ABSTRACT
L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.
ABSTRACT
Vitamin B2 is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant E. coli BL21.
ABSTRACT
In this study, the biosynthesis of phycocyanin ß-subunit (CpcB) in Escherichia coli BL21 was investigated, and its antioxidant activity and application in anti-browning of fresh-cut apples was explored. Four genes (cpcB, cpeS, hox1 and pcyA) involved in the biosynthesis of CpcB were cloned and transformed into E. coli BL21 by constructing recombinant plasmid pETDuet-5. The positive transformant was screened by ampicillin resistance. The analysis of SDS-PAGE and zinc fluorescence spectrum showed that CpcB was successfully expressed in E. coli BL21 with a molecular weight of 21 kDa. The purified CpcB had a maximum absorption peak at 615 nm, and its maximum florescence emission wavelength was 640 nm. It exhibited a stronger ability to scavenge four free radicals than Vc. The color change in fresh-cut apples was obviously delayed by the CpcB treatment. These results suggest that CpcB may be used as a potential anti-browning agent for food preservation.
