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Metabolic bottlenecks of Pseudomonas taiwanensis VLB120 during growth on d-xylose via the Weimberg pathway 台湾假单胞菌(Pseudomonas taiwanensis VLB120)通过魏姆伯格途径在二木糖生长过程中的代谢瓶颈
IF 5.2 Q1 Engineering Pub Date : 2024-06-01 DOI: 10.1016/j.mec.2024.e00241
Philipp Nerke, Jonas Korb, Frederick Haala, Georg Hubmann, Stephan Lütz

The microbial production of value-added chemicals from renewable feedstocks is an important step towards a sustainable, bio-based economy. Therefore, microbes need to efficiently utilize lignocellulosic biomass and its dominant constituents, such as d-xylose. Pseudomonas taiwanensis VLB120 assimilates d-xylose via the five-step Weimberg pathway. However, the knowledge about the metabolic constraints of the Weimberg pathway, i.e., its regulation, dynamics, and metabolite fluxes, is limited, which hampers the optimization and implementation of this pathway for bioprocesses. We characterized the Weimberg pathway activity of P. taiwanensis VLB120 in terms of biomass growth and the dynamics of pathway intermediates. In batch cultivations, we found excessive accumulation of the intermediates d-xylonolactone and d-xylonate, indicating bottlenecks in d-xylonolactone hydrolysis and d-xylonate uptake. Moreover, the intermediate accumulation was highly dependent on the concentration of d-xylose and the extracellular pH. To encounter the apparent bottlenecks, we identified and overexpressed two genes coding for putative endogenous xylonolactonases PVLB_05820 and PVLB_12345. Compared to the control strain, the overexpression of PVLB_12345 resulted in an increased growth rate and biomass generation of up to 30 % and 100 %, respectively. Next, d-xylonate accumulation was decreased by overexpressing two newly identified d-xylonate transporter genes, PVLB_18545 and gntP (PVLB_13665). Finally, we combined xylonolactonase overexpression with enhanced uptake of d-xylonate by knocking out the gntP repressor gene gntR (PVLB_13655) and increased the growth rate and biomass yield by 50 % and 24 % in stirred-tank bioreactors, respectively. Our study contributes to the fundamental knowledge of the Weimberg pathway in pseudomonads and demonstrates how to encounter the metabolic bottlenecks of the Weimberg pathway to advance strain developments and cell factory design for bioprocesses on renewable feedstocks.

利用微生物从可再生原料中生产增值化学品是实现可持续生物经济的重要一步。因此,微生物需要有效利用木质纤维素生物质及其主要成分,如木糖。台湾假单胞菌(Pseudomonas taiwanensis VLB120)通过五步魏姆伯格途径同化二木糖。然而,人们对魏姆伯格途径的代谢限制(即其调节、动态和代谢通量)了解有限,这阻碍了该途径在生物过程中的优化和实施。我们从生物量增长和途径中间产物的动态两方面描述了台湾褐藻 VLB120 的魏姆伯格途径活性。在批量培养过程中,我们发现中间产物 d-xylonolactone 和 d-xylonate 积累过多,这表明 d-xylonolactone 的水解和 d-xylonate 的吸收存在瓶颈。此外,中间产物的积累与二木糖的浓度和细胞外 pH 值密切相关。为了解决这些明显的瓶颈问题,我们发现并过表达了两个编码假定内源性木糖醇内酯酶 PVLB_05820 和 PVLB_12345 的基因。与对照菌株相比,过表达 PVLB_12345 可使生长率和生物量产生率分别提高 30% 和 100%。接下来,通过过表达两个新发现的尼龙酸盐转运体基因 PVLB_18545 和 gntP(PVLB_13665),减少了尼龙酸盐的积累。最后,我们通过敲除gntP抑制基因gntR(PVLB_13655),将木尼醇内酯酶的过表达与增强对d-木尼醇酸盐的吸收结合起来,使搅拌罐生物反应器中的生长速率和生物量产量分别提高了50%和24%。我们的研究丰富了假单胞菌魏姆伯格途径的基础知识,并展示了如何突破魏姆伯格途径的代谢瓶颈,从而推动菌株开发和细胞工厂设计,促进可再生原料的生物加工。
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引用次数: 0
Bioproduction of methylated phenylpropenes and isoeugenol in Escherichia coli 在大肠杆菌中生物生产甲基化苯丙烯和异丁香酚
IF 5.2 Q1 Engineering Pub Date : 2024-05-15 DOI: 10.1016/j.mec.2024.e00237
Jeremy Chua, Erik K.R. Hanko, Andrew Yiakoumetti, Ruth A. Stoney, Jakub Chromy, Kris Niño G. Valdehuesa, Katherine A. Hollywood, Cunyu Yan, Eriko Takano, Rainer Breitling

Phenylpropenes are a class of natural products that are synthesised by a vast range of plant species and hold considerable promise in the flavour and fragrance industries. Many in vitro studies have been carried out to elucidate and characterise the enzymes responsible for the production of these volatile compounds. However, there is a scarcity of studies demonstrating the in vivo production of phenylpropenes in microbial cell factories. In this study, we engineered Escherichia coli to produce methylchavicol, methyleugenol and isoeugenol from their respective phenylacrylic acid precursors. We achieved this by extending and modifying a previously optimised heterologous pathway for the biosynthesis of chavicol and eugenol. We explored the potential of six S-adenosyl l-methionine (SAM)-dependent O-methyltransferases to produce methylchavicol and methyleugenol from chavicol and eugenol, respectively. Additionally, we examined two isoeugenol synthases for the production of isoeugenol from coniferyl acetate. The best-performing strains in this study were able to achieve titres of 13 mg L−1 methylchavicol, 59 mg L−1 methyleugenol and 361 mg L−1 isoeugenol after feeding with their appropriate phenylacrylic acid substrates. We were able to further increase the methyleugenol titre to 117 mg L−1 by supplementation with methionine to facilitate SAM recycling. Moreover, we report the biosynthesis of methylchavicol and methyleugenol from l-tyrosine through pathways involving six and eight enzymatic steps, respectively.

苯丙烯是一类由多种植物合成的天然产品,在香料和香精工业中大有可为。已经进行了许多体外研究,以阐明负责生产这些挥发性化合物的酶的特性。然而,在微生物细胞工厂中体内生产苯丙烯的研究却很少。在本研究中,我们改造了大肠杆菌,使其能够从各自的苯丙氨酸前体中生产甲基茶维素、甲基丁香酚和异丁香酚。我们通过扩展和修改以前优化的异源途径来实现茶维醇和丁香酚的生物合成。我们探索了六种依赖于 S-腺苷-l-蛋氨酸(SAM)的 O-甲基转移酶分别从茶维醇和丁香酚生成甲基茶维醇和甲基丁香酚的潜力。此外,我们还研究了两种异丁香酚合成酶,以从针叶乙酸酯中生产异丁香酚。本研究中表现最好的菌株在喂食适当的苯丙氨酸底物后,能达到 13 mg L-1 甲基茶维醇、59 mg L-1 甲基丁香酚和 361 mg L-1 异丁香酚的滴度。通过补充蛋氨酸促进 SAM 循环,我们能够将甲基丁香酚滴度进一步提高到 117 mg L-1。此外,我们还报告了由 l-酪氨酸通过分别涉及六个和八个酶步骤的途径生物合成甲基茶酚和甲基丁香酚的情况。
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引用次数: 0
Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium 通过并行进化谷氨酸棒状杆菌直接硫化途径中的两种酶来工程化蛋氨酸辅助营养大肠杆菌,使其能够在最小培养基中复原
IF 5.2 Q1 Engineering Pub Date : 2024-05-10 DOI: 10.1016/j.mec.2024.e00236
Matan Gabay , Inbar Stern , Nadya Gruzdev , Adi Cohen , Lucia Adriana-Lifshits , Tamar Ansbacher , Itamar Yadid , Maayan Gal

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichia coli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

蛋氨酸的生物合成依赖于多种酶的顺序催化。大肠杆菌是科研和工业中用于蛋白质生产和工程的主要细菌,它利用由 L-高丝氨酸 O-琥珀酰转移酶、胱硫醚 gamma 合成酶和胱硫醚 beta 裂解酶催化的三步反式硫化途径将 L-高丝氨酸转化为 L-高半胱氨酸。然而,大多数细菌采用两步直接硫化途径,涉及 L-高丝氨酸 O-乙酰转移酶和 O-乙酰高丝氨酸巯基酶。我们以前曾发现,大肠埃希氏菌株(MG1655)缺失了编码 L-高丝氨酸 O-琥珀酰转移酶的 metA 和编码胱硫醚γ合成酶的 metB,但可以通过引入编码 L-高丝氨酸 O-琥珀酰转移酶的 metX、编码 L-高丝氨酸 O-乙酰转移酶的基因 metX 和编码 O-乙酰高丝氨酸巯基酶的基因 metY,从而改变大肠杆菌蛋氨酸生物合成代谢途径,使其直接硫化。然而,从谷氨酸棒杆菌(Corynebacterium glutamicum)中引入 metX 和 metY 无法补充蛋氨酸辅助营养。在此,我们以谷氨酸棒杆菌的 metX 和 metY 为基础生成了一个随机基因文库,并将其转化到缺乏 metA 和 metB 基因的蛋氨酸辅助营养大肠杆菌菌株中。通过多次富集循环,我们成功分离出能够在 M9 最小培养基中生长的活性克隆。在进化的蛋氨酸自养型大肠埃希菌中,显性的 metX 突变为 L315P 和 H46R。有趣的是,我们发现只编码野生型 MetY 酶 438 个氨基酸中 106 个 N 端的 metY 基因具有功能,并能支持蛋氨酸辅助营养体的生长。将新基因重新克隆到原始质粒中并转化到蛋氨酸辅助营养大肠杆菌中验证了其功能。这些结果表明,定向酶进化能够在大肠杆菌蛋氨酸直接硫化途径中快速、同步地设计出新的活性变体,从而实现高效互补。
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引用次数: 0
Improving 5-(hydroxymethyl)furfural (HMF) tolerance of Pseudomonas taiwanensis VLB120 by automated adaptive laboratory evolution (ALE) 通过实验室自动适应性进化(ALE)提高台湾假单胞菌 VLB120 对 5-(羟甲基)糠醛(HMF)的耐受性
IF 5.2 Q1 Engineering Pub Date : 2024-05-10 DOI: 10.1016/j.mec.2024.e00235
Thorsten Lechtenberg, Benedikt Wynands, Moritz-Fabian Müller, Tino Polen, Stephan Noack, Nick Wierckx

The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse P. taiwanensis VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding mexT preventing expression of the associated efflux pump mexEF-oprN. This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms.

5-(hydroxymethyl)furfural (HMF)醛对循环生物经济具有重要意义。它是一种可再生的平台化学品,可转化为一系列有用的化合物,以替代石油产品,如绿色塑料单体 2,5-呋喃二甲酸(FDCA)。然而,它也表现出微生物毒性,例如阻碍了木质纤维素水解物的高效生物技术价值化。因此,迫切需要耐受性更强、适用于全细胞生物催化的生物。在这里,我们通过机器人辅助的适应性实验室进化(ALE)技术,设计出了一种氧化缺陷衍生物,它是一种天然强健的新兴生物技术主力 P. taiwanensis VLB120。通过删除 HMF 氧化酶,首次实现了在醛的持续选择压力下的进化,产生了在有毒物质存在下生长特性持续改善的菌株。对进化克隆的基因组测序发现,编码 mexT 的 LysR 型转录调节器发生了功能缺失突变,从而阻止了相关外排泵 mexEF-oprN 的表达。有了这些知识,即使在 HMF 氧化机制活跃或过度表达的背景下,也能逆向工程培育出耐醛性更强的菌株,这证明了两种不同耐醛机制的协同效应。
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引用次数: 0
Bottom-up parameterization of enzyme rate constants: Reconciling inconsistent data 酶速率常数的自下而上参数化:调和不一致的数据
IF 5.2 Q1 Engineering Pub Date : 2024-04-23 DOI: 10.1016/j.mec.2024.e00234
Daniel C. Zielinski , Marta R.A. Matos , James E. de Bree , Kevin Glass , Nikolaus Sonnenschein , Bernhard O. Palsson

Kinetic models of metabolism are promising platforms for studying complex metabolic systems and designing production strains. Given the availability of enzyme kinetic data from historical experiments and machine learning estimation tools, a straightforward modeling approach is to assemble kinetic data enzyme by enzyme until a desired scale is reached. However, this type of ‘bottom up’ parameterization of kinetic models has been difficult due to a number of issues including gaps in kinetic parameters, the complexity of enzyme mechanisms, inconsistencies between parameters obtained from different sources, and in vitro-in vivo differences. Here, we present a computational workflow for the robust estimation of kinetic parameters for detailed mass action enzyme models while taking into account parameter uncertainty. The resulting software package, termed MASSef (the Mass Action Stoichiometry Simulation Enzyme Fitting package), can handle standard ‘macroscopic’ kinetic parameters, including Km, kcat, Ki, Keq, and nh, as well as diverse reaction mechanisms defined in terms of mass action reactions and ‘microscopic’ rate constants. We provide three enzyme case studies demonstrating that this approach can identify and reconcile inconsistent data either within in vitro experiments or between in vitro and in vivo enzyme function. We further demonstrate how parameterized enzyme modules can be used to assemble pathway-scale kinetic models consistent with in vivo behavior. This work builds on the legacy of knowledge on kinetic behavior of enzymes by enabling robust parameterization of enzyme kinetic models at scale utilizing the abundance of historical literature data and machine learning parameter estimates.

代谢动力学模型是研究复杂代谢系统和设计生产菌株的理想平台。鉴于可以从历史实验和机器学习估算工具中获得酶动力学数据,一种直接的建模方法是逐个酶收集动力学数据,直到达到所需的规模。然而,这种 "自下而上 "的动力学模型参数化一直很困难,原因有很多,包括动力学参数的差距、酶机制的复杂性、从不同来源获得的参数之间的不一致性以及体外-体内差异。在此,我们提出了一种计算工作流程,用于对详细的质量作用酶模型的动力学参数进行稳健估算,同时考虑到参数的不确定性。由此产生的软件包被称为 MASSef(质量作用化学计量模拟酶拟合软件包),可以处理标准的 "宏观 "动力学参数,包括 Km、kcat、Ki、Keq 和 nh,以及以质量作用反应和 "微观 "速率常数定义的各种反应机制。我们提供了三个酶案例研究,证明这种方法可以识别并调和体外实验中或体外与体内酶功能之间不一致的数据。我们进一步展示了参数化酶模块如何用于建立与体内行为一致的通路尺度动力学模型。这项工作利用大量历史文献数据和机器学习参数估计,实现了大规模酶动力学模型的稳健参数化,从而在酶动力学行为知识遗产的基础上更上一层楼。
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引用次数: 0
2-Stage microfermentations 2 级微发酵
IF 5.2 Q1 Engineering Pub Date : 2024-04-08 DOI: 10.1016/j.mec.2024.e00233
Shuai Li , Zhixia Ye , Eirik A. Moreb , Romel Menacho-Melgar , Maximillian Golovsky , Michael D. Lynch

Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of protocols for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, by detailing the approach to protocol development, these methods are also adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.

以细胞为基础的工厂可以生产多种产品。DNA 合成和基因组编辑技术的进步大大简化了这些工厂的设计和建造。生成数百甚至数千个用于评估的细胞工厂菌株变体变得前所未有的容易。这些进步更加需要标准化、高通量的方法来评估这些设计。为了实现这一目标,我们之前报道了工程大肠杆菌菌株的开发和相关的两阶段生产流程,以简化和标准化菌株工程、评估和放大。这种方法将生长(第 1 阶段)与生产(第 2 阶段)分离开来。磷酸盐耗竭是停止生长和诱导异源表达的触发器。在此,我们详细介绍了在两阶段微发酵中对工程大肠杆菌菌株进行评估的方案开发。这些方案可随时用于评估生产各种蛋白质和小分子产品的菌株。此外,通过详细介绍方案开发的方法,这些方法还可适用于其他细胞宿主,以及具有各种额外触发器的其他两阶段过程。
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引用次数: 0
Building blocks needed for mechanistic modeling of bioprocesses: A critical review based on protein production by CHO cells 生物过程机理建模所需的基础模块:基于 CHO 细胞生产蛋白质的重要综述
IF 5.2 Q1 Engineering Pub Date : 2024-02-29 DOI: 10.1016/j.mec.2024.e00232
Yusmel González-Hernández, Patrick Perré

This paper reviews the key building blocks needed to develop a mechanistic model for use as an operational production tool. The Chinese Hamster Ovary (CHO) cell, one of the most widely used hosts for antibody production in the pharmaceutical industry, is considered as a case study. CHO cell metabolism is characterized by two main phases, exponential growth followed by a stationary phase with strong protein production. This process presents an appropriate degree of complexity to outline the modeling strategy. The paper is organized into four main steps: (1) CHO systems and data collection; (2) metabolic analysis; (3) formulation of the mathematical model; and finally, (4) numerical solution, calibration, and validation. The overall approach can build a predictive model of target variables. According to the literature, one of the main current modeling challenges lies in understanding and predicting the spontaneous metabolic shift. Possible candidates for the trigger of the metabolic shift include the concentration of lactate and carbon dioxide. In our opinion, ammonium, which is also an inhibiting product, should be further investigated. Finally, the expected progress in the emerging field of hybrid modeling, which combines the best of mechanistic modeling and machine learning, is presented as a fascinating breakthrough. Note that the modeling strategy discussed here is a general framework that can be applied to any bioprocess.

本文回顾了开发用作生产操作工具的机理模型所需的关键构件。中国仓鼠卵巢(CHO)细胞是制药业生产抗体最广泛使用的宿主之一,本文以该细胞为案例进行研究。CHO 细胞的新陈代谢有两个主要阶段:指数增长期和静止期,前者可产生大量蛋白质。这一过程具有适当的复杂性,因此需要概述建模策略。本文分为四个主要步骤:(1) CHO 系统和数据收集;(2) 代谢分析;(3) 数学模型的制定;最后,(4) 数值求解、校准和验证。整个方法可以建立目标变量的预测模型。根据文献,目前建模的主要挑战之一在于理解和预测自发的新陈代谢转变。触发新陈代谢转变的可能因素包括乳酸和二氧化碳的浓度。我们认为,铵也是一种抑制产物,应进一步研究。最后,本文介绍了混合建模这一新兴领域的预期进展,它结合了机理建模和机器学习的优点,是一项引人入胜的突破。请注意,本文讨论的建模策略是一个通用框架,可应用于任何生物过程。
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引用次数: 0
A new concept of biocatalytic synthesis of acrylic monomers for obtaining water-soluble acrylic heteropolymers 生物催化合成丙烯酸单体以获得水溶性丙烯酸杂聚合物的新概念
IF 5.2 Q1 Engineering Pub Date : 2023-12-19 DOI: 10.1016/j.mec.2023.e00231
Konstantin V. Lavrov , Anna O. Shemyakina , Elena G. Grechishnikova , Tatyana V. Gerasimova , Tatyana I. Kalinina , Andrey D. Novikov , Tatyana E. Leonova , Ludmila E. Ryabchenko , Telman A. Bayburdov , Alexander S. Yanenko

Rhodococcus strains were designed as model biocatalysts (BCs) for the production of acrylic acid and mixtures of acrylic monomers consisting of acrylamide, acrylic acid, and N-alkylacrylamide (N-isopropylacrylamide). To obtain BC strains, we used, among other approaches, adaptive laboratory evolution (ALE), based on the use of the metabolic pathway of amide utilization. Whole genome sequencing of the strains obtained after ALE, as well as subsequent targeted gene disruption, identified candidate genes for three new amidases that are promising for the development of BCs for the production of acrylic acid from acrylamide. New BCs had two types of amidase activities, acrylamide-hydrolyzing and acrylamide-transferring, and by varying the ratio of these activities in BCs, it is possible to influence the ratio of monomers in the resulting mixtures. Based on these strains, a prototype of a new technological concept for the biocatalytic synthesis of acrylic monomers was developed for the production of water-soluble acrylic heteropolymers containing valuable N-alkylacrylamide units. In addition to the possibility of obtaining mixtures of different compositions, the advantages of the concept are a single starting reagent (acrylamide), more unification of processes (all processes are based on the same type of biocatalyst), and potentially greater safety for personnel and the environment compared to existing chemical technologies.

Rhodococcus 菌株被设计为生产丙烯酸以及由丙烯酰胺、丙烯酸和 N-烷基丙烯酰胺(N-异丙基丙烯酰胺)组成的丙烯酸单体混合物的模型生物催化剂(BC)。为了获得 BC 菌株,我们采用了适应性实验室进化(ALE)等方法,其基础是利用酰胺的代谢途径。通过对适应性实验室进化(ALE)后获得的菌株进行全基因组测序以及随后的靶向基因干扰,我们发现了三种新型酰胺酶的候选基因,这些基因有望用于开发从丙烯酰胺生产丙烯酸的 BCs。新的 BCs 具有两种酰胺酶活性,即丙烯酰胺水解活性和丙烯酰胺转移活性,通过改变 BCs 中这两种活性的比例,可以影响所得混合物中单体的比例。在这些菌株的基础上,开发出了丙烯酸单体生物催化合成新技术概念的原型,用于生产含有宝贵的 N-烷基丙烯酰胺单元的水溶性丙烯酸杂聚合物。除了可以获得不同成分的混合物外,该概念的优势还在于只需一种起始试剂(丙烯酰胺),工艺更加统一(所有工艺都基于同一种生物催化剂),而且与现有的化学技术相比,对人员和环境可能更加安全。
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引用次数: 0
Rewiring Saccharomyces cerevisiae metabolism for optimised Taxol® precursors production 重组酿酒酵母代谢优化紫杉醇®前体生产
IF 5.2 Q1 Engineering Pub Date : 2023-11-15 DOI: 10.1016/j.mec.2023.e00229
Behnaz Nowrouzi , Pablo Torres-Montero , Eduard J. Kerkhoven , José L. Martínez , Leonardo Rios-Solis

Saccharomyces cerevisiae has been conveniently used to produce Taxol® anticancer drug early precursors. However, the harmful impact of oxidative stress by the first cytochrome P450-reductase enzymes (CYP725A4-POR) of Taxol® pathway has hampered sufficient progress in yeast. Here, we evolved an oxidative stress-resistant yeast strain with three-fold higher titre of their substrate, taxadiene. The performance of the evolved and parent strains were then evaluated in galactose-limited chemostats before and under the oxidative stress by an oxidising agent. The interaction of evolution and oxidative stress was comprehensively evaluated through transcriptomics and metabolite profiles integration in yeast enzyme-constrained genome scale model. Overall, the evolved strain showed improved respiration, reduced overflow metabolites production and oxidative stress re-induction tolerance. The cross-protection mechanism also potentially contributed to better heme, flavin and NADPH availability, essential for CYP725A4 and POR optimal activity in yeast. The results imply that the evolved strain is a robust cell factory for future efforts towards Taxol© production.

酿酒酵母已被方便地用于生产紫杉醇®抗癌药物的早期前体。然而,紫杉醇(Taxol)途径的第一个细胞色素p450 -还原酶(CYP725A4-POR)氧化应激的有害影响阻碍了酵母的充分进展。在这里,我们进化出一种抗氧化应激的酵母菌株,其底物taxadiene的滴度提高了三倍。进化菌株和亲本菌株在氧化应激前和氧化应激下在半乳糖限制的趋化剂中进行了性能评估。在酵母酶约束的基因组尺度模型中,通过转录组学和代谢物谱整合,全面评估了进化与氧化应激的相互作用。总体而言,进化菌株表现出呼吸改善,溢出代谢物产生减少和氧化应激再诱导耐受性。交叉保护机制也可能有助于提高血红素、黄素和NADPH的利用率,这是酵母中CYP725A4和POR优化活性所必需的。结果表明,进化菌株是一个强大的细胞工厂,为未来紫杉醇©生产的努力。
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引用次数: 0
Engineering Yarrowia lipolytica for the biosynthesis of geraniol 工程解脂耶氏菌生物合成香叶醇
IF 5.2 Q1 Engineering Pub Date : 2023-11-02 DOI: 10.1016/j.mec.2023.e00228
Ayushi Agrawal , Zhiliang Yang , Mark Blenner

Geraniol is a monoterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Microbial production has largely used model organisms lacking favorable properties for monoterpene production. In this work, we produced geraniol in metabolically engineered Yarrowia lipolytica. First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were tested based on previous reports of activity. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by co-expressing with MVA pathway enzymes tHMG1 and IDI1. Truncated CrGES (tCrGES) produced the most geraniol and thus was used for further experimentation. The initial strain was obtained by overexpression of the truncated HMG1, IDI and tCrGES. The acetyl-CoA precursor pool was enhanced by overexpressing mevalonate pathway genes such as ERG10, HMGS or MVK, PMK. The final strain overexpressing 3 copies of tCrGES and single copies of ERG10, HMGS, tHMG1, IDI produced approximately 1 g/L in shake-flask fermentation. This is the first demonstration of geraniol production in Yarrowia lipolytica and the highest de novo titer reported to date in yeast.

香叶醇是一种单萜类化合物,在食品、化妆品、制药等行业有着广泛的应用。微生物生产在很大程度上使用了缺乏单萜烯生产有利特性的模式生物。在这项工作中,我们在代谢工程的解脂耶氏菌中生产香叶醇。首先,在前人报道的基础上,对两种植物源性香叶醇合成酶(GES)进行了活性检测。通过与MVA途径酶tHMG1和IDI1共表达,检测了GES野生型和截断突变体(不靶向叶绿体的信号肽)。截断的CrGES (tCrGES)产生的香叶醇最多,因此用于进一步的实验。通过过表达截断的HMG1、IDI和tCrGES获得初始菌株。乙酰辅酶a前体库通过过表达甲羟戊酸途径基因如ERG10、HMGS或MVK、PMK而增强。最终菌株在摇瓶发酵中过表达3份tCrGES和单份ERG10、HMGS、tHMG1、IDI,产量约为1 g/L。这是首次证明在解脂耶氏菌中产生香叶醇,也是迄今为止在酵母中报道的最高的新生滴度。
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引用次数: 1
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Metabolic Engineering Communications
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