Flavonoid 3β-hydroxylase mutant with improved catalytic activity and its application
A mutant and hydroxylase technology, applied in the field of genetic engineering, can solve the problems of limited application, low conversion efficiency and yield of eriodictyol, and achieve the effect of improving capacity
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Embodiment 1
[0047] Example 1: Directed evolution of SmF3'H
[0048] Starting from the plasmid pY26-P05 (pY26-P INO1 -SmF3′H-P TDH1 -SmCPR) as template, SmF3'H was subjected to directed evolution using the error-prone PCR kit GeneMorph II EZClone (Agilent, CA, US). Primers SmF3'Hm-F and SmF3'Hm-R were used to amplify and randomly mutate SmF3'H, while primers 9.4k-F and 9.4k-R were used to amplify the vector backbone from plasmid pY26-P05. The PCR product was purified and recovered by precipitation. The randomly mutated SmF3'H sequence shares a homology arm of about 40 bp with the linearized vector backbone DNA fragment for homologous recombination in Saccharomyces cerevisiae (for homologous recombination methods, see Gibson, D.G., Synthesis of DNA fragments in yeast by one -step assembly of overlappingoligonucleotides. 2009, Nucleic Acids Res 37(20), 6984-90.). The mutant SmF3′H and the linearized vector backbone were mixed to 50 μL (2:1, mol / mol, about 2-3 μg in total), and the mixed ...
Embodiment 2
[0050] Example 2: Screening and application of mutations
[0051] 10,000-20,000 single colonies were randomly selected from the mutant library for high-throughput screening, and the obtained high-yielding strains were re-screened in shake flasks, and the strains with increased yield were identified and sequenced to detect mutation sites.
[0052] For the strains in the directed evolution library, use 48 deep-well plates for cultivation: use the automatic colony picking instrument QPix420 to automatically inoculate the colonies on the plates into 48 deep-well plates. Add 1.5mL YNB liquid medium to each well, and the medium contains a final concentration of 250mg·L -1 of naringenin. The deep-well plate was transferred to a well-plate shaker (Zhichu, Shanghai, China), and incubated at 30° C., 220 rpm for 48 hours. Place the deep well plate on the table for 2 hours to pellet the cells and the supernatant can be used for high-throughput screening. Figure 4 For the high-throughp...
Embodiment 3
[0058] Example 3: Fermentation optimization of mutant strains
[0059] This mutant vector pY26-P05mut (pY26-P INO1 -SmF3′H R344S -P TDH1 The 453rd amino acid of SmCPR in -SmCPR) was mutated to valine to obtain plasmid pY26-P05mut12 (pY26-P INO1 -SmF3′H R344S -P TDH1 -SmCPR I453V ), the plasmid pY26-P05mut12 was transformed into strain C800 to obtain strain C800P05mut12.
[0060] Pick at least 10 single colonies of strain C800P05mut12 and inoculate them in a 250 mL shake flask containing 20 mL of YNB liquid medium, and culture at 30° C. and 220 rpm for 16-18 h to obtain a seed medium. The seed medium was transferred to a 250 mL shake flask containing 20 mL of fresh YPD liquid medium at 2 mL / 100 mL, and the production of eriodictyol was detected after culturing at 30° C. and 220 rpm for 72 hours. At 0h, 12h, 24h and 36h, 375 mg·L-1 naringenin was added to YPD medium, respectively.
[0061] Fermentation optimization of strain C800P05mut12 at the 5-L fermenter level: Seed cu...
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