Genetic code expansion enables the site-specific installation of noncanonical amino acids (ncAAs) into proteins, but its limited efficiency in eukaryotes remains a major barrier to broader application. Here we establish a visual, plug-and-play screening platform to evolve 18S ribosomal DNA in Saccharomyces cerevisiae and identify ribosomal variants that improve ncAA incorporation. The best-performing strain, designated ribo-hyper, increased ncAA-dependent GFP production by 2.9-fold relative to the wild-type rDNA strain and enhanced incorporation across distinct orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Characterization of ribo-hyper showed that global translation activity and cellular growth were moderately reduced. Proteomic analysis further revealed changes in amino acid biosynthesis, translation-related proteins and stress-response pathways, indicating that the engineered ribosome reshapes cellular translation homeostasis. Perturbation of translation quality-control pathways, including the ribosome-rescue factors Dom34 and Hbs1 and the core mRNA exosome component Ski6, reduced ncAA-containing protein output, whereas disruption of ribosome quality-control factor Rqc2 had little effect. These findings support a role for ribosome rescue and associated mRNA turnover in efficient ncAA incorporation in the ribo-hyper strain. Together, our results establish eukaryotic ribosome engineering as a viable strategy for improving genetic code expansion in yeast.
Nonreducing polyketide synthase Preu6 catalyzes didepside formation by interaction of starter acyl transferase and thioesterase (TE) domains. Here, we show that an aberrant transcript, preu6β, is generated to encode the TE-deficient protein via alternative splicing in A5SS pattern. Upon heterologous expression in Saccharomyces cerevisiae BJ5464-NpgA, coupled with in vitro chemical and enzymatic assays, Preu6β affords compound 9, the first natural orsellinic acid (OA) derivative with a pantetheine (PANT) moiety. We also demonstrate that TE inactivation represents a previously unrecognized mechanism that governs the selective formation of OA derived-PANT products.
Predicting cellular metabolism from molecular data is a central challenge in systems biology, with direct implications for engineering microbial cell factories. Genome-scale metabolic models (GEMs) provide a mechanistic foundation for such predictions, but their accuracy is limited by an inability to account for the finite catalytic capacity of the proteome. Enzyme-constrained GEMs (ecGEMs) address this by incorporating enzyme turnover numbers and proteome allocation constraints. Hybrid neural-mechanistic models like Artificial Metabolic Network (AMN) and Metabolic-Informed Neural Network (MINN) have sought to combine the flexibility of machine learning with the structure of GEMs, yet they rely on conventional, unconstrained metabolic networks, allowing flux predictions to violate enzyme capacity constraints and pushing models toward biologically unrealistic solution spaces. Here, we introduce the Enzyme-Informed Neural Network (EINN), a conceptual framework which relies on ecGEMs to constrain the neural network. We implement two variants of EINN, ecAMN and ecMINN, using Escherichia coli (E. coli) ecGEMs built with the GECKO 3.0 toolbox, and systematically evaluate their performance. Compared to their unconstrained counterparts, ecAMN significantly improves training stability and predictive accuracy by eliminating convergence to poor local minima, while ecMINN reduces overfitting and achieves lower error rates through mechanistic integration of proteomic data as flux bounds. Together, these results show that constraining the neural network's solution space with enzymatic capacity limits is a more effective hybrid modeling foundation than relying on conventional GEMs alone.
The red yeast Rhodotorula toruloides is a robust lipid producer, and its biotechnology applications have been expanded through genetic engineering based on the non-homologous end joining (NHEJ) pathway. As the NHEJ pathway leads to random integration, it remains laborious in phenotypic assessment and challenging for rational strain design. To develop more efficient genetic tools for R. toruloides based on the homologous recombination (HR) pathway, we conducted a systematic evaluation of its DNA repair machinery. Overexpression of HR-related proteins, such as ScRad52, RtRad54 and ScSae2, increased the HR efficiency from less than 0.1% to 3.6%. In addition, knockout of RtKU70 and RtKU80 elevated the HR efficiency to 4.2% and 5.3%, respectively, but reduced resistance to mutagenic stress. Accordingly, we combined the two aforementioned strategies, which yielded an HR efficiency of 5.9%. Although this combined strategy failed to further elevate HR efficiency as expected, it partially alleviated the hypersensitivity to mutagenic stress caused by Ku protein inactivation. Remarkably, when the CRISPR-Cas9 system was used to introduce double-strand breaks (DSBs), HR efficiency was dramatically increased to 16%, and further overexpression of ScRAD52 led to higher HR efficiency of 75-95% across multiple genomic loci. These results will facilitate advanced genetic engineering of R. toruloides for producing value-added compounds and provide a valuable reference for the genetic manipulation of other oleaginous basidiomycetous yeasts.
The inherent conflict between cellular growth and product synthesis, arising from limited resource allocation and metabolic burden, fundamentally constrains the performance of microbial chassis. Traditional strategies that focus solely on flux intensification or knockout of competing pathways often face diminishing returns and may compromise cellular fitness, motivating the exploration of alternative engineering paradigms. Emerging approaches aim to modulate the growth-production trade-off across temporal, network-level, and spatial dimensions, thereby reframing this trade-off from a fixed physiological barrier into a tunable design parameter. This review systematically summarizes four complementary strategies: temporal decoupling via dynamic genetic circuits and phase-segregated cultivation; network-level metabolic resource optimization through redistribution of carbon flux, cofactors, and energy; spatial reorganization using enzyme colocalization, organelle compartmentalization, and modular microbial consortia; and digital and intelligent control frameworks that integrate real-time sensing, artificial intelligence (AI)-based prediction, and digital twin-enabled closed-loop optimization. The key insight is that decoupling does not eliminate competition but rather reschedules, redistributes, or relocalizes it, thereby achieving high production without sacrificing cellular robustness. Collectively, these multi-dimensional strategies transform the growth-production trade-off into a programmable engineering variable. Future efforts should focus on simplifying dynamic circuits, improving long-term genetic stability under industrial conditions, developing tools for non-model hosts, and engineering generally recognized as safe (GRAS) organisms with predictable trade-off management, paving the way for next-generation microbial cell factories that are both productive and resilient.
1,6-Hexanediol (1,6-HDO) is an important chemical platform widely used in polymer and pharmaceutical industries, while its toxicity remains a major bottleneck limiting efficient microbial production. This study demonstrates that 1,6-HDO exhibited strong inhibitory effects on Escherichia coli, reducing the maximum OD600 to approximately 50% of the control level under 10 g/L stress. Transcriptomic analysis was performed under 1,6-HDO stress, identifying 779 differentially expressed genes enriched in pathways related to energy metabolism, sulfur metabolism and amino acid metabolism. To systematically identify functional targets associated with tolerance, genome-wide screening using the ASKA overexpression library identified 93 tolerance-related genes that improved E. coli growth under 1,6-HDO stress by 0.06- to 3.17-fold. Notably, multiple targets were functionally associated with energy and amino acid metabolism, consistent with the transcriptomic analysis. Furthermore, co-expression of rumA and yhbO showed the greatest improvement in growth, reaching a biomass of 9.43 under 1,6-HDO stress, which was 2.61-fold higher than that of the control strain. Collectively, this study systematically elucidates the global stress response and identifies key determinants contributing to tolerance, providing valuable targets for developing robust microbial cell factories for efficient 1,6-HDO bioproduction.
In this work, selective pressure was applied in E. coli to enrich the population expressing recombinant proteins and suppress the emergence of low-expression phenotypes. To this end, plasmids were constructed containing two reporter genes, a degradable green fluorescent protein (GFP) and a stable red fluorescent protein (RFP), positioned upstream of either a gentamicin acetyltransferase gene or a d-serine deaminase gene. Flow cytometry analysis revealed that gentamicin selection prevented the formation of bimodal populations and maintained predominantly homogeneous expression profiles, accompanied by up to 12-fold and 10-fold increases in RFP and GFP fluorescence, respectively. These results suggest that selective pressure favoured the enrichment and maintenance of highly expressing phenotypes while preserving operon functionality. Transcriptomic analysis indicated extensive physiological adaptation under gentamicin selection, including changes in translation-related functions and increased transcript abundance of the genes of interest. To establish an antibiotic-free strategy, d-serine was employed as an alternative selective agent. Detoxification of d-serine by d-serine deaminase similarly reduced population heterogeneity and resulted in predominantly single-expression populations with up to 4-fold and 6-fold higher RFP and GFP fluorescence, respectively. Furthermore, d-serine was evaluated as the sole nitrogen source, combining selective pressure with an auxotrophic strategy and yielding up to 6-fold and 15-fold increases in RFP and GFP fluorescence, respectively. These findings support the use of selective pressure to reduce recombinant expression heterogeneity and suppress low-expression subpopulations. Additionally, we highlighted the potential use of d-serine and similar toxic substrates to simultaneously function as selective agents, inducers, and sources of essential metabolites through detoxification.
Paracoccus denitrificans is a heterotrophic nitrifying-aerobic denitrifying bacterium widely used in wastewater treatment and as a model system for studying electron transport chains, making it a promising chassis for environmental synthetic biology. Precise translational control of gene expression is essential for engineering desired traits in this organism, yet the design of functional ribosome binding sites (RBS) in P. denitrificans remains hindered by limited understanding of their sequence-activity relationships. To address this gap, we systematically profiled 1335 native RBS sequences via integrated transcriptomic and proteomic analyses. We found that RBS strength is predominantly governed by a purine-rich Shine-Dalgarno motif located 5-8 bp upstream of the start codon, with specific A/G patterns in this region serving as key determinants. Building on this dataset, we developed and compared CNN, BiLSTM, and Transformer models for RBS strength prediction; among them, the CNN achieved the highest predictive correlation (Pearson r = 0.68). Additionally, a WGAN-GP framework was implemented to generate novel RBS sequences, which were subsequently filtered and evaluated by the prediction framework to enable the design of RBSs with user-specified strengths. Experimental validation showed a relatively strong correlation between predicted and measured strengths (Pearson r = 0.75). This work establishes the first deep learning-enabled RBS design platform for P. denitrificans, offering a robust tool for precise translational regulation and advancing synthetic biology applications in environmental biotechnology.
Selenoproteins, characterized by the presence of selenocysteine (Sec) residues, are widely distributed across all domains of life. The unique attributes of selenoproteins are conferred by Sec, which is genetically encoded into nascent polypeptide chains through the recoding of the UGA codon. Human glutathione peroxidase 1 (GPx1) is a selenoprotein, which plays a crucial role in maintaining redox homeostasis. However, its complex recoding mechanism presents a major challenge for efficient selenoprotein expression in Escherichia coli. In this study, we engineered a functional amber suppression system that consists of a cysteinyl-tRNA synthetase variant (M38-18) and its cognate tRNACys variant (M1-1). These components were developed through directed evolution of the native E. coli pair. Using this system in E. coli C321.ΔA.exp, we successfully achieved recombinant expression of GPx1 and a GPx1 mutant, GPx1-C-S-49TAG featuring serine substitutions for all cysteine residues, with enzymatic activities of around 420 U/mg and 290 U/mg, respectively. This work provides an alternative platform for selenoprotein production in bacteria and establishes an innovative strategy for the scalable biosynthesis of GPx and related therapeutics.
d-phenylglycine (D-PHG) is a valuable building block extensively used in the synthesis of β-lactam antibiotics and other high-value pharmaceuticals. However, its conventional chemical production relies on expensive starting materials and harsh reaction conditions, while existing biotechnological approaches remain constrained by low production efficiency. In the present work, we established a novel de novo biosynthetic pathway for D-PHG production from glucose in Escherichia coli by reconstructing a streamlined route from the native shikimate pathway intermediate phenylpyruvate. The pathway integrates 4-hydroxymandelate synthase, an FMN-dependent S-mandelate dehydrogenase that avoids H2O2 formation, and a highly specific d-phenylglycine aminotransferase, enabling efficient D-PHG production. Systematic metabolic engineering strategies, including elimination of competing pathways, enhancement of precursor supply, optimization of amino group donor availability, and reinforcement of NADPH regeneration, increased the D-PHG titer to 6.98 g/L in shake-flask cultivation. Further scale-up in a 3-L fed-batch fermentation achieved 9.83 g/L, representing the highest reported titer for de novo D-PHG biosynthesis from glucose to date. This work establishes an efficient and sustainable biosynthetic route to D-PHG and provides a foundation for its large-scale industrial manufacturing.
Recombinant measles virus (rMeV) vectors are promising platforms for vaccine development against emerging infectious diseases due to their safety, stability, and potent immunogenicity. However, conventional rMeV rescue systems frequently exhibit low efficiency, thereby constraining their scalability and throughput. In this study, we developed a modular, helper-virus-free and high-efficiency rescue platform based on an orthogonal transcription system utilizing orthogonal promoters and engineered RNA polymerases fused to an mRNA capping enzyme. This innovative system facilitated robust cytoplasmic manufacture of both genomic and auxiliary components, eliminating the need for helper virus co-infection (such as modified vaccinia virus) and enhancing rescue efficiency by more than 50-fold relative to traditional rescue approaches. Utilizing this technology, we demonstrated the versatility of the platform by successfully generating six rMeV-based vaccine antigen candidates from influenza virus, Pseudomonas aeruginosa, and Brucella spp. All rescued vaccine candidates exhibited stable transgene expression, sustained replication, and strong antigen production. Immunization studies in golden Syrian hamsters verified that the vaccine candidates elicited high titers of neutralizing and antigen-specific antibodies without any observable adverse effects. These results demonstrate that our orthogonal transcription-based platform facilitates the efficient and safe production of rMeV vectors and provides a proof-of-concept methodological framework for the rapid development of vaccine candidates.
Dalbavancin is a potent next-generation lipoglycopeptide antibiotic, but the biosynthesis of its essential precursor, A40926B0, is severely hindered in its native producer Nonomuraea gerenzanensis. Complex competing metabolic pathways and strict transcriptional repression in this strain lead to critical industrial bottlenecks, including extreme product heterogeneity and notoriously low yields. To address these challenges, this study presents a systematic strategy to construct a high-producing strain and optimize the fermentation process for A40926B0. First, random mutagenesis of the strain N. gerenzanensis IPB-9 was performed using a 10 MeV high-energy electron linear accelerator. Screening identified mutant M0318, achieving a 43.6% increase in A40926B0 titer (808.8 mg/L). Concurrently, RNA-Seq analysis of N. gerenzanensis L70 was processed and seven endogenous strong promoters were quantitatively characterized and validated by using an eGFP reporter system and RT-qPCR to expand the genetic toolbox. Applying this toolkit, the strongest identified promoter (orf1597*p) was engineered to overexpress dbv3, the core pathway-specific positive regulator, in the M0318 chassis. This rational intervention further boosted A40926B0 production to 1.03 g/L. Furthermore, a comprehensive bioprocess optimization was executed. Following single-factor evaluations of shake-flask parameters, a Box-Behnken design (BBD)-based response surface methodology (RSM) was employed for systematic medium formulation, successfully elevating the titer to 1.41 g/L. Finally, this optimized process was successfully scaled to a 15 L bioreactor and the final A40926B0 titer reached 2.27 g/L, representing a 303.2% increase over the initial process, laying a foundation for the industrial-scale production of A40926B0.
β-cypermethrin (β-CY) is a widely used pyrethroid insecticide, yet the regulatory mechanisms that determine microbial degradation efficiency remain unclear. Here, we examined the role of the global nutrient-responsive regulator CodY in β-CY degradation by Bacillus cereus GW-01 using a wild-type strain, an in-frame codY deletion mutant, and a complemented strain. Loss of codY shortened the growth lag phase under β-CY stress and accelerated β-CY removal across 50-200 mg/L, reducing the apparent half-life from 3.61 to 12.62 d in the wild type to 2.42-9.61 d in ΔcodY. Complementation largely restored the wild-type phenotype, confirming CodY as a negative regulator of β-CY dissipation. Transcriptomic and physiological analyses showed that ΔcodY reallocated cellular functions toward branched-chain amino acid metabolism, transport, redox adjustment, envelope remodeling, adhesion, and biofilm formation. Consistently, the mutant exhibited higher cell-surface hydrophobicity, stronger auto-aggregation, enhanced biofilm formation, increased superoxide dismutase (SOD) activity, and lower lipid peroxidation. In soil microcosms, ΔcodY also outperformed the wild type in both non-sterilized and sterilized soils, shortening β-CY half-lives by 13.5% and 22.8%, respectively. Community profiling further showed that ΔcodY altered bacterial and fungal succession during remediation, with stronger early selection, later bacterial richness recovery, and a more modular co-occurrence network. These results show that CodY restricts β-CY degradation by constraining both catabolic readiness and surface-associated stress adaptation. Targeting global regulatory nodes may therefore improve microbial remediation of hydrophobic pesticide residues in soil.
Regulatory sequences are commonly characterized using fluorescent reporters, yet how N-terminal coding context shapes these measurements has not been systematically quantified. Here, we evaluated the impact of N-terminal fusion length (45-180 bp) from four genes (lacZ, icd, zwf, bfp) on GFP reporter expression driven by 15 different promoter-RBS combinations in E. coli with normalized fluorescence, enzymatic activity assays and transcription analysis for a representative subset of constructs. Our results demonstrate that N-terminal fusion critically determines the reliability of regulatory-sequence characterization in target-gene-specific coding contexts, with strong gene- and length-dependent effects. Fusions as short as 45 bp failed to rescue context-sensitive cases. However, among the tested fusion lengths, fusions of 90 bp or longer achieved strong correlations (mean R2 > 0.75) between reporter fluorescence and target protein activity. Among the factors examined, N-terminal mRNA secondary structure showed a closer association with these fusion-length-dependent effects than transcription or translation initiation changes. This practical, context-preserving fusion strategy provides cost-effective guidance for scalable and accurate regulatory sequence profiling.
Industrial enzymes are widely used in diverse applications, but low productivity limits their further widespread utilization. This research aimed to develop high-performance alkaline protease (AprE) expression strains of Bacillus licheniformis through element optimization and modular engineering. Firstly, the aprE gene expression cassette was systematically optimized through element engineering. To minimize host background interference, five large gene fragments were deleted from the genome of B. licheniformis DW2. This expression cassette and genome-reduced strain resulted in 5.77-, 4.84- and 1.31-fold increases in the activities of alkaline protease, nattokinase and chitinase, respectively. Crucially, metabolomics analysis then served as the pivotal discovery tool, revealing that high expression of AprE was constrained by insufficient precursor amino acids and excessive metabolic overflow. Subsequently, the amino acid biosynthesis, energy metabolism, overflow metabolism, and cell membrane/wall modules of the strain were successively modified. The final AprE expression host DM6E10 achieved a remarkable enzyme activity of 34,343 U/mL, with a maximum activity of 107,100 U/mL in a 5-L bioreactor. This study built an efficient cell factory for AprE production and provided insights for the optimization of other protein expression hosts.
Intron-mediated enhancement (IME) is a potent endogenous mechanism for boosting gene expression, yet few IME elements function efficiently across distantly related plant species. Here, we characterize the first intron of the rice OsNrx3 gene (OsNrx3i1) as a candidate IME element. OsNrx3i1 significantly enhanced gene expression in rice protoplasts and stable transgenic plants, and also showed detectable activity in the dicot Nicotiana benthamiana in transient assays. The enhancement effect was position-dependent, requiring insertion within the 5' transcribed region, and functionally distinct from typical enhancers. We further identified a repressive upstream open reading frame (uORF) in its native sequence; disruption of this uORF and embedding the intron into an optimized 5' UTR scaffold, unlocked its full potential. Benchmarking against established IME elements (cat-1 intron, AtTub6i1, OsSodCc1i1) revealed that OsNrx3i1 performs competitively in monocots, but all tested introns exhibited context-dependent and host-specific efficacy. When engineered into minimal synthetic promoters, OsNrx3i1 increased their activity by up to 65-fold in rice protoplasts. This work presents OsNrx3i1 as a promising and engineerable genetic module, providing a foundation for its future exploration and application in plant synthetic biology.
An ancient cyanobacterium has evolved through endosymbiosis to form extant chloroplasts of eukaryotic algae and higher plants. During this process, most genes have been transferred to the nuclear genomes. The chloroplast gene sets of different photosynthetic species are relatively conserved yet still exhibiting differences among different species, suggesting a dynamic process and divergence of chloroplast-to-nucleus gene transfer events. Here, based on a comparison of 15 representative green lineage species with sequenced chloroplast genomes, we selected two genes, atpB and rbcL, from a set of 41 conserved genes for chloroplast-to-nucleus gene transfer test. Using the green alga Chlamydomonas reinhardtii as a model organism, we expressed these two genes in the nuclear genome in the corresponding chloroplast mutant background. We demonstrate that transferring atpB and rbcL to the nucleus sustains photoautotrophic growth at different levels. The atpB-TN (transfer-to-nucleus) strains retained photoautotrophic growth to a substantial extent, whereas the rbcL-TN strains were able to grow photoautotrophically only under 5% CO2 and not under ambient air (∼0.04% CO2). The nucleus-encoded proteins accumulated to levels substantially lower than those of the chloroplast-expressed proteins, reaching only 7∼10% (for ATPB) and less than 6% (for RBCL) of the wild-type level. Although both two proteins were relocalized to the chloroplast and assembled into respective ATP synthase and Rubisco complexes, their low abundance appears to account for the incomplete restoration of photosynthetic capability. This study provides a framework for progressively transferring more chloroplast protein-coding genes to the nucleus towards achieving a minimal chloroplast genome in a green algal chassis.
Amyrins arе pеntacyclic tritеrpеnoids with valuablе pharmacеutical and cosmеtic applications; howеvеr, thеir low abundancе in plants and complеx еxtraction procеssеs hindеr sustainablе supply. Hеrе, Yarrowia lipolytica was еnginееrеd to еnhancе α- and β-amyrin biosynthеsis using mеtabolic and еnzymе еnginееring stratеgiеs. First, thе multifunctional amyrin synthasе gеnе (CrMAS) from Catharanthus rosеus was intеgratеd into a superior squalene-producing chassis, еstablishing α-amyrin and β-amyrin production. Squalene epoxidase (ERG1) displayed low native activity and represented a major rate-limiting step in squalene conversion. Structure-guided engineering identified key activity-regulating residues, where T202V improved hydrophobic complementarity and substrate stabilization, and M308L relieved steric constraints at the substrate access tunnel. The T202V/M308L mutant enhanced catalytic efficiency, increasing squalene conversion and reducing its accumulation. Sеcond, thе copy numbеrs of ERG1 and CrMAS wеrе optimizеd, and еnhancеd NADPH supply increased α- and β-amyrin production, while decreasing squalene accumulation. Finally, thе glucosе ratio was optimizеd, and rеstoration of auxotrophic markеrs lеd to improvеd α-amyrin and β-amyrin titеrs of 220.81 ± 6.74 mg/L and 85.41 ± 2.61 mg/L, rеspеctivеly, through fed-batch fermentation in a 5 L fermenter, the titer 1.7 g/L α-amyrin and 0.5 g/L β-amyrin, which is the highest reported to date in Y. lipolytica. This study provides an effective framework for overcoming enzymatic bottlenecks and achieving sustainable microbial production of high-value pentacyclic triterpenoids.
