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Project Description

Background

In Denmark today, less than half the waste produced is recycled, which means that more than 3.5 million tons get burned off each year. We have abundant waste streams from the industry such as glycerol from biodiesel production, byproducts from rapeseed production, used cooking oil and ordinary household waste.

Cell factories are becoming an increasing factor in the industry today, where different microorganisms are utilized to produce various compounds from therapeutics, organic acids, food additives etc. Currently however, the sustainability of these industrial processes is limited by the narrow substrate range of the organisms used. The most common feeds in use are simple carbohydrates such as glucose produced by enzymatic hydrolysis from edible plants such as maize, rice and wheat.

It is widely established that the dimorphic yeast Yarrowia lipolytica grows well on a broad range of substrates such as alcohols, fatty acids, glycerol as well as on simple sugars in complex mixtures, whereas the conventional and widely used yeast Saccharomyces cerevisiae only grows well on a very limited amount of substrates such as glucose. Furthermore, the protein modification and secretion systems of Y. lipolytica gives rise to a higher potential as a cell factory for production of a variety of therapeutics, food additives etc. Both of the species are fast growing, which contributes to the final efficiency as cell factories.

Nonetheless, Y. lipolytica has not been applied in industry as widely as S. cerevisiae due to lack of tools for genetic engineering and as genetic manipulation has been tedious and time consuming.

Aim

This project aims to develop the chassis for a versatile and efficient cell factory that can transform abundant waste streams into valuable products.

Methods

1. Substrate screening

To confirm the ability of Y. lipolytica for efficient utilization of an impure mixture of compounds, various waste streams will be investigated as a substrate. We chose mixtures of fats, present in biodiesel waste or vegetation waters from rapeseed oil production, as well as sugars, which are present in molasses or brewery waste. In order to demonstrate the versatility of Y. lipolytica, we are going even further and ferment homogenized household waste.

2. Product

As a proof of concept we aim to demonstrate the production of both an extracellular heterologous protein and an engineered metabolite, and show how a valuable product can be produced by our cell factory utilizing waste streams. We will implement a codon optimized version of the human proinsulin gene along with a native Y. lipolytica promoter and secretion signal into Y. lipolytica. Using an already constructed plasmid with S. cerevisiae optimized genes from the bacterium Erwinia uredovora encoding four enzymes, we will implement the biosynthesis pathway of beta-carotene in Y. lipolytica by using the K152005 biobrick.

3. Molecular toolbox

This project tries to solve the lack of the well-proven tools for Y. lipolytica. We will develop a standardized genetic toolbox, including CRISPR/Cas9-mediated genome editing. The molecular toolbox will bring new opportunities such as an introduction of new pathways, adjusting waste utilization and targeting genetic manipulations.

Reference tryout

Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit. Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.

Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.^2,3 Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.