Metabolic engineering最新文献

Reconstruction of arginine deiminase pathway sustains a higher-energy state in mammalian cells 精氨酸脱亚胺酶途径的重建维持了哺乳动物细胞的高能量状态

IF 8.4 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-02-10 DOI: 10.1016/j.ymben.2026.02.004

Mauro Torres, Matthew Reaney, Kate Meeson, Devika Kalsi, Leon P. Pybus, Alan J. Dickson

引用次数: 0

Engineering Halomonas bluephagenesis for High-Efficiency Biosynthesis of Pyruvate 丙酮酸高效生物合成的工程产蓝盐单胞菌

IF 8.4 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-02-08 DOI: 10.1016/j.ymben.2026.02.003

Kang Wang, Zonghao Zhang, Zhongnan Zhang, Fuqing Wu, Guo-Qiang Chen

引用次数: 0

Engineering xylose catabolism in the yeast Komagataella phaffii 法菲黑马酵母木糖分解代谢的工程研究

IF 8.4 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-02-07 DOI: 10.1016/j.ymben.2026.02.001

Kun Zhang, Xin Ni, Peng Cai, Yongjin J. Zhou

引用次数: 0

Multisite Cre-lox recombination enables regulatory mechanism elucidation and systematic engineering of echinocandin B biosynthesis in Aspergillus nidulans 多位点Cre-lox重组实现棘球白菌B生物合成调控机制阐明及系统工程

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-22 DOI: 10.1016/j.ymben.2026.01.009

Youchu Ma , Yue Zhang , Dongfang Zhang , Yue Tang , Wanyu Zhang , Liyuan Yue , Wenqing Lou , Chao Meng , Yanling Li , Xiulai Chen , Fanglong Zhao

Echinocandin B (ECB), a cyclic lipohexapeptide for synthesizing antifungal drugs, is produced by the nonribosomal peptide synthetase gene cluster in Aspergillus nidulans. However, industrial production remains limited by the inefficiency of production capacity, primarily due to the complexity of the biosynthetic pathway and the absence of multi-gene regulatory tools in filamentous fungi. Here, we established an orthogonal Cre-lox-based platform enabling single-site insertion of up to 30 kb and simultaneous dual-site integration of 10 kb DNA fragments in A. nidulans. Through precursor supplementation and targeted gene overexpression, we identified key enzymatic bottlenecks in the precursor biosynthetic pathway, including the oxygenases AniF, AniK, AniG, and the acyl-AMP ligase AniI. Combinatorial overexpression of these genes acted synergistically to increase ECB titers. We further addressed bottlenecks in natural amino acid biosynthesis by overexpressing feedback-resistant mutants of Hom3 (L-Thr pathway) and LeuC (L-Leu pathway). Additionally, we uncovered a temperature-dependent regulation mechanism whereby low temperature (25 °C) concurrently upregulates both the ECB biosynthetic gene cluster and odeA gene, encoding Δ12-oleic acid desaturase, thereby increasing linoleic acid availability for ECB production. Leveraging our multisite DNA-integration platform to rewire expression of these key genes, we increased ECB production to 3.5 ± 0.2 g/L in a 5-L fed-batch bioreactor, a 2.3-fold improvement that represents the highest titer reported in the literature to date. Our orthogonal dual-site integration strategy and the systematic optimization approach provide a valuable framework for metabolic engineering of complex natural products in filamentous fungi.

棘白菌素B （Echinocandin B， ECB）是一种合成抗真菌药物的环脂六肽，由细粒曲霉非核糖体肽合成酶基因簇产生。然而，工业生产仍然受到生产能力低下的限制，这主要是由于丝状真菌生物合成途径的复杂性和缺乏多基因调控工具。在这里，我们建立了一个基于cre -lox的正交平台，可以在A. nidulans中单位点插入最多30 kb的DNA片段，同时在双位点整合10 kb的DNA片段。通过前体补充和靶向基因过表达，我们确定了前体生物合成途径中的关键酶瓶颈，包括加氧酶AniF、AniK、AniG和酰基- amp连接酶AniI。这些基因的组合过表达协同作用增加了ECB滴度。我们通过过表达Hom3 （L-Thr通路）和LeuC （L-Leu通路）的反馈抗性突变体进一步解决了天然氨基酸生物合成的瓶颈。此外，我们发现了一种温度依赖的调节机制，即低温（25°C）同时上调ECB生物合成基因簇和odeA基因，编码Δ12-oleic酸去饱和酶，从而增加ECB生产的亚油酸可用性。利用我们的多位点dna整合平台重新连接这些关键基因的表达，我们将ECB的产量提高到3.5±0.2 g/L，在5-L进料批式生物反应器中，提高了2.3倍，代表了迄今为止文献报道的最高滴度。我们的正交双位点整合策略和系统优化方法为丝状真菌复杂天然产物的代谢工程提供了一个有价值的框架。

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引用次数: 0

Genome minimization of a Burkholderia bacterial host 伯克氏菌宿主基因组的最小化

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-17 DOI: 10.1016/j.ymben.2026.01.008

Bruno S. Paulo, Sean B. Romanowski, Adjo E. Kadjo, Vitor B. Lourenzon, Alessandra S. Eustáquio

Genome minimization, including the deletion of endogenous gene clusters that encode natural products, is a common strategy to improve the yield of heterologous products. We have been interested in developing Burkholderia sp. FERM BP-3421 as an alternative bacterial host. Instead of indiscriminately deleting gene clusters, which may have deleterious effects, we guided our efforts using transcriptomics data from production cultures. The genome of FERM BP-3421 is subdivided into two chromosomes and two plasmids. The top transcribed gene clusters were those encoding polyketide-nonribosomal peptide spliceostatins on plasmid p1 and nonribosomal peptide selethramide on chromosome 1. Deletion of the spliceostatin cluster had been shown to improve titers of the ribosomal peptide capistruin, whereas we showed that deletion of the selethramide cluster had no effect on capistruin titers. We next targeted the two endogenous plasmids using a CRISPR-Cas12a strategy, resulting in an 11 % reduction in genome size. The plasmid cured strains showed improved growth and 20–40 % increased production of capistruin depending on whether one or both plasmids were deleted. However, deletion of p2 alone negatively affected the heterologous production of two distinct polyketide-nonribosomal peptides. The p2⁻ strain produced only 5–23 % of the glidobactin A and megapolipeptin A titers compared to the wild type, respectively, whereas titers were restored to wild type levels in the p1⁻ p2⁻ strain. The observation that p2 appears to contain functions that support polyketide-nonribosomal peptide biosynthesis was unexpected and sets the stage for future studies aimed at identifying these functions and further enabling engineering efforts that may be widely applicable to other strains.

基因组最小化，包括删除编码天然产物的内源基因簇，是提高异源产物产量的常用策略。我们一直有兴趣开发伯克霍尔德氏菌sp. FERM BP-3421作为替代细菌宿主。我们使用来自生产培养的转录组学数据来指导我们的工作，而不是不加选择地删除可能产生有害影响的基因簇。FERM BP-3421的基因组被细分为两条染色体和两个质粒。转录最多的基因簇是编码质粒p1上的聚酮-非核糖体肽剪接他汀和1号染色体上的非核糖体肽selethramide的基因簇。剪切抑素簇的删除已被证明可以提高核糖体肽capistruin的滴度，而我们发现selethramide簇的删除对capistruin滴度没有影响。接下来，我们使用CRISPR-Cas12a策略靶向两个内源性质粒，导致基因组大小减少11%。质粒固化菌株的生长得到改善，capistrin的产量增加了20 - 40%，这取决于是否删除了一个或两个质粒。然而，p2的单独缺失会对两种不同的聚酮-非核糖体肽的异种生产产生负面影响。与野生型相比，p2 -菌株分别仅产生5 - 23%的glidobactin A和巨脂肽A滴度，而p1 - p2 -菌株的滴度恢复到野生型水平。观察到p2似乎包含支持聚酮-非核糖体肽生物合成的功能，这是出乎意料的，并为未来的研究奠定了基础，旨在确定这些功能，并进一步使工程努力可能广泛适用于其他菌株。

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引用次数: 0

Investigating overflow metabolism in heterotrophic cultures of the green alga Chromochloris zofingiensis zoofingiensis绿绿藻异养培养中溢流代谢的研究

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-16 DOI: 10.1016/j.ymben.2026.01.007

Michelle Meagher , Dimitrios J. Camacho , Sean D. Gallaher , Sabeeha S. Merchant , Nanette R. Boyle

Chromochloris zofingiensis is of interest for its ability to perform a reversible trophic switch in the presence of glucose that is characterized by a shutdown of photosynthesis and an accumulation of energy storage metabolites. Previous work has shown that this trophic switch is accompanied by overflow metabolism and the production of lactate in aerobic conditions. This trophic switch is not observed in nutrient replete media. We utilized isotopically assisted metabolic flux analysis to characterize intracellular flux distributions that are associated with different metabolic phenotypes observed in this organism in different media formulations in light and dark conditions. The results of this analysis showed that low iron cultures have no flux through carbon fixation reactions, and that the carbon flux entering the TCA cycle in these cultures is approximately 40 % lower than that in iron replete cultures grown heterotrophically. This analysis was complemented with transcriptomics data collected for C. zofingiensis grown in iron limited conditions to provide further evidence towards the negative impact of iron limitation on both photosynthetic and respiratory activity. Overflow metabolism allows this alga to compensate for the lower energy production that results from iron limitation. This work highlights how nutrient availability can lead to changes in the metabolism of C. zofingiensis.

zoofingiensis因其在葡萄糖存在下进行可逆营养开关的能力而受到关注，其特征是光合作用的关闭和能量储存代谢物的积累。先前的研究表明，在有氧条件下，这种营养转换伴随着溢出代谢和乳酸的产生。在营养丰富的培养基中没有观察到这种营养转换。我们利用同位素辅助代谢通量分析来表征与不同代谢表型相关的细胞内通量分布，这些分布在不同的培养基配方中，在光照和黑暗条件下观察到。分析结果表明，低铁培养物没有碳固定反应的通量，并且在这些培养物中进入TCA循环的碳通量比异养培养的富铁培养物低约40%。该分析与在铁限制条件下生长的zofingiensis的转录组学数据相补充，为铁限制对光合和呼吸活性的负面影响提供了进一步的证据。溢出代谢允许这种藻类补偿由于铁限制而导致的较低的能量生产。这项工作强调了营养的可用性如何导致梭菌代谢的变化。

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引用次数: 0

Metabolic engineering of Escherichia coli for the high-level production of putrescine 大肠杆菌代谢工程高产腐胺的研究。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-16 DOI: 10.1016/j.ymben.2026.01.006

Yoo-Sung Ko , Je Woong Kim , Alisher Nazarbekov , Gi Bae Kim , Sang Yup Lee

Putrescine is an important platform chemical used in the manufacture of engineering plastics. To support the development of a sustainable plastics industry, microbial production of putrescine from renewable resources has attracted increasing attention. In this study, we report the development of an engineered Escherichia coli strain capable of efficiently producing putrescine. To overcome the limitation caused by putrescine toxicity, the previously developed XQ52 strain (a W3110-derived putrescine-producing strain) was subjected to adaptive laboratory evolution, resulting in the AXQ52 strain, which produced 61.7 g/L of putrescine in fed-batch fermentation. This titer surpassed the natural tolerance threshold of E. coli. Genome sequencing of the AXQ52 strain revealed mutations that improved cellular fitness under high putrescine concentrations. Further production improvements were achieved by fine-tuning the expression of phosphoenolpyruvate carboxylase gene, introducing a heterologous ornithine acetyltransferase, and disrupting glutamate decarboxylase. The final engineered strain produced 72.7 g/L of putrescine with a yield of 0.25 g/g glucose and a productivity of 1.28 g/L/h, representing the highest microbial putrescine production reported to date from a simple carbon source.

腐胺是制造工程塑料的重要平台化学品。为了支持可持续塑料工业的发展，利用可再生资源微生物生产腐胺已引起越来越多的关注。在这项研究中，我们报道了一种能够有效生产腐胺的工程大肠杆菌菌株的发展。为了克服腐胺毒性的限制，将之前开发的XQ52菌株（w3110衍生的腐胺产生菌株）进行适应性实验室进化，得到了在分批补料发酵中产生61.7 g/L腐胺的AXQ52菌株。该滴度超过了大肠杆菌的自然耐受阈值。AXQ52菌株的基因组测序揭示了在高腐胺浓度下提高细胞适应性的突变。通过微调磷酸烯醇丙酮酸羧化酶基因的表达、引入异源鸟氨酸乙酰转移酶和破坏谷氨酸脱羧酶，进一步提高了产量。最终的工程菌株产生了72.7 g/L的腐胺，产率为0.25 g/g葡萄糖和1.28 g/L/h，代表了迄今为止报道的从简单碳源产生的最高微生物腐胺产量。

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引用次数: 0

Novel routes for bioproduction of delta lactone aroma compounds 生物合成δ内酯芳香化合物的新途径。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-14 DOI: 10.1016/j.ymben.2026.01.005

Sonali Srivastava , Aakash Chandramouli , Payal Gupta , Abdur Rahman Manzer , Rahul Choudhury , D. Srinivasa Reddy , Syed Shams Yazdani , Siddhesh S. Kamat , Debasisa Mohanty , Vinay K. Nandicoori , Rajesh S. Gokhale

Delta lactones are fatty acid-derived aroma compounds that hold tremendous commercial value. As consumer demand for natural flavours continues to rise, the bioproduction of δ-lactones, including δ-decalactone and δ-dodecalactone, is attracting substantial interest. Our study brings forth a novel approach to the bioproduction of δ-lactones from glucose, deviating from existing methods that primarily rely on the biotransformation of fatty acids. The high cost of fatty acid raw material constrains the commercial viability of this traditional approach. We engineered surface-lipid producing type I polyketide synthase (PKS) from Mycobacterium smegmatis by incorporating macrolactone thioesterase (TE) domain. Two out of three fusion constructs produced an appropriately engineered PKS-TE fusion protein that facilitated the synthesis of δ-lactones. When grown on glucose as the sole carbon source, recombinant E. coli expressing the engineered PKS-TE fusion protein successfully made δ-lactones ranging from C8-C18 acyl chains. Our research further highlights the potential of Mycobacterium smegmatis as a cell factory for producing fatty acid-based δ-lactones. By genetically designing and engineering Mycobacterium smegmatis to express PKS-TE fusion protein, we achieved bioproduction of various δ-lactones. Batch fermentation of the engineered E. coli strain fed with 2 % glucose produced 786 mg/L of δ-dodecalactone and 444 mg/L of δ-decalactone at 120 h, underscoring the efficacy of our approach. Thus, this study is the first to demonstrate a methodology for redirecting primary metabolic intermediates towards δ-lactone biosynthesis in engineered bacteria, enabling the use of inexpensive and renewable feedstocks.

内酯是脂肪酸衍生的芳香化合物，具有巨大的商业价值。随着消费者对天然香料的需求不断上升，δ-内酯的生物生产，包括δ-癸内酯和δ-十二内酯，正在吸引大量的兴趣。我们的研究提出了一种从葡萄糖生物生产δ-内酯的新方法，偏离了现有的主要依赖于脂肪酸生物转化的方法。脂肪酸原料的高成本限制了这种传统方法的商业可行性。我们通过整合大内酯硫酯酶（TE）结构域，从耻垢分枝杆菌中设计了产生表面脂质的I型聚酮合成酶（PKS）。三种融合构建中有两种产生了适当的PKS-TE融合蛋白，促进了δ-内酯的合成。当以葡萄糖为唯一碳源生长时，表达工程PKS-TE融合蛋白的重组大肠杆菌成功地产生了C8-C18酰基链的δ-内酯。我们的研究进一步强调了耻垢分枝杆菌作为生产脂肪酸基δ-内酯的细胞工厂的潜力。通过对耻垢分枝杆菌进行基因设计和工程，使其表达PKS-TE融合蛋白，实现了多种δ-内酯的生物生产。以2%葡萄糖为饲料的工程大肠杆菌分批发酵120 h， δ-十二内酯产量为786 mg/L， δ-十二内酯产量为444 mg/L，验证了该方法的有效性。因此，这项研究首次展示了一种方法，可以将初级代谢中间体转向工程细菌中的δ-内酯生物合成，从而使用廉价和可再生的原料。

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引用次数: 0

Precise biosynthesis of β-1,2-glucan from cellulosic materials by in vitro metabolic engineering 体外代谢工程技术在纤维素材料中精确合成β-1,2-葡聚糖。

IF 6.8 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-10 DOI: 10.1016/j.ymben.2026.01.004

Yunjie Li , Yujiao Lu , Pingping Han , Qingqing Guo , Shuo Wang , Zhongyi Jiang , Yi-Heng P. Job Zhang

The biosynthesis of polysaccharides with precisely defined structures, such as tunable degree of polymerization (DP) and low polydispersity index (PDI), remains a significant challenge in microbial cell factories due to their intricate endogenous metabolic networks. In vitro metabolic engineering (ivME) has emerged as a promising alternative, offering simplified pathway design and easy process optimization. In this study, ivME was utilized for the precise synthesis of β-1,2-glucans from β-1,4-linked cellobiose. A four-enzyme system comprising cellobiose phosphorylase, β-1,2-oligoglucan phosphorylase, glucose oxidase, and catalase operated under pH self-neutralized conditions, efficiently producing β-1,2-glucan at a concentration of 31.9 ± 0.4 g/L with a high molar yield (93.3 ± 1.3 %) and a rapid productivity of 4.0 ± 0.1 g/L/h. β-1,2-Glucans with tunable DPs (75–531) and narrow molecular weight distributions (PDI as low as 1.2) were synthesized by adjusting primer concentration, enzyme loadings, and reaction time. The low PDI values of β-1,2-glucans were attributed to the smart pathway design, the careful selection of β-1,2-oligoglucan phosphorylase, and the use of sophorose as the primer. The DP values were mainly influenced by the concentration and type of primers with sophorose outperforming glucose. This strategy of direct glycosidic bond rearrangement from β-1,4 to β-1,2 linkages without coenzymes (e.g., CoA, NAD, ATP, UTP) or external energy input provided a new route for lignocellulosic biomass utilization and significantly enhanced the capabilities of ivME for the production of tailored polysaccharides.

生物合成具有精确定义结构的多糖，如可调聚合度（DP）和低多分散指数（PDI），由于其复杂的内源性代谢网络，在微生物细胞工厂中仍然是一个重大挑战。体外代谢工程（ivME）作为一种很有前途的替代方法，提供了简化的途径设计和易于优化的过程。在本研究中，利用ivME从β-1,4-连接的纤维素二糖中精确合成β-1,2-葡聚糖。由纤维素二糖磷酸化酶、β-1,2-低聚葡聚糖磷酸化酶、葡萄糖氧化酶和过氧化氢酶组成的四酶体系在pH自中和的条件下高效地生产β-1,2-葡聚糖，浓度为31.9±0.4 g/L，摩尔产率高达93.3±1.3%，快速产量为4.0±0.1 g/L/h。通过调整引物浓度、酶载量和反应时间，合成了DPs可调（75-531）、分子量分布窄（PDI低至1.2）的β-1,2-葡聚糖。β-1,2-葡聚糖之所以具有较低的PDI值，主要归功于其巧妙的途径设计，精心选择β-1,2-低聚葡聚糖磷酸化酶，并选用苦参作为引物。DP值主要受引物浓度和类型的影响，其中槐糖优于葡萄糖。这种无需辅酶（如CoA、NAD、ATP、UTP）或外部能量输入，直接将糖苷键从β-1,4重排到β-1,2键的策略为木质纤维素生物质利用提供了一条新途径，并显著提高了ivME生产定制多糖的能力。

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引用次数: 0

Omics analyses decoding mechanisms underlying the self-flocculating phenotype of yeast cells and stress tolerance for robust production. 组学分析了酵母细胞自絮凝表型和抗逆性的解码机制。

IF 8.4 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic engineering

Pub Date : 2026-01-07 DOI: 10.1016/j.ymben.2026.01.003

Xue Zhang,Yang Dai,Xin-Qing Zhao,Chen-Guang Liu,Zhuo Wang,Feng-Wu Bai

A unique self-flocculating yeast strain SPSC01 was developed through protoplast fusion for fuel ethanol production with high product titers. In this study, we conducted comparative multi-omics analyses on SPSC01 to elucidate mechanisms underlying its self-flocculating phenotype and associated stress tolerance, the most desirable merit for robust production in industry. Leveraging two cutting-edge third-generation sequencing technologies, we achieved a gapless high-quality and chromosome-level assembly for the genome of SPSC01 and its parental strains as well. Through comprehensive genome analyses, we identified 25 unique genes that are absent in the parental strains, along with 13 novel genes with unknown functions. The self-flocculation of yeast cells is driven by the copy number of genetic variations and significantly upregulated transcription of FLO genes. Mutations in both cis- and trans-regulatory elements contribute to the constitutive expression of FLO1 and its derivative genes, a prerequisite for developing the self-flocculating phenotype. Notably, we discovered a novel small protein G12 that harbors a zinc finger domain, and its overexpression substantially enhanced ethanol production of engineered yeast strains. Furthermore, alterations in metabolic pathways with ergosterol, glutathione, amino acid, and glycerophospholipid are implicated for developing tolerance to ethanol and major inhibitors acetic acid and furfural that are released during the pretreatment of lignocellulosic biomass. The progress provides strategies for engineering yeast cell factories with robustness through rational designs to produce biofuels and bio-based chemicals with high product titers and productivities, in particular for the biorefinery of lignocellulosic biomass for sustainable socioeconomic development.

通过原生质体融合培养出一株独特的自絮凝酵母菌SPSC01，用于生产高滴度的燃料乙醇。在这项研究中，我们对SPSC01进行了比较多组学分析，以阐明其自絮凝表型和相关抗逆性的机制，这是工业上健壮生产最理想的优点。利用两种先进的第三代测序技术，我们实现了SPSC01及其亲本菌株基因组的无间隙高质量和染色体水平组装。通过全面的基因组分析，我们鉴定出25个亲本株中缺失的独特基因，以及13个功能未知的新基因。酵母细胞的自絮凝是由遗传变异的拷贝数和FLO基因转录的显著上调驱动的。顺式和反式调控元件的突变有助于FLO1及其衍生基因的组成表达，这是发展自絮凝表型的先决条件。值得注意的是，我们发现了一种含有锌指结构域的新型小蛋白G12，它的过表达大大提高了工程酵母菌株的乙醇产量。此外，麦角甾醇、谷胱甘肽、氨基酸和甘油磷脂代谢途径的改变与对乙醇和主要抑制剂醋酸和糠醛的耐受性有关，这些抑制剂在木质纤维素生物质预处理过程中释放。这一进展为工程酵母细胞工厂提供了策略，通过合理的设计来生产具有高产品滴度和生产率的生物燃料和生物基化学品，特别是木质纤维素生物质的生物炼制，以实现可持续的社会经济发展。

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引用次数: 0