JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2022
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IF 5.895
Pingping Zhou,Chunlei Yue,Yuchen Zhang,Yan Li,Xinyi Da,Xiuqi Zhou,Lidan Ye
Rosmarinic acid as a polyphenolic compound has great values in the pharmaceutical, cosmetic, and food industries. To achieve efficient biosynthesis of rosmarinic acid, the major obstacles such as imbalanced metabolic flux among branching pathways and substrate promiscuity of pathway enzymes should be eliminated. Here, a rosmarinic acid producing Saccharomyces cerevisiae strain was constructed by introducing codon optimized d-lactate dehydrogenase gene mutant (OD-LDHY52A), 4-coumarate CoA ligase gene (OPc4CL2), and rosmarinic acid synthase gene (OMoRAS) into a previously constructed caffeic acid hyper-producer. To identify the metabolic bottleneck, the substrate specificity of OPc4CL2 and OMoRAS was figured out by bioconversion experiments and HPLC–MS/MS analysis. Subsequently, the byproducts formation was alleviated by removing prephenate dehydratase and tuning down the expression level of OPc4CL2. The final strain YRA113-15B produced 208 mg/L rosmarinic acid in a shake-flask culture (a 63-fold improvement over the initial strain), which was the highest rosmarinic acid titer by engineered microbial cells reported to date. This work provides a promising platform for fermentative production of rosmarinic acid and offers a strategy to overcome the intrapathway competition.
研究领域:
生物技术
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代谢工程
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制药
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PubMed
高影响因子
METABOLIC ENGINEERING
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2025
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IF 6.8
Xin Jin,Ruxin Hao,Hannuo Shen,Zhu Liu,Sumeng Wang,Qingsheng Qi,Quanfeng Liang
The design of synthetic driving forces for biosynthetic pathway is crucial for directing carbon flux toward the target product. Optimizing cellular redox status is one of the key strategies for constructing microbial cell factories. In this study, we attempt to create a novel redox imbalance force-driven (RIFD) strategy to direct carbon flow toward the target synthetic pathway. Initially, we increased the NADPH pool through a strategy of “open source and reduce expenditure” employing four approaches to achieve excessive NADPH levels and growth inhibition: (I) the expression of cofactor-converting enzymes, (II) the expression of heterologous cofactor-dependent enzymes, (III) the expression of enzymes involved in the NADPH synthesis pathway, and (IV) reduced NADPH wastage by knocking down non-essential genes that consume NADPH in vivo. Next, multiple automated genome engineering (MAGE) techniques were employed to evolve redox-imbalanced engineered strains and drive the metabolic flux to L-threonine production. Finally, we developed a NADPH and L-threonine dual-sensing biosensor, combined it with Fluorescence-Activated Cell Sorting (FACS), and a high-yield (0.65 g/g) L-threonine-producing strain with a titer of 117.65 g L −1 was obtained. This research presents a general approach to increasing the production of cofactor-related products. Utilizing redox imbalance forces to drive metabolic flow toward the target product, it is possible to increase production while simultaneously restoring cell growth.
研究领域:
合成生物学
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代谢工程
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系统生物学
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生物技术
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生物工程
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PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2021
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IF 3.978
Yiwei Dai,Chenchen Li,Luhua Zheng,Bo Jiang,Tao Zhang,Jingjing Chen
In our previous study, a whole-cell biocatalytic system involving α-glucan phosphorylase , phosphoglucomutase , glucose 6-phosphate isomerase, tagatose 1,6-bisphosphate aldolase , and phosphoglycolate phosphatase , was constructed to produce d -tagatose from maltodextrin . However, the biotransformation efficiency of this whole-cell catalyst was low because of the unbalanced ratio of each enzyme and metabolic flux of the intermediates. Therefore, in this study, the biosynthesis of d -tagatose was enhanced by optimizing vectors and improving the expression of rate-limiting enzymes through the construction of multi-copy genes to regulate expression levels. The conversion rate increased from 20.8% after 24 h to 25.2% after 3 h using 10 g/L maltodextrin as the substrate. Furthermore, the genes in the bypass pathways in Escherichia coli , pfka and zwf , were deleted to increase the accumulation of intermediates. The strain ER-2GatZ (Δ p Δ z ) produced 3.383 g/L d -tagatose using 10 g/L substrate after 3 h, which was 1.34 times that of the wild strain. This work exemplifies the use of modular engineering to enhance d -tagatose production using whole-cell catalysts.
研究领域:
生物技术
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代谢工程
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微生物学
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生化工程
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DOI
高影响因子
Analytical Methods
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2023
·Vol. 15
·期 42
·5666-5673
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IF 3.1
Yameng Zhao,Ruoning Fan,Chuyao Wang,Sen Xu,Long Xie,Jinghua Hou,Wen Lei,Jianjun Liu
Metabolic flux analysis (MFA) using stable isotope labeled tracers is a powerful tool to estimate fluxes through metabolic pathways. It finds applications in studying metabolic changes in diseases, regulation of cellular energetics, and novel strategies for metabolic engineering. Accurate and precise quantification of the concentration of metabolites and their labeling states is critical for correct MFA results. Utilizing an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system, an analytical method for simultaneously quantifying the concentration of sugar metabolites and their mass isotopologue distribution (MID) was developed. The method performs with good linearity and coefficient of determination (R2) > 0.99, while the detection limit ranged from 0.1 to 50 mg L−1. Seven sugar metabolites were detected in a labeled Brevibacterium flavum sample using the method. The detected quantities ranged from 6.15 to 3704.21 mg L−1, and 13C abundance was between 12.77% and 66.67% in the fermentation fluid and 16.28% and 91.93% in the bacterial body. Overall, the method is efficient, accurate, and suitable for analysis of labeled sugar metabolites in 13C MFA studies.
研究领域:
分析化学
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代谢组学
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微生物学
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DOI
PubMed
高影响因子
Food Bioscience
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2024
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IF 4.8
Chao Wang,Wang Ma,Luwei Xu,Zhiyun Wei,Ke Tang,Jingwen Zhou,Jian Chen
Flavonoids, including dihydroflavonols and anthocyanins, are phenolic compounds with significant biological activity, playing a crucial role in the sensory characteristics and health benefits of wine. In this study, we selected the naringenin-producing strain HB52 （ Saccharomyces cerevisiae ） as the starting strain and introduced synthetic pathways for dihydroflavonols and anthocyanins, achieving de novo synthesis of various flavonoid compounds. To further optimize flavonoid production, we employed several strategies, including overexpressing 5-enolpyruvylshikimate 3-phosphate synthase to enhance metabolic flux, integrating NADPH regeneration genes, and using citric acid/isocitric acid transporter genes to increase the levels of cofactors. Additionally, organelle engineering was utilized to strengthen the β-oxidation pathway, thereby elevating the levels of precursors such as acetyl-coenzyme A (CoA) and malonyl-CoA. Engineered strains significantly improved their ability to synthesize various flavonoids directly from glucose. In the final engineered strains, the production levels of NAR, DHQ, and DHM in the dihydroflavonol-engineered strains reached 379.2 mg/L, 231.3 mg/L, and 284.8 mg/L, respectively. The anthocyanin-engineered strains achieved the highest yield of anthocyanin synthesized from glucose in S. cerevisiae , reaching 45.7 mg/L (33.4 mg/L for C3G and 12.3 mg/L for D3G). This study highlights the potential of metabolic and organelle engineering in S. cerevisiae to increase flavonoid production, offering new prospects for enhancing sensory quality and health benefits in the wine industry.
研究领域:
代谢工程
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细胞生物学
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生物技术
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生物化学
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葡萄酒科学
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高影响因子
PLANT CELL TISSUE AND ORGAN CULTURE
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2025
·Vol. 162
·期 3
·1-12
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IF 2.4
Li Haihua,Li Jiahe,Liu Ningjin,Li Huishan,Wang Jiarui,Zhang Rulan,Xue Jingyan,Kang Xiaotian,Guan Xinying,Lin Shiya,Zhang Ziyu,Kuang Jiaqi,Qian Chenlei,Zhou Pengfei
In Catharanthus roseus , the inherently low yield of bioactive alkaloids such as vindoline, catharanthine, ajmalicine, and vinblastine remains a major limitation to their broader therapeutic application, particularly in anticancer and antihypertensive treatments. To address this challenge, we developed a novel metabolic flux regulation strategy by combining the enzyme inhibitors lovastatin and naproxen with the abiotic elicitor copper(II)-chitosan (Cu-CS) complex. Compared to controls, treatment with 2 mg/L lovastatin significantly enhanced catharanthine, vindoline, ajmalicine, and vinblastine accumulation in C. roseus CMCs to 2.38, 0.63, 35.3, and 2.33 mg/L, respectively — increases of 48%, 121%, 71%, and 12%. Application of 8 mg/L naproxen further enhanced catharanthine, vindoline, and vinblastine levels to 3.78, 0.67, and 2.14 mg/L, representing increases of 151%, 135%, and 30%, respectively. The Cu-CS complex at 10 mg/L elevated catharanthine, vindoline, ajmalicine, and vinblastine to 2.13, 0.49, 32.11, and 2.46 mg/L, with corresponding increases of 49%, 63%, 55%, and 36%. Notably, the combined treatment of all three agents synergistically boosted catharanthine, vindoline, and vinblastine production to 5.94, 1.52, and 3.65 mg/L — representing 310%, 390%, and 98% increases over control levels. Quantitative RT-PCR analysis revealed that Cu-CS treatment upregulated the transcription of key genes and transcription factors involved in terpenoid indole alkaloid (TIA) biosynthesis. Overall, this integrated strategy effectively redirects metabolic flux toward enhanced TIA production in C. roseus CMCs, providing a practical and efficient approach for improving the yield of pharmaceutically important alkaloids in plant cell culture systems.
研究领域:
植物生物技术
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次生代谢
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医药生物技术
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分子生物学
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代谢工程
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DOI
高影响因子
Journal of Pharmaceutical Analysis
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2023
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IF 8.8
Baixi Shan,Haoyan Zhou,Congying Guo,Xiaolu Liu,Mingyu Wu,Rao Zhai,Jun Chen
Evidence indicates that metabolic reprogramming characterized by the changes in cellular metabolic patterns contributes to the pathogenesis of pulmonary fibrosis (PF). It is considered as a promising therapeutic target anti-PF. The well-documented against PF properties of Tanshinone IIA (Tan IIA) have been primarily attributed to its antioxidant and anti-inflammatory potency. Emerging evidence suggests that Tan IIA may target energy metabolism pathways, including glycolysis and tricarboxylic acid (TCA) cycle. However, the detailed and advanced mechanisms underlying the anti-PF activities remain obscure. In this study, we applied [U- 13 C]-glucose metabolic flux analysis (MFA) to examine metabolism flux disruption and modulation nodes of Tan IIA in PF. We identified that Tan IIA inhibited the glycolysis and TCA flux, thereby suppressing the production of TGF-β1- dependent extracellular matrix and the differentiation and proliferation of myofibroblasts in vitro . We further revealed that Tan IIA inhibited the expression of key metabolic enzyme hexokinase 2 (HK2) by inhibiting phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/hypoxia-inducible factor 1α (HIF-1α) pathway activities, which decreased the accumulation of abnormal metabolites. Notably, we demonstrated that Tan IIA inhibited ATP citrate lyase (ACLY) activity, which reduced the collagen synthesis pathway caused by cytosol citrate consumption. Further, these results were validated in a mouse model of bleomycin-induced PF. This study was novel in exploring the mechanism of the occurrence and development of Tan ⅡA in treating PF using 13 C-MFA technology. It provided a novel understanding of the mechanism of Tan IIA against PF from the perspective of metabolic reprogramming.
研究领域:
药理学
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代谢组学
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肺医学
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细胞生物学
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生物化学
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PubMed
高影响因子
Nature Communications
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2020
·Vol. 11
·期 1
·1-15
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IF 12.121
Zheng Di-Wei,Hong Sheng,Zhang Qiu-Ling,Dong Xue,Pan Pei,Song Wen-Fang,Song Wen,Cheng Si-Xue,Zhang Xian-Zheng
Global alterations in the metabolic network provide substances and energy to support tumor progression. To fuel these metabolic processes, extracellular matrix (ECM) plays a dominant role in supporting the mass transport and providing essential nutrients. Here, we report a fibrinogen and thrombin based coagulation system to construct an artificial ECM (aECM) for selectively cutting-off the tumor metabolic flux. Once a micro-wound is induced, a cascaded gelation of aECM can be triggered to besiege the tumor. Studies on cell behaviors and metabolomics reveal that aECM cuts off the mass transport and leads to a tumor specific starvation to inhibit tumor growth. In orthotopic and spontaneous murine tumor models, this physical barrier also hinders cancer cells from distant metastasis. The in vivo gelation provides an efficient approach to selectively alter the tumor mass transport. This strategy results in a 77% suppression of tumor growth. Most importantly, the gelation of aECM can be induced by clinical operations such as ultrasonic treatment, surgery or radiotherapy, implying this strategy is potential to be translated into a clinical combination regimen.
研究领域:
癌症研究
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生物医学工程
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组织工程
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药学
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DOI
PubMed
高影响因子
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