Metabolic engineering最新文献_第4页

Corrigendum to “Microbial production of propionic acid through a novel β-alanine route” [Metabol. Eng. (2026) 219–231 93] “微生物通过一种新的β-丙氨酸途径生产丙酸”的勘误表[代谢]。Eng。（2026） 219-231 93]

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-12 DOI: 10.1016/j.ymben.2025.11.008

Da-Hee Ahn , Yoo-Sung Ko , Cindy Pricilia Surya Prabowo , Sang Yup Lee

引用次数: 0

Engineered plants for the production of the antioxidants arbutin and gallate 改造植物生产抗氧化剂熊果苷和没食子酸酯

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-10 DOI: 10.1016/j.ymben.2025.11.009

Sami Kazaz , Yu-Ton Chen , Senri Yamamoto , Yang Tian , Chien-Yuan Lin , Dylan Chin , İrem Pamukçu , Ibraheem Mohammed Al Shammaa , Yusuf Selman Akbas , Monikaben Nimavat , Emine Akyuz Turumtay , Edward E.K. Baidoo , Albert P. Kausch , Yuki Tobimatsu , Aymerick Eudes

The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.

莽草酸途径是生物合成许多有价值化学物质的重要代谢途径。在这项研究中，我们通过微生物酶的表达，在植物中设计了莽草酸途径，以产生两种重要的抗氧化剂没食子酸酯和熊果苷。工程途径利用芳香化合物原儿茶酸酯和4-羟基苯甲酸酯作为代谢中间体。通过在烟叶中的瞬时表达，我们首次确定了从choris酸盐或3-脱氢莽草酸盐生产没食子酸盐的生物合成途径。利用过量产生原儿茶酸的遗传背景和假单胞菌4-羟基苯甲酸酯羟化酶PobA的突变版本，在拟南芥中实现了没食子酸酯的产生。利用过量产生4-羟基苯甲酸酯的遗传背景和表达假丝酵母菌的单加氧酶MNX1，在拟南芥中获得了熊果苷的产生。拟南芥转基因品系的没食子酸酯和苦杏仁苷积累量分别在干重% (dwt%) 0.25和0.93范围内。利用高粱进行大规模的植物生产，成熟的转基因植物中由中间的4-羟基苯甲酸酯产生的没食子酸酯和熊果苷的滴度分别达到0.58 dwt%和0.50 dwt%，超过了在自然产生这些化合物的植物中通常观察到的水平。用甲醇溶剂从植物组织中很容易地提取没食子酸酯和熊果苷。对无提取物生物量的分析显示，只有微量的没食子酸酯及其前体4-羟基苯甲酸酯和原儿茶酸酯与细胞壁交联，表明它们主要以可溶性共轭形式储存在液泡中。本研究提出了利用植物宿主生态生产没食子酸酯和熊果苷的替代合成路线。

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引用次数: 0

Advancing arabinose-based bioproduction in Yarrowia lipolytica by integrating metabolic engineering and adaptive laboratory evolution 整合代谢工程和适应性实验室进化，推进以阿拉伯糖为基础的多脂耶氏菌生物生产

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-08 DOI: 10.1016/j.ymben.2025.11.006

Razieh Rafieenia , Jing Fu , Piotr Hapeta , Marko Storch , Rodrigo Ledesma-Amaro

The oleaginous yeast, Yarrowia lipolytica has gained interest as a biotechnological chassis to produce foods, chemicals, pharmaceuticals, and biofuels. To reduce production costs and sustainability, inexpensive and abundant feedstocks such as lignocellulose must be used for bioproduction. Since lignocellulosic biomass contains components that cannot be utilised by Y. lipolytica, it is important to use engineering biology to enable their utilisation. L-arabinose is the second most abundant pentose in lignocellulose after xylose. However, it has received much less attention than xylose as a bioresource. In the present study, we first engineered Y. lipolytica to grow on L-arabinose as the sole carbon source. We used several wild-type and engineered strains to express the multigene arabinose cassette. Second, we used adaptive laboratory evolution to improve the utilisation of arabinose by the engineered strains. Third, we enabled the production of β-carotene from arabinose by expressing a β-carotene cassette in the evolved strain. Using minimal YNB medium supplemented with 20 g/l of arabinose as the sole carbon source resulted in the complete utilisation of L-arabinose within 120 h. In bioreactors, a β-carotene production of 418.89 mg/l was achieved with the complete utilisation of 60 g/l of L-arabinose. This study is the first to engineer L-arabinose utilisation in Y. lipolytica, opening new avenues for biomanufacturing using alternative carbon sources.

产油酵母，脂解耶氏酵母作为生产食品、化学品、药品和生物燃料的生物技术基础已经引起了人们的兴趣。为了降低生产成本和可持续性，必须将廉价和丰富的原料（如木质纤维素）用于生物生产。由于木质纤维素生物质含有不能被聚脂y菌利用的成分，因此利用工程生物学使其得到利用是很重要的。l -阿拉伯糖是木质纤维素中含量第二丰富的戊糖，仅次于木糖。然而，作为一种生物资源，它受到的关注远远少于木糖。在本研究中，我们首先设计了以l -阿拉伯糖为唯一碳源的脂肪瘤菌。我们用几种野生型和工程菌株表达了多基因阿拉伯糖盒。其次，我们使用适应性实验室进化来提高工程菌株对阿拉伯糖的利用。第三，我们通过在进化的菌株中表达β-胡萝卜素盒，使阿拉伯糖生产β-胡萝卜素成为可能。使用添加20 g/l阿拉伯糖作为唯一碳源的最小YNB培养基，在120 h内完全利用了l -阿拉伯糖。在生物反应器中，完全利用60 g/l阿拉伯糖，β-胡萝卜素的产量达到418.89 mg/l。这项研究首次设计了l -阿拉伯糖在聚脂菌中的利用，为使用替代碳源的生物制造开辟了新的途径。

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引用次数: 0

Engineered non-canonical reductive TCA pathway drives high-yield succinic acid biosynthesis in Yarrowia lipolytica 工程非规范还原TCA途径驱动多脂耶氏菌高产琥珀酸生物合成

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-06 DOI: 10.1016/j.ymben.2025.11.003

Huilin Tao, Aomei Hao, Xiaoyue Pan, Yutao Zhong, Zhiyong Cui, Qingsheng Qi

The reductive tricarboxylic acid (rTCA) cycle is a crucial metabolic pathway employed in the microbial production of C4-dicarboxylic acids, especially succinic acid (SA). However, the inherent redox constraints associated with this cycle pose significant limitations on the yields of SA. Here, we address this critical bottleneck by engineering a non-canonical reductive TCA (Nc-rTCA) pathway in oleaginous yeast Yarrowia lipolytica. Our approach substitutes the NADH-dependent conversion of oxaloacetate to fumarate in the native rTCA cycle with an engineered cascade utilizing aspartate aminotransferase, aspartate ammonia-lyase, and glutamate dehydrogenase, effectively decoupling SA synthesis from NADH limitations. This NADPH-dependent module resulted in a remarkable 1.28-fold increase in fumarate titer. Further metabolic optimization in the engineered strain Ncr12 minimized malate byproduct formation, achieving an SA titer of 98.16 g/L with a high yield of 0.91 g/g glucose in 5-L bioreactors. Importantly, the Nc-rTCA pathway demonstrated potential for industrial application, yielding 74.34 g/L SA at 0.98 g/g glucose from lignocellulosic hydrolysate and 117.74 g/L SA at 0.74 g/g from glycerol. Our findings address the longstanding redox imbalance issues that have challenged rTCA-based engineering and establish a scalable platform for bio-based C4-dicarboxylic acid production.

还原性三羧酸（rTCA）循环是微生物生产c4 -二羧酸，特别是琥珀酸（SA）的重要代谢途径。然而，与此循环相关的固有氧化还原约束对SA的产率造成了重大限制。在这里，我们通过在产油酵母解脂耶氏菌中设计非规范还原TCA （Nc-rTCA）途径来解决这一关键瓶颈。我们的方法用利用天冬氨酸转氨酶、天冬氨酸解氨酶和谷氨酸脱氢酶的工程级联代替了天然rTCA循环中NADH依赖的草酰乙酸转化为富马酸，有效地将SA合成与NADH限制解耦。这种nadph依赖模块导致富马酸滴度显著增加1.28倍。对工程菌株Ncr12进行了进一步的代谢优化，最大限度地减少了苹果酸副产物的生成，在5-L生物反应器中，SA滴度达到98.16 g/L，葡萄糖产量达到0.91 g/g。重要的是，Nc-rTCA途径显示出了工业应用的潜力，从木质纤维素水解物中以0.98 g/g葡萄糖产生74.34 g/L SA，从甘油中以0.74 g/g产生117.74 g/L SA。我们的研究结果解决了长期存在的氧化还原不平衡问题，该问题挑战了基于rtca的工程，并建立了一个可扩展的生物基c4 -二羧酸生产平台。

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引用次数: 0

Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida 预测crispr介导的基因下调对恶臭假单胞菌可持续航空燃料前体生产的增强

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-06 DOI: 10.1016/j.ymben.2025.11.007

Ian S. Yunus , David N. Carruthers , Yan Chen , Jennifer W. Gin , Edward E.K. Baidoo , Christopher J. Petzold , Hector Garcia Martin , Paul D. Adams , Aindrila Mukhopadhyay , Taek Soon Lee

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

CRISPR干扰（CRISPRi）已成为重定向代谢通量以增强生物生产的一种有价值的工具。然而，它的应用往往受到两个挑战的制约：(1)合理地识别有效的基因下调靶点；(2)高效地构建多路CRISPRi系统。在本研究中，我们通过将计算优先级工具与构建多路CRISPRi系统的通用组装方法集成来解决这两个挑战。FluxRETAP （Flux-Reaction Target priority）准确地鉴定了敲除导致恶臭假单胞菌KT2440异戊二醇滴度大幅增加的基因靶标，优于传统的非计算、途径引导的靶标选择。通过敲除PP_4118 （α-酮戊二酸脱氢酶基因），获得了最高的异戊二醇滴度，接近1.5 g/L。使用VAMMPIRE (Versatile Assembly Method for MultiPlexing crispr -mediated downREgulation)，可以精确组装包含多达5个sgRNA阵列的CRISPRi构建体，减少上下文依赖性，实现一致的、不依赖于位置的基因下调。FluxRETAP和VAMMPIRE的整合有可能通过快速识别crispr介导的敲低和敲低组合来推进代谢工程，从而提高生物生产滴度，并可能适用于其他微生物系统。

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引用次数: 0

Disruption of acyl-acyl carrier protein (acyl-ACP) synthetase in cyanobacteria impairs lipid remodeling as revealed by acyl-ACP measurements 蓝藻中酰基酰基载体蛋白（酰基acp）合成酶的破坏损害脂质重塑，这是由酰基acp测量显示的

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-05 DOI: 10.1016/j.ymben.2025.11.004

Juthamas Jaroensuk , Joshua P. Abraham , Baltazar E. Zuniga , Hawkins S. Shepard , Michael Wei , Russell Williams , Stewart A. Morley , Maneesh Lingwan , Jiahong Zhou , Michael A. Jindra , Poonam Jyoti , Bo Wang , Jody C. May , John A. McLean , Jamey D. Young , Brian F. Pfleger , Doug K. Allen

Free fatty acid (FFA) production in bacteria is a key target for metabolic engineering. The knockout of the acyl-ACP synthetase (AAS) prevents reincorporation of FFA into the fatty acid biosynthetic cycle and is widely used to enhance their secretion. However, the role of AAS in membrane lipid remodeling under environmental stress, such as altered temperature, remains poorly understood. In cyanobacteria, temperature shifts are known to affect fatty acid desaturation and membrane fluidity, yet it is unclear whether AAS contributes to these adaptive responses through re-esterification of membrane-released acyl chains. We elucidated unique aspects of fatty acid metabolism in response to temperature changes in biotechnologically relevant microbes with the development of an efficient method for quantifying acyl-ACP intermediates using anion exchange chromatography (AEX). In Escherichia coli, which performs desaturation during fatty acid biosynthesis, we detected saturated and unsaturated acyl-ACPs that confirm biosynthetic pathway operation. In the cyanobacteria, Picosynechococcus sp. PCC 7002 and the Δaas strain, changes between two temperatures were interpreted with support from proteomic and lipidomic analyses and indicated that the AAS is tied to membrane lipid remodeling. Further, polyunsaturated acyl-ACPs were detected in the Δaas strain, which was unexpected because fatty acid synthesis does not produce polyunsaturates in cyanobacteria, suggesting the presence of alternative acyl-activating enzymes or unknown acyl-ACP desaturases. This study highlights the possible link between acyl chain recycling and lipid remodeling in cyanobacteria and demonstrates the utility of AEX-based acyl-ACP profiling in dissecting fatty acid metabolism.

细菌游离脂肪酸（FFA）的产生是代谢工程的关键目标。敲除酰基- acp合成酶（AAS）可阻止FFA重新整合到脂肪酸生物合成循环中，并被广泛用于增强其分泌。然而，在环境应激（如温度改变）下，AAS在膜脂重塑中的作用仍然知之甚少。在蓝藻中，已知温度变化会影响脂肪酸的去饱和和膜流动性，但尚不清楚AAS是否通过膜释放的酰基链的再酯化参与这些适应性反应。我们阐明了生物技术相关微生物中脂肪酸代谢对温度变化的独特响应，并开发了一种使用阴离子交换色谱（AEX）定量酰基acp中间体的有效方法。在脂肪酸生物合成过程中进行去饱和的大肠杆菌中，我们检测到证实生物合成途径运作的饱和和不饱和酰基acps。在蓝藻、皮聚球菌（Picosynechococcus sp. PCC 7002）和Δaas菌株中，两种温度之间的变化得到了蛋白质组学和脂质组学分析的支持，表明AAS与膜脂重塑有关。此外，在Δaas菌株中检测到多不饱和酰基- acp，这是出乎意料的，因为脂肪酸合成在蓝藻中不会产生多不饱和脂肪酸，这表明存在替代酰基活化酶或未知的酰基- acp去饱和酶。这项研究强调了蓝藻中酰基链循环和脂质重塑之间的可能联系，并证明了基于aex的酰基- acp谱分析在剖析脂肪酸代谢中的实用性。

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引用次数: 0

Engineering artificial biosynthetic pathways for efficient microbial production of psilocybin and psilocin 高效微生物生产裸盖菇素和裸盖菇素的工程非天然生物合成途径

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-05 DOI: 10.1016/j.ymben.2025.11.002

Cui Guo , Nguyen N.T. Luu , Maryem M. Adwer , Hemen Hosseinzadeh , Venkatesh Balan , Yajun Yan , Yuheng Lin

Psychedelic-assisted therapy is emerging as a highly promising approach for treating depression, with psilocybin, a psychoactive compound in magic mushrooms, gaining the most recognition for its efficacy in treating post-traumatic stress disorder and treatment-resistant depression. However, its low natural abundance makes extraction costly, necessitating alternative production methods. While engineered microbial production has been explored, dependence on the CYP450 hydroxylase (PsiH) in the natural biosynthetic pathway remains a major bottleneck, limiting production efficiency. Here, we report the design, validation, and optimization of artificial biosynthetic pathways in Escherichia coli that bypass PsiH, enabling efficient psilocybin and psilocin production. De novo biosynthesis of psilocybin achieved record titers of 557.91 mg/L in shake flasks and 2.00 g/L in a bioreactor, outperforming previous microbial engineering efforts. This work demonstrates the great commercial potential of microbial psilocybin production via combinatorial metabolic engineering and synthetic biology approaches.

迷幻剂辅助疗法正在成为一种非常有前景的治疗抑郁症的方法，迷幻蘑菇中的一种精神活性化合物裸盖菇素（psilocybin）因其在治疗创伤后应激障碍和难治性抑郁症方面的功效而获得了最多的认可。然而，它的天然丰度低，开采成本高，需要其他生产方法。虽然已经探索了工程微生物生产，但对天然生物合成途径中CYP450羟化酶（PsiH）的依赖仍然是限制生产效率的主要瓶颈。在这里，我们报道了大肠杆菌中绕过PsiH的人工生物合成途径的设计、验证和优化，使裸盖菇素和裸盖菇素的高效生产成为可能。全新生物合成裸盖菇素的滴度在摇瓶中达到557.91 mg/L，在生物反应器中达到2.00 g/L，优于之前的微生物工程成果。这项工作证明了通过组合代谢工程和合成生物学方法生产微生物裸盖菇素的巨大商业潜力。

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引用次数: 0

De novo production of 1,3-olein-2-palmitin (OPO) and 1-olein-2-palmitin-3-linolein (OPL) by multiplexed reconstruction of lipid metabolism in yeasts 通过酵母脂质代谢的多重重建重新生产1,3-油素-2-棕榈素（OPO）和1-油素-2-棕榈素-3-亚油素(OPL

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-05 DOI: 10.1016/j.ymben.2025.11.005

Chenyang Zhang , Xuan Zhou , Wei Wei , Jiahui Yu , Yaokang Wu , Yanfeng Liu , Jianghua Li , Guocheng Du , Jian Chen , Tongcheng Xu , Xueqin Lv , Xianhao Xu , Long Liu

Human milk fats (HMFs) could facilitate nutrient absorption in the infant gut, with 1,3-olein-2-palmitin (OPO) and 1-olein-2-palmitin-3-linolein (OPL) being the most abundant components. The construction of microbial cell factories has garnered significant interest due to their potential to synthesize HMFs from cheap raw materials. However, the substrate preference of endogenous triglyceride (TAG) synthases and the complex fatty acid (FA) composition limit OPO and OPL synthesis. This study developed a microbial cell factory for OPO and OPL production by reconstructing and fine-tuning the lipid metabolic network in Saccharomyces cerevisiae. First, the TAG biosynthesis pathway of S. cerevisiae was reconstructed, resulting in more than 70 % of palmitic acid (C16:0) in TAG being esterified to the sn-2 position, while simultaneously achieving de novo OPO synthesis. Further optimization of intracellular FA composition improved the OPO proportion in TAG to 26.59 %. De novo synthesis of OPL was achieved by introducing a heterologous synthesis pathway of linoleic acid (C18:2). A push-pull strategy was employed to promote FA and TAG synthesis, resulting in a 3.86-fold increase in TAG production and reaching 81.2 mg/g dry cell weight in shake flask. In a 3-L bioreactor, the engineered strain HF-35 achieved OPO and OPL titers of 85.68 mg/L and 162.30 mg/L, respectively, representing the highest reported titers of OPO and OPL using glucose as the substrate to date. This study demonstrated that regulating lipid metabolism is an effective strategy for specific TAG synthesis and lays the foundation for large-scale production of OPO and OPL.

人乳脂肪（HMFs）有助于婴儿肠道对营养物质的吸收，其中1,3-油素-2-棕榈素（OPO）和1-油素-2-棕榈素-3-亚油素（OPL）含量最高。微生物细胞工厂的建设因其从廉价原料合成HMFs的潜力而引起了极大的兴趣。然而，内源性甘油三酯（TAG）合成酶的底物偏好和复合脂肪酸（FA）组成限制了OPO和OPL的合成。本研究通过重构和微调酿酒酵母的脂质代谢网络，建立了生产OPO和OPL的微生物细胞工厂。首先，重构酿酒酵母TAG的生物合成途径，使TAG中70%以上的棕榈酸（C16:0）酯化到sn-2位置，同时实现从头合成OPO。进一步优化胞内FA组成，使TAG中的OPO比例达到26.59%。通过引入亚油酸的异源合成途径（C18:2），实现了OPL的从头合成。采用推挽策略促进FA和TAG的合成，使TAG产量增加3.86倍，摇瓶中干细胞重达到81.2 mg/g。在3-L生物反应器中，工程菌株hf35的OPO和OPL滴度分别为85.68 mg/L和162.30 mg/L，是迄今为止报道的以葡萄糖为底物的OPO和OPL滴度最高的菌株。本研究表明，调节脂质代谢是特异性TAG合成的有效策略，为OPO和OPL的大规模生产奠定了基础。

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引用次数: 0

From soil to biomanufacturing: Systems-driven metabolic pathway rewiring in non-model bacteria for gram-scale antibiotic production 从土壤到生物制造：系统驱动的代谢途径在非模式细菌中重新布线，用于克量级的抗生素生产

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-11-04 DOI: 10.1016/j.ymben.2025.11.001

Tingfeng Cheng , Suihao Yan , Min Xu , Lei Zhao

Microbial natural products (NPs) are a pivotal reservoir for drugs used in human health and agriculture. Andrimid, a polyketide-non-ribosomal peptide hybrid antibiotic inhibiting bacterial acetyl-CoA carboxylase, shows enormous potential in antibiotic drug development to mitigate antimicrobial resistance. However, industrial-scale manufacturing and downstream development of andrimid are largely prohibited due to its milligram level production in microorganisms. Herein, using an integrative multi-omics approach, we improved the yield of andrimid remarkably from milligram to gram level in a non-model environmental soil bacterium, Erwinia persicina BST187, isolated from the rhizosphere of tomato. Systematic reprogramming of the pathways for carbon source uptake, competing metabolites biosynthesis, supply of essential building blocks including phenylalanine, glycine, valine and malonyl-CoA and cofactor biosynthesis using CRIPSR/Cas9 based gene editing tools, coupled with fine-tuning the transcription of the biosynthetic genes of andrimid, resulted in the generation of the optimal producer, G17. Combined with fermentation optimization, andrimid was produced to a highest level of 1099.42 mg/L with a productivity of 15.3 mg/L/h using a 5 L bioreactor, representing a 628-fold increase compared to the parental strain. This study showcases the genome wide engineering of non-model bacteria and generates a plasmid- and inducer-free E. persicina strain for high-level andrimid production, providing a blueprint for systems-driven metabolic engineering of complex bioactive NPs for biomanufacturing.

微生物天然产物（NPs）是用于人类健康和农业的药物的关键储存库。Andrimid是一种抑制细菌乙酰辅酶a羧化酶的聚酮-非核糖体多肽混合抗生素，在抗生素药物开发中显示出巨大的潜力。然而，工业规模的制造和下游开发在很大程度上是被禁止的，因为它的毫克水平的微生物生产。本研究利用综合多组学方法，从番茄根际分离的非模式环境土壤细菌Erwinia persicina BST187中显著提高了雌雄同体的产量，从毫克到克水平。利用基于CRIPSR/Cas9的基因编辑工具，系统地对碳源吸收、竞争性代谢物生物合成、苯丙氨酸、甘氨酸、缬氨酸和丙二酰辅酶a等必需构建块的供应以及辅助因子的生物合成途径进行了重编程，再加上对雄酰胺生物合成基因的转录进行了微调，最终产生了最佳生产者G17。结合发酵优化，在5 L的生物反应器中，雄酰胺的最高产率为1099.42 mg/L，产率为15.3 mg/L/h，比亲本菌株提高了628倍。本研究展示了非模式细菌的全基因组工程，并产生了一种无质粒和无诱导性的persicina菌株，用于高水平的雄酰胺生产，为生物制造中复杂生物活性NPs的系统驱动代谢工程提供了蓝图。

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引用次数: 0

Industrial-scale production of Lacto-N-tetraose in engineered Bacillus subtilis via modular pathway optimization 通过模块化途径优化在工程枯草芽孢杆菌中工业化生产乳酸- n -四糖

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-10-30 DOI: 10.1016/j.ymben.2025.10.012

Chunhua Wang , Shaoru Hu , Shenglong Wang , Jing Du , Ziyi Zhao , Ziyao Zheng , Huihui Qiu , Xiang Ma , Jun Li , Hao Liu , Mingfeng Cao , Weixia Gao

Lacto-N-tetraose (LNT), an important human milk oligosaccharide with prebiotic benefits, was successfully produced de novo in Bacillus subtilis, establishing this Generally Recognized as Safe (GRAS) organism as a suitable platform for infant nutritional ingredients. A detailed enzyme screening identified three key enzymes: β-1,3-galactosyltransferase from Pseudogulbenkiania ferrooxidans, β-1,3-N-acetylglucosaminyltransferase from Neisseria polysaccharea, and β-galactoside permease (LacY) from Escherichia coli. The co-expression of these enzymes in strain BPPY enabled the first complete biosynthesis of LNT in B. subtilis, achieving a yield of 1.42 g/L in shake-flask cultures. Advanced metabolic engineering strategies, such as disrupting competing pathways, enhancing UDP-GlcNAc/Gal precursor flow, and optimizing heterologous pathways, led to the development of strain BPPY31, which produced 7.83 g/L of LNT, a 5.5-fold increase. To efficiently regulate carbon flux, a cost-effective CRISPR interference (CRISPRi) system was created to downregulate essential competing genes (zwf, pfkA, murAB). The engineered strain BD7 yielded 12.51 g/L in flask cultures and an impressive 80.48 g/L in 5-L fed-batch bioreactors, with only 4.43 g/L of the byproduct lacto-N-triose II, achieving lactose and glucose conversion rates of 92.25 % and 24 %, respectively. This study reports the highest documented LNT titer to date using a GRAS-compliant biomanufacturing platform characterized by precise metabolic regulation, scalability, and significant potential for industrial production of human milk oligosaccharides.

乳酸- n -四糖（LNT）是一种重要的具有益生元益处的母乳低聚糖，成功地在枯草芽孢杆菌（Bacillus subtilis）中重新生产，使这种公认安全的（GRAS）生物成为婴儿营养成分的合适平台。详细的酶筛选鉴定了三个关键酶：来自Pseudogulbenkiania ferrooxidans的β-1,3-半乳糖基转移酶，来自Neisseria多糖区的β-1,3- n -乙酰氨基葡萄糖基转移酶和来自大肠杆菌的β-半乳糖苷渗透酶（LacY）。这些酶在BPPY菌株中的共表达使枯草芽孢杆菌首次完成了LNT的生物合成，摇瓶培养的产量达到1.42 g/L。先进的代谢工程策略，如破坏竞争途径，增强UDP-GlcNAc/Gal前体流动，优化异源途径，导致菌株BPPY31的发展，产生7.83 g/L的LNT，增加了5.5倍。为了有效地调节碳通量，我们创建了一种具有成本效益的CRISPR干扰（CRISPRi）系统来下调必要的竞争基因（zwf, pfkA, murAB）。工程菌株BD7在瓶中培养的产量为12.51 g/L，在5-L进料间歇式生物反应器中的产量为80.48 g/L，副产物乳酸- n -三糖II仅为4.43 g/L，乳糖和葡萄糖的转化率分别为92.25%和24%。本研究报告了迄今为止记录最高的LNT滴度，使用符合格拉斯标准的生物制造平台，其特点是精确的代谢调节，可扩展性，以及人乳低聚糖工业生产的巨大潜力。

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