As an essential chemical, p- hydroxystyrene has attracted much attention and is widely used in the field of semiconductors. However, the current production methods have some disadvantages, such as low molar conversion yield, p- hydroxystyrene toxicity, and high substrate cost. To overcome these problems, an five enzymes cell-free cascade, consisting of alditol oxidase from Streptomyces coelicolor A3 (ScALDO), dehydratase from Paralcaligenes ureilyticus (PuDHT), tyrosine phenol lyase from Citrobacter freundii (CfTPL), tyrosine ammonia lyase from Rhodotorula glutinis (RgTAL) and phenylacrylate decarboxylase from Enterobacter sp. (EsPAD ) , was constructed for the de novo biosynthesis of p- hydroxystyrene from glycerol. Through process strategy optimization, a molar conversion yield of more than 84.2% was finally obtained from 10 mM crude glycerol. It is worth mentioning that the reaction system performed an excellent titer using 50 mM crude glycerol, and the p- hydroxystyrene concentration reached 30.32 mM, which exceeded the maximum titer of fermentation. This showed that our system is robust to p- hydroxystyrene and crude glycerol. In addition, a ‘two-step purification process’ was designed to produce pure p- hydroxystyrene. This is the first study focused on the cell-free biosynthesis of p- hydroxystyrene, and the highest conversion yield and titer thus far was obtained for p- hydroxystyrene de novo biosynthesis.
Yang Wenjuan,Tang Sidi,Xu Rubing,Zhang Lu,Zhou Zihao,Yang Yong,Li Yanyan,Xiang Haibo
Fusarium is a soil-borne pathogen that poses a serious threat to the quality and yield of hundreds of crops worldwide, particularly tobacco production. Using metabolomics technology, we investigated natural metabolites from disease-conducting soil (DCS) and disease-suppressing soil (DSS) of tobacco rhizosphere as fungicides to control tobacco Fusarium wilt (TFW), which is mainly caused by Fusarium oxysporum. Furthermore, the antifungal mechanisms of these natural metabolites were preliminarily elucidated through various assessments, including antifungal activity determination, chemotaxis effect tests, PI staining experiments, and measurements of extracellular conductivity and protein content. Metabolomics results showed that the DCS with three different disease grades (G1, G5 and G9 groups) had significantly higher levels of 15, 14 and 233 differential rhizosphere metabolites (DRMs) and significantly lower levels of 72, 152 and 170 DRMs compared to the DSS (G0 group). According to KEGG pathway analysis, these DRMs were found to be enriched in the caffeine metabolism, biosynthesis of phenylpropanoids, galactose metabolism and tyrosine metabolism, etc. Linustatin, scopoletin and phenylpropiolic acid were picked out from these DRMs and found to have suppressive activity against F. oxysporum through correlation analysis and antifungal experiments. The three DRMs showed strong inhibitory effects on the growth and spore germination of F. oxysporum at concentrations of 0.5 mM or higher in each test period. Furthermore, F. oxysporum showed a phobotaxis effect against these three DRMs at concentrations as low as 0.25 mM. Finally, we found that the three DRMs had an inhibitory effect on F. oxysporum by destroying the integrity of the cell membrane and increasing the membrane permeability of F. oxysporum. This study firstly reports the inhibition activity of phenylpropiolic acid and linustatin on F. oxysporum, providing a practical and environmentally friendly method for biocontrol of TFW by using natural fungicides.
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