Hu-Hu Liu

Squalene, a multifunctional natural compound with diverse bioactivities, has significant potential in the nutraceutical and health industries. Microbial synthesis using engineered cell factories represents a sustainable alternative to conventional methods of extracting from plants or animals. This study systematically optimized squalene production in Yarrowia lipolytica through integrated metabolic pathway reconstruction and adaptive laboratory evolution. First, overexpression of DGA1 and LRO1 induced lipid droplet expansion and proliferation to achieve efficient steady-state accumulation of intracellular squalene. Second, the catalytic efficiency between ERG20 and SQS was enhanced by fusing ERG20-SQS through an enzyme fusion strategy for increasing the squalene synthesis flux. Then, the expression of ScHMG1, the rate-limiting enzyme of the MVA pathway, was further enhanced to optimize precursor supply. Finally, adaptive laboratory evolution induced by hydrogen peroxide generated the evolved strain SY8–H3, which produced 801.34 mg/L of squalene in shake-flask fermentation and 4.53 g/L via fed-batch fermentation in a 2.4 L bioreactor. This research firstly applies the oxidative stress-driven adaptive evolution to enhance squalene biosynthesis in Y. lipolytica, establishing the reference for synthesizing squalene and its derived compounds in engineered Y. lipolytica.

Prostaglandin F_2α (PGF_2α) is an important lipid mediator with diverse physiological activities, but its reliance on chemical synthesis presents challenges and limitations, driving the need for efficient microbial fermentation. This study engineered the oleaginous yeast Yarrowia lipolytica to establish a microbial platform for the de novo biosynthesis of PGF_2α from glucose. Firstly, in vitro biotransformation of arachidonic acid (ARA) was achieved using engineered Y. lipolytica expressing prostaglandin H synthase and prostaglandin F synthase. Subsequently, the complete biosynthetic pathway was reconstituted in vivo. To overcome the rate-limiting Δ-8 desaturation step, a high-efficiency Δ-8 desaturase from I. galbana was selected, and a Δ9-elongase/Δ8-desaturase fusion protein with a GGGGS linker was constructed. Further systematic metabolic engineering including dual organelle engineering targeting the endoplasmic reticulum and lipid droplets, and enhanced acetyl-CoA supply via carnitine O-acetyltransferase (Cat2) overexpression - a strategy that proved more effective than regulating NADPH generation. Finally, fermentation optimization with the optimized YAO medium significantly boosted PGF_2α production. These combined metabolic engineering strategies enabled Y. lipolytica de novo production of PGF_2α from glucose, reaching a titer of 32.63 μg/L (nearly 3.58 μg/g DCW). Overall, this research offers a promising platform for advancing the green biosynthesis of PGF_2α and other prostaglandins.

Background: Pueraria lobata has a long edible history owing to its special physiological activities. However, research on the processing methods for P. lobata is limited.Methods: In this study, a more suitable boiling process was established by optimizing the heating method, solvent selection, and different processing techniques. In addition, the stir-fried with bran method was used to investigate the changes in heavy metal contents in P. lobata.Results: Compared with the traditional process, the heavy metal content was reduced 96.5% and the isoflavone content was increased 7.5% in the decoction obtained by continuous heating with a ratio of water-to-white wine of 10:1. Meanwhile, we explored the effect of different stir-fried with bran processes on the reduction of lead, cadmium, chromium, copper, and arsenic in P. lobata. The results showed that the use of medium heat for 20 s and 30% bran roasting for 1 min resulted in the lowest overall heavy metal content in P. lobata.Conclusion: This study will provide the reference for reducing heavy metals from P. lobata using different processing methods.

Squalene is a triterpene with various biological applications. However, the conventioneer squalene industry is limited by complex extraction processes and environmental pollution, necessitating an environmentally sustainable solution to the increasing demand for squalene. Microbial synthesis is a potentially green and efficient method of producing squalene. Acetyl-CoA is a key precursor of squalene. First, we investigated the effects of enhanced acetyl-CoA supply on squalene production, lipid content, and total fatty acid content in Yarrowia lipolytica. Then, strain YLACLH2 with a squalene production of 232.29 mg/L was obtained by co-overexpressing YlACL2 and YlHMG1. Subsequently, the squalene production of YLACLH2 was increased to 514.33 mg/L by fermentation engineering, optimizing fermentation conditions including temperature, media volume, C/N ratio, shaker flask type and medium type. Finally, we investigated the synthesis efficiency of squalene in Y. lipolytica by acid-hydrolyzed sugarcane molasses (AHM) and waste cooking oil (WCO) as carbon sources with optimized fermentation conditions. This study showed that Y. lipolytica has the potential to produce squalene industrially using low-cost substrates. Our study findings provide reference for engineering Y. lipolytica to produce squalene using low-cost substrates and in an environmentally sustainable manner.

A bacterial strain A1-3 with iprodione-degrading capabilities was isolated from the soil for vegetable growing under greenhouses at Lhasa, Tibet. Based on phenotypic, phylogenetic, and genotypic data, strain A1-3 was considered to represent a novel species of genus Azospirillum. It was able to use iprodione as the sole source of carbon and energy for growth, 27.96 mg/L (50.80%) iprodione was reduced within 108 h at 25°C. During the degradation of iprodione by Azospirillum sp. A1-3, iprodione was firstly degraded to N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine, and then to (3,5-dichlorophenylurea) acetic acid. However, (3,5-dichlorophenylurea) acetic acid cannot be degraded to 3,5-dichloroaniline by Azospirillum sp. A1-3. A ipaH gene which has a highly similarity (98.72–99.92%) with other previously reported ipaH genes, was presented in Azospirillum sp. A1-3. Azospirillum novel strain with the ability of iprodione degradation associated with nitrogen fixation has never been reported to date, and Azospirillum sp. A1-3 might be a promising candidate for application in the bioremediation of iprodione-contaminated environments.

Lilii Bulbus, an edible Chinese herbal medicine, has a long history in medicine. However, research on effectively boiling Lilii Bulbus is rare. To make the more nutritious Lilii Bulbus soup, the optimized boiling process, using an alternate heating mode by decoction pot carrying a mixture of water and Chinese liquor at the ration of 9:1, was established in this study. Compared to the soup prepared by the daily process, the polysaccharide amount improved by 54%, and the total heavy metals decreased by 33.5% using the optimized boiling process. In addition, the total saponins at 34.3 μg/g were determined in the soup prepared by the optimized process. Meanwhile, the colchicine content in the boiled Lilii Bulbus soup was undetectable using the optimized process. This research performs an optimized boiling process for making Lilii Bulbus soup, and provides a reference for generating high commercial value from Lilii Bulbus soup in the future.