Periplasmic Engineering Enhances Terminal Hydroxylation for Efficient Caffeic Acid Biosynthesis in Escherichia coli.

Engineering glucose uptake for enhanced production of L-tyrosine in Bacillus amyloliquefaciens.

L-Tyrosine is an important aromatic amino acid widely used in dietary supplements and as a precursor for high-value food ingredients. Microbial fermentation presents a sustainable and green production route, but achieving high titers remains challenging due to incomplete understanding of metabolic regulation. In this study, Bacillus amyloliquefaciens was engineered for efficient L-tyrosine production to support its application in functional food development. Systematic investigation of the glucose transport pathway identified two key genes of the non‑phosphotransferase system, namely glcP1 (glucose permease) from B. amyloliquefaciens and glK (glucokinase) from Corynebacterium glutamicum. Overexpression of these genes increased L-tyrosine titers to 395.82 mg/L and 385.37 mg/L, representing 36% and 33% improvements, respectively. Subsequent optimization of expression elements (promoters, terminators, and 5'-UTRs) further enhanced production to 458.27 mg/L. Furthermore, glucose uptake and phosphorylation were reconstructed by combining non‑PTS enhancement with PTS attenuation. The resulting chassis strain (B52) produced 407.16 mg/L, a 43% increase over the wild‑type strain B1. Finally, overexpression of the rate‑limiting enzymes AroA and TyrA raised the titer to 1400.53 mg/L. This study provides effective metabolic engineering strategies for the biosynthesis of L-tyrosine in non-model Bacillus species and offers a promising approach for the sustainable production of amino acids.

Overcoming protocatechuate and catechol accumulation in muconic acid production via adaptive laboratory evolution and metabolic engineering in Pseudomonas putida

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 catechol, not PCA, is the principal bottleneck 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, yield, and strain resilience in muconate bioproduction. Highlights Accumulation of metabolic intermediates was alleviated by adaptive laboratory evolution Sequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation Increased CatA1 expression reduced bottlenecks and improved muconate production

Metabolic Engineering of Yarrowia lipolytica for High-Level de novo Biosynthesis of Xylitol from Glucose.

Xylitol is a highly functional sweetener with extensive applications. Sustainable biosynthesis from glucose is desirable yet metabolically challenging. Here, we engineered Yarrowia lipolytica as a cell factory by constructing a core biosynthetic route via combinatorial screening and multicopy integration of d-arabitol dehydrogenases (ArDH) and an NADPH-dependent xylitol dehydrogenase (XDH) in the robust chassis NBRC1631. To further drive the metabolic flux and alleviate bottlenecks, we employed a synergistic push-and-pull strategy: overexpressing glucose transporters (YH3 and YH4), while upregulating pentose phosphate pathway enzymes (ZWF1 and GND1) to enhance NADPH regeneration, matching the redox demand of the synthetic cascade. Following two-stage pH-controlled fed-batch fermentation in a 3 L bioreactor, the final engineered strain achieved a record-high xylitol titer of 39.0 g/L with a yield of 0.09 g/g glucose. This study establishes a productive platform for microbial de novo xylitol biosynthesis from glucose, offering a green and economically viable route for industrial production.

Enhanced Trehalose Production Through Integrated Chassis and Expression Engineering in Bacillus subtilis

Trehalose is a functional disaccharide widely used in the food, pharmaceutical, and cosmetic industries. It is industrially produced via a dual-enzyme process involving maltoligosaccharide trehalose synthase (MTSase) and maltoligosaccharide trehalose hydrolase (MTHase), with Escherichia coli (E. coli) serving as the expression host. Bacillus subtilis (B. subtilis) is an ideal host for industrial trehalose production due to its generally recognized as safe (GRAS) status and low phage susceptibility. However, engineered B. subtilis strains often exhibit slow growth, low heterologous protein expression, and high fermentation costs, thereby limiting their industrial application. To address these challenges, this study employed a synergistic strategy that combined chassis modification, expression element optimization, and knockout of substrate-competition pathways. First, a tryptophan-independent strain was constructed by reverting the trpC2 mutation to shorten the growth cycle. Next, knockout of flgD, yueB, and integration of E. coli-derived glutamate dehydrogenase (gdhA) significantly enhanced biomass accumulation. Expression of MTSase and MTHase was markedly improved through tandem strong promoters (PHpaII-P36) and ribosome-binding site (RBS) optimization (RBS1), achieving a 10.87-fold and 4.22-fold increase in enzyme activity, respectively. Finally, disruption of the amyE gene reduced non-specific substrate degradation. Using maltodextrin as substrate, the final trehalose conversion rate reached 76%. This study constructed B. subtilis chassis cells that highly express MTHase and MTSase respectively, laying a foundation for subsequent industrial trehalose production.

A recombinant xylose reductase enzyme with biotechnological potential for xylitol production

Construction of Engineered Escherichia coli for Vanillin Production From Glucose: Pathway Module Optimization and Methyl-Donor Regulation.

Vanillin is an important flavor compound widely used in the food, fragrance, and pharmaceutical industries. Current biotransformation processes from ferulic acid or eugenol are limited by high substrate cost and low carbon efficiency, motivating de novo biosynthesis from glucose. This study employed Escherichia coli as the chassis organism to establish a modular vanillin biosynthesis system based on the phenylpropanoid metabolic pathway. Heterologous expression of sam8, sam5, and comt established a biosynthetic module for the sequential conversion of l-tyrosine to p-coumaric acid, then to caffeic acid, and finally to ferulic acid. This module enabled the production of 15.86 mg/L ferulic acid from glucose. Two ferulic acid-to-vanillin modules were compared: a CoA-dependent deacetylation pathway (fcs/ech) and an oxidative decarboxylation pathway (fdc/cso2). With ferulic acid feeding, the deacetylation route produced 445.78 mg/L vanillin, far exceeding the 3.49 mg/L obtained via oxidative decarboxylation. When integrated with the upstream module, the deacetylation pathway enabled de novo vanillin production from glucose at 4.46 mg/L, whereas the oxidative decarboxylation route yielded only 0.46 mg/L, indicating better performance of the former under the tested conditions. Metabolite profiling indicated accumulation of caffeic acid and limited ferulic acid levels, identifying O-methylation and S-adenosyl-L-methionine (SAM) supply as major bottlenecks. Implementation of SAM regeneration modules revealed that mtn overexpression enhanced the vanillin titer by about 3-fold, to 12.36 mg/L, while luxS overexpression had a negligible effect. In summary, this study establishes a functional de novo phenylpropanoid pathway for vanillin in E. coli, underscores the critical role of terminal‑pathway selection, and demonstrates that SAM regeneration effectively improves vanillin production from glucose.