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<p style="font-size: 80%; color: grey;"> Figure 1: Yogurt Mamas - A very successful microenterprise that empowers women and communities.</p>
 
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Revision as of 23:52, 10 November 2020

Calgary: Oviita

Oviita: A community-based approach to vitamin A deficiency
John Cedric Acierto1,2, Michaella Atienza1,2, Alexa Calkhoven1,2, Nimaya De Silva1,2, Allison Guthrie1,2, Sravya Kakumanu1,2, Randy Moore1,2, Arshia Mostoufi1,2, Simran Sandhu1,2, Andrew Symes1,2, Anika Zaman1,2, Mackenzie Sampson1, Christian Emond1, Tian Zhao1, and Mayi Arcellana-Panlilio1

1 Affiliated with the University of Calgary
2 All authors contributed equally

ABSTRACT

As a leading global cause of preventable blindness and mortality, vitamin A deficiency (VAD) is a serious health problem, particularly in developing regions. Oviita aims to equip these vulnerable regions with a sustainable and community-based solution to VAD. Our solution uses a food-safe strain of Yarrowia lipolytica modified to produce beta-carotene, a Vitamin A precursor. By engineering this yeast to produce cellulase, VAD communities can grow it as their own vitamin A supplement using readily-available plant matter as feedstock. To facilitate community integration, we designed bioreactor schematics based on locally-available resources, and made the yeast auxotrophic to ensure safe growth with no environmental risk. We also created a Vitamin A biosensor to improve VAD testing, and included an anthelmintic agent in the yeast to combat poor intestinal health, two contributing factors to VAD. Through these solutions, Oviita aims to be a sustainable and community-based adjunct to global efforts against VAD.
Introduction
An estimated 250 million children worldwide are currently suffering from Vitamin A deficiency (WHO, 2020). Vitamin A deficiency (VAD) is one of the leading causes of preventable childhood mortality and also causes reproductive issues, reduced immune function, vision loss, and in severe cases, xerophthalmia. VAD disproportionately harms some of the world’s most vulnerable populations, particularly pregnant women and children in Southeast Asia and Sub-Saharan Africa.

Figure 1. Prevalence of vitamin A deficiency around the globe.


VAD is perpetuated in communities for three main reasons:
  1. A lack of sustainable Vitamin A supplementation
  2. Minimal community-based implementation
  3. Inadequate healthcare infrastructure

Oviita has six main sub-projects to address these three issues and provide a holistic and comprehensive solution to VAD.

Figure 2. Oviita pipeline - a schematic representation of the different subprojects designed to address each of the three perpetuators of VAD.

Beta-carotene, a precursor to Vitamin A, is a molecule that can be used to meet the body’s Vitamin A needs. Unlike Vitamin A, beta-carotene has no lethal dose, and is therefore completely safe for consumption.

Graciously sent to us by
Dr. Rodrigo Ledesma-Amaro
of Imperial College London, we plan to utilize Y. lipolytica strains already engineered and characterized for beta-carotene production. These beta-carotene producing yeast cells serve as the basis of Oviita, our approach to alleviating VAD.
Cellulase Integration

For Oviita to be successful, it must first be inexpensive and accessible in the communities that need it. By enabling it to break down cellulose, our yeast can be grown using local agricultural waste such as straw, leaves, and rice hulls as feedstock.

Dr. Charles Mather
, an anthropologist, was vital in advising us on the existing use of cereal crops for yeast cultivation in West Africa. Plant wastes are a prime source of feedstock as they often go unused, and are sometimes burned to dispose of the excess.

EXPERIMENTAL DESIGN

There are three classes of cellulase enzymes necessary for cellulose degradation: endoglucanses (EGs), cellobiohydrolases (CBHs), and beta-glucosidases (BGSs). We are engineering three strains of Y. lipolytica to each secrete one of the three cellulase enzymes that will be necessary to break down plant matter (figure 1).

Figure 1. Secretion of three cellulase enzyme classes by one of the three different engineered Yarrowia lipolytica strains.


Each of our cellulase constructs were designed as per the schematic in figure 2. Furthermore, two of our critical cellulases were modelled and engineered, to have better function in Y. lipolytica. The constructs will be assembled together via Gibson Assembly, and transformed into Y. lipolytica using protocols and advice provided by
Dr. Ledesma-Amaro.
Once our transformations are confirmed by sequencing and protein secretion confirmed using ELISA, we will test our strains' cellulase activity on pure cellulose and crude plant biomass using a DNS assay.

RESULTS

Meanwhile, we decided to do experiments to test the general idea of Y. lipolytica growing on cellulose with cellulase. When we tested the growth of Y. lipolytica with direct plant biomass and 5mg/mL cellulase, growth was sustained for 2 days, after which there was a significant drop. We believe this is due to decreased access of the cellulase to the cellulose over time (figure 2).
Modelling
Modelling goes here
Bioreactor Design
To bring Oviita into communities, we needed to develop a system capable of culturing our yeast that was simple, effective, inexpensive, and flexible in design. With these criteria in mind, our strategy relies on two bioreactor designs: a Laboratory Adaptable Bioreactor (LAB) and a Field Adaptable Bioreactor (FAB).

DESIGN CONSIDERATIONS

The LAB mimics conditions that we expect in the field, providing data from an array of sensors that will influence our FAB design. To develop our LAB, we collected information from literature, mathematical models and our HP contacts, and conducted small experiments, such as the media’s change in pH as the yeast grows and how the yeast settles over time. With the LAB constructed, we are now simulating multivariable, real-world conditions to further our modelling and form predictions, with the aim of generating a range of variables that the FAB design can operate within.

Figure 1. Lab-adapted bioreactor (left) and CAD model of Filed-adapted bioreactor (right).


Rather than a rigid FAB design, we intend to provide users with a flexible design and ideal growth ranges for variables such as temperature and aeration rate. Locals can then construct the FAB to fit their vision with the resources they have at hand. In addition to providing design starting-points and information for each variable, we will develop an effective training program to enable our users to optimize Y. lipolytica growth and yield.

Figure 2. Bioreactor design workflow


Biocontainment
Current biocontainment strategies used in the industry revolve around the use of auxotrophic microorganisms where a constant supplementation of metabolites is required to keep the organism alive. Such methods, however, are unsustainable and unreliable especially for products that are meant to be implemented outside of the laboratory space. Therefore, we designed a robust, yet sustainable biocontainment system to tackle this issue using advice given by
Dr. Robert Mayall.


EXPERIMENTAL DESIGN

Our system used two strains of S. cerevisiae cultured together in a syntrophic community. Each yeast strain was auxotrophic for an amino acid the other strain overproduced, resulting in the strains being required to stay together in order to survive and proliferate.

Figure 1: Yogurt Mamas - A very successful microenterprise that empowers women and communities.



Our system used two strains of S. cerevisiae cultured together in a syntrophic community. Each yeast strain was auxotrophic for an amino acid the other strain overproduced, resulting in the strains being required to stay together in order to survive and proliferate.

RESULTS

To test the efficacy of our biocontainment system, we grew the complementary yeast strains in media lacking the required amino acids and showed that strains were able to set up an obligatory mutualistic relationship and support each other’s growth over two days.

We also tested the ability of the environment to support the growth of each of our yeast strains. We determined that the yeast strains were unable to grow in environmental samples such as soil and river water, indicating a low likelihood of microorganisms escaping into the environment. Given the success of our auxotrophic system in S. cerevisiae, we plan to integrate this system into Y. lipolytica in the near future.
Proposed Implementation

Our proposed implementation strategy focuses primarily on two things: easy cultivation and preparation of the Oviita yeast and increasing access to the yeast through microenterprises. As Y. lipolytica is generally recognized as food-safe, it needs to only be heat-killed (cooked) before being safely ingested. To help integrate the yeast in with local cuisine, we researched and compiled a collection of local recipes that would complement the yeast.

We also believe that a successful organization relies on a network of people at all levels. After speaking with the founders of Western Heads East, an organization that empowers groups of women to start small enterprises that monetize probiotic yogurt, we decided to implement a similar model into our organization.

Figure 1: Yogurt Mamas - A very successful microenterprise that empowers women and communities.

Their success has proven that these microenterprises, when combined with our health product and training, can cultivate positive change, stimulate economic development, and foster good health within their communities. This model also enhances the sustainability of our organization, as it not only provides an efficient means of distribution, but also encourages the consumption of our product. With help from our partners, community health workers, and a local university research group in our pilot area, we will develop a training program designed to provide these women with the tools and knowledge they need to succeed. This training will include information on vitamin A deficiency, our bioreactor, and entrepreneurship. From there, we hope to expand this program to a greater number of local women’s groups, as well as other disenfranchised groups.

Deworming
According to
Dr. Dia Sanou
, a public health nutritionist, intestinal parasite infection runs rampant and impedes micronutrient and vitamin absorption in our target communities. Typically, vitamin A supplementation is coupled with biannual deworming, but lack of access to clean water and proper footwear results in the parasites returning. To address this, we plan to engineer Y. lipolytica to produce thymol, an anthelmintic agent found in thyme leaves.

Figure 1. Intestinal parasites (left) and the molecular structure of thymol (right).


EXPERIMENTAL DESIGN

A terpene synthase and a cytochrome P450 assembled using Gibson assembly will be transformed into our chassis through lithium acetate transformation (figure 2).

Figure 2. Synthesis of thymol.


After, an SDS-PAGE analysis will be performed to confirm protein expression, alongside GC/MS to verify and quantify γ-terpinene and thymol production. When we met with
Dr. Samir Gupta
, a respirologist who has worked on VAD and deworming initiatives, he emphasized the importance of efficacy and safety of the compound before implementation. Thus, upon successful thymol production we will test its efficacy on Caenorhabditis elegans (the gold standard for anthelmintic testing) and Ascaris lumbricoides (a common intestinal parasite in our target demographic). These tests will help us determine the optimal dosage of thymol for ingestion, and inform future iterations of our constructs to increase or decrease its expression.
VAD Testing
The Randle’s Cell Testing Device is a field-based diagnostic test that attempts to improve upon current methods used to identify vitamin A deficiency in an individual. According to
Banda Ndiaye,
the Deputy Regional Director for Health of Nutritional International Africa, decision makers needed more data to inform VAD intervention. This test uses aptamers to cause retinol binding protein in a blood sample to bind to the electrodes. The resultant electrical changes in the electrodes enable the quantification of vitamin A levels. This year’s focus was on constructing an impedance measurement circuit capable of quantifying the impedance changes that would occur when a blood sample containing retinol is tested.

DESIGN CONSIDERATIONS

With help from
Dr. Colin Dalton,
Thomas Ljinse,
and
Sultan Khetani,
we were able to develop a prototype. In order to do this, we sourced a commercial integrated circuit, the AD5933 by Analog Devices, and interfaced it with the popular microcontroller, the Arduino Uno. Using the Arduino IDE, an extensively documented code was written to calibrate the AD9533 to run impedance spectroscopy. This code was designed for customization and modularity to facilitate the use of the impedance analyzing device for a broad range of future applications in addition to its intended use. As a proof of concept, we used the AD5933, measured the known capacitive and resistive loads with a high degree of accuracy, and also conducted a frequency sweep on an aqueous solution (figure 2).

Figure 2. Impedance analysis circuit measuring a 1nF capacitive load featuring the arduino uno, and AD9533 on the pmod IA (left) and the Randle's Cell Testing Device measuring impedance of an aqueous solution (right).


We intend to first verify our legal capacity to work with our selected aptamer. We will then proceed to verify how selective the aptamer is to retinol binding protein, and immobilize this aptamer to an electrode to then test the impedance measurement circuit’s ability to quantify retinol binding protein levels.
References
World Health Organization. (2020). Micronutrient Deficiencies. Nutrition. https://www.who.int/nutrition/topics/vad/en/.

Acknowledgements

We would like to extend heartfelt thanks to our sponsors who made our work on this project possible.

Figure 1. The logos of our sponsors, whose support made our work on this project possible.