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Revision as of 04:34, 21 October 2019

ShanghaiFLS_China: Home

Experiments

The Optimization of the Metabolic Pathways of P. pastoris in Medicine Production via Methanol Fermentation

Methanol is a major byproduct of the coal industry. Engineered Pichia pastoris GS115, a strain of methylotrophic yeast, is capable of converting methanol into medical compounds such as the insulin precursor and lovastatin. However, in such P. pastoris, the metabolism of methanol is highly specific and results in significant oxygen consumption and heat generation, which have limited its industrial applications. We aim to address this issue by improving the methanol conversion rate in P. pastoris by re-engineering its homogeneous circuits expressing the transcription factors that would up-regulate the expression of AOX1, the protein allowing it to metabolize methanol. Through our multiple rounds of experiments, we eventually acquired strains that are capable of yielding an up to 20% increase in total GFP production per gram methanol compared to the wildtype. Based on our modeling, such strains should have an (percent) decrease in heat generation per unit product and a (percent) decrease in oxygen consumption compared to the wildtype.

Design Experiment Results Parts

Outside the Lab

Demonstrate

When we asked professors and industrial leaders whether our project would be successful in the industry, they were not optimistic. However, due to constraints in our experimental settings, we were not able to demonstrate through experiments how our designs would work. This problem led us to construct a model, modifying existent formulas from literary works, to demonstrate this possibility. The rate of oxygen consumption and heat emission are two main factors to be considered in industrial processes, and our project mainly looks at methanol consumption. When we looked at the three factors separately in three models below, they all prove adequate possibilities of industrial production.

Model

To get an idea of how much more efficient our constructs will be compared with the wild-type P. pastoris, we modeled our constructs based on various statistics published in Wang et al., 2016, Shi et al., 2019 and Liang et al., 2012. Based on these data, we calculated basic parameters for each promoter and transcription factor in our three constructs, specifically, the activity of a promoter under the effect of 1 unit transcription factor. As it turned out, we relatively successfully calculated the eventual results of our experiment.

Human Practices

From the start of our project, we actively consulted peers, research professionals, industry experts, and the general public for advice and guidance. We first met up with Dr. Cai from ECUST, who introduced us to Pichia pastoris GS115, which we decided to base our project upon. We also interviewed two industry leaders. One of them, Dr. Hu, CEO of GTL Biotech, illustrated to us the great potential of methanol as a carbon source, which happens to be what P. pastoris is tailored to metabolize. After designing our experiments, we referred to the opinion of the public for evaluations of acceptability, feasibility and general impact via a questionnaire. We also became aware that industrializing our project design will be difficult and decided to use a model to demonstrate its effectiveness.