Design-build-test-analyze workflow for strain optimization

Biotechnological processes for production of chemicals from renewable feedstocks have a potential to replace the petrochemical routes in the future. This requires efficient cell factories that can convert feedstock into product with high titer, rate and yield. Due to complexity of biological systems, our capability to precisely predict and reconstruct the genotype that will lead to the desired production strain is rather limited*.

We used design-build-test-analyze approach to engineer baker’s yeast Saccharomyces cerevisiae for production of non-native 3-hydroxypropionic acid (3HP). 3HP can be chemically dehydrated into acrylic acid and thus can serve as a biosustainable building block for acrylate-based products (diapers, acrylic paints, acrylic polymers, etc). We set up a strain development workflow, which consists of: (i) advanced synthetic biology platform that allows semi-high-throughput construction of yeast strains, (ii) screening in 96-deep-well plates using feed-in-time medium to simulate fed-batch process, (iii) automated adaptive laboratory evolution platform, (iv) –omic characterization of strain and genome-scale modeling. This workflow enabled us to develop S. cerevisiae strains that could produce 3HP with titer above 14 g·L^-1 with 14% C-mol·C-mol^-1 glucose yield and volumetric productivity above 0.24 g·L^-1·h^-1 in a fed-batch process on minimal medium at pH 5. With this performance the strains represent a good starting point for developing a yeast-based 3HP production process. We also deciphered the mechanism for 3HP tolerance using adaptive laboratory evolution followed by genome re-sequencing, transcriptomics and functional analyses. 

*Borodina I & Nielsen J (2014). “Advances in metabolic engineering of yeast for production of chemicals”. Biotechnol J. 9(5):609-620.