Team:Technion-Israel/Design

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On this chapter, we will describe each of the parts featured in our system's design and the reasons for which we decided to include them.

Secretory Signal Peptides

Secreted proteins and enzymes in B. subtilis contain signal peptides. The signal peptides are short peptides, composed of 18-35 amino acids and containing a consensus sequence of Ala-X-Ala at the C-terminus region of the peptide. Cleavage by signal peptidase occurs at the carboxy-terminal of Alanine and allows the secretion of the protein [1][2]

In our project, we decided to use the signal peptide AmyE (alpha amylase) which showed high secretion rates of foreign proteins in previous researches [3][4], for the secretion of both our enzymes. To further improve our system, we have performed experiments using a library of signal peptides, which were purchased from the TaKaRa company. The library contains 173 types of B. subtilis-derived secretory signal peptides, increasing the chance to find signal peptides which will enable the secretion and activity of our enzymes.

Enzymes

The process of honey production is complex and involves various proteins secreted by the bees. Although the content of many of them may vary, there are three main enzymes that take part in the core process of honey production:invertase, glucose oxidase and catalase[5]. The concentrations of the enzymes may vary among different honey samples [6], yet all three enzymes are essential for the creation of honey.

Invertase

Invertase is an enzyme that catalyzes the cleavage of sucrose into fructose and glucose.

Genetic circuit

Figure 1: Illustration of invertase enzymatic reaction

Since cleaving the sucrose is the very first step in the creation of honey, we have chosen to use a variation of invertase that is reported to be highly active, which originates from the mold species Aspergillus niger[7]. This type of invertase has been widely investigated and is known to be relatively stable[6]. Its Km and Vmax values are 0.062mM and 0.013 Mole/minute, respectively[8]. This invertase works as a dimer, each monomer the size of 75 kDa[9] and its optimal pH and temperature are 5 and 60⁰C respectively[8].

Although the optimal conditions for maximal reaction rate are different in both pH and temperature, we have found that the enzymatic activity of Aspergillus niger invertase can still be detected as the temperature drops to 32-37⁰C. We have also found that the activity can be detected on a relatively wide pH range: from 4 to 7 [9].

Glucose Oxidase

Glucose oxidase (GOx) catalyzes the oxidation of glucose using molecular oxygen to create hydrogen peroxide and D-glucono lactone.

Genetic circuit

Figure 2: Illustration of glucose oxidase enzymatic reaction

Since D-glucono lactone spontaneously converts to gluconic acid, the activity of GOx is associated with pH decrease. Due to the acidic and unstable nature of the enzyme's products, we have chosen to regulate the production of glucose oxidase when reaching high gluconic acid and hydrogen peroxide concentration.

We have chosen Aspergillus niger GOx since it is widely researched. It has a dimeric structure[10], and uses FAD as co-factor[11]. Its homodimeric size is 160 kDa[12].

The optimal conditions for enzymatic activity are 25-30⁰C and pH of 5.5-6. The catalytic constants of A. niger GOx Vmax and Km were evaluated to be 7.1 μMole/minute and 7.1 mM[12].

Catalase

Catalase is an enzyme that decomposes hydrogen peroxide to create water and molecular oxygen.

Genetic circuit

Figure 3: Illustration of catalase enzymatic reaction

Its native role in organisms is to protect the cell from oxidative stress due to high levels of free radical and strong chemicals of strong oxidative nature such as hydrogen peroxide. In our system, the catalase is used for both protecting the bacteria from oxidative stress and preventing the accumulation of hydrogen peroxide in our honey.

The catalase enzyme is native to the Bascillus subtilis Bacteria and is partially secreted from the cells[13]. Moreover, the B. subtilis genome encodes for multiple catalases[14]. Their sizes vary from 250-387 kDa, according to the encoding gene.

The Honey Circuit

Honey is a combination of fructose, glucose, sucrose, and other sugars as well as vitamins, antioxidants, hydrogen peroxide, proteins, and aromatic flavors.

The hydrogen peroxide, as previously stated, has an important role as an antibacterial agent in honey, in addition to the high osmotic pressure caused by the high sugar concentration.

Some bacteria can survive in the presence of hydrogen peroxide, to a certain extent, through the secretion of catalase enzyme. Among them is Bacillus subtilis, our chosen bacteria for honey production.

Despite the bacterial survival at certain hydrogen peroxide concentrations, the catalase activity is limited. Therefore, controlling the hydrogen peroxide production is essential for bacterial growth.

This regulation can be achieved by expressing the Glucose Oxidase (GOx) enzyme, which breaks down glucose into D-glucono-1,5-lactone and hydrogen peroxide, under the control of a hydrogen peroxide-sensitive promoter. At high hydrogen peroxide levels, the enzyme expression is inhibited, therefore the production of hydrogen peroxide as a result of enzymatic activity decreases.

Besides regulating hydrogen peroxide levels, our "Honey Circuit" has another important role, which is to maintain a constant sugar composition that simulates the bee honey content. Without the intervention of our circuit – for example, by using commercial enzymes – neither the suitable hydrogen peroxide levels nor the correct sugar composition could be obtained. While commercial invertase will reduce the sucrose concentration, producing glucose and fructose, GOx will degrade the glucose with no regulation. Such a system will ignore the desired fructose to glucose ratio by endlessly decreasing the concentration of glucose and producing a high concentration of hydrogen peroxide, which may eventually kill the bacteria. Thus, the "Honey Circuit" is crucial for the process.

The backbone used to build the system was taken from a commercial plasmid called pBE-S (by TaKaRa) and is designed specifically for the secretion of proteins from B. subtilis.

The first plasmid we created contains a signal peptide fused to the invertase gene to enable the enzyme secretion by the bacteria. This enzyme, as previously mentioned, breaks down sucrose into glucose and fructose. As the concentration of sucrose in honey is low, this enzyme does not need to be regulated and it degrades as much sucrose as possible.

Genetic circuit

Figure 4: Illustration of pBE-S containing the signal peptide AmyE and the gene for invertase

In natural honey, as previously stated, the concentration of fructose is higher than glucose due to the presence of the GOx enzyme. Our goal is to achieve similar fructose to glucose ratio using a regulated synthetic circuit.

Therefore, we created another plasmid containing a promoter that senses the level of hydrogen peroxide and is activated at a specific concentration, called pKat[15]. When activated, the LacI gene is being transcribed and represses the Lac promoter. In turn, this causes the repression of transcription of the GOx gene fused to AmyE signal peptide.

GOx, in the presence of oxygen, catalyzes the degradation of glucose which causes an increase in the concentration of hydrogen peroxide. The presence of hydrogen peroxide represses the transcription of GOx and therefore the degradation of glucose. When the glucose concentration is lower, less hydrogen peroxide is created, which restricts the repression of the entire synthetic circuit.

Genetic circuit

Figure 5: Illustration of pBE-S containing the “Honey Circuit”

Combining these two plasmids, we mimic the natural process that takes place in the bee stomach, achieving an auto-regulated synthetic pathway that produces biosynthetic honey.

The System

To ensure the "Honey Circuit" could sense molecule concentration in the honey production process, we need to enable the transfer of molecules, such as hydrogen peroxide and selected proteins, between the “unprocessed honey mixture” and the engineered bacteria membranes. Another requirement is to separate the bacteria at the end of the process. To overcome these obstacles, we designed a unique capsule that entraps the bacteria inside a membrane, which acts as a selective barrier separating the bacteria from the processed honey mixture. Next, we collaborated with an Israeli company named Bio-castle, which developed a small membrane capsule suited for growing and caging microorganisms for pollutes degradation. Together we modified one of the company products to obtain our designed capsule.

The capsule [16] entraps the microorganisms under optimal growing conditions and coated with a semi-permeable membrane allowing particles less than 0.78µm in diameter pass across the membrane. That way, the microorganisms in the honey process are maintained in a distinct volume, while the secreted enzymes are defused into the external solution, and small molecules, such as hydrogen peroxide, can enter the capsule. Therefore, the bacteria can “sense” the mixture and activate the “Honey Circuit” we have designed to secrete enzymes according to the mixture status. At the end of the enzymatic process, we can pull out the capsules from the sugar mixture to get a bacteria-free solution that is ready for the final step: drying.

Genetic circuit

Figure 6: Illustration of BioCastle capsule

Scale-up of our system can be achieved by evaluation of the enzymatic activity and enzyme secretion in each capsule, per volume. Following that, we can adjust the capsules number needed for each volume to achieve the desired composition in the external solution.

After the enzymatic degradation, our mixture contains about 70% of water. Naturally, in this phase, honeybees swing their wings and using the air for drying, which consequently decreases the water content to a desired 17%, as stated in the description chapter.

Our team chose to mimic the natural process, using pharmaceutical tray dryer[17]. Applying this drying technique, we can dry our mixture without changing the temperature or affecting the sugar's properties.

References
  1. von Heijne G. 1985. Signal sequences. The limits of variation. Journal of Molecular Biology 184:99–105.
  2. Lüke I, Handford JI, Palmer T, Sargent F. 2009. Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB. Archives of Microbiology 191(12):919–925.
  3. Joliff G, Edelman A, Klier A, Rapoport G. 1989. Inducible secretion of a cellulase from Clostridium thermocellum in Bacillus subtilis. Applied and Environmental Microbiology 55(11):2739–2744.
  4. Ohmura K, Shiroza T, Nakamura K, Nakayama A, Yamane K, Yoda K, Yamasaki M, Tamura G. 1984. A Bacillus subtilis secretion vector system derived from the B. subtilis α-amylase promoter and signal sequence region, and secretion of Escherichia coli β-lactamase by the vector system. Journal of Biochemistry 95:87–93.
  5. Alvarez-Suarez JM, Tulipani S, Romandini S, Bertoli E, Battino M. 2010. Contribution of Honey in Nutrition and Human Health: a Review. Mediterr J Nutr Metab 3:15–23.
  6. Persano Oddo L, Piazza MG, Pulcini P. 1999. Invertase Activity in Honey. Apidologie 30:57-65.
  7. Nadeem H, Rashid MH, Siddique MH, Azeem F, Muzammil S, Javed MR, Ali MA, Rasul I, Riaz M. 2015. Microbial invertases: A review on kinetics, thermodynamics, physiochemical properties. Process Biochemistry. 50(8):1202-1210.
  8. L’Hocine L, Wang Z, Jiang B, Xu S. 2000. Purification and partial characterization of fructosyltransferase and invertase from Aspergillus niger AS0023. Journal of Biotechnology. 81:73-84.
  9. Goosen C, Yuan XL, Van Munster JM, Ram AFJ, Van Der Maarel MJEC, Dijkhuizen L. 2007. Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity. Eukaryotic Cell. 6(4):674-681.
  10. Hecht HJ, Kalisz HM, Hendle J, Schmid RD, Schomburg D. 1993. Crystal Structure of Glucose Oxidase from Aspergillus niger Refined at 2·3 Å Reslution. Journal of Molecular Biology. 229:153-172.
  11. Zayats M, Katz E, Willner I. 2002. Electrical contacting of glucose oxidase by surface-reconstitution of the apo-protein on a relay-boronic acid-FAD cofactor monolayer. Journal of the American Chemical Society. 124(10): 2120-2121.
  12. Katherine R. Frederick, Tung J, Richard S. Emerick F, Chamberlain SH, Vasavada A, Rosenberg S. 1990. Glucose Oxidase from Aspergillus niger. The Journal of Biological Chemistry. 265:3793–3802.
  13. Naclerio G, Baccigalupi L, Caruso C, De Felice M, Ricca E. 1995. Bacillus subtilis vegetative catalase is an extracellular enzyme. Applied and Environmental Microbiology. 61(12):4471-4473.
  14. Loewen PC, Switala J. 1987. Multiple Catalases in Bacillus subtilis. Journal of Bacteriology. 169:3601–3607.
  15. Herbig AF, Helmann JD. 2001. Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA. Molecular Microbiology. 41(4):849-859.
  16. Kurzbaum E, Raizner Y, Cohen O, Suckeveriene RY, Kulikov A, Hakimi B, Iasur Kruh L, Armon R, Farber Y, Menashe O. 2017. Encapsulated Pseudomonas putida for phenol biodegradation: Use of a structural membrane for construction of a well-organized confined particle. Water Research 121:37–45.
  17. Misha S, Mat S, Ruslan MH, Sopian K, Salleh E. 2013. Review on the application of a tray dryer system for agricultural products. World Applied Sciences Journal 22:424–433.





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Department of Biotechnology & Food Engineering
Technion – Israel Institute of Technology
Haifa 32000, Israel

    • igem.technion.2019@gmail.com