Zhi-Qiang Liu

Combinatorial Metabolic and Process Engineering for Enhanced GA4+7 Production and Tunable GA4/GA7 Ratio in Fusarium fujikuroi.

Gibberellins (GAs) are ubiquitous phytohormones that regulate plant growth and are widely used in agriculture. Among these, GA3 and GA4+7 are the only commercially available products, yet GA4+7 commands a much higher price than GA3, primarily due to its low titer in the industrial fungus Fusarium fujikuroi. To engineer a high-yielding GA4+7 producer, we first deleted p450-3 to block the conversion of GA4 and GA7 to GA1 and GA3. This led to the accumulation of GA4+7 at 0.934 g/L, a 37-fold increase over the wild-type strain, albeit with over half reduction in total GA accumulation. Using this Δp450-3 mutant as a platform, we combined metabolic engineering (overexpressing rate-limiting enzymes) with process optimization (pH, medium composition and fermentation duration). These combinatorial interventions synergistically boosted GA4+7 production. Under optimized conditions, the engineered strain achieved a final titer of 6.08 g/L (a 6.51-fold increase over the Δp450-3 parent), comprising 2.13 g/L GA4 and 3.96 g/L GA7, representing 5.30- and 7.44-fold increases, respectively. Preliminary optimization in a 10 L fermenter yielded 3.51 g/L of GA4+7. Finally, a solid-state fermentation system was developed on wheat bran, yielding 17.34 g GA4+7 per kg and enabling green, in-house, in-situ gibberellin production. In addition to substantially increasing the GA4+7 titer and total GA accumulation, this study demonstrated that the GA4/GA7 ratio can be modulated through both molecular and fermentation strategies.

Reconstruction and metabolic regulation of O-succinyl-L-homoserine biosynthesis pathway in Escherichia coli W3110.

O-Succinyl-L-homoserine (OSH) plays a pivotal role in L-methionine biosynthesis. Microbial cell factories for high-yield OSH production have been progressively optimized, achieving substantial improvements in fermentation titers. In this study, a multi-step progressive optimization strategy was adopted to construct a high-yield OSH-producing strain. First, the feedback inhibition of the key enzyme HST was relieved, and the key genes involved in the byproduct metabolic pathways were knocked out. This modification enabled engineered strain to produce 9.77 ± 0.27g/L OSH in shake-flask fermentation. Second, ribosome binding site (RBS) engineering, promoter engineering, and dynamic metabolic regulation were integrated to strengthen and balance the intracellular supply of the two core precursors, L-homoserine and succinyl-CoA. These strategies greatly increased the OSH titer to 18.54 ± 0.03g/L. Finally, global optimization of cofactor and energy optimization was carried out to further enhance strain performance, and the engineered strain OSHM40 achieved the OSH titer of 20.15 ± 0.21g/L via shake-flask cultivation, and 104.09 ± 2.06g/L in a 5-L bioreactor under fed-batch fermentation, with a sugar-acid conversion rate of 64.99% and a volumetric productivity of 1.43g/L/h. Notably, the OSH titer and sugar-acid conversion rate of this strain represent the highest levels reported to date among all plasmid-free OSH-producing strains. The plasmid-free system constructed in this study effectively avoids the plasmid-induced metabolic burden and genetic instability. This work demonstrates the prominent advantages and great application potential of plasmid-free modular engineering for the efficient biosynthesis of OSH and other high-value amino acids.

Enhancing d-Pantothenic Acid Production in Corynebacterium glutamicum via Integrating Dynamic Regulation With Structure-Guided Protein Engineering of Ketopantoate Reductase.

This study establishes a robust chassis for sustainable DPA production and provides a generalizable framework for engineering other valuable biochemicals.

Enhanced Adenosine Biosynthesis in Paecilomyces hepiali Through Strain Evolution and Transcriptomic Insights.

Adenosine is a major bioactive nucleoside and quality marker in cordyceps-derived fungal products, but its efficient biosynthesis in Paecilomyces hepiali remains limited by insufficient strain performance and incomplete understanding of metabolic regulation. Here, we developed an integrated strategy combining protoplast-based strain evolution, medium optimization, and transcriptomic analysis to enhance adenosine biosynthesis in P. hepiali. Efficient protoplast preparation was achieved using 48 h seed cultures digested with 1% driselase and 1% yatalase at 28°C for 7 h, and 40 s ultraviolet irradiation was selected for mutant library construction. A stable mutant, P. hepiali A3, produced 65.32 mg/L adenosine, representing a 31.62% increase over the parental strain. Subsequent response surface optimization identified maltose, peptone, and aspartic acid as key nutritional factors, increasing the adenosine titer to 170.41 mg/L in shake flasks and 191.36 mg/L in a 5-L bioreactor. Comparative transcriptomic analysis revealed extensive metabolic remodeling involving central carbon metabolism, ribose precursor supply, purine nucleotide metabolism, and sterol biosynthesis. Upregulation of ribose-5-phosphate isomerase RPIB and downregulation of ADA related to purine degradation were associated with enhanced purine nucleoside accumulation. These results provide transcriptomic insights into adenosine biosynthesis and establish a practical framework for improving fungal nucleoside cell factories.

Transaminases engineering and their cascade systems for natural product synthesis.

Beyond direct pathway engineering: reprogramming Fusarium fujikuroi from a GA3 producer into a GA4+7 factory.

This study achieves indirect metabolic reprogramming not via conventional pathway engineering, but by targeting a membrane transport bottleneck and employing a putative post-transcriptional silencing mechanism.

Active Pocket Engineering of d-Tagatose 4-Epimerase for Improved Catalytic Performance and Efficient Cascade Synthesis of d-Tagatose from d-Glucose.

d-Tagatose is a rare hexose sugar with excellent properties, and its synthesis catalyzed by d-tagatose 4-epimerase (T4E) represents a competitive novel pathway. In this study, EbT4E derived from the Eubacteriales bacterium was screened and systematically characterized. By reshaping the microenvironment of the active pocket, mutant M3(S131D/H410W/T279S) was constructed, which showed a 3.89-fold higher conversion rate compared with the wild-type (WT) enzyme. Kinetic parameter analysis and molecular dynamics (MD) simulations revealed that M3 had enhanced substrate affinity, hydrogen bond network, charge properties, and channel accessibility. Finally, the conversion rates of d-fructose to d-tagatose catalyzed by the purified M3 enzyme and M3 whole-cell catalysts reached 29.46% and 26.2%, respectively. Additionally, the dual-enzyme cascade reaction of M3 with glucose isomerase (GI) TEGI-M-L38M-V137L was constructed, achieving a 13.16% yield of d-tagatose from d-glucose. This study demonstrates that EbT4E-M3 is a promising biocatalyst for d-tagatose production, laying the foundation for its subsequent industrial application.