Development of 5-Aminolevulinic Acid Production in Escherichia coli by Expression Modular Design and Metabolic Regulation

Abstract

5-Aminolevulinic acid (5-ALA) is an essential intermediate in organisms and has also considerable applications in the agricultural and medical field Beyond the applications as biodegradable insecticide and promoting factor for crops 5-ALA has been approved by the US Food and Drug Administration (FDA) as an intraoperative optical imaging agent for suspected high-grade gliomas Thanks to the advance in genetic and metabolic engineering biosynthesis of 5-ALA in microorganisms has become a sustainable process for efficient production In this study several modules for expression systems of ALA synthase from Rhodobacter sphaeroides (RsHemA) a non-specific ALA exporter (RhtA) and chaperones were first developed and discussed As the co-factor of ALA synthase (ALAS) 5-ALA production was enhanced by the key cofactor pyridoxal phosphate (PLP) which was supplied by expressing genes pdxK and pdxY or direct addition With final concentration of 30 ?M PLP the 5-ALA titer could increase from 1 35 g/L to 2 44 g/L However inclusion bodies of RshemA served as an obstacle; thus DnaKJ and GroESL chaperones were introduced to refold the insoluble proteins However only GroESL chaperones could significantly solve the issue of inclusion bodies In dual plasmid system the 5-ALA titer was further enhanced to 3 46 g/L While with integrated groESL cluster in chromosme the 5-ALA production reached 4 01 g/L even with no obvious expression of chaperones As gene deactivation is a common strategy for redistributing the carbon fluxes oriented to target product instead of bypass pathways deactivation of ldhA pta sucC and pck genes were discussed Silencing either pck or pta was more beneficial for improving 5-ALA production However sucC-defect caused inpaired cell growth To adjust the GroESL expressions an all-in-one plasmid encoding RshemA rhtA and groESL was further been constructed Coupling with the effect of all-in-one plasmid system and gene deactivation the 5-ALA titer reached 5 11 g/L and 5 23 g/L with knock-out of pck and pta respectively Lastly plasmid-free strain called RrGI was constructed with expectation of being a robust and marker-free strain Surprisingly it was found robust for 5 53-folds enhancement on 5-ALA titer Thus the aspect of relative transcription levels metabolites analysis and cell morphology were discused to clarify the criticle difference of plasmid-free strain In accordance with the transcription level of genes involved in glycolysis TCA cycle and recombinant expression the energy consumption of RrGI strain may be less due to the much less transcription level of recombinant protein but similar protein expression Therefore 5-ALA yield was significantly higher than other strains even with similar consumption of glycine succinate and glycine In addition the morphology analysis also showed the poor cell integrity of plasmid-harboring strains Generally the plasmid-free strain (i e integration of gene cluster to the chromosme) RrGI could perform higher 5-ALA titer by reduced energy consumption for transcription thus lead to 7 47 g/L titer and 0 588 g/L/h productivity Besides RrGI strain could show more stable ALA production for three generations compared to plasmid-harboring RsrG strain

Date of Award	2020
Original language	English
Supervisor	I-Son Ng (Supervisor)