1.Microbial ecological agent

Microbial ecological agent is a general term about one type of substance preparation with the function of adjustment of microecological disorders, maintaining microecological balance to improve the health of the host of normal flora and their metabolites and selective promotion of the growth of normal flora ,under the guidance of microecological theory . At present, microbial ecological agent are divided into three categories: probiotics, prebiotics and synbiotics.

1.1 Classification of microbial ecological agent

1.1.1 Probiotics

Tannock^[1] defined probiotics as live microbial cell preparations and their metabolites that, when ingested in certain quantities, beneficially affect the host by improving intestinal balance of the intestinal tract . Currently, hundreds of probiotics have been identified, and the main probiotics used in clinical practice include Lactobacillus and Bifidobacterium.

1.1.2 Prebiotics

A non-digestible food component^[2] selectively promote the activity or growth and reproduction of beneficial intestinal flora, thereby improving the intestinal environment and playing a role in enhancing host health and growth promotion. The earliest prebiotics discovered were bifidogenic factors, and later it was found that a variety of indigestible oligosaccharides can be used as prebiotics, and the most common oligosaccharides are lactulose, sucrose oligosaccharide, cottonseed oligosaccharide and oligomaltose, etc. In the past, oligosaccharides were narrowly regarded as prebiotics, and functional oligosaccharides were the most researched, but now the research on prebiotics is not only limited to oligosaccharides, but also many proteins, vitamins and amino acids can be regarded as prebiotics.

1.1.3 Synbiotics

It is a microecological preparation combining probiotics and prebiotics, and some of them also add some vitamins and trace elements. Its characteristic is to play the role of probiotics and prebiotics at the same time to regulate the imbalance of intestinal flora. The application of this kind of preparation has a tendency to increase day by day.

1.2Mechanism of action of microbial ecological agent

1.2.1Mechanism of action of probiotics

(1)Improve the intestinal microenvironment

Under normal circumstances, animal intestinal microbial populations and their numbers are in a dynamic equilibrium, when the body is affected by certain stress factors, this balance may be destroyed, resulting in imbalance of the proportion of the body flora. For example , aerobic bacteria, such as Escherichia coli increase, and protein decomposition to produce amines, ammonia and other harmful substances. And Waller^[3] et al, suggests that intake of Bifidobacterium lactis may reduce colonic pH, while production of other short-chain fatty acids may stimulate intestinal motility. Haiying Zhao^[4] et al. experimentally concluded that probiotic preparation can effectively improve the intestinal flora imbalance of cirrhotic patients and reduce blood ammonia, faecal ammonia and faecal pH. It can be seen that probiotic metabolism can produce organic acids to stimulate intestinal peristalsis, thus neutralising the harmful substances such as amine and ammonia produced by intestinal flora dysbiosis, lowering the pH value of the intestinal lumen, inhibiting the growth and reproduction of harmful bacteria, and enabling probiotics to dominate in the competition among bacterial species.

(2) Enhancement of the body's immune ability

Yi Hu^[5] et al. found that the addition of probiotics to the feed of the shrimp Penaeus vannamei resulted in different degrees of increase in haematocrit, lysozyme, serum protein concentration, serum phenol oxidase, serum superoxide dismutase and serum total antioxidant power. Wenming Zhang^[6] and others also proposed that Lactobacillus can guide the non-specific immunomodulatory effect, stimulate intestinal immune cells, especially enhance the function of macrophages to stimulate the specific immune response and strengthen the intestinal immunity and disease resistance.

(3) Produces various enzymes and improves the activity of digestive enzymes.

Chunyang Zhang^[7] et al. found that feeding broiler chickens with probiotic agents prepared by protease-producing probiotic bacteria could effectively increase their protease quantity and viability. Hui Liu^[8] et al. proved that probiotics could improve nutrient apparent digestibility. It can be seen that probiotics can promote the digestive and absorptive capacity of the host intestine.

(4) Promoting the maturation of intestinal physiology

Chengyao Yu^[9] et al. found that probiotics can significantly change the structure of the small intestinal mucosa of broiler chickens, specifically manifested as the lengthening of the length of the mucosal villi and deepening of the traps, and these changes apparently help to improve the digestive and absorptive function of the intestinal tract.

(5)Improve intestinal sensory-motor function

Intestinal flora not only regulates intestinal activities, but also modulates host brain functions and behaviours through the influence of the gut-brain axis^[10] with obvious characteristic effects on the enteric nervous system.

1.2.2Mechanism of action of prebiotics

(1) Competition for pathogenic bacteria binding sites

Research has shown that pathogenic bacteria (E. coli, Salmonella, Bacillus cereus, etc.) have chitin-like structures on their cell surfaces or villi, which can recognise and bind to the "specific sugar" receptors in the animal intestinal wall cells, develop and multiply, leading to disease. And prebiotics such as mannose oligosaccharide and pathogenic bacteria in the intestinal wall receptor are very similar. So it can compete with the pathogenic bacteria platform, so that the pathogenic bacteria can not be combined with the animal's intestinal wall, also can not get the nutrients needed for growth and lose the ability to cause disease. Since oligosaccharides are not broken down by the digestive tract, they can carry pathogenic bacteria out of the intestines with the faeces.^[11]

(2)Promote the growth and reproduction of beneficial flora

The intake of prebiotics inulin or oligofructose can be fermented in the human intestinal tract, and their GFn-type and Fn-type molecules are the selective growth substrates for the beneficial bacteria bifidobacteria in the human body, which can multiply their proliferation^[12] improve the competitiveness of beneficial bacteria in the intestinal flora,and improve the intestinal microenvironment.

(3) Enhancement of immune function

On the one hand, prebiotics promote the proliferation of beneficial bacteria, which have a strong immunostimulatory effect^[13]; on the other hand, prebiotics such as mannan oligosaccharide and fructooligosaccharides can act as immune stimulating factors to activate the body's immune system and enhance the body's immune response to antigens.^[14]

1.3 Application of microbial ecological agent

With people's continuous research on microbial ecological agent, people are not satisfied with the use of a single microbial ecological agent, but more inclined to the use of synbiotics, composite micro-ecological preparations, drug association and other diversified use, and is widely used in the feed, drugs, health care products and other fields.

1.3.1 Feed additives

At present, microecological preparations are widely used in the feed industry because of their characteristics of no residue and no drug resistance, and are expected to replace antibiotics. There are 12 kinds of feed and microbial additives announced by the Ministry of Agriculture of China in 1999, but the ones with more applications are Bacillus, Saccharomyces and Lactobacillus^[15]. A large number of experiments have proved that the addition of microecological preparations to feed can effectively promote the digestive and absorptive capacity of animal intestines, regulate the balance of intestinal flora, improve immunity, promote growth, and be able to reduce the amount of feed inputs to reduce the cost of feeding^[16][17]. And the compound microecological preparation has better promotion effect^[18].

1.3.2 Clinical treatment

Due to the extensive use of broad-spectrum antibiotics and immunosuppressants in recent years, the imbalance of the organism's microecosystem is often caused, which triggers more and more diseases^[19]. Therefore, many studies have been conducted on the combination of microecological agents with commonly used drugs in the treatment of existing diseases such as antibiotics^[20], Kangfuxin fluid^[21] and others to optimise the treatment and improve the efficacy. The study shows that taking this combination therapy has fewer adverse reactions, higher safety, and better efficacy, which is conducive to the long-term treatment of the disease.

1.3.3 Microecological preparation health products

With the increase of people's attention to their own intestinal health,there are the rapid development of micro-ecological preparations of health care products line. Forms of them now are capsules, microcapsules type of frozen working bacterial powder, as well as tablets, aqueous (oral liquid), yoghurt or cold drinks and others. China's Ministry of Health in 2001 made a clear list of nine "probiotic strains that can be used in health food"^[22]. Although micro-ecological reagents can theoretically regulate the balance of intestinal micro-ecology, the industry has been plagued by the phenomenon of consumers ignorantly purchasing them due to technological limitations and false or excessive publicity by merchants.

1.4 Challenges of microbial ecological agent

Research and practical applications have shown that microecological agents are effective in promoting intestinal health and are efficacious, less adverse effects, and safe for use as drugs for the treatment and prevention of various intestinal diseases^[23] which has great potential for development. However, the current research can only recognise the utility of microecological agents, but not their specific mechanism of action, i.e., how to improve the symptoms of specific diseases, whether they can achieve accurate colonisation of live bacteria, and how to achieve personalised selection of microecological agents for the treatment of patients with different changes in intestinal flora^[24] and whether the resistance of strains in microecological preparations has more advantages than disadvantages or more disadvantages than advantages^[25] and other problems are yet to be solved by people.

2. Gut bacteria

The human intestinal tract hosts a huge number and variety of microorganisms, mainly bacteria, collectively known as the gut bacteria, whose total number of cells is 10 times the total number of cells in the human body, with a number of 1014 and more than 1,000 species. Under normal circumstances, the intestinal flora maintains a dynamic balance and maintains intestinal health, but the balance of the intestinal flora may be broken by pathogenic bacteria due to a variety of factors, triggering various types of diseases.

2.1 Normal gut bacteria

2.1.1 Classification of normal gut bacteria

(1) According to the relationship with the host: mostly pathogenic bacteria for passing bacteria, mainly Aspergillus, Pseudomonas and Clostridium; parthenogenetic aerobic bacteria as the main conditional pathogenic bacteria, which can cohabit with the host, mostly Enterobacteriaceae, enterococci; specialised anaerobic bacteria as the main physiological bacterial flora, which can cohabit with the host, and its resident bacterium mainly for bifidobacteria, bacillus-like bacteria, digestive coccus.

It is worth noting that resident bacteria are a factor that prevents transient bacteria from colonising.^[26]

(2) According to the spatial distribution of bacteria (three biological layers): the deep layer is close to the surface of the mucous membrane and adheres to the mucous membrane epithelial cells to form a bacterial biofilm of the bacterial flora known as the membrane flora, which is mainly composed of Bifidobacterium and Lactobacillus; in the middle layer, there are anaerobic bacteria such as Faecal bacillus, Streptococcus pepticus, Rhizobium weirongii and Bacillus eubiquitans; in the surface layer, bacteria can be swam known as the luminal bacterial flora, which is mainly aerobic and parthenogenous aerobes, such as Escherichia coli and Enterococcus faecium.

The two main types of bacteria in the deep layer are the most physiological significance, which is beneficial and harmless to human body.

2.1.2 Physiological role of normal gut bacteria

The dynamic balance of gut bacteria plays an indispensable role in maintaining human health, especially the normal function of the gastrointestinal tract.

(1) Biological antagonism of the flora barrier

On the one hand, the bacteria themselves, such as Bifidobacterium bifidum and Lactobacillus lactis, are ecologically stable, which can resist and exclude the invasion of exogenous pathogenic bacteria and the invasion of intestinal mucosal cells.^[27] On the other hand, gut bacteria can secrete many beneficial compounds, such as short-chain fatty acids^[28] etc. can adhere to the mucosal surface of the digestive tract to block the invasion of pathogenic microorganisms through immune response.

(2) Safeguard or promote immune function

In addition to influencing mucosal immune responses, intestinal flora can also act as antigenic substances, which not only non-specifically promote the development and maturation of immune organs, but also specifically and continuously stimulate the immune system to generate an immune response, with immune-enhancing functions.^[29]

(3)Metabolism and synthesis of substances

Gut bacteria can secrete a variety of enzymes, participate in the metabolism of the three major nutrients in the human body, promote intestinal digestion and absorption function, regulate PH in the intestinal tract, thus promoting the proliferation of beneficial bacteria, effectively alleviating the symptoms of abdominal pain or constipation, etc.^[30].

2.2 Abnormal gut bacteria and diseases

In general, the gut bacteria, the host and the external environment maintain a dynamic equilibrium state, but due to certain factors such as geographic environment, living habits, diseases, etc. the balance can be broken, forming gut bacterial dysbiosis. This can lead to disease or aggravate the condition, which in turn promotes dysbiosis, forming a vicious circle. As the main interest group of our project is the elderly, the following talk about the situation is mainly the relationship between elderly basic or common disease such as cerebrovascular disease, diabetes^[31] and immunity decline due to ageing and gut bacterial dysbiosis .

2.2.1 Diabetes and gut bacterial dysbiosis

The gut bacteral of diabetic patients is dysbiotic to varying degrees, with particularly significant changes in the abundance of the phylum Thick-walled Bacteria and the phylum Mycobacterium. The dysbiosis of gut bacteria can reduce insulin sensitivity and abnormal energy metabolism through altering the host intestinal mucosal barrier, affecting the synthesis of short-chain fatty acids, abnormal bile acid metabolism and other mechanisms, which ultimately lead to diabetes.^[32]

2.2.2 Hypertension and gut bacterial dysbiosis

Gut bacteria and its metabolites can affect blood pressure by influencing lipid metabolism and the neuromodulatory system. And the nervous system also affects the balance of gut bacteria, while hypertension genetic susceptibility genes can also play a role in the pathogenesis of hypertension by regulating gut bacteria.^[33]

2.2.3 Alzheimer's disease and gut bacterial dysbiosis

Gut bacteria plays a crucial role in regulating the brain-gut axis, and the microbe-gut-brain axis is a bidirectional communication system. Dysbiosis of gut bacteria can prompt microglia to secrete pro-inflammatory factors, causing neuroinflammation and accelerating dysfunction in neurodegenerative diseases, including AD.^[34]

2.2.4 Stress and gut bacterial dysbiosis

External stimuli such as changes in geographic environment^[35], surgical trauma^[36] and so on may lead to hypoxia, hypotension, infection, which will change in the metabolism of gut bacteria in the presence of stress conditions. Under high intensity or prolonged stress stimulation may lead to small intestinal mucosal damage, gut bacteria imbalance and bacterial translocation, and intestinal mucosal barrier disruption.

2.2.5 Decline in immunity and gut bacterial dysbiosis

When the body's immunity declines, the invasion of exogenous pathogenic bacteria will be enhanced, breaking the dynamic balance of normal gut bacteria. And experiments show that^[37] dysbiosis of the gut bacteria causes a decrease in serum immunoglobulins, leading to further disturbances in the body's immunity.

2.3 Regulation of gut bacteria

At present, China's medical treatment of gut bacterial dysbiosis mainly includes microecological preparation regulation and traditional Chinese medicine polysaccharide regulation. Of course, because many diseases are related to intestinal flora dysbiosis, it is also necessary to actively treat the original disease. It is worth noting that broad-spectrum antibiotics are used in the treatment of many primary diseases^[38] and immunosuppressants, which happens to also lead to gut bacterial dysbiosis . This is back-and-forth conflict, which may be one of the reasons why many diseases do not achieve the desired therapeutic effect.

2.3.1 Methods of regulating gut bacteria

(1) Microecological agents regulation

Clinical microecological agents that have been put into use in the application as drugs are still probiotics^[39] such as Bacillus Licheniformis cupsules, whose main ingredient is Bacillus licheniformis, and Changtai Oral Liquid, whose main ingredient is Bifidobacterium tetrandraeum. But there are more and more people began to study the microecological preparations combined with other drugs to treat the disease, such as microecological preparations combined with aminosalicylic acid drugs for the treatment of ulcerative colitis^[40]. Microecological agents combined with antimicrobial drugs in the treatment of paediatric acute infectious diarrhoea^[41] and so on.

(2) Regulation of Chinese medicine polysaccharide

TCM polysaccharides are natural polysaccharides found in Chinese herbs, which are widely available, stable, easier to preserve, less expensive, and well-targeted. Compared with conventional treatments, TCM polysaccharides are less toxic and safer. Its mechanism of action is to promote the growth of beneficial bacteria in the intestinal flora, and partially inhibit the growth of pathogenic bacteria.^[42]

2.3.2 Effects on related diseases

At present, it is found that both microecological preparations and or traditional Chinese medicine polysaccharides have a more significant effect on the regulation of intestinal flora. It is mainly reflected in increasing the number of beneficial intestinal bacteria such as Bifidobacterium and Lactobacillus, and decreasing the number of anaerobic bacteria to influence the intestinal microecology^[43], to affect related diseases. However, how to use the selection of different microecological preparations and traditional Chinese medicine polysaccharides to target the intestinal flora, the optimal way of administration and the optimal dosage of administration in the clinic still need to be further researched.^[44][45]

3.Gut-related healthcare products

With the growth of the country's per capita GDP, the purchasing power of residents has been rapidly improved, and people's concern for their own health has also prompted people to purchase and use health care products.In the first quarter of 2021, as the main consumer group in the healthcare products market, nearly 80% of Chinese seniors said they had purchased healthcare products.^[46] Considering item relevance, here we mainly discuss gut-related health products.

3.1 Current status of gut-related nutraceuticals

At present, there are two main types of gut-related health products: one is to supplement probiotics, prebiotics-based microecological agents that we mentioned earlier health products to regulate gut bacteria; the other is to contain rhubarb, aloe vera, senna and other stimulating ingredients to stimulate the intestinal tract in order to promote intestinal peristalsis^[47] such as the market common intestinal cleansing tea, intestinal cleansing fruit.

3.2 Effectiveness of gut-related health products

Many elderly people always have high expectation on the effect of health food, and even use them as medicines, but in fact, health food is not equal to medicines. It is defined in the General Standard for Health Food as food claiming to have specific health care functions or for the purpose of supplementing vitamins and minerals, i.e., but not for the purpose of treating disease.^[48] The false or excessive publicity in the industry has also led to the blind purchase of many elderly people who do not know the reasoning, which in turn induced diseases^[49]. As many of the health care products under the banner of intestinal toxin cleansing and beauty care are essentially the same as laxatives, long-term abuse of this type of laxative to stimulate the intestinal tract, which may lead to colorectal melioidosis.^[50] And untreated colonic nigropathy will increase the risk of intestinal polyps.

4. Summary

The pathogenic mechanism of gut bacterial dysbiosis is still not completely clear, but it is clear that there are many diseases that have a close relationship with gut bacterial dysbiosis, and these diseases will in turn affect the stability of gut bacteria, so the two are causative of each other. It can be seen that the treatment of these diseases on the one hand to catch the primary disease, on the other hand, we should also pay attention to the intervention of the regulation of intestinal flora. This also requires that the current treatment of functional constipation needs to be individualised because the underlying disease in the elderly varies from person to person, and of course functional constipation may lead to intestinal flora dysbiosis, which in turn leads to complications. These three relationships are interacting with each other.

Regarding the treatment of functional constipation, the use of microecological agents and herbal polysaccharides to regulate the intestinal flora and thus treat functional constipation does have the advantages of fewer side effects and higher safety than the traditional therapy (laxative-based), which can effectively improve the health index of the elderly.^[51]However, at present, people are mainly concerned about the effect of their use, and the research on the mechanism has not yet been in-depth, such as the in vivo colonisation of live bacteria, how to achieve personalised treatment for patients with different changes in intestinal flora, the optimal mode of administration and the optimal dosage of administration, etc., which still need to be further researched.

The effect of intestinal health products has more uncertainty, but from the psychological point of view of the majority of the elderly willing to buy, in the face of intestinal problems such as constipation, compared with going to the hospital, the elderly are more willing to buy products similar to health care, which increases the feasibility of the effective promotion of the products of our project.

5. Inspiration

(1) If we can ensure the colonisation of our engineered bacteria, we can expect to achieve a long-term effect against constipation.

(2)Whether our engineered bacteria will affect the dynamic balance of intestinal flora and increase the incidence of other diseases after passing through the intestinal tract or after colonisation is debatable.

(3) From the perspective of treating constipation alone, if our bacteria have a certain degree of safety, i.e., after colonisation, they will not cause other diseases, nor will the treatment effect be greatly reduced due to the patient's other pre-existing diseases. Then can we say that our project can simplify the complexity of constipation treatment to a certain extent, i.e. we do not need to consider the individual selection of bacteria compared to probiotics, etc.?

(4) We believe that it is necessary to educate the elderly about the intestinal tract and microbiology. On the one hand, it can reduce the incidence of fraud in the purchase of health care products, and on the other hand, it can make the elderly more assured and more willing to try or accept our products.

On 5 March, which is the Day of Learning Lei Feng, the school organised an activity to learn from Lei Feng. After his death in the line of duty and accident, Lei Feng's service to the people was reported again and again. That is why we Chinese have decided to designate 5 March as Lei Feng Memorial Day, on which we will carry out various activities to help others and give back to the society.

On 5th March this year, Li Yiming and some of the students from our team went to Shanghai Yangpu District Zhengmin Elderly Home to do some things for the elderly, such as helping the caregivers to clean up, performing shows for the elderly, chatting and walking with them. During the activities of respecting and helping the elderly in the nursing home, the team members, in accordance with the original intention of medical students, thought about whether they could make use of their medical knowledge and the broad professional platform of the school to provide medical advice to the elderly, and answer as much as possible the problems that bother them in their daily life. Through in-depth communication with the staff of the nursing home, Li Yiming found that there was a common problem among the elderly in the nursing home - constipation. After talking with the caregivers, the caregivers pointed out that the common causes of constipation among the elderly in our nursing home are usually as follows: firstly, the elderly are uncomfortable because their children often cannot meet them due to their busy work schedule; secondly, the elderly lack of exercise due to their age; and thirdly, most of the elderly in our nursing home have underlying illnesses, such as diabetes mellitus, constipation due to lesions and medication, and so on. Constipation caused by dietary factors can be effectively avoided by carers through proper diet. However, it is not the constipation itself that worries the staff most, but the other problems caused by constipation. Through communication with the nurses, the team learnt that one elderly person suffered from haemorrhoids due to constipation, which not only caused pain to the elderly, but also increased the difficulty in caring for them. According to the nurses, they were worried that constipation would lead to more serious cardiovascular diseases, such as stroke and cerebral haemorrhage, with unimaginable consequences. We enquired about the use of medication and the effectiveness of the medication. According to the staff, the treatment was effective for most of the elderly. However, there were still some elderly people who could not express their emotions clearly, so the caregivers could not accurately judge the effectiveness of the treatment. In response to this problem, our team of students wanted to know if it was possible to design a product that can treat constipation to improve intestinal health.

After our survey and interview data, we found that the incidence of chronic constipation among Chinese adults is 4 to 10%, which means that about 90 million people in China suffer from constipation. In addition, the prevalence of constipation gradually increases with age. According to research, the prevalence of chronic constipation is as high as 23% in people over 70 years old and 38% in people over 80 years old. Based on the large population of constipation, we would like to design a product for constipation. Currently, non-pharmacological treatments for constipation take a long time and are slow to work. Drugs for constipation that exist on the market need to be taken on time and in quantity, which bring distress to forgetful people to take the medication, especially for the elderly forgetful group and busy office workers.Our team is working hard to design products that can colonise the gut for a long period of time and exert a therapeutic effect.

In order to achieve our goal, we have done many HP work involving multiple fields one after another.

Q: How does TCM dialectically treat constipation?

A:Constipation is actually a symptom, which can be recognised as a disease in TCM. In modern medicine, this symptom occurs in many diseases, such as severe constipation in diabetic patients at a later stage, and long-term constipation in constipated patients with irritable bowel, including the elderly who are prone to suffer from constipation as mentioned in your project. From the point of view of TCM diagnosis is very complex, constipation is located in the intestines, but it is closely related to the internal organs, such as the spleen and stomach, liver and kidneys.

However, the diagnosis is still based on cold, heat, deficiency and solidity, especially to distinguish between deficiency and solid first. If it is determined that the constipation is solid, and then divided into cold and heat, whether it is caused by cold accumulation or heat knot, but also may be caused by stagnation of qi and qi depression transforming into fire. The process of stool formation and discharge relies mainly on the peristalsis of the large intestine, which is considered to be the role of qi in TCM. Its formation also depends on the heat of the large intestine and the operation of fluids, that is, the TCM saying of increasing water to move the boat. If the large intestine is too cold, peristalsis and the role of qi to promote will weaken. Constipation symptoms will appear when the stool stops accumulating for a long time. Constipation is manifested partly by dry stools, partly by long periods of time without defecation, all related to heat and cold. There is also a part of the person is deficiency constipation. Qi deficiency type, such as the elderly defecation weakness, is treated to replenish qi; blood deficiency type, such as postpartum women blood loss, will appear constipation in a short period of time; yang deficiency type, such as the elderly lack of energy, fear of cold, defecation weakness, discharged stool soft and not dry; Yin deficiency type, lasting a long time, such as diabetic patients in the late stage of wasting who are not enough yin. And they can not discharge the feces, with hypertension, dry mouth and tongue redness.

Q: What are the main methods or means of treating constipation in TCM?

A: There are many means of treating constipation in TCM, which can be broadly divided into internal and external treatments. Internal treatment is dietary therapy and medication; external treatment is non-pharmacological therapy, such as acupuncture, moxibustion, massage. Modern medicine will use laxatives. TCM also have diarrhoeal methods, such as "typhoid fever" in the honey decoction guide, that is, the honey boiled to make a suppository plugged.

Q: Compared with Western medicine taking laxatives, or external use of corkscrew and other methods, what are the advantages of TCM treatment?

A: TCM treatment not only leads to diarrhoea to treat constipation, but also prevents constipation. For example, if constipation is caused by heat in the large intestine, after the feces arrives, its fluid is fried and absorbed, so at this time, in addition to the conductive but also to drain the heat, and here to drain the heat is not diarrhoeal drugs, but clearing the heat and detoxification or clearing the heat and fire medicines. If it is because of the patient's emotional tension then is difficult to dissolve stools, i.e., constipation caused by stagnation of qi, we will use the method of regulating qi, detoxifying the liver and relieving depression. In modern medicine, intestinal flora is also a way to prevent constipation, and long-term use can relieve symptoms. There is no absolute superiority or inferiority between Chinese and Western medicine, just that the method of TCM may be more diverse in comparison, and the window of treatment is a little bit forward.

Q: If our project is to use the principles of synthetic biology, based on E. coli to design a set of preventive, therapeutic and other multi-functional as one of the engineering bacteria. When constipation occurs, the engineered bacterium senses the decrease of butyrate content in the intestines and secretes a certain drug to regulate the intestinal function, promote intestinal peristalsis, and play a therapeutic role. What do you think are the advantages and disadvantages of this project? What can be improved in the future?

A: If you want to do prevention, then the advantage of this project is that it is dynamic change, not limited to the treatment of constipation. However, the time it takes for this engineered bacteria to work is uncertain. For example, if it is taken orally once a day, how much butyrate is sensed by the engineered bacteria in the intestines, will it start to work right away, and how long does it take for this to work. At present, your project focuses more on the therapeutic effect, and the preventive effect is not obvious. Constipation doesn't occur when there isn't a massive drop in butyrate levels, but you haven't pinpointed where the window is for how much this drops to. The therapeutic window may be the key to the problem. For example, rhubarb used in TCM can only be taken in a single dose, like a corkscrew, but constipation can still form again. But TCM can treat and prevent constipation by determining the cause and characteristics of constipation and prescribing the right medicine. The highlight of the project should be prevention, but how to achieve prevention is difficult and complicated, not just because of changes in one or two indicators. If you want to achieve long-term colonisation, it is difficult to engineer bacteria to feel and give timely feedback. After all, there may be only one target to focus on during the experiment, but in fact, the engineered bacteria itself may have different targets.

a. An uncle

Q: Have you ever had difficulty in passing stools, usually once every few days?

A: No, I'm not constipated but I have diarrhoea.

Q:What methods have you used to solve diarrhoea, such as some proprietary Chinese medicines, laxatives, Chinese herb , or capsules containing probiotics. How effective are they?

A:I'll take some gut-regulating health care products, which I think are effective.

Q:What kind of medicine or method would you like to use to relieve constipation? For example, capsule, or some drinks ? Or how would you like to improve the existing medication, such as reducing the number of times to take the medicine? Or can you take it once for a longer period of time?

A:I think whether it's for constipation or diarrhoea, taking capsules always gives the feeling of taking medication as if you're ill. It give me an uncomfortable feeling. Moreover, the less you take of the medicine, the better. After all , they will always be side effects.

Q:Have you ever heard of what synthetic biology is, or have you heard of any bioengineering/synthetic biology applications?

A:No, I have never heard of it and do not know much about it, but I know about genetic modification. Because there have been a lot of news reports on its negative impacts before, I do not think that the public acceptance of it was not high at present.If you want to get more people to accept your product, you must be able to allay people's fears about it.

b. An auntie

Q: Auntie, how much vegetables and fruits do you usually eat? What do you like to eat?

A:I like to eat lighter meals, like some people can't handle boiled greens but I love them!

Q:Have you ever had difficulty in passing stools, usually once every few days?

A:At the moment I don't have serious constipation problems, and you know I eat a healthy diet.

Q:Have you or your friends or relatives ever suffered from constipation?

A:Yes, my mother-in-law, she's old and has a very serious constipation problem.

Q: What methods have you or they used to solve constipation, such as some proprietary Chinese medicines, laxatives, Chinese herb , or capsules containing probiotics. How effective are they?

A:I would occasionally cook some food contributing to defecation for my mother-in-law, but the effect is only short-lived. And my mother-in-law’s son is a pharmaceutical representative, so she now takes western medicine for constipation, but the improvement is not obvious, and the long-term constipation problem still exists.

Q: If you or your relatives or friends can try a capsule with modified E. coli bacteria, which can relieve constipation for a few weeks, would you be willing to try it?

A: I support this idea of you university students and think it is very meaningful, because I think that the elderly are more or less troubled by constipation, and there is really no good solution at present. However, due to the numerous deceptive incidents of health care products and so on, I only believes in the products of the formal channels of the country and don't believe in the advertisements and direct sales. If the products of our project have gained national recognition after maturity, I would be willing to recommend it to the elderly in my family to have a try.

Q: What do you think of the security design of this project?

A: I think your idea of using engineered bacteria to treat constipation is very good and innovative. However, at present, the research and development of live bacterial drugs is strictly controlled by the state. The application and marketing of intestinal engineered bacteria need to be strictly approved, and intestinal engineered bacteria as drugs need to follow the principle of edible. The lack of safety considerations in your project will limit the subsequent production and use of engineered bacteria. If you want to know more about it, you can check out the relevant standards and rules.

Q: So what do you think we can do to improve safety?

A: We know that the use of drugs in the treatment process is always patient-centered, in the application of drugs should first consider what impact on the patient's body will have, whether the treatment effect on the patient's discomfort is more than the side effects brought by the drug. The design of your existing safety module focuses on how to prevent contamination of the environment by engineered bacteria, which can be induced to kill at 37 degrees Celsius. However, it did not consider whether there would be side effects when using the engineered bacteria, whether patients could voluntarily stop using the engineered bacteria, and whether the patients would be affected after stopping the use of the engineered bacteria. At the same time, the acceptance of your "live bacterial therapy" in patients with constipation is not high, so you should work hard to improve safety, but also to make patients more reassured. In addition to preventing engineered bacteria from contaminating the environment, you can also consider adding induced containment routes.

Q: What is your overall impression of our project? Prior to this, we have improved our safety module in view of the safety concerns of our target elderly group. What do you think of the safety module of our project?

A: Your theme is quite good. It picks a common but real need and tries to provide a solution. At present, with the aging trend in China and the change of people's living habits, the number of constipated people is increasing and the market demand is relatively large. Your safety module takes into account both environmental protection and in vivo induced containment, and the design is more complete.

Q: We are considering how to deliver engineering bacteria into the human body through a suitable carrier. For this purpose, we have designed two options. One, use capsules as the carrier to take the engineered bacteria. Another option is to use yoghurt as the carrier and add the engineered bacteria into yoghurt, so that the user can take the engineered bacteria by drinking the yoghurt.

A: I notice that you have done a survey on more acceptable forms of medication for the elderly, and this idea of thinking about the target population is good. In my opinion, both carriers have some viability.

Oral administration is easy, but as the target organ is the gut, given the acidic conditions of the stomach, you need to consider how to ensure that the product can withstand the low pH environment in the stomach. This issue is directly related to dosage, i.e. how much E. coli can be delivered to the target organ. If capsules are used, there are more relevant and proven technologies on the market today and the protocols are already more mature. I love yoghurt too, and it's normal for people to prefer this form. However, if you use yoghurt, you may need to add some barrier protection for the engineered bacteria rather than adding them directly to the yoghurt. Unlike capsules, which can be made enteric, you also need to consider whether your engineered bacteria can survive the stomach acid and not affect their function in the gut. You may want to consider embedding the engineered bacteria in an encapsulated form.

Also, if you end up choosing this carrier, you will need to consider shipping conditions. Yoghurt, unlike capsules, is more demanding in terms of temperature and environment, and how to get the yoghurt to the customer without loss of flavour is also an issue to be considered.

Q: Yes, there is still quite a bit of room for improvement in our product. So from the perspective of a consultant in the pharmaceutical industry, do you think there is anything else we should be aware of if our product?

A: I think you might need to clarify the category of your product. Is it a food supplement or a drug that you are trying to develop? Please be aware of the differences in regulatory requirements, time and funding required for product development.

Q: Hello, I would like to start with a general introduction of our program. Constipation is a common problem in modern life, with a prevalence rate of about 6%, i.e., 1 in 16 people suffer from constipation. At present, there are five main treatment options for constipation, namely general therapy, drug therapy, surgery, Chinese medicine and biofeedback therapy. However, these therapies are often characterized by a long period of time, excessive impact on the patient's life, lack of obvious therapeutic effects, and a high degree of self-consciousness in taking the medication. Therefore, our team decided to use synthetic biology to design an engineered bacterium that integrates prevention, monitoring, and treatment using E. coli EcN as a chassis to help patients with functional constipation to improve their constipation problems, and we plan to modify it to be added into yogurt, so that the patients can intervene in constipation subconsciously in their daily diets. We plan to design sensing, metabolism, and safety modules that will enable the engineered bacteria to express 5-HT based on the level of butyrate in the intestines of constipated patients to promote intestinal peristalsis and secretion for the treatment of intestinal problems. We have also designed safety modules that are adapted to cryopreservation and transportation, and drug-controllable, which provide a guarantee for the safety of the engineered bacteria.

Interview Questions:

Q: Legally what are health products, what are foods, what are medicines, and what is the difference between them?

Answer: General food refers to all kinds of finished products and raw materials for human consumption or drinking. Ordinary food is consumed by the general public, from which the human body receives various types of nutrients and meets the sensory requirements of colour, aroma, taste and shape. There is no recommended intake for common foods, nor is there any applicable or unapplicable groups.

The National Standard for Food Safety Health Food stipulates that health food refers to food claiming to have specific health functions or for the purpose of supplementing vitamins and minerals. That is, it is suitable for consumption by specific groups of people. It has the function of regulating the body. It does not aim at treating diseases and does not cause any acute, sub-acute or chronic harm to the human body. That is to say, health food has the attributes of food, but due to its unique health care function, it is different from ordinary food; it is stipulated that there is a recommended intake, and long-term consumption within the range of recommended intake will not produce side effects, and it is safe; according to the different health care functions, there are applicable and inapplicable groups, but it cannot replace drugs.

In addition, the Food Safety Law of the People's Republic of China stipulates that the State shall exercise strict supervision and management over special foods such as health foods, formulas for special medical purposes and infant formulas. In other words, health food is a kind of special food.

According to the Drug Administration Law of the People's Republic of China Article 2 of the Law defines medicines as follows: Medicines referred to in this Law are substances, including traditional Chinese medicines, chemical medicines and biological products, that are used for the prevention, treatment and diagnosis of human diseases, and for the purpose of regulating the physiological functions of human beings, and for which there are specific indications or functional prescriptions, as well as usage and dosage.

Q: We have learnt that some elderly people will use probiotic products or yoghurt to relieve constipation, so are products containing probiotics health supplements or food products, and is yoghurt containing probiotics generally health supplements or food products?

A: Different probiotic products are classified according to their ingredients and functions. On the top of the package or label of health food, there must be a health food logo (commonly known as the "blue hat"), and the label, instructions need to be clear on the serving size, the applicable population, the inapplicable population and other instructions, and need to clearly state that the product can not replace drugs. If a probiotic product complies with the relevant provisions of the "Regulations on Declaration and Review of Probiotic Health Food (for Trial Implementation)", and is declared and approved through the health food route, it will be given a health food production approval number and allowed to use the health food logo, which is a health food product. Generally, yoghurt containing probiotics is declared for production approval through the ordinary food route, and is classified as ordinary yoghurt food.

Q: Through previous community surveys, we have learnt that for constipation, most of the elderly people prefer to use health supplements and food to relieve their symptoms, and are resistant to medication. For this reason, we have designed to incorporate engineered bacteria into yoghurt to improve the taste and experience of using it, and to consider using it as a health supplement or food. However, by definition, our product is designed to treat constipation, regulate human physiology, and has a prescribed dosage, which seems to be more in line with pharmaceuticals. What do you think?

A: Constipation involves many indications and the target of the drug is not clear. Toxicity testing and safety evaluation of drugs are important prerequisites for new drug development. Often, drug product development is focussed on a narrow field of indications, which makes it easy to demonstrate safety and efficacy and obtain regulatory approval. Once a product is approved and placed on the market, then to expand the indication, more data needs to be generated to support the additional requirements. The most typical indication is therapy, which has an urgent need and is more readily accepted.

However, given your research and the realities of the present day where older people are resistant to forms of medication, your design is very thoughtful. I am providing you with some relevant laws for your reference.

The probiotic strains of your project are genetically modified. According to the "Regulations on the Safety of Agricultural Genetically Modified Organisms", agricultural genetically modified organisms refers to animals, plants, microorganisms and their products that utilize genetic engineering technology to change the genome composition and are used in agricultural production or processing of agricultural products, and your product can belong to Article 2 thereof "(2) Genetically modified animal and plant and microorganism products;", which is in line with the Regulations. of which you need to comply with the Administrative Measures for the Safety Evaluation of Agricultural Genetically Modified Organisms.

In addition, according to the "new resource food management approach" Article 2 of the new resource food "(3) new varieties of microorganisms used in food processing", your products can also be applied to this approach; as for the products made into health care products or pharmaceuticals, depending on the declaration of what category, if the health care products according to the review, according to the "probiotic class Health food declaration and review provisions (for trial implementation)" Article 15: genetically modified strains shall not be used for health food. Then it is difficult to pass. There are only 10 kinds of probiotics that can be used for health food in the above Provisions, such as Bifidobacterium bifidum, Bifidobacterium infantis, Lactobacillus Germanicus Bulgarianus and so on. At present, many scientific research institutions in China have carried out research on the cultivation and application of genetically modified varieties of traditional Chinese medicine, but Article 36, paragraph 1 of the newly revised "Code of Practice for the Quality Management of the Production of Chinese Materia Medica" of the State Administration of Pharmaceutical Administration in conjunction with the State Administration of Traditional Chinese Medicine and Forestry and Herbaceous Departments of the State Food and Drug Administration of the People's Republic of China (SARFT) in 2022 stipulates that polyploid or haploid varieties, interspecies hybrid varieties and genetically modified varieties resulting from artificial interventions are prohibited; and the second paragraph stipulates that: if it is necessary to use non-traditional The second paragraph stipulates that if it is necessary to use non-traditional interspecies grafting materials, mutant varieties (including physical, chemical, space mutation, etc.) and other biotechnology breeding varieties, enterprises should provide sufficient risk assessment and experimental data to prove that the new varieties are safe, effective and quality-controllable. Therefore, although the use of genetically modified raw materials for pharmaceuticals has a certain research basis and broad prospects for application, the state based on ensuring the safety of people's lives and drug safety point of view, in the law is still strictly prohibited and restricted, but to achieve a certain level of safety as food and drug base source use is also possible. Specifically depends on the safety level of your product subsequent certification, safety level certification can refer to the "Management Measures for the Safety Evaluation of Agricultural Genetically Modified Organisms", the specific safety certification need to contact the relevant departments responsible for the evaluation work.

Q:Hello, Mr Wang! We would like to design an engineered bacterium that can initiate the regulation of subsequent genes by sensing a certain substance in the human intestinal tract, and then alleviate the symptom of constipation. Regarding the substance to be sensed, we initially designed two options, methane and butyrate, to sense the state of constipation, and what the teacher thinks about the choice of these two substances?

A: I would like to know what made you choose these two substances for the initial design. Since you chose them, the first thing that is clear is that these two substances are closely related to the occurrence of constipation, right? Have you considered whether other non-constipation factors may affect the changes of these two substances in the body? In the case of methane, it is a product of intestinal bacterial activity. In addition to constipation, which may lead to elevated levels of methane in the gut, a number of other digestive problems and gastrointestinal discomforts may also lead to elevated levels of methane in the gut. This may even be just a normal physiological phenomenon, such as a high-fibre diet and active bowel movements.

Q: Yeah, teacher. After reviewing a lot of literature, we found that relevant experiments show that methane excretion is closely related to chronic constipation, and when the organism is constipated, the concentration of methane gas in the intestines will increase; at the same time, because constipation will lead to changes in the intestinal flora, the concentration of butyrate in the intestines will decrease. Based on the above principles, we have designed two options, but we would like to know which one you think is more appropriate.

A: From the lines you have designed now, I think the methane one is the simpler one. The advantage of simplicity is that it is easy to implement and makes gene expression more efficient. This is definitely helpful for your subsequent expression, if the gene line in front is too complicated, then the efficiency of the substance you want to produce in the end will definitely be compromised. If you guys want to treat constipation, then the amount of production of the drug used to treat constipation must be a requirement, otherwise it wouldn't work. But there are certainly advantages to complex circuits, and many complex circuits regulate a higher degree of refinement than simple ones.It does require a high degree of fine tuning if you are regulating metabolism in the internal environment of the human body. However, I still suggest you to simplify the gene circuits. Your project is just starting now, and too much complexity is not conducive to your research. If your project goes well, you can design some complex circuits to improve the fineness of regulation.

Q: OK, we will go back and improve this part. Do you have any comments on our current line design?

A: Well, actually, as I mentioned earlier, it's about the influence of other factors on your lines. Like some other diseases besides constipation and the intestinal flora may be altered by various interfering factors. Will these factors affect the changes in the concentration of your sensory receptors, leading to a reduction in the utility of your sensory modules. After all, human beings are in a complex external environment, and there are many internal regulatory factors, which may affect your designed gene line. How to make these factors not affect the functioning of your designed gene line is a question that needs to be pondered over. Therefore, you need to consider many factors to make a comprehensive assessment, in order to better design and optimise the circuit.

Q: As an anorectal doctor, do you usually receive patients with constipation? Will we pay attention to the patient's constipation in clinical work?

A: Constipation is a common disease in anorectal surgery. Our center has carried out a national survey of constipation, and is also the first research unit of constipation in China and the leading unit of the first surgical treatment guidelines for constipation in China, which shows the importance and authority of clinical work on constipation.

Q: Changhai Hospital is a big hospital, so it may not have a patient to anorectal treatment of constipation problem, if there are patients with constipation problem, is it mostly secondary?

A: Yes, mostly primary constipation.

Q: What kind of treatment would you recommend for constipation?

A: Psychological and life intervention, laxative treatment, intestinal probiotics treatment, psychotropic drug treatment, etc.

Q: What do you think are the pros and cons of this project?

A: The study design is good, but many of the statements are not scientific, for example:

1) we need to pay attention to when the patient has constipation, we first consider not to give him constipation drugs, but to consider the patient's organic lesions, whether there is colorectal cancer, and so on.

2) The color module in the project will affect the doctor's examination of the patient's stool; And having blue stools can be challenging for patients, who may not accept their stools as blue.

3) Symptoms of constipation usually begin to appear over a period of several days to about a week. If constipation is not relieved for a long time, it may lead to severe symptoms, such as abdominal pain, bloating, nausea, vomiting, loss of appetite, headache, etc. In more severe cases, there will be water and electrolyte balance disorders and intestinal obstruction, and even life-threatening. Therefore, the engineered bacteria need to sense the constipation state of the body in a short time to treat it in time.

Q: Hello, Professor. We have designed an engineered bacterium that senses changes in butyrate levels in the intestines and secretes a substance to treat constipation. Now we want to select a substance to treat constipation. I would like to ask your opinion about this. Do you think that substances produced naturally in the body or those chemically synthesised drugs are better as therapeutic drugs?

A: I think that naturally occurring substances in the body have some potential advantages over chemically synthesised drugs as therapeutic agents. Although the specific advantages will vary depending on the nature of the disease and the drug. However, substances produced naturally in the body are usually more readily accepted, absorbed and utilised by the body. They are less likely to trigger allergic reactions, as the body has developed a degree of immune tolerance to them. They are also usually less toxic because they are widely available in nature and interact with biological systems. In contrast, some chemically synthesised drugs may require more chemical processing to ensure their safety. A more important point is that certain natural substances can intervene directly in the underlying cause of a disease, rather than just alleviating symptoms. Such treatments are usually more likely to produce long-term and lasting results.

Q:Okay, so does the professor have any other comments on our project?

A:I would like to ask, since you are doing engineered bacteria, have you considered the overgrowth of the bacterial population. You should know that the number of bacteria in the human intestinal tract accounts for 90% of the total number of bacteria in the human body. Under normal circumstances, they are in a dynamic equilibrium system by themselves. Will the growth and multiplication of your engineered bacteria upset this balance and bring about greater side effects?

Q: So, about this problem, do you have any ideas for a better solution?

A: I think you should consider the extent to which the colonisation can cause major disruption to the balance of the human gut. Think of ways to monitor this situation and then take certain measures to avoid it.

Q: Hello aunties and uncles, we are the university students who came to do the bowel science lecture and interview last time. The last interview on constipation had a great impact on our project, and we realised that the elderly group has a high demand for the safety of the project. After we went back, we interviewed many experts and professors, and according to their suggestions, we made many adjustments and modifications to the safety module of the project, and the improvement effect is quite satisfactory. At the same time, we interviewed investment consultants in the pharmaceutical industry, and got certain affirmation and effective suggestions for the project, and we also interviewed the staff of the Food and Drug Administration, so as to clarify the positioning of our project from the perspective of laws and regulations. We also have a special safety team responsible for the safety of the entire project, including laboratory safety. It can be said that our product safety has been comprehensively improved. Do you feel more assured about the safety of our products now?

A1: You are very attentive! You put our needs in your heart and have done so much work. I am quite satisfied, thank you.

A2: The problem of constipation itself is more common. You really want to help us solve this problem for the elderly, and the work done is very practical. I feel that the safety of this product should be a big challenge for you, but you have not avoided it, and you have gone to interview the experts and professors to solve the problem, which is very touching to me.

A3: If the project has improved, then there is certainly more assured. After all, this is the issue we are most concerned about, but if your project is finally approved by the state and the product is listed, then we will certainly dare to eat with confidence. The girls and boys have been great!

A4: Looking forward to the early realisation of your project. If it works well, we will recommend it to people around us, cheers.

Q: When we came to interview a few uncles and aunts before, they said they didn't like taking medicines. Therefore, we finally decided to take yoghurt and capsules to meet different people's needs after searching for information, experimenting and discussing. The capsules are similar to those on the market. Only different from the traditional capsule inside the drug, our capsule inside the modified engineering bacteria. The specific method of yoghurt is to bury the engineering bacteria, which is equivalent to giving the engineering bacteria a coat of membrane, and then add the engineering bacteria to the yoghurt, so that you can drink the engineering bacteria by drinking yoghurt. What do the uncles and aunts think of this way?

A1: Oh, I like this way, my husband and I already have the habit of drinking yoghurt every day. We feel more comfortable in our intestines after drinking yoghurt, and the yoghurt tastes better, so I support this form.

A2: I also think it is quite good. Eating capsules always feel that I have a lot of problems, normal people do not like to take medication, right? Drinking yoghurt will make me feel better and less psychologically stressed, otherwise taking capsules always feels like a constant reminder of my age and illnesses.

A3:I don't share the same opinion with both of them. I feel that no matter whether it is a capsule or yoghurt, ultimately effect is on the line. As for which one to choose, which effect is good to use which.

Abstract

Synthetic biology is a new discipline that combines life science observational analysis methods and engineering design thinking, and converges through information technology and engineering technology, so that human beings can design, modify and even synthesize biological systems with specific functions from scratch through engineering methods.^[1]In recent years, with the advancement of science and technology, various synthetic biology-related patented technologies have developed, and many emerging synthetic biology achievements have emerged. When new achievements in synthetic biology research have come out repeatedly, companies have also accelerated the pace of application and development of synthetic biology, forming a series of products, and the output value of synthetic biology into the market is also increasing year by year.^[2]According to SynBio Beta statistics, the global financing scale of companies in the field of synthetic biology was nearly $4 billion in 2018, and $3.1 billion in 2019. In 2020, this value reached US$7.8 billion, an annual increase of 1.5 times, and 74 synthetic biology companies completed listing.^[3]This review intends to analyze the process of synthetic biology products from R&D to marketing, important products and key enterprises of synthetic biology, and the commercial risks of synthetic biology products, so as to provide reference for the industrial application development of synthetic biology.

The specific process from the development of synthetic biological products to the market

1.1 Research and development phase

At this stage, the team conducts in-depth research and development work, including theoretical research, laboratory testing, optimization and improvement. The goal of this phase is to screen from a large number of experimental results and obtain products with commercial potential. This process often relies on laboratories, especially synthetic biology engineering platforms (R&D centers) or infrastructure, which is the foundation and guarantee for completing the "design-build-test-learn" closed loop of synthetic biology.^[4]The development process of synthetic biology requires the development and application of enabling technologies such as DNA synthesis technology and standardized component design, which requires a lot of automated trial and error, far beyond the ability of traditional labor-intensive research paradigms, and requires the support of transformative engineering research platforms.^[5]

1.2 Clinical trial stage

If the product is developed for use in the pharmaceutical field, clinical trial is an indispensable step. In the past pharmaceutical marketing process, clinical trials were usually divided into three stages: preclinical trials, clinical trial stages I, II and III. The purpose of clinical trials is to evaluate the safety and efficacy of products and to obtain clinical data to support the marketing application. Premarket safety evaluation of drugs generally serves two purposes.^[6]The first is to determine whether the sponsor has completed all reasonable tests used to evaluate the safety of the drug, and the second is to determine whether the drug is reasonably safe based on the available safety data. However, unlike drugs that already have a complete process, the clinical trial process for many synthetic biology products for medical purposes is not yet clear. In 2016, the US FDA issued a guideline clarifying how some live biotherapeutic products (LBPs) that are legally available as food or dietary supplements can be used in research, and explaining what production requirements researchers must meet in early-stage clinical trials in order to use probiotics as drugs.^[7]

1.3 Get approved

Once the clinical trial phase is completed, the team can submit a marketing application. This requires providing relevant application materials to relevant government agencies (such as FDA, CFDA, etc.), such as research data, drug safety evaluation reports, etc. The government agency will review the application and decide whether to approve the product based on clinical data, evaluation of the efficacy and safety of the product, etc. In the UK, for example, clinical trial data approved by the ethics committee and the UK Food and Drug Administration under its Drugs Act and the Clinical Trials Regulations for Human Use are required.^[8]When marketing approval is obtained, the team can begin production and quality control work. This includes developing production processes, setting up production lines, carrying out quality inspections, etc., to ensure the quality and safety of products.

1.4 Marketing & Sales

When the product is ready, the team can do marketing and sales work to get the product to market and be widely accepted. However, when drugs and products enter the market, they still face post-marketing evaluation to explore the clinical effect of the product and implement feedback on use.^[9]It should be noted that there may be differences in the R&D and marketing requirements for synthetic biology products in different countries and regions. In addition, we must ensure that the relevant laws, regulations and ethical norms are observed during the development process.

2. Synthetic biology product categories and their characteristics

Synthetic biology and its competition focus on solving problems for local people, and in the current shortage of resources and serious environmental pollution, synthetic biology shoulders the burden and provides different outcomes and solutions for social-ecological, environmental-economic development. So far, there are a large number of synthetic biology enterprises, roughly divided into three categories, the first is the traditional chemical and energy that has been established for longtime; the second is companies related to genetic engineering and medical treatment, trying new synthetic biology products in a small field; and the third is professional enterprises focusing on synthetic biology technology and patent research and development.^[10]At present, the synthetic biology industry has played an effective role in the fields of medical health, green energy, beauty and daily chemicals, energy materials, and food, changing the original production mode of poor environment and low efficiency.

2.1 Healthcare

The medical and health industry is the most important field of synthetic biology, which has a wide range of applications in the medical and health field, including cell immunotherapy, medical consumables, in vitro testing, drug ingredient production and pharmaceutical enzymes. Steady advances of synthetic biology have enabled scientists to use genetically engineered cells, rather than small molecules or biological agents, as a basis for developing new therapies.^[11]Cells with synthetic gene circuits can control the localization, timing, and dose of therapeutic activity in response to specific disease biomarkers, becoming powerful new weapons in the fight against disease.^[12]

For example, implantable cells engineered with synthetic feedback circuits have been used to regulate thyroid hormones^[13]and blood glucose^[14]for hyperthyroidism and diabetes, respectively. Traditional approaches to treating hyperthyroidism with antithyroid drugs or thyroidectomy can lead to hypothyroidism,^[15]to address this, Saxena et al. A synthetic feedback circuit using thyroid hormone-binding nuclear receptors that drive the expression of negative regulators of thyroid hormone production upon ligand binding is designed. In this case, the engineered cells are all microencapsulated and implanted in mice, and circulating thyroid hormones or blood glucose are restored to physiological levels in the experimentally induced pathology.

In addition to the therapeutic field, a variety of products have been developed for the monitoring of human health. For example, a team of researchers demonstrated a non-invasive sensor placed on the forearm, which can be used to detect continuous skin signal intensity to detect blood glucose levels and thus enable disease monitoring in people with high blood sugar.^[16]

The rapid research and development of vaccines can also be seen in the credit of synthetic biology technology. Bio factory in synthetic biology technology systems can rapidly and massively screen vaccine prototypes and accelerate vaccine delivery time through DNA editing tools, high-throughput screening systems, iterative engineering in the "design-build-test-learn" cycle and machine learning algorithms.^[17]

The research and development, innovation and marketing of synthetic biology products are expected to further help the prevention, diagnosis and treatment of tumors, malaria, strain infections and other diseases.

2.2 Green energy

Synthetic biology energy products refer to the synthesis of raw materials such as agricultural, forestry wastes and organic wastes generated in daily life into energy products through artificially constructed synthetic biology engineering cells, which is the process of "turning waste into treasure".

Synthetic biology converts renewable biomass into fuel through fermentation engineering, reducing carbon emissions and creating new green energy options. Specifically, synthetic biology overcomes the technical problems of biomass fuel by designing, modifying, reconstructing or manufacturing biomolecules, biological components, biological reaction systems, metabolic pathways and processes, and even cells and biological individuals of the entire biological activity.

Synthetic biology can use chassis cells such as yeast and E. coli to produce clean, efficient and sustainable chemicals and bioenergy products using carbon dioxide and other substances as raw materials, and realize the gradual replacement of non-renewable resources. For example, microorganisms modified by synthetic biology methods can convert biomass into ethanol proteins^[18] Sanjay Kumar's team at the Indian Institute of Technology discovered the fastest growing strains of biofuels, Synechococcus, UTEX2973, known as PCC 6803 and C.sinuosa PCC7942 which have been successfully used in biofuel production.^[19]

Synthetic biology energy products that have been commercialized include bioethanol, biodiesel and bioaliphatic hydrocarbons. For example, the US company LanzaTech uses synthetic bacteria to make ethanol based on exhaust gases such as carbon dioxide and methane produced during the plant's production process^[20]Deep Branch uses microorganisms to convert industrially emitted carbon dioxide into high-value protein to produce clean and sustainable fish and poultry feed, reducing its carbon footprint by up to 75%.^[21]

2.3 Daily make-up

The application of synthetic biology in the field of daily cosmetics has instantly opened up a new world in the raw material market for cosmetic products that are limited by raw materials. Through synthetic biology technology, raw materials such as collagen and squalane that have limited natural access channels and cannot be used on a large scale in the field of cosmetics can be "artificially manufactured", which greatly solves the situation of insufficient supply of raw materials and high prices.

Experimental studies have shown that squalane as an effective carrier can achieve anti-aging effect by enhancing the retention time of the skin, increasing the physiological function of phenolic compounds, and increasing the physiological function of phenolic compounds,^[22]In addition, it is also used in vaccine components，It is currently used for numerous vaccine and drug delivery emulsions due to its stability-enhancing properties and biocompatibility^[23]However, squalane is extracted from the liver of deep-sea sharks in very small amounts and causes ecological damage. Biosynthetic squalane can solve this problem almost perfectly. Genetic manipulation of E. coli has focused on the introduction of key MEP enzymes (such as squalene synthase, SQS) from other squalene-producing microorganisms, via plasmid insertion Further optimization was achieved by the introduction of the full MVA pathway, with modified SQS and HGMR enzymes, from S. cerevisiae into E. coli, yielding up to 230 mg/L of squalene^[24]. The foreign company Amyris successfully synthesized squalane with its own technology. Based on the synthesis process of squalene in synthetic biology, the domestic enterprise Morphic Bio can also use microorganisms as the chassis to produce high-purity squalene, which is later converted into squalane by hydrogenation. In addition, Statetron also has the production capacity of a variety of small molecule peptides, and has solved key mass production problems, and currently sells about 30 kinds of synthetic biological substances.

2.4 Environmental protection

In the field of environment, many of the technical achievements of synthetic biology are still in the laboratory stage, and there has been a lot of progress in laboratory research in environmental monitoring, pollutant control, restoration of natural environment and garbage treatment and utilization.^[25]

Since 1989, degradable strains through natural screening and domestication of microorganisms, and the consequent development of a large number of efficient, low-cost, environmentally friendly bioremediation technologies, have been widely used to remove pollutants from contaminated farmland, groundwater, rivers, lakes and oceans. In the late 80s of the 20th century, the United States first used bioremediation technology to successfully clean up the large-scale pollution caused by the oil spill of the Valdez tanker in Alaskan waters. In 2010, microbial remediation technology once again played an important role in the Gulf of Mexico oil spill^[26]. In China, in 2016, a variety of petroleum hydrocarbon degrading bacteria were used to remediate heavy oil pollution in Xiamen ，and the total degradation rate of petroleum pollutants reached 99.7%, and the degraded oil sludge reached the standard of relandfill.^[27]

A well-known example of the commercial application of synthetic biology products in the environmental field is gene-driven mosquitoes, that is, the use of synthetic biology technology to change mosquito genes to force their population to collapse, and ultimately achieve the purpose of preventing mosquito-borne diseases and purifying the environment [28]. In addition, heavy metal microbial sensors have also made great breakthroughs. A well-known example is the modification of bacteria through synthetic biology technology to make whole-cell microbial sensors that can detect the content of arsenic in water.^[29]

3. The main business risks of synthetic biological products

3.1 Biosecurity risks of synthetic biology products

Biosecurity risks are defined as unauthorized access, loss, theft, misuse, diversion or intentional release. For many years, the biosecurity risks of synthetic biology have focused mainly on the theft and misuse of fixed, current, and intangible assets in laboratories, including synthetic biology components (minimal genomes, orthogonal biological systems/xenobiology, blasts, etc.) with SynBio products and SynBio technology^[30] There may be a biological weapons risk, as it may be used to reconstruct known pathogens, make existing pathogens more dangerous, or create new pathogens for certain purposes.^[31]Once stolen, illegally transported or misused, they can pose a biosecurity risk to all parties, including people, communities and the environment. The potential misuse of these results could lead to serious bioterrorism, biological warfare or biological attacks that may originate from genetically engineered organisms.^[32]

3.2 Synthetic biology involves ethical issues in science and technology

The ethical issues of synthetic biology involve a wide range of scientific and technological issues due to different application fields, but the most important is the ethical issues involving human society. The first is whether life should be synthesized? Once new organisms are produced through synthesis, the form of life is no longer the process and form of natural evolution, life is no longer the only product of nature, technology and artificial, it may be some kind of entity between life and machine. Moreover, the introduction of new elements into organisms through synthetic biology techniques may cause infection and immune rejection, while also destroying the integrity and regularity of nature and breaking the boundary between natural and unnatural.^[33]Secondly, due to the "technical rationality" and huge commerciality inherent in synthetic biotechnology, once liberalized and integrated with the current social production system, it may pose a serious threat to human free will and the dignity of life, and even lead to the aggravation of social stratification,^[34]and synthetic biology, as an interdisciplinary field, requires bioengineers.^[35]doctors and other parties participate together, so it will inevitably lead to the distribution of intellectual property benefits^[36]and if criminals research illegal drugs through synthetic biology, it will pose a huge threat to human life and health safety.^[37]

3.3 Potential risks to the environment of synthetic biology products

The commercialization of synthetic biology in areas such as the environment, agriculture, and chemical products could pose significant potential dangers to the environment. In synthetic biology labs, scientists often design a large number of safety modules to ensure that synthetic biology products enter the self-destruct program after leaving the lab. However, after commercialization, the production of large-scale synthetic biology products faces a higher test in the complex natural environment.^[38]The genome of the modified engineered bacteria is different from the bacterial genome in nature, and when the modified engineered bacteria enter the natural environment, it may mutate and infiltrate its modified DNA into the natural organism. Unknowable effects on the original natural gene pool.^[39]In addition, the complex biological structure of living organisms, the behavior that can produce autonomy, and the characteristics of self-reproduction. Esvelt's team constructed a mathematical model to assess the impact on ecosystems of individuals modified by CRISPR technology released back into the wild, and the results showed that there was a high risk: the modified genes may spread to areas free of invasive alien species, thereby destroying the region's own well-established ecosystem.^[40]

3.4 Potential risks to human health from synthetic biology products

Synthetic biology products in food, agriculture, medical and other fields often act directly or indirectly on the human body. Including gene expression products, recombinant genetic material, artificial gene elements, etc. on the human body is still unknown. Products whose safety is not yet clear can pose significant risks once used by consumers. Therefore, synthetic biology products need to go through strict and complete laboratory safety testing and clinical trial processes from R&D to marketing, and subsequent strict supervision. In addition, some modifications to the environment and agricultural products may also pose a threat to human health, and genetically modified organisms may bring new health and safety problems to humans, such as. In the case of altered gene drives through synthetic biology techniques to prevent mosquitoes from transmitting dengue virus, modified mosquitoes cannot host dengue viruses, but their sensitivity to carrying other viruses may be enhanced.^[41]

4. Future directions

Synthetic biology has made rapid progress, but compared with the gradual maturity of the underlying technology, the synthetic biology industry is still in a very "young" stage. In addition to the problems inherent in emerging industries such as opening up markets, synthetic biology brings changes to the entire industrial chain, requiring a high degree of cooperation between upstream and downstream chains, so it takes a certain amount of time to wait for the industry and the market to adapt. At the same time, as the main consumers, there is still a lack of understanding of synthetic biology, and the scientific and technological ethical risks and commercial risks also need to be solved. Although there are a series of problems, with the continuous attention of society to biomanufacturing, as well as the influx of technology and talents, the rapid development of the synthetic biology industry is unstoppable. In the foreseeable future, more and more synthetic biology companies will emerge, and a new round of "industrial revolution" will play a role in overcoming major difficulties such as population and health, resources and environment, energy and materials in the future. At the same time, the government and relevant regulatory departments need timely supporting legal support and supervision.

Abstract

At present, because of its advantages like precision and high efficiency, genetically engineered bacteria have become one of the hot spots in the research of genetic engineering therapy at home and abroad. Its primary purposes include the construction of genetically engineered bacteria for intestinal flora disorders and delivery to the gut, and the use of CRISPR-Cas9 to build genetically engineered bacteria that can express specific killing effects, but the safety and ethics of sending genetically engineered bacteria to the human body and making it settle and work are still questionable. This paper focuses on the construction and the therapeutic application of genetically engineered bacteria and its risks and challenges.

Using DNA recombination technology, gene editing tools such as CRISPR-Cas9 can build genetically engineered bacteria that can specifically express target genes. The genetically engineered bacteria have been widely used in the high-density fermentation industry to produce insulin, bacterial lipase and other target proteins on a large scale. In recent decades, there has been great interest in bacteria at home and abroad, and gene editing through synthetic biology is used to construct genetically engineered bacteria that can treat diseases.

Selection of genetically engineered bacteria

The selection of host bacteria commonly used include Escherichia coli, corynebacterium glutamicum, Bacillus subtilis, Streptomyces and Yeyeria liposis, etc. The common advantages are the small size of cells, rapid reproduction, vigorous metabolism, fast expression of products, and less existing genetic material in cells, so the operation is relatively convenient. Taking Escherichia coli as an example, there are BU series, K802 series, and MG1655 laboratory strains that are widely used at present. Some strains, such as DH10B, are suitable for cloning genomic DNA and are often used to construct gene banks or cDNA libraries. However, for treatment, we need Escherichia coli to express a certain amount of proteins or other substances with biological regulatory activity. At this time, we need to select special expression strains and construct plasmids. ZY-312 strain which is currently under research is a good choice. TOP10 strains of engineered Escherichia coli constructed by Saeidi^[2]and Gupta^[3] and SK08 series, which are the first batch of viable bacteria drugs to enter the clinics are good examples.

Construction of genetically engineered bacteria

In the case of E. coli, we need to introduce the target gene and make it moderately expressed. The first step is to obtain the target gene in vitro, the specific methods include restriction enzyme direct separation, library screening, in vitro amplification and artificial synthesis. The target gene is obtained by enzyme digestion and amplified by PCR or RT-PCR. The second step is to connect the target gene with the carrier plasmid to form the recombinant DNA molecule, and the third step is to introduce the recombinant DNA molecule into the recipient cell and induce its expression. The expression of target genes requires inducible promoters. In fact, promoters are inhibited most of the time. The more commonly used induction stages include IPTG, DE3-lysogen and T7 promoter expression systems. Considering that there is a certain level of background expression in the cell itself, it is not practical to give additional glucose in the human body. Studies have shown that the use of carriers with LacⅠ promoter can inhibit the expression of the background, but does not affect the expression of the target protein after subsequent IPTG induction.^[4]The fourth step is to screen and detect the target genes, and some researchers have developed expression systems that do not rely on antibiotics and are not affected by foreign toxins. The fifth step is to deliver the prepared host cells into the human body, control their reproduction rate and stabilize the expression of the target genes.

Clinical application of genetically engineered bacteria

Dr. Yang Liu isolated ZY-312 strain from the gut of healthy infants, and studies have shown that the strain and its metabolites can promote macrophages to phagocytic pathogens and regulate the inherent immune system.^[8]SYNB1618, an engineered bacterial drug developed by Synlogic, an American synthetic biology company, can significantly reduce the level of phenylalanine in serum of patients with phenylketonuria.^[1]The incidence of colitis in China is increasing, the incidence of men and women are similar, the main symptoms are intestinal inflammation, continuous or intermittent diarrhea, abdominal pain, long-term recurrence is easy to treat. Chen Guoqiang's team developed EcN Escherichia coli that can overexpress 3HB in response to this disease, and the obtained recombinant bacterium EcNL4 can colonize the intestine and synthesize drugs using food as raw materials. The drugs are released directly in situ, reducing the off-target effect of the drugs, eliminating the influence of the first pass effect, and can be automatically administered for a long time. It has better efficacy than using alone.^[5]

Problems and reflections on clinical application

It is very important to evaluate the safety of engineered bacteria. When selecting the appropriate engineered bacteria, it is necessary to consider its growth rate in the human body, whether it can symbiosis with the native flora, and whether a small percentage of people will be infected or experience immune rejection. Another disadvantage of E. coli expression is antibiotic-dependent, drug-resistant gene problems. Professor Bi Yujing proposed the study of whole genome sequencing, including genotype identification, enterotoxin-producing gene analysis, virulence factor analysis, drug resistance gene analysis and passage stability analysis. In addition, some virulence factors can also be found through database comparison, and resistance genes can be tested by in vitro drug sensitivity test. The stability of passage is also an urgent problem to be solved in genetically engineered bacteria, and it is also an important index to measure whether bacteria meet the needs of subsequent industrialization. Other safety assessments have been proposed, including cell adhesion and cytotoxicity. Whether the engineered bacteria itself , its constitutive expression, or the dead bacteria will have adverse effects on the human body remain unknown. For immunocompromised population, immune deficiency mice can be used as a model, but the specific safety still needs further research.^[6]

In terms of ethical issues, in addition to the six ethical principles proposed by ERBC, the use of genetically engineered bacteria for treatment should be communicated and discussed as far as possible with cross-fields, especially stakeholders. The acceptability of such live bacteria therapy to patients is different, and their opinions on mode of administration should also be taken into account.

Prospect

Since Genentech announced the successful production of insulin from recombinant genetically engineered bacteria, genetically engineered bacteria and related technologies have developed rapidly. Compared with the first generation of probiotics, the advantages of genetically engineered bacteria embrace automatic delivery of drugs according to demand, long-term use and rapid onset. However, genetically engineered bacteria inevitably bring new strains and enzyme genes to the human body, and their safety and interaction with the human body should be particularly considered.^[7]Once the drug delivery bacteria mutate or produce adverse consequences, a suicide switch or negative regulatory mechanism should be designed. To transform design to clinical medicine, a large number of experiments and reliable data are still needed.