"Star-Chlamy"

Due to natural hazards such as desertification, salinization, and soil erosion, a large amount of arable land loses its productivity every year around the world. However, although arable land is constantly decreasing, the rate of global population growth is accelerating, and the demand for food is constantly rising. Against this backdrop, the food crisis is spreading everywhere, and it is urgent to solve the global arable land crisis.

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Figure 0.1.1 Schematic diagram of the cultivated land crisis.

In the picture above, we can see due to the continuous expansion of urbanization, a large amount of arable land has been requisitioned or converted to other uses, resulting in a continuous decrease in the area of arable land. In addition, due to the lack of environmental protection awareness and over-exploitation, cultivated land in some areas has also been seriously polluted and damaged, resulting in a decline in the quality of cultivated land and making it impossible to continue farming.

Based on this, our Tsinghua-TFL team proposed the Star-Chlamy solution - using synthetic biology methods to transform Chlamydomonas reinhardtii into a possible alternative source of food in the future.
The name "Star-Chlamy" has two meanings:
 for one thing, we aim to cultivate a superstar in food production, for another, the production we choose is starch (also broken as Starch-lamy).

Our goals are:

Figure 0.1.2 An overview of Our Project“Star-Chlamy”
  • To change the traditional agricultural paradigm by reducing the dependence of agricultural production on land resources
  • To increase grain production by improving the efficiency of photosynthesis
  • To improve nutritional value by changing the composition ratio of food.

    • Our chassis Organism: Chalmydomonas Reinhardtii

    Figure 1.1.1
    Chlamydomonas reinhardtii, chlamy in short, is a unicellular eukaryotic photosynthetic algae and a well-established model organism with a clear genetic background.
    In our project, we aim to propose a potential solution to the eukaryotic photosynthetic global land crisis by exploring a potential food source with this chassis.
    Chlamydomonas reinhardtii has many advantages:
  • Does not occupy arable land
  • High photosynthetic efficiency
  • High carbon sequestration efficiency based on CCM mechanism
  • Growth cycle is shorter than higher plants

    • Therefore, we chose Chlamydomonas reinhardtii as our chassis organism. We designed a series of genetic operations based on the characteristics of the metabolism pathways in Chlamydomonas reinhardtii to make it a more ideal food source, the modified strains called Star-Chlamy.

    Engineering-A Superstar in Food Production

    Modification of Starch Metabolic Pathway

    Chlamy with High Output of Starch

    Figure 2.1.1 An overview of starch metabolic pathway design
    By genetic manipulation, we have genetically edited several key enzyme genes in the starch metabolic pathway of Chlamydomonas reinhardtii, thereby achieving an increase in starch production in the algae:
  • According to the principle of "broaden the source of income and economize on expenditure", we introduce a more efficient modified AGPase(G336D) encoded by glgC from Escherichia coli as the target point in the starch synthesis pathway.
  • We knock out the key enzyme genes GWD1 and GWD2 in the starch decomposition pathway.
  • Besides, We established a reliable method for detecting starch content in Chlamydomonas reinhardtii.

    • At the same time, the starch metabolism pathway and the lipid metabolism pathway have significant crosstalk. Since starch synthesis and lipid synthesis compete with each other, we cultured Chlamydomonas in the presence of cerulenin, a lipid inhibitor.

    Figure 2.1.2 An overview of metabolic engineering design for starch metabolic pathways


    Figure 2.1.3 Qualitative and quantitive starch content of control and cerulenin treatment group

    It can be seen from the figure that the starch content of cerulenin treatment group was significantly higher than the control group, which meant that the inhibition of oil synthesis pathway is effective to improve starch yield in the future.


    2-Chlamy with High Ratio of Amylose

    When we initially embarked on our project focused on starch synthesis, we conducted an interview with a member [ The speaker requested anonymity.] of the China Starch Industry Association [ a national, cross-regional, and cross-sector industry organization, also a non-profit social organization]. He emphasized the importance of not only producing a product with social value but also enhancing its economic viability. In light of this valuable guidance, we made the decision to optimize the amylose and amylopectin ratio, which holds greater commercial potential.

    Actually, with the continuous emergence of cheap fast food, people's diet is becoming more and more unhealthy all over the world. And the incidence rate of obesity, hyperglycemia and other diseases is rising. Due to difficult digestion of amylose, it can avoid the rapid rise of blood sugar and reduce the absorption of energy, without significantly changing the taste of food. Thus, amylose is more friendly to people's blood sugar and blood lipids, and therefore has higher nutritional value and greater commercial potential.

    Figure 2.1.4 The prevalence of fast food with unbalanced nutrition has posed an increasingly serious challenge to people's health

    In order to make our Star-Chlamy a healthier food source, we regulated the metabolic pathway of starch by knocking out the key enzyme genes SBE2 and SBE3 that are responsible for the conversion of amylose to amylopectin, thereby increasing the proportion of amylose in starch and thus improving the nutritional value of Star-Chlamy strains.

    Figure 2.1.5 Knockout of SBE results in efficient accumulation of amylose in Chlamy.

    We have performed different kinds of experiments to verify the increase of amylose content and proportion in our mutants:



    Figure 2.1.6 Qualitative and quantitive starch content of control and cerulenin treatment group


    It can be seen from the figure above that the amylose content of 3 kinds of mutants were higher than wild type:

    Figure 2.1.7 Percent of Amylose to total starch

    It can be seen from the figure above that amylose perception of all mutant were higher than wild type:

    Figure 2.1.8 Phenotypic characterization of starches produced by mutant sbe2-2;sbe3-3

    In wild type cells, the surface of starch particle was round and smooth like that in the previous article[ Deletion of BSG1 in Chlamydomonas reinhardtii leads to abnormal starch granule size and morphology ]. But in mutant cells the particle was folded and angular, which may show that the perception of amylose produced by the mutants we got increased significantly.




    Figure 2.1.9 Phenotypic characterization of starches produced by mutant sbe2-2;sbe3-3


    It can be seen from the figure above that the starch content of mutant gwd1-1 and gwd2-1 were higher than wild type, but gwd1-1 was significant while gwd2-1 was not. sbe2-3 and sbe2-3;sbe3-5 were significantly higher than wild type. Sbe3-4 was lower than wild type but not significant. Agp4 was significantly lower than wild type.

    Transition of Our Ideas

    At the same time, in order to adapt our Star-Chlamy to industrialization and improve the efficiency of industrial transformation, we targeted the engineering cell's photosynthesis (light reaction and carbon fixation reaction) for regulation, further improving the efficiency of Chlamydomonas photosynthesis.

    ChlF: Extended light harvesting of Chlamy

    Terrestrial cyanobacteria lives in an environment under near-infrared light(720nm) because of shading by plants or because of their associations with soil crusts, benthic mat communities, or dense cyanobacterial blooms. For evolution pressure, these cyanobacteria have evolved a novel far-red light photoacclimation (FaRLiP) that enable them to use far-red light (FRL) for photosynthesis.
    Chlamydomonas only uses near-red light but not far red light for photosynthesis.
    Therefore, the near-red light attenuation in the depth of runway ponds, similar to the living environment of terrestrial cyanobacteria, becomes a serious problem for Chlamy, which leads to decrease of the photosynthetic efficiency.
    In order to solve this problem, we adopted the means of synthetic biology by expressing ChlF, the chlorophyll F synthetic enzyme from cyanobacteria to introduce FaRLiP-absorbing chlorophyll into Chlamydomonas, trying to check whether the far-red-light-absorbing chlorophyll will extend the light harvesting spectrum of Chalmy into far red area (700 to 800 nm) and improve their photosynthetic efficiency.

    Figure 2.1.1 Introduce of FaRLiP in Chlamy by expressing ChlF.
    RLiP: Red light photoacclimation; FaRLiP:Far-red light photoacclimation

    Broaden Application Scenarios of CCM

    The photosynthetic process of Chlamydomonas reinhardtii in aquatic ecosystems must overcome the low availability of carbon dioxide due to slow diffusion of carbon dioxide in water.

    The enzyme RuBisCO, which fixes carbon dioxide, has low affinity for it, and therefore the photosynthetic efficiency of algae is highly dependent on the CCM (Carbon dioxide-Concentrating Mechanism).

    The algal CCM involves sequential actions of inorganic carbon (Ci) transporter proteins and carbonic anhydrase enzymes located in different cell regions, which results in active accumulation of carbon dioxide at the RuBisCO level.
    Carbon dioxide concentration is one of the main environmental factors that affect the response of the CCM mechanism, which regulates its response by affecting the expression of proteins related to the CCM mechanism. Specifically, high concentration of this gas inhibits the expression of CCM genes, while low concentration of it promotes the expression of CCM-related genes. Therefore, in actual industrial production, the CCM mechanism does not continuously function due to the high concentration of carbon dioxide provided.
    In practical industrial production, we often try to increase the concentration of carbon dioxide artificially (this can be done at low cost by using carbon dioxide waste gas from other factories) to increase production. So we need to modify the regulatory mechanism of CCM pathway in Chlamydomonas reinhardtii to contact inhibition under high concentrations of carbon dioxide[1].

    Figure 2.2.1 Schematic diagram of CCM pathway

    Our project selects the major transcription factor CIA5 as the target, which undergoes phosphorylation modification at the C-terminus under high carbon dioxide concentrations, thereby activating its transcriptional activity and up regulating the expression of over 90% of CCM-related genes. By overexpressing CIA5 in Chlamydomonas reinhardtii, we allow the CCM mechanism to continuously respond to even high concentration carbon dioxide , thereby greatly increasing carbon fixation efficiency.

    Contributions to Plant Synthetic Biology

    3.1 Our Favourite Part - SGT1

    Unlike chassis such as E coli, which have high expression efficiency for exogenous proteins, the low expression efficiency of exogenous proteins and the resulting low screening efficiency for exogenous protein expression have been a disadvantage of using Chlamydomonas reinhardtii as chassis.
    Many iGEM teams that use Chlamydomonas reinhardtii as chassis have been plagued by this issue, and many of their experimental designs have failed to proceed to subsequent verification due to screening failure.
    To address this issue, we propose our solution: Our favourite basic part SGT1. SGT1 provides a more efficient method for screening exogenous protein expression, greatly reducing the workload, and can largely help future iGEM teams that want to use Chlamydomonas reinhardtii as chassis.

    Figure 3.1.1 Design of our favourite part SGT1


    In traditional methods for screening exogenous protein expression, WB is commonly performed after labeling the target exogenous protein with a tag (such as YFP, HA) to confirm whether the exogenous protein is expressed.
    However, due to the low efficiency of expressing exogenous genes in Chlamydomonas reinhardtii, this process often requires a significant amount of effort and cost.
    Therefore, we urgently need a simpler method to screen for exogenous gene expression in Chlamydomonas reinhardtii.

    To address the problem of screening for exogenous gene expression in Chlamydomonas reinhardtii, we utilize sgt1 as a screening marker and construct a plasmid for efficient screening of exogenous gene expression in Chlamydomonas reinhardtii.
    SGT1/SUGT1, a co-chaperone of HSP90, is involved in multiple cellular activities including cullin E3 ubiquitin ligase activity，multiple protein folding and complex formation process. What’s more, it is hypothesized that this co-chaperone might play an auxiliary role in the synthesis of foreign protein.Chlamydomonas mutants of sgt1 have cilia-related defects, thus they are agglomerated in TAP medium and unable to move.

    Figure 3.1.2 Chlamydomonas mutants of SGT1 are agglomerated in medium and unable to move
    GOI: Gene of interest;

    In our design, SGT1-mutated Chlamydomonas reinhardtii exhibits a non-motile phenotype. The target foreign gene(GOI, gene of interest) and SGT1 share the same promoter and initiation codon, ensuring that the expression levels of the target foreign protein(POI, protein of interest) and SGT1 are equivalent. Finally, by performing microscopic examination of the SGT1 motility phenotype complementation, we achieve efficient screening for foreign protein expression in Chlamydomonas reinhardtii.

    Figure 3.1.3 An overview of principle of foreign protein expression screening by SGT1 marker
    GOI: gene of interest; POI: protein of interest

    All in all, our Basic Part SGT1 provides a more efficient method for screening foreign protein expression, greatly reducing the workload and greatly assisting iGEM teams that want to use Chlamydomonas reinhardtii as a chassis organism in the future. We hope that through our efforts, more teams will use Chlamydomonas reinhardtii as a chassis organism, and tap into its unlimited potential in iGEM, which was limited by the expression efficiency of foreign proteins in the past.



    Click here to see more about "our favourite part SGT1"

    3.2 More Possibilities - Other Parts

    To achieve the synthetic biological transformation of Chlamydomonas reinhardtii, we also constructed multiple parts of Chlamydomonas reinhardtii photosynthesis, including the light reaction, carbon fixation reaction, and downstream carbon flow distribution. These parts meet the standardized part requirements of the iGEM official website. Future iGEM teams interested in Chalmydomonas Reinhardtii can further learn about and obtain guidance for using these parts in our part pages.