Metabolic engineering最新文献_第7页

Reconstruction of a resource balance analysis model of Clostridium thermocellum examines the metabolic cost of glycolytic and cellulosome enzymes 热梭菌资源平衡分析模型的重建研究了糖酵解和纤维素酶的代谢成本。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-09-08 DOI: 10.1016/j.ymben.2025.09.001

Thomas C. Willis , Wheaton L. Schroeder , Daven B. Khana , Xuejun Qian , Sanjeev Dahal , Daniel Amador-Noguez , Costas D. Maranas

Clostridium thermocellum is an increasingly well-studied organism with considerable advantages for consolidated bioprocessing towards ethanol production. Here, a genome-scale resource balance analysis (RBA) model of C. thermocellum, ctRBA, is reconstructed based on a recently published stoichiometric model (iCTH669), global proteomics, and ¹³C MFA datasets to analyze proteome allocation and the burden imposed on metabolism with regard to ethanol yield and titer. Glycolytic and fermentation enzyme concentrations were accurately quantified by the model, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and acetaldehyde-alcohol dehydrogenase (AdhE) having predicted and measured higher concentrations relative to other enzymes in glycolysis and fermentation. The metabolic burden associated with the formation of the cellulosome, the enzyme complex responsible for carbon source degradation and solubilization, was assessed and found to be consequential in constraining ethanol yield and titer, but not biomass formation. Putative enzyme substitution strains were modeled, with each strain replacing a single enzyme in C. thermocellum with a variant that uses more favorable cofactors. Strains substituting GAPDH and phosphofructokinase (PFK) predicted 30 % and 86 % increases in maximum theoretical ethanol yield and titer, respectively, a result unavailable to typical stoichiometric modeling. Model ctRBA acts as a predictive tool for assessing the effect of genetic perturbations on proteome allocation and ethanol yield and titer.

热胞梭菌是一种研究越来越深入的生物，在乙醇生产的强化生物处理中具有相当大的优势。本文基于最近发表的化学计量模型（iCTH669）、全球蛋白质组学和13C MFA数据集，重建了热cellum （ctRBA）的基因组尺度资源平衡分析（RBA）模型，以分析蛋白质组分配以及乙醇产量和滴度对代谢的影响。通过该模型，糖酵解和发酵酶的浓度被精确量化，甘油醛-3-磷酸脱氢酶（GAPDH）、磷酸甘油激酶（PGK）和乙醛-醇脱氢酶（AdhE）预测和测量的浓度相对于糖酵解和发酵中的其他酶更高。与纤维素（负责碳源降解和溶解的酶复合体）的形成相关的代谢负担被评估，并发现在限制乙醇产量和滴度方面是重要的，但不是生物量的形成。对假定的酶替代菌株进行建模，每个菌株用使用更有利的辅因子的变体替换C. thermocellum中的单个酶。取代GAPDH和磷酸果糖激酶（PFK）的菌株预测最大理论乙醇产量和滴度分别提高30%和86%，这一结果无法通过典型的化学计量模型得到。ctRBA模型作为一种预测工具，用于评估遗传扰动对蛋白质组分配、乙醇产量和滴度的影响。

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引用次数: 0

Systems metabolic engineering of Klebsiella pneumoniae for high-level 1,3-propanediol production 高水平1,3-丙二醇生产肺炎克雷伯菌的系统代谢工程。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-09-01 DOI: 10.1016/j.ymben.2025.08.012

Shaolun Zhang , Fan Zhang , Jiake Sun , Hailang Yu , Peng Sun , Jia Liu , Xiaomin Li , Guipeng Hu , Jing Wu , Cong Gao , Liming Liu

1,3-Propanediol (1,3-PDO) is an essential monomer used in the synthesis of polytrimethylene terephthalates. However, the microbial production of 1,3-PDO is limited by product tolerance and Vitamin B12 (VB₁₂) supplementation. In this study, FMME-KP—a Klebsiella pneumoniae strain with a 1,3-PDO titer of 63.4 g/L. Through pathway reprogramming, the 1,3-PDO titer of strain FMME-14 was increased by 49.1 %. To enhance strain tolerance, a 1,3-PDO biosensor was developed to screen for high-yield strains during adaptive laboratory evolution. Reverse metabolic engineering of genes ydaM^L63V and pgaD improved the 1,3-PDO tolerance of strain FMME-38 by 62.5 %, leading to a 15.9 % increase in 1,3-PDO production. By enhancing the supply of cofactors VB₁₂ and NADH, strain FMME-51 produced 138.6 g/L 1,3-PDO, with a yield of 0.52 g/g within 48 h, without the need for external VB₁₂ supplementation. Additionally, this strain produced 122.7 g/L of 1,3-PDO using crude glycerol as a substrate. To the best of our knowledge, this is the highest reported titer and yield of microbial 1,3-PDO production.

1,3-丙二醇（1,3- pdo）是合成聚对苯二甲酸三亚甲基酯的重要单体。然而，1,3- pdo的微生物生产受到产品耐受性和维生素B12 （VB12）补充的限制。本研究FMME-KP-a肺炎克雷伯菌1,3- pdo滴度为63.4 g/L。通过途径重编程，菌株FMME-14的1,3- pdo滴度提高了49.1%。为了提高菌株的耐受性，开发了一种1,3- pdo生物传感器，用于筛选实验室适应性进化过程中的高产菌株。通过基因ydaML63V和pgaD的反向代谢工程，菌株FMME-38对1,3- pdo的耐受性提高了62.5%,1,3- pdo产量提高了15.9%。通过增加辅因子VB12和NADH的供给，菌株FMME-51在48 h内产生138.6 g/L 1,3- pdo，产量为0.52 g/g，无需外部补充VB12。此外，该菌株以粗甘油为底物产生122.7 g/L的1,3- pdo。据我们所知，这是报道的微生物1,3- pdo生产的最高滴度和产量。

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引用次数: 0

Biosynthesis of 2,5-pyridinedicarboxylate from glucose via p-aminobenzoic acid in Escherichia coli 利用对氨基苯甲酸在大肠杆菌中由葡萄糖合成2,5-吡啶二羧酸酯

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-08-25 DOI: 10.1016/j.ymben.2025.08.011

Akinobu Katano , Ayana Mori , Daisuke Nonaka , Yutaro Mori , Shuhei Noda , Tsutomu Tanaka

Pyridine carboxylic acids, because of their structural similarity to aromatic carboxylic acids, have garnered increasing attention as alternative compounds in chemical synthesis. However, their broader utilization has been limited by challenges in biosynthetic production. In this study, we developed a metabolic pathway for biosynthesizing 2,5-pyridinedicarboxylate (2,5-PDCA) from glucose from p-aminobenzoate (PABA). The heterologous expression of 4-amino-3-hydroxybenzoate 2,3-dioxygenase (AhdA) in Escherichia coli enabled the conversion of 0.5 g/L of 4-amino-3-hydroxybenzoate (4A3HBA) into 0.47 g/L of 2,5-PDCA. Subsequent systematic evaluation of p-hydroxybenzoate hydroxylase (PobA) variants and optimization of pobA and ahdA co-expression facilitated the development of a 2,5-PDCA biosynthetic module for efficient production from PABA. Incorporating this module into a PABA biosynthesis pathway enabled direct 2,5-PDCA production from glucose. Further enhancements were achieved by increasing metabolic flux through the shikimate pathway and optimizing sodium pyruvate supplementation. Under optimized conditions, we achieved a titer of 1.84 g/L in test-tube cultures after 72 h and 10.6 g/L in bioreactor fermentation after 144 h. Overall, this study introduces a valuable strategy for the microbial production of pyridine carboxylates and establishes a promising platform for broader applications in aromatic compound biosynthesis.

吡啶羧酸由于其结构与芳香羧酸相似，在化学合成中作为替代化合物受到越来越多的关注。然而，它们的广泛利用受到生物合成生产挑战的限制。在这项研究中，我们开发了一个代谢途径，以葡萄糖为原料从对氨基苯甲酸酯（PABA）生物合成2,5-吡啶二羧酸（2,5- pdca）。4-氨基-3-羟基苯甲酸2,3-双加氧酶（AhdA）在大肠杆菌中的异源表达使0.5 g/L的4-氨基-3-羟基苯甲酸（4A3HBA）转化为0.47 g/L的2,5- pdca。随后对对羟基苯甲酸羟化酶（PobA）变异进行了系统评估，并优化了PobA和ahdA的共表达，促进了2,5- pdca生物合成模块的开发，从而有效地从PABA中生产。将该模块整合到PABA生物合成途径中，可以直接从葡萄糖中生产2,5- pdca。通过增加莽草酸途径的代谢通量和优化丙酮酸钠的补充，进一步增强了功能。在优化的条件下，试管培养72 h后滴度为1.84 g/L，生物反应器发酵144 h后滴度为10.6 g/L。总之，本研究为微生物生产吡啶羧酸酯提供了有价值的策略，并为芳香族化合物生物合成的广泛应用建立了一个有前景的平台。

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引用次数: 0

A metabolic engineering strategy for producing poly-(3-hydroxyoctanoic acid) in Escherichia coli from glycerol 利用甘油在大肠杆菌中生产聚- 3-羟基辛酸的代谢工程策略

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-08-22 DOI: 10.1016/j.ymben.2025.08.009

Shivangi Mishra, Ke Xu, Madeline K. Kuckuk, William T. Cordell, Néstor J. Hernández-Lozada, Brian F. Pfleger

Poly(3-hydroxyoctanoate) (PHO) is a medium-chain-length PHA with low crystallinity and high elongation to break ratio, unlike the brittle short-chain-PHAs like PHB. These properties make PHO a promising candidate for industrial and biomedical applications. In this study, we demonstrated the production of PHO in Escherichia coli from a renewable and inexpensive glycerol feedstock by engineering fatty acid synthesis and β-oxidation to create a pool of 2,3-octenoyl-CoAs. In this base strain, E. coli ΔfadRABIJ, an (R)-specific enoyl-CoA hydratase (phaJ) and a PHA synthase (phaC) were expressed to produce PHO. Bioprospecting phaJ and phaC homologs from Pseudomonas aeruginosa and fadD homolog from Pseudomonas putida implicated a combination of phaJ2, phaC2, and ^PpfadD genes yielded the highest PHO content from exogenously fed octanoate. Finally, when a single copy of a previously described C₈-specific thioesterase mutant CpFatB1.2-M4-287 was integrated into the chromosome of E. coli ΔfadRABIJ, the resulting E. coli strain NHL18 was capable of producing 3.69 ± 0.146 g/L of octanoic acid. Subsequently, the integration of PHA synthesis genes in NHL18 resulting in strain SM23 allowed the cell to accumulate 15 % cell dry weight of PHO with a final titer of 1.54 ± 0.234 g/L from glycerol in fed-batch fermentation.

聚3-羟基辛酸酯（PHO）是一种低结晶度、高断裂伸长率的中链PHA，不同于PHB等脆性短链PHA。这些特性使PHO成为工业和生物医学应用的有前途的候选者。在这项研究中，我们证明了大肠杆菌通过工程脂肪酸合成和β-氧化产生2,3-辛烯酰辅酶a池，从可再生和廉价的甘油原料生产PHO。在大肠杆菌ΔfadRABIJ中，表达了一种(R)特异性烯酰辅酶a水合酶（phaJ）和一种PHA合成酶（phaC）来产生PHO。从铜绿假单胞菌的phaJ和phaC同源物和恶臭假单胞菌的fadD同源物中发现，外源性辛酸盐中PHO含量最高的是phaJ2、phaC2和PpfadD基因的组合。最后，将先前描述的c8特异性硫酯酶突变体CpFatB1.2-M4-287的单个拷贝整合到大肠杆菌ΔfadRABIJ的染色体上，得到的大肠杆菌菌株NHL18能够产生3.69±0.146 g/L的辛酸。随后，在NHL18中整合PHA合成基因，产生菌株SM23，使细胞在补料分批发酵中积累了15%细胞干重的PHO，最终滴度为1.54±0.234 g/L。

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引用次数: 0

Lipid accumulation in nitrogen and phosphorus-limited yeast is caused by less growth-related dilution 氮磷限制酵母中的脂质积累是由较少的生长相关稀释引起的

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-08-22 DOI: 10.1016/j.ymben.2025.08.010

Xi Li , Daniel R. Weilandt , Felix C. Keber , Arjuna M. Subramanian , Shayne R. Loynes , Christopher V. Rao , Yihui Shen , Martin Wühr , Joshua D. Rabinowitz

Oleaginous yeasts are used commercially to produce oleochemicals and hold potential also for biodiesel production. In response to nitrogen or phosphorous limitation, oleaginous yeasts accumulate lipids in the form of triacylglycerols. Previous work has investigated potential mechanisms by which nutrient limitation induces lipid biosynthesis without verifying whether lipid biosynthesis flux is actually enhanced. Here we show, using ¹³C-glucose tracing, that in nitrogen or phosphorous limitation, lipid accumulation occurs without consistent increases in biosynthetic flux. Instead, the main driver of increased lipid pools is decreased growth-related dilution. This conclusion holds across two divergent oleaginous yeasts: Rhodotorula toruloides and Yarrowia lipolytica. Quantitative proteomics shows a substantial proteome reallocation in response to nitrogen and phosphorous limitation, with ribosomal proteins strongly downregulated, while lipid enzymes are preserved but not consistently upregulated in absolute quantity. Thus, nutrient limitation, rather than triggering greatly enhanced lipid synthesis, results in roughly sustained lipid enzyme levels and biosynthetic flux. Due to slower lipid dilution by cell division, this suffices to drive marked lipid accumulation.

产油酵母在商业上用于生产油脂化学品，也具有生产生物柴油的潜力。由于氮或磷的限制，产油酵母以三酰基甘油的形式积累脂质。以前的工作研究了营养限制诱导脂质生物合成的潜在机制，但没有验证脂质生物合成通量是否实际上增强。在这里，我们表明，使用13c -葡萄糖示踪，在氮或磷限制下，脂质积累发生在生物合成通量不一致增加的情况下。相反，脂质池增加的主要驱动因素是与生长相关的稀释度降低。这一结论适用于两种不同的产油酵母：环形红酵母和多脂耶氏酵母。定量蛋白质组学显示，在氮和磷的限制下，蛋白质组发生了实质性的再分配，核糖体蛋白被强烈下调，而脂质酶被保留，但在绝对数量上不一致上调。因此，营养限制，而不是触发大大增强脂质合成，导致大致维持脂质酶水平和生物合成通量。由于细胞分裂对脂质稀释较慢，这足以驱动显著的脂质积累。

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引用次数: 0

Pan-reactome analysis of Streptomyces strains reveals association and disconnection between primary and secondary metabolism 链霉菌菌株的泛反应组分析揭示了初级和次级代谢之间的联系和脱节

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-08-20 DOI: 10.1016/j.ymben.2025.08.005

Byung Tae Lee , Omkar S. Mohite , Mun Su Kwon , Hahk-Soo Kang , Tilmann Weber , Sang Yup Lee , Hyun Uk Kim

Secondary metabolites have crucial medicinal and industrial applications, but their alignment with primary metabolism remains unclear. As secondary metabolism depends on primary metabolism for precursor supply, we present a pan-reactome analysis of 242 Streptomyces strains to investigate their association and disconnection. This analysis includes phylogenetic grouping of the strains using genome data, and uniform manifold approximation and projection (UMAP) analysis of their genome-scale metabolic models (GEMs) and biosynthetic gene cluster (BGC) data, which represent biochemical reactions in primary and secondary metabolism. Subsequent correlation analysis of the preprocessed GEM and BGC data showed a Pearson correlation coefficient of 0.54, revealing both metabolic association and disconnection. In particular, among 47 precursors of polyketides, nonribosomal peptides, and hybrids, nine precursors required by these BGCs were predicted to be non-producible due to missing genes in primary metabolism or BGCs. The pan-reactome analysis facilitates the identification of precursor availability and metabolic gaps, providing insights into secondary metabolite biosynthesis.

次生代谢产物具有重要的医学和工业应用，但它们与初级代谢的一致性尚不清楚。由于次级代谢依赖于初级代谢提供前体，我们对242株链霉菌进行了泛反应组分析，以研究它们的关联和分离。该分析包括利用基因组数据对菌株进行系统发育分组，并对它们的基因组尺度代谢模型（GEMs）和生物合成基因簇（BGC）数据进行均匀流形近似和投影（UMAP）分析，这些数据代表了初级和次级代谢中的生化反应。随后对预处理后的GEM和BGC数据进行相关分析，Pearson相关系数为0.54，表明代谢相关和分离。特别是，在聚酮、非核糖体肽和杂交体的47种前体中，由于初级代谢或bgc中缺失基因，预计这些bgc所需的9种前体无法产生。泛反应组分析有助于识别前体可利用性和代谢间隙，为次级代谢物的生物合成提供见解。

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引用次数: 0

Advances in metabolic engineering of Vibrio natriegens as an unconventional host for biotechnology 作为生物技术非常规宿主的营养弧菌代谢工程研究进展

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-08-18 DOI: 10.1016/j.ymben.2025.08.008

Maurice Hädrich , Josef Hoff , Bastian Blombach

The exploitation of Vibrio natriegens as an unconventional host for biotechnology has progressed rapidly. This development is not only a result of the remarkable high growth rate of this marine bacterium on different substrates but is also possible due to good handling properties, a versatile metabolism and its inherent natural competence – features that have facilitated the development of a sophisticated genetic engineering and synthetic biology toolbox. The availability of robust metabolic and regulatory data enables a model-based quantitative description of metabolic routes and accelerates rational metabolic engineering of the facultative anaerobic bacterium. As reviewed here, numerous examples, ranging from small-molecule production over cell-free protein synthesis to bioremediation render V. natriegens a promising next-generation host for biotechnological applications.

营养弧菌作为一种非传统的生物技术宿主的开发进展迅速。这一发展不仅是由于这种海洋细菌在不同基质上的显著高生长速度，而且由于良好的处理性能、多功能代谢和其固有的自然能力——这些特征促进了复杂基因工程和合成生物学工具箱的发展。可靠的代谢和调控数据的可用性使得基于模型的代谢途径定量描述和加速兼性厌氧细菌的合理代谢工程成为可能。正如本文所述，从小分子生产到无细胞蛋白质合成，再到生物修复，许多例子都使营养弧菌成为生物技术应用的下一代寄主。

引用次数: 0

A genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome 中国仓鼠卵巢细胞基因组级CRISPR缺失筛选揭示了编码和非编码基因组的重要区域

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-08-18 DOI: 10.1016/j.ymben.2025.08.007

Federico De Marco , Ivy Rose Sebastian , Antonino Napoleone , Alexander Molin , Markus Riedl , Nina Bydlinski , Krishna Motheramgari , Mohamed K. Hussein , Lovro Kramer , Thomas Kelly , Thomas Jostock , Nicole Borth

The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated “dark matter” includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties.

生物制药部门依靠CHO细胞来研究生物过程，并作为生产生物治疗药物的首选宿主。同时，CHO细胞基因组组装的进展为开发复杂的基因工程策略提供了见解。虽然这些研究大多集中在编码基因上，对转录的非编码rna（如microrna和lncrna）也有一些兴趣，但仍然缺乏精确检查其余90%基因组及其对细胞表型影响的全基因组系统研究。这个未注释的“暗物质”包括调控元件和其他可能对细胞行为至关重要的基因组特征。在这项研究中，我们部署了一个基因组规模的CRISPR筛选平台，其中有112,272对引导rna，针对14,034个基因组区域，完全删除150 kb长的片段。该平台能够执行阴性筛选，选择性地鉴定死亡细胞，以确定细胞存活所必需的区域。通过使用配对grna，我们克服了传统移码策略的内在局限性，传统移码策略可能对非编码基因组影响很小或没有影响。这项研究揭示了427个对CHO细胞存活至关重要的区域，其中许多区域目前缺乏基因注释或已知功能。对于这些区域，我们给出了它们的注释状态、转录活性和注释的染色质状态。个别删除了选定的区域，特别是不具备上述所有条件的区域，以确认其重要性。这项工作为哺乳动物基因组的很大一部分提供了新的视角，这些基因组在传统上很难研究，因此被忽视了。值得注意的是，其中一些区域的缺失对细胞是致命的，这表明它们编码了关键的调节功能。对这些功能更好的全基因组理解可以为改进生物过程相关特性的工程细胞开辟新的途径。

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引用次数: 0

Retrofitting Escherichia coli for de novo production of rare L-sorbose from abundant D-glucose 改造大肠杆菌，使其从丰富的d -葡萄糖中重新生产稀有的l -山梨糖。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-08-16 DOI: 10.1016/j.ymben.2025.08.006

Jayce E. Taylor , Trevor Gannalo , Bryant Luu , Dileep Sai Kumar Palur , Augustine Arredondo , Ian C. Anderson , Twisha Dasgupta , John Didzbalis , Justin B. Siegel , Shota Atsumi

Monosaccharides exist in either “D” or “L” conformations, with L-sugars being much less abundant in nature and therefore classified as “rare sugars.” Rare sugars hold significant potential due to their unique interactions with biological systems, offering health, food, and crop benefits. One such sugar, L-sorbose, serves as a critical precursor to Vitamin C and offers a low-calorie, moderately sweet alternative to table sugar, being 60–70 % as sweet but with only 25 % of the caloric value. However, the broader study and application of rare sugars, including L-sorbose, are constrained by their high cost and limited availability. To address this challenge, we developed a biosynthetic strategy to convert the abundant and inexpensive D-sugar D-glucose into the rare L-sugar L-sorbose using microbial production. By utilizing phosphorylation and dephosphorylation steps to thermodynamically drive carbon flux, efficient production of 14.5 g L⁻¹ L-sorbose was achieved under test tube conditions. Additionally, this pathway results in the co-production of D-sedoheptulose, a non-sweet, rare sugar shown to inhibit C6 sugar consumption in humans by modulating energy metabolism. The dual production of L-sorbose and D-sedoheptulose presents unique opportunities for applications in food and health sciences. This study demonstrates microbial production as a promising platform for rare L-sugar biosynthesis and provides a generalizable strategy for converting abundant D-sugars into underexplored L-sugars. Expanding access to L-sugars enables deeper investigations into their biological functions, metabolic pathways, and industrial applications. By advancing both fundamental sugar metabolism research and microbial production strategies, this study broadens the scope of rare sugar utilization.

单糖以“D”或“L”构象存在，L糖在自然界中的含量要少得多，因此被归类为“稀有糖”。稀有糖具有巨大的潜力，因为它们与生物系统的独特相互作用，提供健康，食品和作物效益。其中一种糖，l -山梨糖，是维生素C的关键前体，提供了一种低热量、中等甜味的替代食糖，甜味为60-70%，但热量只有食糖的25%。然而，包括l -山梨糖在内的稀有糖的广泛研究和应用受到其高成本和有限可用性的限制。为了解决这一挑战，我们开发了一种生物合成策略，利用微生物生产将丰富而廉价的d -糖d -葡萄糖转化为稀有的l -糖L-sorbose。利用磷酸化和去磷酸化步骤热力学驱动碳通量，在试管条件下实现了14.5 g L-1 l -海马糖的高效生产。此外，这一途径导致D-sedoheptulose的共同产生，D-sedoheptulose是一种非甜的稀有糖，通过调节能量代谢来抑制人体对C6糖的消耗。l -山梨糖和d -糖庚糖的双重生产为食品和健康科学的应用提供了独特的机会。本研究证明微生物生产是稀有l糖生物合成的一个有前途的平台，并为将丰富的d糖转化为未开发的l糖提供了一种通用策略。扩大对l糖的获取，可以更深入地研究它们的生物学功能、代谢途径和工业应用。本研究通过推进糖代谢基础研究和微生物生产策略，拓宽了稀有糖利用的范围。

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引用次数: 0

Metabolic engineering of Corynebacterium glutamicum for increased cis, cis-muconate production from plant-derived p-hydroxycinnamates via deregulated pathway flux and increased CoA intermediate availability 谷氨酸棒状杆菌的代谢工程，通过解除调控的途径通量和增加辅酶a中间体的可用性，增加植物源的对羟基肉桂酸的顺式、顺式肉桂酸的产量。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2025-08-12 DOI: 10.1016/j.ymben.2025.08.004

Fabia Weiland, Kyoyoung Seo, Franka Janz, Marius Grad, Lea Geldmacher, Michael Kohlstedt, Judith Becker, Christoph Wittmann

Lignocellulosic biomass represents a promising renewable feedstock for sustainable biochemical production, with p-hydroxycinnamates emerging as key aromatic building blocks derived from agricultural residues and grassy plants. C. glutamicum has recently been engineered to produce cis, cis-muconate (MA), a high-value platform chemical used in biobased plastics, resins, and specialty chemicals. However, unlike other aromatics, the metabolism of the p-hydroxycinnamates p-coumarate, ferulate, and caffeate in MA-producing C. glutamicum is inefficient, limiting MA production performance. Here, we discovered that p-hydroxycinnamate metabolism, encoded by the phd operon, is repressed by the local repressor PhdR under glucose-rich conditions, while the global regulator GlxR activates the pathway in the absence of glucose. The deregulated C. glutamicum MA-10 lacking phdR exhibited an up to 98-fold increase in the conversion of p-coumarate, ferulate, and aromatic mixtures derived from plant waste into MA. Transcriptomic and metabolomic analyses revealed strong induction of the phd operon in strain MA-10 and a marked increase in intracellular aromatic CoA-esters and acetyl-CoA, indicating enhanced flux through the p-hydroxycinnamate degradation pathway. ¹³C-tracer studies demonstrated a substantial contribution of aromatic side-chain carbon to central metabolic pathways, supporting biomass formation and enabling MA production even in the absence of sugars. Additionally, MA-10 showed broadened substrate flexibility, degrading cinnamate into MA and methoxylated cinnamates into valuable benzoate derivatives. The strain also successfully converted aromatics from real straw lignin hydrolysates into MA. Our findings reveal the potential of targeted regulatory engineering to optimize C. glutamicum for lignin valorization. The newly developed strain MA-10 provides a robust platform for the biobased production of MA from lignocellulosic feedstocks, paving the way for sustainable and economically viable biorefinery processes.

木质纤维素生物质代表了一种有前途的可持续生化生产的可再生原料，对羟基肉桂酸酯作为从农业残留物和禾草植物中提取的关键芳香基石。C. glutamicum最近被设计用于生产cis， cis-muconate (MA)，这是一种高价值的平台化学品，用于生物基塑料，树脂和特种化学品。然而，与其他芳香烃不同的是，对羟基肉桂酸、对香豆酸、阿魏酸和咖啡酸在产生MA的C. glutamum中的代谢效率很低，限制了MA的生产性能。在这里，我们发现由phd操纵子编码的对羟基肉桂酸代谢在富含葡萄糖的条件下被局部抑制因子PhdR抑制，而全局调节因子GlxR在缺乏葡萄糖的情况下激活该途径。缺乏phdR的不受调控的谷氨酰胺MA-10在从植物废物中提取的对香豆酸盐、阿魏酸盐和芳香混合物转化为MA的能力增加了98倍。转录组学和代谢组学分析显示，菌株MA-10的phd操纵子被强烈诱导，细胞内芳香辅酶a酯和乙酰辅酶a显著增加，表明通过对羟基肉桂酸降解途径的通量增强。13c示踪研究表明芳香侧链碳对中心代谢途径的重要贡献，支持生物量的形成，即使在没有糖的情况下也能产生MA。此外，MA-10表现出更宽的底物柔韧性，将肉桂酸降解为MA，并将甲氧基化的肉桂酸转化为有价值的苯甲酸衍生物。该菌株还成功地将真正的秸秆木质素水解产物中的芳烃转化为MA。我们的研究结果揭示了靶向调控工程优化谷氨酰胺木质素增值的潜力。新开发的菌株MA-10为木质纤维素原料的生物基生产MA提供了一个强大的平台，为可持续和经济上可行的生物炼制工艺铺平了道路。

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