Team:SCUT-ChinaA/Safety
Biosafety
a. Safety
It is a state whose simplest definition is that there is no danger. The more detailed definition means protection from various types of failures, damages, mistakes, accidents, injuries or other reluctances. These include many aspects, such as body, social, spiritual, financial, political, emotional, professional, psychological and educational. Safety can also be defined as the ability to control a specific identified hazard so that the risk is below an acceptable level, thus reducing the likelihood of health or economic loss. Security can include protection of people or belongings.
In some guarantees or insurances, safety can define the function quality of an item or organization, and this function is harmless. The goal is to ensure that the item or organization only performs the functions that were originally defined.
In some cases, security is a concept of relativity. Sometimes it is impossible to eliminate all risks, or it is not feasible because of its difficulty and its cost. In this case, safety means that the risks and damages of people or items are low, within acceptable limits, and manageable.
b. Biosafety
Based on the possible adverse effects of biotechnology development, the concept of biosafety has been proposed. Biosecurity generally refers to the potential threat to the ecological environment and human health caused by the development and application of modern biotechnology, and a series of effective prevention and control measures taken against it.
Biological hazard: Also known as “biohazard”, it refers to biological or biological substances that are harmful to humans and animals. These materials include but are not limited to: animals, plants, microorganisms, viruses, and tissue sections containing pathogens, body fluids, solid waste, and exhaled breath.
Substances with biological hazards are represented by an internationally common pattern: “☣”.
The term and its symbols are often used as warnings to alert people who may be exposed to biologically hazardous substances to take appropriate protective measures.
Biological hazard level
Level 1:
It is less harmful to humans and animals. The main measures are wearing gloves and paying attention to face protection during contact, washing hands after contact and cleaning the exposed table and utensils.
Listed at this level are Bacillus subtilis, Escherichia coli, chickenpox and so on.
Level 2:
The hazards to humans and animals are moderate or contagious.
Listed at this level are pathogens of hepatitis B, hepatitis C, influenza, Lyme disease, Salmonella, and so on.
Level 3:
The hazards to humans and animals are high, but there are still methods for inhibition.
Listed at this level are pathogens such as anthrax, SARS, HIV, West Nile encephalitis, tuberculosis, yellow fever, and so on.
Level 4:
The highest risk to humans and animals has not been found in any effective vaccine or treatment.
Viruses that have been identified at this level are hemorrhagic fever diseases such as Ebola, Hanta, and Lhasa.
To deal with this level of biological hazard, it is necessary to have a laboratory (BSL4 or P4) that meets the standards of the fourth level. Such laboratories must have extremely strict access control and must be negatively isolated to avoid leak when it is damaged. Workers and items to be treated must be isolated (such as placing the item in a glove box with a negative pressure or wearing a full and independent gas supply gown).
Level 1 Basic laboratory - Teaching of the first level of biosafety, research GMT is not required; open laboratory.
Level 2 Basic laboratory - Primary biosafety level primary health service; Diagnose, research GMT Plus protective clothing, biohazard mark open laboratory, and biosafety cabinet for prevention of possible aerosols.
Level 3 Protection laboratory - Level 3 biosafety level special diagnosis, Research adding special protective clothing, entry system, directional airflow BSC and/or all other basic laboratory equipment required for secondary biosafety protection.
Level 4 Highest protection laboratories - Level 4 biosafety research Levels; Hazardous pathogens research; Add airlocked entrances and exit showers to all other basic laboratory equipment required for the Level 3 biosafety; Special treatments for contaminated items in Class III BSC or Class II BSCs; Wears positive pressure suits; Double door autoclave (through wall) and air filtration equipment.
c. Biosafety assurance
In October 2016, the team members began to enter the laboratory to study the experimental procedures to ensure that all experiments were implemented within a controlled safety range and could cope with any conditions that occurred during the experiment to prevent accidents.
Experimental design: The engineering bacteria we modifyed can only live on uracil medium, preventing it from leaking and polluting the environment.
We strictly abide by the laboratory safety rules and conduct experiments in accordance with standard laboratory requirements. In accordance with the regulations, the waste is placed in a designated container for sterilization, so as to ensure that no strain remains.
http://www2.scut.edu.cn/biology/2012/1210/c3483a69988/page.htm
http://www.chinabaike.com/z/keji/shiyanjishu/2011/0116/166965.html
[1] United States: https://www.fda.gov/default.htm
[2] EU: http://www.efsa.europa.eu/
Biological development
a. Project prospects
With the development of biotechnology, especially the rapid development of synthetic biology in recent years, more and more sustainable production of energy, chemicals and natural products can be achieved through metabolic remodeling of microorganisms. Among them, perfume compounds are an important class of chemicals, which play an important role in the food, chemical, pharmaceutical and other industries, and have a huge and rapidly growing market demand.
Among them, quinone fragrances have become an important high-end spice that has attracted much attention due to its unique botanical aroma and potential medical properties. The production methods currently used are mainly chemical methods, direct extraction methods and biosynthesis methods. Although most of the flavors and fragrances are currently synthesized by chemical methods, however, for terpenoids, chemical methods have bottlenecks that are difficult to synthesize or have low yields and poor quality. The direct extraction method is far from satisfying human needs because most plants grow slowly and are restricted by strict geographical and climatic conditions, resulting in difficult product purification and low yield.
In theory, any naturally occurring terpenoid fragrance can be fermentatively produced by constructing a corresponding synthetic route in a suitable microbial cell. Currently, it is mainly reconstituted in traditional E. coli and Saccharomyces cerevisiae to produce target products, and most of the terpenoids can only obtain simple intermediates by microbial heterogeneous synthesis, and the yield of most products is still limited to milligrams per liter. The level of production has led to low overall production, low production capacity, and difficulty in industrialization. Among them, limonene is a very important anthraquinone fragrance. Similarly, limonene encounters chemical synthesis, which is complicated in process and has a great impact on the environment. Therefore, in combination with the characteristics of products and microorganisms, select appropriate hosts and develop corresponding genetic manipulation tools and methods. On the one hand, it helps to understand the metabolic regulation of organisms and form excellent chassis cells, on the other hand, it is beneficial to promote the commercial production process of biosynthesis of scented spices.
In previous studies, Yarrowia.lipolytica is a non-traditional yeast with great industrial application potential and is currently used in the industrial production of single-cell proteins, single-cell lipids and organic acids. Yarrowia.lipolytica has several advantages over other common industrial microorganisms and has been proven to be useful in the production of a variety of fine chemicals such as organic acids, sugar alcohols, DHA, carotenoids and the like. Numerous studies have shown that Yarrowia.lipolytica has the potential to become a good cell chassis, especially due to the synthesis of secondary metabolites derived from acetyl-CoA (such as terpenoids). However, there are few studies on the synthesis of terpenoids for this yeast, and the genetic manipulation tools and methods are relatively simple and inefficient.
In response to this bottleneck that needs to be broken, the project uses the high-efficiency biosynthesis of the important quinone-flavored limonene as a starting point, and the Yarrowia.lipolytica, which has been widely proven to be an excellent acetyl-CoA derivative, is a highly efficient strain. Enzyme self-assembly system.
The compound is an important part of the flavor. It is also a major ingredient in a large number of high-end flavors and fragrances, mainly found in plants. Terpenoids belong to the class of compounds, and their research is also within the scope of research on terpenoids. The synthesis of terpenoids is carried out via mevalonate pathway, MVA pathway, or 2C-methyl-D-erythritol-4-phosphate pathway, MEP pathway, to obtain isopentenyl pyrophosphate and dimethylallyl pyrophosphate, followed by anthraquinones. The synthase catalyzes the formation of the corresponding backbone by IPP and DMAPP, and synthesizes different terpenoids via cytochrome P450 catalysis or other carboxyl, hydroxyl and glycosylation modifications. Due to the complexity of its metabolic regulation network, it involves many layers and a wide range, which is the difficulty of current research. Therefore, through the study of the important anabolic regulation of terpenoids, it is helpful to promote the discovery of the mechanism of metabolic regulation of the steroids.
In addition to genetic engineering and optimization, optimization of the fermentation process is also an important strategy to increase the yield of terpenoids. E. coli recombinants can be fermented to yield up to 2.7 g/L limonene, and 435 mg/L in glucose, as optimized by carbon source and using glycerol as a substrate. Through the optimization of the medium carbon source and introduction into the dodecane/water two-phase system, the yield of the recombinant E. coli myrcene producing myrcene reached 58 mg/L. In addition, using the fed-batch fermentation strategy, the Pseudomonas putida engineering strain producing linear monoterpene flavoring geranyl acid can ferment and produce 193 g/L geranic acid.
At present, the research on microbial heterogeneous synthesis of scented spices has made some breakthroughs in recent years, but still faces many problems to be solved. Among them, the choice of chassis cells is one of the key factors for the success of microbial synthesis of terpenoid compounds. At present, the most commonly used steroid-like chassis cells are model microorganisms such as Escherichia coli and Saccharomyces cerevisiae, but they are often not realized in the expression of some key enzymes, protein folding and effective cell localization. Recently, a few primitive organisms with relatively simple genetic manipulations, such as Nicotiana benthamiana, ginseng root, and Physcomitrella patens, have been applied to the heterologous synthesis of some complex terpenoids. However, the genetic manipulation and production conditions of these plant cells still have this great limitation. Up to now, the yield of most engineering strains is still limited to the level of milligrams per upgrade, so the development of a more productive chassis cell, more efficient approach to assembly methods and adaptation of artificial biological systems is the synthesis of microbes and spices. The key to industrial applications.
Y.lipolytica is a non-traditional yeast with great industrial application potential. It is currently used in the production of single-cell proteins, single-cell lipids and organic acids, and has great potential in the production and application of terpenoids. Compared with other common industrial microorganisms, Yarrowia.lipolytica has several advantages: 1) the genome-wide sequence of many strains; 2) high biomass, satisfying GRAS; 3) extensive carbon source spectrum, capable of The use of hydrophilic and hydrophobic carbon sources, no significant inhibition of glucose metabolism; 4) strong protein expression, low glycosylation level, can carry out intracellular and extracellular expression and surface display; 5) rapid metabolic rate, acetyl-CoA Strong synthetic ability; 5) It has been proven to be useful in the production of a variety of fine chemicals, especially functional fatty acids or acetyl-CoA derivatives, which has great application prospects. Acetyl-CoA is the synthetic precursor of terpenoids. Currently, two products, DHA and EPA, produced by Yarrowia.lipolytica engineering bacteria have been commercialized. And more products are moving towards industrial production scale, such as the production of acetyl-CoA-derived carotene is currently the highest level in the eukaryotic system. Y.lipolytica is also used in the expression of modified enzymes in the synthesis of perfumes for whole cell catalysis, such as for better conversion and selectivity in the hydroxylation of the outer cyclomethyl; and for the catalysis of limonene Perillate. Furthermore, Yarrowia.lipolytica has fat granules which can regulate the distribution of fat-soluble steroidal compounds in cells, thereby reducing the toxicity of products to cells and increasing yield. Therefore, Yarrowia.lipolytica exhibits strong industrial biotechnology application potential and potential as a scorpion spice chassis cell.
In addition, in eukaryotes, intracellular cells control the interaction of metabolic reactions and metabolites through compartmentalized membrane organelles, which can isolate toxic metabolites, direct the enzymatic reaction, and ensure a suitable microenvironment for enzymatic reactions. Such as Ph, redox state. Studies have shown that by targeting the hospital pathway to peroxisomes or mitochondria, the yield of the target product can be significantly increased, and the accumulation of intermediates or by-products can be reduced.
b. China's biological development status and prospects
After the “11th Five-Year Plan” and “Twelfth Five-Year Plan” policies and support, China's industrial biotechnology entered a period of rapid development. The central government has invested and guided the local and enterprise funding, providing financial support for industrial biotechnology research and development and industrialization.
During the “Twelfth Five-Year Plan” period, the Ministry of Science and Technology passed the precise deployment of the national “863” program, and arranged 15 projects in key areas such as advanced bio-manufacturing of major chemical products, microbial genome breeding, and industrial enzyme molecular transformation. 650 million yuan, self-raised funds of more than 2.2 billion yuan, the total funding of more than 2.8 billion yuan. The project undertakers cover a wide range of innovative entities such as enterprises, research institutes and universities, among which 62% are led by enterprises. In addition to the key breakthroughs in the implementation of core technologies in the above key areas, significant progress has been made in key technologies such as industrial biocatalysis technology, biorefinery technology, modern fermentation engineering technology, and green bioprocessing technology. At the same time, 30 major products have been developed in the field and industrialized (including 4 to 5 biological materials, 7 to 9 C 2 to C 4 chemical materials, 14 to 15 fine chemicals, 2 to 3 species). Energy products and 4 to 5 green biotechnology). The main representative results include the biochemical production of fumaric acid and its derivatives, the chemical-enzymatic method for the preparation of chiral pyrethroids in all organic solvents, the biotransformation of phytosterols to produce androstenediones, the bioasymmetric synthesis of chiral alcohols, and the genome. Rational design promotes the upgrading of the amino acid industry, the biosynthesis of biopolymer materials and the efficient biosynthesis of succinic acid.
Through a large number of national key laboratories, engineering technology research centers, industrial technology innovation strategic alliances and industrial bases, the industry, research and research resources of the industrial biotechnology industry have been optimized and integrated, significantly improving the industry's independent innovation capability and international competitiveness.
For example, Tianjin Industrial Biotechnology Research Institute is the first in the world to realize the full bio-production of L-alanine and industrialization. The production cost and energy consumption are reduced by more than 40% compared with the traditional process, and the wastewater volume is reduced by 90%. As of June 2017, the technology has added an additional value of 500 million yuan to Huaheng Bio, with a foreign exchange of 40 million US dollars, and the international market share of products exceeded 70%, which led to the formation of a new industrial chain of alanine chemical industry. For example, the Institute of Physics and Chemistry of the Chinese Academy of Sciences has strong enzymatic gelatin technology and industrialization advantages. The enzymatic gelatin technology developed by it has shortened the production cycle from 50 to 60 days to less than 3 days, and the water consumption per ton of glue is 400~ 600 tons reduced to 200 tons, the product with a freezing strength of 200Bloom g or more is increased from 50% to 60% of the traditional alkali process to 100%, the production workshop area is reduced by 30%, the labor consumption is reduced by 10%, and the production cost per ton of rubber Reduce by 10%. In September 2016, the technology was put into operation in Ningxia Xinhaoyuan Biotechnology Co., Ltd., which realized the production of 3,000 tons/year. It marks that China's self-developed enzymatic bone gelatin production technology has undergone pilot and industrial demonstrations. Commercial operation stage.
At present, China's economic development has entered a new normal, and is in the stage of growth rate change, structural optimization and power transformation. With the tightening of land, energy, resources and environmental constraints, the growth rate has shifted from high speed to medium and high speed. At the same time, the structural supply and demand of China's real economy is unbalanced. Therefore, it is extremely urgent to cultivate new growth drivers. With the implementation of the “five in one” overall layout and the “four comprehensive” strategic layout, the development concept of innovation, coordination, green, openness and sharing has been deepened, and the structural reform of the supply side has been gradually promoted, whether it is bulk fermentation. The upgrading of the industry, or the embedded application of the chemical industry, must be implemented as a key task of “going capacity, reducing costs, and supplementing short boards”.
At present, China's large-scale fermentation products such as amino acids, vitamins and organic acids rank first in the world; bioenergy replaces fossil energy in the year with more than 33 million tons of standard coal, which is in the forefront of the world; realized organisms such as ethylene glycol, butanol and ethylene. The production of biological materials such as bio-plastics and bio-chemical fibers has begun to take shape. Pantothenic acid, acrylamide and nisin account for 50% to 70% of the world market, and raw material consumption and waste discharge are reduced by more than 50%. After the vigorous development during the “Twelfth Five-Year Plan” period, the output value of the main products of China's industrial biotechnology industry has exceeded 550 billion yuan, with an average annual growth rate of more than 8%. In 2016, the output of major products in China's fermentation industry reached 26.29 million tons, an increase of 8.3% compared with 2015, which reversed the situation of low level in recent years. Among them, amino acids achieved rapid growth, starch sugar, enzyme preparations, yeast, functional fermented products maintained steady growth, the polyol industry grew slightly, and the organic acid industry experienced negative growth. In 2016, the export volume of major export products reached 4.08 million tons, a year-on-year increase of 18.6%, much higher than the 3.3% increase in 2015. Affected by factors such as the decline in raw corn prices, the export volume of starch sugar, lysine, lactic acid and sodium gluconate achieved double-digit growth. MSG, citric acid, polyol and yeast remained stable growth due to the continuous decline in export prices. Enzyme production showed a negative growth. The scale of industrial biotechnology enterprises has been continuously expanded, and the industrial concentration has been further enhanced. The production capacity of the top 6 enterprises of some major product capacity accounts for more than 80% of the national production capacity; the industrial cluster area shows the distribution of raw material production areas.
However, China's industrial biotechnology industry still faces challenges. Although the output of China's bulk fermentation products occupies an international leading position, it is competitive with high resource consumption, high energy consumption and low labor cost. The level of production strains is still relatively low; although Chinese enterprises are in emerging bio-industries (such as organisms) Fuel, bio-based chemicals, bio-materials have a certain foundation, but the production technology of new products is relatively scarce; China mainly produces high value-added chemicals (such as fine chemicals, pharmaceutical chemicals and intermediates) by chemical methods. The pollution is serious; foreign companies monopolize most of the domestic market in the field of enzyme preparation, while domestic enterprises not only produce more limited enzymes, but also have low production levels; high-pollution industries represented by textile, paper, metallurgy and mining are wide coverage, consume energies heavily, and of serious pollution.
