Production Of3-hydroxybutanone By Systems Metabolic Engineering In Candida Glabrata

In this dissertation, a pyruvate producer, a multi-vitamin (thiamine, biotin, pyrodoxin andnicotinic acid) auxotroph Candida glabrata strain, CCTCC M202019was employed as amodel system to produce the valuable chemical of3-hydroxybutanone from pyruvate. Thiscan be realized by combination of genome scale metabolic model (GSMM), systemsmetabolic engineering, cofactor metabolic engineering and sub-cellular metabolic engineering.Additionally, the present study also investigated the mechanisms that protected C. glabratafrom3-hydroxybutanone stress, and provided an alternative strategy for rationally improvingthe growth performance of eukaryotes under high environmental stress. The main results weredescribed as follows:1. GSMM iNX804of C. glabrata and constraints-based methods were applied to analyze theeffects of modified models on3-hydroxybutanone production, in which the reactionsrepresenting the enzymatic activities of α-acetolactate synthetase (ALS) andα-acetolactate decarboxylase (ALDC) were added into iNX804for flux balance analysisby the FBA algorithm. As a result, compared to the wild-type strain, the theoreticalproduction rate of3-hydroxybutanone was increased by0.24mmol g DCW-1h-1. Basedon an in silico strategy, a synthetic, composite metabolic pathway involving two distinctenzymes, acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC), wasconstructed, leading to the accumulation of3-hydroxybutanone incrased to1.14g L-1in C.glabrata. Additionally, further genetic modifications (deletion and expression of genes)and regulation of intracellular NADH level were applied to increase the carbon flux of theheterologous pathway. Consequently, a higher production of3-hydroxybutanone (3.67g L-1) was obtained with C. glabrata.2. Subcellular metabolic engineering was applied to engineer C. glabrata for3-hydroxybutanone production. With the aid of mitochondrial targeting sequences(MTS)-CoxIV, a heterologous3-hydroxybutanone pathway was targeted into themitochondria to realize the accmulation of3-hydroxybutanone. Compared to thecytoplasmic pathway, the production of3-hydroxybutanone and its yield on pyruvate andbiomass were increased by16.1%,13.3%and20%by mitochondrial pathway. Meanwhile,overexpressing mitochondrial pyruvate carrier (MPC) was also applied to enhance thetransporation of mitochondrial pyruvate with97%, and then increase the concentration ofmitochondrial pyruvate. As a result, the strain comprising the combination of the heterologous pathway and MPC could yield approximately3.26g L-1of3-hydroxybutanone and0.19g g-1on pyruvate.3. Based on the known regulatory and metabolic information, acetaldehyde and thiaminewere fed to identify the key nodes of carboligase activity reaction (CAR) pathway andprovide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase,acetaldehyde dehydrogenase, and pyruvate decarboxylase were selected to be manipulatedfor strengthening the CAR pathway. Following the rational metabolic engineering, theengineered strain exhibited increased3-hydroxybutanone biosynthesis (2.24g L-1). Inaddition, through in Silico simulation and redox balance analysis, NADH was identified asthe key factor restricting higher3-hydroxybutanone production. Consequently, bycombining the rational metabolic engineering and cofactor engineering, the3-hydroxybutanone-producing C. glabrata was improved stepwise, the final3-hydroxybutanone production was further increased to7.33g L-1.4. To construct a robust yeast strain, the physiological and biochemical characteristics of C.glabata were investigated to resist3-hydroxybutanone stress. The results suggested thatthe high3-hydroxybutanone concentration could significantly decrease cell viablitiy,inhibit cell growth, increase cellular ROS level and decrease the production of cellularATP, and then accelerated cell apoptosis induces. Furthermore, the combination ofRT-PCR and metabolic engineering were applied to demonstrate enhancement ofmitochondrial fusion could significantly increase the cell tolerance for3-hydroxybutanonestress through inhibiting ROS production, increasing intracellular ATP production, andmaitaining the banlance of mitochondrial membrane potential. The results provided analternative strategy for rationally improving the growth performance of eukaryotes underhigh environmental stress, and also enlarged the knowledge of the mechanism of celltolerance through process of energy-related metabolic pathways.