17: Turning Trees into Plexiglass - Synthetic Biology For Production of Green Foods and Products

Burgers by Impossible Foods

Agriculture has transformed the planet.  About 50% of all land (other than deserts and ice shields and comprising an area equal to the Americas) is used for agriculture, most of which is for animal production.  In 2015, the US contributed about 8% of the world's total greenhouse gas emissions from food, with China at around 14%.  Contributions to food production include agriculture, land use change, and supply chain emissions (transport, packaging, food processing, retail, cooking, and waste).  

Estimates show that wild mammals comprise only about 6% of the total mass of all mammals (including people, livestock, and pets) on the planet).  In a parallel finding, the mass of "stuff" (plastics, metals, asphalt, concrete, etc) created by humans now exceeds the entire biomass of the planet!

Most of the agricultural land is used to produce meat and milk for human consumption. Collectively, cattle by far require the most land use, as shown in the interactive graph of Our World In Data in Figure \(\PageIndex{1}\) below, which shows how many square meters are required to produce 1000 kcals (1000 cal in the dietary sense) of food from each food type listed.  The graph is similar when the measure is land use per 100 grams of protein produced.

Figure \(\PageIndex{1}\):  Land use of foods/1000 kcals.  https://ourworldindata.org/land-use-diets

The number of animals slaughtered daily worldwide is unbelievably high, as shown below in Table \(\PageIndex{1}\).

Table \(\PageIndex{1}\): Animals slaughtered each day for food.  Max Roser (2023) - “How many animals get slaughtered every day?” Published online at OurWorldInData.org. Retrieved from: 'https://ourworldindata.org/how-many-...ered-every-day' [Online Resource]

The global percentage of all GHG emissions from the agricultural sector is about 35%, as illustrated in Figure \(\PageIndex{2}\) below.  Averages for some specific countries are also shown.

Figure \(\PageIndex{2}\):   https://ourworldindata.org/grapher/f...SA~IND~RUS~POL

The percentage of greenhouse gas emissions from all aspects of food products through the entire food chain is about 25%.  Figure \(\PageIndex{3}\) below shows the best estimate of global greenhouse gas per sector in 2016. The number today would probably differ little from these.

Figure \(\PageIndex{3}\):  Breakdown of global greenhouse gas emissions in 2016.  Hannah Ritchie (2020) - “Sector by sector: where do global greenhouse gas emissions come from?” Published online at OurWorldInData.org. Retrieved from: 'https://ourworldindata.org/ghg-emissions-by-sector' [Online Resource].  

An expanded analysis of the approximate 25% of global emissions that arise from food is shown in Figure \(\PageIndex{4}\) below.

Figure \(\PageIndex{4}\): Global greenhouse gas emission from food production. Hannah Ritchie (2019) - “Food production is responsible for one-quarter of the world’s greenhouse gas emissions,” Published online at OurWorldInData.org. Retrieved from: 'https://ourworldindata.org/food-ghg-emissions' [Online Resource]

Making plant-based foods that taste more like meat, if people would eat them, could have a large effect on greenhouse gas emissions and climate change. One example is the Impossible Burgers and other similar meats from Impossible Foods.  They have soy leghemoglobin, a monomeric heme-binding protein found in root nodules in legumes, to give the appearance and taste of blood in meat.  As a single-chain heme-binding protein, it has a high affinity for O₂, similar to animal myoglobin.  The high affinity derives from very high on-rates for binding O₂ (almost diffusion-controlled at around 2x10⁸ s^-1, and an off rate of around 20 s^-1.  This high affinity keeps O₂ bound, which would otherwise inhibit nitrogenase and nitrogen fixation by root-associated microbes.  The heme is important for positive tastes when we eat red meat.   Plant-based burgers containing leghemoglobin require much less land and lead to far lower greenhouse gas emissions. 

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of the alignment of sperm whale myoglobin (1MBO) and soy leghemoglobin (1BIN).

Figure \(\PageIndex{5}\): Alignment of sperm whale myoglobin (1MBO, cyan) and soy leghemoglobin (1BIN, magenta). (Copyright; author via source). Click the image for a popup or use this external link:  https://structure.ncbi.nlm.nih.gov/i...4pUi1S6qFtGn4A

The leghemoglobin in the Impossible burgers is produced in yeast.  Large amounts of heme are required to produce leghemoglobin in yeast, which is also produced in the engineered cells by introducing the appropriate genes.  The heme synthesis pathways (described in Chapter 22.3) for C4 (humans, animals, fungi, and purple non-sulfur phototrophic bacteria top) and C5 (archaea, plants, and other bacteria) for heme synthesis are shown in Figure \(\PageIndex{6}\) below.  The succinyl-CoA is derived from the citric acid cycle.

Figure \(\PageIndex{6}\): Top - heme synthesis pathway for C4 (humans, animals, fungi, and purple non-sulfur phototrophic bacteria top).  Bottom-heme synthesis pathway for C5 (archaea, plants, and other bacteria).   Heme biosynthetic pathway. Wikimedia Commonsile: Heme-Synthesis-Chemical-Details-Mirror (top) and  Heme pathway in E. coli. Zhang, J., Kang, Z., Chen, J. et al. Optimization of the heme biosynthesis pathway for producing 5-aminolevulinic acid in Escherichia coli. Sci Rep 5, 8584 (2015). https://doi.org/10.1038/srep08584. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ (bottom).

Succinyl-CoA (C4 pathway) and α-ketoglutarate (C5 pathway) are derived from the citric acid cycle.  The key precursor 5-aminolevulinate, ALA, needs to be elevated by engineering the C4 or C5 pathways, with the C5 pathways generally producing more ALA in engineered E. Coli.

Leghemoglobin from soy (species name Glycine max) can also be synthesized in the methylotrophic (uses methanol as a sole carbon source) yeast Pichia pastoris, often used for recombinant protein expression. Three groups of enzymes are needed"

• (group 1: porphobilinogen synthase (PBGS)
• group 2: uroporphyrinogen III synthase (UROS), uroporphyrinogen III decarboxylase (UROD), coproporphyrinogen III oxidase (CPO)
• group 3: Ala synthase (ALAS), protoporphyrinogen oxidase (PPO), and ferrochelatase (FECH)

Transcription of these genes in P. pastoris can be controlled by using the methanol-induced alcohol oxidase (AOX1) promoter, which is often used to achieve high expression of recombinant proteins. 

Figure \(\PageIndex{7}\) below shows a more detailed representation of the heme synthesis pathway.

Figure \(\PageIndex{7}\): The biosynthetic pathway of heme. Su, H.; Chen, X.; Chen, S.; Guo, M.; Liu, H. Applications of the Whole-Cell System in the Efficient Biosynthesis of Heme. Int. J. Mol. Sci. 2023, 24, 8384. https://doi.org/10.3390/ijms24098384.  Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

The two biosynthetic pathways of 5-aminolevulinic acid (bolded to indicate its importance) are shown in green (C4 pathway) and red (C5 pathway). The three downstream synthetic pathways of heme are marked with blue (CPD), indigo (SHD), and purple (PPD). Solid lines indicate single reactions, and dashed lines indicate more than two. The names of genes encoding the individual enzymes are in italics, and some reactions have alternative genes. The abbreviations of the corresponding enzymes are shown in the grey rectangle. See Table 1 for a list of names and abbreviations for heme synthesis enzymes

Figure \(\PageIndex{8}\) below complements this figure and shows the synthetic biology strategies to enhance heme production.  

Figure \(\PageIndex{8}\):  Synthetic biology strategies to enhance heme production. Green, orange, and red color blocks indicate genes that need to be up-regulated down-regulated, and knocked out, respectively.  Su, H. et al., ibid.

Computational tools such as AI can help design new pathways and novel enzymes to enhance production. It is becoming easier to transfer large pathways into yeast. 

The sales of alternative meat products have decreased recently. This decrease probably stems from more vocal opposition from the meat industry and peoples' reticence to eat what many consider ultra-processed foods.  High-protein and nutritious burgers can be made very simply with beans and grains.

Figure \(\PageIndex{10}\):  Production pathway of acrylates using fossil fuel and renewable resources. Souza, L.R.d.; Whitfield, B.; Al-Tabbaa, A. Biobased Acrylate Shells for Microcapsules Used in Self-Healing of Cementitious Materials. Sustainability 2022, 14, 13556. https://doi.org/10.3390/su142013556.  Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

When the alcohol is methanol, the final product is methylacrylate (MA).  The structure of the cyclic acrylate monomer feedstock used to polymerize plexiglass (lucite) is methylene-butyrolactone (MBL), whose structure is shown in Figure \(\PageIndex{11}\) below.  

Figure \(\PageIndex{11}\): Structure of methyl methacrylate and its lactone (a cyclic ester)

Methyl methacrylate can undergo a free radical polymerization in the presence of an initiator (In^.), as shown below in Figure \(\PageIndex{12}\).

Figure \(\PageIndex{12}\): Mechanism of free radical polymerization of MMA

This reaction can form large polymers like plexiglass.  The market for acrylic acid, the feedstock for plexiglass, is estimated to reach 12 million metric tons by 2030.

MBL, the lactone of MMA, is made in tulips from pathways that are not completely elucidated. It can also be used as a feedstock for the polymerization of plexiglass.  Figure \(\PageIndex{13}\) shows the polymerization products from MMA and MBL. 

Figure \(\PageIndex{13}\): Structure of poly-MMA and poly-MBL

Using synthetic biology and advanced computational methods, plexiglass can be made from biological sources instead of fossil fuels.  To accomplish this, Azerda has designed synthetic pathways from millions of potential metabolic pathways (using a software package called Scylax™) and intelligently redesigned key enzymes to maximize their catalytic potential for the synthesis of MBL (using the software Archytas™). They used high-throughput DNA and protein analyses to maximize expression. Finally, they engineered expression strains and downstream purification processes to maximize the final output of MBL.  In summary, the key steps in the process were:

• identifying a pathway from millions of reactions in databases of pathways that could produce MBL from simple sugars through a fermentation process;
• engineering pathway enzymes to significantly increase catalytic efficiency and decrease inhibition;
• producing test quantities of the products in cell strains;
• scaling up production to levels needed for purification and reactions of the MBL
• purifying sufficient amounts of MBL from large fermentation broths
• making the desired product (plexiglass, for example) from the feedstock.

Strains of bacteria, yeast, and filamentous fungi were modified to meet the above criteria. The ultimate substrate for the process was a lignocellulosic hydrolysate, so in the end, the process converts trees into plexiglass (incredible to think about)! Of course, it is also amazing that CO₂ from the air, water, and minerals/ions from the soil can become a tree!

Starting with just a detectable product level, the process was continually improved and scaled to yield 5 g/L of broth, which is getting close to the 20 g/L required for commercial viability.  Figure \(\PageIndex{14}\) below shows plexiglass created from the lignocellulosic stock!

Figure  \(\PageIndex{14}\): Plexiglass made from biosourced MBL.  

Table \(\PageIndex{2}\) below compares the key physical properties of the polymers from Arzeda's PMBL compared to literature values for fossil-fuel-based PMBL and PMMA.

Table \(\PageIndex{2}\): https://www.energy.gov/sites/default...-korkegian.pdf