Snapshot at Saccharomycopsis fibuligera metabolism for industrial terpenoid production and bioengineering.

High-Level Terpene Production via a Novel Actinomycetota-Derived MVA Pathway in E. coli.

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.

Toward Complex Polyketide Biosynthesis in Yarrowia lipolytica via Metabolic Reprogramming

Polyketides are among the most structurally diverse and therapeutically important classes of natural products, serving as antibiotics, anticancer agents, agrochemicals, and industrial pigments. Their structural complexity and limited natural availability have driven the development of microbial biosynthetic platforms as scalable and sustainable alternatives to traditional extraction or chemical synthesis. Yarrowia lipolytica, a non-conventional and metabolically versatile yeast, has emerged as a promising alternative chassis for polyketide biomanufacturing, owing to its streamlined central metabolism, high acetyl-CoA availability, and exceptional physiological robustness. Recent advances in metabolic engineering and synthetic biology have enabled extensive rewiring of Y. lipolytica metabolism to support the high-yield production of complex, high-value polyketides. This review summarizes state-of-the-art strategies, from classical metabolic rewiring to emerging approaches such as organelle engineering and subcellular compartmentalization. We further highlight representative case studies of polyketide biosynthesis in Y. lipolytica, critically assess current limitations, and explore future directions to establish this organism as a programmable, industrially viable platform for polyketide production.

Metabolic Flux and Growth Profiling of Megasphaera cerevisiae for Medium‐Chain Fatty Acid Synthesis

ABSTRACT Megasphaera cerevisiae is a well‐known beer spoilage organism, capable of producing undesirable flavours and turbidity. Although the biosynthesis of medium‐chain fatty acids (MCFAs) has been extensively studied in different Megasphaera species, the metabolic behaviour of M. cerevisiae in controlled environments remains largely unexplored. This study examines the MCFAs production from diverse substrates and reports flux analyses of core metabolism for the first time using a genome‐scale model. The results suggest that acetate stimulates butyrate production but not caproic acid production. Butyrate supplementation, either alone or in combination with acetate, promoted CA synthesis. Lactate supplementation primarily led to the formation of propionic acid and acetic acid. The metabolic network model was manually curated and validated against the different experimental data. The metabolic flux results showed that butyrate production facilitated via the reverse β‐oxidation (RBO) pathway, with a minor contribution from the fatty acid synthesis (FAS) pathway. Conversely, CA synthesis was mainly synthesised through the FAS pathway, irrespective of the substrate used. The pathway analyses results highlighted the critical role of hydrogen production in M. cerevisiae metabolism, particularly under conditions where lactate is utilised. Collectively, these findings offer novel insights into the metabolic versatility and pathway preferences of M. cerevisiae .

The isopentenol utilization pathway: enzyme engineering, metabolic integration, and applications in microbial terpenoid biosynthesis.

Terpenoids are among the most structurally diverse and commercially important natural products, with applications in pharmaceuticals, flavors, fragrances, and biofuels. Controllable supply of the universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) remains a central limitation for microbial terpenoid production. The native mevalonate (MVA) and methylerythritol phosphate (MEP) pathways are deeply integrated with sterol homeostasis, central carbon metabolism and redox balance. Their distributed regulation, cofactor demands and scale-dependent bottlenecks complicate further intensification. The isopentenol utilization pathway (IUP) offers an orthogonal alternative: a compact, ATP-only, alcohol-fed bypass converting exogenous isoprenol and prenol to IPP and DMAPP in two kinase steps plus isomerization. This review treats the IUP as a configurable precursor module rather than a single pathway variant. Enzyme-level sections cover entry-kinase and isopentenyl phosphate kinase structure, kinetics, and engineering, including the kinetic imbalance that places most flux control in the first phosphorylation step in canonical C5 configurations, together with the feeding, energetic and downstream conditions under which control shifts elsewhere. Host-level sections examine expression hierarchies, construct design, host choice and compartmentalization, and the interaction between IUP flux, toxicity management, and native precursor pathways. Cell-free cascades and techno-economic analysis then identify the regimes, and the quantitative margins, within which an alcohol-fed bypass outperforms further MVA or MEP optimization.

Metabolic Engineering of Pichia pastoris for S-Adenosylmethionine Overproduction via Transcriptomic-Guided Energy Reallocation.

S-Adenosylmethionine (SAM) is a high-value biomolecule with critical applications in nutraceuticals, pharmaceuticals, and health supplements. However, the detailed metabolic mechanism by which sodium citrate promotes SAM production in Pichia pastoris has not yet been elucidated. Here, a comprehensive time-series transcriptomic analysis revealed that sodium citrate profoundly influences gene expression across multiple pathways. Sodium citrate supplementation redirects carbon flux toward oxidative energy metabolism by upregulating amino acid biosynthesis, translation, and glycolysis. Crucially, the transcriptional upregulation of argininosuccinate synthase (ARG1) and argininosuccinate lyase (ARG4) was identified as a key node driving fumarate-mediated TCA cycle anaplerosis and ATP supply. This foundational analysis led to the identification of ARG4 as a key metabolic engineering target. Overexpression of ARG4 significantly improved SAM production, achieving a 56.46% increase in shake flasks and 9.06 g/L SAM (a 22.10% improvement) in 500 mL fermenters compared to control strains. Integrated physiological and metabolic flux analysis (MFA) demonstrated that ARG4 overexpression redirects metabolic flow, channeling energy substrates toward SAM synthesis, effectively maintaining cellular respiratory metabolism and alleviating energy limitations in the late fermentation phase. This work establishes a novel strategy for enhancing product synthesis by restructuring energy allocation rather than merely increasing overall energy supply.

Enhanced terpenoid synthesis in Alternaria alstroemeriae PPL7 via co-fermentation with Paeonia lactiflora roots: integrated metabolomic and transcriptomic analysis