Metabolic Engineering Communications最新文献_第2页

13C-metabolic flux analysis of Saccharomyces cerevisiae in complex media 酵母在复杂培养基中的13c代谢通量分析

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-04-01 DOI: 10.1016/j.mec.2025.e00260

Hayato Fujiwara , Nobuyuki Okahashi , Taisuke Seike , Fumio Matsuda

Saccharomyces cerevisiae is often cultivated in complex media for applications in food and other biochemical production. However, ¹³C-metabolic flux analysis (¹³C-MFA) has been conducted for S. cerevisiae cultivated in synthetic media, resulting in a limited understanding of the metabolic flux distributions under the complex media. In this study, ¹³C-MFA was applied to S. cerevisiae cultivated in complex media to quantify the metabolic fluxes in the central metabolic network. S. cerevisiae was cultivated in a synthetic dextrose (SD) medium supplemented with 20 amino acids (SD + AA) and yeast extract peptone dextrose (YPD) medium. The results revealed that glutamic acid, glutamine, aspartic acid, and asparagine are incorporated into the TCA cycle as carbon sources in parallel with glucose consumption. Based on these findings, we successfully conducted ¹³C-MFA of S. cerevisiae cultivated in SD + AA and YPD media using parallel labeling and measured amino acid uptake rates. Furthermore, we applied the developed approach to ¹³C-MFA of yeast cultivated in malt extract medium. The analysis revealed that the metabolic flux through the anaplerotic and oxidative pentose phosphate pathways was lower in complex media than in synthetic media. Owing to the reduced carbon loss by the branching pathways, carbon flow toward ethanol production via glycolysis could be elevated. ¹³C-MFA of S. cerevisiae cultured in complex media provides valuable insights for metabolic engineering and process optimization in industrial yeast fermentation.

酿酒酵母通常在复杂的培养基中培养，用于食品和其他生化生产。然而，对酿酒酵母在合成培养基中培养的13c -代谢通量分析（13C-MFA），对复杂培养基下的代谢通量分布了解有限。本研究将13C-MFA应用于复杂培养基培养的酿酒酵母，量化其中心代谢网络中的代谢通量。在添加20种氨基酸（SD + AA）和酵母提取液蛋白胨葡萄糖（YPD）的合成葡萄糖（SD）培养基中培养酿酒酵母。结果表明，谷氨酸、谷氨酰胺、天冬氨酸和天冬酰胺作为碳源被纳入TCA循环，与葡萄糖消耗平行。基于这些发现，我们成功地利用平行标记法对SD + AA和YPD培养基培养的酿酒酵母进行了13C-MFA分析，并测量了氨基酸摄取率。此外，我们将该方法应用于麦芽提取物培养基中培养的酵母13C-MFA。分析结果表明，复合培养基中戊糖磷酸脱色和氧化途径的代谢通量低于合成培养基。由于分支途径减少了碳损失，通过糖酵解向乙醇生产的碳流量可能会增加。复杂培养基培养的酿酒酵母13C-MFA为工业酵母发酵代谢工程和工艺优化提供了有价值的见解。

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引用次数: 0

Production of borneol, camphor, and bornyl acetate using engineered Saccharomyces cerevisiae 利用工程酿酒酵母生产冰片、樟脑和冰片醋酸酯

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-03-31 DOI: 10.1016/j.mec.2025.e00259

Masahiro Tominaga , Kazuma Kawakami , Hiro Ogawa , Tomomi Nakamura , Akihiko Kondo , Jun Ishii

Microbial production of bicyclic monoterpenes is of great interest because their production primarily utilizes non-sustainable resources. Here, we report an engineered Saccharomyces cerevisiae yeast that produces bicyclic monoterpenes, including borneol, camphor, and bornyl acetate. The engineered yeast expresses a bornyl pyrophosphatase synthase from Salvia officinalis fused with mutated farnesyl pyrophosphate synthase from S. cerevisiae and two mevalonate pathway enzymes (an acetoacetyl-CoA thiolase/hydroxymethylglutaryl-CoA [HMG-CoA] reductase and an HMG-CoA synthase) from Enterococcus faecalis. The yeast produced up to 23.0 mg/L of borneol in shake-flask fermentation. By additionally expressing borneol dehydrogenase from Pseudomonas sp. TCU-HL1 or bornyl acetyltransferase from Wurfbainia villosa, the engineered yeast produced 23.5 mg/L of camphor and 21.1 mg/L of bornyl acetate, respectively. This is the first report of heterologous production of camphor and bornyl acetate.

微生物生产双环单萜烯是非常有趣的，因为它们的生产主要利用不可持续的资源。在这里，我们报告了一种工程酿酒酵母产生双环单萜，包括冰片，樟脑和龙脑酯醋酸酯。该工程酵母表达一种来自鼠尾草的龙脑基焦磷酸酶合成酶、一种来自酿酒酵母的突变法尼基焦磷酸酶和两种来自粪肠球菌的甲羟戊酸途径酶（乙酰乙酰辅酶- coa硫酶/羟甲基戊二酰辅酶[HMG-CoA]还原酶和一个HMG-CoA合成酶）。在摇瓶发酵中，酵母产生高达23.0 mg/L的冰片。此外，通过表达假单胞菌TCU-HL1的冰片脱氢酶或长绒Wurfbainia villosa的冰片乙酰转移酶，工程酵母的樟脑产量分别为23.5 mg/L和21.1 mg/L。本文首次报道了樟脑和醋酸龙脑酯的异种生产。

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引用次数: 0

Synthetic pathways for microbial biosynthesis of valuable pyrazine derivatives using genetically modified Pseudomonas putida KT2440 利用转基因恶臭假单胞菌KT2440合成有价吡嗪衍生物的微生物合成途径

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-03-30 DOI: 10.1016/j.mec.2025.e00258

Vytautas Petkevičius, Justė Juknevičiūtė, Domas Mašonis, Rolandas Meškys

Using engineered microbes for synthesizing high-valued chemicals from renewable sources is a foundation in synthetic biology, however, it is still in its early stages. Here, we present peculiarities and troubleshooting of the construction of novel synthetic metabolic pathways in genetically modified work-horse Pseudomonas putida KT2440. The combination of this microbial host and heterologous expressed non-heme diiron monooxygenases enabled de novo biosynthesis of 2,5-dimethylpyrazine (2,5-DMP) carboxylic acid and N-oxides as target products. A key intermediate, 2,5-DMP, was obtained by using Pseudomonas putida KT2440Δ6 strain containing six gene deletions in the L-threonine pathway, along with the overexpression of thrA^S345F and tdh from E. coli. Thus, the carbon surplus was redirected from glucose through L-threonine metabolism toward the formation of 2,5-DMP, resulting in a product titre of 106 ± 30 mg L⁻¹. By introducing two native genes (thrB and thrC from P. putida KT2440) from the L-threonine biosynthesis pathway, the production of 2,5-DMP was increased to 168 ± 20 mg L⁻¹. The resulting 2,5-DMP was further derivatized through two separate pathways. Recombinant P. putida KT2440 strain harboring xylene monooxygenase (XMO) produced 5-methyl-2-pyrazinecarboxylic acid from glucose as a targeted compound in a product titre of 204 ± 24 mg L⁻¹. The microbial host containing genes of PmlABCDEF monooxygenase (Pml) biosynthesized N-oxides – 2,5-dimethylpyrazine 1-oxide as a main product, and 2,5-dimethylpyrazine 1,4-dioxide as a minor product, reaching product titres of 82 ± 8 mg L⁻¹ and 11 ± 2 mg L⁻¹ respectively.

利用工程微生物从可再生资源中合成高价值化学品是合成生物学的基础，然而，它仍处于早期阶段。在这里，我们介绍了在转基因工作马恶臭假单胞菌KT2440中构建新的合成代谢途径的特点和故障排除。该微生物宿主与异种表达的非血红素二铁单加氧酶结合，使2,5-二甲基吡嗪（2,5- dmp）羧酸和n-氧化物作为靶产物重新生物合成。利用含有l -苏氨酸途径中6个基因缺失以及大肠杆菌中thrAS345F和tdh过表达的恶臭假单胞菌KT2440Δ6菌株，获得了关键中间体2,5- dmp。因此，碳过剩通过L-苏氨酸代谢从葡萄糖重定向到2,5- dmp的形成，导致产物滴度为106±30 mg L−1。在L-苏氨酸生物合成途径中引入两个天然基因（来自p.p putida KT2440的thrB和thrC），将2,5- dmp的产量提高到168±20 mg L−1。得到的2,5- dmp通过两个不同的途径进一步衍生化。含有二甲苯单加氧酶（XMO）的重组恶臭p.p . putida KT2440菌株以葡萄糖为目标化合物生产5-甲基-2-吡嗪羧酸，产品滴度为204±24 mg L−1。含有PmlABCDEF单加氧酶（Pml）基因的微生物宿主以n -氧化物- 2,5-二甲基吡嗪1-氧化物为主要产物，以2,5-二甲基吡嗪1,4-二氧化为次要产物，产物滴度分别为82±8 mg L−1和11±2 mg L−1。

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引用次数: 0

Metabolic growth-coupling strategies for in vivo enzyme selection systems 体内酶选择系统的代谢生长偶联策略

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-02-12 DOI: 10.1016/j.mec.2025.e00257

Tobias B. Alter , Pascal A. Pieters , Colton J. Lloyd , Adam M. Feist , Emre Özdemir , Bernhard O. Palsson , Daniel C. Zielinski

Whole-cell biocatalysis facilitates the production of a wide range of industrially and pharmaceutically relevant molecules from sustainable feedstocks such as plastic wastes, carbon dioxide, lignocellulose, or plant-based sugar sources. The identification and use of efficient enzymes in the applied biocatalyst is key to establishing economically feasible production processes. The generation and selection of favorable enzyme variants in adaptive laboratory evolution experiments using growth as a selection criterion is facilitated by tightly coupling enzyme catalytic activity to microbial metabolic activity. Here, we present a computational workflow to design strains that have a severe, growth-limiting metabolic chokepoint through a shared class of enzymes. The resulting chassis cell, termed enzyme selection system (ESS), is a platform for growth-coupling any enzyme from the respective enzyme class, thus offering cross-pathway application for enzyme engineering purposes. By applying the constraint-based modeling workflow, a publicly accessible database of 25,505 potential and experimentally tractable ESS designs was built for Escherichia coli and a broad range of production pathways with biotechnological relevance. A model-based analysis of the generated design database reveals a general design principle that the target enzyme activity is linked to overall microbial metabolic activity, not just the synthesis of one biomass precursor. It can be observed that the stronger the predicted coupling between target enzyme and metabolic activity, the lower the maximum growth rate and therefore the viability of an ESS. Consequently, growth-coupling strategies with only suboptimal coupling strengths, as are included in the ESS design database, may be of interest for practical applications of ESSs in order to circumvent overly restrictive growth defects. In summary, the computed design database, which is accessible via https://biosustain.github.io/ESS-Designs/, and its analysis provide a foundation for the generation of valuable in vivo ESSs for enzyme optimization purposes and a range of biotechnological applications in general.

全细胞生物催化有助于从可持续的原料（如塑料废物、二氧化碳、木质纤维素或植物性糖源）中生产广泛的工业和制药相关分子。在应用生物催化剂中识别和使用高效酶是建立经济可行的生产工艺的关键。在以生长为选择标准的适应性实验室进化实验中，酶的催化活性与微生物代谢活性紧密耦合，促进了有利酶变体的产生和选择。在这里，我们提出了一种计算工作流程，通过共享一类酶来设计具有严重的生长限制代谢瓶颈的菌株。由此产生的底盘细胞，被称为酶选择系统（ESS），是一个生长偶联的平台，从各自的酶类的任何酶，从而提供交叉途径应用于酶工程的目的。通过应用基于约束的建模工作流，为大肠杆菌和广泛的与生物技术相关的生产途径建立了一个可公开访问的数据库，其中包含25,505种潜在的和实验可处理的ESS设计。对生成的设计数据库进行基于模型的分析，揭示了一个通用的设计原则，即目标酶的活性与整体微生物代谢活性有关，而不仅仅是一种生物质前体的合成。由此可见，靶酶与代谢活性之间的耦合越强，ESS的最大生长速率越低，因此生存力越低。因此，仅具有次优耦合强度的增长耦合策略，如ESS设计数据库中所包含的，可能对ESS的实际应用感兴趣，以避免过度限制的增长缺陷。总之，可通过https://biosustain.github.io/ESS-Designs/访问的计算设计数据库及其分析为产生有价值的体内ESSs提供了基础，用于酶优化目的和一系列生物技术应用。

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引用次数: 0

Co-consumption for plastics upcycling: A perspective 塑料升级回收的共同消费：一个视角。

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2024-12-12 DOI: 10.1016/j.mec.2024.e00253

Michael Weldon, Sanniv Ganguly, Christian Euler

The growing plastics end-of-life crisis threatens ecosystems and human health globally. Microbial plastic degradation and upcycling have emerged as potential solutions to this complex challenge, but their industrial feasibility and limitations thereon have not been fully characterized. In this perspective paper, we review literature describing both plastic degradation and transformation of plastic monomers into value-added products by microbes. We aim to understand the current feasibility of combining these into a single, closed-loop process. Our analysis shows that microbial plastic degradation is currently the rate-limiting step to “closing the loop”, with reported rates that are orders of magnitude lower than those of pathways to upcycle plastic degradation products. We further find that neither degradation nor upcycling have been demonstrated at rates sufficiently high to justify industrialization at present. As a potential way to address these limitations, we suggest more investigation into mixotrophic approaches, showing that those which leverage the unique properties of plastic degradation products such as ethylene glycol might improve rates sufficiently to motivate industrial process development.

日益严重的塑料报废危机威胁着全球生态系统和人类健康。微生物塑料降解和升级再循环已成为应对这一复杂挑战的潜在解决方案，但其工业可行性和局限性尚未得到充分描述。在这篇视角论文中，我们回顾了有关微生物降解塑料和将塑料单体转化为高附加值产品的文献。我们的目的是了解目前将这两者结合成单一闭环工艺的可行性。我们的分析表明，目前微生物塑料降解是 "闭环 "的限速步骤，所报道的降解率要比塑料降解产物的升级循环途径低几个数量级。我们还发现，无论是降解还是升级再循环，其速率都不足以证明目前的工业化是合理的。作为解决这些局限性的一种潜在方法，我们建议对混养方法进行更多的研究，研究表明，利用乙二醇等塑料降解产物的独特性质的混养方法可能会提高降解率，从而推动工业化工艺的开发。

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引用次数: 0

From plastic waste to bioprocesses: Using ethylene glycol from polyethylene terephthalate biodegradation to fuel Escherichia coli metabolism and produce value-added compounds 从塑料垃圾到生物处理：利用聚对苯二甲酸乙二醇生物降解为大肠杆菌代谢提供燃料并产生增值化合物。

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2024-12-01 DOI: 10.1016/j.mec.2024.e00254

Alexandra Balola, Sofia Ferreira, Isabel Rocha

Polyethylene Terephthalate (PET) is a petroleum-based plastic polymer that, by design, can last decades, if not hundreds of years, when released into the environment through plastic waste leakage. In the pursuit of sustainable solutions to plastic waste recycling and repurposing, the enzymatic depolymerization of PET has emerged as a promising green alternative. However, the metabolic potential of the resulting PET breakdown molecules, such as the two-carbon (C2) molecule ethylene glycol (EG), remains largely untapped. Here, we review and discuss the current state of research regarding existing natural and synthetic microbial pathways that enable the assimilation of EG as a carbon and energy source for Escherichia coli. Leveraging the metabolic versatility of E. coli, we explore the viability of this widely used industrial strain in harnessing EG as feedstock for the synthesis of target value-added compounds via metabolic and protein engineering strategies. Consequently, we assess the potential of EG as a versatile alternative to conventional carbon sources like glucose, facilitating the closure of the loop between the highly available PET waste and the production of valuable biochemicals. This review explores the interplay between PET biodegradation and EG metabolism, as well as the key challenges and opportunities, while offering perspectives and suggestions for propelling advancements in microbial EG assimilation for circular economy applications.

聚对苯二甲酸乙二醇酯（PET）是一种基于石油的塑料聚合物，按照设计，当通过塑料废物泄漏释放到环境中时，它可以持续使用几十年，如果不是几百年的话。为了寻求塑料废物回收和再利用的可持续解决方案，PET的酶解聚合已成为一种有前途的绿色替代方案。然而，所产生的PET分解分子的代谢潜力，如二碳（C2）分子乙二醇（EG），在很大程度上仍未开发。在这里，我们回顾和讨论了现有的自然和合成微生物途径的研究现状，这些途径使大肠杆菌能够吸收EG作为碳和能量来源。利用大肠杆菌的代谢多样性，我们探索了这种广泛使用的工业菌株的可行性，利用EG作为原料，通过代谢和蛋白质工程策略合成目标增值化合物。因此，我们评估了EG作为葡萄糖等传统碳源的多功能替代品的潜力，促进了高可用性PET废物和有价值的生化物质生产之间的闭环。本文探讨了PET生物降解与EG代谢之间的相互作用，以及主要挑战和机遇，同时为推动微生物EG同化在循环经济应用中的进展提供了观点和建议。

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引用次数: 0

Cutting-edge developments in plastic biodegradation and upcycling via engineering approaches 通过工程方法在塑料生物降解和升级回收方面的前沿发展

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2024-12-01 DOI: 10.1016/j.mec.2024.e00256

Zeinab Rezaei, Amir Soleimani Dinani, Hamid Moghimi

The increasing use of plastics has resulted in the production of high quantities of plastic waste that pose a serious risk to the environment. The upcycling of plastics into value-added products offers a potential solution for resolving the plastics environmental crisis. Recently, various microorganisms and their enzymes have been identified for their ability to degrade plastics effectively. Furthermore, many investigations have revealed the application of plastic monomers as carbon sources for bio-upcycling to generate valuable materials such as biosurfactants, bioplastics, and biochemicals. With the advancement in the fields of synthetic biology and metabolic engineering, the construction of high-performance microbes and enzymes for plastic removal and bio-upcycling can be achieved. Plastic valorization can be optimized by improving uptake and conversion efficiency, engineering transporters and enzymes, metabolic pathway reconstruction, and also using a chemo-biological hybrid approach. This review focuses on engineering approaches for enhancing plastic removal and the methods of depolymerization and upcycling processes of various microplastics. Additionally, the major challenges and future perspectives for facilitating the development of a sustainable circular plastic economy are highlighted.

塑料使用量的增加导致大量塑料废物的产生，对环境构成严重威胁。将塑料升级为增值产品为解决塑料环境危机提供了一个潜在的解决方案。最近，各种微生物及其酶已被确定为具有有效降解塑料的能力。此外，许多研究已经揭示了塑料单体作为生物升级循环的碳源的应用，以产生有价值的材料，如生物表面活性剂、生物塑料和生物化学品。随着合成生物学和代谢工程领域的发展，构建高性能的塑料去除和生物升级利用微生物和酶是可能的。塑料增值可以通过提高吸收和转化效率、工程转运体和酶、代谢途径重建以及使用化学-生物杂交方法来优化。本文综述了增强塑料去除的工程方法以及各种微塑料的解聚和升级回收方法。此外，还强调了促进可持续循环塑料经济发展的主要挑战和未来前景。

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引用次数: 0

Genetically encoded biosensors for the circular plastics bioeconomy 用于循环塑料生物经济的基因编码生物传感器。

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2024-12-01 DOI: 10.1016/j.mec.2024.e00255

Micaela Chacón, Neil Dixon

Current plastic production and consumption routes are unsustainable due to impact upon climate change and pollution, and therefore reform across the entire value chain is required. Biotechnology offers solutions for production from renewable feedstocks, and to aid end of life recycling/upcycling of plastics. Biology sequence/design space is complex requiring high-throughput analytical methods to facilitate the iterative optimisation, design-build, test-learn (DBTL), cycle of Synthetic Biology. Furthermore, genetic regulatory tools can enable harmonisation between biotechnological demands and the physiological constraints of the selected production host. Genetically encoded biosensors offer a solution for both requirements to facilitate the circular plastic bioeconomy. In this review we present a summary of biosensors developed to date reported to be responsive to plastic precursors/monomers. In addition, we provide a summary of the demonstrated and prospective applications of these biosensors for the construction and deconstruction of plastics. Collectively, this review provides a valuable resource of biosensor tools and enabled applications to support the development of the circular plastics bioeconomy.

由于气候变化和污染的影响，目前的塑料生产和消费路线是不可持续的，因此需要对整个价值链进行改革。生物技术为可再生原料的生产提供了解决方案，并帮助塑料的报废回收/升级回收。生物序列/设计空间是复杂的，需要高通量的分析方法来促进合成生物学的迭代优化、设计-构建、测试-学习（DBTL）周期。此外，遗传调控工具可以使生物技术需求和选定的生产宿主的生理限制之间的协调。基因编码的生物传感器为促进循环塑料生物经济提供了一个解决方案。在这篇综述中，我们总结了迄今为止报道的对塑料前体/单体有反应的生物传感器。此外，我们还概述了这些生物传感器在塑料构造和解构方面的应用。总的来说，这篇综述提供了生物传感器工具的宝贵资源，并使应用能够支持循环塑料生物经济的发展。

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引用次数: 0

Reconstruction and analyses of genome-scale halomonas metabolic network yield a highly efficient PHA production 基因组尺度卤单胞菌代谢网络的重建和分析产生了高效的 PHA 生产

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2024-11-19 DOI: 10.1016/j.mec.2024.e00251

Luhui Zhang , Xinpei Sun , Jianwen Ye , QianQian Yuan , Xin Zhang , Fei Sun , Yongpan An , Yutong Chen , Yuehui Qian , Daqian Yang , Qian Wang , Miaomiao Gao , Tao Chen , Hongwu Ma , Guoqiang Chen , Zhengwei Xie

In pursuit of reliable and efficient industrial microbes, this study integrates cutting-edge systems biology tools with Halomonas bluephagenesis TD01, a robust halophilic bacterium. We generated the complete and annotated circular genome sequence for this model organism, constructed and meticulously curated a genome-scale metabolic network, achieving striking 86.32% agreement with Biolog Phenotype Microarray data and visualize the network via an interactive Electron/Thrift server architecture. We then analyzed the genome-scale network using vertex sampling analysis (VSA) and found that productions of biomass, polyhydroxyalkanoates (PHA), citrate, acetate, and pyruvate are mutually competing. Recognizing the dynamic nature of H. bluephagenesis TD01, we further developed and implemented the hyper-cube-shrink-analysis (HCSA) framework to predict effects of nutrient availabilities and metabolic reactions in the model on biomass and PHA accumulation. We then, based on the analysis results, proposed and validate multi-step feeding strategies tailored to different fermentation stages. This integrated approach yielded remarkable results, with fermentation culminating in a cell dry weight of 100.4 g/L and 70% PHA content, surpassing previous benchmarks. Our findings exemplify the powerful potential of system-level tools in the design and optimization of industrial microorganisms, paving the way for more efficient and sustainable bio-based processes.

为了追求可靠高效的工业微生物，本研究将前沿的系统生物学工具与蓝光单胞菌（Halomonas bluephagenesis TD01）--一种强健的嗜卤细菌--进行了整合。我们为这种模式生物生成了完整的注释环状基因组序列，构建并精心策划了基因组尺度的代谢网络，与生物表型芯片数据的一致性达到惊人的 86.32%，并通过交互式 Electron/Thrift 服务器架构实现了网络的可视化。然后，我们利用顶点取样分析（VSA）对基因组尺度网络进行了分析，发现生物量、多羟基烷酸（PHA）、柠檬酸盐、乙酸盐和丙酮酸盐的生成是相互竞争的。考虑到 H. bluephagenesis TD01 的动态性质，我们进一步开发并实施了超立方体-水槽分析（HCSA）框架，以预测模型中营养物质利用率和代谢反应对生物量和 PHA 积累的影响。然后，我们根据分析结果，提出并验证了针对不同发酵阶段的多步骤喂养策略。这种综合方法取得了显著的成果，发酵后细胞干重达到 100.4 克/升，PHA 含量达到 70%，超过了以前的基准。我们的研究结果体现了系统级工具在设计和优化工业微生物方面的强大潜力，为更高效、更可持续的生物基工艺铺平了道路。

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引用次数: 0

Selective production of the itaconic acid-derived compounds 2-hydroxyparaconic and itatartaric acid 选择性生产衣康酸衍生化合物 2-羟基衣康酸和酒石酸

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2024-11-16 DOI: 10.1016/j.mec.2024.e00252

Philipp Ernst , Felicia Zlati , Larissa Kever , Astrid Wirtz , Rainer Goldbaum , Jörg Pietruszka , Benedikt Wynands , Julia Frunzke , Nick Wierckx

There is a strong interest in itaconic acid in the medical and pharmaceutical sectors, both as an anti-bacterial compound and as an immunoregulator in mammalian macrophages. Fungal hosts also produce itaconic acid, and in addition they can produce two derivatives 2-hydroxyparaconic and itatartaric acid. Not much is known about these two derivatives, while their structural analogy to itaconate could open up several applications. In this study, we report the production of these two itaconate-derived compounds. By overexpressing the itaconate P450 monooxygenase Cyp3 in a previously engineered itaconate-overproducing Ustilago cynodontis strain, itaconate was converted to its lactone 2-hydroxyparaconate. The second product itatartarate is most likely the result of the subsequent lactone hydrolysis. A major challenge in the production of 2-hydroxyparaconate and itatartarate is their co-production with itaconate, leading to difficulties in their purification. Achieving high derivatives specificity was therefore the paramount objective. Different strategies were evaluated including process parameters such as substrate and pH, as well as strain engineering focusing on Cyp3 expression and product export. 2-hydroxyparaconate and itatartarate were successfully produced from glucose and glycerol, with the latter resulting in a higher derivatives specificity due to an overall slower metabolism on this non-preferred carbon source. The derivatives specificity could be further increased by metabolic engineering approaches including the exchange of the native itaconate transporter Itp1 with the Aspergillus terreus itaconate transporter MfsA. Both 2-hydroxyparaconate and itatartarate were recovered from fermentation supernatants following a pre-existing protocol. 2-hydroxyparaconate was recovered first through a process of evaporation, lactonization, and extraction with ethyl acetate. Subsequently, itatartarate could be obtained in the form of its sodium salt by saponification of the purified 2-hydroxyparaconate. Finally, several analytical methods were used to characterize the resulting products and their structures were confirmed by nuclear magnetic resonance spectroscopy. This work provides a promising foundation for obtaining 2-hydroxyparaconate and itatartarate in high purity and quantity. This will allow to unravel the full spectrum of potential applications of these novel compounds.

医学和制药领域对伊塔康酸有着浓厚的兴趣，它既是一种抗菌化合物，也是哺乳动物巨噬细胞的免疫调节剂。真菌宿主也能产生衣康酸，此外，它们还能产生两种衍生物--2-羟基衣康酸和酒石酸。目前对这两种衍生物的了解还不多，而它们与衣康酸的结构相似，可以开发出多种应用。在本研究中，我们报告了这两种伊它康酸衍生化合物的生产情况。通过在先前设计的过量生产伊它康酸的乌斯替拉克菌株中过表达伊它康酸 P450 单加氧酶 Cyp3，伊它康酸被转化为其内酯 2-hydroxyparaconate 。第二种产物酒石酸盐很可能是随后内酯水解的结果。生产 2-hydroxyparaconate 和 itatartarate 的一个主要挑战是它们与 itaconate 的共生，这导致了它们的纯化困难。因此，实现高衍生物特异性是首要目标。对不同的策略进行了评估，包括底物和 pH 值等工艺参数，以及侧重于 Cyp3 表达和产物输出的菌株工程。从葡萄糖和甘油中成功生产出了 2-hydroxyparaconate 和 itatartarate，其中后者的衍生物特异性更高，这是因为这种非首选碳源的整体代谢速度较慢。衍生物特异性可通过代谢工程方法进一步提高，包括用土曲霉伊他康酸转运体 MfsA 交换本地伊他康酸转运体 Itp1。按照已有的方案，从发酵上清液中回收了 2-羟基天冬氨酸和酒石酸。2-hydroxyparaconate 首先通过蒸发、内酯化和乙酸乙酯萃取过程回收。随后，通过对纯化的 2-hydroxyparaconate 进行皂化，可以得到钠盐形式的酒石酸衣塔酯。最后，我们使用了多种分析方法来表征所得到的产物，并通过核磁共振光谱法确认了它们的结构。这项工作为获得高纯度和高数量的 2-羟基对位乌头酸盐和酒石酸盐奠定了良好的基础。这将有助于全面了解这些新型化合物的潜在应用领域。

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