Ying Lin

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.

Pyomelanin has been extensively applied in various fields. However, the yield of pyomelanin isolated from natural producers is low. Engineering microbial biosynthesis is considered a sustainable and economically feasible alternative. We utilize engineered Komagataella phaffii to increase the yield of homogentisic acid by 66-fold by balancing three different biosynthetic modules and further synthesizing pyomelanin by oxidative polymerization. During this process, we establish a pyomelanin color screening system and apply this system to perform directed evolution of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase and semirational design modification of hydroxyphenylpyruvate dioxygenase. We report the difference of pyomelanin between oxidative polymerization under alkaline conditions and that under laccase treatment. After high-density fermentation in a 5-L fermenter for 204 h, strain Pyo29 achieve the highest titer (70.5 ± 0.7 g/L). Finally, we demonstrate the potential application value of pyomelanin. This study provides a feasible solution for high-yield pyomelanin biosynthesis with substantial industrial production.

Traditional microbial research has focused on C₄₀ carotenoids. Recent reports showed that novel C₃₀ carotenoid compounds, found in certain bacterial microorganisms, exhibited antioxidant properties similar to or greater than those of their C₄₀ counterparts. However, the biosynthesis yields were relatively low. In this study, an artificial 8-step pathway for de novo mevalonate biosynthesis in Yarrowia lipolytica was constructed, increasing squalene titer to 1235.79 mg/L. This led to the development of a high-production squalene platform strain capable of supplying sufficient precursors for C₃₀ carotenoid biosynthesis. We identified the low substrate conversion rate as the main limitation through a systematic research. The bottleneck was addressed through protein and compartmentalization engineering, resulting in 90.948 mg/L C₃₀ carotenoid production (11.091 mg/g DCW). Ultimately, by incorporating these approaches, we developed strains capable of producing 2686.77 mg/L C₃₀ carotenoids with a cellular content of 35.05 mg/g DCW in bioreactor fermentations, a significant improvement over the initial strain and the highest ever reported for C₃₀ carotenoids.

Traditional Chinese herbal medicines are characterized by multi-component synergy and have garnered significant attention in the healthcare research. The Immune Enhancing Oral Liquid, formulated with Polygonum multiflorum Thunb. and Morinda officinalis How extracts, has demonstrated biological activities including gut microbiota modulation. To systematically evaluate its safety profile, toxicity, and immune-enhancing efficacy, this study assessed toxicological and immunomodulatory parameters in murine models through the following protocols: acute toxicity testing; genotoxicity assays; 28-day oral toxicity study; and immunological function assessments encompassing cellular immunity, humoral immunity, non-specific immunity, and cytotoxic function. Results indicated that the immune enhancing oral liquid showed no obvious acute toxicity, genotoxicity, or 28-day oral toxicity. Furthermore, the immune enhancing oral liquid exhibited no adverse effects on murine hematopoietic or hepatic functions. Crucially, the immune enhancing oral liquid significantly enhanced immune responses, as evidenced by promoted lymphocyte proliferation, elevated serum hemolysin levels, augmented macrophage phagocytic activity, and heightened NK cell activity. Immune enhancing oral liquid was recognized as safe and non-toxic, and the study provides a scientific basis for the application of the oral liquid, validating the effectiveness of the combination of traditional herbal wisdom and modern pharmacology.

Patatin, a prominent food protein derived from potatoes, is renowned for its exceptional nutritional value. Patatin has been characterized for its diverse physiological attributes, including esterase activity, antioxidative properties, cholesterol-lowering effects, and high lysine content, alongside notable physicochemical traits such as foaming, emulsification, and gelation capabilities. Conventional methods for patatin extraction are fraught with inefficiencies, elevated costs, and detrimental impacts on protein structural and functional integrity. Herein, we leveraged an optimized strategy integrating an expression cassette toolbox and regulation of protein secretion to harness Komagataella phaffii as the expression host and achieved an expression level of 3.2 g per litre (g/L) in a 5-Litre bioreactor, which is the highest yield of patatin production using engineered bacteria and funguses that has been reported thus far. In this study, we innovatively refined the endogenous promoter P_CAT1, and its efficacy in driving heterologous protein expression under methanol induction surpassed that of the conventional AOX1 promoter. Furthermore, crucial nodes for patatin heterologous expression in yeast were identified, substantially curtailing the production costs associated with patatin synthesis.

The methylotrophic yeast Pichia pastoris (P. pastoris) exhibits remarkable capability for methanol-driven protein biosynthesis, positioning it as an attractive platform for carbon-neutral biomanufacturing utilizing methanol as a renewable feedstock. However, challenges arising from methanol metabolism, particularly the accumulation of toxic formaldehyde intermediates, significantly hinder efficient methanol biotransformation. To address this limitation, we implemented a metabolic engineering strategy involving dual knockout of alcohol oxidase genes (aox1 and aox2) combined with glycerol co-substrate supplementation. Using enhanced green fluorescent protein (EGFP) as a model heterologous product, we demonstrated that the ΔAOX1/2 strain achieved superior protein productivity in glycerol-methanol co-feeding cultures. Under optimized conditions (0.5% methanol + 0.4% glycerol), the engineered strain attained a biomass density of 38.5 (OD₆₀₀) and EGFP fluorescence intensity of 494,723 units, representing improvements of 32.8% and 53.6%, respectively, compared to the wild-type (WT) strain cultivated with 1% methanol alone. Transcriptome profiling revealed that the observed enhancement in protein synthesis originated from optimized methanol utilization through coordinated upregulation of both assimilatory and dissimilatory metabolic modules. This study demonstrates that alcohol oxidase suppression coupled with glycerol co-metabolism constitutes an effective strategy to alleviate methanol-derived metabolic stress while enhancing heterologous protein yields in P. pastoris.

Morus nigra L. is rich in anthocyanins and other active ingredients, but its extraction residues pose a burden on the environment. In the present study, Morus nigra L. extraction residue resource utilization was achieved through liquid fermentation of Schizophyllum commune, with the aim of enhancing anthocyanin solubilization and evaluating anti-inflammatory activity. Response surface methodology was used to optimize fermentation parameters and quantify anthocyanin fractions by HPLC. The anti-inflammatory effect was evaluated using the lipopolysaccharide-induced inflammation model of human foreskin fibroblast (BJ cell), and the interaction of cyanidin-3-O-glucoside (C3G) with NLRP3, a key target of the pyroptosis pathway, was resolved by molecular docking. Our results indicated that the optimal conditions (substrate 3.4%, inoculum 9%, time 50 h) enabled the total anthocyanin to reach 85.1 μg/mL, of which the C3G content was elevated to 66.7 μg/mL (release efficiency of 83.9%). The fermented filtrate effectively promoted BJ cell proliferation and inhibited the lipopolysaccharide-induced inflammatory response, with the pyroptosis signaling pathway playing a significant role. Molecular docking confirmed that C3G binds strongly to the NLRP3 protein. This technology provides a new strategy for high-value utilization of Morus nigra L. residues and the development of natural anti-inflammatory drugs.