Construction of microbial cell factories often requires extensive reconfiguration of metabolic networks, which frequently compromises cell growth. Here, we engineered E. coli to produce dencichine (β-ODAP), a plant-derived hemostatic agent. Knocking out serB blocks the competing L-serine pathway, causing L-serine auxotrophy and severely impairing cell growth. This growth defect was only partially restored by supplementation with L-serine or L-glycine, suggesting the presence of additional growth-limiting factors. Through adaptive laboratory evolution and reverse engineering, we further uncovered that overexpression of serA to enhance the supply of the precursor L-2,3-diaminopropionate led to accumulation of the toxic byproduct 2-hydroxyglutarate (2-HG) from α-ketoglutarate. To resolve these two growth constraints, we developed a growth-phase-dependent dual-dynamic regulation circuit. This circuit gradually activates serA expression and progressively represses serB expression as cells enter the stationary phase, thereby reducing 2-HG accumulation and alleviating L-serine auxotrophy. Combined with pathway balancing and cofactor optimization, the final engineered strain produced 13.46 g L-1 of β-ODAP with a yield of 0.32 g g-1 in 3-L bioreactors. This study reveals a toxicity mechanism in serine-pathway engineering and provides a dynamic regulation strategy applicable to the biosynthesis of serine-pathway-derived metabolites.
Yu Zhang, Jun Wu, Jinghua Yu et al.· The Innovation Life· 0 citations
Methanol is a promising renewable C1 feedstock for sustainable single‐cell protein (SCP) production. However, its inherent cytotoxicity and metabolic trade‐offs between cell growth and protein synthesis remain significant bottlenecks. Here, we established an “evolutionary‐rational” dual‐driven paradigm to construct a high‐yield Pichia pastoris chassis. Through UV mutagenesis and adaptive laboratory evolution, we developed a highly tolerant strain A40, capable of growing in 70 g/L methanol. Notably, at 30 g/L methanol, A40 achieved a 3.4‐fold higher maximum biomass than the wild‐type. Whole‐genome resequencing and reverse genetics revealed that this superior performance stems from a multi‐gene synergistic network rather than a single dominant mutation. To further optimize SCP production, we rationally co‐overexpressed nitrogen assimilation genes (GLN1, GDH1) and a translation elongation factor (PpeEF3) in the A40 background. This targeted metabolic engineering effectively redirected carbon flux toward protein biosynthesis. The engineered strain A40‐2Ge3 achieved a peak intracellular crude protein content of 67.9% and a 51.3 g/L total titer in a 5‐L bioreactor, representing a 23.1% increase over the wild‐type strain. Collectively, this study provides deep insights into the synergistic mechanisms of methanol adaptation and establishes an efficient, scalable strategy for sustainable SCP production from C1 feedstocks.
Chong Xie, Cheng-Chao Zhu, Jun-Ze Liu et al.· Biotechnology Journal· 0 citations
Developing sustainable single-cell protein (SCP) from non-food feedstocks offers a promising strategy to address the escalating global demand for sustainable nutrition. However, the lack of industrially robust platforms capable of cost-efficient multi-substrate assimilation remains a key bottleneck. Here, we discover a Cyberlindnera jadinii strain, CGMCC34730, which can efficiently utilize diverse non-food carbon sources (e.g., acetate, ethanol, and xylose) for SCP production. Metabolic analysis reveals the assimilation mechanisms for these substrates, notably identifying the reductive glycine pathway as central to formic acid (FA) utilization. Scaled-up production in a 5-L bioreactor demonstrates excellent performance, with ethanol- and acetate-driven cultures reaching protein contents of 68.60 and 67.34% and volumetric productivities of 8.91 and 7.90 t m–3 y–1, respectively. The resulting SCP surpasses soybean meal and approaches the quality of fish meal, exhibiting superior essential amino acid indices alongside elevated carbohydrate and B-vitamin contents. Furthermore, we validate a circular bioeconomy model by converting electrochemically synthesized 13C-labeled acetate into SCP. Techno-economic analysis confirms that acetate-based fermentation offers optimal cost-effectiveness for industrial deployment. In summary, this GRAS-certified platform establishes a highly efficient and economically viable route for the sustainable production of high-value SCP from non-food substrates.
Daphnetin is a clinically established coumarin, but its native biosynthetic pathway remains elusive. In this study, a 2-oxoglutarate-dependent dioxygenase (2OGD), AtS8H, was identified that hydroxylates umbelliferone (UMB) to daphnetin , enabling the design of an artificial biosynthetic pathway. However, the pathway efficiency was constrained by the poor solubility of AtS8H and the low catalytic activity of the upstream 2OGD enzyme IbC2'H. To enhance AtS8H solubility, we developed an integrated strategy combining ProteinMPNN-guided sequence redesign with surface charge engineering. The obtained quintuple mutant S8H 2-6 exhibited significantly improved solubility and a 7.2-fold increase in catalytic efficiency. For IbC2'H, we developed a fluorescence-based high-throughput screening method, and a quadruple mutant C6 was obtained by directed evolution, which displayed a 2.7-fold higher kcat and a 3.2 °C improvement in thermal stability. Implementing both engineered enzymes into an optimized Escherichia coli strain enabled the de novo production of daphnetin at a titer of 46 mg/L. This work reports, to the best of our knowledge, the first microbial de novo production of daphnetin from a simple carbon source and demonstrates an integrated enzyme engineering approach that synergistically refines biosynthetic pathways for efficient microbial production.
Shunmin Ji, Chong Xie, Yanyan Wang et al.· ACS Synthetic Biology· 0 citations
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