Synthetic and Systems Biotechnology最新文献_第8页

Efficient synthesis of l-DOPA in Escherichia coli via cofactor and enzyme engineering 利用辅助因子和酶工程在大肠杆菌中高效合成左旋多巴

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-12 DOI: 10.1016/j.synbio.2025.09.011

Lihao Deng , Jurong Ping , Zhuoyuan Liu , Kai Linghu , Heng Zhang , Xiaoyu Shan , Weizhu Zeng , Jianghua Li , Jingwen Zhou

The global incidence of Parkinson's disease continues to rise. Levodopa (l-DOPA) is the core therapeutic drug, and efficient and sustainable production methods are needed. However, the complex metabolic pathways and the low catalytic efficiency of enzymes limit biosynthesis of l-DOPA in microorganisms. To address this issue, this study significantly enhanced the production efficiency of l-DOPA through a multi-dimensional, integrated metabolic and enzyme engineering approach. Firstly, the de novo synthesis pathway for l-DOPA was established through optimization of the promoter, ribosome-binding site (RBS), plasmid copy number, and tighly accurately regulating the expression level of key enzymes. Secondly, combined with metabonomic analysis, carbon metabolic flow was diverted, increasing the l-DOPA titer by 36.7 %. Glucose dehydrogenase (BmgdH) and gluconate kinase (gntK) were introduced to construct a cofactor regeneration system, which synergistically enhanced the supply of NADH and FADH₂, increasing the l-DOPA conversion rate by 18 %. Next, the substrate tunnel of 4-hydroxyphenylacetic acid-3-monooxygenase subunit B (HpaB) was subjected to rational design, and mutant T292A significantly expanded the substrate channel, improved catalytic efficiency, and decreased l-tyrosine by 87 %. Finally, through the process optimization in a 5 L bioreactor (involving phased pH control and induction timing adjustment) achieved an l-DOPA titer of 60.73 g/L, the highest reported to date for de novo microbial synthesis. This research offers a novel approach for industrial biosynthesis of l-DOPA, and broadens engineering concepts for efficient synthesis of aromatic compounds.

帕金森氏症的全球发病率持续上升。左旋多巴（l-DOPA）是核心治疗药物，需要高效、可持续的生产方法。然而，复杂的代谢途径和酶的低催化效率限制了微生物对左旋多巴的生物合成。为了解决这一问题，本研究通过多维、综合的代谢和酶工程方法，显著提高了左旋多巴的生产效率。首先，通过优化启动子、核糖体结合位点（RBS）、质粒拷贝数，严格准确调控关键酶的表达水平，建立l-DOPA从头合成途径。其次，结合代谢组学分析，改变了碳代谢流量，使左旋多巴滴度提高了36.7%。引入葡萄糖脱氢酶（BmgdH）和葡萄糖酸激酶（gntK）构建辅助因子再生体系，协同增强NADH和FADH2的供应，使l-DOPA转化率提高18%。接下来，对4-羟基苯基乙酸-3-单加氧酶亚基B （HpaB）的底物通道进行合理设计，突变体T292A显著扩大了底物通道，提高了催化效率，使l-酪氨酸降低了87%。最后，通过在5l生物反应器中进行工艺优化（包括分阶段pH控制和诱导时间调节），获得了60.73 g/L的L - dopa滴度，这是迄今为止报道的微生物从头合成的最高滴度。本研究为左旋多巴的工业生物合成提供了新的途径，拓宽了芳香族化合物高效合成的工程概念。

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引用次数: 0

Enhanced PrrAB system activation and restricted farnesyl pyrophosphate diversion underlie high coenzyme Q10 accumulation in Rhodobacter sphaeroides HY01 在球形红杆菌HY01中，PrrAB系统激活增强和法尼脂基焦磷酸转移受限是辅酶Q10高积累的基础

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-12 DOI: 10.1016/j.synbio.2025.09.012

Xinwei He , Huangwei Wang , Mindong Liang , Weishan Wang , Biqin Chen , Dan Li , Lixin Zhang , Gao-Yi Tan

The industrial Rhodobacter sphaeroides HY01 accumulates an exceptionally high level of coenzyme Q₁₀ (Q₁₀), but the underlying mechanisms remain incompletely understood. Given the central role of Q₁₀ in respiratory electron transport, previous observation of reduced expression of cbb₃-type cytochrome c oxidase genes in HY01 suggested a potential mechanistic link. In this study, we found that cbb₃ oxidase activity in HY01 was only 21.8–32.8 % of that in the wild-type 2.4.1, and restoring this activity led to a 64.4 % decrease in Q₁₀ accumulation, demonstrating a strong inverse correlation. This correlation was found to be mediated by the activation of the PrrAB two-component regulatory system, which is negatively regulated by cbb₃ oxidase. However, disruption of cbb₃ oxidase in 2.4.1 alone was insufficient to reproduce the high Q₁₀ accumulation phenotype, indicating that additional factors may be required. Previous research also revealed restricted synthesis of geranylgeranyl diphosphate (GGPP) in HY01, which likely reduces the diversion of the Q₁₀ precursor farnesyl diphosphate (FPP). Reconstituting this metabolic constraint in wild-type strain, combined with fine-tuning of PrrAB system activation, resulted in up to a 218.0 % increase in Q₁₀ accumulation, achieving a level nearly identical to HY01. Combining mechanistic investigation and inverse metabolic engineering, this study demonstrates that the high Q₁₀ accumulation in HY01 results from the synergistic effects of enhanced PrrAB activation and restricted FPP diversion, providing new insights into the key factors underlying high-level Q₁₀ accumulation in R. sphaeroides.

工业球形红杆菌HY01积累了异常高水平的辅酶Q10 (Q10)，但其潜在的机制尚不完全清楚。鉴于Q10在呼吸电子传递中的核心作用，先前观察到HY01中cbb3型细胞色素c氧化酶基因表达减少，表明可能存在潜在的机制联系。在本研究中，我们发现HY01的cbb3氧化酶活性仅为野生型2.4.1的21.8 - 32.8%，恢复该活性导致Q10积累减少64.4%，表现出很强的负相关关系。这种相关性被发现是通过激活PrrAB双组分调控系统介导的，该系统由cbb3氧化酶负调控。然而，仅在2.4.1中破坏cbb3氧化酶不足以重现高Q10积累表型，这表明可能需要其他因素。先前的研究还发现，在HY01中，geranylgeranyl diphosphate （GGPP）的合成受到限制，这可能减少了Q10前体farnesyl diphosphate （FPP）的转移。在野生型菌株中重建这种代谢约束，并结合PrrAB系统激活的微调，导致Q10积累增加了218.0%，达到与HY01几乎相同的水平。本研究结合机制研究和逆向代谢工程研究，证明了在HY01中Q10的高积累是PrrAB激活增强和FPP转移受限的协同作用的结果，为探究球棘草Q10高积累的关键因素提供了新的见解。

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引用次数: 0

MicroDFBEST: A dCas12b-derived dual-function base editor with programmable editing characteristics for microbial genetic engineering MicroDFBEST：一种dcas12b衍生的双功能碱基编辑器，具有可编程编辑特性，用于微生物基因工程

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-12 DOI: 10.1016/j.synbio.2025.09.013

Wen-Liang Hao , De-Zhi Geng , Yu-Feng Liu , Lai-Chuang Han , Zhe-Min Zhou , Wen-Jing Cui

Base editors (BEs) enable precise genome editing, but their use in microbes remains limited by restricted mutagenesis capabilities and narrow editing windows. Here, we reported MicroDFBEST, a novel dual-function base editor (DFBE) for microbes, by fusing the high-activity deaminases evoCDA1 and TadA9 with nuclease-deficient Cas12b from Bacillus hisashii (dBhCas12b). This engineered system enables simultaneous C-to-T and A-to-G editing within a 26–33 nt window, the broadest range reported for microbial DFBEs. The editing characteristics of MicroDFBEST can be easily adjusted by changing fusion protein expression and editing generations to create diverse mutant libraries. We show that the MicroDFBEST system enables both flexible gene expression modulation via random promoter (P_ylbP) diversification and targeted protein evolution through mutational hotspot scanning in native genomic contexts. This study offers a versatile platform enabling in situ gene regulation (e.g., biosynthetic gene clusters activation) and protein evolution (e.g., chassis optimization), with broad synthetic biology utility.

碱基编辑器（BEs）能够实现精确的基因组编辑，但它们在微生物中的应用仍然受到有限的诱变能力和狭窄的编辑窗口的限制。在这里，我们报道了MicroDFBEST，一种新的微生物双功能碱基编辑器（DFBE），通过将高活性脱氨酶evoCDA1和TadA9与来自hisashii芽孢杆菌的核酸酶缺陷Cas12b （dBhCas12b）融合。该工程系统能够在26 - 33nt的窗口内同时进行C-to-T和a -to- g编辑，这是目前报道的微生物DFBEs中范围最广的。通过改变融合蛋白表达和编辑代数，可以轻松调整MicroDFBEST的编辑特性，从而创建不同的突变文库。我们发现MicroDFBEST系统既可以通过随机启动子（PylbP）多样化灵活地调节基因表达，也可以通过突变热点扫描在原生基因组环境中实现靶向蛋白进化。该研究为原位基因调控（如生物合成基因簇激活）和蛋白质进化（如底盘优化）提供了一个多功能平台，具有广泛的合成生物学实用性。

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引用次数: 0

Whole-cell biosensors with regulatory circuits based on a synthetic dual-input promoter enabling the highly sensitive detection of ultra-trace Cd2+ 具有基于合成双输入启动子的调节电路的全细胞生物传感器，能够高度灵敏地检测超痕量Cd2+

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-11 DOI: 10.1016/j.synbio.2025.09.008

Yuke Qin , Shuting Hu , Yibin Yang , Xiaoqiang Jia

The research on whole-cell biosensors tailored for trace and ultra-trace detection remains limited and the biosensors based on natural bacterial heavy metal resistance mechanisms have common issues of low sensitivity. In this study, we designed a promoter P_T7-cadO using the Cadmium ions (Cd²⁺)-binding protein binding site cadO with the T7 promoter at first and constructed a single-input whole-cell biosensor, which was named CP100, whose detection limit for Cd²⁺ met the WHO requirement, yet its response and sensitivity were quite low. We further introduced the lacI and lac operator (lacO) as the signal amplifier to construct a dual-input promoter P_{T7-cadO-lacO-cadO} and developed a biosensor named LC100, whose response and sensitivity were significantly improved, but background leakage became a new problem. Then we redesigned the gene circuit based on the regulatory circuit LCPM-2, which has the structure of “CadR-P_J23100-P_{T7-cadO-lacO-cadO}-mRFP1-LacI”, with the LacI protein as the autoregulatory negative feedback model and finally obtained the biosensor named LC100-2, which achieved the detection of ultra-trace Cd²⁺ (0.00001–0.02 nM), with the sensitivity of 3748.22 times that of CP100. Moreover, LC100-2 demonstrated excellent specificity to Cd²⁺ among four other divalent metal ions and good anti-interference capability in the mixed divalent metal ions system. The results of the real water sample tests demonstrated that precise quantitative detection of Cd²⁺ with a final concentration of 0.001–0.02 nM could be achieved by adding only a small volume of the sample (1 μL). This finding showed promising application potential in the field of trace detection. Additionally, the regulatory circuit LCPM-2 based on the unique dual-input promoter enhanced responses and reduced background leakage simultaneously, thereby providing an innovative strategy for the design and simplification of biosensors’ circuits.

针对痕量和超痕量检测的全细胞生物传感器的研究仍然有限，基于天然细菌重金属抗性机制的生物传感器普遍存在灵敏度低的问题。本研究首先利用镉离子（Cd2+）结合蛋白结合位点cadO与T7启动子设计了启动子PT7-cadO，构建了单输入全细胞生物传感器CP100，该传感器对Cd2+的检出限符合WHO要求，但其响应和灵敏度较低。我们进一步引入lacI和lac算子（lacO）作为信号放大器，构建双输入启动子PT7-cadO-lacO-cadO，并开发了LC100生物传感器，其响应和灵敏度得到了显著提高，但背景泄漏成为新的问题。然后基于结构为“CadR-PJ23100-PT7-cadO-lacO-cadO-mRFP1-LacI”的调控电路LCPM-2重新设计基因电路，以LacI蛋白为自调控负反馈模型，最终获得命名为LC100-2的生物传感器，实现了超微量Cd2+ (0.00001-0.02 nM)的检测，灵敏度为CP100的3748.22倍。LC100-2对其他四种二价金属离子中的Cd2+具有良好的特异性，在混合二价金属离子体系中具有良好的抗干扰能力。实际水样测试结果表明，只需加入少量样品（1 μL），就可以实现对Cd2+的精确定量检测，最终浓度为0.001 ~ 0.02 nM。这一发现在痕量检测领域具有广阔的应用前景。此外，基于独特双输入启动子的调控电路LCPM-2增强了响应，同时减少了背景泄漏，从而为生物传感器电路的设计和简化提供了一种创新策略。

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引用次数: 0

Efficient trans-aconitic acid production using systematically metabolic engineered Escherichia coli 利用系统代谢工程大肠杆菌高效生产反式乌头酸

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-09 DOI: 10.1016/j.synbio.2025.09.009

Leilei Guo , Yi Cheng , Xiaoxu Tan , Hongxu Zhang , Hairong Yu , Wenjia Tian , Mingyuan Liu , Weikang Sun , Kaiyu Gao , Tianyi Jiang , Qianjin Kang , Wensi Meng , Yidong Liu , Zhaoqi Kang , Chuanjuan Lü , Chao Gao , Ping Xu , Cuiqing Ma

trans-Aconitic acid (TAA) is a versatile platform biochemical exhibiting extensive applications. In this work, Escherichia coli W3110 (DE3) was metabolic modified for the de novo biosynthesis of TAA. Firstly, a pyruvate accumulation chassis strain E. coli W3110-P6 was constructed through deletion of six byproducts generation genes. Secondly, the aconitate isomerase was screened from four candidates and co-overexpressed with TAA transporter to construct heterologous TAA biosynthetic pathway in E. coli W3110-P6. Thirdly, the genes pyc^P458S encoding a feedback-insensitive pyruvate carboxylase and gltA encoding an allosterically unaffected citrate synthase were overexpressed, and aceA encoding the isocitrate lyase was deleted, to increase precursor supply and TAA generation of the recombinant E. coli. Finally, the fermentation condition of the obtained strain E. coli W3110-TAA08 was optimized. TAA at a concentration of 37.32 g/L was generated within 40 h, with a yield of 0.76 g/g glucose and a productivity of 0.93 g/L/h.

反式乌头酸（TAA）是一种用途广泛的生物化学平台。本研究对大肠杆菌W3110 （DE3）进行了代谢修饰，用于TAA的从头生物合成。首先，通过删除6个副产物产生基因，构建丙酮酸积累底盘菌株W3110-P6。其次，从4个候选物中筛选出乌头酸异构酶，与TAA转运蛋白共过表达，在大肠杆菌W3110-P6中构建异源TAA生物合成途径。再次，将编码反馈不敏感型丙酮酸羧化酶的基因pycP458S和编码变构不影响型柠檬酸合成酶的基因gltA过表达，删除编码异柠檬酸裂解酶的基因aceA，以增加重组大肠杆菌前体供应和TAA的生成。最后，对所得菌株W3110-TAA08的发酵条件进行了优化。在40 h内生成浓度为37.32 g/L的TAA，产率为0.76 g/g葡萄糖，产率为0.93 g/L/h。

{"title":"Efficient trans-aconitic acid production using systematically metabolic engineered Escherichia coli","authors":"Leilei Guo , Yi Cheng , Xiaoxu Tan , Hongxu Zhang , Hairong Yu , Wenjia Tian , Mingyuan Liu , Weikang Sun , Kaiyu Gao , Tianyi Jiang , Qianjin Kang , Wensi Meng , Yidong Liu , Zhaoqi Kang , Chuanjuan Lü , Chao Gao , Ping Xu , Cuiqing Ma","doi":"10.1016/j.synbio.2025.09.009","DOIUrl":"10.1016/j.synbio.2025.09.009","url":null,"abstract":"<div><div><em>trans</em>-Aconitic acid (TAA) is a versatile platform biochemical exhibiting extensive applications. In this work, <em>Escherichia coli</em> W3110 (DE3) was metabolic modified for the de novo biosynthesis of TAA. Firstly, a pyruvate accumulation chassis strain <em>E. coli</em> W3110-P6 was constructed through deletion of six byproducts generation genes. Secondly, the aconitate isomerase was screened from four candidates and co-overexpressed with TAA transporter to construct heterologous TAA biosynthetic pathway in <em>E. coli</em> W3110-P6. Thirdly, the genes <em>pyc</em><sup>P458S</sup> encoding a feedback-insensitive pyruvate carboxylase and <em>gltA</em> encoding an allosterically unaffected citrate synthase were overexpressed, and <em>aceA</em> encoding the isocitrate lyase was deleted, to increase precursor supply and TAA generation of the recombinant <em>E. coli</em>. Finally, the fermentation condition of the obtained strain <em>E. coli</em> W3110-TAA08 was optimized. TAA at a concentration of 37.32 g/L was generated within 40 h, with a yield of 0.76 g/g glucose and a productivity of 0.93 g/L/h.</div></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"11 ","pages":"Pages 110-116"},"PeriodicalIF":4.4,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145096564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A rapid combinatorial assembly method for gene cluster characterisation illuminates glidobactin biosynthesis 一种用于基因簇表征的快速组合组装方法阐明了glidobactin的生物合成

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-08 DOI: 10.1016/j.synbio.2025.09.010

Mark J. Calcott , Jonas Kröger , David F. Ackerley

Heterologous expression of natural product gene clusters in tractable hosts offers great promise for achieving sustainable production of nature-inspired drugs. However, it is common for the roles of some genes in a cluster to be unclear, and this can make it difficult to identify the minimal gene set required to produce the desired molecule. Typically, the function of unknown genes is inferred by time-consuming reductionist techniques, e.g. single and multiple gene knockouts in the native producer, followed by phenotype analysis. Here, we instead present a rapid combinatorial method to assemble individual genes involved in glidobactin biosynthesis into a collection of partial or complete clusters in a heterologous host. Following up with mass spectrometry allowed identification of the minimum genes required for compound production. We applied this synthetic biology approach to characterise the glidobactin gene cluster, for which previous gene knockout studies had yielded conflicting results. In the process, we showed that an added intrinsic advantage of combinatorial assembly is the generation of multiple strains that produce potentially desirable analogues in addition to glidobactin.

自然产物基因簇在可处理宿主中的异源表达为实现自然启发药物的可持续生产提供了巨大的希望。然而，在一个基因簇中，一些基因的作用通常是不清楚的，这可能会使识别产生所需分子所需的最小基因集变得困难。通常，未知基因的功能是通过耗时的还原技术来推断的，例如，在本地生产者中敲除单个和多个基因，然后进行表型分析。在这里，我们提出了一种快速组合方法，将参与glidobactin生物合成的单个基因组装成异源宿主中部分或完整簇的集合。随后用质谱法鉴定了化合物生产所需的最小基因。我们应用这种合成生物学方法来表征glidobactin基因簇，之前的基因敲除研究产生了相互矛盾的结果。在这个过程中，我们发现组合组装的一个额外的内在优势是产生多种菌株，这些菌株除了glidobactin外，还能产生潜在的理想类似物。

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引用次数: 0

Systems metabolic engineering of dual pathways with stage-specific activation for high-level 5-aminolevulinic acid production in Escherichia coli 大肠杆菌生产高水平5-氨基乙酰丙酸的双途径与阶段特异性激活的系统代谢工程

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-08 DOI: 10.1016/j.synbio.2025.09.007

Yanghao Liu , Changgeng Li , Yu Miao , Zhichao Chen , Tang'en Shi , Ling Ma , Siyu Zhao , Zichen Yu , Lanxiao Li , Qingyang Xu

5-Aminolevulinic acid (5-ALA) is an important non-proteinogenic amino acid with wide applications in agriculture and medicine. To achieve high-level microbial production, this study established a dual-pathway reconstruction strategy in Escherichia coli by integrating the endogenous C5 pathway with an inducible exogenous C4 pathway. Multi-copy overexpression of gltX, hemA, and hemL, combined with enhanced glutamate supply and the introduction of a non-oxidative glycolysis pathway, effectively increased the C5 pathway flux and carbon efficiency, while product toxicity was significantly alleviated by strengthening efflux mechanisms and oxidative stress tolerance. A quorum sensing–based regulatory system was employed to dynamically regulate hemB expression, thereby balancing cell growth and 5-ALA biosynthesis, and a controlled glycine feeding strategy specifically activated the C4 pathway during the later fermentation stage, forming a stage-specific dynamic activation mechanism. Promoter regulation of sucC/sucD expression and enhanced endogenous PLP biosynthesis further stabilized the C4 flux. Fed-batch fermentation in a 5 L bioreactor resulted in a final 5-ALA titer of 37.34 g/L, demonstrating the industrial potential of this systems metabolic engineering strategy combining dual pathways and stage-specific activation control.

5-氨基乙酰丙酸（5-ALA）是一种重要的非蛋白质原性氨基酸，在农业和医学上有着广泛的应用。为了实现高水平的微生物生产，本研究通过整合内源性C5途径和可诱导的外源性C4途径，在大肠杆菌中建立了双途径重建策略。gltX、hemA和hemL的多拷贝过表达，结合谷氨酸供应的增加和非氧化糖酵解途径的引入，有效地增加了C5途径的通量和碳效率，同时通过加强外排机制和氧化应激耐受性，显著减轻了产物毒性。利用群体感应调控系统动态调控hemB表达，平衡细胞生长和5-ALA的生物合成，调控甘氨酸取食策略在发酵后期特异性激活C4通路，形成阶段性动态激活机制。启动子对sucC/sucD表达的调控和内源性PLP生物合成的增强进一步稳定了C4通量。在5l生物反应器中分批补料发酵，最终5- ala滴度为37.34 g/L，证明了该系统代谢工程策略结合双途径和阶段特异性激活控制的工业潜力。

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引用次数: 0

Engineering riboflavin-overproducing Bacillus subtilis via pathway gene overexpression 通过途径基因过表达工程制造核黄素过量的枯草芽孢杆菌

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-05 DOI: 10.1016/j.synbio.2025.09.005

Sijia Wang , Qiyao Zhu , Chuan Liu , Huina Dong , Miaomiao Xia , Zhaoxia Jin , Dawei Zhang

Overexpression of genes at key metabolic nodes frequently enhances the yield of target products. However, this often leads to widespread issues with growth defect and plasmid instability. This study utilizes the riboflavin over-producer Bacillus subtilis U3 as a model to systematically investigate the impact of overexpressing riboflavin operon genes on strain growth and plasmid structural instability. Overexpression of genes in rib operon enhances target product yields by 13.2 % in U3, but the engineered strains suffer growth defects and plasmid instability. Frameshift mutations in the ribD gene were found to significantly reduce the loss of operon gene fragments by 16.7 %. Furthermore, this study investigates the application of the Respiration Activity Monitoring System (RAMOS) in evaluating the growth of metabolically engineered strains. RAMOS serves as a medium optimization tool and pre-fermentation screening platform. Specifically, guanine supplementation increased biomass by 11.1 % in zwf-overexpressing strains, whereas histidine, uracil, and tryptophan supplementation could improve the biomass of purF-overexpressing strain by 71.1 %. This approach provides a methodological framework for resolving growth defects in metabolic engineering.

关键代谢节点基因的过表达往往能提高目标产物的产量。然而，这往往导致生长缺陷和质粒不稳定的广泛问题。本研究以过量产生核黄素的枯草芽孢杆菌U3为模型，系统研究过表达核黄素操纵子基因对菌株生长和质粒结构不稳定性的影响。在U3中，过表达rib操纵子基因可使目标产物产量提高13.2%，但工程菌株存在生长缺陷和质粒不稳定。发现ribD基因的移码突变显著减少了16.7%的操纵子基因片段的丢失。此外，本研究还探讨了呼吸活动监测系统（RAMOS）在代谢工程菌株生长评价中的应用。RAMOS作为培养基优化工具和发酵前筛选平台。其中，添加鸟嘌呤可使zwf过表达菌株的生物量提高11.1%，而添加组氨酸、尿嘧啶和色氨酸可使purf过表达菌株的生物量提高71.1%。这种方法为解决代谢工程中的生长缺陷提供了一种方法学框架。

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引用次数: 0

Identification of a novel β-1,4-galactosyltransferase from Haemophilus parainfluenzae for efficient biosynthesis of lacto-N-neotetraose in Escherichia coli 副流感嗜血杆菌β-1,4-半乳糖转移酶在大肠杆菌中高效合成乳酸-n -新四糖的鉴定

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-05 DOI: 10.1016/j.synbio.2025.09.006

Dan Liu , Chenhui Ying , Yingqi Ping , Shanquan Liang , Qiaojuan Yan , Zhengqiang Jiang

As an important member of human milk oligosaccharides (HMOs), lacto-N-neotetraose (LNnT) has been recognized as a promising HMO with many physiological activities for infants. β-1,4-Galactosyltransferases play a vital role in the final step of LNnT biosynthesis in microbial cell factory. Herein, we identified a novel β-1,4-galactosyltransferase (Hpa-GalT) from Haemophilus parainfluenzae for the efficient biosynthesis of LNnT in E. coli BL21 star(DE3). After overexpression of the key genes in the synthetic pathway, the titer of LNnT reached 1.86 g/L in shake flask fermentation. Moreover, a variant A186P/E146Q of Hpa-GalT was obtained by molecular modification, resulting in an increase of the LNnT titer to 2.15 g/L. Subsequently, a series of genes related to competitive pathways were deleted, including lacZ, ugd, wcaJ, and wecB, the engineered strain produced a LNnT titer of 2.23 g/L. After optimization of LNnT transport along with LNT II utilization, the engineered strain AH31 produced 2.93 g/L of LNnT in shake flask fermentation. Furthermore, it produced a maximum titer of 32.6 g/L of total LNnT and 27.1 g/L of extracellular LNnT in a 5-L fermentor with the productivity for 0.52 g/L/h. Overall, this study provides a novel β-1,4-galactosyltransferase and lays a foundation for the efficient biosynthesis of LNnT.

乳清-n -新四糖（LNnT）作为人乳寡糖（HMOs）的重要成员，具有多种婴儿生理活性，是一种很有发展前景的低聚糖。在微生物细胞工厂中，β-1,4-半乳糖转移酶在LNnT生物合成的最后一步起着至关重要的作用。在此，我们从副流感嗜血杆菌中鉴定出一种新的β-1,4-半乳糖转移酶（Hpa-GalT），用于大肠杆菌BL21星（DE3）中高效的LNnT生物合成。合成途径关键基因过表达后，摇瓶发酵LNnT滴度达到1.86 g/L。通过分子修饰获得Hpa-GalT的A186P/E146Q变体，使LNnT滴度提高到2.15 g/L。随后，lacZ、ugd、wcaJ、wecB等一系列与竞争通路相关的基因被删除，工程菌株的LNnT滴度为2.23 g/L。优化LNnT运输和利用后，工程菌株AH31摇瓶发酵的LNnT产量为2.93 g/L。在5-L的发酵罐中，最大滴度为总LNnT 32.6 g/L，胞外LNnT 27.1 g/L，产率为0.52 g/L/h。总之，本研究提供了一种新的β-1,4-半乳糖转移酶，为LNnT的高效生物合成奠定了基础。

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引用次数: 0

Engineering Yarrowia lipolytica as a yeast cell factory for the de novo production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) 利用酵母细胞工厂改造脂化耶氏菌，重新生产聚（3-羟基丁酸-co-4-羟基丁酸）

IF 4.4 2区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Synthetic and Systems Biotechnology

Pub Date : 2025-09-04 DOI: 10.1016/j.synbio.2025.09.002

Jinming Liu, Jia Yan, Zhiyong Cui, Qingsheng Qi

The oleaginous yeast Yarrowia lipolytica presents a promising chassis for the production of biodegradable plastic polyhydroxyalkanoates (PHAs) due to its advantages in precursor supply capabilities and cost-effective substrate utilization. However, Y. lipolytica has primarily been engineered for the synthesis of homopolymeric PHAs, such as poly (3-hydroxybutyrate) (P3HB), which have limited material properties and application potential. In this study, the succinate dehydrogenase-deficient Y. lipolytica strains were utilized as hosts for the de novo biosynthesis of copolymer poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). By employing a strategy of subcellular partitioning, poly (4-hydroxybutyrate) (P4HB) was synthesized in the cytoplasm using 4-hydrobutyrate (4HB) derived from mitochondrial sources as a monomer. We then introduced the biosynthetic pathway for 3-hydroxybutyryl-CoA to facilitate P34HB accumulation. Importantly, the ratio of 4HB monomer in P34HB could be adjusted from 9.17 mol% to 45.26 mol% by varying medium components and carbon sources. Under fed-batch fermentation conditions in a 5 L bioreactor, the engineered strain achieved a P34HB titer of 18.61 g/L representing 19.18 % of cell dry weight (CDW). This study not only demonstrates the potential of Y. lipolytica for P34HB synthesis, but also expands the monomer pool available for PHAs in yeast cell factories.

产油酵母聚脂耶氏酵母由于其前体供应能力和低成本底物利用的优势，为生产可生物降解塑料聚羟基烷酸酯（PHAs）提供了一个有前途的基础。然而，聚脂Y. lipolytica主要用于合成均聚pha，如聚（3-羟基丁酸酯）（P3HB），其材料性质和应用潜力有限。在这项研究中，利用琥珀酸脱氢酶缺陷的聚脂Y. lipolytica菌株作为宿主，对共聚物聚（3-羟基丁酸盐-co-4-羟基丁酸盐）（P34HB）进行了重新生物合成。采用亚细胞分配策略，以线粒体来源的4-羟基丁酸酯（4HB）为单体，在细胞质中合成聚（4-羟基丁酸酯）（P4HB）。然后，我们引入了3-羟基丁基辅酶a的生物合成途径，以促进P34HB的积累。重要的是，通过改变介质成分和碳源，P34HB中4HB单体的比例可以从9.17 mol%调整到45.26 mol%。在5 L生物反应器中分批补料发酵条件下，工程菌株的P34HB滴度为18.61 g/L，占细胞干重（CDW）的19.18%。这项研究不仅证明了聚脂Y. lipolytica合成P34HB的潜力，而且扩大了酵母细胞工厂中pha可用的单体库。

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