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

Bringing Mould to Work: Upgrading Citrus Peel and Sugar Beet Pulp

by Peter Richard

For regular households, mould in the food is hardly considered good news. For us research scientists it’s another story. Researching the mould on your onion can open the way to produce chemicals from agricultural wastes that can replace chemicals made from fossil oil.

115 million tons of citrus fruit and 270 million tons of sugar beet are produced annually worldwide which leaves a lot of citrus peel and sugar beet pulp. These raw materials are rich in pectin but of not much use. Sugar beet pulp is dried and used as animal feed. It has to be dried since it otherwise would rot; however drying is very energy consuming. Citrus peel can also be used as animal feed but is often just dumped which is problematic because of the odour. The pectin from citrus peel is sometimes extracted and used as a gelling agent in food, such as jams or marmalades. For this application only 40 – 50 thousand tons are used, i.e. there is a lot of pectin in the world that is unused.

OnionThe mould Aspergillus niger, the black mould, is a common contaminate of foods. If you find black spots on the onion in your kitchen, this is most likely Aspergillus niger. This mould is however also a useful microorganism that is used to produce citric acid. Since the 1930’s citric acid is produced in industrial scale using Aspergillus niger. It is used in food as an acidifier and especially much in soft drinks. Almost all the commercial citric acid, which is currently about 1.6 million tons per year is produced by this mould.

Aspergillus niger ferments also very efficiently pectin which is a polymer made out of D-galacturonic acid monomers. The mould produces pectinase enzymes that hydrolyse the pectin to D-galacturonic acid (oxidised sugar) which is then used as an energy source for growth. We thought that if we want to make something useful out of pectin or D-galacturonic acid, this is a suitable organism. We succeeded to genetically modify the mould so that it would convert D-galacturonic acid to different products, for example galactaric acid which is also called mucic acid.

With the engineered mould the process to produce galactaric acid is very simple. Galactaric acid is not soluble in water at acidic pH which facilitates the downstream processing. The substrates are all soluble and the mould operates well at acidic conditions.

Currently galactaric acid is produced chemically by oxidising the sugar D-galactose using nitric acid. This process is environmentally challenging. Galactaric acid is currently used in skincare products.  In the past, i.e. in the 1930’s it was also used as a food component in self-rising flour. In Mobile, a city in the south of the USA, D-galactose from a wood processing plant was oxidised chemically to produce about 600 tonnes galactaric acid per year.

Besides these applications galactaric acid can be converted chemically to other compounds like adipic acid or furan dicarboxylic acid, FDCA. Adipic acid is used for Nylon. FDCA can also be polymerised. The polymer has good gas barrier properties and can be used for bottles for carbonated drinks. This could replace the PET bottles that are currently used.

Reference: Mojzita et al.Metabolic engineering of fungal strains for conversion of D-galacturonate to meso-galactarate. Appl Environ Microbiol. 2010, 76(1):169-75.

100_0046If you want more information about the fungal pathways and D-galacturonic acid catabolism contact the author Dr. Peter Richard. He works at VTT Technical Research Centre of Finland Ltd. as Principal Scientist in Metabolic Engineering research team. His main interests are metabolic engineering of yeast and mould for the production of fuels and chemicals. peter.richard@vtt.fi