Jianyong Zheng

Metabolomics analysis reveals 6-benzylaminopurine as a stimulator for improving lipid and DHA accumulation of Aurantiochytriumsp.

Abstract BACKGROUND Phytohormones are chemical messengers that have a positive effect on biodiesel production of microalgae at low concentrations. However, the effect of phytohormone 6‐benzylaminopurine on lipid and docosahexaenoic acid (DHA) production in marine DHA‐producer Aurantiochytrium has never been reported. In this study, a GC‐MS‐based metabolomics method combined with a multivariate analysis is applied to reveal the metabolic mechanism of 6‐benzylaminopurine enhancing production of lipid and DHA in Aurantiochytrium sp.YLH70. RESULTS In total, 71 metabolites were identified by GC‐MS. The PCA model revealed that 76.9% of metabolite variation was related to 6‐benzylaminopurine treatment, and overall metabolomics profiles between the 6‐benzylaminopurine and control groups were clearly discriminated. Forty‐six metabolites identified by the PLS‐DA model were responsible for responding to 6‐benzylaminopurine. Metabolic analysis showed that 6‐benzylaminopurine could accelerate the rate of utilization of glucose in Aurantiochytrium sp. YLH70, and the metabolic flux from glycolysis, TCA cycle and mevalonate pathway to fatty acids biosynthesis was promoted. Moreover, the anti‐stress mechanism in Aurantiochytrium sp.YLH70 might be induced by 6‐benzylaminopurine. CONCLUSION Metabolomics is a suitable tool to discover the metabolic mechanism for improving lipid and DHA accumulation in a microorganism. 6‐benzylaminopurine has the potential to stimulate lipid and DHA production of Aurantiochytrium sp.YLH70 for industrial purposes.

A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.

Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity

This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λex 330 nm and λem 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process.

Bio-preparation of (R)-DMPM using whole cells of Pseudochrobactrum asaccharolyticum WZZ003 and its application on kilogram-scale synthesis of fungicide (R)-metalaxyl

Methyl (R)‐N‐(2,6‐dimethylphenyl)alaninate ((R)‐DMPM) is a key chiral intermediate for the production of (R)‐metalaxyl, which is one of the best‐selling fungicides. A new strain, Pseudochrobactrum asaccharolyticum WZZ003, was identified as a biocatalyst for the enantioselective hydrolysis of (R,S)‐DMPM. The key parameters including pH, temperature, rotation speed and substrate concentrations were optimized in the enantioselective hydrolysis of (R,S)‐DMPM. After the 48 h hydrolysis of 256 mM (R,S)‐DMPM under the optimized reaction conditions, the enantiomeric excess of product (e.e.p) was up to 99% and the conversion was nearly 50%. Subsequently, the unhydrolyzed (S)‐DMPM was converted to (R,S)‐DMPM through the n‐butanal‐catalyzed racemization. Furthermore, stereoselective hydrolysis of (R,S)‐DMPM catalyzed by whole cells of P. asaccharolyticum WZZ003 was scaled up to kilogram‐scale, offering (R)‐MAP‐acid with 98.6% e.e.p and 48.0% yield. Moreover, (R)‐metalaxyl was prepared at kilogram scale after subsequent esterification and coupling reactions. Therefore, a practical production process of (R)‐DMPM and (R)‐metalaxyl with the prospect of industrialization was developed in this study.