫色,但也有品种为白色,其原因尚不清楚。通过前人的研究,主要有三种情况造成果实中不含花青素或含量较低,一是花青素结构基因的表达受到抑制或缺失,二是控制花青素合成基因表达的转录因子受到抑制,三是缺失负责花青素液泡转运的基因。目前,CHS、CHI、F3H和DFR基因的突变体已被证实。此外,在蛇莓的白果品种中,ANS基因的表达受到下调或被阻断。在洋葱中也发现了类似情况。此外,在葡萄、苹果、中国月桂果和红梨中,花青素合成基因的表达受到了MYB转录因子的调控。本研究的目的是为了探究哪些花青素合成基因和花青素的合成相关。我们克隆了4个花青素合成基因,分别是Ma CHS、Ma F3H、Ma F3’H和Ma DFR,以及本实验室已报道的基因Ma PAL、Ma CHI和Ma ANS基因,检测了它们在白果和红果的不同组织中表达,以探究花青素合成的基因的表达模式。Ma DFR和Ma ANS基因在紫果品种的桑椹中表达量较高。随着桑椹的发育,花青素的含量变化和Ma DFR,Ma ANS和Ma CHS基因的表达量密切相关。雌花的外层在光诱导下转变成红色,主要是由于Ma CHS,Ma DFR和Ma ANS基因的表达量被上调。本研究中所获得的花青素合成基因可能参与控制桑树花青素合成,可以通过上调他们的表达来提高花青素的含量。我们希望能够为今后深入解析桑椹花青素合成的机制和改良桑椹花青素的积累提供依据。

关键词： phenylalanine ammonia lyase chalcone isomerase chalcone synthase dihydroflavonol reductase flavanone-3hydroxylase flavanone-3′-hydroxylase anthocyanidin synthase

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