Team:SCUT-FSE-CHINA/Description
Description
Background
What is industrial fermentation?
Industrial fermentation is the intentional use of fermentation by various microorganisms to convert raw materials into some useful products for humans.
Application of industrial fermentation
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Fermented products have applications as food, medicine, chemical industry, agriculture, bio-energy as well as environmental protection. Nowadays, industrial fermentation processes are increasingly popular.
Food
Fermented foods are among the oldest processed foods and have formed a traditional part of the diet in almost all country for millennia. Today they continue to form major sectors of food processing industry, including baked products, alcoholic drinks, yogurt, cheese and soy products among many others. The use of mild conditions of pH and temperature often produce various food with the nutritional properties and sensory characteristics that cannot be achieved by other methods, with a low energy consumption as well as relatively low capital and operating costs. Many other substances occupied by food industry such as some food additives, L-malic acid, citric acid, glutamic acid, are generated by fermentation process.
Medicine
Industrial biotech experience in fermentation technology started in the field of single cell protein production and then moved on to the developments of fermentation technologies producing antibiotics, amino acids, antitumor agents, statins, vitamins and steroids. The use of microbial fermentation can provide new means for the development of new drugs, improving drug efficacy and reducing the
side effects of drugs. The biological diversity of microbes provides abundant alternative strains for the researchers. Based on fermentation engineering technology, a wide variety of drugs such as recombinant human growth hormone, recombinant hepatitis B vaccine, interleukin-2, Factor VIII-related Antigen and so on have been developed. Microbiological drugs account for more than 20% of the global pharmaceutical market, and for more than 35% of the Chinese mainland market. Antibiotics, as the most widely used drugs of clinical treatment in China, account for about a quarter of the total clinical medication.
Chemical Industry
Each year, new products are added to the list of chemical compounds derived from fermentation. Several vitamins now are produced routinely, employing fermentation steps in their synthesis. Outstanding examples are vitamin C. B2, B12, D and β-Carotene. Fermentative syntheses of the amino acids L-lysine and L-glutamic acid also are being carried out commercially. Some of the more interesting fermentation process are the valuable polymer biomaterials such as PHA (polyhydroxyalkanoates), which can also be used as a biomedical material and a biodegradable packaging material, with both good biocompatibility, biodegradability and thermal workability of plastics. Other examples in this field are the production of abundant enzymes and single cell protein.
Agriculture
Important agricultural uses are found for the new fermentation products gibberellin, a plant growth regular; and crystalline inclusions for a species of Bacillus are being used as specific insecticides in another agricultural application. Besides, a wide variety of agro industrial waste products can be fermented to use as food for animals, especially ruminants. Fungi have been employed to break down cellulosic wastes to increase protein content and improve in vitro digestibility.
Energy
Fermentation is the main source of ethanol in the production of ethanol fuel. Common crops such as sugar cane, potato, cassava and corn are fermented by yeast to produce ethanol which is further processed to become fuel.
Bio-hydrogen production is a newly developed bio-technology depending on microbial fermentation, with renewable anaerobic microorganisms as the bioreactor. Using the industrial and agricultural organic waste as the fermentation substrate, it helps to lower energy consumption and economic cost, which has great application prospects and development potential.
Environmental protection
The application of microbial fermentation technology is an effective way to realize harmlessness, reduction and resource utilization.
In the process of sewage treatment, sewage is digested by enzymes secreted by bacteria. Solid organic matters are broken down into harmless, soluble substances and carbon dioxide. Liquids that result are disinfected to remove pathogens before being discharged into rivers or the sea or can be used as liquid fertilizers. Digested solids, known also as sludge, is dried and used as fertilizer. Gaseous byproducts such as methane can be utilized as biogas to fuel electrical generators. One advantage of bacterial digestion is that it reduces the bulk and odor of sewage, thus reducing space needed for dumping.
Industrial fermentation also involves in utilizing waste. For example, there are annually over 4.5 million tons of organic matter in paper mill waste produced in China, which can be used as the substrate of the biogas fermentation and generate 16.8 kilowatt hour of electricity. Meanwhile, due to the huge amounts of SSL (sulfite spent liquor), waste from paper production, it was attractive to search for an added value use of this paper mill waste stream. Thus, SSL was commercially used in alcohol fermentation in the 1940s and later on in the production of protein biomass (single cell protein) for animal feed.
How to make the industrial fermentation products become more economically competitive? (Discussion on the economics of the fermentation.)
However, Industrial biotechnology has not developed as fast as expected due to some challenges. Especially for its high production cost compare to the chemical technology. Reducing the production cost can make these products become more economically competitive and maximize the profit. Actually it is one of the prime concerns in this industrial fermentation era.
Production costs are mainly composed of the following aspects:
It can be easily noted that the energy consumption accounts for a big part in entire production cost, while most of the energy is occupied by the sterilization processes. To achieve sterility condition, high temperature as well as high pressure are always used to make the whole fermenter system under a sterile condition. That means the energy-intensive sterilization process is actually a cost-intensive process. Additionally, better sterilization facilities are associated with higher economic cost. Therefore, it is easily to realize that sterilization process contributes to the high cost of bio-products. There is no doubt that if the fermentation process can achieve open(unsterilized) culture will greatly reduce the cost of production.
How do people generally achieve open(unsterilized) fermentation processes?
In nature, there are extreme environments where most of the organisms can not survive, such as high temperature, low temperature, high acid, high alkali, high salt, high toxicity, hypertonic, high pressure, drought or high radiation intensity and the other. Microorganisms that rely on these extreme environments for normal growth and reproduction of microorganisms are known as the extreme microorganisms. Extremophile is a hot issue as many researchers are trying to find out useful bacterial species with proper characteristics and their specific applications. Researchers are working on building genetic manipulation systems in extremophile to develop, transform and utilize important functional materials obtained from these bacteria. Extreme environments are actually suitable for open (unsterile) fermentation processes as it inhibits the growth of normal bacteria and the contamination of undesired bacteria.
However, limitations of utilizing extremophile include the followings:
- Time-consuming: it may take a long time for screening the desirable strains for specific production of bio-products in extreme environments. Meanwhile, it is difficult to isolate and cultivate them in the laboratory.
- Higher economic cost: the extreme environments and complex nutrients required for the growth of extremophiles leads to the higher cost in the cultivating facilities and materials.
- Lower production rate: the extreme microorganisms may grow much slower than normal bacteria, which can result in decreased production efficiency.
What are we doing?
In order to obtain high-quality fermentation products with low cost, achieve maximum profits and sustainable development, by integrating zymology and economics, we try to introduce a “robust strain” for developing the fermentation process in the open (unsterile) culture. If well conducted, open fermentation processes will lead to reduction of production cost in industrial fermentation.
Design
How do we achieve open culture in industrial fermentation processes?
Obviously, to achieve open fermentation process, the specific engineering bacteria must be easily grow and yield bio-products efficiently during the long cultivations without any risk of microbial contamination and phages infection, even under ‘unsterile’ condition. It means that sterilization is not required as microbial contamination and phages infection is unlikely to happen.
Here, we introduce the metabolic pathway of formamide and phosphite (N&P Pathway) into the host to fit our special designed medium to avoid microbial contamination and apply CRIPSPR/Cas9 system for the host to attain phage resistance ability. Eventually, an open fermentative process can achieve.
① N&P Pathway:
In normal medium, the nitrogen and phosphorus sources are usually NH4+ and HPO42-, which can be easily utilized by most of the microbes. Under this situation, the unmodified Escherichia coli and other undesired microbes can both grow in the open(unsterile) culture. So it is difficult to avoid contamination of bacteria. In order to resolve the problem of microbial contamination in fermentation process, we have constructed an expression vector possessing formamidase gene as well as phosphite dehydrogenase gene. Consequently, as we transform the plasmid into the E. coli. This new engineering E. coli can utilize nitrogen and phosphorus source by hydrolyzing formamide to ammonia and oxidizing phosphite to phosphate for its own utilization and growth. Consequently, the basal MOPS medium in the presence of formamide and phosphite, but lacking of the basic nutritional components of NH4+ and HPO42-, makes the engineering E. coli grow and multiply while other microbes will be "starved" to death due to have no ability to assimilate formamide and phosphite. In a word, in this particular MOPS medium, the engineering E. coli would be hopefully considered as a promising bacteria to take advantage of formamide and phosphite as nutrient substance to become the dominant bacteria.
② CRIPSPR/Cas9:
In the evolutionary process, bacteria have developed an immune system--CRISPR. CRISPR/Cas immunity is a natural process of some bacteria and archaea species.
Based on this, We used a targeted gene editing strategy by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize the anti-phage ability of our engineering E. coli. In this method, the 20-bp complementary region (N20) with the requisite PAM (NGG) matching genomic loci of interest was programmed directly into a heterologously expressed CRISPR array, and fused crRNA and tracrRNA as a single synthetic guide RNA (sgRNA) transcript obviated the need for processing the transcribed CRISPR array (pre-crRNA) into individual crRNA components.
For genome editing, we assembled a two-plasmid system, in which the cas9 gene and the sgRNA directing it to the targeted region were separated in the pCas and pTarget, respectively. pTargetF consists of the sgRNA sequence, the N20 sequence and the multiple restriction sites. The N20 sequences were selected according to the principle that they should be the most conserved among related genomes from common phages, to ensure a wider range of adaptive immunity of our engineering E. coli BL21(DE3).
The initial aim of our design was to develop a methodology about anti-phage, so we started from the most available T7 phage and attempted to select target N20 from the genome of T7 phage.
First, we aimed at essential proteins of the phage in its infection to host bacteria. Then, we searched the protein coding sequences to find any occurrences of the PAM site NGG. Potential N20 sequences adjacent to the PAM site would be further considered if they were the most conserved among related genomes from the T7 phage, to ensure a wider range of adaptive immunity of our engineering E. coli BL21(DE3).
The two-plasmid system functions when the viral DNA invaded. The Cas9 protein form a complex with the sgRNA and then sgRNA direct the Cas9 protein to the corresponding sequence of the selected N20 in the invaded DNA. The Cas9 recognizes the PAM adjacent to the target gene and use its dual HNH and RuvC endonuclease domains to cleaves the DNA, so as to stop the virus in its invasion process.
③ Robust System:
After successfully building up the recombinant E. coli BL21(DE3)(pGEX-for-ptx) and BL21(DE3) (pCas-pTarget-N20), we aimed at combining these two systems together to endue the E. coli with the abilities to utilize formamide and phosphite to be dominant during the cultivation, as well as resist the phage. To construct the robust system, three plasmids including the pCas, pTarget-N20 and pGEX-for-ptx had been co-transfected into BL21(DE3), resulting in the recombinant strain BL21(DE3) (pCas-pTarget-pGEX-for-ptx), called Robust E. coli.
How to conduct open(unsterilized) fermentation processes by using Robust E. coli.?
Picture 1. Contents of carbon, nitrogen and phosphorus in several common fermentation substrates.
In the first step of preparation of fermentation substrates, the raw materials containing biomass is separated into pieces by physical methods. The main products will subsequently be subjected to microbiological or chemical methods for starch gelatinization and saccharification. In industrial feedstocks showed in Picture 1, the contents of nitrogen and phosphors are much less than that of carbon. So, essential nitrogen and phosphorus supplements are required for normal fermentation. In our design, raw materials that have comparable limiting phosphorus and nitrogen contents will be supplied with formamide (HCO-NH2) and phosphite(PO33-), making them feasible for the growth of our Robust E. coli and further fermentation process.
