Engineering Microbiology最新文献

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Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica 脂溶耶氏菌基因编辑CRISPR/Cas9系统的优化

Engineering Microbiology

Pub Date : 2025-06-01 Epub Date: 2025-03-18 DOI: 10.1016/j.engmic.2025.100193

Jianhui Liu , Yamin Zhu , Jin Hou

Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility. Efficient genome editing tools are essential for advancing its biotechnological applications. Although CRISPR/Cas9 technology has been applied in Y. lipolytica, achieving a consistently high editing performance remains challenging owing to the low homologous recombination efficiency and variability in system components. In this study, we optimized CRISPR/Cas9-mediated genome editing in Y. lipolytica to enhance its editing efficiency. Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression, we achieved a gene disruption efficiency of 92.5 %. The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5 %. KU70 deletion in the Cas9 system increased the integration efficiency to 92.5 %, and Rad52 and Sae2 overexpression boosted homologous recombination. The introduction of Cas9^{D147Y, P411T} (iCas9) enhanced the efficiency of both gene disruption and genome integration. This study provides a powerful tool for efficient gene editing in Y. lipolytica, which will accelerate the construction of yeast cell factories.

由于其健壮性和代谢的多功能性，多脂耶氏菌是一种很有希望产生有价值化学物质的宿主。高效的基因组编辑工具对于推进其生物技术应用至关重要。尽管CRISPR/Cas9技术已经应用于脂质体Y. lipolytica，但由于同源重组效率低和系统组分的可变性，实现始终如一的高编辑性能仍然具有挑战性。在本研究中，我们优化了CRISPR/ cas9介导的脂质体Y. lipolytica基因组编辑，以提高其编辑效率。使用RNA聚合酶III启动子SCR1-tRNA表达sgRNA，我们实现了92.5%的基因破坏效率。tRNA-sgRNA结构使双基因破坏效率达到57.5%。Cas9系统中KU70的缺失使整合效率提高到92.5%，Rad52和Sae2的过表达促进了同源重组。Cas9D147Y, P411T （iCas9）的引入提高了基因破坏和基因组整合的效率。本研究为酵母细胞工厂的高效基因编辑提供了有力的工具，将加速酵母细胞工厂的建设。

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引用次数: 0

Functional heterologous expression of the reversible Cu-decarboxylase from the lichen, Cladonia uncialis 地衣中可逆cu脱羧酶的功能异源表达

Engineering Microbiology

Pub Date : 2025-06-01 Epub Date: 2025-05-25 DOI: 10.1016/j.engmic.2025.100211

Harman Gill, John L. Sorensen

Despite the isolation of over 1000 known bioactive lichen mycobiont-derived secondary metabolites (SMs), understanding the genetic basis of their biosynthesis remains elusive. Biosynthetic gene clusters (BGCs) have been tentatively linked to chemical structures, with core genes such as polyketide synthases (PKSs) surrounded by accessory genes like decarboxylases. In this study, we focused on a decarboxylase gene from the genome of the lichen cladonia uncialis (named as Cu-decarboxylase) to elucidate its role in SM biosynthesis. A 963 bp gene was cloned from C. uncialis and expressed in Escherichia coli (BL21(DE3) cells using the pQE80L expression vector. The resulting 35 kDa protein was purified by applying a Ni⁺-NTA column using an FPLC system. Functional activity assays revealed the decarboxylation and reversible carboxylation of resorcinol to 2,4-dihydroxybenzoic acid and orcinol to orsellinic acid. This suggests a potential role for this Cu-decarboxylase in SM biosynthesis.

Furthermore, the lack of activity on substrates like anthranilic acid and aniline highlighted the importance of the phenolic OH group in facilitating these reactions. The 3D protein structure was predicted with AlphaFold3, based on sequence similarity with a known decarboxylases and revealed the importance of a zinc cofactor for the catalytic activity of the enzyme. The optimization of the reaction conditions, particularly for orsellinic acid production from orcinol, may enhance conversion rates and offer a viable route for industrial-scale production of bioactive compounds. This study marks the first known instance of functional heterologous expression of a non-codon-optimized gene isolated from lichen in E. coli.

尽管已经分离出超过1000种已知的具有生物活性的地衣分枝生物衍生的次生代谢物（SMs），但对其生物合成的遗传基础的了解仍然难以捉摸。生物合成基因簇（BGCs）已初步与化学结构联系起来，其核心基因如聚酮合成酶（pks）被辅助基因如脱羧酶包围。在这项研究中，我们重点研究了来自衣苔藓的脱羧酶基因（命名为Cu-decarboxylase），以阐明其在SM生物合成中的作用。从棘球绦虫中克隆了一个963 bp的基因，并利用pQE80L表达载体在大肠杆菌BL21（DE3）细胞中表达。所得35kda蛋白用FPLC系统应用Ni+-NTA柱纯化。功能活性测定显示间苯二酚脱羧和可逆羧化生成2,4-二羟基苯甲酸和orcinol生成orsellinic酸。这表明该cu脱羧酶在SM生物合成中具有潜在的作用。此外，在邻氨基苯酸和苯胺等底物上缺乏活性，突出了酚羟基在促进这些反应中的重要性。基于与已知脱羧酶的序列相似性，用AlphaFold3预测了三维蛋白结构，并揭示了锌辅助因子对酶的催化活性的重要性。对反应条件的优化，特别是对从orcinol生产orsellinic酸的优化，可以提高转化率，并为工业规模生产生物活性化合物提供一条可行的途径。该研究标志着从大肠杆菌中分离出的非密码子优化基因的功能性异源表达的第一个已知实例。

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引用次数: 0

Engineering thermotolerant microbial strains via TrRCC1 overexpression for efficient bioethanol production 通过TrRCC1过表达工程耐热微生物菌株高效生产生物乙醇

Engineering Microbiology

Pub Date : 2025-06-01 Epub Date: 2025-05-26 DOI: 10.1016/j.engmic.2025.100212

Tingting Chen, Xiao He, Xinyan Zhang, Tian Tian, Jian Cheng, Tingting Long, Yonghao Li

Efficient conversion of corn stover to bioethanol via simultaneous saccharification and fermentation (SSF) is a promising strategy for sustainable biofuel production. A major current barrier to this process is the limited thermotolerance of Saccharomyces cerevisiae, which hampers its performance under the high-temperature conditions required for efficient SSF. In this study, we identified TrRCC1, a gene from Trichoderma reesei, as a candidate for improving microbial stress resistance. Overexpression of TrRCC1 in both T. reesei Rut C30 and S. cerevisiae BY4741 significantly enhanced thermotolerance. In T. reesei Rut C30, TrRCC1 overexpression improved heat resistance and increased cellulase production by 2.5-fold compared to the wild-type strain. In S. cerevisiae BY4741, TrRCC1 overexpression resulted in enhanced thermotolerance and a 21.8 % increase in ethanol production during SSF of corn stover. The ethanol concentration achieved in the SSF process with TrRCC1-overexpressing S. cerevisiae was 44.1 g/L, which was a notable improvement over control strain production. These findings highlight the potential of TrRCC1 as a key gene for engineering microbial strains with improved stress resistance to enhance the efficiency of bioethanol production from lignocellulosic biomass.

通过同步糖化和发酵（SSF）将玉米秸秆高效转化为生物乙醇是一种有前途的可持续生物燃料生产策略。目前该工艺的主要障碍是酿酒酵母有限的耐热性，这阻碍了其在高效SSF所需的高温条件下的性能。在这项研究中，我们从里氏木霉（Trichoderma reesei）中鉴定出TrRCC1基因作为提高微生物抗逆性的候选基因。TrRCC1在T. reesei Rut C30和S. cerevisiae BY4741中过表达均能显著增强耐温性。在T. reesei Rut C30中，TrRCC1过表达提高了抗热性，纤维素酶产量比野生型提高了2.5倍。在酿酒酵母BY4741中，TrRCC1过表达导致玉米秸秆SSF期间耐温性增强，乙醇产量增加21.8%。过表达trrcc1的酿酒酵母在SSF过程中获得的乙醇浓度为44.1 g/L，比对照菌株产量有显著提高。这些发现突出了TrRCC1作为工程微生物菌株的关键基因的潜力，这些菌株具有更好的抗逆性，可以提高木质纤维素生物质生产生物乙醇的效率。

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引用次数: 0

Erratum to “Regulation of protein thermal stability and its potential application in the development of thermo-attenuated vaccines” [Engineering Microbiology 4 (2024) 100162] “蛋白质热稳定性的调控及其在热减毒疫苗开发中的潜在应用”的勘误[工程微生物学4 (2024)100162]

Engineering Microbiology

Pub Date : 2025-06-01 Epub Date: 2025-03-25 DOI: 10.1016/j.engmic.2025.100204

Maofeng Wang , Cancan Wu , Nan Liu , Xiaoqiong Jiang , Hongjie Dong , Shubao Zhao , Chaonan Li , Sujuan Xu , Lichuan Gu

引用次数: 0

Superoxide-mediated O2 activation drives radical cyclization in ergot alkaloid biosynthesis 超氧化物介导的O2活化驱动麦角生物碱生物合成中的自由基环化

Engineering Microbiology

Pub Date : 2025-06-01 Epub Date: 2025-05-08 DOI: 10.1016/j.engmic.2025.100207

Yuanyuan Jiang , Zhong Li , Shengying Li

Conventional heme enzymes utilize iron–oxygen intermediates to activate substrates and drive reactions. Recently, Chen et al. discovered a novel NADPH-independent superoxide mechanism of heme catalase EasC, which facilitates an O₂-dependent radical oxidative cyclization reaction during ergot alkaloid biosynthesis. This enzyme coordinates superoxide-mediated catalysis by connecting spatially distinct NADPH-binding pocket and heme pocket via a slender tunnel, offering a novel perspective on the catalytic mechanisms of heme enzymes in nature.

传统的血红素酶利用铁氧中间体来激活底物并驱动反应。最近，Chen等人发现了一种新的不依赖nadph的血红素过氧化氢酶EasC的超氧化机制，该机制促进了麦角生物碱生物合成过程中o2依赖的自由基氧化环化反应。该酶通过细长的通道连接空间上不同的nadph结合口袋和血红素口袋，从而协调超氧化物介导的催化作用，为研究自然界中血红素酶的催化机制提供了新的视角。

引用次数: 0

Advances in synthetic microbial ecosystems approach for studying ecological interactions and their influencing factors 合成微生物生态系统方法研究生态相互作用及其影响因素的进展

Engineering Microbiology

Pub Date : 2025-06-01 Epub Date: 2025-03-26 DOI: 10.1016/j.engmic.2025.100205

Wei Jiang , Sumeng Wang , Fei Gu , Xiaoya Yang , Qingsheng Qi , Quanfeng Liang

Investigating ecological interactions within microbial ecosystems is essential for enhancing our comprehension of key ecological issues, such as community stability, keystone species identification, and the manipulation of community structures. However, exploring these interactions proves challenging within complex natural ecosystems. With advances in synthetic biology, the design of synthetic microbial ecosystems has received increasing attention due to their reduced complexity and enhanced controllability. Various ecological relationships, including commensalism, amensalism, mutualism, competition, and predation have been established within synthetic ecosystems. These relationships are often context-dependent and shaped by physical and chemical environmental factors, as well as by interacting populations and surrounding species. This review consolidates current knowledge of synthetic microbial ecosystems and factors influencing their ecological dynamics. A deeper understanding of how these ecosystems function and respond to different variables will advance our understanding of microbial-community interactions.

研究微生物生态系统内的生态相互作用对于提高我们对关键生态问题的理解至关重要，例如群落稳定性、关键物种鉴定和群落结构的操纵。然而，在复杂的自然生态系统中探索这些相互作用证明是具有挑战性的。随着合成生物学的发展，合成微生物生态系统的设计因其降低了复杂性和增强了可控性而受到越来越多的关注。在合成生态系统中已经建立了各种生态关系，包括共生、互食、互惠、竞争和捕食。这些关系往往依赖于环境，并受到物理和化学环境因素以及相互作用的种群和周围物种的影响。本文综述了目前合成微生物生态系统及其生态动力学影响因素的研究进展。更深入地了解这些生态系统如何发挥作用并对不同变量作出反应，将促进我们对微生物群落相互作用的理解。

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引用次数: 0

Cytochrome P450-catalyzed allylic oxidation of pentalenene to 1-deoxypentalenic acid in pentalenolactone biosynthesis 五烯内酯生物合成中细胞色素p450催化烯丙基氧化五烯烯生成1-脱氧五烯酸

Engineering Microbiology

Pub Date : 2025-06-01 Epub Date: 2025-04-05 DOI: 10.1016/j.engmic.2025.100206

Jing Li , Chengde Zhang , Shiwen Wu , Jiao Xue , Ke Chen , Zixin Deng , Dongqing Zhu

Pentalenolactone is a sesquiterpene antibiotic from Streptomyces. Its biosynthetic pathway has been elucidated, except for the oxidation of pentalen-13-al to 1-deoxypentalenic acid. In this study, we show that cytochrome P450 pentalenene oxygenase catalyzed the formation of 1-deoxypentalenic acid. Ferredoxin XNR_5179 and ferredoxin reductase XNR_4478 from S. albus are suitable redox proteins for pentalenene oxygenase. The biosynthetic pathway presented fills a gap in the biosynthetic pathway of pentalenolactone and provides an example of cytochrome P450 enzyme activity being affected by redox proteins.

戊烯内酯是一种来自链霉菌的倍半萜类抗生素。除戊烯-13-al氧化为1-脱氧戊烯酸外，其生物合成途径已被阐明。在这项研究中，我们发现细胞色素P450五烯加氧酶催化1-脱氧五烯酸的形成。白螺旋体中的铁氧化还蛋白XNR_5179和铁氧化还蛋白还原酶XNR_4478是适合于五烯加氧酶的氧化还原蛋白。所提出的生物合成途径填补了戊烯内酯生物合成途径的空白，并提供了细胞色素P450酶活性受氧化还原蛋白影响的一个例子。

引用次数: 0

The Kongming defense: Host-pathogen battles take a new face 孔明防御：宿主-病原体的战斗有了新的面貌

Engineering Microbiology

Pub Date : 2025-06-01 Epub Date: 2025-05-10 DOI: 10.1016/j.engmic.2025.100209

Dongchun Ni

Bacteria employ diverse immune systems, such as CRISPR-Cas, to fend off phage infections. A recent study uncovered the unprecedented mechanistic features of the Kongming bacterial defense system, which uniquely exploits phage-derived enzymes to synthesize deoxyinosine triphosphate (dITP), thereby triggering host immunity through NAD+ depletion. In response, some phages have evolved countermeasures to disrupt dITP synthesis, highlighting the ongoing evolutionary arms race between hosts and pathogens. This discovery not only deepens our understanding of bacterial defense strategies but also paves the way for new insights in biomedical research and synthetic biology.

细菌利用多种免疫系统，比如CRISPR-Cas，来抵御噬菌体感染。最近的一项研究揭示了孔明细菌防御系统前所未有的机制特征，该系统独特地利用噬菌体衍生酶合成脱氧肌苷三磷酸（dITP），从而通过NAD+消耗触发宿主免疫。作为回应，一些噬菌体进化出了破坏dITP合成的对策，这凸显了宿主和病原体之间正在进行的进化军备竞赛。这一发现不仅加深了我们对细菌防御策略的理解，而且为生物医学研究和合成生物学的新见解铺平了道路。

引用次数: 0

Applications of bacteriophages in precision engineering of the human gut microbiome 噬菌体在人体肠道微生物群精密工程中的应用

Engineering Microbiology

Pub Date : 2025-03-01 Epub Date: 2025-01-06 DOI: 10.1016/j.engmic.2025.100189

Xiaoxian Kuang , Juntao Shen , Linggang Zheng , Yi Duan , Yingfei Ma , Elaine Lai-Han Leung , Lei Dai

As our understanding of the role of the gut microbiome in human diseases deepens, precision engineering of the gut microbiome using bacteriophages has gained significant attention. Herein, we review the recent advances in bacteriophage-mediated modulation of the gut microbiome, discuss approaches at the ecological and genetic levels, and summarize the challenges and strategies pertinent to each level of intervention. Drawing on the structural attributes of bacteriophages in the context of precision engineering, we examined the latest developments in the field of phage administration. Gaining a nuanced understanding of microbiome manipulation will yield tailored strategies and technologies. This could revolutionize the prevention and treatment of diseases linked to gut pathogens and offer new avenues for the therapeutic use of bacteriophages.¹

随着我们对肠道微生物群在人类疾病中的作用认识的加深，利用噬菌体进行肠道微生物群的精密工程得到了极大的关注。在此，我们回顾了噬菌体介导的肠道微生物组调节的最新进展，讨论了生态和遗传水平的方法，并总结了与每个水平的干预相关的挑战和策略。在精密工程背景下，利用噬菌体的结构属性，我们研究了噬菌体管理领域的最新发展。获得对微生物组操纵的细致理解将产生量身定制的策略和技术。这可能会彻底改变与肠道病原体有关的疾病的预防和治疗，并为噬菌体的治疗用途提供新的途径

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Modification of essential factors mediating post-translational processing for high-quality protein expression in Penicillium 介导青霉高质量蛋白表达翻译后加工的关键因子的修饰

Engineering Microbiology

Pub Date : 2025-03-01 Epub Date: 2025-03-04 DOI: 10.1016/j.engmic.2025.100194

Demin Guo , Shengfang Zhao , Jie Chen, Shuhui Han, Yangtao Li, Yu Chen, Shengbiao Hu, Yibo Hu

The formation of mature proteins requires complex post-translational modification and processing. Efficient post-translational processing machinery is beneficial for the high-quality expression of proteins. To comprehensively evaluate the role of post-translational mediating factors (PTMFs) in protein synthesis, two reporter strains expressing a homologous protein, Amy15A, and a heterologous protein, TaEG, were constructed in Penicillium oxalicum. Three PTMFs including a conserved basic leucine zipper transcription factor, HacA; an endoplasmic reticulum chaperone-binding protein, BipA; and a protein disulfide isomerase, PdiA, were individually overexpressed in the both reporter strains. The findings showed that overexpression of these PTMFs enhanced the enzymatic activity of both homologous and heterologous proteins. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that, upon overexpression of the PTMFs, heterologous protein secretion remained stable or slightly increased, whereas that of homologous proteins remained unchanged or decreased. Neither the vegetative growth rate nor reporter transcription levels accounted for these variations in protein production or enzymatic activity. Conclusively, this study suggests that PTMFs play a positive role in protein expression and can be leveraged to optimize filamentous fungal chassis cells in the future.

成熟蛋白的形成需要复杂的翻译后修饰和加工。高效的翻译后加工机制有利于蛋白质的高质量表达。为了综合评价翻译后介导因子（PTMFs）在蛋白质合成中的作用，我们在草酸青霉中构建了表达同源蛋白Amy15A和异源蛋白TaEG的两个报告菌株。三个PTMFs包括保守的基本亮氨酸拉链转录因子，HacA；内质网伴侣结合蛋白；蛋白二硫异构酶PdiA在两种报告菌株中均有过表达。结果表明，这些PTMFs的过表达增强了同源和异源蛋白的酶活性。然而，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示，过表达PTMFs后，外源蛋白分泌保持稳定或略有增加，而同源蛋白分泌保持不变或减少。无论是营养生长速度还是报告转录水平都不能解释这些蛋白质生产或酶活性的变化。综上所述，本研究表明PTMFs在蛋白表达中发挥积极作用，未来可用于优化丝状真菌底盘细胞。

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