Team:ICJFLS/Design
After several times of brain storm and integration of the creative design of bacterial cellulose in 2020 IGEM, we decided to synthesize amylose from bacteria to make straw because of its disposable and biodegradable, better useful characteristics and, most important, saving food.
1.Features of the amylose (why we choose the amylose produced by bacteria)
Starch has a wide range of applications in food and non-food based industries. It is the main reserve polysaccharide of cereal grains roots, and tubers and a main energy source in the human diet. Starch consists of two polysaccharides: almost linear amylose containing glucose residues joined by α-1,4-glycoside bonds and highly branched amylopectin containing glucose residues connected by α-1,4-glycoside and α-1,6-glycoside linkages. Starch commonly contains about 20 to 30% amylose and 70 to 80% amylopectin. The amylose content greatly affects the functional characteristics of starch, such as pasting, gelatinization, retrogradation, syneresis, and so on, and becomes a major factor controlling almost all physicochemical properties of starch. Starch is perhaps the most important polymeric carbohydrate in terms of its functionality that imparts to products used in diverse industries.
High amylose maize starch granules resist human amylolytic digestion and are referred to as resistant starch and a source of dietary fiber with beneficial health effects. The enzyme resistance of which is the result of it containing crystalline amylose. Resistant starches are used in some food applications such as making starch straw.
In the present, the amylose obtained through the isolation of starch is complicated. Moreover, several organic compounds, such as nitroalkanes, thymol, t-butyl alcohol, and dimethyl sulfoxide, are involved to form a precipitatable complex with amylose during the separation of starch. These treatments not only have the potential risk to healthy, but also are costly and environmentally unfriendly, with thermo- or chemical reactions.
That’s why we choose the amylose produced by bacteria to make amylose straw, which is cheaper and environmentally unfriendly. More importantly, it saves food.
2. The applications of amylose at present
At present, amylose has played a very important role in many industries. In the food industry, it can be used as puffed food, low-fat food, food packaging materials, food additives and so on. In terms of environmental protection, the plastic made by amylose is degradable, which is of great significance to solve the current white pollution and protect the environment. In addition, it has played a certain role in the medical and pharmaceutical industry, light industry and textile industry.
2.1 Food additives
Because amylose has gel setting property, the suitable ratio of amylose and amylopectin in starch can make as additive for canned, beverage, oral liquid, and so on. Under the treatment with additive, the liquid phase is not stratified, solid liquid phase is stable and no sedimentation, and the taste is good. As a coagulant for sausage and ice cream, it has a delicate and delicate taste. It can be used for cake and cheese to make products soft and delicious.
2.2 Functional food
In recent years, high amylose is used as a substitute for oil and cream to solve the problem of high fat in fast food, such as replacing a large amount of cream in ice cream and some sandwich biscuit oil with amylose to make "low-fat food". High amylose food is also an ideal food for diabetics. It also prevents gallstone formation and lowers cholesterol.
2.3 Packaging materials
High amylose has the properties of condensation and easy formation of stable gel properties. It can be used as a tough food packaging film. The film has good isolation for oxygen and grease, and is suitable for product protection because of its good deployment. Its acetic acid derivative can also be used as textile fiber, sizing, fiber polishing, adhesive, binding material, etc. In addition, the adhesive is non-toxic and water-soluble. It is also used in medicaments, bandages, sutures and other medical products.
2.4 Biodegradable plastics
The plastic resin can be made by amylose with other polymers, and then processed into plastic. High amylose plastic has biodegradability and is used as agricultural covering film. Because 90% of its composition is amylose, it can decompose naturally in a very short time and do not pollute the environment. Therefore, it can solve the increasingly serious white pollution to the environment, which is a very pollution problem. This feature of amylose is suite for making straw.
Based on our knowledge, there is few reports that amylose produced by bacteria was used to make straw.
3. synthesis pathway of amylose in plant
In plant, Starch biosynthesis is controlled by isoforms of five enzymes. Adenosine 5′ diphosphate (ADP) pyrophosphorylase (AGPase) produces ADP-Glc (“Glc” = glucose) as the first step in starch biosynthesis. ADPGlc is elongated by starch synthases (SSs). There are number of SS isoforms including granule bound starch synthase 1 (GBSS1), SSI, SSII-1, SSII-2, SSII-3, SSIII-1, SSIII-2, SSIV-1, and SSIV-2. Amylopectin synthesis involves multiple isoforms of SSs, starch branching enzymes (SBEs) and starch debranching enzymes (DBEs). SBE snips (1→4)-α linked chains and then adds a snipped chain to form (1→6)-α linkage branch points. The enzymes in amylose synthesis are mainly GBSS1 and one or more SBEs.
Therefore, amylose was synthesized by 2 mainly enzymes: AGPase and GBSS1. SBE is not necessary since we do not hope have branches in amylose.
Fig. 1. The pathway of amylose synthesis
4. How amylose will be produced in E. coli?
In order to produce amylose in E. coli, we plan to clone the genes related to amylose synthesis based on sequences of Arabidopsis thaliana, and transfer them to the engineered E. coli, hoping to be able to produce amylose using E. coli.
There are 2 genes related to the synthesis of amylose, which were ADP glucose pyrophosphorylase (AGPase) containing 2 subunits ADG1 and APL1, and granule-bound starch synthase 1 (GBSS1).
AGPase catalyzes Glucose-6-phosphate (Glu-6-P) converted to adenosine diphosphate glucose (ADPG), which is a key enzyme in amylose synthesis and catalyzes the first, rate limiting step in amylose biosynthesis.
The ADP glucose pyrophosphorylase gene contains 2 subunits, ADG1 and APL1. ADG1encodes the small subunit responsible for catalytic activity, and APL1 encodes the large subunit responsible for regulatory role of ADP-glucose pyrophosphorylase.
Another enzyme for the amylose synthesis is GBSS1, which belongs to UDP-Glycosyltransferase super family protein.
GBSS1 is an enzyme that catalyzes the transfer of glucose from to glucose-containing polysaccharides with α1,4-linkages, which is the main gene controlling amylose synthesis.
5. Design of our project
Based on the pathway of amylose synthesis (Fig. 1), we were going to clone 3 genes (ADG1, APL1 and GBSS1) required for amylose synthesis and construct the expressed vectors and transfer them to the E. coli.
We constructed 2 expressed vectors (pETDuet-1-ADG1-APL1 and pET-28a (+)-GBSS1) as follows:
The vector pETDuet-1-ADG1-APL1 was constructed by two steps.
First, we used vector pETDuet-1 and ADG1 gene to construct the plasmid vector of pETDuet-1-ADG1 by EcoRI and PstI digested and ligated method (Fig. 2).
Fig.2. The vector construction of pETDuet-1-ADG1
Second, the APL1 genes were inserted to vector pETDuet-1-ADG1 by restriction enzyme (NdeI and Xhol) digestion and ligated method. We got the vector pETDuet-1-ADG1-APL1 (Fig. 3).
Fig.3. The vector construction of pETDuet-1-ADG1-APL1
We constructed another vector pET-28a(+)-GBSS1 using pET-28a(+) and GBSS1 gene by restriction enzyme (EcoRI and Xhol) digestion method (Fig. 4).
Fig.4. The vector construction of pET-28a(+)-GBSS1
The two expressed vectors of pETDuet-1-ADG1-APL1 and pET-28a(+)-GBSS1 were co-transferred to E. coli, and induced by isopropyl-β-D-thiogalactoside (IPTG). Under the supply of substrate of glucose, amylose was produced by E. coli (Fig. 5).
Fig.5. The schematic of amylose synthesis in E. coli
In order to get more amylose, different conditions were tested.
6. Combination with our project in 2020 IGEM
Another advantage of this project is to combine with bacterial cellulose projects performed by our group in 2020 IGEM.
Besides amylose, making straw also needs cellulose acting as supporting role. In 2020 IGEM, we cloned 6 protein or enzymes genes (Glucose hexokinase, Phosphoglucomutase, UDP-glucose pyrophosphorylase, Bacterial cellulose synthase, CcpAx protein, Endo-β-1,4-glucanase(CMCax) and β-glucosidase) required for cellulose synthesis, and transferred them to the E. coli. And cellulose was successfully expressed. Therefore, combining with 2020 IGEM achievements, the amylose straw will be made by amylose and cellulose in the coming years. That is amazing.
7. Innovation of our project
7.1 We did not find research that using amylose produced by E. coli to make straw in the published papers. This project can work together with 2020 IGEM achievement we did. Maybe these are the biggest novelties of our project.
7.2 The amylose straw is friendly environment, avoid white pollutions.
7.3 The most important thing of amylose straw produced by E. coli is saving food, which is great importance for poor people who are always in hungry.
References:
[1]Tang H, Fan S, Li Y, Dong S. Amylose: Acetylation, Optimization, and Characterization. J Food Sci. 2019;84(4):738-745. doi:10.1111/1750-3841.14487
[2]Doblado-Maldonado AF, Gomand SV, Goderis B, Delcour JA. Methodologies for producing amylose: A review. Crit Rev Food Sci Nutr. 2017;57(2):407-417. doi:10.1080/10408398.2014.954030
[3]Matsuki J, Park JY, Shiroma R, et al. Characterization of starch granules in rice culms for application of rice straw as a feedstock for saccharification. Biosci Biotechnol Biochem. 2010;74(8):1645-1651. doi:10.1271/bbb.100257
[4]Zhu J, Yu W, Zhang C, et al. New insights into amylose and amylopectin biosynthesis in rice endosperm. Carbohydr Polym. 2020; 230:115656. doi: 10.1016/j.carbpol.2019.115656