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.
Abstract
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.
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.
Jing-Wen Jia, Zheng-Bin Zhang, Ming-Han Li et al.· Biotechnology and Bioenginee...· 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.
Metabolic bioengineering has emerged as a transformative approach for reshaping plant defense by targeting intrinsic biosynthetic pathways to enhance immunity in modern agriculture. Moving beyond proof-of-concept metabolomics to broad-spectrum programmable pathway engineering addresses gaps in plant rational design and optimizes resilience in response to diverse environmental cues. This review aims to comprehensively highlight the transition of innovative approaches to phenolics, alkaloids, flavonoids, terpenoids, and benzoxazinoids, inferring adaptive reprogramming that mediates the growth-defense balance and functions as molecular sentinels in plants. Furthermore, decoding the volatile metabolome reveals a dynamic signaling interface that influences defense responses and stress-induced plant-microbe interactions, with the shikimate, jasmonate, and salicylate pathways functioning as central hubs for microbial deterrence and priming immune memory. Recent developments in multi-scalar genome-editing strategies, including CRISPR-driven combinatorial edits, enzyme orthogonalization, fluxomics, and spatially resolved multi-omics, reconfigure central and specialized metabolic fluxes toward improved defense function and regulation. Additionally, emerging tools, such as WUSCHEL2 and BABY BOOM transcriptional modules, and artificial engineering strategies integrating deep learning model-driven predictions facilitate rapid development of synthetic genetic circuits and support a predictive engineering of plants. Moreover, Mass spectrometry imaging (MSI) in spatial metabolomics enables to obtain structures and locations of unidentified endogenous metabolites within cells and tissues. Overall, this review emphasizes a diverse array of primary and secondary metabolites, spanning molecular concepts to recent advances in plant immune mechanisms. It also illustrates new frontiers in programmable metabolic engineering that accelerate the understanding of plant-microbe-metabolite cross-talks, offering strategies to improve plant resistance and advance sustainable agricultural solutions.
P. Raghuraman, Seonjoo Park· Frontiers in Plant Science· 0 citations
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
Xylitol is a highly functional sweetener with extensive applications. Sustainable biosynthesis from glucose is desirable yet metabolically challenging. Here, we engineered Yarrowia lipolytica as a cell factory by constructing a core biosynthetic route via combinatorial screening and multicopy integration of d-arabitol dehydrogenases (ArDH) and an NADPH-dependent xylitol dehydrogenase (XDH) in the robust chassis NBRC1631. To further drive the metabolic flux and alleviate bottlenecks, we employed a synergistic push-and-pull strategy: overexpressing glucose transporters (YH3 and YH4), while upregulating pentose phosphate pathway enzymes (ZWF1 and GND1) to enhance NADPH regeneration, matching the redox demand of the synthetic cascade. Following two-stage pH-controlled fed-batch fermentation in a 3 L bioreactor, the final engineered strain achieved a record-high xylitol titer of 39.0 g/L with a yield of 0.09 g/g glucose. This study establishes a productive platform for microbial de novo xylitol biosynthesis from glucose, offering a green and economically viable route for industrial production.
Bingbing Liu, Xi Yao, Jianping Lin et al.· Journal of Agricultural and...· 0 citations
Yarrowia lipolytica is a promising industrial host, yet its metabolic engineering potential remains limited by insufficient genetic tools. Here, we engineered a synthetic Transcription Activation Toolkit (TAT) based on LacI–VPRH, a chimeric protein fusing the prokaryotic DNA-binding domain LacI with the eukaryotic activation domain VPRH. Systematic optimization of LacO copy numbers and core promoter composition achieved up to 205-fold gene activation. The TAT platform was further expanded to construct bidirectional expression systems and enable multiplexed gene control. Applied to resveratrol biosynthesis via a “push-pull” strategy, CRISPR/Cas9-mediated integration of TAT-controlled synthetic promoters upregulated the shikimate pathway genes aroM10 and aroC alongside the rate-limiting enzyme ST1, achieving a shake-flask titer of 2.715 g/L─the highest reported to date. Additionally, an IPTG-inducible “turn-on” system (TAT-2.0) incorporating the antiLacI9 mutant was developed for small-molecule-responsive transcriptional control. Collectively, the modular TAT system provides a versatile strategy for precise metabolic pathway optimization in Y. lipolytica.
Yunhe Li, Wen-Ping Wei, Ping Zhang et al.· Journal of Agricultural and...· 0 citations
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