In an increasingly plastic-dependent society, the need to keep up with rising plastic demands has led global plastic production to reach 370 million tons in 2021[1]. Plastic production is expected to triple by 2060[2] and produce 56 gigatons of carbon dioxide emissions by 2050[3].
The staggering statistics have ultimately caused great urgency to address this issue. Japan’s embrace of a culture of high hygiene consciousness and ease of buying single-use plastics has prompted the need to tackle plastic over usage.
An emerging and promising alternative to conventional petrochemical plastics is polyhydroxyalkanoates or PHAs[4]. PHAs are biodegradable polyesters synthesized by certain bacteria to store excess carbon and energy. While promising, the current limitations and challenges regarding PHA are the high costs associated with production. Notably, the available carbon sources and extraction methods for biopolymers are the leading factors that drive the costs of PHAs to be six times that of conventional plastics[5].
Such polyesters are similar to petrochemical polymers including polypropylene and polystyrene, making them a suitable alternative to petroleum-based plastics[6]. Researchers from the RIKEN Laboratory in Japan found that modifying PHA-producing proteins could further improve bioplastic yield, concentration, and production rate. These modifications could enhance PHA quality and molecular weight[7].
PHAs are based from hydroxycarboxylic and hydroxyacid polymers. Produced by soil bacteria, PHA degrades when exposed to other bacterias. The physical properties of PHAs differ with its structure, For example, a longer monomer chain grants the PHA more flexibility giving them real world applications in today’s markets: garbage bags, food packaging, diapers, surgical sutures, and more[8].
PHB is a derivative of PHA and is the most widely used member. PHB production follows two steps: fermentation and extraction. Bacteria store PHB in their cells during fermentation to store sugar and during extraction, the produced PHB is taken out of the bacteria PHB possesses better physical properties than polypropylene for food packaging, it is completely nontoxic, and biodegrades in about a year in the wild[9].
P(3HB) is a biopolyester synthesized from bacteria for carbon and energy storage. P(3HB) possesses a wide range of molecular masses, biodegradability, and thermoplastic properties which allow it to be used in today’s markets and manufacturing. And, P(3HB) has strong biocompatibility due to being made from natural materials which makes it promising for medical applications. Said properties are heavily affected by P(3HB)’s molecular mass and thus the wide range of molecular masses allows for many different P(3HB)s to exist[10]. Furthermore, being water-insoluble, P(3HB)s are resistant to hydrolytic degradation, meaning that they are durable bioplastics[11].
Acid-derived PLAs are bioplastics made from the ionic polymerization of lactic acid. They’re soluble in organic solvents, transparent, and are hydrophobic[12]. However, the physical properties of PLAs depend on how they were produced: glass transition temperature, melting temperature, tensile strength, have a positive relationship with molecular weight and these properties are affected by the co-monomer, crystallinty, and molecular weight. PLA degrades slower meaning it has a longer shelf life and there are no toxic chemicals produced during degradation. However, compared to P(3HB), PLA has a substantially slow degradation rate. Limitations of PLA include poor mechanical properties and poor thermal resistance[13].
Lactic acid can be produced using bacteria and fungi. Lactic acid producing bacteria (LAB) have received wide interest because of their high growth rate and product yield. However, LAB have complex nutrient requirements because of their limited ability to synthesize B-vitamins and amino acids, making supplementation of sufficient nutrients such as yeast extracts to media is necessary. This expensive downstream process increases the overall cost of production of lactic acid using lactic acid producing bacteria[14].
PHA production costs are currently six times higher than conventional plastics and roughly one third of these costs are attributed to extraction methods[15]. Current methods of PHA extraction are enzymatic digestion and chemical extraction[16]. Chemical solvents are excessively consumed, nearly 3 times higher than petrochemical plastics, which contributes to high costs of disposal and regeneration and can have negative environmental effects[17].
Earlier on in our research, we learned that Polyhydroxyalkanoate synthases (PhaC) are the key enzymes for biopolyester synthesis[18].
This enzyme consists of a N-terminal domain and a C-terminal domain that help stabilize enzymatic activity, substrate specificity, PHA molecular weight, the expression of PhaC, and the ability of PhaC to bind to PHA granules. The C-terminal catalytic domain consists of a cap subdomain, a substrate entrance channel, an active site, and a product egress tunnel.
With this information protein modeling became a huge field that opened up for exploration. Possibly mutating PhaC could provide benefits for the expression of PHA. Looking into different protein modeling programs the team was able to find 7 different residues for point mutations that could potentially be used to increase PHA expression.
An operon of three genes, phaA, phaB, and phaC encode proteins for the production of PHAs and according to various research articles, rearranging the genes can manipulate the yield and molecular weight of PHA granules. In this context, according to a 2016 review by Takeharu Tsuge, higher molecular weight correlates with a stronger material.
The order phaCAB and phaCBA resulted in the greatest P(3HB) content (wh%) but had the lowest molecular weight. Contrary, the order phaBCA had an increased molecular weight with a relatively high P(3HB) yield.
Alongside this, there appears to be a negative correlation between the molecular weight and PHA synthase (phaC) activity. This means that when phaC has a lower enzymatic activity, the molecular weight of produced PHB granules is higher. And, with higher phaC activity, the yield of PHB increased. Meanwhile, higher phaA and phaA had no correlation with molecular weight or content.
Researchers from Nanjing Agricultural University tested error-prone PCR (epPCR), which can be utilized to cause random mutations in selected genes. And though the impacts would be random at first, finding the optimal location to mutate a protein may lead to more enhanced properties. After obtaining different mutated versions of the same protein, gel electrophoresis can be conducted to separate the optimal protein[19].
The Type I Secretion System (T1SS) is a direct transportation-based secretion pathway from the intracellular space (cytoplasm) to the extracellular matrix. Gram-negative bacteria extensively utilize this method of secretion, including E. coli.
We looked at hemolysin A (HlyA) secretion primarily for E. coli, since it is a well-documented secretion system that is endogenous to E. coli. Three channel proteins assist T1SSs: inner membrane proteins (HlyB and HlyD), periplasmic adaptor proteins, and an outer membrane protein (TolC).
Folding rates
Researchers from the University of Paris-Sud found that lowering the substrate folding rates of the MalE binding protein increased secretion efficiency in Type 1 Secretion System. Conducting a folding mutation caused the MalE protein, which normally lacks an export signal and is poorly secreted by the Hly system, to make the T1SS secrete more of the protein. Additionally, iGEM ASIJ-Tokyo initially researched ways in which this could be implemented in order to enhance PHA production from the phasin protein.
Overexpressing HlyBD
iGEM Team Edinburgh experimented with overexpressing HlyBD. Results showed that overexpressing HlyBD alone hinders cell growth but by cotransforming the host with plasmids for PHA-production and secretion, overexpression of HlyBD becomes beneficial for cell growth[20]. Team Edinburgh also concluded that overexpression of HlyBD works with a phaCAB operon; however, comparative results were not included[21].
The Type II Secretion System is a direct transportation-based secretion pathway from the periplasm, the space between the inner and outer membranes of a cell, and the extracellular matrix. While T1SSs are able to directly transport materials from the cytoplasm to the extracellular matrix, T2SSs require a second channel protein system to bring the tagged proteins to the periplasm for secretion out of the cell. T2SSs use two types of inner membrane channels for this purpose: the general secretory (Sec) pathway and the twin-arginine translocation (Tat) pathway. The Sec pathway prefers unfolded proteins for secretion, and the Tat pathway prefers folded proteins.
General secretory (Sec) pathway
The protein of interest in the general secretory pathway is first synthesized as a precursor containing an N-terminal signal peptide that is cleavable. This precursor is targeted and transported through the inner membrane of the cell by a complex that contains the Sec transcolon. Finally, the signal peptide is cleaved off and the mature protein, free to fold, is released into the periplasm. The protein then requires more cellular machinery to translocate across the outer membrane. In the sec pathway, the protein does not enter its fully folded, active configuration until it is within the periplasmic space[22].
Twin-arginine translocation (Tat) pathway
As mentioned above, there are certain protein export systems that allow the secretion of large proteins from within gram negative bacteria such as E.Coli. The twin-arginine transolactation (Tat) protein system, with the use of TatA, TatB, and TatC proteins, creates a pathway through the celluclaar membrane wide enough for substrates (such as PHA) to pass through[23].
One study utilized this Tat pathway to increase the oxygen available for better PHB production. This was done by transporting bacterial Vitreoscilla hemoglobin (VHb) into E.coli, showing high yields of overall PHA production[24].
Extracellular Secretion Mechanism
Type II Secretion systems usually involve 12~16 different proteins that constitute a “secreton,” which accepts substrates from either the Sec or Tat pathway. When a potential ligand is detected by the architecture of the T2SS, ATP hydrolysis leads to changes in the hexamer of a protein called GspE located in the cytoplasm, initiating a reaction that creates a large tower of pseudopilin subunits that pushes the exoprotein out of the periplasm[25].
In addition to the Type 1 and Type 2 Secretion Systems, we questioned whether we could use vesicles to transport bioplastics directly out of the cell. We came across the Cas9 protein that could be used to create a OMV-CRISPR-Cas9 system in gram-negative bacteria including E.Coli[26]. This system would induce the creation of a vescicle, which can “be degraded easily while preserving the shape and bioactivity of sensitive Cas9 proteins within, as well as single guide RNAs (sg-RNAs)[27].”
The 2021 Moscow Institute of Physics and Technology iGEM team found the Extracellular Vesciale (EV) system which uses the GAG protein, which would recognize RNA and correctly package it into its own vesicle[28]. However, we assume that an entire protein, in our case, phasin, would be too large to fit within its own vesicle, making this EV system unfeasible for our project. Furthermore, this team utilized the potential of OMVs in the biomedical field, yet the question still remains as to whether OMVs could be studied in conjunction with PHB[29].
In 2022, Sangho Koh and other researchers from the Tokyo University of Agriculture found that when observing foaming on PHB producing cultures, there is a correlation between PHB accumulation and membrane vesicles, or MVs[30]. Essentially, the biogenesis of MVs was triggered by the accumulation of PHB granules in E. Coli, and the amount of MVs released was correlated to the amount of PHB accumulated. This led them to theorize that a trigger for MV formation may be PHB accumulation, and since PHB accumulation increases as glucose content in the cell increases, MV formation may correlate with the concentration of glucose externally added[31].
In their observations of the formation of MVs, they noticed that there was a mixture of single layer outer membrane vesicles, or OMVs, and multilayer outer/inner membrane vesicles, or OIMVs[32]. The researchers speculated that it could be beneficial to take advantage of the OIMVs by using them as cargo to encapsulate intracellularly polymerized products of interest such as protein, nucleic acids and polyesters like PHB. Their findings were the first to connect MVs and PHBs, raising numerous questions as to what effect their relationship could have on the secretion of PHB, and how the MV secretion system could be better controlled to efficiently secrete matter from the cell[33].
There have been a number of iGEM teams that have experimented with OMVs in the past. This includes Iiser 2022, who aimed to improve chemotherapy by making drug delivery systems targeted more specifically at cancer cells[34]. They developed a two-step verification system consisting of two target biomarkers for cancerous cells. This way the drug is only activated to kill the cell when both markers are present. Their delivery system involved two separate extracellularly loaded OMVs, one loaded with the drug and the other with an activating enzyme[35].
In February 2023, researchers from the University of Kent described a novel system for the secretion of tagged proteins from MVs in e. Coli[36]. Their goal was to create a system that allows for convenient production of toxic or insoluble proteins in the cell (or other properties that made proteins difficult to produce or secrete), and they engineered a simple peptide tag which causes the cell to generate high yields of recombinant proteins of varying sizes and allows for long-term storage in the cell. These single peptide tags are now known as vesicle nucleating peptides, or VNps, works at a much larger volume than fermentation cultures, is flexible in that it is capable of functioning in different strains of E. Coli, and is compatible with a range of plasmids, promoters, and induction levels[37].
The researchers further explored the possibility of improving the tag itself by making simple modifications to the VNp amino acid sequence. Their results found that they could enhance protein yields, enhance vesicular export over a wider range of culture temperatures, and shorten the sequence to just 20 residues[38]. In a paper outlining the methodology and procedures of the bacterial production of recombinant proteins using VNps, Eastwood and others at the University of Kent identified three VNp variants[39]:
Due to the novelty of this system, the researchers suggested using all three VNp variants for any kind of protein, and stated that, “This simple and cost-effective recombinant protein tool is likely to have a positive impact upon biotechnology and medical industries as well as discovery science.”
Taking this literature into consideration, iGEM ASIJ took the chance to observe the intersectionality between PHB and VNps, and inquired as to whether the VNp secretion process could be refined and better controlled to improve the efficiency of PHB secretion in E. Coli.
Due to the novelty of this system, the researchers suggested using all three VNp variants for any kind of protein, and stated that, “This simple and cost-effective recombinant protein tool is likely to have a positive impact upon biotechnology and medical industries as well as discovery science.”
Taking this literature into consideration, iGEM ASIJ took the chance to observe the intersectionality between PHB and VNps, and inquired as to whether the VNp secretion process could be refined and better controlled to improve the efficiency of PHB secretion in E. Coli.
Team Imperial from 2013 listed criteria for optimal PHA production, including the degradation and synthesis of P(3HB) and the degradation of PLA[40]. Additionally, an article in 2016 published by researcher Takeharu Tsuge found that different types of bacteria produce different molecular weights of PHA[41].
PHA accumulates under imbalanced nutrient conditions, such as excess carbon but limited nitrogen availability, and is degraded under starvation conditions to maintain cellular energy homeostasis. Leading on from Imperial’s and Tsuge’s paper, we found four main candidates: E. coli, R. eutropha, Bacillus Cereus, and S. cerevisiae.
E. coli is well suited to PHA production primarily due to its absence of phaZ, a PHA depolymerase enzyme[42]. Furthermore, while some strains of bacteria require nutrient starvation in order to produce PHAs, E.coli accumulates PHAs during the growth phase instead. These benefits, while considering the disadvantages of other strains of bacteria, made Ecoli the optimal chassis for this year’s ASIJ-Tokyo project[43].
R. eutropha is a well-known native producer of P(3HB). It is capable of accumulating PHA at over 90% of the dry cell weight while in most bacterial cells its ~30-50%. Short-chain-length monomers (C3-C5) are polymerized using a class I PHA synthase. Deletion of several PHA depolymerase genes allows the production of high-molecular-weight PHA in R. eutropha[44].
Bacillus Cereus is a gram positive bacteria with a naturally occurring PHB-producing strain, optimized for producing PHA bioplastics at a low cost. Unfortunately, due to its sporous nature, we were unable to proceed with this option. Bacillus species are advantageous over other bacteria because they do not contain lipopolysaccharides external layers, thereby making extraction of PHAs much easier than with other bacteria[45].
S. cerevisae, also known as baker’s yeast, is effective since it can withstand acidic environments. These cells may be optimal for lactic acid production through LDH gene expression, which can be polymerized into PHB and PLA bioplastics[46]. However, as a yeast, S. Cerevisae would require substantial amounts of gene editing, increasing possible errors[47].
Our team is interested in further investigating methods to decrease bioplastic production costs by utilizing food waste as an alternative biofuel source. Unfortunately, due to time constraints, we were unable to develop an efficient method that would allow us to do so. Nevertheless, the following text provides an overview of our approach to biosynthesizing P(3HB) from food waste.
Saccharification:
Food waste is primarily composed of starch, a polysaccharide made up of chains of monosaccharides[48]. Given that our desired source of energy is glucose, a monosaccharide, saccharification of simulated municipal food waste is first necessary. This is achieved through enzymatic digestion to complete the saccharification of all starch variants into usable glucose by hydrolysis[49]. Specifically, utilizing alpha-amylase to break down polysaccharides into dextrins, and glucoamylase to further break down dextrin into usable glucose[50].
Separation:
Once saccharification is completed, fat-soluble molecules (fatty acids) and ionic compounds (electrolytes, amino acids, organic acids, salts) must be removed from neutral molecules (including carbohydrates) through a solid-liquid solution to the liquid filter system[51]. A secondary separation is required to further separate the 3 types of molecules now in liquid form. This can be completed by applying centrifugal sedimentation[52].
Glucose Isolation:
To further isolate glucose from here, filtration of solids through filters and enzymatic digestion ensure that starches are no longer present. To ensure we use the food waste found within glucose as biofuel, an area of further research is required within this specific area of developing a method to utilize glucose.
Conversion to Acetyl-CoA:
The purified glucose from the simulated concentration of municipal food waste must then be converted to acetyl-CoA through cellular respiration- a pathway that is commonly used by most organisms to produce P(3HB)[53].
Through a series of enzymatic reactions, one molecule of glucose is converted into two molecules of pyruvate, plus two ATP molecules. The reaction described is the first half of glycolysis, a series of biochemical reactions that occur in the cytoplasm of cells to break down glucose into pyruvate. Glycolysis is the first stage of cellular respiration and is anaerobic, meaning it does not require oxygen[54].
Pyruvate Oxidation:
Pyruvate is oxidatively decarboxylated to form acetyl-CoA by two enzymes: a metalloenzyme called pyruvate ferredoxin oxidoreductase (POR) and pyruvate formate lyase (PFL)[55]. Essentially, the pyruvate is decarboxylated (a CO2 is removed), which generates enough energy and the hydrogens, I guess, to reduce a NAD+ molecule to NADH and H+. Finally, a Coenzyme A complex is attached to the end that the carboxyl group was removed from to yield acetyl CoA[56].
Biosynthesis of P(3HB):
The final step of the process is the biosynthesis of P(3HB). Take the 2 acetyl-CoA molecules and use acetyl-CoA acetyltransferase (PhaA) to bind them together. Then, NADPH-dependent acetoacetyl-CoA reductase or R-3-hydroxybutyryl-CoA dehydrogenase converts the resulting acetoacetyl-CoA and a hydrogen ion into a NADP+ molecule[57]. The PHA synthase then attaches the 3HB-CoAs together to create P(3HB) our target bioplastics.
Areas of Future Improvement:
A possible improvement that can be made to this section is an overexpression and a bypass that several studies have claimed to increase concentrations of acetyl-CoA. The method involves overexpressing the enzyme pantothenate kinase and allowing for a PDH (pyruvate dehydrogenase) bypass. I don’t know exactly what this does, though, because we haven’t really gravitated towards this. Furthermore, I don’t wanna overcomplicate our final plasmid, so let’s not deal with this for now.
A potential exploration component: Changing the gene order of the three enzymes to achieve a balance in expression that grants us good yield without sacrificing molecular weight. We plan to test the balance between molecular weight and yield based on enzymes. This will be completed by testing phaCAB and phaBCA to name a few.
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