Bioplastics

In an increasingly plastic-dependent society, the need to keep up with the 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 a 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 our iGEM team to tackle plastic over usage.

An emerging and promising alternative to conventional petrochemical plastics is polyhydroxyalkanoates or PHAs^[4]. PHAs are biodegradable polyesters synthesized within some bacteria to store excess carbon and energy. While promising, the current limitations and challenges regarding PHA are the high cost 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].

Currently, standard methods of PHB extraction include enzymatic digestion and direct chemical extraction^[6]. Both involve toxic chemicals, such as chloroform, and contribute to 30% of total production costs^[7]. As an alternative to these unsustainable harvestation methods, we sought to develop a model for PHB secretion in engineered bacteria in aiming to reduce downstream processing costs.

General Secretion of PHA:

Traditional secretion systems in E. coli operate through secretion tags that attach to specific proteins of interest^[8]. Phasin is a surface protein on PHA granules that functions as one of the main proteins responsible for regulating granule size. Past teams and research have conventionally used phasin as a target protein for an indirect method of secretion of PHAs^[9]. Secretion tags can be attached to the phasin protein, allowing membrane-bound proteins recognize these tags as a signal for secretion, therefore, expelling both the tag and the bonded PHA^[10].

Past and present research touches upon but fails to fully explore the potential of secretion. In project ESPHA, we aimed to collect comprehensive data and analysis for comparing various secretion systems found through literature review.

Traditional, non-invasive secretion methods include type I^[11] and type II secretion systems^[12]. These secretion methods utilize peptide tags can be fused with the phasin surface proteins of PHA and recognized by transport proteins to secrete the PHA without having to rely on external injections^[13].

Type 1 Secretion:

Type I bacterial secretion systems operates by transporting products directly from the cytoplasm within the cell membrane into the extracellular matrix^[14]. The products are then transported through a single protein channel to the outer membrane. However, the relatively small secretion channels favor the secretion of unfolded proteins, which leads to a decrease in PHA secretion and its overall product quality and efficiency^[15]. To combat this particular limitation, we used the HlyA tag and overexpressed HlyB and HlyD which are important membrane proteins that could allow for HlyA secretion, thus, allows for bigger granules of PHA to be secreted^[16].

Overexpressing HlyBD may result in the creation of an increased number of pores for PHA secretion, which would be a huge advantage if and only if the PHA granules are small enough to pass through the

Type II Secretion:

Type II Secretion Systems use a two-part process that involves secretion from the periplasm—the space between the inner and outer membranes of prokaryotic cells—to the extracellular space^[17]. Simply put, when the correct substrate is detected by the secretion system, it activates a signal that consumes 2 ATP to extend a piston-like structure and pushes proteins outside the cell.

Specifically, there are two inner membrane channel proteins response to transport PHA: general secretion pathway (Sec) and twin-arginine translocation pathway (Tat)^[18]. Each system has their own unique advantages. The Sec sequence has a signal (pre) sequence, a signal sequence, and a leader sequence. This particular sequence secretes unfolded proteins cotranslationally, which allows for quick turnover from production to secretion^[19]. The Tat sequence has the signal peptide sequence (S/T)RRXFLK, and transports after the protein folds in the periplasm^[20]. This allows for the timeframe where the cell is preparing for secretion but is not necessarily secreting, where the cell may be transferred to a different medium or viewed to see how secretion is occurring. Additionally, the Tat sequences prefer folded proteins, so it is possible that larger granules will be able to pass through the Tat system more easily than the Sec system^[21]. Current limitations in the size of the granule that can be secreted due to the nature of both inner and outer membrane channels using a relatively small pore.

We looked into the viability of three tags: Gene III used by 2009 Utah State, the first team ever to look into secreting PHAs, OmpA, and TorA. Gene III, and OmpA use the Sec system while the TorA system uses Tat^[22]. Since we're using Gene III to verify the effectiveness of the 2009 protocol, using one other Sec and a Tat system allows us to compare the effectiveness of Sec and Tat systems in secreting PHA.

We later decided to use solely the Tat secretion system, due to its nature in preferring folded proteins. Thus, we selected TorA as our secretion tag for Type 2 secretion.

Vesicle Nucleating Peptides:

Vesicle Nucleating Peptides work by attaching the VNp tag to a target protein, where the VNp tag induces the creation of a vesicle in the inner membrane, pinches off and forms an extracellular vesicle^[23]. This is a highly advantageous secretion method given that the PHA granules won't be required to flow through a narrow channel and suffer a loss in yield or quality due to size. There are currently 3 VNps of which we know the full sequence: VNp2, VNp6, and VNp15^[24]. We decided to use VNp6 since it had the highest secretion efficiency out of the three when used as a fusion protein tag. Future teams could look into comparing the secretion efficiency for phasin of VNp2, 6, and 15 to see if VNp6 truly is the most efficient option for phasin and PHA secretion.

Foodwaste

Japan Context:

“もったいない” (mottainai), roughly translating to “too good to waste” is a Japanese phrase that embodies a culture of no-waste. With many daily applications, the phrase plays an integral role in promoting minimal waste in Japan at the individual level. For instance, because it is "もったいない," individuals are inspired to savor every last grain of rice in their bowl.

Similarly, because it is もったいない, people avoid discarding items that still hold potential utility. Thus the phrase contributes to a broader effort to reduce waste and make the most of available resources.

However, despite its deep integration into Japanese culture, Japan finds itself among the countries producing the most food waste^[25]. This is largely attributed to aesthetics. On an industrial scale, produce that is slightly unaesthetic will be immediately discarded, as they are unwanted and will not sell^[26]. According to The Center for Environmental Science in Saitama, it was estimated that a staggering 22.2% of food was being wasted before reaching the table.^[27]

Global Context:

On a global scale, 1.6 billion tons of food is wasted annually, 1.3 billion tons of which is considered edible^[28]. Equally as concerning as the immediate implications of wasted food are the environmental effects. According to the UN, 8-10% of global greenhouse emissions can be attributed to food waste^[29]. As such, food waste emits nearly the same amount of emissions as cars or five times the emissions of planes^[30].

Conclusively, food waste is an issue that must be urgently addressed. Project ESPHA aims to do this by utilizing food waste as a biofuel source to produce bioplastics in the form of PHA. By leveraging food waste which exists in overabundance, we hope to promote the idea of “もったいない” while contributing to environmental sustainability and resource efficiency.

Unfortunately, due to time constraints, we were unable to develop an efficient method that would allow for food waste to be used as a biofuel source. Nevertheless, the extended literature review provides an overview of our approach to biosynthesizing P(3HB) from food waste.