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[前沿资讯 ] 天津工业生物所在谷氨酸棒杆菌全基因组规模筛选工业生产相关功能元件方面取得新突破 进入全文
中科院天津工业生物技术研究所
近日，中国科学院天津工业生物技术研究所实现了谷氨酸棒杆菌全基因组规模单基因过表达库的自动化构建与高通量筛选，鉴定了多个影响渗透压耐受性和氨基酸产量的新型功能元件。该研究利用天津工生所生物铸造厂，构建了覆盖谷氨酸棒杆菌全基因组99.7%的单基因过表达库，并建立了资源共享网站。同时，该研究通过全自动、实时生长曲线测定建立了新型功能元件的高通量筛选方法；通过全文库筛选获得了15个可提高渗透压耐受和L-赖氨酸产量的新元件，如新型转录调控因子和DNA修复蛋白，并揭示了渗透压耐受新机制。进一步，研究对近400个膜转运蛋白子库进行筛选，获得了在谷氨酸棒杆菌和大肠杆菌中高效、特异的新型L-苏氨酸外排蛋白。研究将新型外排蛋白应用于L-苏氨酸生产菌株改造，实现了目前最高水平的谷氨酸棒杆菌L-苏氨酸生产。
[学术文献 ] Metabolic engineering of Corynebacterium glutamicum: Unlocking its potential as a key cell factory platform for organic acid production 进入全文
Biotechnology Advances
Corynebacterium glutamicum, a well-studied industrial model microorganism, has garnered widespread attention due to its ability for producing amino acids with a long history. In recent years, research efforts have been increasingly focused on exploring its potential for producing various organic acids beyond amino acids. Organic acids, which are characterized by their acidic functional groups, have diverse applications across industries such as food, agriculture, pharmaceuticals, and biobased materials. Leveraging advancements in metabolic engineering and synthetic biology, the metabolic pathways of C. glutamicum have been broadened to facilitate the production of numerous high-value organic acids. This review summarizes the recent progress in metabolic engineering for the production of both amino acids and other organic acids by C. glutamicum. Notably, these acids include, amino acids (lysine, isoleucine, and phenylalanine), TCA cycle-derived organic acids (succinic acid, alpha-ketoglutaric acid), aromatic organic acids (protocatechuate, 4-amino-3-hydroxybenzoic acid, anthranilate, and para-coumaric acid), and other organic acids (itaconic acid and cis, cis-muconic acid).
[学术文献 ] Metabolic engineering of Corynebacterium glutamicum for the production of pyrone and pyridine dicarboxylic acids 进入全文
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Environmental concerns from plastic waste are driving interest in alternative monomers from bio-based sources. Pseudoaromatic dicarboxylic acids are promising alternatives with chemical structures similar to widely used petroleum-based aromatic dicarboxylic acids. However, their use in polyester synthesis has been limited due to production challenges. Here, we report the fermentative production of five pseudoaromatic dicarboxylic acids, including 2-pyrone-4,6-dicarboxylic acid (PDC) and pyridine dicarboxylic acids (PDCAs: 2,3-, 2,4-, 2,5-, and 2,6-PDCA), from glucose using five engineered Corynebacterium glutamicum strains. A platform C. glutamicum chassis strain was constructed by modulating the expression of nine genes involved in the synthesis and degradation pathways of precursor protocatechuate (PCA) and the glucose-uptake system. Comparative transcriptome analysis of the engineered strain against wild-type C. glutamicum identified iolE (NCgl0160) as a target for PDC production. Optimized fed-batch fermentation conditions enabled the final engineered strain to produce 76.17 ± 1.24 g/L of PDC. Using this platform strain, we constructed 2,3-, 2,4-, and 2,5-PDCA-producing strains by modulating the expression of key enzymes. Additionally, we demonstrated a previously uncharacterized pathway for 2,3-PDCA biosynthesis. The engineered strains produced 2.79 ± 0.005 g/L of 2,3-PDCA, 494.26 ± 2.61 mg/L of 2,4-PDCA, and 1.42 ± 0.02 g/L of 2,5-PDCA through fed-batch fermentation. To complete the portfolio, we introduced the 2,6-PDCA biosynthetic pathway to an L-aspartate pathway–enhanced C. glutamicum strain, producing 15.01 ± 0.03 g/L of 2,6-PDCA in fed-batch fermentation. The metabolic engineering strategies developed here will be useful for the production of pseudoaromatic chemicals.
[学术文献 ] Adaptive laboratory evolution recruits the promiscuity of succinate semialdehyde dehydrogenase to repair different metabolic deficiencies 进入全文
NATURE COMMUNICATIONS
Promiscuous enzymes often serve as the starting point for the evolution of novel functions. Yet, the extent to which the promiscuity of an individual enzyme can be harnessed several times independently for different purposes during evolution is poorly reported. Here, we present a case study illustrating how NAD(P)+-dependent succinate semialdehyde dehydrogenase of Escherichia coli (Sad) is independently recruited through various evolutionary mechanisms for distinct metabolic demands, in particular vitamin biosynthesis and central carbon metabolism. Using adaptive laboratory evolution (ALE), we show that Sad can substitute for the roles of erythrose 4-phosphate dehydrogenase in pyridoxal 5’-phosphate (PLP) biosynthesis and glyceraldehyde 3-phosphate dehydrogenase in glycolysis. To recruit Sad for PLP biosynthesis and glycolysis, ALE employs various mechanisms, including active site mutation, copy number amplification, and (de)regulation of gene expression. Our study traces down these different evolutionary trajectories, reports on the surprising active site plasticity of Sad, identifies regulatory links in amino acid metabolism, and highlights the potential of an ordinary enzyme as innovation reservoir for evolution.
[学术文献 ] Machine learning-assisted amidase-catalytic enantioselectivity prediction and rational design of variants for improving enantioselectivity 进入全文
Nature Communications
Biocatalysis is an attractive approach for the synthesis of chiral pharmaceuticals and fine chemicals, but assessing and/or improving the enantioselectivity of biocatalyst towards target substrates is often time and resource intensive. Although machine learning has been used to reveal the underlying relationship between protein sequences and biocatalytic enantioselectivity, the establishment of substrate fitness space is usually disregarded by chemists and is still a challenge. Using 240 datasets collected in our previous works, we adopt chemistry and geometry descriptors and build random forest classification models for predicting the enantioselectivity of amidase towards new substrates. We further propose a heuristic strategy based on these models, by which the rational protein engineering can be efficiently performed to synthesize chiral compounds with higher ee values, and the optimized variant results in a 53-fold higher E-value comparing to the wild-type amidase. This data-driven methodology is expected to broaden the application of machine learning in biocatalysis research.
[前沿资讯 ] 中科院天津工业生物所等通过开发从头合成途径提高NMN产量 进入全文
中国科学院天津工业生物技术研究所
中国科学院天津工业生物技术研究所毕昌昊研究员带领的合成生物技术研究团队和张学礼研究员带领的微生物代谢工程团队合作，通过系统工程化改造大肠杆菌，成功开发了大肠杆菌中NMN的从头合成途径，有效提高了NMN的体内代谢产量。首先，研究人员通过基因编辑技术敲除了pncC和nadR基因，实现较原始菌株超过100倍的NMN产量提升；随后，研究人员对NMN 从头合成途径进行了优化，并将其与 NadV 介导的 NMN 生物合成途径整合，并引入两种转运蛋白增强了NAM的吸收和NMN的外排，将 NMN 产量提升至约1300 μM；最后，通过优化改造PRPP合成酶，进一步提升NMN产量，在摇瓶水平发酵24小时后超过 3000μM。该工作为NAD+补救途径及其在大肠杆菌能量代谢中的作用提供了新的见解，同时也有助于进一步推动合成生物学在生物制药和健康产业应用中的发展。
