Fusarium venenatum is the most widely used filamentous fungus for biomass-based microbial protein production mainly used as meat analogs. However, insufficient iterative genome editing and lack of synthetic biology tools for fine-tuning gene expression limited engineering of F. venenatum for improved traits. Here, replicon engineering-based modular synthetic biology toolkit in F. venenatum was established. Specifically, stable and curable mitochondrial DNA-derived replicons were firstly identified and characterized. The curable replicon MT07 enabled CRISPR/Cas9 genome editing system with efficient plasmid curing, shortening curing time from 22 to 8 days. Based on genome editing system, six neutral genomic integration sites were identified for gene integration expression. In contrast, the stable replicon MT071 enabled stable episomal expression system construction, facilitating the characterization of modular transcriptional and translational regulatory elements, including 21 promoters, 8 terminators, 9 Kozak sequences, and 2A peptide-mediated polycistronic expression modules, yielding 662.9-fold gene expression dynamic range. As a proof of concept, this toolkit was applied to engineering the chassis cell and construct 3-hydroxy-3-methylbutyrate (HMB) biosynthetic pathway, enabling efficient co-production of microbial protein and HMB, with titers reaching 36.2 g/L and 6.2 g/L, respectively. Overall, this work establishes a replicon engineering-driven synthetic biology platform to engineer filamentous fungi for sustainable food biomanufacturing.

Muconic acid is an industrially valuable molecule that can be biologically produced from diverse biogenic and waste-derived feedstocks, including sugars and lignin- and plastic-derived aromatic compounds. However, accumulation of protocatechuate (PCA) has been observed in multiple microbes engineered for muconate production when the PCA decarboxylase, AroY, is used. This raises the question of whether PCA decarboxylation represents a rate-limiting step and how this bottleneck might be alleviated, especially given the toxicity and reactivity of PCA and catechol intermediates. To address this, we performed adaptive laboratory evolution (ALE) on a strain of Pseudomonas putida originally engineered for muconate production from aromatic compounds, but with catBC restored, to select for improved conversion of PCA and, in separate lineages, 4-hydroxybenzoate. Contrary to our expectations, the predominant beneficial mutations localized to the catA1 cassette encoding catechol 1,2-dioxygenase, rather than aroY or its associated cofactor biosynthesis genes. Transcriptomic analysis revealed elevated catA1 expression in evolved isolates from ALE, and introduction of these mutations improved productivity in strains designed for muconate production from both aromatic and sugar substrates. Quantitative proteomics and biochemical assays demonstrated that the mutations also led to increased CatA1 protein abundance and modest enhancements in catalytic efficiency, respectively, with strain phenotypes largely driven by high CatA1 levels and potentially synergistic kinetic improvements. Additional reverse-engineering studies identified variants with modest effects on muconate accumulation, including those with potential to enhance biosynthesis of the prenylated FMN cofactor of AroY. Collectively, these results indicate that CatA, not AroY, is the main rate-controlling step in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining our understanding of pathway limitations and offering new strategies for improving rate and strain resilience in muconate bioproduction.

Regulation of nitrogenase, the enzyme that converts nitrogen gas (N2) into ammonium (NH4[+]), typically involves a conserved set of regulatory proteins across diverse N2-fixing (diazotrophic) bacteria. However, the interactions and relative influence of these regulators can vary between species. Thus, one cannot make assumptions about nitrogenase regulation when working with uncharacterized diazotrophs like Vibrio natriegens, a γ-proteobacterium of growing interest for synthetic biology. Little is known about V. natriegens nitrogenase regulation, which could be used to exploit inexpensive N2 for various applications, including NH4[+] production. Here, we characterized the roles of several annotated V. natriegens nitrogenase regulatory proteins in response to NH4[+] versus N2. Using functional genomics, targeted mutations, and reporter assays, we identified a typical regulatory hierarchy where the two-component system NtrBC governs a nitrogen-scavenging regulon that includes NifA, the transcriptional activator of nitrogenase genes. Unlike other diazotrophic γ-proteobacteria, NifA was sufficient to activate nitrogenase gene expression, as a mutant lacking NtrBC grew normally with N2 after a lag phase. Thus, NtrBC was dispensable, but still important for timely nitrogenase expression. Furthermore, NtrBC was negatively regulated by the nitrogen-responsive PII proteins GlnB and GlnK; disruption of both PII proteins led to NtrBC-dependent nitrogenase overactivity, marked by NH4[+] excretion. Redundant repression of NtrBC by GlnB and GlnK more closely resembles that of non-diazotrophic E. coli than other diazotrophic γ-proteobacteria. Together, our findings provide a framework for V. natriegens nitrogenase regulation that can be leveraged for applications like NH4[+] production.

Malonyl-CoA is a major extender unit in polyketide biosynthesis; however, in Escherichia coli, it is predominantly consumed by fatty acid biosynthesis, limiting its availability for heterologous polyketide production. Here, we engineered a fatty acid biosynthesis-β-oxidation carbon-recycling pathway (FABOX pathway) that redirects fatty acid biosynthetic flux toward free fatty acid formation while reinforcing β-oxidation, thereby recycling carbon through acetyl-CoA to support malonyl-CoA-dependent polyketide biosynthesis. Application of this strategy to three malonyl-CoA-dependent polyketides, 1,3,8-trihydroxyanthraquinone (AQ256), phloroglucinol, and flaviolin, increased AQ256 and phloroglucinol production, whereas no clear improvement was observed for flaviolin. [13]C-malonate tracing was consistent with β-oxidation-associated recycling of malonate-derived carbon into acetyl-CoA and extensive incorporation of malonate-derived carbon into AQ256. Here, we show that, although its effectiveness depends on the product and cultivation conditions, the FABOX pathway provides a mechanistically distinct flux-reconfiguration strategy for enhancing malonyl-CoA-dependent polyketide biosynthesis, complementing conventional precursor-supply-based approaches.

Sustainable economy and social development require a paradigm shift in the modes of production of edible proteins. Komagataella phaffii is a preferred host for food protein expression. However, its methanol-dependent production mode and host improvement with exogenous bioelements pose challenges for the manufacturing and approval of bulk food proteins. In this study, we report an endogenous streamlined promoter-regulator integrated transcriptional (ESPRIT) system for efficient expression on non-methanol substrates in K. phaffii. A strong and simplified PAOX1 variant, PAA13-t13, was obtained through core promoter mining and streamlining of upstream regulatory sequences, with a 57% reduction in promoter length and expression increased to 3.4-fold that of PAOX1 under methanol conditions. Overexpression of the transactivator Mit1 enabled PAA13-t13 to drive high-level expression on glucose, glycerol, and ethanol. Endogenous zinc-finger transactivators were screened from genome, and their activation regions were fused with the DNA-binding domain of Mit1, resulting in 25 synthetic transactivators. Combinatory function of the engineered endogenous promoter and transactivator established the ESPRIT system, which achieved 6.8-fold and 10.1-fold expression levels on glucose that of methanol-inducible PAOX1 on methanol and constitutive PGAP on glucose, respectively. It also allowed efficient production of three food proteins under methanol-free conditions. In the bioreactor, the ESPRIT system produced 5.14 g/L β-lactoglobulin and 1.95 g/L brazzein on glucose, achieving 3.0-fold and 1.9-fold as compared to the commercial methanol-dependent PAOX1 system, respectively. These results established the ESPRIT system as an alternative expression platform for efficient food protein production from non-methanol substrates in K. phaffii.

Chinese hamster ovary (CHO) cells are the dominant host for therapeutic protein production, yet intra- and inter-clonal heterogeneity in manufacturing phenotypes, and the underlying metabolic and secretory circuitry, remain poorly defined at single-cell resolution. Using Secretion EnCoded single-cell sequencing (SEC-seq), we simultaneously measure transcriptomes and secreted IgG in single cells from a parental production cell line and five CHO clones, each varying in cell-specific productivity. Endpoint IgG mRNA abundance and accumulated IgG secretion are only moderately coupled across single cells, indicating that recombinant transcript abundance, sampled at a single time point, alone does not explain intra-clonal secretion heterogeneity. By integrating SEC-seq with single-cell metabolic and secretory task scoring, we find that CHO cells accommodating recombinant protein expression burden have more active translation-associated pathways and suppressed energy-intensive endogenous secreted protein processing. Three high-secreting clones converge on this translation-focused state but differ in their subpopulation composition and energy/redox transcriptomic programs coupled to IgG output: one highly productive clone shows a low-growth, glycolytic, NAD/one-carbon-associated and UPR-activated program; a second shows increased oxidative phosphorylation and fatty-acid β-oxidation, and a third shows more modest central-carbon and mitochondrial lipid-metabolism. Genes such as Aldoa, Ndufab1, Acsl5, and Mthfd2 showed clone-specific correlations with IgG, linking glycolysis, mitochondrial respiration, fatty-acid metabolism, and redox to secretion. Together, these results demonstrate that SEC-seq can resolve IgG-coupled metabolic-secretory signatures within and between CHO clones, providing a framework to identify subpopulation and circuit features to engineer or select for improved recombinant protein production.

While nitrogen fertilizers are widely used in agricultural production, their application incurs significant environmental and energetic costs. In contrast, some crops are less dependent on these fertilizers because they engage in symbioses with rhizobia, nitrogen-fixing bacteria that provide ammonium to the plant in exchange for carbon. However, the carbon cost associated with nitrogen fixation can negatively impact crop yields. Improving the efficiency of this metabolic process could alleviate this impact on crop productivity. Mathematical models can help us quantitatively explore metabolic behavior and identify potential targets for metabolic engineering. In this work, we developed a kinetic model of determinate root nodule metabolism, where this symbiotic exchange of carbon from the plant and nitrogen from the bacteria occurs. We used this model to evaluate how the predicted metabolic behavior differs between inefficient and efficient nodules, and to identify potential engineering targets for improving nitrogen fixation efficiency and rate. We show that the enzymes phosphoenolpyruvate carboxylase and pyruvate kinase have significant influence on the predicted rate and efficiency of nitrogen fixation, especially when their expression is varied in combination with oxidative Pentose Phosphate Pathway enzymes like glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase. The model predicts that pairing a 3-fold decrease in glucose-6-phosphate dehydrogenase activity along with either a 3-fold increase in phosphoenolpyruvate carboxylase activity or decrease in pyruvate kinase activity could increase nitrogen fixation rate by 8.82% while improving nitrogen fixation efficiency by 10.99%.

Bioreactor process conditions have a significant effect on Chinese Hamster Ovary (CHO) cell metabolism. However, there exists very limited literature on incorporating process conditions in mathematical models of CHO cell metabolism. To address this limitation, guided by recently published experimental data, we have curated a compact stoichiometric network, including 19 canonical amino acids, and formulated phenotype-driven kinetic expressions to develop a kinetically constrained dynamic flux balance analysis (dFBA) model. The dFBA model incorporates Critical Process Parameters (CPPs), notably bioreactor pH, basal and feed media nutrient composition, feeding times, and inoculation cell densities to predict metrics of bioreactor performance: cell growth rates, antibody titers, and nutrient and metabolite profiles. The dFBA model was trained on diverse fed-batch data of the CHO VRC01 cell line to regress the kinetic parameters. The model's utility was demonstrated through experimentally validated model predictions of CHO-cell performance in intensified fed-batch cultures, perfusion cultures, and cultures with different media. Experimentally validated predictions of a culture with high initial cell density and increased feed addition (intensified fed-batch culture) showed that mAb titers similar to fed-batch culture can be achieved with shorter culture duration. Similarly, experimentally validated predictions of perfusion bioreactor performance showed that coupling historical fed-batch data with computational tools can be leveraged to predict continuous biomanufacturing performance. We thus demonstrate that the developed mathematical model can simulate culture performance across multiple operating modes and process conditions beyond those used for parameter regression for the CHO VRC01 cell line used in this study.

End-product inhibition represents a major challenge in the microbial synthesis of various value-added chemicals. Cadaverine, a key monomer for polyamide synthesis, exhibits severe cytotoxicity, limiting its high-titer biosynthesis. Here, transcriptomic analysis and genome-wide library screening were integrated to systematically elucidate the cytotoxic mechanisms of cadaverine in Escherichia coli (E. coli) and identify beneficial genes for enhanced tolerance and overproduction. Transcriptomic analysis revealed that high concentrations of cadaverine disrupted cell membrane integrity and impaired oxidative phosphorylation, leading to redox imbalance and reactive oxygen species (ROS) accumulation. Subsequent genome-wide screening further confirmed these toxicity mechanisms and uncovered crucial cellular defense strategies. Functional validation highlighted the important role of NikR, UbiE, and YcbX in enhancing membrane integrity, restoring respiratory function and ROS homeostasis, or scavenging 6-N-hydroxylaminopurine (6-HAP) to prevent DNA damage. Among these, YcbX emerged as the most effective target for improving production. Consequently, we constructed a robust E. coli strain by implementing a dynamic regulation system for YcbX expression under cadaverine-responsive promoters, which significantly enhanced cadaverine biosynthesis to 87.2 g/L (a 46.8% enhancement). This work provides an in-depth understanding of cadaverine toxicity and tolerance, offering valuable targets and strategies for the rational design of high-performance microbial cell factories for diamines.

Multi-copy integration is a core strategy for redirecting metabolic flux toward target compounds. However, its application has been hampered by the absence of methods for systematically identifying native multi-copy genomic loci. To overcome this, we developed a computational procedure for genome-wide identification of such loci. Theoretically, this method is potentially applicable to any genome-sequenced species as it only requires the genomic assembly of the target species as input. Applying the procedure to Kluyveromyces marxianus, we identified four groups of loci (KmCS1-4). Combining these loci-KmCS1-4 and the traditional 26S rDNA-with 14 markers with graded selection strengths, we established a versatile multi-copy integration toolkit comprising 70 plasmids. Each plasmid exhibits a unique integration pattern, collectively forming an integration profile. This profile serves as a manual, enabling users to select appropriate tools tailored to the expression requirements of rate-limiting enzymes in their pathways. Applying representative plasmids exhibiting low-, medium-, and high-copy integration patterns to lycopene biosynthesis modules resulted in lycopene titers of 3.5, 6.8 and 40.5 mg/L, corresponding to 2, 6 and 9 genomic copies, respectively, demonstrating a positive correlation between lycopene titers, genomic copy numbers and integration patterns, which highlights the versatility of the toolkit and its supporting manual. Our study not only provides a broadly applicable methodology for genome-wide identification of multi-copy loci, but also an efficient integration platform for K. marxianus.

CYP11A1-mediated catalysis is the rate-limiting step in the de novo biosynthesis of progesterone in yeast, and its relatively low catalytic efficiency largely results from insufficient electron transfer efficiency. In this study, we addressed this bottleneck by systematically reconstructing the electron transfer system (ETS) of CYP11A1. We found that, beyond its native Class I-type ETS (Adx/AdR), CYP11A1 can also function with diverse Class II-type ETSs (CPRs) across species. Screening identified the combination of CYP11A1 from Sus scrofa (SsCYP11A1) and CPR from Absidia orchidis (AoCPR) as the most effective pair. Furthermore, fusion of the two proteins via a flexible GGGS linker significantly enhanced catalytic efficiency. Subsequently, single-site mutational scanning of SsCYP11A1 using the protein language model ESM-2 identified the F75S variant, which increased progesterone titer by 2.43-fold compared to the wild type. Further analysis integrating residue flexibility prediction and site-directed mutagenesis revealed that the F75S mutation improves overall catalytic efficiency by strengthening interfacial interactions between SsCYP11A1 and AoCPR and stabilizing a catalytically favorable conformation. In addition, deletion of the vacuolar iron transporter CCC1 increased the availability of [2Fe-2S] clusters in the yeast cytosol, thereby enhancing Adx functionality and enabling the coordinated operation of Class I-type and Class II-type ETSs. Ultimately, in a 5 L bioreactor fermentation system, the engineered yeast chassis achieved a progesterone titer of 1594.15 ± 71.36 mg/L, representing the highest reported level of de novo progesterone production in yeast to date.

β4-galactosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs), enhancing complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Despite its therapeutic importance, galactosylation remains the most variable glycosylation motif due to its sensitivity to cell culture conditions. Here, we describe a dual genetic engineering strategy applied to two mAb-producing CHO cell lines, DP12 and VRC01, to simultaneously overcome the cellular machinery and metabolic bottlenecks that limit β4-galactosylation. The first engineering event knocks out COSMC, the chaperone required for core 1 β-1,3-galactosyltransferase 1 activity, to redirect UDP-Gal consumption from O-linked β3-galactosylation towards mAb Fc N-linked β4-galactosylation. The second event overexpresses β-1,4-galactosyltransferase 1 (β4GalT1) to augment cellular galactosylation machinery. Each modification was characterised individually (COSMC- and GalT+) and in combination (C-/GT+) across both cell lines in batch and fed-batch cultures. The combined C-/GT+ strategy consistently achieved greater than 90% mAb Fc β4-galactosylation, irrespective of host cell line or culture mode. Metabolic characterisation confirmed that both engineering events alleviate their respective bottlenecks: COSMC knockout redirects UDP-Gal flux and β4GalT1 overexpression increases N-galactosylation capacity. The C-/GT+ strategy also reduced production of Man5 glycans, which accelerate serum clearance and pose immunogenicity risks. Metabolic profiling suggests that the COSMC knockout may attenuate UTP consumption and contributes to reduced Man5 production. C-/GT+ glycoengineering had no negative impact on mAb titre. Our results establish the C-/GT+ dual glycoengineering strategy as a robust approach for consistently achieving high mAb galactosylation across diverse cell culture conditions, with the additional benefit of reduced Man5 glycans.

L-tryptophan is a high-value aromatic amino acid widely used in the food, feed, and pharmaceutical industries. However, large-scale microbial production is constrained by insufficient precursor supply and limited strain tolerance to high product concentrations. In this study, modular metabolic engineering was first employed to enhance the availability of key precursors, including shikimate, serine, and glutamine, yielding strain TRPJ-13 with a 34.6% increase in L-tryptophan titer. To enhance strain tolerance, an indigo-based high-throughput reporter system was constructed and coupled with genome-scale overexpression library screening, leading to the identification of soxS as a tolerance-conferring target. Mechanistic analysis demonstrated that soxS upregulated lpxC to enhance lipopolysaccharide biosynthesis, thereby reinforcing membrane integrity and improving L-tryptophan tolerance. Combinatorial engineering of soxS and lpxC generated strain TRPJ-23, which increased L-tryptophan tolerance by 74.8% and L-tryptophan titer by 10.3%. Furthermore, YicL was identified as a novel transmembrane protein involved in L-tryptophan transport that effectively promoted L-tryptophan efflux, further increasing the titer by 9.0%. After fermentation optimization, strain TRPJ-28 produced 74.3 g/L L-tryptophan in a 5-L bioreactor, with a yield of 0.26 g/g and a productivity of 1.24 g/L/h. In a 1000-L pilot-scale bioreactor, TRPJ-28 reached a titer, yield, and productivity of 70.4 g/L, 0.25 g/g, and 1.17 g/L/h, respectively. This study provides new engineering insights for developing industrially promising L-tryptophan-producing strains.

Artificial membraneless organelles (MLOs) are emerging as spatial organizers in synthetic biology, yet their applications remain largely confined to post-translational regulation. Here, we engineer synthetic condensates that act as modular hubs for targeted mRNA sequestration, enabling programmable post-transcriptional control in Escherichia coli. By incorporating orthogonal RNA-binding protein-aptamer interaction into the compartment, the designed MLOs can enhance translation through the integration of the translation machinery or repress specific pathways by isolating target transcripts. Tailoring condensate properties allows flexible functional optimization. We demonstrate rescue of bacterial cell division by sequestering a division inhibitor mRNA, and significantly improve the production of lacto-N-tetraose and lacto-N-neotetraose through compartmentalization or suppression of competing metabolic genes. Our work expands the functional repertoire of synthetic MLOs beyond enzyme scaffolding to encompass RNA-centric regulation, offering an adaptable strategy for reprogramming cellular functions in synthetic biology and biomanufacturing.

In this study, we investigated how methionine availability influences the phenotype and metabolism of the methionine-auxotrophic E. coli ΔmetA strain. To identify metabolic and physiological changes, the strain was cultured under two conditions: i) a methionine excess condition, where 1 mM of methionine was added to the medium; and ii) a methionine limited condition, where the medium did not contain any methionine, but instead methionine was fed slowly to maintain methionine concentration below 1 μM. To quantify metabolic fluxes, isotopic tracers [1,2-[13]C]glucose and [1,6-[13]C]glucose were applied and [13]C-metabolic flux analysis ([13]C-MFA) was performed. To obtain acceptable fits of the labeling data the metabolic network model had to be updated. First, secretion reactions for four amino acids, i.e. glutamate, threonine, lysine and glycine, were added, which were found to accumulate in the medium under methionine limitation, both under growth and non-growth conditions. Second, a reaction was added to one-carbon metabolism that explicitly captures the incorporation of labeled methyl carbon derived from glucose into methionine. This cycle has thus far been overlooked in [13]C-MFA models, however, our results suggest that taking the methylation of methionine into consideration is critical for successfully implementation of [13]C-MFA. Under methionine limitation, we observed: 1) reduced oxidative pentose phosphate pathway flux; 2) significant increase in the TCA cycle flux and anaplerotic flux into the TCA cycle; 3) activation of a normally dormant pathway from threonine to glycine; 4) secretion of glutamate, threonine, lysine and glycine; and 5) recycling of 15% of intracellular methionine through the methylation cycle. In contrast, when the ΔmetA strain was grown in the presence of excess methionine, all of the unique features of this strain were masked and the flux phenotype simply reflected wild-type E. coli phenotype with only minor flux changes directly related to the gene knockout itself. We also performed co-culture experiments using ΔmetA and ten different auxotrophic E. coli strains, and cultured the same strains on spent medium from ΔmetA culture. We found that the growth behavior of the co-cultures matched with the growth behavior observed for the auxotrophic strains grown on ΔmetA's spent medium. Taken together, this study presents a robust, high-resolution approach to dissect nutrient-limited metabolism and provides novel insights that can advance our understanding of syntrophic interactions in microbial communities.