Metabolic engineering最新文献

The faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis. DptE 巴豆酰化对达托霉素生物合成初始流程的龙头旋钮效应。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-12 DOI: 10.1016/j.ymben.2024.11.003

Wen-Li Gao, Lie Ma, Meng-Han Li, Wei-Feng Xu, Chen-Fan Sun, Qing-Wei Zhao, Xin-Ai Chen, Zhong-Yuan Lyu, Yong-Quan Li

We propose here that acylation modification of actinomycete proteins is a restrictive system that limits the excessive synthesis of secondary metabolites, its mechanism has not been clearly elucidated before. We used crotonylation as an example to investigate the acylation effect in the daptomycin biosynthesis by Streptomyces roseosporus. Our experiments revealed abundant crotonylation of numerous secondary metabolic enzymes in Streptomyces roseosporus, a daptomycin producer. DptE, which initiates daptomycin biosynthesis, is crotonylated at K454. We experimentally identified the corresponding DptE crotonyltransferase Kct1 and decrotonylase CobB. Further studies consistently confirmed that decrotonylation increases DptE activity. Decrotonylation functions like loosening a faucet knob, increasing substrate channel throughput and the initial flow of daptomycin biosynthesis. Moreover, DptE catalytic activity was enhanced via K454 and neighboring residues K184 and Q420 mutation, increasing daptomycin yield by 132%; daptomycin biosynthesis related metabolism activities also increased. Substrate channel prediction revealed 38% higher throughput for mutant DptE (K454I/K184Q/Q420N) than crotonylated DptE. Molecular dynamics (MD) simulations revealed significant increases in flexibility and substrate affinity of the mutant. In summary, we elucidated the faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis and adopted decrotonylation to generate high-yield industrial strains.

我们在此提出，放线菌蛋白质的酰化修饰是一种限制次生代谢产物过度合成的约束系统，但其机制尚未得到明确阐明。我们以巴豆酰化为例，研究了玫瑰孢链霉菌在达托霉素生物合成过程中的酰化效应。我们的实验发现，作为达托霉素生产者的玫瑰孢链霉菌（Streptomyces roseosporus）中的许多次级代谢酶都存在大量的巴豆酰化作用。启动达托霉素生物合成的 DptE 在 K454 处被巴豆酰化。我们通过实验确定了相应的 DptE 巴豆酰基转移酶 Kct1 和脱巴豆酰基酶 CobB。进一步的研究一致证实，脱质子酰化增加了 DptE 的活性。脱质子酰化的作用就像松开水龙头旋钮，增加了底物通道的吞吐量和达托霉素生物合成的初始流量。此外，通过 K454 及其邻近残基 K184 和 Q420 的突变，DptE 的催化活性得到了增强，使达托霉素的产量增加了 132%；达托霉素生物合成相关的代谢活动也有所增加。底物通道预测显示，突变 DptE（K454I/K184Q/Q420N）的吞吐量比巴豆化 DptE 高 38%。分子动力学（MD）模拟显示，突变体的灵活性和底物亲和力显著提高。总之，我们阐明了 DptE 巴豆酰化对达托霉素生物合成初始流程的龙头旋钮效应，并通过去巴豆酰化生成了高产工业菌株。

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引用次数: 0

Versatile Xylose and Arabinose Genetic Switches development for Yeasts. 为酵母开发多功能木糖和阿拉伯糖基因开关。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-11 DOI: 10.1016/j.ymben.2024.11.004

Shuhui Guo, Juhua Du, Donghan Li, Jinghui Xiong, Ye Chen

Inducible transcription systems are essential tools in genetic engineering, where tight control, strong inducibility and fast response with cost-effective inducers are highly desired. However, existing systems in yeasts are rarely used in large-scale fermentations due to either cost-prohibitive inducers or incompatible performance. Here, we developed powerful xylose and arabinose induction systems in Saccharomyces cerevisiae, utilizing eukaryotic activators XlnR and AraR^A from Aspergillus species and bacterial repressors XylR and AraR^R. By integrating these signals into a highly-structured synthetic promoter, we created dual-mode systems with strong outputs and minimal leakiness. These systems demonstrated over 4000- and 300-fold regulation with strong activation and rapid response. The dual-mode xylose system was fully activated by xylose-rich agricultural residues like corncob hydrolysate, outperforming existing systems in terms of leakiness, inducibility, dynamic range, induction rate, and growth impact on host. We validated their utility in metabolic engineering with high-titer linalool production and demonstrated the transferability of the XlnR-based xylose induction system to Pichia pastoris, Candida glabrata and Candida albicans. This work provides robust genetic switches for yeasts and a general strategy for integrating activation-repression signals into synthetic promoters to achieve optimal performance.

诱导转录系统是基因工程中必不可少的工具，在这种系统中，人们非常需要严格的控制、强大的诱导性和快速的反应，同时还需要具有成本效益的诱导剂。然而，由于诱导剂成本过高或性能不兼容，现有的酵母系统很少用于大规模发酵。在这里，我们利用来自曲霉的真核激活剂 XlnR 和 AraRA 以及细菌抑制剂 XylR 和 AraRR，在酿酒酵母中开发出了强大的木糖和阿拉伯糖诱导系统。通过将这些信号整合到高度结构化的合成启动子中，我们创建了具有强大输出和最小泄漏的双模式系统。这些系统的调控能力分别超过 4000 倍和 300 倍，并且具有很强的激活能力和快速反应能力。富含木糖的农业残留物（如玉米芯水解物）可完全激活双模式木糖系统，该系统在泄漏性、诱导性、动态范围、诱导率以及对宿主生长的影响方面均优于现有系统。我们通过高滴度芳樟醇的生产验证了它们在代谢工程中的实用性，并证明了基于 XlnR 的木糖诱导系统在 Pichia pastoris、Candida glabrata 和 Candida albicans 中的可移植性。这项工作为酵母提供了强大的基因开关，并为将激活-抑制信号整合到合成启动子中以实现最佳性能提供了通用策略。

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引用次数: 0

Heterologous biosynthesis of betanin triggers metabolic reprogramming in tobacco 异源生物合成甜菜宁引发烟草代谢重编程

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.11.002

Xun Jiang , Zhuoxiang Zhang , Xiuming Wu , Changmei Li, Xuan Sun, Fengyan Wu, Aiguo Yang, Changqing Yang

Engineering of a specialized metabolic pathway in plants is a promising approach to produce high-value bioactive compounds to address the challenges of climate change and population growth. Understanding the interaction between the heterologous pathway and the native metabolic network of the host plant is crucial for optimizing the engineered system and maximizing the yield of the target compound. In this study, we performed transcriptomic, metabolomic and metagenomic analysis of tobacco (Nicotiana tabacum) plants engineered to produce betanin, an alkaloid pigment that is found in Caryophyllaceae plants. Our data reveals that, in a dose-dependent manor, the biosynthesis of betanin promotes carbohydrate metabolism and represses nitrogen metabolism in the leaf, but enhances nitrogen assimilation and metabolism in the root. By supplying nitrate or ammonium, the accumulation of betanin increased by 1.5–3.8-fold in leaves and roots of the transgenic plants, confirming the pivotal role of nitrogen in betanin production. In addition, the rhizosphere microbial community is reshaped to reduce denitrification and increase respiration and oxidation, assistant to suppress nitrogen loss. Our analysis not only provides a framework for evaluating the pleiotropic effects of an engineered metabolic pathway on the host plant, but also facilitates the development of novel strategies to balance the heterologous process and the native metabolic network for the high-yield and nutrient-efficient production of bioactive compounds in plants.

对植物的专门代谢途径进行工程改造是生产高价值生物活性化合物以应对气候变化和人口增长挑战的一种前景广阔的方法。了解异源途径与宿主植物原生代谢网络之间的相互作用对于优化工程系统和最大限度地提高目标化合物的产量至关重要。在这项研究中，我们对烟草（Nicotiana tabacum）植物进行了转录组、代谢组和元基因组分析，这些植物经工程改造后能产生一种存在于石竹科植物中的生物碱色素--甜菜宁。我们的数据显示，在剂量依赖的情况下，甜菜宁的生物合成会促进叶片的碳水化合物代谢，抑制氮代谢，但会增强根部的氮同化和代谢。通过提供硝酸盐或铵盐，转基因植物叶片和根部的甜菜宁积累增加了 1.5∼3.8 倍，证实了氮在甜菜宁生产中的关键作用。此外，根瘤微生物群落也发生了改变，减少了反硝化作用，增加了呼吸作用和氧化作用，从而抑制了氮的损失。我们的分析不仅为评估工程代谢途径对宿主植物的多效应提供了一个框架，还有助于开发新的策略，在异源过程和本地代谢网络之间取得平衡，从而在植物中高产、高效地生产生物活性化合物。

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引用次数: 0

Applying metabolic control strategies to engineered T cell cancer therapies 将代谢控制策略应用于工程化 T 细胞癌症疗法

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.10.009

Andrea C. Fox, John Blazeck

Chimeric antigen receptor (CAR) T cells are an engineered immunotherapy that express synthetic receptors to recognize and kill cancer cells. Despite their success in treating hematologic cancers, CAR T cells have limited efficacy against solid tumors, in part due to the altered immunometabolic profile within the tumor environment, which hinders T cell proliferation, infiltration, and anti-tumor activity. For instance, CAR T cells must compete for essential nutrients within tumors, while resisting the impacts of immunosuppressive metabolic byproducts. In this review, we will describe the altered metabolic features within solid tumors that contribute to immunosuppression of CAR T cells. We'll discuss how overexpression of key metabolic enzymes can enhance the ability of CAR T cells to resist corresponding tumoral metabolic changes or even revert the metabolic profile of a tumor to a less inhibitory state. In addition, metabolic remodeling is intrinsically linked to T cell activity, differentiation, and function, such that metabolic engineering strategies can also promote establishment of more or less efficacious CAR T cell phenotypes. Overall, we will show how applying metabolic engineering strategies holds significant promise in improving CAR T cells for the treatment of solid tumors.

嵌合抗原受体（CAR）T 细胞是一种表达合成受体的工程免疫疗法，能识别并杀死癌细胞。尽管 CAR T 细胞在治疗血液肿瘤方面取得了成功，但对实体瘤的疗效有限，部分原因是肿瘤环境中的免疫代谢特征发生了改变，阻碍了 T 细胞的增殖、浸润和抗肿瘤活性。例如，CAR T 细胞必须在肿瘤内争夺必需的营养物质，同时抵御免疫抑制代谢副产物的影响。在这篇综述中，我们将描述实体瘤内导致 CAR T 细胞免疫抑制的代谢特征的改变。我们将讨论关键代谢酶的过度表达如何增强 CAR T 细胞抵抗相应肿瘤代谢变化的能力，甚至将肿瘤的代谢特征恢复到抑制性较弱的状态。此外，代谢重塑与 T 细胞的活性、分化和功能有着内在联系，因此代谢工程策略也能促进建立更有效或更无效的 CAR T 细胞表型。总之，我们将展示应用代谢工程策略如何在改善 CAR T 细胞治疗实体瘤方面大有可为。

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引用次数: 0

A multiscale hybrid modelling methodology for cell cultures enabled by enzyme-constrained dynamic metabolic flux analysis under uncertainty 在不确定条件下通过酶约束动态代谢通量分析实现细胞培养的多尺度混合建模方法。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.10.013

Oliver Pennington , Sebastián Espinel Ríos , Mauro Torres Sebastian , Alan Dickson , Dongda Zhang

Mammalian cell cultures make a significant contribution to the pharmaceutical industry. They produce many of the biopharmaceuticals obtaining FDA-approval each year. Motivated by quality-by-design principles, various modelling methodologies are frequently trialled to gain insight into these bioprocesses. However, these systems are highly complex and uncertain, involving dynamics at different scales, both in time and space, making them challenging to model in a comprehensive and fully mechanistic manner. This study develops a machine-learning-supported multiscale modelling framework of cell cultures, linking the macroscale bioprocess dynamics to the microscale metabolic flux distribution. As a relevant biopharmaceutical case study, we consider the production of Trastuzumab by Chinese Hamster Ovary (CHO) cells in batch. A macroscale hybrid model is constructed by integrating macro-kinetic and machine-learning approaches. Enzyme-constrained Dynamic Metabolic Flux Analysis (ecDMFA) is adopted to calculate flux distributions based on the dynamic predictions of the hybrid model. Uncertainty estimation of the multiscale model is conducted through bootstrapping. Judging from experimental data, our hybrid model can reduce the modelling error of the macroscale dynamics to 8.0%; a 70% reduction from the purely mechanistic model. In addition, the predicted dynamic flux distribution aligns with observations seen in literature, highlighting important metabolic changes throughout the process. Model uncertainty is maintained at a low level, demonstrating the trustworthiness of the predictions. Overall, our comprehensive modelling framework has the potential to facilitate the development of digital twins in the biopharmaceutical industry.

哺乳动物细胞培养物为制药业做出了重大贡献。每年获得美国食品及药物管理局批准的许多生物制药都是由它们生产的。在设计质量原则的推动下，人们经常尝试各种建模方法来深入了解这些生物过程。然而，这些系统具有高度复杂性和不确定性，涉及不同尺度的时间和空间动态，因此以全面和完全机械的方式建模具有挑战性。本研究开发了一个机器学习支持的细胞培养多尺度建模框架，将宏观生物过程动力学与微观代谢通量分布联系起来。作为一个相关的生物制药案例研究，我们考虑了中国仓鼠卵巢（CHO）细胞批量生产曲妥珠单抗的情况。通过整合宏观动力学和机器学习方法，我们构建了一个宏观混合模型。基于混合模型的动态预测，采用酶约束动态代谢通量分析（ecDMFA）计算通量分布。多尺度模型的不确定性估计是通过引导法进行的。从实验数据来看，我们的混合模型可以将宏观尺度动态的建模误差降低到 8.0%，比纯力学模型降低了 70%。此外，预测的动态通量分布与文献观测结果一致，突出了整个过程中重要的新陈代谢变化。模型的不确定性保持在较低水平，证明了预测的可信度。总之，我们的综合建模框架有望促进生物制药行业数字孪生的发展。

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引用次数: 0

Engineering peroxisomal surface display for enhanced biosynthesis in the emerging yeast Kluyveromyces marxianus 在新兴酵母 Kluyveromyces marxianus 中设计过氧化物酶体表面显示，以增强生物合成。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.10.014

Shane Bassett, Jonathan C. Suganda, Nancy A. Da Silva

The non-conventional yeast Kluyveromyces marxianus is a promising microbial host for industrial biomanufacturing. With the recent development of Cas9-based genome editing systems and other novel synthetic biology tools for K. marxianus, engineering of this yeast has become far more accessible. Enzyme colocalization is a proven approach to increase pathway flux and the synthesis of non-native products. Here, we engineer K. marxianus to enable peroxisomal surface display, an enzyme colocalization technique for displaying enzymes on the peroxisome membrane via an anchoring motif from the peroxin Pex15. The native KmPex15 anchoring motif was identified and fused to GFP, resulting in successful localization to the surface of the peroxisomes. To demonstrate the advantages for pathway localization, the Pseudomonas savastanoi IaaM and IaaH enzymes were co-displayed on the peroxisome surface; this increased production of indole-3-acetic acid 7.9-fold via substrate channeling effects. We then redirected pathway flux by displaying the violacein pathway enzymes VioE and VioD from Chromobacterium violaceum, increasing selectivity of proviolacein to prodeoxyviolacein by 2.5-fold. Finally, we improved direct access to peroxisomal acetyl-CoA and increased titers of the polyketide triacetic acid lactone (TAL) by 2-fold through concurrent display of the proteins Cat2, Acc1, and the type III PKS 2-pyrone synthase from Gerbera hybrida relative to the same three enzymes diffusing in the cytosol. We further improved TAL production by up to 2.1-fold through engineering peroxisome morphology and lifespan. Our findings demonstrate that peroxisomal surface display is an efficient enzyme colocalization strategy in K. marxianus and applicable for improving production of a wide range of non-native products.

非常规酵母马氏酵母（Kluyveromyces marxianus）是一种用于工业生物制造的前景广阔的微生物宿主。随着最近基于 Cas9 的基因组编辑系统和其他用于 K. marxianus 的新型合成生物学工具的开发，这种酵母的工程设计变得更加容易。酶共定位是一种行之有效的方法，可提高通路通量和非本地产物的合成。在这里，我们对 K. marxianus 进行了工程改造，以实现过氧化物酶体表面显示，这是一种通过过氧化物酶 Pex15 的锚定基团在过氧化物酶体膜上显示酶的共定位技术。本机 KmPex15 锚定基序已被确定并与 GFP 融合，从而成功定位到过氧化物酶体表面。为了证明路径定位的优势，我们在过氧化物酶体表面共同展示了沙瓦氏假单胞菌的 IaaM 和 IaaaH 酶；通过底物通道效应，吲哚乙酸的产量增加了 7.9 倍。然后，我们通过展示来自长春花癣菌（Chromobacterium violaceum）的紫草素（violacein）途径酶 VioE 和 VioD，重新定向了途径通量，使前紫草素对原脱氧紫草素的选择性提高了 2.5 倍。最后，通过同时展示来自非洲菊的 Cat2、Acc1 和 III 型 PKS 2-pyrone 合成酶，我们改善了过氧物酶体乙酰-CoA 的直接获取途径，并将多酮类化合物三乙酸内酯（TAL）的滴度提高了 2 倍，而这三种酶在细胞质中扩散。通过对过氧物酶体形态和寿命的改造，我们进一步将 TAL 产量提高了 2.1 倍。我们的研究结果表明，过氧物酶体表面显示是 K. marxianus 中一种有效的酶共定位策略，适用于提高多种非本地产品的产量。

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引用次数: 0

Generation of a Vibrio-based platform for efficient conversion of raffinose through Adaptive Laboratory Evolution on a solid medium 通过在固体培养基上进行自适应实验室进化，生成基于弧菌的高效棉子糖转化平台。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.11.001

Sunghwa Woo , Yong Hee Han , Hye Kyung Lee , Dongyeop Baek , Myung Hyun Noh , Sukjae Han , Hyun Gyu Lim , Gyoo Yeol Jung , Sang Woo Seo

Raffinose, a trisaccharide abundantly found in soybeans, is a potential alternative carbon source for biorefineries. Nevertheless, residual intermediate di- or monosaccharides and low catabolic efficiency limit raffinose use through conventional microbial hosts. This study presents a Vibrio-based platform to convert raffinose efficiently. Vibrio sp. dhg was selected as the starting strain for the Adaptive Laboratory Evolution (ALE) strategy to leverage its significantly higher metabolic efficiency. We conducted ALE on a solid minimal medium supplemented with raffinose to prevent the enrichment of undesired phenotypes due to the shared effect of extracellular raffinose hydrolysis among multiple strains. As a result, we generated the VRA10 strain that efficiently utilizes raffinose without leaving behind degraded di- or monosaccharides, achieving a notable growth rate (0.40 h⁻¹) and raffinose consumption rate (1.2 g/g_dcw/h). Whole genome sequencing and reverse engineering identified that a missense mutation in the melB gene (encoding a melibiose/raffinose:sodium symporter) and the deletion of the two galR genes (encoding transcriptional repressors for galactose catabolism) facilitated rapid raffinose utilization. The further engineered strain produced 6.2 g/L of citramalate from 20 g/L of raffinose. This study will pave the way for the efficient utilization of diverse raffinose-rich byproducts and the expansion of alternative carbon streams in biorefinery applications.

棉子糖是一种大量存在于大豆中的三糖，是生物炼油厂的潜在替代碳源。然而，残留的中间二糖或单糖以及较低的分解效率限制了传统微生物宿主对棉子糖的利用。本研究提出了一种基于弧菌的棉子糖高效转化平台。弧菌 dhg 被选为自适应实验室进化（ALE）策略的起始菌株，以利用其显著较高的代谢效率。我们在添加了棉子糖的固体最小培养基上进行了适应性实验室进化，以防止由于多个菌株之间共享胞外棉子糖水解效应而导致不期望的表型富集。因此，我们产生了 VRA10 菌株，它能高效利用棉子糖而不留下降解的二糖或单糖，实现了显著的生长速度（0.40 h-1）和棉子糖消耗率（1.2 g/gdcw/h）。通过全基因组测序和逆向工程发现，melB 基因（编码美拉比糖/棉子糖：钠交感器）的错义突变和两个 galR 基因（编码半乳糖分解的转录抑制因子）的缺失促进了棉子糖的快速利用。进一步改造的菌株能从 20 克/升的棉子糖中产生 6.2 克/升的柠檬醛酸。这项研究将为高效利用富含棉子糖的各种副产品和扩大生物精炼应用中的替代碳流铺平道路。

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引用次数: 0

Engineering Halomonas bluephagenesis for synthesis of polyhydroxybutyrate (PHB) in the presence of high nitrogen containing media 在高含氮培养基条件下合成聚羟基丁酸（PHB）的蓝光单胞菌工程技术

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.10.012

Zhongnan Zhang , Mingwei Shao , Ge Zhang , Simian Sun , Xueqing Yi , Zonghao Zhang , Hongtao He , Kang Wang , Qitiao Hu , Qiong Wu , Guo-Qiang Chen

The trade-offs exist between microbial growth and bioproduct synthesis including intracellular polyester polyhydroxybutyrate (PHB). Under nitrogen limitation, more carbon flux is directed to PHB synthesis while growth is inhibited with diminishing overall carbon utilization, similar to the suboptimal carbon utilization during glycolysis-derived pyruvate decarboxylation. This study reconfigured the central carbon network of Halomonas bluephagenesis to improve PHB yield theoretically and practically. It was found that the downregulation of glutamine synthetase (GS) activity led to a synchronous improvement on PHB accumulation and cell growth under nitrogen non-limitation condition, increasing the PHB yield from glucose (g/g) to 85% of theoretical yield, PHB titer from 7.6 g/L to 12.9 g/L, and from 51 g/L to 65 g/L when grown in shake flasks containing a rich N-source, and grown in a fed-batch cultivation conducted in a 7-L bioreactor also containing a rich N-source, respectively. Results offer better metabolic balance between glucose conversion efficiency and microbial growth for economic PHB production.

微生物生长与生物产品合成（包括细胞内聚酯聚羟丁酸（PHB））之间存在权衡。在氮限制条件下，更多的碳通量被导向 PHB 合成，同时生长受到抑制，总体碳利用率降低，这与糖酵解衍生的丙酮酸脱羧过程中的次优碳利用率类似。本研究重新配置了蓝光单胞菌的中心碳网络，从理论和实践上提高了 PHB 产量。研究发现，下调谷氨酰胺合成酶（GS）活性可同步改善氮无限制条件下的PHB积累和细胞生长，在含有丰富氮源的摇瓶中生长和在同样含有丰富氮源的7升生物反应器中进行分批进料培养时，PHB产量分别从葡萄糖（克/克）提高到理论产量的85%，PHB滴度从7.6克/升提高到12.9克/升，PHB滴度从51克/升提高到65克/升。结果表明，葡萄糖转化效率和微生物生长之间的代谢平衡更好，有利于经济地生产 PHB。

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引用次数: 0

Not all cytochrome b5s are created equal: How a specific CytB5 boosts forskolin biosynthesis in Saccharomyces cerevisiae 并非所有细胞色素 b5 都是相同的：特定的 CytB5 如何在酿酒酵母中促进福斯克林的生物合成。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.10.008

Victor Forman , Dan Luo , Sotirios C. Kampranis , Dan Stærk , Birger Lindberg Møller , Irini Pateraki

Cytochrome B5s, or CytB5s, are small heme-binding proteins, ubiquitous across all kingdoms of life that serve mainly as electron donors to enzymes engaged in oxidative reactions. They often function as redox partners of the cytochrome P450s (CYPs), a superfamily of enzymes participating in multiple biochemical processes. In plants, CYPs catalyze key reactions in the biosynthesis of plant specialized metabolites with their activity dependent on electron donation often from cytochrome P450 oxidoreductases (CPRs or PORs). In eukaryotic microsomal CYPs, CytB5s frequently participate in the electron transfer process although their exact role remains understudied, especially in plant systems. In this study, we assess the role of CytB5s in the heterologous biotechnological production of plant specialized metabolites in yeast. For this, we used as a case-study the biosynthesis of forskolin - a bioactive diterpenoid produced exclusively from the plant Coleus forskohlii. The complete biosynthetic pathway for forskolin is known and includes three CYP enzymes. We reconstructed the entire forskolin pathway in the yeast Saccharomyces cerevisiae, and upon co-expression of the three CytB5s - identified in C. forskohlii transcriptomes - alleviation of a CYP-related bottleneck step was noticed only when a specific CytB5, CfCytB5A, was used. Co-expression of CfCytB5A in yeast, in combination with forskolin pathway engineering, resulted in forskolin production at titers of 1.81 g/L in a bioreactor. Our findings demonstrate that CytB5s not only play an important role in plant specialized metabolism but also, they can interact with precision with specific CYPs, indicating that the properties of CytB5s are far from understood. Moreover, our work highlights how CytB5s may act as indispensable components in the sustainable microbial production of plant metabolites, when their biosynthetic pathways involve CYP enzymes.

细胞色素 B5s 或 CytB5s 是一种小型血红素结合蛋白，在生物界无处不在，主要作为电子供体供参与氧化反应的酶使用。它们通常是细胞色素 P450s（CYPs）的氧化还原伙伴，CYPs 是参与多种生化过程的酶超家族。在植物中，CYPs 催化植物特殊代谢物生物合成过程中的关键反应，其活性依赖于细胞色素 P450 氧化还原酶（CPRs 或 PORs）的电子捐赠。在真核微粒体 CYPs 中，CytB5s 经常参与电子传递过程，但它们的确切作用仍未得到充分研究，尤其是在植物系统中。在本研究中，我们评估了 CytB5s 在酵母异源生物技术生产植物特殊代谢物中的作用。为此，我们以福斯可林的生物合成为案例进行了研究，福斯可林是一种生物活性二萜类化合物，专门从植物鞘氨醇中提取。据了解，福斯可林的完整生物合成途径包括三种 CYP 酶。我们在酿酒酵母中重建了整个福斯可林的合成途径，在福斯可林转录组中发现了三种 CytB5，当它们共同表达时，只有在使用特定的 CytB5（CfCytB5A）时，才会发现与 CYP 相关的瓶颈步骤有所缓解。在酵母中联合表达 CfCytB5A 与福斯克林途径工程相结合，可在生物反应器中以 1.81 克/升的滴度生产福斯克林。我们的研究结果表明，CytB5s 不仅在植物特化代谢中发挥着重要作用，而且还能与特定的 CYPs 精确地相互作用，这表明人们对 CytB5s 的特性还知之甚少。此外，我们的工作还突显了当植物代谢物的生物合成途径涉及 CYP 酶时，CytB5s 如何成为可持续微生物生产植物代谢物过程中不可或缺的组成部分。

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引用次数: 0

Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids 改善挥发性脂肪酸分解代谢的裸冠突炎菌适应性实验室进化和代谢工程

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2024-11-01 DOI: 10.1016/j.ymben.2024.10.011

Eric C. Holmes, Alissa C. Bleem, Christopher W. Johnson, Gregg T. Beckham

Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.

将大量废物流生物转化为高附加值产品，将成为不断发展的生物经济不可或缺的组成部分。挥发性脂肪酸（VFAs）（如丁酸盐、戊酸盐和己酸盐）是一种新兴的、前景广阔的废物衍生原料，可用于微生物碳升级再循环。裸冠突铜绿菌 H16 是将 VFAs 转化为各种生物产品的有利宿主，因为它具有多样化的碳代谢、易于进行代谢工程以及可用于工业规模。在这里，我们报告了一种提高 C. necator 产品滴度的常见策略--删除聚羟基丁酸（PHB）生物合成操作子--会导致 VFA 底物上的显著生长缺陷。通过适应性实验室进化，我们确定了调节基因 phaR、由 H16_A1372/H16_A1373 编码的双组分反应调节器-组氨酸激酶对以及由 H16_A2296-A2298 编码的三方转运体组件的突变是改善 VFA 底物生长的原因。缺失 phaR 和 H16_A1373 会导致 NADH 丰度显著降低，同时涉及碳代谢、电子载体平衡和氧化应激耐受性的基因表达也会发生巨大变化，这可能是这些工程菌株生长改善的原因。这些结果让我们深入了解了 PHB 生物合成在碳和能量代谢中的作用，并强调了调控因子 PhaR 在全球调控网络中的关键作用。通过组合突变，我们产生了对 VFAs 有显著生长改善的平台菌株，这能使废物衍生的 VFA 底物更好地转化为目标生物产品。

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