Protein phase separation for enhanced production of 3-hydroxypropionate and polyhydroxybutyrate by Halomonas.

The oleaginous yeast Yarrowia lipolytica is an outstanding chassis for producing high-value chemicals. Its highly active β-oxidation pathway generates a substantial pool of acetyl-CoA within peroxisomes; however, this abundant reservoir remains largely underutilized. Here, we developed a peroxisomal surface-display platform in Y. lipolytica, enabling enhanced biosynthesis of acetyl-CoA-derived chemicals. We first identified three peroxisomal membrane-targeting signals, derived from Pex3, Pex22, and Pex26 for protein display on the cytosolic face of the peroxisome. By anchoring the carnitine acetyl-CoA transferase Cat2 via these membrane-targeting signals, peroxisomal acetyl-CoA was redirected to the cytosol, increasing acetyl-CoA level and mevalonate production up to 2.4-fold and 2.9-fold, respectively. Further engineering improved squalene titers by 47% to 1.62 g/L. The same platform applied to 3-hydroxypropionic acid biosynthesis improved its production by 22% to 12.85 g/L. Our study establishes a peroxisomal surface display platform for harnessing subcellular acetyl-CoA and provides a versatile toolkit for subcellular engineering in Y. lipolytica.

Vibrio natriegens, a fast-growing bacterium, has emerged as a promising next-generation microbial platform for microbiology and biological engineering. While an expanding toolkit of genetic parts and genome engineering methods has been established, strategies for precise and predictable control of gene expression remain limited. Here, we report a dCas9-based tunable CRISPR interference (CRISPRi) system that enables multilevel transcriptional regulation in V. natriegens. By engineering the tetraloop and flanking regions of single-guide RNA (sgRNA), we constructed a synthetic sgRNA library that modulates the binding affinity between sgRNA and dCas9. The resulting sgRNA variants exhibited modular repression behavior across multiple protospacer targets. We further demonstrated the utility of this tunable CRISPRi system in metabolic engineering applications by redirecting intracellular carbon flux. Tunable repression of endogenous genes led to a 2.2-fold increase in 3-hydroxypropionic acid (3-HP) production and a 1.5-fold increase in lycopene production. Collectively, this work provides a simple and effective strategy for tunable gene regulation in V. natriegens and expands its potential as a versatile platform for the production of value-added chemicals.

Efficient microbial production of platform chemicals requires precise regulation of metabolic fluxes. However, static control strategies─including constitutive overexpression and multi-inducer systems requiring manual optimization─cannot adapt in real time to changing intracellular states and therefore hinder fine-tuning of cofactor and precursor pools. Here, we constructed a product-responsive regulatory circuit in Escherichia coli that coordinates two key biosynthetic determinants─NADPH regeneration and malonyl-CoA allocation─in response to intracellular 3-hydroxypropionic acid (3-HP) accumulation. Following initial IPTG induction of the 3-HP biosynthesis module, intracellular 3-HP accumulation activates the downstream product-responsive regulatory circuit, enabling coordinated modulation of NADPH regeneration and malonyl-CoA allocation without additional external intervention. The final engineered strain, SBH40, achieved a 3-HP titer of 3.0 g/L and a yield of 0.19 g/g glucose, representing an approximately 5-fold improvement in both metrics relative to a nonregulated control strain (0.64 g/L; 0.04 g/g glucose). These results demonstrate that a single-input product-responsive circuit can autonomously coordinate multiple metabolic nodes after pathway initiation, offering a practical and modular framework for dynamic regulation of cofactor- and precursor-intensive biosynthesis.

The Ogura-type male sterile cytoplasm is commonly used in Brassicaceae plants, but its sterility mechanism still requires further investigation. The flower buds of Ogura CMS line ZJCMS1A and its maintainer line ZJCMS1B based on their length < 2mm, 2mm-3mm, 3mm-4mm, grouped A2, A3, A4 and B2, B3, B4, respectively. Anthers were excised from flower buds of each group for transcriptome and metabolome sequencing. Differential comparison analysis, Venn analysis, Multi-omics Integration KEGG enrichment analysis, and pathway network analysis were performed on the data from different groups. The results showed that the abnormal propanoate metabolic pathway in ZJCMS1A was closely related to pollen abortion. The content of 3-hydroxypropionic acid (3-HP) in the propanoate metabolic pathway was higher in ZJCMS1A than in ZJCMS1B in the three comparisons A2 vs B2, A3 vs B3, and A4 vs B4. Furthermore, the content of 3-HP in ZJCMS2A flower buds was also higher than in ZJCMS2B. Quantification of 3-HP content revealed that the content in the stems, leaves, and flower buds of ZJCMS1A was higher than that in the corresponding tissues of ZJCMS1B. The 3-HP content in the flower buds of ZJCMS1A was 8.1% higher than that in ZJCMS1B. Treatment of rapeseed plants with 20mM and 50mM 3-HP showed that starch accumulation in pollen grains was affected. These results indicated that pollen abortion in Ogura-type CMS is closely related to abnormalities in the propanoate metabolic pathway, particularly the abnormal accumulation of 3-HP, which may participate in the pollen abortion process.

Enhancing 3-hydroxypropionic acid production in Komagataella phaffii via methanol and formate co-feeding.

3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

The selective electrooxidation of biomass-derived 1,3-propanediol (1,3-PDO) to high-value 3-hydroxypropionic acid (3-HP) offers a sustainable route for chemical synthesis but is severely hindered by competitive C–C bond cleavage and sluggish reaction kinetics. Here, we propose an electronic structure tuning strategy through interfacial engineering, constructing a Pd–Ni(OH)2 heterostructure to enhance the catalytic performance of Pd nanoparticles. The resulting catalyst exhibits exceptional activity toward the 1,3-PDO oxidation reaction (POR) in alkaline media, requiring a low potential of only 0.645 V (vs. RHE) to achieve a current density of 100 mA cm−2 while retaining a high faradaic efficiency of ∼94.9%. Comprehensive characterization and density functional theory (DFT) calculations reveal that strong interfacial interaction induces a downshift in the d-band center of Pd. This electronic restructuring optimizes the adsorption energetics of key intermediates, facilitating rapid desorption of 3-HP from active Pd sites and thereby suppressing overoxidation and carbon backbone degradation. A membrane electrode assembly (MEA) electrolyzer towards the POR demonstrates robust stability, retaining faradaic efficiencies above 90% during continuous operation. Furthermore, we demonstrate an integrated synthesis–energy device by coupling the POR workflow with the hydrogen evolution reaction (HER) and oxygen reduction reaction (ORR). This hybrid system, powered by the multifunctional Pd–Ni(OH)2 catalyst, enables flexible temporal deployment via a mode-switching scheme between daytime (HER‖POR) and nighttime (ORR‖POR). Collectively, this work elucidates an effective strategy for selective biomass valorization through electronic structure tailoring, offering an appealing multidimensional solution for sustainable electrochemical synthesis.

Selective oxidation of 3-hydroxypropionic acid to malonic acid over Pd/C: Mechanistic and kinetic study

PurposePreoperative anti-vascular endothelial growth factor (VEGF) therapy in proliferative diabetic retinopathy (PDR) may exacerbate fibrovascular contraction, posing significant challenges. This study aims to characterize proteo-metabolomic changes induced by anti-VEGF therapy, validate key candidates on orthogonal analytical platforms, and provide functional evidence for the lead molecule.MethodsAqueous humor from 25 patients with PDR with simple vitreous hemorrhage (VH; n = 13) or VH with tractional retinal detachment (VH + TRD; n = 12) underwent data-independent acquisition (DIA) proteomics and widely targeted metabolomics before and 7 days after intravitreal aflibercept. Molecules altered in both cross-sectional and longitudinal comparisons were classified as “aggravation-type” or “improvement-type.” Aggravation-type molecules were validated by parallel reaction monitoring/selected reaction monitoring (PRM/SRM) in an independent cohort (cataract controls, n = 10; simple VH, n = 5; VH + TRD, n = 5), and GALNS was functionally assessed by siRNA knockdown in fibroblasts.ResultsIntersection analysis identified 38 molecules (5 proteins and 33 metabolites): 35 improvement-type and 3 aggravation-type (CAST, GALNS, and 3-hydroxypropanoic acid [3-HPA]). Independent validation using PRM proteomics and SRM metabolomics confirmed that only GALNS was robustly validated across both cross-sectional (P = 0.016) and longitudinal (P = 0.049) comparisons, whereas CAST and 3-HPA did not reach statistical significance in validation. GALNS exhibited a biphasic pattern—elevated in PDR relative to cataract controls yet depleted in VH + TRD and further declined post-treatment. GALNS knockdown in fibroblasts upregulated α-SMA and collagen I, accelerated migration, and enhanced contractility.ConclusionsThis multi-omics study, reinforced by independent validation, reveals GALNS depletion as a potential pro-fibrotic molecule following anti-VEGF therapy in PDR, offering a promising candidate for perioperative risk stratification. CAST and 3-HPA remain exploratory candidates requiring validation in larger cohorts.

Microbial cell factories are vital for the sustainable production of chemicals, fuels, and pharmaceuticals, yet their performance depends on the precise control of gene expression to balance metabolic flux between cell growth and production. While the regulation of metabolic networks through constitutive gene expression can promote high-level production, it also imposes a metabolic burden that adversely affects cellular growth. Here, we developed a versatile and high-efficiency inducible platform for the methylotrophic yeast Pichia pastoris, which enables dynamic gene expression for 3-hydroxypropionic acid (3-HP) production from sole methanol. We first constructed an anhydrotetracycline (aTc) inducible promoter library of diverse strengths modified from the constitutive promoter PGCW14. The promoter library consisted of 32 inducible promoters with the activated strengths ranging from 2.5% to 84.7% of PGCW14. Second, we dynamically regulated key genes in the 3-HP biosynthesis pathway by aTc inducible promoters, including malonyl-CoA reductase gene (MCR) from Chloroflexus aurantiacus with strong promoters RM5 and RM6, endogenous malonyl-CoA carboxylase gene ACC1 with the very strong promoter RM1, and transmembrane permease PpMFS with the medium promoter RM12. The final engineered strain SHP18 produced 3.7 g/L 3-HP in a shake flask by using methanol as the sole carbon source. This modular aTc inducible promoter toolkit provided finely graded transcriptional control in P. pastoris, which offers a broadly applicable strategy for cell factory design.

The bio-based platform chemical 3-hydroxypropionic acid (3-HP) has attracted considerable attention as a precursor for the production of various value-added chemicals, motivating extensive efforts to biosynthesize it using engineered microbial cell factories. Among the known pathways, the cascade of glycerol dehydratase (GDHt) and α-ketoglutaric semialdehyde dehydrogenase (KGSADH) offers high theoretical yield and strong industrial potential. However, the implementation of this pathway as a freely diffusing two-enzyme system faces two major limitations. The reactive intermediate 3-hydroxypropionaldehyde (3-HPA) is cytotoxic, and diffusion of this intermediate into the surrounding bulk phase leads to intermediate loss and reduced catalytic coupling between the two enzymes. Here, we report a cell-free biosynthetic platform for 3-HP production from glycerol featuring a designer two-enzyme complex composed of GDHt and KGSADH. Each enzyme was rationally fused to complementary Cys-engineered α-helical domains that enable selective heterodimer formation, bringing the two enzymes into close proximity and minimizing diffusion of the labile intermediate into the bulk solution. Under optimized conditions, the self-organized enzyme cascade achieved more than a 17-fold increase in 3-HP yield and a twofold increase in productivity relative to the free enzyme mixture. These findings demonstrate how precise molecular engineering of enzyme organization can substantially enhance the efficiency of cascade biocatalysis and provide a promising strategy for cell-free production of 3-HP.

Engineering aspartate metabolism improves β-alanine-based 3-hydroxypropionic acid production in Saccharomyces cerevisiae.

Xylose metabolic engineering of Issatchenkia orientalis for 3-hydroxypropionic acid production from cellulosic hydrolysate without nutrient supplementation.

A bacterial-derived quorum-sensing platform enables dynamic metabolic control in yeast.

Microbial biochemical production can suffer reduced growth and productivity from intracellular product accumulation, which can be mitigated by minimizing product import. Limited understanding of import-related genes, especially for non-native products, has hindered this approach in strain development. We developed a workflow to identify genes involved in 3-hydroxypropionic acid (3-HP) import. We constructed a genome-wide overexpression library coupled with a 3-HP-responsive fluorescent biosensor and used flow cytometry to isolate narQ-overexpressing strains with a 3.0-fold higher fluorescence signal. Transcriptome profiling under NarQ overexpression revealed a distinct set of membrane-associated genes (acrD, mliC, and pgaABCD) that were transcriptionally upregulated, and functional tests confirmed that their overexpression enhanced 3-HP import while their deletion in producing strains increased 3-HP titers by up to 21% compared with the control strain. This study provides a systematic workflow for identifying import-related genes directly from genomic DNA, advancing the development of more efficient microbial production platforms.

Cardiac ischemia induces substantial metabolomic reprogramming, which dysregulates cardiomyocytes (CMs) and non-myocyte stromal cell populations. The stromal cells derived from epicardial adipose tissue (EAT) and ventricle are critical for extracellular matrix (ECM) remodeling, paracrine signaling, and myocardial homeostasis. However, the metabolomic content and responses of EAT-derived stromal cells (EATDS) and ventricular stromal cells (VSCs) remain unknown. This study employed untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics to characterize ischemia-driven metabolic reprogramming in EATDS and VSCs harvested from swine hearts. Ischemia was simulated using the standard ischemic buffer (pH 6.2) for 2h. Metabolomic screening revealed 65 and 68 metabolites, respectively, for EATDS and VSCs. Results revealed extensive downregulation of amino acid biosynthesis, redox pathways, and mitochondrial metabolism, alongside selective upregulation of glycolytic and cofactor-associated metabolites. Pathway enrichment analyses indicated significant suppression of the TCA cycle, one-carbon metabolism, glutathione cycling, and branched-chain amino acid degradation, reflecting impaired bioenergetic and antioxidant capacity. Adaptive responses included the enrichment of glycolysis, β-alanine, and glyoxylate/dicarboxylate metabolism, consistent with metabolic plasticity under hypoxic conditions. Network-based analyses linked these metabolic shifts to inflammatory pathways. Functional assays demonstrated that sarcosine, pyroglutamic acid, and 3-hydroxypropionic acid modulate the gene expression of cardiac regenerative biomarkers, including GATA4, Nkx2.5, TROP-I, LGALS1, TBX5, and IRX4. These findings suggest that ischemia-induced metabolomic changes exert transcriptional control over cardiac remodeling programs, emphasizing the regulatory potential of metabolite-gene interactions. Such an integrated metabolomic transcriptional response highlights novel therapeutic targets for modulating cellular resilience and heart regeneration following ischemic heart disease.

The oxaloacetate (OAA) pathway represents a promising biosynthetic route to produce 3-hydroxypropionic acid (3-HP), comprising two steps: the decarboxylation of OAA to malonic semialdehyde, followed by its reduction to 3-HP. A thiamine diphosphate (ThDP)-dependent α-keto acid decarboxylase was identified as a potential bottleneck in this pathway due to its low catalytic efficiency toward the non-natural substrate OAA. In this study, rational protein engineering is employed to enhance the catalytic efficiency of KdcA. By rearranging the interaction network within the enzyme's binding pocket, variants S286R and S286K are developed, exhibiting 4.6-fold and 6.2-fold increases in activity, respectively, compared to wild-type KdcA (WT). Further reduction of the binding pocket volume leads to the creation of enhanced variants S286K/V461I/M538Y and S286K/F381W/V461I/M538Y, which display significantly lower Km values (6.6 and 6.0 mM, respectively) relative to those of WT (Km > 20 mM), along with up to about 120-fold increases in catalytic efficiency (kcat/Km). When the variant S286K/V461I/M538Y is integrated into Escherichia coli (E. coli), 3-HP production reaches 1.6 ± 0.2 mM in shake flask cultures. This study demonstrates the effectiveness of protein engineering in overcoming enzymatic bottlenecks to improve biochemical production.

Bioconversion of green macroalgae Chaetomorpha antennina for microbial growth and production of 3-hydroxypropionic acid from glycerol using engineered Escherichia coli.

3-Hydroxypropionic acid recovery from fermentation broth through novel downstream processing: Technoeconomic analysis.