Jul 2026· Journal of Agricultural and Food Chemistry· 0 citations· 45 references
Medicine
Abstract
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.
Cyanidin-3-O-glucoside (C3G) is a water-soluble, value-added natural flavonoid with extensive applications in the nutraceutical and cosmetic industries. However, its efficient bioproduction is hampered by intermediate instability, metabolic imbalance and enzyme kinetic constraints. Here, we successfully constructed an efficient biosynthesis pathway from the dihydroquercetin (DHQ) to C3G in Escherichia coli through a multilevel engineering strategy. Initially, the integration of glutathione S-transferase (GST) redirected the metabolic flux towards target cyanidin formation. To minimize the dissipation of labile intermediates, pathway enzymes were spatially organized within a protein cage to enhance cascade efficiency. Furthermore, genomic integration of Glycine max sucrose synthase established an in-situ UDP-glucose regeneration module to ensure a continuous precursor supply for the final glycosylation step. After these pathway-level optimizations, the key enzyme dihydroflavonol 4-reductase (FaDFR) emerged as a new rate-limiting bottleneck due to substrate inhibition under increased DHQ loading. Structure-guided and evolution-informed engineering generated FaDFR variants with improved high-substrate tolerance, as supported by in vitro activity profiling and molecular dynamics simulations. Through combined pathway and enzyme engineering, the G130C-containing strain achieved a C3G titer of 1.34 g/L, representing a 23-fold improvement over the GST-assisted baseline strain. Our platform enables efficient, value-added C3G production and provides a promising framework for constructing downstream pathways toward structurally diverse anthocyanin derivatives.
Retinol, a derivative of vitamin A with potent antioxidant and therapeutic properties, is in high market demand. In response to the low productivity of conventional methods, metabolic engineering has been explored for microbial retinol production. However, systematic engineering strategies for high‐level retinol synthesis in Komagataella phaffii remain limited. In this study, the methylotrophic yeast K. phaffii was developed as an engineered chassis for efficient de novo retinol biosynthesis. Based on a previously constructed β‐carotene‐producing strain, β‐carotene‐15,15′‐dioxygenase (Blh) and retinol dehydrogenase (RDH12) were screened and introduced to establish the synthetic pathway of retinol. To increase precursor supply, key genes in the β‐carotene biosynthetic pathway were overexpressed. The mevalonate (MVA) pathway was further optimized, and central carbon metabolism was reprogrammed to enhance metabolic flux toward retinol. Transport engineering was also performed to improve retinol secretion. Several candidate transporters were overexpressed, and the protein encoded by chr1‐4_0619 in K. phaffii was identified as an endogenous retinol transporter. The final engineered strain produced 3.38 g/L retinol with BHT supplementation in fed‐batch fermentation using a 1.5 L bioreactor. This work represents de novo microbial synthesis of retinol from a one‐carbon feedstock, demonstrating the formidable potential of K. phaffii as a sustainable chassis for retinol production.
Caffeic acid (CA) is a valuable phenylpropanoid with applications in food, pharmaceutical, and chemical industries. Microbial production of CA is often limited by the terminal hydroxylation catalyzed by 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, we constructed a de novo CA biosynthetic pathway in Escherichia coli and enhanced production through systematic metabolic and spatial engineering. Optimization of l-tyrosine supply and HpaBC expression increased CA production to 61.0 mg/L. However, further enhancement of glucose uptake and precursor supply was insufficient, indicating that hydroxylation remained a major limitation. To address this, HpaBC was relocated to the periplasm via the Tat pathway, increasing CA production 4.9-fold to 299.1 mg/L with reduced byproduct formation. Further lpp+14 mediated periplasmic remodeling and fermentation optimization increased shake-flask production to 463.7 mg/L. Finally, fed-batch fermentation achieved 5.1 g/L CA in a 3 L bioreactor. This study highlights periplasmic engineering as an effective strategy for improving oxidation-dependent phenylpropanoid biosynthesis.
Shangyi Wang, Yuqi Zhuo, Jamila A Tuly et al.· Journal of Agricultural and...· 0 citations
Forskolin, a labdane-type diterpenoid isolated from Coleus forskohlii, exhibits therapeutic potential for osteoporosis, cardiovascular diseases, and metabolic syndrome. Its rising nutraceutical demand and limited natural availability have driven synthetic biology approaches for sustainable production. Although significant efforts have been devoted to upstream pathway optimization, the improvement of the downstream pathway still faces challenges due to the complex metabolic network and the low catalytic activity of cytochrome P450s (P450s). In this study, we elucidated the biosynthetic network involved in forskolin production, in which three P450s mediate multi-site oxidation, providing critical pathway insights for forskolin biosynthesis. Based on this, we reconstructed an efficient biosynthetic pathway of forskolin in yeast and subsequently optimized its production efficiency through multidimensional engineering strategies including central carbon flux optimization, rate-limiting enzyme engineering, P450 electron transfer chain reinforcement, and fermentation optimization. The final strain achieved the production of 2.7 g/L forskolin in a 5-L bioreactor, which represents the highest titer reported to date. This study establishes a microbial platform for forskolin production and provides advancements in the complex network of plant natural product biosynthesis.
Meiling Jiang, Hao Tang, Ying Ma et al.· Bioresource Technology· 0 citations
Metabolic engineering of Talaromyces pinophilus through promoter optimization, multicopy integration, and protease deletion enables efficient α-amylase production from lignocellulosic biomass, achieving 26 712 U/mL in bioreactor fermentation.
Jing Zeng, Jianjun Guo, Shuaiwen Zhang et al.· Journal of Industrial Microb...· 0 citations
Erythritol is a natural zero-calorie sweetener with potential for sustainable healthy diets. While yeasts can convert biodiesel-derived glycerol into erythritol, the low production and by-product accumulation limit its industrial commercialization. Here, we isolated different morphologically Yarrowia lipolytica mutants with higher erythritol production through adaptive laboratory evolution under hyperosmotic stress, in which mutant Z exhibited superior growth performance and membrane-related genetic variants compared to the parent strain. We then systematically engineered strain Z to optimize the flux towards erythritol by improving glycerol utilization, reducing the synthesis of competing sugar alcohols, complementing auxotrophic markers, and boosting precursor supply. After fermentation condition optimization and two-stage fed-batch fermentation in a 5-L bioreactor, the final engineered strain Z12 produced 250.76g/L erythritol from pure glycerol and 232g/L erythritol from crude glycerol, which is the highest reported titers for both feedstocks. This study demonstrates the effective integration of adaptive evolution with metabolic remodeling for efficient erythritol biosynthesis.
Ling-Xuan Zhao, Jiawei Li, Ya-Ting Wang et al.· Journal of Biotechnology· 0 citations
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