The sustainable microbial production of high-value terpenoids like longifolene represents a critical frontier in green manufacturing, yet it is often hindered by systemic metabolic inefficiencies in microbial hosts. To address this, we present a comprehensive systems metabolic engineering framework in Saccharomyces cerevisiae that integrates three complementary modules to overcome the key bottlenecks of enzyme catalysis, cofactor supply, and precursor availability. First, we enhanced the catalytic module by employing the RII dimerization/docking domain-RI anchoring disruptor (RIDD-RIAD) peptide-mediated enzyme assembly and structure-guided Pinus sylvestris longifolene synthase (psTPS) variant screening, ‘which improved whole-cell longifolene production under the tested conditions. Second, to resolve nicotinamide adenine dinucleotide phosphate (NADPH) cofactor imbalance, we engineered a dual-regeneration system by introducing an exogenous formate dehydrogenase (FDH) and simultaneously amplifying endogenous pentose phosphate pathway (PPP) flux. Third, we reprogrammed central carbon metabolism by integrating the non-oxidative glycolysis (NOG) pathway and deleting the transcriptional repressors DOS2 and YNR063W, and was associated with a larger steady-state cytosolic acetyl-CoA pool, the essential terpenoid precursor. This multi-pronged strategy culminated in a highly optimized strain that achieved a titer of 92.84 mg/L in shake-flask cultures. Critically, the process was successfully scaled to a 5 L bioreactor under fed-batch conditions, yielding 4181.51 mg/L total longifolene at 204 h (3484.79 mg/L extracellular and 696.72 mg/L cell-associated), the highest total titer among the microbial reports identified in Supplementary Table S4. Differences in product recovery and cultivation protocols limit direct process comparisons. Our work demonstrates that coordinated optimization of enzyme assembly, cofactor regeneration, and carbon-flow reprogramming can substantially improve longifolene biosynthesis in S. cerevisiae. This study provides a longifolene-focused case study for modular pathway optimization, while broader application of this strategy to other terpenoids or natural products will require further pathway-specific validation.
Engineering of nonribosomal peptide synthetases (NRPSs) is frequently limited by loss of biosynthetic productivity following domain recombination, particularly within the initiation module, where interdomain compatibility plays a critical yet underexplored role. Although substrate selectivity can often be reprogrammed by replacing the starter condensation (Cs) domain, such modifications are typically accompanied by severe reductions in productivity, imposing a major bottleneck in NRPS engineering. To produce long-chain surfactin homologs, we replaced the native Cs domain of the surfactin synthetase SrfAA with a homologous Cs domain from a long-chain lipopeptide-producing NRPS. This enabled incorporation of long-chain fatty acyl substrates but resulted in a substantial decrease in production titer. Systematic analyses suggested that this productivity loss is primarily associated with suboptimal interdomain compatibility within the engineered initiation module rather than intrinsic deficiencies of the substituted Cs domain. By combining targeted mutagenesis of the Cs domain with interdomain engineering and fine-tuning of the carrier (T1) domain, we achieved recovery of production through combinatorial optimization. Notably, improved productivity was largely achieved without altering acyl-chain specificity, supporting the view that catalytic efficiency can be enhanced independently of substrate selection. These findings suggest that productive reprogramming of NRPS initiation modules requires coordinated optimization of both domain-specific properties and interdomain compatibility.
Synthetic consortia of Escherichia coli and Saccharomyces cerevisiae offer a powerful division-of-labor strategy for chemical biosynthesis. However, maintaining the stability of cross-species populations remains a major challenge. Here, we report an interspecific autonomous population-control platform for dynamically regulating Escherichia coli and Saccharomyces cerevisiae consortia. We found that the growth rate disparity between these two microorganisms leads to the suppression of yeast growth ultimately resulting in population imbalance and low biosynthesis. To overcome it, an E. coli auto-induction lysis circuit and a S. cerevisiae-driven cross-species lysis circuit were developed using salicylate-mediated intercellular signaling pathway to dynamic control the growth of E. coli in the consortia. These systems successfully increased the final yeast proportion from 7% to 89% and 44.5%, respectively, effectively improving population stability of E. coli-S. cerevisiae consortia. As a proof-of-concept, we applied these lysis circuits to a division-of-labor system for caffeic acid biosynthesis and showed that they increased caffeic acid production, by 115% and 73%, respectively. This work establishes a versatile, auto-regulation platform for cross-species consortia control and will contribute to the bioproduction of different chemicals.
Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher intracellular ATP concentration and a model-predicted 2.5-fold increase in apparent ATP excess over anabolic demand. Pyruvate and acetate secretion by CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L−1 muconate at 1.1 g L−1 h−1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.
Large-scale industrial bioprocesses are characterized by spatial and temporal inhomogeneities that expose microorganisms to fluctuating bioprocess conditions. These gradients can strongly influence cellular physiology, thereby affecting bioprocess robustness and product formation. Although phenotypic population heterogeneity under such conditions has been described, the underlying protein-level mechanisms remain poorly understood. In this study, the impact of environmental fluctuations on the proteome of an L-phenylalanine producing Escherichia coli strain was investigated. Samples were collected from cultivations in a well-mixed stirred-tank bioreactor and a two-compartment bioreactor operated at mean hydraulic residence times of τ = 103s and τ = 404s. Global proteome patterns were analyzed using principal component analysis (PCA), followed by differential protein abundance analysis and functional interpretation by gene ontology (GO) enrichment and clusters of orthologous groups (COG)-based analysis. PCA identified residence time in the bypass as the dominant factor shaping proteome variation. Prolonged exposure to limiting conditions at τ = 404s led to a distinct clustering of proteome profiles, clearly separated from both the stirred-tank bioreactor and the shorter residence time condition. GO enrichment analysis revealed increased abundance of proteins associated with stress response during the batch and product formation phases, and proteins related to cell communication during biomass production. Consistently, COG-based analysis showed a shift from growth- and production-associated proteins toward maintenance and stress adaptation in cells from the two-compartment bioreactor, with this shift becoming more pronounced at longer residence times.
