Global Problem - Land Crisis

Hunger is one of the sustainable challenges the world is facing, and the staggering number of people suffering from hunger is alarming. According to the latest research by the Food and Agriculture Organization (FAO) of the United Nations, there are 828 million people in the world who are undernourished. In this situation, however, food production is increasingly under immense pressure.

Figure 1.1 FAO data on global hunger [1]

Apart from natural disasters, the main cause of the global food crisis is the scarcity of arable land. Up to today, the most common agricultural paradigm is still highly dependent on land. According to FAO, over 95% of the world's crop production comes from land. However, this high dependency also means that food production faces the same threat as arable land. Due to natural hazards such as desertification, salinization, and soil erosion, a significant amount of arable land loses its productivity every year. Many countries resort to deforestation to maintain arable land area, leading to a substantial reduction in global forest coverage. Despite these efforts, it is still insufficient to meet the growing food demand of the global population, with a decreasing per capita arable land area.

Figure 1.2 Changes in per capita arable land area over time (data from FAO) [2]

However, this problem not only exists in the present but may become even more severe in the future. According to FAO's document How to Feed the World in 2050, by 2050, the world's population will reach 9.1 billion, which is 34 percent higher than today. It is estimated that annual cereal production will need to rise to about 3 billion tons from the current 2.1 billion tons. The increasing demand for agricultural produce, both new and traditional, will further strain already scarce agricultural resources. Agriculture will not only be forced to compete for land and water with expanding urban settlements but will also be required to serve on other major challenges: adapting to and mitigating climate change, preserving natural habitats, and maintaining biodiversity. To meet these demands, it is urgent to solve the global arable land crisis and discover new methods that can produce more from less land.

Fig 1.3 Global progress in food consumption (data from FAO) [3]

Our Solution

Therefore, the key question to address is how to produce more food with limited arable land, thus tackling the land crisis. Based on this, our Tsinghua-TFL team propose a potential solution - using synthetic biology methods to transform Chlamydomonas reinhardtii into a possible alternative source of food in the future. Our goal is to create a means of agricultural production that can utilize non-arable land while simultaneously increasing grain production by improving photosynthetic efficiency. Additionally, we aim to improve the nutritional value by altering the composition ratio of food.
Chlamydomonas reinhardtii, also known as chlamy, is a unicellular eukaryotic photosynthetic algae. It possesses several advantages, making it an ideal candidate for our project:

Figure 1.4
As a potential food source:
  • Do not require arable land.
  • High photosynthetic efficiency.
  • High carbon fixation efficiency based on the CCM (Carbon Concentrating Mechanism) mechanism.
  • Growth cycle is shorter compared to higher plants.

    • As a model organism:
  • Accomplished complete genome sequencing.
  • With well-established gene editing and genetic transformation system.
  • With extensive research on CCM.

    • Therefore, we have chosen Chlamydomonas reinhardtii as our chassis and designed a series of bioengineering operations based on its inherent characteristics to enhance its potential as a food source.

    After thorough research, we have decided to target starch and design a land-free agriculture paradigm. Globally, most cereal crops are primarily composed of starch. Approximately 2 billion out of the estimated 3 billion tons of annual grain production worldwide consist of starch molecules. Starch is a crucial energy source for humans and an important industrial raw material. By developing a land-independent method for starch production, we are able to achieve sustainable development in the field of starch agriculture and address the challenges of land scarcity and global hunger.

    Fig 1.5 Distribution of arable crop types [4]

    To accomplish this, we have modified the starch synthesis pathway in Chlamydomonas reinhardtii to enable large-scale starch production, which we refer to as Star-Chlamy. The name "Star-Chlamy" holds two meanings: firstly, we aim to cultivate a superstar in food production, and secondly, we focus on starch production.

    About "Star-Chalmy"

    Modification of Starch Metabolic Pathway

    About the Starch Amount

  • To achieve high starch production in Chlamydomonas reinhardtii, we targeted the rate-limiting enzyme AGPase (ADP-glucose pyrophosphorylase) and introduced a more efficient AGPase encoded by glgC from Escherichia coli to the mutant chlamy strain knockout of the endogenous one.
  • We also knocked 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.

    • Fig 2.1.1


    About the Amylose Content

    During the process of HP, we were inspired by communication with the China Starch Industry Association (visit HP page to read more) that changing the composition ratio of starch adds more value and economic significance. Chemically, starch consists of two types of anhydroglucose polymers called amylose and amylopectin. Amylose is relatively long and slightly branched, while amylopectin consists of numerous shorter highly branched chains. High amylose starch (HAS) is a resistant starch that can ferment and produce various short-chain fatty acids (SCFA) in the colon through the action of intestinal microflora, providing several health benefits. HAS also enhances insulin sensitivity, lowers blood glucose levels, improves metabolic health, and shows potential for treating obesity, diabetes, and other diseases. Therefore, we regulated the starch composition in Chlamydomonas reinhardtii to produce strains with high production of amylose.


    Figure 2.1.2 Starch amylose and amylopectin structure [5]

    To increase the proportion of amylose in Chlamydomonas reinhardtii, we targeted the pathway responsible for the conversion of amylose to amylopectin. By knocking out the starch branching enzyme (SBE) in Chlamydomonas reinhardtii, we promote the accumulation of amylose.

    Transition of Further Ideas

    For further validating the feasibility and effectiveness of our project, we interviewed Xiao Yibo, the CEO of the microalgae synthetic biology company, PROTOGA BIOTECH. He emphasized the need to improve the actual cultivation efficiency of microalgae in terms of light energy conversion for commercialization of starch production. Therefore, we made two additional design modifications, targeting both the light and dark reactions of photosynthesis in Chlamydomonas. These modifications aim to broaden the spectrum of light captured by photosynthetic pigments and promote the aggregation of carbon dioxide near the rubisco enzyme, thus enhancing photosynthesis and starch yield in Chlamydomonas.

    Figure 2.1.3 Schematic representation of light and dark reactions

    Extend light harvesting of Chlamy

    In runway ponds for industrial production and cultivation, Chlamydomonas in the upper layer uses near-red light for photosynthesis. Therefore, similar to the living environment of terrestrial cyanobacteria, a serious problem of near-red light attenuation in the depth of runway ponds exists, which leads to serious decrease of the photosynthetic efficiency of Chlamydomonas in the depth of runway ponds. In order to solve this problem, we adopted the means of synthetic biology. We tried to express enzyme ChlF from cyanobacteria to introduce FaRLiP into Chlamydomonas, thereby extending their light harvesting into far red (700 to 800 nm) and improving their photosynthetic light absorbance efficiency

    Figure 2.2.1 Introduce of FaRLiP in Chlamy by expressing ChlF.
    RLiP: Red light photoacclimation; FaRLiP:Far-red light photoacclimation

    Broaden the application scenarios of CCM

    CCM, short for Carbon Concentrating Mechanism, represents a pathway that specially enrich carbon dioxide for photosynthesis in green algae such as Chlamydomonas reinhardtii. Through a series of coordinated membrane transporters and carbonic anhydrase, extracellular CO2 is actively transported into the pyrenoid structure within chloroplasts against its concentration gradient.

    CCM pathway is an adaptation of green algae to the changes of carbon dioxide concentration in the water environment. In wild-type Chlamydomonas reinhardtii cells, a shift to limiting CO2 conditions induces expression of more than 50 genes, most of which encodes proteins in CCM pathway, thereby enhancing the CCM process and improving the utilization of inorganic carbon during cellular photosynthesis.

    CIA5, a transcription factor protein found in Chlamydomonas cells, plays a pivotal role in activating downstream gene expression related to CCM [6][7]. Under limited CO2 condition, the chemical modification of CIA5 changes, thus CIA5 locates at the nucleus and functions as a transcription factor for CCM genes. However, under high CO2 condition, the modification of CIA5 is not favorable to its transcription factor activity, resulting as a low CCM efficiency.

    Figure 2.2.2 Schematic diagram of CCM pathway


    It has been reported that the CIA5 protein gene with a deletion of 54 amino acids in the C terminus can complement the CIA5 mutant phenotype [8], which suggests that the C terminus of CIA5 may function as the upstream CO2 sensing domain and regulate its own transcription factor activity. Therefore, we planned to transfer an artificial CIA5 mutant with a C-terminal truncation (CIA5-C-del) into the wild type Chlamydomonas reinhardtii to allow it to function as a transcription factor under any CO2 concentration.

    The Future

    Project

    Figure 3.1

    In summary, we have proposed microalgae as a next-generation, sustainable, low-cost, and high-yield starch producing system, providing a solution to the food crisis. Our project is a great example of applying synthetic biology (the integration of biology and engineering) to solve real-world problems. Through the Design-Build-Test-Learn cycle, we have engineered the chassis, Chlamydomonas reinhardtii, to produce a large quantity of high-quality starch. This reduces the reliance on land resources in agricultural production and paves the way for addressing issues such as land scarcity and global hunger.

    Implementation

    Star-Chlamy, as a product combining principles of genetics and engineering, holds great potential for industrialization and forms the foundation for the concept of creating a light-drive cell-based starch factory. In fact, we have opened up the possibility of a sustainable farming that does not rely on arable land. Our project may not only assist to solve the issues described earlier but also contribute to mitigating climate change, generating new employment opportunities, and establishing sustainable industrial facilities to some extent. In the future, with advancements in chassis development and engineering optimization, productivity can increase and costs will reduce, thus our vision of constructing a phototrophic cell starch synthesis factory may become a reality.

    Figure 3.2 Schematic diagram of light-drive cell-based starch factory