Metabolic Engineering Communications最新文献

Model-guided chemical environment and metabolic network design to couple pathways with cell fitness 模型引导的化学环境和代谢网络设计与细胞适应度耦联途径

IF 4.1 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-12-01 DOI: 10.1016/j.mec.2025.e00267

Natalia Kakko von Koch , Tuula Tenkanen , Sandra Castillo , Virve Vidgren , Tino Koponen , Kristoffer Krogerus , Merja Penttilä , Paula Jouhten

Heterologous compound production is a complex trait since the native metabolic fluxes supplying the precursors, redox power, and energy are under multilevel cellular regulation. Improving complex traits using targeted engineering needs combinatorially charting the complex genetic underpinnings. While this is laborious, adaptive laboratory evolution (ALE) has been used to improve many traits of microbial strains that are of application relevance such as tolerance of harsh conditions and nutrient utilization. However, in contrast to such traits, heterologous production can seldom be intuitively coupled with cellular fitness.

Here, a novel method EvolveXGA was developed for genome-scale metabolic model guided design of strategies combining chemical environments and genetic engineering of the metabolic network to allow ALE of desired traits. Adaptive evolution of traits occurs when the co-variance between the traits and fitness involves a genetic dependency like a flux coupling would indicate. Thus, combinations of chemical environments and metabolic network structures were searched using a genetic algorithm to identify those that render desired traits (i.e., sets of metabolic fluxes) flux-coupled with fitness. The search was performed for the production of 29 heterologous compounds in yeast Saccharomyces cerevisiae. Strategies for coupling the production routes of 13 compounds with fitness were found with four metabolic reaction knock outs and three components in the chemical environment. In addition, strategies for fitness-coupling native fluxes involved in the production was found for the remaining compounds. In addition, a model-guided strategy was implemented for fitness-coupling of heterologous glycolic acid (GA) synthesis in S. cerevisiae via oxaloacetase, oxalyl-CoA synthetase, and oxalyl-CoA reductase (i.e., oxalate pathway). ALE was performed and evolved populations and isolated clones were characterized using whole-genome sequencing and quantitative metabolite analysis. Three out of six isolates had better GA yield from glucose than a non-optimized control strain expressing the oxalate pathway and glyoxylate reductase.

EvolveXGA generalizes metabolic model-guided design of strategies to couple production routes with cell fitness. The strategies bring optimizing heterologous production in engineered microbial cells in the realm of ALE. Slow and expensive strain optimization is a major hinder of novel processes using engineered microbial cells reaching industrial realization. Thus, EvolveXGA contributes to biotechnological solutions for the brighter future.

异源化合物的产生是一个复杂的特性，因为提供前体、氧化还原力和能量的天然代谢通量受到多层次的细胞调节。利用目标工程改进复杂性状需要组合绘制复杂的遗传基础。虽然这很费力，但适应性实验室进化（ALE）已被用于改善微生物菌株的许多特性，这些特性与应用相关，如对恶劣条件的耐受性和营养利用。然而，与这些性状相反，异源生产很少能直观地与细胞适应度相结合。本文开发了一种新的方法EvolveXGA，用于基因组尺度的代谢模型指导设计策略，将化学环境和代谢网络的基因工程相结合，以实现所需性状的ALE。当性状和适应度之间的协方差涉及遗传依赖时，性状的适应性进化就发生了，就像通量耦合所表明的那样。因此，使用遗传算法搜索化学环境和代谢网络结构的组合，以确定那些呈现所需特征（即代谢通量集）通量耦合适应度的特征。对酿酒酵母中29种异源化合物的生产进行了研究。通过4个代谢反应敲除和化学环境中的3个组分，找到了13个化合物与适应度耦合的生产路线。此外，还发现了剩余化合物生产过程中适宜耦合的天然通量策略。此外，采用模型引导策略，通过草酸途径（草酸途径），对酿酒酵母中异源乙醇酸（GA）的合成进行适应度偶联。利用全基因组测序和定量代谢物分析对进化种群和分离克隆进行了表征。6株菌株中有3株表达草酸途径和乙醛酸还原酶的菌株比未优化的对照菌株有更好的葡萄糖GA产量。EvolveXGA推广了代谢模型指导的策略设计，将生产路线与细胞适应度结合起来。这些策略为ALE领域的工程微生物细胞的异种生产带来了优化。缓慢和昂贵的菌株优化是利用工程微生物细胞实现工业实现的新工艺的主要障碍。因此，EvolveXGA致力于为更光明的未来提供生物技术解决方案。

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引用次数: 0

Substrate stiffness-dependent metabolic reprogramming of iPSC-derived cardiomyocytes on physiological PDMS polymers ipsc衍生的心肌细胞在生理PDMS聚合物上的代谢重编程

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-07-08 DOI: 10.1016/j.mec.2025.e00266

Leena Patel , Bryan P. Marzullo , Jonathan Barlow , Himani Rana , Amar J. Azad , Patricia Thomas , Daniel A. Tennant , Katja Gehmlich

Many cardiac pathologies are characterised by increased stiffness of the myocardium, due to excess deposition of extracellular matrix (ECM) proteins and structural remodelling, impacting the behaviour of cardiomyocytes (CMs). Metabolism of CMs shifts in cardiac pathologies, with the healthy heart primarily utilising fatty acids as its source of energy production, whilst the diseased heart switches to utilise glucose. The shift in metabolic source with stiffness of the myocardium has not been investigated.

To investigate the effect of ECM stiffnesses on iPSC-CM metabolism, iPSC-CMs were cultured on polydimethylsiloxane (PDMS) substrates of healthy and fibrotic stiffness (20 kPa and 130 kPa respectively) and plastic. Cellular metabolism of iPSC-CMs was assessed through isotope-labelled mass spectrometry with central carbon tracing as well as real-time cellular bioenergetics using extracellular flux analysis. Key metabolic genes were investigated at transcript and protein level, with proteomics analysis conducted to identify protein profiles on substrate stiffnesses.

Mass spectrometry data revealed greater utilisation of glucose in iPSC-CMs cultured on plastic compared to softer PDMS substrates, indicating greater glycolytic activity. Extracellular flux analysis demonstrated greater lactic acid efflux from iPSC-CMs cultured on plastic substrates, reflective of increased glycolytic flux and a shift towards aerobic glycolysis as the primary source of ATP synthesis. This study revealed culture of iPSC-CMs on traditional cell culture plastics or glass coverslips displaying pathological metabolism, highlighting the use of physiological substrates for metabolic investigation.

许多心脏疾病的特征是由于细胞外基质（ECM）蛋白的过度沉积和结构重塑，影响心肌细胞（CMs）的行为，导致心肌硬度增加。在心脏疾病中，CMs的代谢会发生变化，健康的心脏主要利用脂肪酸作为其能量产生的来源，而患病的心脏则转而利用葡萄糖。代谢源随心肌硬度的变化尚未被研究。为了研究ECM刚度对iPSC-CM代谢的影响，我们将iPSC-CM培养在健康和纤维化刚度（分别为20 kPa和130 kPa）和塑性的聚二甲基硅氧烷（PDMS）基质上。iPSC-CMs的细胞代谢通过中心碳示踪的同位素标记质谱法和细胞外通量分析的实时细胞生物能量学进行了评估。在转录物和蛋白质水平上研究了关键代谢基因，并进行了蛋白质组学分析，以确定底物刚度的蛋白质谱。质谱数据显示，与较软的PDMS底物相比，在塑料上培养的iPSC-CMs中，葡萄糖的利用率更高，表明糖酵解活性更高。细胞外通量分析表明，在塑料基质上培养的iPSC-CMs有更大的乳酸外排，这反映了糖酵解通量的增加和有氧糖酵解作为ATP合成的主要来源的转变。本研究揭示了iPSC-CMs在传统细胞培养塑料或玻璃盖上的培养，显示了病理代谢，强调了生理底物在代谢研究中的应用。

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引用次数: 0

Improving recombinant protein secretion in Aspergillus nidulans by targeting the N-glycosylation machinery 利用n -糖基化机制改善球状曲霉重组蛋白分泌

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-06-01 DOI: 10.1016/j.mec.2025.e00264

Jaqueline Aline Gerhardt , Marcelo Ventura Rubio , Cesar Rafael Fanchini Terrasan , Natalia Sayuri Wassano , Aryadne Rodrigues , Fernanda Lopes de Figueiredo , Everton Paschoal Antoniel , Fabiano Jares Contesini , Artur Hermano Sampaio Dias , Uffe Hasbro Mortensen , Munir Salomão Skaf , André Damasio

Filamentous fungi are cell factories traditionally used for enzyme production in various industrial sectors, including food and beverages, biopolymers, biofuels, and animal feed. Despite significant progress in optimizing enzyme production, challenges related to cost-effectiveness persist. Genes involved in the fungal secretory pathway have been modified to address productivity barriers, including post-translational modifications such as N-glycosylation of proteins. N-glycosylation can significantly affect protein stability, production yield, and functionality. This study investigated the isolated and combined deletion of genes involved in N-glycan assembly on protein production in Aspergillus nidulans. To test this hypothesis, we utilized CRISPR/Cas9 technology to knock out 14 genes related to N-glycan assembly (AN5888, AN11802, AN5346, AN6874, AN5725, AN7425, algC, algI, algL, algF and AN5748) and protein quality control (clxA, gtbA, and AN4623), resulting in eight viable mutants. Next, we integrated a GH3 beta-xylosidase encoding gene (bxlb; AN8401) into these mutants and the reference strain for constitutive expression and secretion. Single deletion of most target genes did not affect protein secretion and fungal growth. Interestingly, the specific activity of BxlB in the secretome of single mutants was influenced by culture time, while BxlB secretion remained unaffected. Conversely, the combined deletion of algC and algI increased BxlB secretion, whereas the kinetic parameters remained unaffected relative to the enzyme produced by the reference strain. Multiple deletions of algC, algF, and algI did not affect BxlB secretion but reduced catalytic efficiency. After analyzing the secretomes of double and triple mutant strains produced on plant biomass using mass spectrometry, we observed that these knockouts reduced the overall secretion of a specific set of carbohydrate-active enzymes (CAZymes). Other clusters were upregulated in the mutant strains, indicating severe secretome alterations. Overall, the combined deletion of algC and algI may be a promising strategy for increasing the secretion of recombinant proteins in A. nidulans while also enhancing downstream processes, such as protein purification, by reducing the protein background in the secretome of the mutant strain.

丝状真菌是细胞工厂，传统上用于各种工业部门的酶生产，包括食品和饮料、生物聚合物、生物燃料和动物饲料。尽管在优化酶生产方面取得了重大进展，但与成本效益相关的挑战仍然存在。参与真菌分泌途径的基因已经被修改，以解决生产力障碍，包括翻译后修饰，如蛋白质的n -糖基化。n -糖基化可以显著影响蛋白质的稳定性、产量和功能。本研究研究了中性曲霉蛋白生产中n -聚糖组装相关基因的分离和组合缺失。为了验证这一假设，我们利用CRISPR/Cas9技术敲除了14个与n -聚糖组装相关的基因（AN5888、AN11802、AN5346、AN6874、AN5725、AN7425、algC、algI、algL、algF和AN5748）和蛋白质质量控制相关的基因（clxA、gtbA和AN4623），得到了8个活的突变体。接下来，我们整合了一个GH3 β -木糖苷酶编码基因(bxlb；AN8401)注入这些突变体和参考菌株进行组成性表达和分泌。大多数靶基因的单一缺失不影响蛋白质分泌和真菌生长。有趣的是，单突变体分泌组中BxlB的比活性受培养时间的影响，而BxlB的分泌不受影响。相反，algC和algI的联合缺失增加了BxlB的分泌，而相对于参考菌株产生的酶，动力学参数没有受到影响。algC、algF和algI的多次缺失不影响BxlB的分泌，但降低了催化效率。利用质谱分析了植物生物量产生的双突变株和三突变株的分泌组后，我们观察到这些基因敲除减少了一组特定碳水化合物活性酶（CAZymes）的总体分泌。其他簇在突变株中上调，表明严重的分泌组改变。总的来说，联合删除algC和algI可能是一种很有希望的策略，可以增加a . nidulans中重组蛋白的分泌，同时通过减少突变菌株分泌组中的蛋白质背景，增强下游过程，如蛋白质纯化。

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引用次数: 0

Multi-step pathway engineering in probiotic Saccharomyces boulardii for abscisic acid production in the gut 益生菌博氏酵母菌肠道脱落酸生产的多步骤途径工程

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-06-01 DOI: 10.1016/j.mec.2025.e00263

Femke Van Gaever , Paul Vandecruys , Yasmine Driege , Seo Woo Kim , Johan M. Thevelein , Rudi Beyaert , Jens Staal

The plant hormone abscisic acid (ABA) has gained attention for its role in animals and humans, particularly due to its protective effects in various immune and inflammatory disorders. Given its high concentrations in fruits like figs, bilberries and apricots, ABA shows promise as a nutraceutical. However scalability, short half-life and cost limit the use of ABA-enriched fruit extracts and synthetic supplements. In this study, we propose an alternative ABA administration method to overcome these challenges. We genetically engineered a strain of the probiotic Saccharomyces boulardii to produce and deliver ABA directly to the gut of mice. Using the biosynthesis pathway from Botrytis cinerea, four genes (bcaba1-4) were integrated into S. boulardii, enabling ABA production at 30 °C, as previously described in Saccharomyces cerevisiae. Introducing an additional cytochrome P450 reductase gene resulted in a 7-fold increase in ABA titers, surpassing previous ABA-producing S. cerevisiae strains. Supplementation of the ABA-producing S. boulardii in the diet of mice (at a concentration of 5 × 10⁸ CFU/g) led to effective gut colonization but resulted in low serum ABA levels (approximately 1.8 ng/mL). The absence of detectable serum ABA after administration of the ABA-producing probiotic through oral gavage, prompted further investigation to determine the underlying cause. The physiological body temperature (37 °C) was identified as a major bottleneck for ABA production. Modifications to enhance the mevalonate pathway flux improved ABA levels at 37 °C. However, additional modifications are needed to optimize ABA production before testing this probiotic in disease contexts in mice.

植物激素脱落酸（ABA）因其在动物和人类中的作用而受到关注，特别是由于其对各种免疫和炎症疾病的保护作用。鉴于其在无花果、越桔和杏子等水果中的高浓度，ABA有望成为一种营养保健品。然而，可扩展性、半衰期短和成本限制了富含aba的水果提取物和合成补充剂的使用。在这项研究中，我们提出了一种替代的ABA管理方法来克服这些挑战。我们对一株益生菌博氏酵母菌进行了基因工程改造，使其能够直接产生ABA并将其输送到小鼠的肠道。利用灰霉病菌（Botrytis cinerea）的生物合成途径，将4个基因（bcaba1-4）整合到S. borlardii中，使其能够在30°C下生产ABA，正如之前在酿酒酵母（Saccharomyces cerevisiae）中所述。引入一个额外的细胞色素P450还原酶基因导致ABA滴度增加7倍，超过了以前产生ABA的酿酒葡萄球菌菌株。在小鼠日粮中添加产生ABA的博氏弧菌（浓度为5 × 108 CFU/g）可有效定植肠道，但导致血清ABA水平较低（约1.8 ng/mL）。通过灌胃给予产生ABA的益生菌后，血清中没有可检测到的ABA，这促使进一步调查以确定潜在的原因。生理体温（37℃）被认为是ABA产生的主要瓶颈。增强甲羟戊酸途径通量的修饰提高了37°C时ABA水平。然而，在测试这种益生菌在小鼠疾病背景下的ABA产量之前，还需要进行额外的修改。

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引用次数: 0

Improvement of D-lactic acid production from methanol by metabolically engineered Komagataella phaffii via ultra-violet mutagenesis 紫外诱变诱导代谢工程法菲Komagataella phaffii甲醇产d -乳酸的研究

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-05-17 DOI: 10.1016/j.mec.2025.e00262

Yoshifumi Inoue, Kaito Nakamura, Ryosuke Yamada, Takuya Matsumoto, Hiroyasu Ogino

Methanol has attracted attention as an alternative carbon source to petroleum. Komagataella phaffii, a methanol-assimilating yeast, is a useful host for the chemical production from methanol. A previous study successfully constructed a metabolically engineered K. phaffii GS115/S8/Z3 strain capable of producing D-lactic acid from methanol. In this study, we aimed to develop a strain with improved D-lactic acid production by applying ultra-violet mutagenesis to the D-lactic acid-producing strain, GS115/S8/Z3. The resulting mutant strain DLac_Mut2_221 produced 5.38 g/L of D-lactic acid from methanol, a 1.52-fold increase compared to the parent strain GS115/S8/Z3. The transcriptome analysis of the mutant DLac_Mut2_221 identified 158 differentially expressed genes, providing insights into key mechanisms contributing to enhanced D-lactic acid production. Metabolic engineering strategies for K. phaffii based on the knowledge gained from this study will contribute to improving the productivity of various useful chemicals from methanol.

甲醇作为一种可替代石油的碳源已引起人们的关注。法菲Komagataella phaffii是一种甲醇同化酵母，是甲醇化工生产的有益宿主。先前的研究成功构建了代谢工程的K. phaffii GS115/S8/Z3菌株，能够从甲醇中产生d -乳酸。本研究通过对产d乳酸菌株GS115/S8/Z3进行紫外诱变，获得一株产d乳酸能力较强的菌株。突变菌株DLac_Mut2_221从甲醇中产生5.38 g/L的d -乳酸，比亲本菌株GS115/S8/Z3增加了1.52倍。突变体DLac_Mut2_221的转录组分析鉴定了158个差异表达基因，为促进d -乳酸生成的关键机制提供了见解。基于本研究获得的知识的法菲氏菌代谢工程策略将有助于提高甲醇中各种有用化学物质的生产力。

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引用次数: 0

Engineering Pseudomonas putida for production of 3-hydroxyacids using hybrid type I polyketide synthases 利用杂交I型聚酮合成酶生产3-羟基酸的工程恶臭假单胞菌

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications

Pub Date : 2025-04-02 DOI: 10.1016/j.mec.2025.e00261

Matthias Schmidt , Aaron A. Vilchez , Namil Lee , Leah S. Keiser , Allison N. Pearson , Mitchell G. Thompson , Yolanda Zhu , Robert W. Haushalter , Adam M. Deutschbauer , Satoshi Yuzawa , Lars M. Blank , Jay D. Keasling

Engineered type I polyketide synthases (T1PKSs) are a potentially transformative platform for the biosynthesis of small molecules. Due to their modular nature, T1PKSs can be rationally designed to produce a wide range of bulk or specialty chemicals. While heterologous PKS expression is best studied in microbes of the genus Streptomyces, recent studies have focused on the exploration of non-native PKS hosts. The biotechnological production of chemicals in fast growing and industrial relevant hosts has numerous economic and logistic advantages. With its native ability to utilize alternative feedstocks, Pseudomonas putida has emerged as a promising workhorse for the sustainable production of small molecules. Here, we outline the assessment of P. putida as a host for the expression of engineered T1PKSs and production of 3-hydroxyacids. After establishing the functional expression of an engineered T1PKS, we successfully expanded and increased the pool of available acyl-CoAs needed for the synthesis of polyketides using transposon sequencing and protein degradation tagging. This work demonstrates the potential of T1PKSs in P. putida as a production platform for the sustainable biosynthesis of unnatural polyketides.

工程型I型聚酮合成酶（t1pks）是一个潜在的小分子生物合成的变革性平台。由于其模块化的性质，t1pks可以合理设计，以生产广泛的散装或特种化学品。虽然在链霉菌属微生物中对异源PKS表达的研究最多，但最近的研究主要集中在对非本地PKS宿主的探索上。在快速发展的工业相关东道国进行化学品生物技术生产具有众多的经济和物流优势。凭借其利用替代原料的天然能力，恶臭假单胞菌已成为可持续生产小分子的有前途的主力。在这里，我们概述了恶臭杆菌作为表达工程化t1pks和生产3-羟基酸的宿主的评估。在建立了工程T1PKS的功能表达后，我们利用转座子测序和蛋白质降解标记成功地扩大和增加了合成多酮所需的可用酰基辅酶a库。这项工作证明了p.p utida中t1pks作为可持续生物合成非天然聚酮的生产平台的潜力。

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引用次数: 0

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