Team:BIT-China/Background
Background
With the rapid development of modern biotechnology, fermentation engineering has
made great progress in microbial breeding, biosynthesis, regulation mechanism
and the development of microbial resources, which is laying a solid foundation
for the emergence of the concept of cell factory. Using DNA recombination
technology to construct genetically engineered bacteria to complete industrial
production has become the best choice for more and more manufacturers.
Genetically engineered bacteria not only have extremely low cost and very
efficient output, but also can greatly improve the fermentation level of
products, and can also introduce foreign genes into them to produce products
that could not be produced by original engineered bacteria. It is estimated that
the global market for fermented products has 12 billion to 13 billion US
dollars, of which 46% are antibiotics, 16.3% are amino acids, 13.2% are organic
acids, 10% are enzymes and 14.5% are other fermented products
Significance of resource allocation
In current methods, many strategies have been used to increase productivity of cell factories, such as transcription of genetic materials, regulation of translation, gene coupling and decoupling, alteration of metabolic pathways, changes of key enzymes, and changes of substrate concentration. But if we want cells to function differently at different times and allocate specific resources to specific genes producing target products, resources need to be allocated reasonably.
Significance of transcriptional resource allocation
The application of refined cell factories to achieve Bio-Green manufacturing of energy, medicine, chemicals as well as efficient and sustainable production requires proper allocation of resources that can be useful to cells. However, there are many kinds of resources that can be utilized by cells. Why do we choose those of transcription to manage? Transcription is an important part of the central law and the first step in gene expression. Catalysts and enzymes are decisive factors affecting the occurrence of intracellular metabolic reactions and their reaction rates. When the catalytic properties of the enzyme are constant, the amount of the enzymes determines the reaction rate. The amount of enzymes depends on the transcription and translation of the corresponding genes in cells. Here, we define transcription resources as substrates (nucleotides, etc.)that are directly involved in transcription and substances (transcription factors, RNA polymerase, etc.) that assist in transcription. In refined cell factories, the amount of transcriptional resources available to cells are limited, and we need to make efficient use of them. The production model we designed is that cells grow at a high speed in the prophase, and transcriptional resources are allocated to the metabolism of cell growth. When the number and size of cell factories grow to the appropriate scale, the focus of transcriptional resource allocation shifts from cell growth to cell production. It is a kind of new cell factory with high intelligence and refinement. Such cell factory can minimize the consumption of resources needed for their own growth and accelerate their growth during the growth period, while most of the resources are used to synthesize target products during the production period, thus saving resources and increasing production. For industrial production, our transcription resource allocation system can also save costs and efficiently produce more target products.
Quorum sensing
Bacteria can sense the concentration changes of a small molecular weight chemical
signaling molecule secreted by other bacteria growing with them in the growing
environment. This phenomenon is called Quorum Sensing (QS), which can promote
the communication between bacterial individuals and coordinate the behavior of
groups. These signaling molecules secreted by bacteria can regulate their
biological behavior, so they are called self-inducible factors. Once the
concentration of self-inducible factor reaches a certain threshold, the quorum
sensing will activate or inhibit the expression of some target genes, thus
regulating the biological behavior of bacteria. Quorum sensing is involved in
regulating various biological behaviors of bacteria, such as bioluminescence,
cell membrane formation, cell differentiation, extracellular polysaccharide
formation, exercise, antibiotic production, etc
The QS signaling molecule of Gram-negative bacteria is also known as autoinducer (AI1). AHLs consist of a conservative hydrophobic head of the homoserine lactone ring and a variable hydrophilic tail of the amide side chain, which determines the diversity of AHLs. The differences between AHLs are mainly reflected in the presence or absence of side chains of amides, the difference of substituted groups (hydrogen, hydroxyl or carbonyl) at the third carbon atom of amides, and the presence or absence of one or more unsaturated bonds in side chains. The difference of AHLs is formed by the acyl side chains of different acyl carrier proteins bound by homoserine in the synthesis process. The difference of AHLs is due to the combination of homoserine with acyl side chains of different acyl carrier proteins in the synthesis process. AHLs have short amide side chains that allow them to pass passively into and out of bacterial cell wall. This is different from AHLs and AIP (signal molecules of Gram-positive bacteria) with long amide side chains, which cross bacterial cell membranes through an active transport mechanism.
LuxI and LuxR proteins are involved in quorum sensing in most gram-negative
bacteria. LuxI protein is an autoinducer synthase, which can synthesize signal
molecules AHLs. LuxR protein is an autoinducer receptor in cytoplasm and also a
transcription activating element. With the proliferation of AHLs outside the cell,
its density also accumulates with the increase of cell density. When the density
of this signal molecule accumulates to the critical density, it binds to LuxR.
And the binding complex can activate gene transcription. Since LuxR protein only
binds to specific AHLs, LuxI / LuxR system is also mainly used for intraspecific
quorum sensing. However, LuxR protein that bind several AHLs have been reported.
For example, Salmonella SdiA protein are mainly related to bacterial
interspecific induction
σ factor expression system and T7 expression system
σ factor and core enzymes are the components of RNA polymerases. σ factor itself can’t initiate transcription, but can specifically tow RNA polymerase to specific promoter binding sites. The σ factor expression system has the advantages of no cytotoxicity and no leaked expression, and it has many optional promoters and a wide range of gene expression intensity.
T7 RNA polymerase-dominated transcription is very active. RNA polymerase-driven transcription in host cells can not compete with T7 RNA polymerase. Almost all transcription in cells occurs rapidly due to the activation of T7 RNA polymerase.
Lycopene
Lycopene, also known as ψ-carotene, belongs to isoprene compounds. Lycopene is a kind of non-circular planar polyunsaturated fatty hydrocarbon. Its molecular structure formula contains 11 conjugated double bonds and 2 non-conjugated double bonds. Lycopene is the most effective antioxidant in carotenoids. It regulates the metabolism of cholesterol and has the functions of anti-cancer and improving immunity. Lycopene is widely used in specific diet, medicine and cosmetics.
Significance for Industrial Production
Our project gives bacteria the wisdom to judge their own density and transform between growth and production. This has great practical significance.
Firstly, compared with the high density culture technology commonly used in
industry, it is no longer necessary to monitor the bacterial density in the tank
in real time, which reduces the cost of technology
Secondly, our strain can utilize industrial materials more efficiently, achieve the best balance in growth rate and production rate, increase the production of target products, avoids the inhibition of metabolic byproduct on recombinant cells, and reduces the oxygen supply restriction in fermentation tank.
Finally, the idea of allocating transcriptional resources in our project provides a feasible direction for future industrial intelligent production and lays a foundation.
If the allocation of transcriptional resources can be achieved to the extreme, the secondary metabolites in cells will only be our target products. Resources will be more concentrated on the target products, which will further improve efficiency and further reduce the waste of resources.
Our iGEM project this year can also be used as a standardized component for technical integration with previous iGEM projects. That is to say, we hope to integrate the various functions implemented in previous iGEM projects, such as microorganism intelligent response and regulation of temperature, pH and redox state(I'M HeRE, 2013, pH controller, 2015, who can get an A, 2018), intelligent protection of engineering bacteria technical safety (E.co-Lock, 2014), and automatic elimination of low-yield engineering bacteria (P-SLACKiller, 2016), in order to achieve a more intelligent and refined cell factory, further energy saving and emission reduction and increase production.
In the application of industrial production, our engineering bacteria can be further domesticated to adapt to the industrial production of large fermenter environment, and then applied to industrial production. We can increase the negative feedback system to improve the stability of the system. According to the different rates and environments required for industrial production, we can change or mutagenesis based on the sequence library of T7 and σ factors, and modify the critical density of strain for switching between growth and production, in order to meet the production needs of different products. According to the different target products needed in industrial production, we can change the key enzyme sequence after T7 and σ expression system to produce other industrial products besides lycopene and hope this strategy more widely used in production.
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