Lysozyme Secretion in S. cerevisiae for Fermentation on Anabaena sp. Biomass and Development of the Model MPRC. A Proof-of-Concept Study for Sustainability of Human Life on Mars

Abstract

Prospective long-duration space exploration missions require onsite production to sustain human life, due to both economic and technical challenges. Life is a modular and self-replicating technology with a wide range of production capabilities. S. cerevisiae and other fungi can produce a lot of useful products for space applications. However, S. cerevisiae is heterotrophic and need to be fed a substrate to grow and produce. Cyanobacterial biomass has been proposed as a potential substrate but has previously only been used as such after pretreatment of the biomass. In this proof of concept study, I have engineered S. cerevisiae to express and secrete enzymes with lytic capabilities, lysozymes (EC: 3.2.1.17), to lyse the space application relevant cyanobacterium Anabaena sp. to be able to use it as a substrate. 159 relevant lysozyme candidates were identified from which ten were tested for compatibility with the hypothesized functionality. The alpha mating factor se- cretion tag and the p426GDP and cPOTud expression platforms in BY4742 and CEN.PK.530.1CK were used to test the selected ten lysozymes. Six out of ten lysozymes were successfully expressed in S. cerevisiae strains with promising enzy- matic activity, albeit with reduced S. cerevisiae fitness. Expression of the final candi- date, Lysozyme C Chinese softshell turtle (L4), had a significant lytic effect on frozen Anabaena sp. biomass. L4, in particular, significantly reduced the fitness of S. cere- visiae CEN.PK.530.1CK by 11% lower endpoint OD600 and 15% lower growth rate. S. cerevisiae growth measurements in cyanobacterial media using optical density is not applicable, as cyanobacterial absorbance overshadows any changes due to S. cere- visiae growth. Therefore, a new over-deterministic linear model named MPRC was created from flow cytometry data. MPRC was shown to predict CEN.PK.530.1CK growth in Anabaena sp. containing medium with a 98% relative accuracy. Us- ing MPRC, an endpoint reduction of cell count of CEN.PK.530.1CK at significant 22% was measured in the presence of Anabaena sp. biomass in normal media. CEN.PK.530.1CK with L4 insert in it’s cPOTud plasmid, and thus expressed and secreted of L4 significantly increased the endpoint cell count by 9% relative to the empty plasmid in normal media with 3% added Anabaena sp. biomass. Similarly, L4 expressing CEN.PK.530.1CK had a significant 124% higher endpoint cell count in low nitrogen content media and a significant 71% higher endpoint cell count in no glucose content media, both with 3% added Anabaena sp. biomass to the media. Thus, the Anabaena sp. biomass seems to be able to provide both a nitrogen- and a glucose- source that can facilitate CEN.PK.530.1CK growth, but further testing is needed to fully understand the performance of the system.