Biochemical and Structural Studies of Microbial Enzymes for the Biosynthesis of 1,3-butanediol

Protein design approach for systems metabolic engineering entails discovery of novel enzymes and protein engineering for improving biocatalysts activity. It allows us to design and optimize cellular metabolism for the generation of novel metabolic pathways, aiming at the production of chemicals from renewable biomass. This thesis is aimed at two topics: the enzyme discovery and engineering for optimization of the biosynthetic pathway for 1,3-butanediol (1,3BDO), and the in vivo demonstration of the protein design in E. coli and cyanobacteria. The 1,3BDO pathway consists of three steps: pyruvate, a natural metabolite, is converted into acetaldehyde by pyruvate decarboxylase (PDC); it is followed by aldol condensation using 2-deoxyribose-5- phosphate aldolase (DERA), yielding 3-hydroxybutanal (3-HB); an aldo-keto reductase (AKR) catalyzes the reduction of 3-HB to produce 1,3BDO. Thus, we specifically investigated novel DERA and AKR in this thesis. First, PA1127 from Pseudomonas aeruginosa was identified based on the activity on 3-HB. The AKR was biochemically and structurally studied, providing insights into further applications with a remarkable substrate promiscuity. Next, BH1352 from Bacillus halodurans was identified with a high activity of aldol condensation. As the DERA-catalyzed condensation was the rate-limiting step, we rationally designed site-directed mutagenesis for enhancing 1,3BDO synthesis from DERA-AKR coupled reaction; two of the mutations showed 2.6 times higher in vitro activity compared to BH1352 wildtype. The two mutations were then introduced to E. coli fermentation for 1,3BDO biosynthesis from glucose. The Phe160Tyr single mutation exhibited a 5-fold increase in 1,3BDO titer, and the Phe160Tyr/Met173Ile double mutation displayed a 6-fold increase in 1,3BDO production with a 7-fold improvement in glucose yield compared to the wildtype. Finally, the biosynthetic pathway was introduced into Synechococcus elongatus PCC 7942 for the photosynthetic conversion of CO2 into 1,3BDO. Although we were not able to obtain the strain of cyanobacterial 1,3BDO production yet, the cyanobacterial engineering system was established, and the heterologous expression of the pathway genes was demonstrated via genetic engineering of Synechococcus elongatus PCC 7942.Ph.D.2020-04-30 00:00:0