This article was submitted to Synthetic Biology, a section of the journal Frontiers in Bioengineering and Biotechnology
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Biological chemical production has gained traction in recent years as a promising renewable alternative to traditional petrochemical based synthesis. Of particular interest in the field of metabolic engineering are photosynthetic microorganisms capable of sequestering atmospheric carbon dioxide. CO2 levels have continued to rise at alarming rates leading to an increasingly uncertain climate. CO2 can be sequestered by engineered photosynthetic microorganisms and used for chemical production, representing a renewable production method for valuable chemical commodities such as biofuels, plastics, and food additives. The main challenges in using photosynthetic microorganisms for chemical production stem from the seemingly inherent limitations of carbon fixation and photosynthesis resulting in slower growth and lower average product titers compared to heterotrophic organisms. Recently, there has been an increase in research around improving photosynthetic microorganisms as renewable chemical production hosts. This review will discuss the various efforts to overcome the intrinsic inefficiencies of carbon fixation and photosynthesis, including rewiring carbon fixation and photosynthesis, investigating alternative carbon fixation pathways, installing sugar catabolism to supplement carbon fixation, investigating newly discovered fast growing photosynthetic species, and using new synthetic biology tools such as CRISPR to radically alter metabolism.
It is well established that rising atmospheric CO2 levels are the primary cause for unprecedented climate change impacting the globe (
Despite the burgeoning interest around these photosynthetic microorganisms, the field still faces many challenges that have yet to be addressed. The primary concern is the inefficient nature of photosynthesis and CO2 fixation. Attempts to improve upon the central carbon fixation enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) have been met with little success (
The focus of this review will be on the recent methods employed to overcome the supposed shortcomings of photosynthetic organisms ranging from rewiring carbon metabolism and photosynthesis, introducing additional carbon substrates, generating other chassis organisms capable of superior carbon sequestration, studying faster growing variants of cyanobacteria, and developing new tools via synthetic biology.
Efforts have been made to overcome the intrinsic shortcomings of photosynthetic microorganisms by rewiring metabolism related to carbon fixation and photosynthesis. While efforts to improve RuBisCO have not been met with much success, current research has shifted focus towards rerouting metabolism to improve overall photosynthetic efficiency by focusing on key aspects of the Calvin-Benson cycle or the photosynthetic electron transport chain (PETC). One strategy used to harness the excess energy being lost by the PETC in cyanobacteria involved overexpressing the protein OmcS (
An inherent drawback of RuBisCO is its promiscuous nature, when RuBisCO undergoes oxygenase activity a costly side pathway known as photorespiration occurs where the oxygenase product is recycled back into usable metabolism consuming energy and losing CO2 in the process. As much as 30% of energy produced by photosynthesis has been observed to be lost through photorespiration in plants (
It should be noted that photorespiration is not the sole pathway responsible for carbon inefficiencies, many metabolic processes include steps where CO2 is lost to the environment. An important way to engineer microorganisms for sustainability involves carbon conservation, focusing on rerouting metabolism to circumvent decarboxylation reactions (
In contrast to research centering on canonical CO2 fixation, investigations into
Many of these
Another approach to increase chemical production capacity is to supplement CO2 with carbohydrates as an auxiliary carbon source for the Calvin-Benson cycle, thereby making the organism photomixotrophic. By re-engineering glucose catabolism to direct carbon flux into the Calvin-Benson cycle, more ribulose-1,5-bisphosphate can be supplied to RuBisCO, accelerating CO2 fixation. This ultimately results in faster growth and production of downstream targets, as well as a six-fold increase in titer once metabolism was rewired to accommodate photomixotrophy (
Most research done in this field focuses on using just a handful of species that have traditionally been used as model organisms to study the mechanics of photosynthesis. With increasing interest in using photosynthetic organisms for industrial production there have been efforts to uncover new species that are faster growing and more receptive to engineering. In the realm of cyanobacterial chemical production, species like 2973 and
In cyanobacteria, traditional genomic modifications are a labor-intensive task and limiting in nature due to the polyploidal nature of these organisms and the need for antibiotic resistance markers (
The overall task of metabolic engineering in cyanobacteria has been made dramatically more efficient thanks to the advent of CRISPR gene editing which allows for markerless edits (
Other work on CRISPR technologies in cyanobacteria includes the use of CRISPR inhibition (CRISPRi) by using dead Cas9 (dCas9) (
While many challenges remain and must be overcome to enable widespread adoption of photosynthetic chemical production hosts, the above studies suggest that there are myriad avenues of research that can get closer to this goal. The renewed interest in the field due to the ongoing climate crisis has spurred efforts to improve and adopt these microorganisms as a sustainable alternative for traditional petroleum-based synthesis. Many of the challenges in this field revolve around the intrinsic inefficiencies of carbon fixation and photosynthesis. While engineering RuBisCO remains an interesting target for improving carbon fixation, it has proven to be highly resistant to traditional engineering and decades of research would suggest that it is next to impossible to improve. Focusing on engineered carbon fixation pathways is a more promising route towards improving the carbon sequestration ability of cyanobacteria. Other research into improving the efficiency of photosynthesis by introducing alternative pathways downstream of the PETC for the production of chemical products is a prime example of how we can engineer these microbes to make full use of excess reducing potential from the PETC. The aforementioned approaches aim to enhance our understanding of the inefficiencies related to carbon fixation and photosynthesis while also representing some of the more novel approaches being undertaken by the field of synthetic biology. The discovery of new synthetic biology tools and investigation into faster growing cyanobacteria is also expanding the field of photosynthetic microbial research to make photosynthetic microorganisms a more viable alternative to petroleum based chemical production.
Of the discussed challenges for synthetic biology in cyanobacteria, improving the rate and efficiency of carbon fixation seems to be the most difficult, however, this task also holds the most promise. While RuBisCO is resistant to direct engineering strategies, adding additional carbon fixation modules can enhance the viability of cyanobacteria as a chemical production chassis. Further research into
Strategies for improving efficiency of carbon fixation and photosynthesis in cyanobacteria. Shown in red are carbon sources for metabolism in cyanobacteria, in blue are pathways of interest to synthetic biology and metabolic engineering, solid lines represent native pathways and dashed lines represent pathways of interest for improvement using synthetic biology.
As new synthetic biology tools become available for cyanobacteria, high throughput screening will allow for rapid progress to be made within this field. The advent of CRISPR technology has had profound effects on research in a wide variety of fields but is relatively new to cyanobacteria. Additionally, faster growing species of cyanobacteria are rising in popularity and more have yet to be discovered. While these newly discovered cyanobacteria are efficient production hosts in their own right, they also inform the field on future targets for modification. Understanding the inherent differences among these organisms is vital to improving our understanding of carbon fixation and photosynthesis. It stands to reason that research into discovering more of these faster growing species, as well as studying the known cyanobacterial variants will provide insight and guidance for future work in this field. While improving photosynthetic production hosts has been historically difficult, the studies described in this work point to a promising future.
TT, JG, JP, and SA wrote the manuscript.
This work was supported by the National Science Foundation (CBET-1902014).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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