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.

Carbon Fixation to Combat Climate Change Carbon dioxide (CO2) has been associated with the rise of greenhouse gasses and harmful emissions into the atmosphere, resulting in the onset of climate change [4]. Carbon fixation cycles exist to balance this continual release of carbon into the environment, allowing organisms to metabolize carbon and recycle it into the biosphere [4]. The primary method of carbon 6 absorption is the Calvin–Benson–Bassham (CBB) pathway in plants/algae, consisting of 29 reactions and 13 enzymes [4]. Even under optimal conditions, the CBB cycle is limited by the inefficiency of the primary enzyme, RuBiSO, which is a slow catalyst [4]. In order to increase efficiency, scientists can combine enzymes from organisms and design synthetic CO2 fixation pathways in host organisms that have more favorable thermodynamics and kinetics [5]. A study of evolution and ecology has revealed crucial enzymes and intermediaries that Earth’s first organisms used to metabolize carbon [4]. Some natural carbon fixation pathways of interest are rTCA, Wood-Ljundahl, Natural Reductive Glycine, etc. Similarly, there are synthetic pathways like CETCH, SACA, and Reductive Glycine which function at higher rates of fixation [4]. A major challenge is trying to successfully implement these fixation pathways in vivo [5]. The inserted enzymes may interact with the complex network of reactions in the host, which could result in secondary reactions and toxicity [5]. To avoid this, it is crucial to find new methods of screening, testing, and optimizing the CO2 pathways under fixed conditions [5]. 

One method that can be employed is the use of a minimal cell as a screening platform for metabolic pathways. Ever since genomes have been first investigated, many bacterial models have been created in order to discover ways to identify essential and nonessential genes in a cell. Due to the redundancy of many gene functions, creating a model that defines which genes are essential to the viability of the cell has been a challenge [6]. To overcome this, the J. Craig Venter Institute worked to experimentally define a minimal cellular genome by designing and constructing one. The complete genome of Mycoplasma genitalium was designed and built from scratch by chemically synthesizing oligonucleotides and stepwise recombination in vitro and in Saccharomyces cerevisiae (yeast), and using genetic manipulation to assemble the genome as a  plasmid inside the yeast cell [7]. This process led to the birth of JCVI-syn1.0, the first cell controlled by a synthetic genome that would be the framework for the current minimal cell strain that we intend to use [4]. A reduction from 901 genes in JCVI-syn1.0 to 473 genes yielded the strain JCVI-syn3.0, which was deemed the smallest genome of any free-living organism [8]. JCVI-syn3.0 contains all the genes of JCVI-syn1.0 that are necessary for growth, both essential and quasi-essential [6]. In the creation of JCVI-syn3.0, the non-essential genes were individually removed to the greatest extent possible without jeopardizing cell survivability due to simultaneous knockouts. However, some seemingly non-essential genes were retained for the convenience of genome design and construction [7]. These initial variants led to the formation of the minimal cell that we plan to use for our project. This minimal cell, known as JCVI-syn3B, was synthetically derived from a bacterial cell called Mycoplasma mycoides. JCVI-syn3B is a cell with a genome that has been reduced to 493 protein coding genes, which has been labeled the smallest genome that can be grown in a laboratory culture [6] . Developed by the J. Craig Venter Institute, all of the non-essential genes were removed, only leaving the necessary genetic pathways needed for cell viability, such as for survival and replication or reproduction [8]. Using such a minimized genome gives us a platform with which to determine what components can be categorized as core to physiological cellular processes [8]. Through the creation of this bacterium, scientists now have a model of unicellular life that more broadly reflects known organisms and consists of what we know as the simplest free-living system [8]. 

Naturally occuring carbon fixation pathways normally consist of many reactions and intermediaries which causes it to be an inefficient cycle for carbon sequestration [9]. Its complexity can be reduced by designing a minimized artificial CO2 fixation pathway, such as the pyruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase (POAP) cycle [9]. This synthetic pathway can absorb two CO2 molecules per cycle, converting it into one oxalate molecule while using two ATP molecules. The POAP cycle functions best at 50ºC in anaerobic conditions, and has a fixation rate of 8 nmol CO2 per minute per mg of CO2 fixing enzymes [10]. This four-step reaction cycle is the shortest pathway created until now and consists of only four reactions, making it easy to insert into a host organism [10]. However, some of the challenges with this pathway include using a system of multiple enzymes and relying on exogenous energy sources [10].