Reprogramming
Komagataella phaffii
Cell Factory for High-Yield
Production of 3S,3′S-Astaxanthin
via Multiplex Metabolic Engineering and Its Application
Aug 2026· ACS Synthetic Biology· 0 citations· 48 references
TL;DR
The synthesis of 3S,3′S-astaxanthin was successfully and effectively applied in shrimp farming for color enhancement and antioxidant effects and will pave the way for astaxanthin industrial production.
Abstract
Astaxanthin is a keto-carotenoid with high added value. In this study, we aimed to biosynthesize 3S,3′S-astaxanthin efficiently and sustainably from the renewable single-carbon (C1) feedstock methanol through multiplex metabolic engineering strategies in Komagataella phaffii. First, the K. phaffii cell-free terpene synthesis system was established successfully and applied to evaluate the astaxanthin synthase combinations rapidly. We then systematically engineered K. phaffii for the overproduction of 3S,3′S-astaxanthin from methanol by tuning the carotenoid synthesis module rationally, optimizing the precursor supply and carotenoid storage globally, thereby achieving a significant elevation in astaxanthin content from 3.910 mg/g to 7.513 mg/g. Thereafter, key node enzyme assembly, branch route reconstruction, and cofactor engineering were employed to further improve astaxanthin accumulation, achieving a significant increase of astaxanthin content to 11.397 mg/g. Finally, the astaxanthin production reached 4.75g/L under fed-batch fermentation, which is the highest astaxanthin level reported in an engineered microbe to date. In addition, the synthesized astaxanthin was successfully and effectively applied in shrimp farming for color enhancement and antioxidant effects. These results demonstrate the potential of K. phaffii as a promising platform for sustainable green production of value-added terpenoid compounds from organic one-carbon feedstocks and will pave the way for astaxanthin industrial production.
This study demonstrates the combined optimization of isozyme combination and environmental stress to elevate the synthesis of astaxanthin and other carotenoids in D. salina, providing new research ideas and experimental evidence for the future construction of high-yield engineered algal strains.
Hao Zhang, Yifan Kong, Yaping Shao et al.· World Journal of Microbiolog...· 0 citations
Astaxanthin is a high-value carotenoid used in nutrition, cosmetics, pharmaceuticals, and biotechnology, but its poor water dispersibility and oxidative stability limit its formulation. Here, we engineered yeast Yarrowia lipolytica for the efficient production of glycosylated astaxanthin. Introducing zeaxanthin glucosyltransferase CrtX from Pantoea ananatis into an astaxanthin-producing strain enabled the synthesis of astaxanthin glucosides, confirmed by HR-ESI-MS, 1H NMR, and 2D NMR. Engineering UDP-glucose metabolism by reducing competing pathways and enhancing UDP-glucose biosynthesis improved the astaxanthin glucoside production. The best strategy comprised inactivation of the glycogen synthase gene GSY1 combined with multicopy integration of PGM1 (phosphoglucomutase) and UGP1 (UDP-glucose pyrophosphorylase), achieving 17.6 mg/L of astaxanthin monoglucoside and 25.1 mg/L of diglucoside in test-tube cultures. Fed-batch fermentation with glucose yielded 141.6 mg/L of astaxanthin monoglucoside. When corn syrup was used as a low-cost, sustainable carbon source, the production shifted toward astaxanthin diglucoside, reaching 134.3 mg/L. These results represent the highest glycosylated astaxanthin titers reported to date.
M. O. Taratynova, Ivan M. Tarasov, Iuliia M. Fedyaeva et al.· Journal of Agricultural and...· 0 citations
Efficient biosynthesis of 1,3-PDO, a key bio-based chemical, depends on precise regulation of the host metabolic network. In this study, a heterologous CRISPR-Cas12a genome editing system was established and systematically optimized in Klebsiella pneumoniae, enabling efficient and stable genome editing (75-100% efficiency). Using on this platform, by-product pathways were reduced through multi-gene deletions (frdA, poxB, adhE, ldhA, glpK, ptsG and dhaM) to enhance the yield of 1,3-PDO from glycerol. By heterologously expressing GPD1/GPP2 and optimizing promoter, a metabolic network for the "glucose-glycerol-1,3-PDO" pathways was reconstructed. Cometabolism studies indicated that low concentrations of xylose and arabinose as co-substrates enhanced conversion efficiency of glycerol, whereas glucose alleviated metabolic competition. Under cometabolism of glucose and glycerol, the engineered strain K. pneumoniae S2 ΔABEAKGM-1-2 produced 1003.7 mmol/L (76.4 g/L) of 1,3-PDO with a yield of 0.83 mol/mol glycerol, an overall molar yield of 0.78 mol/mol based on total substrate consumption, and a productivity of 27.9 mmol/L/h. When lignocellulosic hydrolysate was used as co-substrate, 981.1 mmol/L (74.7 g/L) of 1,3-PDO was produced with a yield of 0.77 mol/mol glycerol and a productivity of 27.3 mmol/L/h. This study achieved efficient redirection of carbon flux toward 1,3-PDO through systematic metabolic engineering, providing valuable strain resources and technical guidance for the sustainable and cost-effective biomanufacturing of bio-based 1,3-PDO.
Li Wang, Ming-Yang Zhao, Yuan-Ming Ye et al.· Bioresource Technology· 0 citations
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.
This study demonstrates a successful paradigm for engineering a newly isolated strain toward robust, high-titer and cost-competitive PHA production across lab-to-industry scales.
Peng Liu, Xinying Xie, Yi-Hao Deng et al.· bioRxiv· 0 citations
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.
