Environmental destruction due to anthropogenic carbon dioxide emissions may have reached a state of no return. There are global environmental changes occurring without any new emissions [1]. The reversal of this destructive sequence requires a significant and sustained effort to remove carbon dioxide from the atmosphere. In regards to synthetic biology, carbon removing solutions can be found in the form of genetically engineered organisms containing carbon fixation pathways.These projects aim to develop organisms to grow at a sufficiently large scale to fix atmospheric carbon dioxide.

Synthetic carbon fixation pathways are constructed with reactions from existing natural pathways to objectively maximize carbon fixation rate. Some synthetic carbon fixation pathway designs aim for minimal complexity in order to simplify in vivo implementation [2]. Complex thermodynamics and changing metabolic states of a living cell present challenges when designing an in vivo carbon fixation pathway that is competitive with natural fixation rates, essential for cell survival, and allows the cell to grow at a reasonable rate. However, in-vitro carbon fixation has had many successes, and the challenge remains in the translation of an in-vitro carbon fixing pathway to in vivo.

The JCVI-UCSD iGEM team's primary objective is to insert a synthetic carbon fixation pathway into a biological chassis. The synthetic carbon fixation pathway that was chosen to be inserted is the POAP (pyruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase) pathway. The POAP pathway is an in vitro carbon fixation pathway that consists only of four reactions, enzymes, and metabolites [3]. The POAP pathway was intriguing to use as the implemented synthetic carbon fixation pathway because the low reaction count could lead to greater accessibility to optimization of the pathway.

A cell is defined to be minimal if its genome contains the least amount of genes needed for survival and replication. The JCVI minimal cell (JCVI-Syn3B), is derived from Mycoplasma mycoides and only comprises 493 genes [4]. The minimal cell is a compelling in vivo chassis for engineering synthetic biological functions due to the simplified metabolic framework. Simpler metabolics will minimize the risk of inserted reaction pathways aiding existing metabolic functions instead of conducting the carbon fixation cycle [2]. The minimal cell’s appeal of being a biological chassis is further enhanced by the cre-lox system within the minimal cell genome.

Of the four enzymes within the POAP pathway, pyruvate synthase places a thermal constraint on the operating conditions of the minimal cell. Pyruvate synthase operates in the correct direction within the POAP pathway at 45°C. This poses a problem for the minimal cell because a normal minimal cell cannot withstand temperatures above 37°C. Instead of looking for variants of pyruvate synthase that could operate at lower temperatures, the JCVI-UCSD team opted to conduct thermal adaptive laboratory evolution (TALE) of the minimal cells. TALE is conducted by serially passaging minimal cell cultures in slowly increasing temperatures. As a result of this process, we would be picking mutant minimal cell cultures that could grow at higher temperatures. These cultures would be continuously pressured to grow at higher temperatures until the minimal cell could withstand the operating conditions of pyruvate synthase. Through TALE, we have successfully evolved the John Craig Venter Institute’s Syn 3.0B minimal cell to become thermally tolerant at a temperature of 45.2°C. Sequencing results of the thermally tolerant minimal cells pushes for the conclusion that the cells did evolve specific mutations for heat tolerance and not adaptations.

The JCVI-UCSD team aims to introduce the POAP pathway into the minimal cell created by the J. Craig Venter Institute. This minimal cell would be a thermally tolerant variant to accommodate the high operating temperature of pyruvate synthase, a key enzyme in the POAP pathway. Ultimately, the JCVI-UCSD designed MACS, a minimal and adapted carbon sequestration cell, using the JCVI minimal cell as the biological chassis and the POAP pathway as the inserted synthetic carbon fixation pathway. We will also report the selection of POAP genes based on temperature and pH constraints, assemble all genes into minimal cell plasmids via Escherichia coli assembly, and the purification and sequencing of assembled plasmids. Further, we will soon begin in-vivo activity screens using GC-MS and LC-MS depending on the metabolites observed to assess success of POAP implementation into the minimal cell.