Systematical Engineering And Construction Of Succinate And 5-aminolevulinate Synthesis Pathway In Escherichia Coli

In the 21st century, as environmental pollution and the increasing depletion of oil resources, the way of sustainable development has attracted much attention. Industrial microorganism as an important support of sustainable development is a key way to solve resource crisis, ecological crisis and the transformation of traditional industries. Currently, industrial microbial technology has penetrated into almost all industrial areas, such as medicine, agriculture, energy, chemicals and environmental protection, and played an increasingly important role. Metabolic engineering has been developed as a powerful tool to optimize the metabolic pathways and increase microbial products. Escherichia coli, one of the best studied microbial model organism, has many incomparable advantages, including clear genetic background, easy to operate, grow fast and easy to cultivate. Accordingly, it is very important to achieve the fundamental change of source of industrial raw materials to produce important industrial products with engineered E. coli.In this study, we firstly constructed a stress induced expression system based on analyzing the way of gene regulation in E. coli. This system was independent of inducer artificially added. When cells enter the late exponential phase, this system is induced quickly and transcribes genes downstream. As an example, we successfully applied this system in PHB production. Fermentation results showed that without any inducer addition, recombinant strain DH5a (pQKZ103) accumulated CDW, PHB concentration and PHB content were 4.1 g/L,3.52 g/L and 85.8 wt.%, respectively. However, the control strain DH5a (pBHR68) with addition of 0.1mM IPTG, CDW, PHB concentration and PHB content were only 2.3 g/L,0.98 g/L and 42.6 wt.%, respectively. Obviously, the results validated that the stress-induced system constructed in this study was much better than the IPTG induced system.After construction of the stress induced system, our work was focused on the analysis of the glucose central metabolic pathway. During production of recombinant protein or other important industrial materials from glucose, about 30 percent of carbon source was driven to acetate synthesis. Accumulation of acetate not only wasted the carbon source, but also poisoned cell growth. How to solve the problem of acetate secretion has attracted much attention. Here, we inactivated a list of related genes ptsG, poxB, pta and iclR, and to analyze its effect on acetate production. In the end, we successfully decreased the acetate accumulation distinctly and constructed an aerobic fermentation platform:E. coli QZ1110. Meanwhile, we constructed a vector for non-coding sRNA expression, aimed to introduce a potential regulation tool in metabolic engineering. As an example, we over-expressed sRNA RyhB in QZ1110 to analyze its effect on glucose central pathway. Cultivation results showed that compared with the control strain, overexpression of RyhB resulted in 7-fold succinate accumulation. Later, we inactivated sdhA gene on chromosome to constructed strain QZ1111, which accumulated 26.4 g/L succinate under aerobic conditions.From the perspective of redox equilibrium, succinate is more oxidized and PHB is more reduced, respectively, than the glucose substrate. Consequently, we introduced the PHB synthesis pathway into QZ1111, to engineer a co-production (succinate and PHB) pathway from glucose. Cultivation results revealed that compared to single succinate fermentation, coproduction strategy was much more efficiency for substrate utilization. Enlightened by biorefinery, we constructed an engineered strain to co-utilize glycerol (or glucose) and fatty acids to produce succinate and polyhydroxyalkanoate (PHA). This method provided a way to utilize the biodiesel wastes:glycerol and fatty acids.Based on the studies about construction of succinate producing strains, my study was concentrated on another important related compound:5-aminolevlinic acid, ALA. In the past years, ALA was synthesized by chemical method and it used widely in agricultural and medical field. Recently, Biotransformation of ALA with C4 synthesis pathway attracted much attention and studies. Succinate and glycine artificially added were catalyzed to ALA by E. coli cells, which overexpressed ALA synthetase. In the present study, we want to engineer a novel ALA fermentation pathway according to the native ALA C5 synthesis pathway. Firstly, cultivation results validate that HemA was the first rate-limiting enzyme of the C5 pathway. Furthermore, we also find that HemL was also the committed enzyme. The engineered strain DAL, which co-overexpressed hemAM and hemL genes accumulated ALA to 2.05 g/L. Meanwhile, cultivation results of overexpression of gltX decreased ALA production. The reason was discovered by RT-PCR that overexpression of gltX up-regulated the transcription of hemB gene, which driven more ALA to the next reactions. To accelerate ALA extracellular transportation and decrease ALA concentration intracellularly, we analyzed the membrane proteins related to amino acid transportation. rhtA gene was chosen and was overexpressed with hemAM and hemL. Fermentation results showed that overexpression of rhtA increased ALA substantially (about 45.9%). The maximal ALA production was achieved (4.13 g/L) from batch fermentation operated in 5L fermentor. In E. coli, hemB encoded HemB, which catalyzed ALA to PBG. To increase ALA accumulation, the C terminal of hemB on chromosome was added a modified ssrA protein degradation tag, which driven HemB to degrade by protease ClpXP with assistant of small protein SspB. However, Degradation of HemB not decreased ALA accumulation significantly (25 mg/L). When inactivation of sspB gene, ALA production was restored (735 mg/L). The interesting results revealed that degradation of HemB was harmful to ALA production.