New, enabling tools for broad utilization of cell factories in sustainable production of chemicals and fuels

By Jussi Jäntti and Dominik Mojzita

Irrespective of the development of oil prices and our technical abilities to retrieve oil from the ground, it is inevitable that in order to fight climate change there is a need to reduce our dependency on fossil fuels and develop sustainable, environmentally friendly production processes using renewable raw materials.

The world we live in has 119 million identified inorganic and organic chemical substances that are either natural or man-made (www.cas.org). The global chemical industry produces roughly 70,000 different chemicals. Oil consumption in Finland is 5.5 liters/day/Finn of which 1 liter/day/Finn contributes raw material for production of chemicals and lubricants (www.oil.fi). Globally, roughly 2 liters/day/person of oil is consumed (www.iea.org).

One way to reduce our oil dependency  is to harness the diversity of biology, especially in the form of microbes to convert waste streams such as pulp and paper industry wood hydrosylates to valuable compounds in bioreactors. This can be achieved when the vast selection of chemical reactions that microbes carry out naturally is combined with novel, designed biochemical pathways. Such pathways can be engineered in an increasing number of microbes using modern genome engineering technologies.

The modified microbes generated in this way can be considered as microbial cell factories that convert a biological raw material into designed end products such as polymer precursors and fuels.

Novel technologies accelerate the development

Significant advances have taken place in our abilities to generate enzymatic production pathways for more and more complex chemicals in microbes due to development of  novel genome engineering techniques (e.g. CRISPR/Cas9), the wealth of DNA sequence data of life on earth, and improved methods in sequence analysis and mathematical modelling of reaction pathways. The first wave of new technologies has already now significantly reduced the time needed for engineering of an industrial, microbial production host. For example, the genome engineering of an industrial yeast strain can now be done over 10 times faster than before. At the same time, these technologies enable engineering of an increasingly broad spectrum of organisms.

Components of a biological system need to be expressed typically at defined levels in order to obtain optimal functionality. This can be important for example for metabolic pathway engineering, where individual genes encoding a production pathway need to be expressed (and the corresponding enzymes produced) in balanced ratios to ensure optimal metabolic flux towards a desired product. Thus, one of the challenges in microbial production host engineering is how to establish specific levels of expression of multiple genes. Furthermore, for many potentially interesting, but currently little used industrial microbes, there are very limited or non-existing tools and/or methods to accomplish controlled expression of heterologous genes. This prohibits efficient use of these hosts in industrial applications.

Need for predictable, stable expression of target genes

An additional level of complexity for the production host engineering comes from the need to maintain predictable and stable gene expression in diverse cultivation conditions or stages of growth. Often, in currently available gene expression systems specific inducing conditions need to be used to achieve desirable expression of target genes. This leads to specific requirements for growth media composition or downstream processing approaches that ultimately result in increased production costs.

In order to achieve predictable and stable expression of target genes in a production organism it is important that the expression of these genes is minimally affected by the intrinsic regulatory mechanisms of the organism. This can be achieved by the use of non-native components (such as promoters and transcription factors) in the engineered gene expression system.

Modular expression system from VTT

We have established a modular expression system that is independent from externally added compound(s) and that enables tight control over a wide range of gene expression levels in the yeast S. cerevisiae (Rantasalo et al. 2016 http://dx.doi.org/10.1371/journal.pone.0148320). A basal, low level expression of a synthetic transcription factor (sTF) is ensured by a core promoter which expression activity is not affected by growth conditions. The sTF is composed of modular DNA binding and gene transcription activating parts that can be used to drive expression of any target gene at tunable levels simply by varying the number of sTF binding sites within the promoter that regulates the expression of the gene of interest.

stf
Importantly, subsequent work has enabled us to extend the published system with a set of novel core promoters and sTFs that can be used to achieve highly tunable expression of target genes, e.g. for optimized metabolic pathways or hydrolytic enzyme mixtures, in a broad range of industrially relevant microbes from various yeasts to filamentous fungi (patent pending).

These systems provide us with novel tools and abilities to engineer eukaryotic microbes for designed purposes using a few, well defined genetic elements. Thus, this system enables us to expand the selection of production organisms that can be effectively engineered for sustainable production of chemicals, fuels and proteins.

If you want to learn more, contact Senior Research Scientist Dominik Mojzita (dominik.mojzita@vtt.fi) or Research Team Leader Jussi Jäntti (jussi.jantti@vtt.fi).

 

jussiDr. Jussi Jäntti leads the Production Host Engineering research team. His team focuses on developing engineering tools for a broad range of eukaryotic microbes. These tools are used to generate cell factories for the sustainable production of chemicals and fuels. jussi.jantti@vtt.fi

 

 

dominikDr. Dominik Mojzita has a strong background in molecular biology and genetic engineering of yeast and filamentous fungi. He has worked on the identification and characterization of novel genes and metabolic pathways, transcriptional and metabolic regulation, genome-wide and gene-specific expression analysis, the production of organic compounds, single-cell analysis, development of synthetic expression tools and the establishment of synthetic control circuits in S. cerevisiae and A. niger. dominik.mojzita@vtt.fi