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2A-linked bi-, tri-, and quad-cistrons for the stepwise biosynthesis of β-carotene, zeaxanthin, and ketocarotenoids in rice endosperm 用于水稻胚乳中β-胡萝卜素、玉米黄质和类酮胡萝卜素逐步生物合成的2a -连接双、三和四顺子
IF 5.2 Q1 Engineering Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2021.e00166
Ye Sol Jeong , Hyung-Keun Ku , Young-Joo Jung , Jae Kwang Kim , Kyoung Bok Lee , Ju-Kon Kim , Sun-Hyung Lim , Dongho Lee , Sun-Hwa Ha

Foot-and-mouth disease virus (FMDV) 2A constructs have been successfully used for the production of “Golden Rice”, a β-carotene producing rice strain. However, to allay public fears and opposition to plants carrying a mammalian pathogenic viral sequence, 2A-like synthetic sequences from Thosea asigna virus and Infectious myonecrosis virus were used to coordinate the coexpression of carotenoid biosynthetic genes. Here, up to four carotenogenic genes encoding PSY, CRTI, BCH and BKT were concatenated and produced β-carotene, zeaxanthin, and ketocarotenoids (astaxanthin and adonixanthin) in transgenic rice seeds displaying color variation due to the difference in carotenoid content and composition.

口蹄疫病毒(FMDV) 2A构建体已成功用于生产“黄金大米”,这是一种产生β-胡萝卜素的水稻品系。然而,为了减轻公众对携带哺乳动物致病性病毒序列的植物的恐惧和反对,研究人员使用了来自异位病毒和传染性肌坏死病毒的2a样合成序列来协调类胡萝卜素生物合成基因的共表达。在本研究中,将编码PSY、CRTI、BCH和BKT的4种胡萝卜素基因串联在一起,在由于类胡萝卜素含量和组成不同而呈现颜色变化的转基因水稻种子中产生β-胡萝卜素、玉米黄质和类酮胡萝卜素(虾青素和阿多黄质)。
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引用次数: 4
Analysis of Yarrowia lipolytica growth, catabolism, and terpenoid biosynthesis during utilization of lipid-derived feedstock 脂源性原料利用过程中多脂耶氏菌生长、分解代谢和萜类生物合成的分析
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00130
Alyssa M. Worland , Jeffrey J. Czajka , Yun Xing , Willie F. Harper Jr. , Aryiana Moore , Zhengyang Xiao , Zhenlin Han , Yechun Wang , Wei Wen Su , Yinjie J. Tang

This study employs biomass growth analyses and 13C-isotope tracing to investigate lipid feedstock utilization by Yarrowia lipolytica. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (<20% of total biomass) and enzyme abundances for product synthesis. Labeling results indicate a segregated metabolic network (the glycolysis vs. the TCA cycle) during co-catabolism of sugars (glucose or glycerol) with fatty acid substrates, which facilitates resource allocations for biosynthesis without catabolite repressions. This study has also examined the performance of a β-carotene producing strain in different growth mediums. Canola oil-containing yeast-peptone (YP) has resulted in the best β-carotene titer (121 ± 13 mg/L), two-fold higher than the glucose based YP medium. These results highlight the potential of Y. lipolytica for the valorization of waste-derived lipid feedstock.

本研究采用生物量生长分析和13c同位素示踪研究了多脂耶氏菌对脂质原料的利用。与葡萄糖相比,最小培养基中的油原料增加了酵母的生物量产量和细胞大小,但降低了其蛋白质含量(占总生物量的20%)和用于产物合成的酶丰度。标记结果表明,在糖(葡萄糖或甘油)与脂肪酸底物的共分解代谢过程中,存在分离的代谢网络(糖酵解与TCA循环),这有助于生物合成的资源分配,而不会抑制分解代谢。本研究还考察了一种β-胡萝卜素生产菌株在不同生长培养基中的表现。含菜籽油酵母蛋白胨(YP)培养基的β-胡萝卜素滴度最高(121 ± 13 mg/L),比以葡萄糖为基础的YP培养基高2倍。这些结果突出了解脂芽孢杆菌在废物衍生的脂质原料增值方面的潜力。
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引用次数: 13
Aerobic acetone-butanol-isopropanol (ABI) fermentation through a co-culture of Clostridium beijerinckii G117 and recombinant Bacillus subtilis 1A1 贝氏梭菌G117与重组枯草芽孢杆菌1A1共培养的好氧丙酮-丁醇-异丙醇(ABI)发酵
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00137
Yonghao Cui , Jianzhong He , Kun-Lin Yang , Kang Zhou

An engineered B. subtilis 1A1 strain (BsADH2) expressing a secondary alcohol dehydrogenase (CpSADH) was co-cultured with C. beijerinckii G117 under an aerobic condition. During the fermentation on glucose, B. subtilis BsADH2 depleted oxygen in culture media completely and created an anaerobic environment for C. beijerinckii G117, an obligate anaerobe, to grow. Meanwhile, lactate produced by B. subtilis BsADH2 was re-assimilated by C. beijerinckii G117. In return, acetone produced by C. beijerinckii G117 was reduced into isopropanol by B. subtilis BsADH2 via expressing the CpSADH, which helped maintain the redox balance of the engineered B. subtilis. In the symbiotic system consisting of two strains, 1.7 ​g/L of acetone, 4.8 ​g/L of butanol, and 0.9 ​g/L of isopropanol (with an isopropanol/acetone ratio of 0.53) was produced from 60 ​g/L of glucose. This symbiotic system also worked when oxygen was supplied to the culture, although less isopropanol was produced (0.9 ​g/L of acetone, 4.9 ​g/L of butanol, and 0.2 ​g/L of isopropanol). The isopropanol titer was increased substantially to 2.5 ​g/L when we increased the inoculum size of B. subtilis BsADH2 and optimized other process parameters. With the Bacillus-Clostridium co-culture, switching from the original acetone-butanol (AB) fermentation to an aerobic acetone-butanol-isopropanol (ABI) fermentation can be easily achieved without genetic engineering of Clostridium. This strategy of employing a recombinant Bacillus to co-culture with Clostridium should be potentially useful to modify traditional acetone-butanol-ethanol fermentation for the production of other value-added chemicals.

在好氧条件下,将表达二醇脱氢酶(CpSADH)的枯草芽孢杆菌1A1 (BsADH2)与beijerinckii C. G117共培养。在葡萄糖发酵过程中,枯草芽孢杆菌BsADH2完全耗尽培养基中的氧气,为专性厌氧菌C. beijerinckii G117的生长创造了厌氧环境。同时,枯草芽孢杆菌BsADH2产生的乳酸被贝氏弧菌G117重新同化。反过来,C. beijerinckii G117产生的丙酮通过表达CpSADH被枯草芽孢杆菌BsADH2还原为异丙醇,这有助于维持工程枯草芽孢杆菌的氧化还原平衡。在由两菌株组成的共生体系中,从60 g/L葡萄糖中产生1.7 g/L丙酮、4.8 g/L丁醇和0.9 g/L异丙醇(异丙醇/丙酮比为0.53)。尽管异丙醇产量较低(丙酮0.9 g/L,丁醇4.9 g/L,异丙醇0.2 g/L),但向培养物提供氧气时,这种共生系统也起作用。通过增加枯草芽孢杆菌BsADH2的接种量和优化其他工艺参数,使异丙醇滴度大幅提高至2.5 g/L。通过芽孢杆菌与梭状芽孢杆菌的共培养,可以在不需要梭状芽孢杆菌基因工程的情况下,很容易地实现由原来的丙酮-丁醇(AB)发酵向需氧丙酮-丁醇-异丙醇(ABI)发酵的转变。这种利用重组芽孢杆菌与梭状芽孢杆菌共培养的策略可能有助于改进传统的丙酮-丁醇-乙醇发酵,以生产其他增值化学品。
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引用次数: 12
Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli 大肠杆菌异源生产聚γ-谷氨酸的膜酶复合物的表征
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00144
Bruno Motta Nascimento, Nikhil U. Nair

Poly-γ-glutamic acid (PGA) produced by many Bacillus species is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme. This study focuses on expressing the enzyme heterologously in the Escherichia coli membrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. In E. coli, we were able to produce high molecular weight PGA polymers with minimal degradation at titers of approximately 13 ​mg/L in deep-well microtiter batch cultures. Using fusion proteins, we observed, for the first time, the association and orientation of the different subunits with the inner cell membrane. These results provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.

聚γ-谷氨酸(PGA)是由许多芽孢杆菌种产生的一种具有许多独特和理想特性的聚合物。然而,负责其合成的多亚基酶复合物PGA合成酶(PGS)尚未在天然环境和重组环境中得到很好的表征。阐明该酶的结构和功能特性对未来改变其催化性能的工程研究至关重要。本研究主要研究了该酶在大肠杆菌膜上的异种表达,并对其定位、取向和活性进行了表征。在大肠杆菌中,我们能够产生高分子量的PGA聚合物,在深孔微滴批培养中,其滴度约为13 mg/L,降解最小。利用融合蛋白,我们首次观察到不同亚基与内细胞膜的结合和取向。这些结果为这种研究较少的酶复合物提供了基本的结构信息,并将有助于未来的基础研究和工程努力。
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引用次数: 4
Enhancement of amino acid production and secretion by Lactococcus lactis using a droplet-based biosensing and selection system 利用微滴生物传感和选择系统增强乳酸乳球菌氨基酸的产生和分泌
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00133
Jhonatan A. Hernandez-Valdes , Myrthe aan de Stegge , Jos Hermans , Johan Teunis , Rinke J. van Tatenhove-Pel , Bas Teusink , Herwig Bachmann , Oscar P. Kuipers

Amino acids are attractive metabolites for the pharmaceutical and food industry field. On one hand, the construction of microbial cell factories for large-scale production aims to satisfy the demand for amino acids as bulk biochemical. On the other hand, amino acids enhance flavor formation in fermented foods. Concerning the latter, flavor formation in dairy products, such as cheese is associated with the presence of lactic acid bacteria (LAB). In particular, Lactococcus lactis, one of the most important LAB, is used as a starter culture in fermented foods. The proteolytic activity of some L. lactis strains results in peptides and amino acids, which are flavor compounds or flavor precursors. However, it is still a challenge to isolate bacterial cells with enhanced amino acid production and secretion activity. In this work, we developed a growth-based sensor strain to detect the essential amino acids isoleucine, leucine, valine, histidine and methionine. Amino acids are metabolites that can be secreted by some bacteria. Therefore, our biosensor allowed us to identify wild-type L. lactis strains that naturally secrete amino acids, by using co-cultures of the biosensor strain with potential amino acid producing strains. Subsequently, we used this biosensor in combination with a droplet-based screening approach, and isolated three mutated L. lactis IPLA838 strains with 5–10 fold increased amino acid-secretion compared to the wild type. Genome re-sequencing revealed mutations in genes encoding proteins that participate in peptide uptake and peptide degradation. We argue that an unbalance in the regulation of amino acid levels as a result of these gene mutations may drive the accumulation and secretion of these amino acids. This biosensing system tackles the problem of selection for overproduction of secreted molecules, which requires the coupling of the product to the producing cell in the droplets.

氨基酸是制药和食品工业领域有吸引力的代谢物。一方面,大规模生产微生物细胞工厂的建设旨在满足氨基酸作为大宗生化原料的需求。另一方面,氨基酸促进了发酵食品风味的形成。关于后者,乳制品(如奶酪)中的风味形成与乳酸菌(LAB)的存在有关。乳酸乳球菌是最重要的乳酸菌之一,被用作发酵食品的发酵剂。某些乳酸菌的蛋白水解活性产生多肽和氨基酸,这些多肽和氨基酸是风味化合物或风味前体。然而,分离具有增强氨基酸生产和分泌活性的细菌细胞仍然是一个挑战。在这项工作中,我们开发了一种基于生长的传感器菌株,用于检测必需氨基酸异亮氨酸、亮氨酸、缬氨酸、组氨酸和蛋氨酸。氨基酸是一些细菌可以分泌的代谢物。因此,通过将生物传感器菌株与可能产生氨基酸的菌株共培养,我们的生物传感器使我们能够鉴定自然分泌氨基酸的野生型乳酸乳杆菌菌株。随后,我们将该生物传感器与基于液滴的筛选方法相结合,分离出3株氨基酸分泌量比野生型增加5-10倍的突变乳酸乳杆菌IPLA838菌株。基因组重测序揭示了编码参与肽摄取和肽降解的蛋白质的基因突变。我们认为,由于这些基因突变,氨基酸水平调节的不平衡可能会驱动这些氨基酸的积累和分泌。这种生物传感系统解决了分泌分子过量产生的选择问题,这需要将产物与液滴中的产生细胞偶联。
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引用次数: 17
De novo production of resveratrol from glycerol by engineering different metabolic pathways in Yarrowia lipolytica 脂解耶氏菌通过不同代谢途径从甘油重新生产白藜芦醇
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00146
Qin He , Patrycja Szczepańska , Tigran Yuzbashev , Zbigniew Lazar , Rodrigo Ledesma-Amaro

Resveratrol is a polyphenol with multiple applications in pharma, cosmetics and food. The aim of this study was to construct Yarrowia lipolytica strains able to produce resveratrol. For this purpose, resveratrol-biosynthesis genes from bacteria and plants were expressed in this host. Since resveratrol can be produced either via tyrosine or phenylaniline, both pathways were tested, first with a single copy and then with two copies. The phenylalanine pathway resulted in slightly higher production in glucose media, although when the media was supplemented with amino acids, the best production was found in the strain with two copies of the tyrosine pathway, which reached 0.085 ​g/L. When glucose was replaced by glycerol, a preferred substrate for bioproduction, the best results, 0.104 ​g/L, were obtained in a strain combining the expression of the two synthesis pathways. Finally, the best producer strain was tested in bioreactor conditions where a production of 0.43 ​g/L was reached. This study suggests that Y. lipolytica is a promising host for resveratrol production from glycerol.

白藜芦醇是一种多酚,在制药、化妆品和食品中有多种应用。本研究的目的是构建能够生产白藜芦醇的多脂耶氏菌菌株。为此,从细菌和植物中提取白藜芦醇生物合成基因在该宿主中表达。由于白藜芦醇既可以通过酪氨酸产生,也可以通过苯胺产生,所以两种途径都进行了测试,首先用一个副本,然后用两个副本。在葡萄糖培养基中,苯丙氨酸途径的产量略高,但在培养基中添加氨基酸时,具有两份酪氨酸途径的菌株产量最高,达到0.085 g/L。当葡萄糖被生物生产的首选底物甘油取代时,在结合两种合成途径表达的菌株中获得了0.104 g/L的最佳结果。最后在生物反应器条件下对最佳产菌进行了试验,产菌量达到0.43 g/L。这一研究表明,脂肪瘤是甘油生产白藜芦醇的一个有希望的宿主。
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引用次数: 14
Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production 开发快速生长的枯草芽孢杆菌以提高生化和重组蛋白的生产
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00141
Yanfeng Liu , Anqi Su , Rongzhen Tian , Jianghua Li , Long Liu , Guocheng Du

Bacillus subtilis is a model Gram-positive bacterium, which has been widely used as industrially important chassis in synthetic biology and metabolic engineering. Rapid growth of chassis is beneficial for shortening the fermentation period and enhancing production of target product. However, engineered B. subtilis with faster growth phenotype is lacking. Here, fast-growing B. subtilis were constructed through rational gene knockout and adaptive laboratory evolution using wild type strain B. subtilis 168 (BS168) as starting strain. Specifically, strains BS01, BS02, and BS03 were obtained through gene knockout of oppD, hag, and flgD genes, respectively, resulting 15.37%, 24.18% and 36.46% increases of specific growth rate compared with BS168. Next, strains A28 and A40 were obtained through adaptive laboratory evolution, whose specific growth rates increased by 39.88% and 43.53% compared to BS168, respectively. Then these two methods were combined via deleting oppD, hag, and flgD genes respectively on the basis of evolved strain A40, yielding strain A4003 with further 7.76% increase of specific growth rate, reaching 0.75 h-1 in chemical defined M9 medium. Finally, bioproduction efficiency of intracellular product (ribonucleic acid, RNA), extracellular product (acetoin), and recombinant proteins (green fluorescent protein (GFP) and ovalbumin) by fast-growing strain A4003 was tested. And the production of RNA, acetoin, GFP, and ovalbumin increased 38.09%, 5.40%, 9.47% and 19.79% using fast-growing strain A4003 as chassis compared with BS168, respectively. The developed fast-growing B. subtilis strains and strategies used for developing these strains should be useful for improving bioproduction efficiency and constructing other industrially important bacterium with faster growth phenotype.

枯草芽孢杆菌(Bacillus subtilis)是一种模式革兰氏阳性菌,在合成生物学和代谢工程中具有重要的工业应用基础。底盘的快速生长有利于缩短发酵周期,提高目标产品的产量。然而,目前还缺乏具有快速生长表型的工程枯草芽孢杆菌。本研究以野生型菌株B. subtilis 168 (BS168)为起始菌株,通过合理基因敲除和适应性实验室进化,构建了速生枯草芽孢杆菌。其中,通过敲除oppD、hag和flgD基因分别获得菌株BS01、BS02和BS03,比BS168的特定生长率提高了15.37%、24.18%和36.46%。通过实验室适应性进化得到菌株A28和A40,其比生长率比BS168分别提高了39.88%和43.53%。然后在进化菌株A40的基础上,通过分别删除oppD、hag和flgD基因,将这两种方法结合起来,产生菌株A4003,比生长率进一步提高7.76%,在化学定义的M9培养基中达到0.75 h-1。最后,检测了快速生长菌株A4003胞内产物(核糖核酸,RNA)、胞外产物(乙酰蛋白)和重组蛋白(绿色荧光蛋白(GFP)和卵清蛋白)的生物生产效率。与BS168相比,快速生长菌株A4003的RNA、乙酰蛋白、GFP和卵清蛋白的产量分别提高了38.09%、5.40%、9.47%和19.79%。所开发的速生枯草芽孢杆菌菌株及其开发策略,对提高生物生产效率和构建其他具有快速生长表型的重要工业细菌具有重要意义。
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引用次数: 10
Development of a DNA double-strand break-free base editing tool in Corynebacterium glutamicum for genome editing and metabolic engineering 谷氨酸棒状杆菌DNA双链无断裂碱基编辑工具的开发及其基因组编辑和代谢工程
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00135
Chen Deng , Xueqin Lv , Jianghua Li , Yanfeng Liu , Guocheng Du , Long Liu

As a traditional amino acid producing bacterium, Corynebacterium glutamicum is a platform strain for production of various fine chemicals. Based on the CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system, gene editing tools that enable base conversion in the genome of C. glutamicum have been developed. However, some problems such as genomic instability caused by DNA double-strand break (DSB) and off-target effects need to be solved. In this study, a DSB-free single nucleotide genome editing system was developed by construction of a bi-directional base conversion tool TadA-dCas9-AID. This system includes cytosine base editors (CBEs): activation-induced cytidine deaminase (AID) and adenine deaminase (ABEs): tRNA adenosine deaminase (TadA), which can specifically target the gene through a 20-nt single guide RNA (sgRNA) and achieve the base conversion of C-T, C-G and A-G in the 28-bp editing window upstream of protospacer adjacent motif. Finally, as a proof-of-concept demonstration, the system was used to construct a mutant library of zwf gene in C. glutamicum S9114 genome to improve the production of a typical nutraceutical N-acetylglucosamine (GlcNAc). The GlcNAc titer of the mutant strain K293R was increased by 31.9% to 9.1 ​g/L in shake flask. Here, the developed bases conversion tool TadA-dCas9-AID does not need DNA double-strand break and homologous template, and is effective for genome editing and metabolic engineering in C. glutamicum.

谷氨酸棒状杆菌作为一种传统的氨基酸生产菌,是生产各种精细化工产品的平台菌株。基于CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9系统,已经开发出能够在谷氨酰胺(C. glutamicum)基因组中进行碱基转换的基因编辑工具。然而,DNA双链断裂(DSB)引起的基因组不稳定性和脱靶效应等问题仍有待解决。本研究通过构建双向碱基转换工具TadA-dCas9-AID,构建了无dsb的单核苷酸基因组编辑系统。该系统包括胞嘧啶碱基编辑器(CBEs):激活诱导胞苷脱氨酶(AID)和腺嘌呤脱氨酶(ABEs): tRNA腺苷脱氨酶(TadA),它们可以通过一个20 nt的单导RNA (sgRNA)特异性靶向基因,在原间隔器邻近基序上游的28 bp编辑窗口实现C-T、C-G和a - g的碱基转换。最后,作为概念验证,该系统被用于构建谷氨酰胺S9114基因组zwf基因突变文库,以提高典型营养保健产品n -乙酰氨基葡萄糖(GlcNAc)的产量。突变株K293R在摇瓶中的GlcNAc滴度提高了31.9%,达到9.1 g/L。本研究开发的碱基转换工具TadA-dCas9-AID不需要DNA双链断裂和同源模板,可有效地用于谷氨酸酵母的基因组编辑和代谢工程。
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引用次数: 10
Pathway engineering of Escherichia coli for one-step fermentative production of L-theanine from sugars and ethylamine 从糖和乙胺一步法发酵生产l -茶氨酸的大肠杆菌途径工程
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00151
Xiaoguang Fan , Tong Zhang , Yuanqing Ji , Jie Li , Keyi Long , Yue Yuan , Yanjun Li , Qingyang Xu , Ning Chen , Xixian Xie

L-theanine is the most abundant free amino acid in tea that offers various favorable physiological and pharmacological effects. Bacterial enzyme of γ-glutamylmethylamide synthetase (GMAS) can catalyze the synthesis of theanine from glutamate, ethylamine and ATP, but the manufacturing cost is uncompetitive due to the expensive substrates and complex processes. In this study, we described pathway engineering of wild-type Escherichia coli for one-step fermentative production of theanine from sugars and ethylamine. First, the synthetic pathway of theanine was conducted by heterologous introduction of a novel GMAS from Paracoccus aminovorans. A xylose-induced T7 RNA polymerase-PT7 promoter system was used to enhance and control gmas gene expression. Next, the precursor glutamate pool was increased by overexpression of native citrate synthase and introduction of glutamate dehydrogenase from Corynebacterium glutamicum. Then, in order to push more carbon flux towards theanine synthesis, the tricarboxylic acid cycle was interrupted and pyruvate carboxylase from C. glutamicum was introduced as a bypath supplying oxaloacetate from pyruvate. Finally, an energy-conserving phosphoenolpyruvate carboxykinase from Mannheimia succiniciproducens was introduced to increase ATP yield for theanine synthesis. After optimizing the addition time and concentration of ethylamine hydrochloride in the fed-batch fermentation, the recombinant strain TH11 produced 70.6 ​g/L theanine in a 5-L bioreactor with a yield and productivity of 0.42 ​g/g glucose and 2.72 ​g/L/h, respectively. To our knowledge, this is the first report regarding the pathway engineering of E. coli for fermentative production of theanine. The high production capacity of recombinant strain, combined with the easy processes, will hold attractive industrial application potential for the future.

茶氨酸是茶叶中含量最多的游离氨基酸,具有多种有利的生理和药理作用。细菌酶γ-谷氨酰甲酰胺合成酶(GMAS)可以催化谷氨酸、乙胺和ATP合成茶氨酸,但由于底物昂贵和工艺复杂,制造成本缺乏竞争力。在这项研究中,我们描述了野生型大肠杆菌一步发酵从糖和乙胺中生产茶氨酸的途径工程。首先,通过异源引入一种新的氨基副球菌GMAS,研究了茶氨酸的合成途径。木糖诱导的T7 RNA聚合酶- pt7启动子系统可增强和控制gmas基因的表达。然后,通过过量表达天然柠檬酸合成酶和引入谷氨酸脱氢酶来增加前体谷氨酸库。然后,为了推动更多的碳通量用于茶氨酸的合成,中断三羧酸循环,引入C. glutamicus的丙酮酸羧化酶作为从丙酮酸中供应草酰乙酸的旁路。最后,从琥珀酸Mannheimia producens中引入了一种节能的磷酸烯醇丙酮酸羧激酶,以提高茶氨酸合成中ATP的产量。通过优化补料分批发酵中盐酸乙胺的添加时间和浓度,重组菌株TH11在5-L生物反应器中产茶氨酸70.6 g/L,产率为0.42 g/g葡萄糖,产率为2.72 g/L/h。据我们所知,这是关于大肠杆菌发酵生产茶氨酸的途径工程的第一篇报道。重组菌株生产能力强,工艺简便,具有广阔的工业应用前景。
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引用次数: 7
Modeling of nitrogen fixation and polymer production in the heterotrophic diazotroph Azotobacter vinelandii DJ 异养重氮营养固氮细菌vinelandii的固氮和聚合物生产模型
IF 5.2 Q1 Engineering Pub Date : 2020-12-01 DOI: 10.1016/j.mec.2020.e00132
Diego Tec-Campos , Cristal Zuñiga , Anurag Passi , John Del Toro , Juan D. Tibocha-Bonilla , Alejandro Zepeda , Michael J. Betenbaugh , Karsten Zengler

Nitrogen fixation is an important metabolic process carried out by microorganisms, which converts molecular nitrogen into inorganic nitrogenous compounds such as ammonia (NH3). These nitrogenous compounds are crucial for biogeochemical cycles and for the synthesis of essential biomolecules, i.e. nucleic acids, amino acids and proteins. Azotobacter vinelandii is a bacterial non-photosynthetic model organism to study aerobic nitrogen fixation (diazotrophy) and hydrogen production. Moreover, the diazotroph can produce biopolymers like alginate and polyhydroxybutyrate (PHB) that have important industrial applications. However, many metabolic processes such as partitioning of carbon and nitrogen metabolism in A. vinelandii remain unknown to date.

Genome-scale metabolic models (M-models) represent reliable tools to unravel and optimize metabolic functions at genome-scale. M-models are mathematical representations that contain information about genes, reactions, metabolites and their associations. M-models can simulate optimal reaction fluxes under a wide variety of conditions using experimentally determined constraints. Here we report on the development of a M-model of the wild type bacterium A. vinelandii DJ (iDT1278) which consists of 2,003 metabolites, 2,469 reactions, and 1,278 genes. We validated the model using high-throughput phenotypic and physiological data, testing 180 carbon sources and 95 nitrogen sources. iDT1278 was able to achieve an accuracy of 89% and 91% for growth with carbon sources and nitrogen source, respectively. This comprehensive M-model will help to comprehend metabolic processes associated with nitrogen fixation, ammonium assimilation, and production of organic nitrogen in an environmentally important microorganism.

固氮是微生物将分子氮转化为氨(NH3)等无机氮化合物的重要代谢过程。这些含氮化合物对生物地球化学循环和基本生物分子(即核酸、氨基酸和蛋白质)的合成至关重要。固氮杆菌是一种研究好氧固氮(重氮化)和产氢的细菌非光合模式生物。此外,重氮营养盐可以生产海藻酸盐和聚羟基丁酸盐(PHB)等生物聚合物,具有重要的工业应用。然而,许多代谢过程,如碳和氮代谢的分配,至今仍不清楚。基因组尺度代谢模型(m -模型)是在基因组尺度上揭示和优化代谢功能的可靠工具。m模型是包含有关基因、反应、代谢物及其关联的信息的数学表示。m -模型可以利用实验确定的约束条件模拟各种条件下的最优反应通量。在这里,我们报道了野生型细菌a . vinelandii DJ (iDT1278)的m模型的发展,该模型由2003种代谢物,2,469种反应和1,278个基因组成。我们利用高通量表型和生理数据验证了该模型,测试了180个碳源和95个氮源。iDT1278在碳源和氮源条件下的生长精度分别达到89%和91%。这种综合的m模型将有助于理解与固氮、铵同化和有机氮生产有关的代谢过程。
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引用次数: 15
期刊
Metabolic Engineering Communications
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