RESUMO
Tyrosol is a natural polyphenolic product that is widely used in chemical, pharmaceutical and food industries. Currently, the de novo synthesis of tyrosol by Escherichia coli suffers from issues such as low cell density and poor yield. Therefore, the phenylpyruvate decarboxylase mutant ARO10F138L/D218G obtained in our previous study was fused with an alcohol dehydrogenase from different microorganisms for fusion expression, and the optimal ARO10F138L/D218G-L-YahK produced 1.09 g/L tyrosol in shake flasks. In order to further improve tyrosol production, feaB, a key gene in the competing pathway of 4-hydroxyphenylacetic acid, was knocked out, and the resulted strain produced 1.26 g/L tyrosol with an increase of 21.15% compared to that of the control. To overcome the low cell density in tyrosol fermentation, the quorum-sensing circuit was used to dynamically regulate the tyrosol synthesis pathway, so as to alleviate the toxic effect of tyrosol on chassis cells and relieve the growth inhibition. Using this strategy, the yield of tyrosol was increased to 1.74 g/L, a 33.82% increase. In a 2 L fermenter, the production of tyrosol in the engineered strain TRFQ5 dynamically regulated by quorum-sensing reached 4.22 g/L with an OD600 of 42.88. Compared with those in the engineered strain TRF5 statically regulated by induced expression, the yield was increased by 38.58% and the OD600 was enhanced by 43.62%. The combination of blocking the competing pathway using gene knockout technology, and reducing the inhibitory effect of tyrosol toxicity on chassis cells through quorum-sensing dynamic regulation increased the production of tyrosol. This study may facilitate the biosynthesis of other chemicals with high toxicity.
Assuntos
Escherichia coli/genética , Produtos Biológicos , Reatores Biológicos , FermentaçãoRESUMO
Carotenoid cleavage dioxygenase (CCD) family is important for production of volatile aromatic compounds and synthesis of plant hormones. To explore the biological functions and gene expression patterns of CsCCD gene family in tea plant, genome-wide identification of CsCCD gene family was performed. The gene structures, conserved motifs, chromosome locations, protein physicochemical properties, evolutionary characteristics, interaction network and cis-acting regulatory elements were predicted and analyzed. Real time-quantitative reverse transcription PCR (RT-qPCR) was used to detect the relative expression level of CsCCD gene family members under different leaf positions and light treatments during processing. A total of 11 CsCCD gene family members, each containing exons ranging from 1 to 11 and introns ranging from 0 to 10, were identified. The average number of amino acids and molecular weight were 519 aa and 57 643.35 Da, respectively. Phylogenetic analysis showed the CsCCD gene family was clustered into 5 major groups (CCD1, CCD4, CCD7, CCD8 and NCED). The CsCCD gene family mainly contained stress response elements, hormone response elements, light response elements and multi-factor response elements, and light response elements was the most abundant (142 elements). Expression analysis showed that the expression levels of CsCCD1 and CsCCD4 in elder leaves were higher than those in younger leaves and stems. With the increase of turning over times, the expression levels of CsCCD1 and CsCCD4 decreased, while supplementary LED light strongly promoted their expression levels in the early stage. The expression level of NCED in younger leaves was higher than that in elder leaves and stems on average, and the expression trend varied in the process of turning over. NCED3 first increased and then decreased, with an expression level 15 times higher than that in fresh leaves. In the late stage of turning over, supplementary LED light significantly promoted its gene expression. In conclusion, CsCCD gene family member expressions were regulated by mechanical force and light. These understandings may help to optimize tea processing techniques and improve tea quality.