Synthetic biology is emerging as a key approach in chemical synthesis, whose efficiency hinges crucially on the direct engineering of metabolic pathways. In this study, we propose a dual-intervention paradigm to reprogram the industrial fungus Fusarium fujikuroi from a default gibberellic acid (GA3) producer into an exclusive factory for the higher-value gibberellin GA4+7, providing a complementary and orthogonal approach to traditional intra-pathway manipulations. First, by introducing Arabidopsis-derived transporters (Npfs and Sweets), we successfully created a thermodynamic sink that actively depletes intracellular GA4/GA7 pools. With the best candidate protein, Sweet1, the parent strain was converted into an exclusive producer of GA4+7 (with GA3 levels undetectable), a conversion driven by the significantly accelerated dissociation rate (Kdis) for GA7. Concurrently, we uncovered a non-canonical, highly specific regulatory mechanism: overexpression of the Sfp-type 4'-phosphopantetheinyl transferase Ppt1 triggered targeted post-transcriptional silencing of up to 99.9 % of P450-3 mRNA, thereby completely silencing GA3 biosynthesis and again yielding an exclusive producer of GA4+7. Synergistic integration of transporter-driven spatial pulling and Ppt1-mediated gene silencing, coupled with fermentation optimization, propelled the final GA4+7 titer to an unprecedented 3.29 g/L (reaching 0.4 g/L for GA4 and 2.89 g/L for GA7, representing 17.39-, 962.33-, and 125.54-fold increases over the parent strain, respectively). 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.

Gibberellic acid (GA3) is a crucial phytohormone involved in plant growth regulation with widespread applications in agriculture and other fields. In this study, GA3 synthesis in an industrial GA3 producer F. fujikuroi strain FF00 was improved with a strategy by reprogramming the regulatory network and overexpressing key genes. Three positive regulators AreA (the nitrogen regulator), Lae1 (the velvet complex component), and Hat1 (the histone modification regulator) were identified by screening regulators affecting GA3 biosynthesis. Mutant FF18-4 with GA3 titer of 2.61g/L in shake-flask fermentation was constructed by co-overexpressing AreA, Lae1, and Hat1 in strain FF00. Comparative transcriptomic analysis revealed that reprogramming of the regulatory network resulted in the down-regulation of two key genes (Ggs2 and Cps/Ks) in the GA3 biosynthetic pathway which reduced the supply of GA3 precursor geranylgeranyl pyrophosphate. Ggs2 and Cps/Ks genes were further co-overexpressed to enhance metabolic flux for GA3 biosynthesis. The GA3 titer of the resulting mutant FF19-5 reached 2.73g/L, which was 49.2% higher than that of strain FF00. Therefore, the GA3 biosynthesis of strain FF00 was significantly improved by metabolic network reprogramming and metabolic balance. The established strategy provided the basis for GA3 over-producer construction and may be helpful for the synthesis of other chemicals with microbial cell factories.

Gene knockdown is a pivotal genetic manipulation technique, particularly when targeting lethal genes or genes involved in product synthesis pathways, where complete gene knockout is not a viable option. This approach is particularly valuable in multinucleate species, such as Fusarium fujikuroi, where generating homogeneous gene knockouts is notoriously difficult. To address these limitations, we first screened a set of repression domains, and then leveraged the optimal candidates to construct a CRISPR/dCas9-mediated knockdown platform for F. fujikuroi. By targeting erg9, which encodes squalene synthase, the first committed enzyme in the mevalonate pathway for ergosterol biosynthesis, we successfully diverted a portion of the metabolic flux from sterol production to gibberellic acid (GA) biosynthesis. This strategy minimizes carbon loss to competing pathways while retaining phenotypically normal growth. Additionally, CRISPR/dCas9-mediated knockdown of the dehydrogenase gene des enhanced GA4 production by 2.62-fold and eliminated the intermediate GA7, generating a GA3+4-producing strain and fine-tuning its metabolic profile. Using our CRISPRi system, we achieved a 70-89 % reduction in erg9 mRNA levels and a 67- 84 % reduction in des mRNA levels. Our findings establish a tailored CRISPRi platform for effective gene repression in F. fujikuroi.

Cytochrome P450 enzymes hold immense potential as biocatalysts for oxidative reactions but often suffer from inefficient electron transfer due to their reliance on transient interactions with redox partners (RPs). To overcome this limitation, we constructed self-sufficient fusion enzymes by fusing the avermectin-oxidizing P450-Ema1-M212A to selected RPs. Linker sequences were primarily optimized experimentally, while computational modeling, including AlphaFold3-predicted multi-protein assemblies and molecular dynamics simulations, was used as an auxiliary tool to assess feasible linker lengths and interpret the structural basis by which optimized linker design enhances electron-transfer efficiency. Based on literature precedent and preliminary structural considerations, a series of fusion constructs with varying linker sequences and lengths was constructed. Molecular dynamics simulations suggested a structural rationale for why a linker length of 9 amino acids exhibited optimal enzymatic activity, as it stabilized a functional conformation, minimized structural fluctuations, and maintained a favorable distance between the heme and Fe₂S₂ clusters for efficient electron transfer. Experimental characterization confirmed that the F4 variant exhibited substantially increased activity over the non-fusion version. Kinetic analysis demonstrated a significantly enhanced kcat/Km ratio, indicating accelerated catalysis without compromising substrate binding. For the synthesis of 4″-oxo-avermectin, a key intermediate of emamectin, the F4 variant achieved 90% conversion within 8 h, far exceeding the performance of the non-fused system. This study establishes an experimentally driven, semi-rational framework for constructing self-sufficient P450 fusion enzymes, highlighting the synergy between computational prediction and protein engineering for developing powerful biocatalysts with high industrial application potential.

Gibberellic acid (GA3) is an important plant growth regulator with rising agricultural demand, yet its industrial production via submerged fermentation using Fusarium fujikuroi remains limited by low space-time yield. In this study, a high-yield mutant was generated through atmospheric and room-temperature plasma (ARTP) and ethyl methanesulfonate (EMS) mutagenesis, coupled with lovastatin-based screening. Process optimization, including early nitrogen supplementation, late-stage pH control, and a two-stage temperature-agitation strategy, further enhanced GA3 synthesis. The mutant (strain 9) achieved a GA3 titer of 2.483 g/L in a 5-L bioreactor, a 33.38

Efficient electron supply and cofactor utilization remain key bottlenecks in cytochrome P450-catalyzed oxidations. Here, we report an electro-assisted system that overcomes these limitations, using the P450 Ema1-catalyzed oxidation of avermectin B1a as a model reaction. Under a constant current of 1 mA, > 96% conversion of 2.5 mM (2.2 g/L) substrate was achieved within 4 h, whereas the non-electrified system reached only 68% conversion after 8 h. The electro-assisted system enhanced catalytic efficiency at low substrate concentrations, while the advantage diminished at higher substrate loadings (≥ 5 mM), likely attributable to H2O2 accumulation, indicating a transition between different kinetic regimes. Prolonged electrical input led to enzyme deactivation, revealing a trade-off between catalytic enhancement and enzyme stability. Notably, transient electrical exposure for 1 h produced a catalytic activity gain that was abolished by catalase, persisted after current removal, and was accompanied by minor spectroscopic changes suggesting localized structural rearrangements rather than global conformational reorganization. Finally, we establish that the NADH/NAD+ ratio, rather than absolute NADH concentration, is the master regulator of catalytic activity, with NAD+ acting as a high-affinity allosteric effector. Our work demonstrates a controllable strategy for enhancing P450-catalyzed reactions by integrating electrical input with cofactor regulation, providing insights for the design of electro-biocatalytic processes.

This study aimed to characterize the G protein-coupled receptor FfGpr1-Gα-AC transduction pathway and its role in regulating gibberellin (GA) metabolism in Fusarium fujikuroi. By constructing Ffgpr1 deletion and constitutively activated FfG2Q204 L mutants, we found that glucose-induced cAMP (cyclic Adenosine MonoPhosphate) synthesis was abolished in Ffgpr1Δ. Bimolecular fluorescence complementation confirmed membrane-localized FfGpr1-FfG2 and FfG2-AC interactions, with intensified fluorescence at septa. Fermentation assays revealed opposing GA3 yields: Ffgpr1Δ produced 21% less GA3 than the wild type, whereas FfG2Q204 L increased yield by 17%. qPCR（quantitative real-time PCR） analysis demonstrated that Ffgpr1Δ upregulated FfCPS/KS, FfP450-2, and FfP450-3 transcription by 6-8-fold while downregulating FfDES by 82%, whereas FfG2Q204 L induced a 6-fold increase in FfCPS/KS mRNA level. Strikingly, FfDES overexpression in Ffgpr1Δ restored GA3 production to wild-type levels but led to GA7 accumulation and suppressed FfP450-3 upregulation, suggesting feedback-regulated metabolic constraints. Glucose-induced cAMP production required FfGpr1. FfGpr1-FfG2 and FfG2-AC interacted on the cell membrane, with enhanced co-localization at the septal region. The FfGpr1-Gα-AC pathway significantly affected GA yield, with complex and noteworthy regulation of GA cluster gene expression.

Using Nakaseomyces glabrata as a model organism, we demonstrate that targeting calcineurin can synergize with caspofungin to induce a quorum sensing (QS) effect mediated by farnesol. This QS effect requires calcineurin deficiency, sub-minimum inhibitory concentration (MIC) levels of caspofungin, and a high-density cell population. Cell growth and biofilm formation were significantly inhibited within a specific range of cell density and sub-MIC caspofungin treatment in the calcineurin mutant. The inhibition of biofilm formation follows the 'paradoxical growth,' showing a concentration-dependent response to caspofungin. We show that high cell density triggers two antagonistic effects: overcoming antibiotic inhibition, which promotes cell propagation, and QS-mediated growth inhibition, which negatively regulates cell proliferation. The QS molecule farnesol was detectable only in the calcineurin mutant, where the transcription of the farnesol synthase Dpp3 was significantly up-regulated, and deletion of DPP3 abolished the QS effect in both spot assay and biofilm formation of the calcineurin mutant. Besides this, we identified a Dpp3-dependent, ergosterol-farnesol metabolism-linked Crz1-independent regulatory mechanism that contributes to the calcineurin-mediated multi-stress resistance. We demonstrate that calcineurin, Dpp3, and caspofungin are all involved in regulating ergosterol metabolism and the transcription of ERG11 and FKS genes, leading to significant changes in membrane and cell wall stress tolerance. The cell wall composition undergoes substantial alterations upon deletion of calcineurin or treatment with caspofungin, while caspofungin also increases the levels of β-glucan and short peptides in the medium, tentatively pointing to the release of QS inducers from the cell wall.

In biosynthesis, while focusing on the productivity of individual compounds, the development of high-efficiency bio-components and universal enabling tools for advancing biosynthesis remains a critical and persistent challenge. Plant-derived Integral Membrane Proteins (IMPs) from two distinct families were heterologously expressed in E. coli, inducing filamentous cell growth, increased membrane permeability, polyploidy, and growth arrest. GFP-tagged IMPs were successfully delivered to the cell membrane. Filamentous cells contained significantly elevated DNA content, and displayed a rough surface morphology, an enlarged periplasmic space, and heightened sensitivity against membrane and cell wall stressors. These findings correspond to significantly altered transcription of genes linked to cell membrane and wall integrity, including those regulating cell division, elongation, DNA replication, and IMP delivery. Notably, the observed cellular toxicity could be modulated by chimeric fusion of the N-terminus and a certain number of hydrophobic transmembrane helices, potentially through α-aggregation-mediated membrane disruption. Finally, we demonstrated that IMP expression enhanced biosynthesis in all six tested scenarios, including biocatalysis, fermentation, and mixed-cell catalysis for the production of diverse chemicals. A plant-IMPs toolkit has been developed for versatile biosynthetic applications in E. coli.

Many years ago, life transitioned from the ocean to land, evolving from halophilic to non-halophilic organisms. Our research indicates that some enzymes from halophiles require salt for soluble expression in E. coli and retain activity within certain salt concentration ranges in the growth medium. The cytoplasmic electrical resistance varies in accordance with the salt concentration in the medium. Further experiments and simulations reveal that the protein structure undergoes dynamic and sophisticated changes under different salt concentrations, affecting soluble expression, surface charge and enzyme activity. This suggests that salt concentrations affect enzyme functionality and potentially influence overall metabolic processes, pointing to a sophisticated adaptive system that operates independently of genetic molecules. Our findings propose insights into a type of environmental cue induced evolution of halophilic microorganisms from the perspective of protein structure. Ultimately, given our extensive marine and other saline resources, our research lays a foundational basis for the development and utilization of halophile-origin enzymes.

Avermectins (AVMs) and their derivatives are the most effective and widely used nematicides, insecticides, and acaricides against endo- and ectoparasites of plants, animals, and humans. Demand for avermectins and their highly effective derivatives has increased due to their high cost-effectiveness and wide range of applications as medicines and crop protection products. Due to the unique structures of these compounds and for industrial production purposes, numerous efforts and strategies have been dedicated to enhancing the production of avermectins and creating new analogues in recent years. Here, we have systemically reviewed the recent studies on the biosynthesis and application of avermectins and their derivatives, including avermectin metabolism and its related bioregulation in Streptomyces avermitilis, approaches for enhancing the bioproduction of avermectins, the structure and toxicology of avermectin derivatives, and future prospects, with a focus on the recent advances in biosynthesis and significance of the superior avermectin derivatives.

AIMS:This study aimed to characterize the G protein-coupled receptor FfGpr1-Gα-AC transduction pathway and its role in regulating gibberellin (GA) metabolism in Fusarium fujikuroi. METHODS AND RESULTS:By constructing Ffgpr1 deletion and constitutively activated FfG2Q204 L mutants, we found that glucose-induced cAMP (cyclic Adenosine MonoPhosphate) synthesis was abolished in Ffgpr1Δ. Bimolecular fluorescence complementation confirmed membrane-localized FfGpr1-FfG2 and FfG2-AC interactions, with intensified fluorescence at septa. Fermentation assays revealed opposing GA3 yields: Ffgpr1Δ produced 21% less GA3 than the wild type, whereas FfG2Q204 L increased yield by 17%. qPCR（quantitative real-time PCR） analysis demonstrated that Ffgpr1Δ upregulated FfCPS/KS, FfP450-2, and FfP450-3 transcription by 6-8-fold while downregulating FfDES by 82%, whereas FfG2Q204 L induced a 6-fold increase in FfCPS/KS mRNA level. Strikingly, FfDES overexpression in Ffgpr1Δ restored GA3 production to wild-type levels but led to GA7 accumulation and suppressed FfP450-3 upregulation, suggesting feedback-regulated metabolic constraints. CONCLUSION:Glucose-induced cAMP production required FfGpr1. FfGpr1-FfG2 and FfG2-AC interacted on the cell membrane, with enhanced co-localization at the septal region. The FfGpr1-Gα-AC pathway significantly affected GA yield, with complex and noteworthy regulation of GA cluster gene expression.

Catalytic activity is undoubtedly a key focus in enzyme engineering. The complicated reaction conditions hinder some enzymes from industrialization even though they have relatively promising activity. This has occurred to some dehydrogenases. Hydroxysteroid dehydrogenases (HSDHs) specifically catalyze the conversion between hydroxyl and keto groups, and hold immense potential in the synthesis of steroid medicines. We underscored the importance of 7α-HSDH activity, and analyzed the overall robustness and underlying mechanisms. Employing a high-throughput screening approach, we comprehensively assessed a mutation library, and obtained a mutant with enhanced enzymatic activity and overall stability/tolerance. The superior mutant (I201M) was identified to harbor improved thermal stability, substrate susceptibility, cofactor affinity, as well as the yield. This mutant displayed a 1.88-fold increase in enzymatic activity, a 1.37-fold improvement in substrate tolerance, and a 1.45-fold increase in thermal stability when compared with the wild type (WT) enzyme. The I201M mutant showed a 2.25-fold increase in the kcat/KM ratio (indicative of a stronger binding affinity for the cofactor). This mutant did not exhibit the highest enzyme activity compared with all the tested mutants, but these improved characteristics contributed synergistically to the highest yield. When a substrate at 100 mM was present, the 24 h yield by I201M reached 89.7%, significantly higher than the 61.2% yield elicited by the WT enzyme. This is the first report revealing enhancement of the catalytic efficiency, cofactor affinity, substrate tolerance, and thermal stability of NAD(H)-dependent 7α-HSDH through a single-point mutation. The mutated enzyme reached the highest enzymatic activity of 7α-HSDH ever reported. High enzymatic activity is undoubtedly crucial for enabling the industrialization of an enzyme. Our findings demonstrated that, when compared with other mutants boasting even higher enzymatic activity, mutants with excellent overall robustness were superior for industrial applications. This principle was exemplified by highly active enzymes such as 7α-HSDH.

Tacrolimus (FK506) is a widely used and clinically important immunosuppressant drug that can be produced by fermentation of Streptomyces tsukubaensis . The industrial strains are typically obtained through multiple rounds of mutagenesis and screening, a labor-intensive process. We have established an efficient yeast cell based screening method for the evolutionary process of high-FK506-yielding strain. The S. tsukubaensis strains of different FK506 yields were tested for zone of growth inhibition of the wild type and calcineurin mutant ( cnb1∆ ) yeast strains. We found that different FK506 yields correspond well to altered yeast growth inhibitions. Based on the combinational inhibition effects of FK506 with different antifungals that have been frequently reported, we also tested the zone of inhibition by addition of fluconazole, amphotericin B and caspofungin to the medium. In the end, for the best screening performance, we systemically evaluated the strategy when different yeast strains and different antifungals were used according to the clarity, size, and divergence of the inhibition circles. Using different yeast strains and antifungals, we successfully broadened the screening spectrum. An efficient high-FK506-yield S. tsukubaensis screening method has been established and optimized.

Fusarium fujikuroi is the microorganism that used for industrial production of gibberellic acids (GAs), a commercially very important plant hormone. A stable, high-yielding F. fujikuroi strain is essential for efficient bio-production. Although, there is a quick development in molecular tools and metabolic engineering research for this microbe in the past decade, the current industrially applying strains are mostly obtained from numerous rounds of mutagenesis and screening, while the industry is still dedicating in strain improvement work based mostly on this method. However, after over half a century's effort, the yield is still maintained at a low level today. We designed an efficient strategy for strain improvement by amphotericin B resistance prescreening and evolutionary engineering. A superior strain was obtained with over 25% increased yield after testing merely 640 isolates. The superior strain was later confirmed to be genetically very stable for GA production. Minimum inhibitory concentration (MIC) assay verified its increased resistance to amphotericin B, whose target ergosterol shares a same precursor with GAs. Decreased ergosterol accumulation indicated the increased GA3 synthesis accompanied with attenuation of metabolic branch flux. In the end, we found the increased GA3 titer of the superior strain also accompanied with faster growing biomass.

With the ban of highly toxic herbicides, such as paraquat and glyphosate, phosphinothricin (PPT) is becoming the most popular broad-spectrum and highly effective herbicide. The current PPT products in the market are usually a racemic mixture with two configurations, the D-type and L-type, of which only the L-PPT has the herbicidal activity. The racemic product is not atom economic, more toxic and may cause soil damage. Asymmetric synthesis of L-PPT has become a research focus in recent years, while biological synthesis methods are preferred for its character of environmental friendly and requiring less reaction steps when being compared to the chemical methods. We have developed a biological synthesis route to produce optically pure L-PPT from D,L-PPT in two steps using 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid as the intermediate. In this study, we expressed the glutamate dehydrogenase and glucose dehydrogenase using Pichia pastoris as the first time. After a series of optimization, the total L-PPT yield reached 84%. The developed synthesis system showed a high potential for future industrial application. Compare to the previous plasmid-carrying-E. coli expression system, the established method may avoid antibiotic usage and provided an alternative way for industrial synthesis of optically pure L-PPT.

传统教育体系下的研究生教育,存在着研究生创新性思维培养不足、学生学习的主动性欠缺等问题,制约着研究生培养质量.新工科背景下亟需改进研究生培养措施,提高研究生培养质量.影响研究生教育创新性思维培养的关键因素包括学生学习自觉性和自主性、专业知识与课题研究的适配度以及导师的作用发挥等.新工科背景下研究生教育创新性思维的培养路径包括树立明确的培养目标,拓展学生思考探索的空间,多途径激发学生的创造性思维等.

Phosphinothricin (PPT) is one of the most prevalently using herbicides. The commercial phosphinothricin products are generally in the form of a racemic mixture, of which only the l-phosphinothricin (L-PPT) gives herbicidal function. Synthesis of optically pure L-PPT by deracemization of D/L-PPT is a promising way to cut down the environmental burden and manufacturing cost. To convert D/L-PPT to L-PPT, we expressed the catalytic enzymes by genomic integration in E. coli. The whole production was implemented in two steps in one pot using four catalytic enzymes, namely d-amino acid oxidase, catalase, glutamate dehydrogenase, and glucose dehydrogenase. Finally, after a series of process optimization, the results showed that with our system the overall L-PPT yield reached 86%. Our study demonstrated a new strategy for L-PPT synthesis, based on enzymes from chromosomal integrated expression, which does not depend on antibiotic selection, and shows a high potential for future industrial application.

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.

Thermostability is considered to be an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostability makes an enzyme more competitive and desirable in industry. However, most natural enzymes show poor thermostability, which restricts their application. Protein structure modification is a desirable method to improve enzyme properties. In recent years, tremendous progress has been achieved in protein thermostability engineering. In this review, we provide a systemic overview on the approaches of protein structure modification for the improvement of enzyme thermostability during the last decade. Structure modification approaches, including the introduction of non-covalent interactions and covalent bonds, increase of proline and/or decrease in glycine, reinforcement of subunit-subunit interactions, introduction of glycosylation sites, truncation and cyclization have been highlighted.