Team:OUC-China
OVerview
Nowadays, strain quality has become a major factor limiting the yield of fermentation industry. The engineering bacteria degenerate into wild-type due to mutation or some unknown endogenous regulation. They have more growth advantages than the production engineering bacteria and gradually occupy resources to become the dominant species. The production genes introduced into the engineering bacteria are copied and lost in the process of DNA replication during cell proliferation. At the same time, some products accumulate in the engineering bacteria, which has a negative feedback regulation effect on the production of enzymes. Moreover, the extraction process of the products by breaking the engineering bacteria is very complicated. We plan to design a software and hardware platform that automatically regulates the production and screening of strains to extract product simply to ensure high-quality strains and improve the fermentation yield.
Number of copies
The introduced production genes are copied and lost in the continuous DNA replication as cells proliferate. So we decided to design a gene circuit that can maintain the number of copies of production genes. We try to knock out a gene necessary for the growth and introduced it into the plasma with a weak expression promoter added before it. If the copy number was too low after the production gene introduced into the engineered bacteria, the engineered bacteria would die. In this project, we knock out phosphofructokinase, which is the key enzyme for engineering bacteria to use glucose in the culture medium. In fact, we successfully constructed a strain of Aureobasidium melanogenum for maintaining the copy number of production genes, and found that it has roughly the same production activity as the strain without PFK.
The activity of strain knocked out PFK
According to the results of measuring the growth curves of strains without PFK gene knockout and three strains with PFK gene knockout in YPD liquid and YPGL liquid medium, it can be seen that the three strains with PFK gene knockout can hardly grow in the medium with glucose as the main carbon source, while in the medium with lactic acid and glycerol as the carbon source, there is only a difference before and after a logarithmic period compared with the strains without PFK gene knockout, At 60h, the same stable period was reached, which showed that the three strains with PFK gene knocked out had roughly the same production activity as the strains without PFK gene knocked out.
Quorum sensing
When microorganisms are used for production, the yield is often reduced due to resource competition between growth and production metabolism. In order to avoid such adverse effects, we can achieve the purpose of increasing the yield by dynamically regulating the growth and production metabolism of engineered microorganisms. Quorum sensing- mediated dynamic control is a very useful strategy to balance the growth of cells and production of metabolites. Unlike most dynamic regulation strategies that require addition of an inducer or need pathway-specific parts such as special transcription factors which respond to products or key intermediate metabolites in metabolic pathways, quorum sensing-based dynamic regulation is pathway-independent and inducer-free. It is triggered when a certain cell density is reached, which is an important parameter in many metabolic engineering applications.
We use cytokinin-mediated quorum sensing circuit from Arabidopsis thaliana makes our engineered microorganisms only reaches a certain population density will start fermentation process, used for decoupling competition between cell growth and fermentation. Cytokinin isopentenyladenine (IP), as quorum sensing molecule, is the product of ATP catalyzed by AtlPT4. When the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the expression of production gene controlled by SSRE promoter. In our experiment,to better characterize SSRE promoter and quorum sensing circuit,we put eGFP gene at the downstream of SSRE promoter.
SSRE promoter
For IP dose–response experiments, all strains were precultured for 8h with terminal OD600 of 0.4, then treated with IP for 0，0.5，1，1.25，1.5h at 28℃, 175 rpm, and the fluorescence intensity of the strains was measured. Different concentrations of IP can increase the expression of SSRE promoter. After rough statistical analysis, the expression intensity of SSRE promoter was significantly correlated with IP concentration,showing that SSRE promoter can fulfill our assumption to regulate the production and growth.
Kill Switch
To enable the engineered microorganism to initiate suicide at low yields, we designed kill switch whose aptamer faction binding to the product would activate ribozyme activity, causing the designed mRNA to self-cleavage and preventing YopE expression.
By using a RNA-based gene-regulatory platform, we design a ribozyme switch linked behind YopE mRNA, it contains a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of hammerhead ribozyme sequence.
Threshold
In order to roughly measure the threshold of kill switch through experiments, we replaced the expression gene controlled by ribozyme switch with eGFP. Because we could not get to wet lab to measure the threshold of our ribozyme switch due to the detection equipment failure,we used the dissociation Constant(Kd) of RNA aptamer to simulate the threshold of our kill switch.
As shown in the result of simulation,the threshold of our ribozyme switch is very low ,in other words,it is very sensitive.
Toxic protein
At the beginning of the project design, we chose Yope as the suicide protein of our engineering bacteria. The YopE cytotoxin of Yersinia pseudotuberculosis an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid-encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. After communicating with other teams, we found that using Yope as the suicide protein in our design not only posed a security threat to the platform's R&D personnel, but also posed a greater security problem to the staff of the microbial factory. After communicating with the DUT-China team to test the sacB toxic protein in their project, we decided to use sacB as the final suicide protein of our platform because of its good killing effect and better biosafety.
Fermentation tank simulation
Because we can't use the large fermentation tank used in the company's production, it is difficult for us to know whether the platform we designed has the function of successfully enabling the engineering bacteria to automatically adjust the production and growth continuously ferment to increase production. In order to make up for this defect, we give full play to the advantages of dry experiment, and use cellular automata to simulate the fluctuation of the engineering bacteria output in the fermentation tank platform design. In the process of visualization, we simulated three groups, representing engineered bacteria introduced CHIP components ,engineered bacteria introduced CHIP components except transport RNA and common engineering bacteria. After the simulation, we collect the output of each group of engineering bacteria, and draw the figure below for analysis. We found that the output of engineered bacteria carrying CHIP is significantly higher than that of common engineering bacteria under the same fermentation conditions, which can explain the function of CHIP components to a certain extent.
The results are very exciting: our platform can enable the engineering bacteria to automatically adjust the production growth and continuously ferment to increase production.In the future, we may need to import all our parts into engineering bacteria to detect the combined expression of platform elements. At the same time, we will also test the productivity of the platform to further upgrade the software.
