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In vivo production of pederin by labrenzin pathway expansion labrenzin通路扩张在体内产生pederin
IF 5.2 Q1 Engineering Pub Date : 2022-06-01 DOI: 10.1016/j.mec.2022.e00198
Dina Kačar , Carmen Schleissner , Librada M. Cañedo , Pilar Rodríguez , Fernando de la Calle , Carmen Cuevas , Beatriz Galán , José Luis García

Pederin is a potent polyketide toxin that causes severe skin lesions in humans after contact with insects of genus Paederus. Due to its potent anticancer activities, pederin family compounds have raised the interest of pharmaceutical industry. Despite the extensive studies on the cluster of biosynthetic genes responsible for the production of pederin, it has not yet been possible to isolate and cultivate its bacterial endosymbiont producer. However, the marine bacterium Labrenzia sp. PHM005 was recently reported to produce labrenzin, the closest pederin analog. By cloning a synthetic pedO gene encoding one of the three O-methyltraferase of the pederin cluster into Labrenzia sp. PHM005 we have been able to produce pederin for the first time by fermentation in the new recombinant strain.

Pederin是一种强效的聚酮毒素,在与pepederus属昆虫接触后会引起严重的皮肤损伤。由于其强大的抗癌活性,菊酯类化合物已引起了医药行业的广泛关注。尽管对产生pederin的生物合成基因簇进行了广泛的研究,但尚未能够分离和培养其细菌内共生生产者。然而,海洋细菌Labrenzia sp. PHM005最近被报道产生labrenzin,这是最接近的同性恋类似物。通过将一个合成的pedO基因克隆到Labrenzia sp. PHM005中,首次在重组菌株中发酵生产了pederin。
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引用次数: 3
Development of an E. coli-based norbaeocystin production platform and evaluation of behavioral effects in rats 基于大肠杆菌的去baeocystin生产平台的建立及其对大鼠行为影响的评价
IF 5.2 Q1 Engineering Pub Date : 2022-06-01 DOI: 10.1016/j.mec.2022.e00196
Alexandra M. Adams , Nicholas A. Anas , Abhishek K. Sen , Jordan D. Hinegardner-Hendricks , Philip J. O’Dell , William J. Gibbons Jr. , Jessica E. Flower , Matthew S. McMurray , J. Andrew Jones

Interest in the potential therapeutic efficacy of psilocybin and other psychedelic compounds has escalated significantly in recent years. To date, little is known regarding the biological activity of the psilocybin pathway intermediate, norbaeocystin, due to limitations around sourcing the phosphorylated tryptamine metabolite for in vivo testing. To address this limitation, we first developed a novel E. coli platform for the rapid and scalable production of gram-scale amounts of norbaeocystin. Through this process we compare the genetic and fermentation optimization strategies to that of a similarly constructed and previously reported psilocybin producing strain, uncovering the need for reoptimization and balancing upon even minor genetic modifications to the production host. We then perform in vivo measurements of head twitch response to both biosynthesized psilocybin and norbaeocystin using both a cell broth and water vehicle in Long-Evans rats. The data show a dose response to psilocybin while norbaeocystin does not elicit any pharmacological response, suggesting that norbaeocystin and its metabolites may not have a strong affinity for the serotonin 2A receptor. The findings presented here provide a mechanism to source norbaeocystin for future studies to evaluate its disease efficacy in animal models, both individually and in combination with psilocybin, and support the safety of cell broth as a drug delivery vehicle.

近年来,对裸盖菇素和其他致幻剂的潜在治疗效果的兴趣显著增加。迄今为止,关于裸盖菇素途径中间体去甲盖菇素的生物活性知之甚少,这是由于体内试验中磷酸化色胺代谢物来源的限制。为了解决这一限制,我们首先开发了一种新的大肠杆菌平台,用于快速和可扩展地生产克级量的去甲黄囊素。通过这一过程,我们将遗传和发酵优化策略与类似构建和先前报道的裸盖菇素生产菌株进行比较,发现即使对生产宿主进行微小的遗传修饰也需要重新优化和平衡。然后,我们在Long-Evans大鼠体内使用细胞培养液和水载体对生物合成的裸盖菇素和去甲盖菇素进行了头抽搐反应的测量。数据显示裸盖菇素有剂量反应,而去甲毛囊素没有引起任何药理反应,提示去甲毛囊素及其代谢物可能对5 -羟色胺2A受体没有很强的亲和力。本文的研究结果为未来的动物模型研究提供了去甲盖菇素来源的机制,以评估其疾病疗效,无论是单独还是与裸盖菇素联合,并支持细胞汤作为药物递送载体的安全性。
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引用次数: 9
Production of human milk fat substitute by engineered strains of Yarrowia lipolytica 溶脂耶氏菌工程菌株生产人乳脂代用品
IF 5.2 Q1 Engineering Pub Date : 2022-06-01 DOI: 10.1016/j.mec.2022.e00192
Govindprasad Bhutada , Guillaume Menard , Rupam Kumar Bhunia , Piotr P. Hapeta , Rodrigo Ledesma-Amaro , Peter J. Eastmond

Human milk fat has a distinctive stereoisomeric structure where palmitic acid is esterified to the middle (sn-2) position on the glycerol backbone of the triacylglycerol and unsaturated fatty acids to the outer (sn-1/3) positions. This configuration allows for more efficient nutrient absorption in the infant gut. However, the fat used in most infant formulas originates from plants, which exclude palmitic acid from the sn-2 position. Oleaginous yeasts provide an alternative source of lipids for human nutrition. However, these yeasts also exclude palmitic acid from the sn-2 position of their triacylglycerol. Here we show that Yarrowia lipolytica can be engineered to produce triacylglycerol with more than 60% of the palmitic acid in the sn-2 position, by expression of lysophosphatidic acid acyltransferases with palmitoyl-Coenzyme A specificity. The engineered Y. lipolytica strains can be cultured on glycerol, glucose, palm oil or a mixture of substrates, under nitrogen limited condition, to produce triacylglycerol with a fatty acid composition that resembles human milk fat, in terms of the major molecular species (palmitic, oleic and linoleic acids). Culture on palm oil or a mixture of glucose and palm oil produced the highest lipid titre and a triacylglycerol composition that is most similar with human milk fat. Our data show that an oleaginous yeast can be engineered to produce a human milk fat substitute (β-palmitate), that could be used as an ingredient in infant formulas.

人乳脂具有独特的立体异构体结构,其中棕榈酸被酯化到三酰基甘油骨架上的中间(sn-2)位置,不饱和脂肪酸被酯化到外侧(sn-1/3)位置。这种结构允许婴儿肠道更有效地吸收营养。然而,大多数婴儿配方奶粉中使用的脂肪来自植物,这就排除了sn-2位置的棕榈酸。产油酵母为人类营养提供了另一种脂质来源。然而,这些酵母也从其三酰甘油的sn-2位置排除棕榈酸。本研究表明,通过表达具有棕榈酰基辅酶A特异性的溶血磷脂酸酰基转移酶,可以对聚脂耶氏菌进行工程改造,使其以超过60%的棕榈酸在n-2位置产生三酰基甘油。在氮限制条件下,在甘油、葡萄糖、棕榈油或混合底物上培养的工程解脂芽孢杆菌菌株可以产生三酰基甘油,其脂肪酸组成类似于主要分子种类(棕榈酸、油酸和亚油酸)的人乳脂肪。在棕榈油或葡萄糖和棕榈油混合物上培养产生最高的脂质滴度和与人乳脂最相似的三酰基甘油组成。我们的数据表明,一种产油酵母可以被改造成一种人类乳脂替代品(β-棕榈酸酯),可以用作婴儿配方奶粉的一种成分。
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引用次数: 7
Thermodynamics contributes to high limonene productivity in cyanobacteria 热力学有助于蓝藻的高柠檬烯生产率
IF 5.2 Q1 Engineering Pub Date : 2022-06-01 DOI: 10.1016/j.mec.2022.e00193
Shrameeta Shinde , Sonali Singapuri , Zhenxiong Jiang , Bin Long , Danielle Wilcox , Camille Klatt , J. Andrew Jones , Joshua S. Yuan , Xin Wang

Terpenoids are a large group of secondary metabolites with broad industrial applications. Engineering cyanobacteria is an attractive route for the sustainable production of commodity terpenoids. Currently, a major obstacle lies in the low productivity attained in engineered cyanobacterial strains. Traditional metabolic engineering to improve pathway kinetics has led to limited success in enhancing terpenoid productivity. In this study, we reveal thermodynamics as the main determinant for high limonene productivity in cyanobacteria. Through overexpressing the primary sigma factor, a higher photosynthetic rate was achieved in an engineered strain of Synechococcus elongatus PCC 7942. Computational modeling and wet lab analyses showed an increased flux toward both native carbon sink glycogen synthesis and the non-native limonene synthesis from photosynthate output. On the other hand, comparative proteomics showed decreased expression of terpene pathway enzymes, revealing their limited role in determining terpene flux. Lastly, growth optimization by enhancing photosynthesis has led to a limonene titer of 19 mg/L in 7 days with a maximum productivity of 4.3 mg/L/day. This study highlights the importance of enhancing photosynthesis and substrate input for the high productivity of secondary metabolic pathways, providing a new strategy for future terpenoid engineering in phototrophs.

萜类化合物是一类具有广泛工业应用的次生代谢物。工程蓝藻是可持续生产商品萜类化合物的一条有吸引力的途径。目前,一个主要的障碍在于低生产力达到工程蓝藻菌株。传统的代谢工程,以改善途径动力学导致有限的成功,在提高萜类化合物的生产力。在这项研究中,我们揭示了热力学作为蓝藻中高柠檬烯生产力的主要决定因素。通过过表达初级sigma因子,长聚球菌工程菌株PCC 7942获得了较高的光合速率。计算模型和湿实验室分析表明,天然碳汇糖原合成和光合产物输出的非天然柠檬烯合成的通量都增加了。另一方面,比较蛋白质组学显示萜烯途径酶的表达减少,揭示了它们在决定萜烯通量方面的作用有限。最后,通过加强光合作用进行生长优化,使柠檬烯在7天内滴度达到19 mg/L,最大产量为4.3 mg/L/d。该研究强调了加强光合作用和底物输入对于提高次生代谢途径的生产力的重要性,为未来光养生物萜类工程提供了新的策略。
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引用次数: 7
De novo biosynthesis of diverse plant-derived styrylpyrones in Saccharomyces cerevisiae 不同植物源苯丙酮在酿酒酵母中的重新生物合成
IF 5.2 Q1 Engineering Pub Date : 2022-06-01 DOI: 10.1016/j.mec.2022.e00195
Yinan Wu , Maple N. Chen , Sijin Li

Plant styrylpyrones exerting well-established neuroprotective properties have attracted increasing attention in recent years. The ability to synthesize each individual styrylpyrone in engineered microorganisms is important to understanding the biological activity of medicinal plants and the complex mixtures they produce. Microbial biomanufacturing of diverse plant-derived styrylpyrones also provides a sustainable and efficient approach for the production of valuable plant styrylpyrones as daily supplements or potential drugs complementary to the prevalent agriculture-based approach. In this study, we firstly demonstrated the heterogenous biosynthesis of two 7,8-saturated styrylpyrones (7,8-dihydro-5,6-dehydrokavain (DDK) and 7,8-dihydroyangonin (DHY)) and two 7,8-unsaturated styrylpyrones (desmethoxyyangonin (DMY) and yangonin (Y)), in Saccharomyces cerevisiae. Although plant styrylpyrone biosynthetic pathways have not been fully elucidated, we functionally reconstructed the recently discovered kava styrylpyrone biosynthetic pathway that has high substrate promiscuity in yeast, and combined it with upstream hydroxycinnamic acid biosynthetic pathways to produce diverse plant-derived styrylpyrones without the native plant enzymes. We optimized the de novo pathways by engineering yeast endogenous aromatic amino acid metabolism and endogenous double bond reductases and by CRISPR-mediated δ-integration to overexpress the rate-limiting pathway genes. These combinatorial engineering efforts led to the first three yeast strains that can produce diverse plant-derived styrylpyrones de novo, with the titers of DDK, DMY and Y at 4.40 μM, 1.28 μM and 0.10 μM, respectively. This work has laid the foundation for larger-scale styrylpyrone biomanufacturing and the complete biosynthesis of more complicated plant styrylpyrones.

植物苯丙酮具有良好的神经保护作用,近年来受到越来越多的关注。在工程微生物中合成每个单独的苯乙烯吡啶酮的能力对于了解药用植物的生物活性及其产生的复杂混合物是重要的。多种植物源苯丙酮的微生物生物制造也为生产有价值的植物苯丙酮提供了一种可持续和有效的方法,作为日常补充剂或潜在的药物补充,以普遍的农业为基础的方法。在本研究中,我们首次在酿酒酵母菌中异质合成了两个7,8饱和的苯基pyro酮(7,8-二氢-5,6-脱氢钾素(DDK)和7,8-二氢阳根素(DHY))和两个7,8不饱和的苯基pyro酮(去甲氧基阳根素(DMY)和阳根素(Y))。虽然植物苯丙酮生物合成途径尚未完全阐明,但我们对最近在酵母中发现的具有高底物混杂性的卡瓦苯丙酮生物合成途径进行了功能重构,并将其与上游羟肉桂酸生物合成途径结合,在不使用天然植物酶的情况下生产了多种植物源苯丙酮。我们通过改造酵母内源性芳香氨基酸代谢和内源性双键还原酶,并通过crispr介导的δ-整合来过表达限速途径基因,从而优化了从头途径。通过这些组合工程的努力,首次获得了3株能够产生多种植物源性苯丙酮的酵母菌,其DDK、DMY和Y的滴度分别为4.40 μM、1.28 μM和0.10 μM。本研究为更大规模的苯乙烯吡咯酮生物制造和更复杂的植物苯乙烯吡咯酮的完全生物合成奠定了基础。
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引用次数: 5
Elimination of aromatic fusel alcohols as by-products of Saccharomyces cerevisiae strains engineered for phenylpropanoid production by 2-oxo-acid decarboxylase replacement 用2-氧基酸脱羧酶置换法去除酿酒酵母生产苯丙醇的副产物芳香醇醚
IF 5.2 Q1 Engineering Pub Date : 2021-12-01 DOI: 10.1016/j.mec.2021.e00183
Else-Jasmijn Hassing, Joran Buijs, Nikki Blankerts, Marijke A. Luttik, Erik A.de Hulster, Jack T. Pronk, Jean-Marc Daran

Engineered strains of the yeast Saccharomyces cerevisiae are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use l-phenylalanine and/or l-tyrosine, generated by the yeast shikimate pathway, as aromatic precursors. The Ehrlich pathway converts these precursors to aromatic fusel alcohols and acids, which are undesirable by-products of yeast strains engineered for production of high-value aromatic compounds. Activity of the Ehrlich pathway requires any of four S. cerevisiae 2-oxo-acid decarboxylases (2-OADCs): Aro10 or the pyruvate-decarboxylase isoenzymes Pdc1, Pdc5, and Pdc6. Elimination of pyruvate-decarboxylase activity from S. cerevisiae is not straightforward as it plays a key role in cytosolic acetyl-CoA biosynthesis during growth on glucose. In a search for pyruvate decarboxylases that do not decarboxylate aromatic 2-oxo acids, eleven yeast and bacterial 2-OADC-encoding genes were investigated. Homologs from Kluyveromyces lactis (KlPDC1), Kluyveromyces marxianus (KmPDC1), Yarrowia lipolytica (YlPDC1), Zymomonas mobilis (Zmpdc1) and Gluconacetobacter diazotrophicus (Gdpdc1.2 and Gdpdc1.3) complemented a Pdc strain of S. cerevisiae for growth on glucose. Enzyme-activity assays in cell extracts showed that these genes encoded active pyruvate decarboxylases with different substrate specificities. In these in vitro assays, ZmPdc1, GdPdc1.2 or GdPdc1.3 had no substrate specificity towards phenylpyruvate. Replacing Aro10 and Pdc1,5,6 by these bacterial decarboxylases completely eliminated aromatic fusel-alcohol production in glucose-grown batch cultures of an engineered coumaric acid-producing S. cerevisiae strain. These results outline a strategy to prevent formation of an important class of by-products in ‘chassis’ yeast strains for production of non-native aromatic compounds.

酵母工程菌株作为芳香化合物如羟基肉桂酸、二苯乙烯和黄酮类化合物的生产平台被广泛研究。生产这些化合物的异源途径使用由酵母莽草酸途径产生的l-苯丙氨酸和/或l-酪氨酸作为芳香前体。埃利希途径将这些前体转化为芳香醇和酸,这是用于生产高价值芳香化合物的酵母菌株的不良副产物。埃利希途径的活性需要四种酿酒葡萄球菌2-氧酸脱羧酶(2- oadc)中的任何一种:Aro10或丙酮酸脱羧酶同工酶Pdc1、Pdc5和Pdc6。消除酿酒酵母的丙酮酸脱羧酶活性并不是直截了当的,因为它在葡萄糖生长过程中对胞质乙酰辅酶a的生物合成起着关键作用。为了寻找不使芳香2-氧酸脱羧的丙酮酸脱羧酶,研究了11个酵母和细菌2- oadc编码基因。来自乳酸克卢维菌(KlPDC1)、马氏克卢维菌(KmPDC1)、多脂耶氏耶氏菌(YlPDC1)、活动单胞菌(Zmpdc1)和重氮营养菌(Gdpdc1.2和Gdpdc1.3)的同源物补充了酿酒酵母Pdc−菌株对葡萄糖的生长。细胞提取物的酶活性测定表明,这些基因编码的活性丙酮酸脱羧酶具有不同的底物特异性。在这些体外实验中,ZmPdc1、GdPdc1.2或GdPdc1.3对苯丙酮酸没有底物特异性。用这些细菌脱羧酶取代Aro10和Pdc1,5,6,完全消除了葡萄糖培养的香豆酸生产酵母菌株的芳香杂醇生产。这些结果概述了一种策略,以防止在“底盘”酵母菌株中形成一类重要的副产物,用于生产非天然芳香化合物。
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引用次数: 0
Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway 温伯格途径克服衣康酸过量大肠杆菌谷氨酸缺失症
IF 5.2 Q1 Engineering Pub Date : 2021-12-01 DOI: 10.1016/j.mec.2021.e00190
Ken W. Lu, Chris T. Wang, Hengray Chang, Ryan S. Wang, Claire R. Shen

Biosynthesis of itaconic acid occurs through decarboxylation of the TCA cycle intermediate cis-aconitate. Engineering of efficient itaconate producers often requires elimination of the highly active isocitrate dehydrogenase to conserve cis-aconitate, leading to 2-ketoglutarate auxotrophy in the producing strains. Supplementation of glutamate or complex protein hydrolysate then becomes necessary, often in large quantities, to support the high cell density desired during itaconate fermentation and adds to the production cost. Here, we present an alternative approach to overcome the glutamate auxotrophy in itaconate producers by synthetically introducing the Weimberg pathway from Burkholderia xenovorans for 2-ketoglutarate biosynthesis. Because of its independence from natural carbohydrate assimilation pathways in Escherichia coli, the Weimberg pathway is able to provide 2-ketoglutarate using xylose without compromising the carbon flux toward itaconate. With xylose concentration carefully tuned to minimize excess 2-ketoglutarate flux in the stationary phase, the final strain accumulated 20 g/L of itaconate in minimal medium from 18 g/L of xylose and 45 g/L of glycerol. Necessity of the recombinant Weimberg pathway for growth also allowed us to maintain multi-copy plasmids carrying in operon the itaconate-producing genes without addition of antibiotics. Use of the Weimberg pathway for growth restoration is applicable to other production systems with disrupted 2-ketoglutarate synthesis.

衣康酸的生物合成是通过三羧酸循环中间体顺-aconitate的脱羧发生的。高效的衣康酸生产者工程通常需要消除高活性的异柠檬酸脱氢酶来保存顺式乌头酸,导致生产菌株中2-酮戊二酸萎缩。为了支持衣康酸发酵过程中所需的高细胞密度,通常需要大量补充谷氨酸或复合蛋白水解物,这增加了生产成本。在这里,我们提出了一种替代方法,通过综合引入来自异种伯克霍尔德菌的Weimberg途径,用于2-酮戊二酸的生物合成,来克服衣康酸生产者的谷氨酸缺失。由于其独立于大肠杆菌的天然碳水化合物同化途径,Weimberg途径能够利用木糖提供2-酮戊二酸,而不会影响衣康酸的碳通量。仔细调整木糖浓度以减少固定相中过量的2-酮戊二酸通量,最终菌株在最小培养基中从18 g/L木糖和45 g/L甘油积累了20 g/L衣康酸。重组Weimberg途径对生长的必要性也使我们能够在不添加抗生素的情况下维持在操纵子中携带产itaconate基因的多拷贝质粒。使用Weimberg途径恢复生长适用于其他2-酮戊二酸合成中断的生产系统。
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引用次数: 4
Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds 工程贝氏不动杆菌ADP1从木质素衍生的芳香族化合物生产甲羟戊酸
IF 5.2 Q1 Engineering Pub Date : 2021-12-01 DOI: 10.1016/j.mec.2021.e00173
Erika Arvay , Bradley W. Biggs , Laura Guerrero , Virginia Jiang , Keith Tyo

Utilization of lignin, an abundant renewable resource, is limited by its heterogenous composition and complex structure. Biological valorization of lignin provides advantages over traditional chemical processing as it occurs at ambient temperature and pressure and does not use harsh chemicals. Furthermore, the ability to biologically funnel heterogenous substrates to products eliminates the need for costly downstream processing and separation of feedstocks. However, lack of relevant metabolic networks and low tolerance to degradation products of lignin limits the application of traditional engineered model organisms. To circumvent this obstacle, we employed Acinetobacter baylyi ADP1, which natively catabolizes lignin-derived aromatic substrates through the β-ketoadipate pathway, to produce mevalonate from lignin-derived compounds. We enabled expression of the mevalonate pathway in ADP1 and validated activity in the presence of multiple lignin-derived aromatic substrates. Furthermore, by knocking out wax ester synthesis and utilizing fed-batch cultivation, we improved mevalonate titers 7.5-fold to 1014 mg/L (6.8 mM). This work establishes a foundation and provides groundwork for future efforts to engineer improved production of mevalonate and derivatives from lignin-derived aromatics using ADP1.

木质素是一种丰富的可再生资源,但其组成的异质性和结构的复杂性限制了其利用。与传统的化学处理相比,木质素的生物增值具有优势,因为它是在环境温度和压力下进行的,并且不使用刺激性化学品。此外,生物漏斗异质底物到产品的能力消除了昂贵的下游加工和原料分离的需要。然而,缺乏相关的代谢网络和对木质素降解产物的低耐受性限制了传统工程模式生物的应用。为了克服这一障碍,我们利用baylyi不动杆菌ADP1,它通过β-酮己二酸途径天然分解木质素衍生的芳香底物,从木质素衍生的化合物中产生甲羟戊酸。我们在ADP1中激活了甲羟戊酸途径的表达,并在多种木质素衍生的芳香底物存在下验证了其活性。此外,通过敲除蜡酯合成并利用补料分批培养,我们将甲羟戊酸滴度提高了7.5倍,达到1014 mg/L(6.8 mM)。这项工作为未来利用ADP1改进木质素衍生芳烃的甲羟戊酸及其衍生物的生产奠定了基础和基础。
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引用次数: 12
Biosensor-based isolation of amino acid-producing Vibrio natriegens strains 基于生物传感器的产氨基酸营养弧菌菌株的分离
IF 5.2 Q1 Engineering Pub Date : 2021-12-01 DOI: 10.1016/j.mec.2021.e00187
Roberto Giuseppe Stella , Philipp Baumann , Sophia Lorke , Felix Münstermann , Astrid Wirtz , Johanna Wiechert , Jan Marienhagen , Julia Frunzke

The marine bacterium Vibrio natriegens has recently been demonstrated to be a promising new host for molecular biology and next generation bioprocesses. V. natriegens is a Gram-negative, non-pathogenic slight-halophilic bacterium, with a high nutrient versatility and a reported doubling time of under 10 min. However, V. natriegens is not an established model organism yet, and further research is required to promote its transformation into a microbial workhorse.

In this work, the potential of V. natriegens as an amino acid producer was investigated. First, the transcription factor-based biosensor LysG, from Corynebacterium glutamicum, was adapted for expression in V. natriegens to facilitate the detection of positively charged amino acids. A set of different biosensor variants were constructed and characterized, using the expression of a fluorescent protein as sensor output. After random mutagenesis, one of the LysG-based sensors was used to screen for amino acid producer strains. Here, fluorescence-activated cell sorting enabled the selective sorting of highly fluorescent cells, i.e. potential producer cells. Using this approach, individual L-lysine, L-arginine and L-histidine producers could be obtained producing up to 1 mM of the effector amino acid, extracellularly. Genome sequencing of the producer strains provided insight into the amino acid production metabolism of V. natriegens.

This work demonstrates the successful expression and application of transcription factor-based biosensors in V. natriegens and provides insight into the underlying physiology, forming a solid basis for further development of this promising microbe.

海洋细菌营养弧菌最近被证明是分子生物学和下一代生物过程的一个有前途的新宿主。V. natriegens是一种革兰氏阴性、非致病性的轻度嗜盐细菌,具有较高的营养多样性,据报道翻倍时间在10分钟以下。然而,V. natriegens还不是一种确定的模式生物,需要进一步的研究来促进它向微生物的转变。在这项工作中,研究了V. natriegens作为氨基酸生产者的潜力。首先,利用谷氨棒状杆菌转录因子为基础的生物传感器LysG在V. natriegens中表达,以方便检测带正电的氨基酸。利用荧光蛋白的表达作为传感器输出,构建并表征了一组不同的生物传感器变体。随机诱变后,利用一种基于lysg的传感器筛选氨基酸产生菌株。在这里,荧光激活的细胞分选能够选择性地分选高荧光细胞,即潜在的生产者细胞。使用这种方法,可以在细胞外获得单个l -赖氨酸、l -精氨酸和l -组氨酸产生物,产生高达1mm的效应氨基酸。产生菌的基因组测序提供了对弧菌氨基酸生产代谢的深入了解。这项工作证明了基于转录因子的生物传感器在V. natriegens中的成功表达和应用,并提供了对潜在生理学的深入了解,为进一步开发这种有前途的微生物奠定了坚实的基础。
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引用次数: 5
Production of raspberry ketone by redirecting the metabolic flux to the phenylpropanoid pathway in tobacco plants 通过将代谢通量重定向到苯丙素途径在烟草植物中产生覆盆子酮
IF 5.2 Q1 Engineering Pub Date : 2021-12-01 DOI: 10.1016/j.mec.2021.e00180
Takao Koeduka , Sachiho Takarada , Koya Fujii , Akifumi Sugiyama , Kazufumi Yazaki , Masahiro Nishihara , Kenji Matsui

Raspberry ketone is one of the characteristic flavors of raspberry fruits, and it is an important and expensive ingredient in the flavor and fragrance industries. It is present at low levels in plant tissues, and its occurrence is limited to a few taxa. In this context, the stable production of nature-identical raspberry ketone using heterologous synthesis in plants hosts has recently garnered the attention of plant biochemists. In this study, we demonstrate the rational switching of the metabolic flow from anthocyanin pigments to volatile phenylbutanoid production via the phenylpropanoid pathway. This shift led to the efficient and stable production of raspberry ketone and its glycosides via heterologous expression of the biosynthetic enzymes benzalacetone synthase (BAS) and raspberry ketone/zingerone synthase 1 (RZS1) in the transgenic tobacco (Nicotiana tabacum ‘Petit Havana SR-1’). Additionally, we achieved improved product titers by activating the phenylpropanoid pathway with the transcriptional factor, production of anthocyanin pigment 1 (PAP1), from Arabidopsis thaliana. We further demonstrated another metabolic-flow switching by RNA interference (RNAi)-mediated silencing of chalcone synthase (CHS) to increase pathway-intermediate p-coumaroyl-CoA in transgenic tobacco for raspberry-ketone production. The redirection of metabolic flux resulted in transgenic lines producing 0.45 μg/g of raspberry ketone and 4.5 μg/g, on the fresh weight basis, of its glycosides in the flowers. These results suggest that the intracellular enforcement of endogenous substrate supply is an important factor while engineering the phenylpropanoid pathway. This strategy might be useful for the production of other phenylpropanoids/polyketides that are produced via the pathway-intermediate p-coumaroyl-CoA, in tobacco plants.

树莓酮是树莓果实的特色香料之一,是香精工业中重要而昂贵的原料。它在植物组织中含量较低,并且只存在于少数分类群中。在这种背景下,利用植物宿主的异源合成稳定生产与自然相同的覆盆子酮最近引起了植物生化学家的关注。在这项研究中,我们证明了代谢流从花青素色素到通过苯丙素途径产生挥发性苯丁烷的合理转换。这一转变使得转基因烟草(Nicotiana tabacum ' Petit Havana SR-1)通过异源表达苯甲丙酮合成酶(BAS)和树莓酮/姜酮合成酶1 (RZS1)高效稳定地生产树莓酮及其苷类。此外,我们还利用拟南芥花青素色素1 (PAP1)的转录因子激活苯丙素途径,从而提高了产品滴度。我们进一步证明了另一种代谢流开关,通过RNA干扰(RNAi)介导的查尔酮合成酶(CHS)沉默,增加转基因烟草中途径中间的对coumaroyl- coa,用于树莓酮的生产。代谢通量的重定向导致转基因系在花中产生0.45 μg/g的覆盆子酮和4.5 μg/g的覆盆子苷(以鲜重计)。这些结果表明,细胞内对内源性底物供应的强制执行是设计苯丙素途径的重要因素。这一策略可能对烟草植物中通过中间体对coumaroyl- coa途径产生的其他苯丙素/聚酮类化合物的生产有用。
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引用次数: 4
期刊
Metabolic Engineering Communications
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