Genome-integrated engineering of Escherichia coli Nissle 1917 enables sustained gut-local biosynthesis of 5-hydroxytryptophan

Metabolic Engineering of Probiotic Saccharomyces boulardii Enables Intestinal 3-Hydroxybutyrate Delivery and Alters Short-Chain Fatty Acid Profiles in Mice

3-Hydroxybutyric acid (3-HB) is a bioactive ketone body involved in the regulation of intestinal inflammation and metabolic homeostasis. Although engineered bacterial probiotics have been developed for localized 3-HB delivery, their susceptibility to antibacterial antibiotics may limit their use during concurrent antibiotic treatment. The probiotic yeast Saccharomyces boulardii offers an alternative host for intestinal 3-HB delivery because of its compatibility with antibacterial antibiotics and the availability of well-established genetic engineering tools. Here, we engineered S. boulardii for 3-HB production using Cas9-mediated genome editing. A heterologous 3-HB biosynthetic pathway was introduced into S. boulardii MYA-797, and endogenous acetyl-CoA and ethanol metabolism was subsequently rewired by overexpressing ACS1, deleting ADH1, and overexpressing ADH7. The optimized strain, SbDY02, produced 1.7 g/L 3-HB under microaerobic conditions. Oral administration of SbDY02 to C57BL/6J mice increased fecal 3-HB and short-chain fatty acid (SCFA) concentrations by 1.89-fold and 1.68-fold, respectively, compared with mice receiving the parental strain. Repeated administration also increased fecal acetate and circulating total SCFAs, butyrate, and propionate. In human colonic epithelial cells, purified 3-HB attenuated lipopolysaccharide-induced p38 MAPK phosphorylation, supporting its direct activity toward inflammation-associated epithelial signaling. To our knowledge, this study provides the first demonstration of a 3-HB-producing probiotic yeast and links central metabolic engineering of S. boulardii with increased 3-HB availability, altered SCFA profiles, and a host-relevant epithelial response.

Systematic modular engineering of genome-integrated Escherichia coli MG1655 for high-level 2'-fucosyllactose production.

2'-Fucosyllactose (2'-FL), the most abundant human milk oligosaccharide (HMO), has attracted considerable interest for its prebiotic and immunomodulatory functions, with broad applications in infant nutrition. In this study, we report the development of a high-yield, genome-integrated 2'-FL-producing strain based on Escherichia coli MG1655 through systematic modular optimization. Starting from a single-copy BKHT strain (MGC06), we first optimized the copy number of the α-1,2-fucosyltransferase (α-1,2-FT) gene BKHT. Subsequently, the GDP-L-fucose supply was enhanced through coordinated genomic integration of the gene clusters cpsG-cpsB and gmd-fcl, while the multidrug efflux transporter gene mdfA was integrated to improve product export and strain robustness. BKHT copy number was then re-evaluated in the optimized background, with four copies yielding the highest production. The final engineered strain, harboring all genetic modifications stably integrated into the chromosome, produced 17.18 g/L 2'-FL in shake-flask culture. In fed-batch fermentation using a 5-L bioreactor, this strain achieved a titer of 154.12 g/L after 60 h, with a productivity of 2.57 g/L/h. Notably, throughout the entire fermentation process, no antibiotics or inducers were supplemented, underscoring the genetic stability and regulatory compliance of this plasmid-free system. To our knowledge, this represents the highest 2'-FL titer reported to date, positioning our engineered strain as a promising candidate for commercial 2'-FL production.

Biosynthesis of 3-Hydroxy-3-methylbutyrate by Probiotic Escherichia coli Nissle 1917

3-Hydroxy-3-methylbutyrate (HMB) is an important nutritional supplement for managing sarcopenia. This study engineered an HMB biosynthetic pathway in the probiotic Escherichia coli Nissle 1917 (EcN). The initial engineered strain exhibited low HMB titers, as carbon flux was primarily diverted toward acetate formation. Deleting the pyruvate oxidase gene (poxB) reduced acetate but caused significant pyruvate accumulation, revealing a severe kinetic bottleneck in endogenous pyruvate dehydrogenase (PDH) activity. Supplementing pantothenic acid (Vitamin B5) profoundly expanded the intracellular coenzyme A (CoA) pool, providing the essential backbone for pathway intermediates. This strategy enhanced PDH flux and improved HMB production by 5.5-fold. Through fed-batch fermentation, the engineered strain achieved an HMB titer of 8.6 g/L with a yield of 0.14 g/g glucose. The results of this study provided a probiotic strain that can synthesize HMB de novo and demonstrated the importance of pantothenic acid availability for EcN metabolic engineering.

Multidimensional metabolic engineering strategies for efficient production of branched-chain amino acids and their derivatives in Escherichia coli and Corynebacterium glutamicum.

Branched-chain amino acids (BCAAs), comprising L-valine, L-leucine, and L-isoleucine, are essential amino acids with extensive applications in food, feed, pharmaceuticals, and cosmetics. Escherichia coli and Corynebacterium glutamicum, the two predominant industrial workhorses have been extensively engineered for high-level BCAAs biosynthesis. This review presents a comprehensive analysis of recent advances in microbial BCAAs production through a Mechanism-Module-Process framework. From the mechanism dimension, the intricate BCAA biosynthetic architectures are delineated, encompassing allosteric feedback inhibition, transcriptional attenuation, and transport system. From the module dimension, modular metabolic engineering strategies are dissected, including precursor supply enhancement, NADPH cofactor rebalancing, biosensor-driven dynamic regulation, and adaptive laboratory evolution for strain robustness. From the process dimension, how oxygen availability reprograms cellular metabolism across aerobic, microaerobic, and anaerobic fermentation is systematically examined, and how two-stage fermentation coupled with cofactor rebalancing resolves the growth-production trade-off to achieve near-theoretical yields is discussed. Furthermore, the biosynthetic strategies and current production status of BCAAs derivatives are summarized, highlighting the importance of multidimensional framework engineering. Collectively, this Mechanism-Module-Process framework provides a holistic roadmap for constructing superior BCAAs cell factories and extending their metabolic potential toward derivative biosynthesis.

Biosensor-driven Evolution and Transcriptome-Guided Chassis Engineering Enhance Endogenous l-Cysteine Supply for Ergothioneine Production.

Ergothioneine (EGT) is a high-value antioxidant for food, pharmaceutical, nutraceutical, and cosmetic applications. Microbial production from renewable feedstocks is promising, but efficient biosynthesis requires coordinated precursor supply. Precursor-supplementation experiments indicated that multiple amino acids may jointly influence EGT production. Based on pathway biochemistry and previous engineering evidence, endogenous l-cysteine supply was selected as one mechanistically relevant engineering target. Here, an Escherichia coli platform for glucose-derived EGT production was developed by combining biosensor-driven evolution and transcriptome-guided chassis engineering. A dual-output l-cysteine-responsive biosensor linked kanamycin resistance and fluorescence, enabling sequential growth-based enrichment and fluorescence-based prioritization. Mutagenesis, microdroplet-assisted adaptive evolution, and single-cell sorting generated an evolved chassis with 47.10% higher extracellular l-cysteine accumulation and 64.49% higher EGT production. Transcriptome-guided bsmA activation further improved production, and the final strain produced 1.20 g/L EGT in a 3 L fed-batch fermentation without additional l-cysteine supplementation.

Tailoring Precise Genomic Integration Toward Isolate-to-Industry Strain Development for Scalable High-Titer Production of Polyhydroxyalkanoate