Design
In biological aspect, BNU_China wants to solve the questions of plasmids’ genetic and structural instability, which directly cause strain’s degeneration and then weaken the function of target genes. These instability and degeneration-related influence can be clearly valued by observing expression-related phenomenon, like the amount of production and enzyme activity.
We firstly design the gene loops described in Fig.1. When engineered strains produce the wanted product, the downstream growth factor will be expressed and accelerate the growth of strains. In opposite situation, the growth factor will not be activated and strains will relatively get the growth inhibition.
1) Exogenous pathway transformed into strains. In this scheme, pathway is consist of gene1 and gene 2. Genes’ co-work can catalyze the precursor to the product.
2) Repressor. Repressor can specifically bind with promoter at the upstream of growth factor. It also can bind with product, then change its conformation and release from promoter, allowing the subsequent genes expressed.
3) Growth factor. It can give engineering bacteria growth advantage, making engineered-strains a relatively dominant species compared with degenerated strains.
In order to use specific experiments to verify our first idea, we separate the loop into two modules. Module 1 is growth factor module aiming at give strains the growth advantage. Module 2 includes a valuable product, a specific allosteric protein, and a negtive promoter. General relationship in module 2 can be clarified by that in β-galactosidase, lac1 repressor and lac promoter.
Module 1
According to literature, expression of glucose dehydrogenase (gdh) can significantly enhance the growth rate of Escherichia coli and Bacillus subtilis. In Bacillus subtilis, when consumed same amount of glucose, the dry weight of the gdh-overexpressed strain is larger. That is to say, gdh-overexpression significantly increases the metabolic efficiency of glucose1-3. Gdh works in the branch of the pentose phosphate pathway. It converts glucose to gluconate, and then gluconate is phosphorylated into 6-P-gluconate and enter the pentose phosphate pathway (Fig.2). This metabolic process generate products includes NADPH, which significantly increases the cell's reducing power and helps cells fight against oxygen free radicals, and different carbon number compounds (C3 to C7), which play an important role in cell damage repair, energy supply and so on (Fig.3). （For example, product ribose-5-phosphate is a precursor to synthetic DNA and RNA, and product glyceraldehyde triphosphate is an important intermediate in glycolysis.）
Module 2
There are some allosteric proteins in E. coli. They can works together with other genes, forming an operon to perform certain function. One of the operons, emrRAB (also known as mprA or marRAB), generally has resistance to some bacteria-harmful uncoupler. emrR, the repressor, can be expressed and bind to its own promoter and inhibit the express of emrA and emrB. When emrR bind with uncoupler like DNP, it is no longer able to bind with its promoter and open or enhance the expression of emrA and emrB. Then cell membrane’s permeability of cell membrane to uncoupler increase can increase and pumped uncouplers out of the cell.
According to literature, salicylic acid (SA) is also one of the allosteric inhibitors of emrR. SA is an important organic product that can be used in the production of cosmetics, preservatives, medical supplies, and so on. Nowadays, commonly used industrial synthetic method to produce SA is the Kolbe-Schmitt method, which is a three-step reaction using phenol as raw material. However, this method costs a lot of energy and quite a long time, and phenol has a carcinogenic effect, which is very harmful to the human body and the environment. Therefore, it is promising to replace chemical synthesis method by biosynthesis method.
In nature, only few strains can produce SA, and they are difficult to be isolated or be screened. Recent years, some laboratories have succeed in SA’s biosynthesis in E.coli by utilizing the shikimate synthesis pathway, with a maximum yield of up to 1 g/L. Chorismate, an intermediate product in the pathway, is catalyzed to iso-branched acid by isochorismate synthase (ICS), and then catalyzed to SA by iso-branched pyruvate lyase (IPL) (Fig.7).
Based on the research results from literature, we have the idea of applying our anti-degeneration loop to produce SA in order to increase production and reduce costs. It will undoubtedly have a positive effect on the industrialized biosynthesis of SA.
Previous experiments and literature have shown that both Pseudomonas putida and Pseudomonas fluorescens contains natural ICS and IPL genes and both genes can express and work in E.coli. Meanwhile, we found pchBA (Part: BBa_J45319), an IPL-ICS genes extracted from Pseudomonas fluorescens, in iGEM2018kit. So we use this pchBA and the positive-feedback loop mentioned above to design the pathway showed in Fig.7. When pchBA is correctly expressed, SA is produced in the bacteria and makes the downstream gdh expressed. The expression of gdh can give bacteria growth advantage, so that the bacteria can produce larger amount of SA. In this way, we construct a positive feedback, allowing a vigorously growth and higher production to bacteria.
2) RBS: ribosome-binding site.
3) T: transcriptional terminator.
4) emrR: repressor protein, which specifically binds to PemrR and blocks the transcription of PemrR. After binding to SA, emrR change its own conformation and is no longer able to bind with PemrR.
5) PemrR: negative promoter, which is unable to transcript after binding to emrR. Only works when SA or uncouplers release the inhibition of emrR.
6) gdh: glucose dehydrogenase. It works as a growth factor that can enhance the pentose phosphate pathway and give the strains growth advantage.