Nucleoside analogues such as vidarabine (Ara-A) are vital therapeutics but are often limited by poor pharmacokinetics. Its phosphorylated form, vidarabine 5′-monophosphate (Ara-AMP), offers improved solubility and is used as a clinical alternative, although it can be dephosphorylated to Ara-A in vivo. Here, we report a modular multienzyme cascade for the sustainable synthesis of Ara-AMP directly from adenosine. The cascade integrates three functional modules: a core module (PNP, PPM, and RpiA) for the conversion of adenosine to Ara-A, a driver module (dNKδ20) for the selective phosphorylation of Ara-A to Ara-AMP, and a support module (PPK) for ATP regeneration from low-cost ADP and polyphosphate. The use of dNKδ20, a mutant deoxyribonucleoside kinase with high selectivity for Ara-A over adenosine, is critical for minimizing the formation of AMP byproduct, which would otherwise complicate downstream processing. Under optimized conditions, the cascade achieved an 80.2% conversion of 1 mM adenosine to Ara-AMP in 4 h using 0.05 mM ADP. The modular design was also applicable to guanosine and inosine, affording the corresponding arabinosyl nucleotides, albeit with lower yields. This work establishes a sustainable biocatalytic platform for nucleoside analogues, exemplifying how enzyme promiscuity, thermodynamic driving, and cofactor recycling enable green pharmaceutical manufacturing.
Komagataella phaffii is a prominent methylotrophic yeast widely used in industrial biotechnology for heterologous protein production, chemical synthesis, and C1 feedstock utilization. However, its industrial application is limited by thermal stress and methanol toxicity, while the relationship between heat adaptation and methanol tolerance remains poorly understood. Here, we obtained a thermotolerant mutant ATH through ARTP mutagenesis followed by adaptive laboratory evolution (ALE) under heat stress (37 °C). ATH tolerated heat and methanol robustly under individual stress conditions, achieving 3-fold and 8-fold higher cell densities than GS115 at 37 °C or in 5% (v/v) methanol, respectively, yet failed under the combined stress of 37 °C and 5% methanol. Using three proteins (GFP, OPH, and ChiA) as reporters, we found that ATH showed higher heterologous expression than GS115 at 33 °C. Physiologically, ATH displayed enhanced cell wall/membrane integrity, reduced ROS accumulation and lower lipid peroxidation (>30% reduction in MDA), and accelerated formaldehyde clearance. Transcriptomics identified 561 shared differentially expressed genes, with coordinated upregulation of heat shock proteins (HSP70/40/90 families; Hsp104 and Hsp70-SSA2), antioxidant enzymes (catalase, peroxiredoxin), and cell wall/membrane remodeling genes, alongside downregulation of amino acid and secondary metabolite biosynthesis. Whole-genome resequencing further distinguished ATH from GS115 by only 19 variants (9 SNPs and 10 InDels), highlighting a compact genetic basis for the evolved phenotype. This work provides a K. phaffii chassis with improved individual heat and methanol tolerance and elucidates the physiological, transcriptional, and genomic mechanism underlying heat–methanol cross-adaptation.
Predictable and tuneable gene expression is essential for advanced mammalian cell engineering yet remains challenged by genomic position effects and copy number variability. This study systematically compares random integration (RI) and targeted integration (TI) of two synthetic lineariser gene circuits, TetR/eGFP_Lin and PhlFd/mCherry_Lin, in CHO-S cells. Both circuits exhibited strong linearity and orthogonality across integration strategies, but integration method and repressor choice produced distinct performance trade-offs. RI increased achievable dynamic range for PhlFd but increased basal leakiness for TetR, whereas TI, via a dual-landing-pad system, constrained copy number, and reduced basal expression and PhlFd expression variability. These findings highlight integration context and circuit architecture as key design parameters informing rational design of synthetic regulators for biopharmaceutical applications.
Komagataella phaffii is a prominent methylotrophic yeast widely used in industrial biotechnology for heterologous protein production, chemical synthesis, and C1 feedstock utilization. However, its industrial application is limited by thermal stress and methanol toxicity, while the relationship between heat adaptation and methanol tolerance remains poorly understood. Here, we obtained a thermotolerant mutant ATH through ARTP mutagenesis followed by adaptive laboratory evolution (ALE) under heat stress (37 °C). ATH tolerated heat and methanol robustly under individual stress conditions, achieving 3-fold and 8-fold higher cell densities than GS115 at 37 °C or in 5% (v/v) methanol, respectively, yet failed under the combined stress of 37 °C and 5% methanol. Using three proteins (GFP, OPH, and ChiA) as reporters, we found that ATH showed higher heterologous expression than GS115 at 33 °C. Physiologically, ATH displayed enhanced cell wall/membrane integrity, reduced ROS accumulation and lower lipid peroxidation (>30% reduction in MDA), and accelerated formaldehyde clearance. Transcriptomics identified 561 shared differentially expressed genes, with coordinated upregulation of heat shock proteins (HSP70/40/90 families; Hsp104 and Hsp70-SSA2), antioxidant enzymes (catalase, peroxiredoxin), and cell wall/membrane remodeling genes, alongside downregulation of amino acid and secondary metabolite biosynthesis. Whole-genome resequencing further distinguished ATH from GS115 by only 19 variants (9 SNPs and 10 InDels), highlighting a compact genetic basis for the evolved phenotype. This work provides a K. phaffii chassis with improved individual heat and methanol tolerance and elucidates the physiological, transcriptional, and genomic mechanism underlying heat–methanol cross-adaptation.
Filamentous fungi harbor numerous silent biosynthetic gene clusters (BGCs), representing an underexplored reservoir of natural products. Chemical inhibition of epigenetic regulators is an effective strategy for activating silent BGCs, but it often lacks specificity and cannot readily link activated clusters to their cognate regulators. Here, we developed a coupled cytosine base editing and auxotrophic selection platform in Aspergillus nidulans to systematically identify epigenetic regulators of silent gene clusters. An sgRNA library targeting 24 core epigenetic regulators was introduced, together with a cytosine base editor, into 21 pyrG-based PKS reporter strains, enabling growth-based selection and direct sgRNA recovery by Sanger sequencing. This screen identified five regulator-cluster activation pairs, including known interactions and two previously uncharacterized mechanisms. Specifically, KdmB represses the AN7084 gene cluster by erasing promoter H3K4me3, whereas Kmt5 silences the cichorine core pks gene through H4K20me3 deposition. This scalable platform enables rapid matching of silent BGCs with their epigenetic regulators and advances chromatin-guided fungal genome mining.
