Li Minghua,Meng Xiumei,Sun Zhiyang,Zhu Chunjie,Ji Huiying
It is well known that cofactors play a key role in the production of different compounds in bioconversion processes, while the high cost of cofactors limits their usage in industrial applications. In the present study, a NADH regeneration system was successfully developed in Lactobacillus plantarum by expressing the fdh gene coding for formate dehydrogenase (FDH) from Candida boidinii. Results indicated that the FDH was expressed with the highest activity of 0.82 U/mg of protein when cells entered early stationary phase. In addition, the expression of FDH increased the intracellular level of NADH and NADH/NAD + ratio in L. plantarum , and therefore, enhanced the NADH-dependent production of 3-phenyllactic acid (PLA) in repeated and fed-batch bioconversions. In brief, the results demonstrate that the NADH regeneration by expressing FDH is a promising strategy for producing NADH-dependent microbial metabolites in L. plantarum .
Polyphenol oxidase is the popular enzyme involved in fruit-vegetable browning and melanin synthesis. In the present paper, L-3-phenyllactic acid, a natural bacteriostatic substance, was investigated as an inhibitor of polyphenol oxidase. The results were demonstrated that the residual enzyme activity of polyphenol oxidase decreased gradually with the increase of the concentration of L-3-phenyllactic acid. The L and ΔE values of the reaction system increased gradually. The reversible mixed-type inhibition mode of L-3-phenyllactic acid was determined by Lineweaver Burk plot. At the same time, the results of fluorescence quenching demonstrated that L-3-phenyllactic acid was a quencher of polyphenol oxidase, and the molecular docking study provided the binding mode of L-3-phenyllactic acid and polyphenol oxidase at the molecular level. L-3-phenyllactic acid decreased the activity of polyphenol oxidase and browning of fresh-cut Agaricus Bosporus. This research first studied the inhibitory effect of L-3-phenyllactic acid on the activity of polyphenol oxidase, and would provide a theoretical foundation for the use of L-3-phenyllactic acid as anti-polyphenol oxidase agents.
3-phenyllactic acid (PLA) has broad anti-fungal activity, however, target sites of PLA on fungal cells and its anti-fungal mechanism of action have been poorly studied. In this study, we explored the inhibition mechanism of Rhizopus oryzae ( R. oryzae ) on rotten lily bulbs by PLA. The minimum inhibitory concentration value of PLA against R. oryzae was 8 mg/mL. We observed the ultrastructure of R. oryzae by scanning electron microscopy and transmission electron microscopy which indicated that PLA did not damage the cell membrane, but destroyed the mitochondria and other organelles. Tandem mass tag proteomes showed that PLA significantly down-regulated ( P < 0.05) the expression of hexokinase (HK), phosphofructokinase (PFK), a-ketoglutarate dehydrogenase (a-KGDH), adenylate kinase (ADK1), Cytochrome C oxidase and NADH dehydrogenase, and up-regulated ( P < 0.05) the expression of mitochondrial ADP/ATP carrier proteins (AAC) and subunit IV (CCIO IV) in glycolysis, tricarboxylic acid cycle or oxidative phosphorylation metabolism. Following these findings, down-regulated HK and a-KGDH activity of aforementioned pathways was shown by enzyme activity assay, and regulated gene expression of ADK1, AAC, CCIO IV and NADH dehydrogenase was further confirmed by real-time quantitative PCR. Central carbon metabolomics showed that citric acid, cis-Aconitic acid, isocitric acid, alpha-Ketoglutaric acid, succinate, fumarate and malic acid of the tricarboxylic acid cycle metabolites were significantly down-regulated ( P < 0.05), and ATP production by oxidative phosphorylation was also significantly reduced ( P = 0.02), resulting in insufficient energy production. Thus, ROS levels increased by 141% of the control values and cytochrome C was released, resulting in gradual cell apoptosis. All data indicated that energy metabolism was the target of PLA against R. oryzae . This was the first study to show that energy metabolism could be the target of PLA against R. oryzae , which could provide a theoretical basis to study the mechanism of fungal inhibition.
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