Abstract

Iron deficiency affects 2 billion people -or over 30% of the world’s population – and can lead to anemia, ill health, and even death [1]. Surprisingly, this deficiency is typically not due to a lack of dietary iron, but rather due to low bio-availability, and thus poor absorption of iron. Phytic acid is a prevalent chelator of iron and other nutrients in food. Our mission is to build a system that breaks down phytic acid in the digestive system, releasing bound iron for the body to absorb. Our solution comprises two engineered components: a module that constitutively produces phytase to break down phytic acid and a pH-sensitive module that causes cells to lyse and release the accumulated phytase in the stomach. If successful, our strain would be a low-cost sustainable solution to preventing iron deficiency without the need for constant supplies of iron supplements.

Purpose

Our team focused on ameliorating nutrient deficiencies in developing countries. The three main nutrient deficiency disorders listed by the World Health Organization are vitamin A deficiency (VAD), iodine deficiency disorder (IDD) and iron deficiency anemia (IDA). We chose to focus on iron deficiency anemia, which is "the most common and widespread nutritional disorder in the world."

Research in low-income countries has demonstrated that iron deficiency arises not from a lack of iron in the diet, but rather from low bioavailability of iron in the diet. Low bioavailability is due to phytate-mediated chelation of iron in a state that the body cannot absorb. Indeed, phytate (or phytic acid) is a major component of plant tissue; it is the principal form of storage of phosphorous in many plants including grains and legumes [2][3][4]. This suggests that iron could be made more readily available without a change in diet by simply dechelating the iron (and other nutrients) from the phytate.

Phytase is an enzyme produced by bacteria and fungi that breaks down phytate. Our goal is to produce a probiotic that will release phytase into the body, thus increasing the bioavailability of iron. Since many communities that experience a high prevalence of iron deficiency already use fermented dairy products as part of their diet [5], our strategy is designed to facilitate implementation in the field by incorporating our engineered probiotic into an existing and accepted cultural practice.

Execution

Engineering E. coli to produce phytase:

Many organisms produce different forms of phytase, including E. coli [7][8]. Although E. coli natively produces phytase, our engineered probiotic concept requires producing the phytase at a higher-than-native level, and selective release of this enzyme into the extracellular environment. Different phytases from different organisms also produce different types of phytases, each of which varies in the rate at which it hydrolyzes phytate as a function of pH.

We sought to clone four different types of phytase from four different organisms: E. coli, Aspergillus niger, Bacillus subtilis, and Citrobacter braakii. Our aim was to create new synthetic biology parts by cloning each into a Biobrick standard backbone, including constructs in which each phytase is expressed from a constitutive promoter.

Method of protein release:

Our strategy is to engineer a probiotic that lyses and releases pre-synthesized phystase only when the probiotic is exposed to the acidic environment of the stomach (pH ~1.35-3.5), since release of iron from phytate in the stomach would enable optimal uptake of this now bioavailable iron. Although E. coli is commonly used for recombinant protein production, its secretion capabilities are finite, and we desire a system in which large quantities of phytase are released quickly before the probiotic and food exit the stomach. Thus, we decided to utilize inducible cell disruption to release pre-synthesized, intracellularly expressed phytase. In order to achieve rapid and conditional disruption via cell death, while ensuring minimal cell disruption prior to the induction of this death program, we decided to utilize bacterial cell lysis induced via intracellular expression of lytic proteins derived from a bacteriophage.

To meet this need, we utilized UC Berkeley’s Lysis Device part (from iGEM 2008), which is very well-characterized on their wiki here [9]. In this inducible lysis system, holin from bacteriophage T4 and lysozyme from bacteriophage T7 are used to produce autolytic E. coli strains. Holin is responsible for forming pores in the inner membrane of bacteria, while lysozyme breaks down the peptidoglycan layer and lyses the cell. Prior to the induction of this death program, anti-holin is constitutively expressed and prevents holin from mediating lysis. Anti-holin is essential for meeting our needs, because preventing lysis should maximize replication of our engineered probiotic during the production phase (e.g., during fermentation of the dairy product) and thereby maximize delivery of phytase cargo to the desired location (the stomach).

Sources:

1. http://www.who.int/nutrition/topics/ida/en/index.html
2. http://www.chalmers.se/chem/EN/divisions/food-science/research
3. http://en.wikipedia.org/wiki/Phytic_acid
4. http://en.wikipedia.org/wiki/Phytate
5. http://www.sciencedirect.com/science/article/pii/S1360138501021045
6. http://www.benthamscience.com/cnf/sample/cnf3-1/D0007NF.pdf
7. http://mic.sgmjournals.org/content/144/6/1565.short
8. http://www.sciencedirect.com/science/article/pii/S0960852400001395
9. https://2008.igem.org/Team:UC_Berkeley/LysisDevice