This study demonstrates a successful paradigm for engineering a newly isolated strain toward robust, high-titer and cost-competitive PHA production across lab-to-industry scales.
Efficient biosynthesis of 1,3-PDO, a key bio-based chemical, depends on precise regulation of the host metabolic network. In this study, a heterologous CRISPR-Cas12a genome editing system was established and systematically optimized in Klebsiella pneumoniae, enabling efficient and stable genome editing (75-100% efficiency). Using on this platform, by-product pathways were reduced through multi-gene deletions (frdA, poxB, adhE, ldhA, glpK, ptsG and dhaM) to enhance the yield of 1,3-PDO from glycerol. By heterologously expressing GPD1/GPP2 and optimizing promoter, a metabolic network for the "glucose-glycerol-1,3-PDO" pathways was reconstructed. Cometabolism studies indicated that low concentrations of xylose and arabinose as co-substrates enhanced conversion efficiency of glycerol, whereas glucose alleviated metabolic competition. Under cometabolism of glucose and glycerol, the engineered strain K. pneumoniae S2 ΔABEAKGM-1-2 produced 1003.7 mmol/L (76.4 g/L) of 1,3-PDO with a yield of 0.83 mol/mol glycerol, an overall molar yield of 0.78 mol/mol based on total substrate consumption, and a productivity of 27.9 mmol/L/h. When lignocellulosic hydrolysate was used as co-substrate, 981.1 mmol/L (74.7 g/L) of 1,3-PDO was produced with a yield of 0.77 mol/mol glycerol and a productivity of 27.3 mmol/L/h. This study achieved efficient redirection of carbon flux toward 1,3-PDO through systematic metabolic engineering, providing valuable strain resources and technical guidance for the sustainable and cost-effective biomanufacturing of bio-based 1,3-PDO.
Li Wang, Ming-Yang Zhao, Yuan-Ming Ye et al.· Bioresource Technology· 0 citations
The synthesis of 3S,3′S-astaxanthin was successfully and effectively applied in shrimp farming for color enhancement and antioxidant effects and will pave the way for astaxanthin industrial production.
The heterologous production of terpene in microbial hosts is often limited by inefficient and unstable pathway expression, creating a major bottleneck for industrial-scale synthesis. While E. coli as a chassis offers significant advantages, such as rapid growth, ease of cultivation, and genetic tractability. Its endogenous supply of terpenoid precursors remains a critical constraint, fundamentally restricting high-yield production. To address this challenge, we developed a genomically integrated Mevalonate (MVA) pathway from Actinomycetota in E. coli BL21(DE3) to enhance terpene precursor supply. Our approach began with an in silico multi-layer global genome mining analysis of 25,261 Actinomycetota genomes to identify a series of MVA pathway enzymes with potentially high catalytic efficiency, created a high-efficiency chassis E. coli MVA platform (ecMVA-1 and ecMVA-2) for terpene precursor synthesis. Its functionality was validated by testing eight distinct TSs. Among them, the fermentation of artemisinin precursor amorphadiene using a 5-liter bioreactor yielded 947.80 mg/L. These results indicated that E. coli (MVA) is well-suited for TS studies in the laboratory as well as holding significant promise for industrial applications. In addition, this in silico approach offers a new perspective for metabolic engineering and provides potential reservoir of diverse chassis for the industrial production of terpenoid-derived compounds.
Wenchao Liu, Xueying Tian, W. Wong et al.· Metabolic Engineering· 0 citations
Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (> 4.5kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (> 16kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (> 15kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.
This work demonstrates that the high-yield production of recombinant human therapeutic glycoproteins bearing homogeneous, humanized N-glycans in engineered yeast offers promising prospects.
Zhenzhen Cheng, Keyu Gong, Yuchao Song et al.· Bioresource Technology· 0 citations
Branched-chain amino acids (BCAAs), comprising L-valine, L-leucine, and L-isoleucine, are essential amino acids with extensive applications in food, feed, pharmaceuticals, and cosmetics. Escherichia coli and Corynebacterium glutamicum, the two predominant industrial workhorses have been extensively engineered for high-level BCAAs biosynthesis. This review presents a comprehensive analysis of recent advances in microbial BCAAs production through a Mechanism-Module-Process framework. From the mechanism dimension, the intricate BCAA biosynthetic architectures are delineated, encompassing allosteric feedback inhibition, transcriptional attenuation, and transport system. From the module dimension, modular metabolic engineering strategies are dissected, including precursor supply enhancement, NADPH cofactor rebalancing, biosensor-driven dynamic regulation, and adaptive laboratory evolution for strain robustness. From the process dimension, how oxygen availability reprograms cellular metabolism across aerobic, microaerobic, and anaerobic fermentation is systematically examined, and how two-stage fermentation coupled with cofactor rebalancing resolves the growth-production trade-off to achieve near-theoretical yields is discussed. Furthermore, the biosynthetic strategies and current production status of BCAAs derivatives are summarized, highlighting the importance of multidimensional framework engineering. Collectively, this Mechanism-Module-Process framework provides a holistic roadmap for constructing superior BCAAs cell factories and extending their metabolic potential toward derivative biosynthesis.
Guihong Zhao, Si-Yu Tian, Jiong-Ran Li et al.· Biotechnology Advances· 0 citations
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