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. 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. [1][2] 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 [3], our strategy is designed to facilitate implementation in the field by incorporating our engineered probiotic into an existing and accepted cultural practice.

1. http://en.wikipedia.org/wiki/Phytic_acid
2. http://www.sciencedirect.com/science/article/pii/S1360138501021045
3. http://www.benthamscience.com/cnf/sample/cnf3-1/D0007NF.pdf

Execution

Engineering E. coli to produce phytase

Many organisms produce many different forms of phytase, including E. coli. Although E. coli natively produces phytase, we planned on contitutively producing the phytase at a higher rate than in the native cell. Different phytases from different organisms also produce different forms of phytase, each which may be more or less effective at hydrolyzing phytate at different pHs.

We decided to attempt to clone four different types of phytase from four different organisms: E. coli, Aspergillus niger, Bacillus subtilis , and Citrobacter braakii. We would clone each out of their respective genome with colony PCR, and the ligate the gene into a Biobrick standard backbone.

Method of protein release

The probiotic we are constructing is intended to be activated at a pH from 1.35 to 3.5 that is specific to the stomach. The probiotic is designed to reside in the stomach and selectively lyse the cloned cells to release the phytase. Despite the fact that E.coli is commonly used for recombinant protein production, its secretion capabilities are rather limited. Thus, it was necessary to utilize cell disruption techniques to procure the intracellularly expressed protein of interest. In order to program cell death, while ensuring minimal cell disruption, it was apt to implement bacterial cell lysis, a technique that can successfully lyse cells through intracellular expression of lytic proteins native to the phage.

In literature, several genetic parts for programmed cell lysis have been studied, including inducible lysis systems. In an inducible lysis system, the T4 phage holin and T7 phage lysozyme genes have been used to produce lytic E.coli strains. Holin is responsible for forming pores in the inner membrane of bacteria, while lysozyme breaks down the peptidoglycan layer and lyzes the cell. Meanwhile, anti-holin is constitutively expressed and it prevents holin from spontaneously allowing lysis. Anti-holin was an imperative part of the construct because ideal lytic systems should be display predictable behavior, instead of randomly lysing at times.