Achieving the highest alpha amylase activity is the main engineering goal of the team this year. We explored the possibilities of Saccharomyces spp. as a cell factory by testing enzymes from different organisms; Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Aspergillus oryzae.

It is sometimes hard to predict which combination of promoter, secretion signal and gene will lead to better yield. Therefore, we aimed to test as many promoters, secretion signal peptides and genes as possible. For this purpose, we used Golden Gate Assembly and took advantage of its modularity to clone our genes into a vector, creating 60 different combinations of promoters, secretion peptides and genes.

The native sequence of alpha amylase codes for a secretion signal that is used in the native host to secrete the enzyme (as these enzymes act extracellularly). To increase the possibilities of achieving higher yields of extracellular protein, besides testing for the native secretion peptide, we isolate the CDS (coding sequence) of alpha amylase and add a different secretion peptide that has been proven to work on yeast^[1]. To know more about the genetic design approach go to the engineering page.

The sequences of alpha amylase from 4 different organisms were codon optimized for the chassis. The basic parts were designed from the native sequence and synthesized de novo. The basic parts can be found in Table 1.

A library of constructs was created (Table 2) in which we included different elements (go to Engineering page) The strategy followed to fuse the different parts can be seen in Figure 1.

Promoters, secretion signals and terminators are taken from previous characterized genetic parts^[1],[2]. 10 promoters were chosen among the 20 promoters described in Lee ME et al. 2015 based on their strength, selecting promoters representing all strengths, from strong to weak. We also decided to include the most characterized secretion signal, as well as a regular terminator, tTDH1. The process to create the set of samples from all the possible combinations achieving an even distribution of all the elements from the different categories can be found in the model page.

1. Obst, U., Lu, T. K., & Sieber, V. (2017). A Modular Toolkit for Generating Pichia pastoris Secretion Libraries. ACS Synthetic Biology, 6(6), 1016–1025. https://doi.org/10.1021/acssynbio.6b00337
2. Lee, M. E., Deloache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol., 4, 975–986. https://doi.org/10.1021/sb500366v