This project aims to establish a modular and programmable synthetic biology platform in E. coli for the high-efficiency production of polyhydroxyalkanoate (PHA) granules with precisely controllable sizes. The core design integrates three functional modules—​​PHA synthesis​​, ​​size regulation​​, and ​​temporal control​​—to achieve spatiotemporal regulation of PHA biosynthesis. By decoupling the synthesis of the PHA core from the subsequent surface assembly process through inducible expression systems, this platform enables dynamic control over granule size and morphology while minimizing metabolic burden.

​​1. Core PHA Synthesis Module

The ​​PHA synthesis module​​, comprising phaA (β-ketothiolase), phaB (acetoacetyl-CoA reductase), and phaC (PHA synthase), serves as the foundational engine for polymer production. This autonomous pathway converts central metabolites (acetyl-CoA) into PHA monomers and polymerizes them into intracellular granules. The phaCAB operon ensures efficient precursor supply and polymerization, significantly outperforming systems relying solely on phaC, which depend on the host’s native metabolic pathways and often lead to lower yields and higher metabolic stress.

​​2. Size-Regulation Module

To control granule size and uniformity, we incorporated ​​hydrophobin-based regulators​​ to achieve programmable control over PHA particle dimensions to meet diverse application requirements.

• ​​PhaP​​: A native phasin that binds to the PHA surface via hydrophobic interactions, suppressing granule coalescence to produce ​​small, monodisperse particles​​.
• ​​ADF3​​: A spider silk protein whose strong hydrophobic domains promote granule fusion, yielding ​​larger aggregates​​.

3. Time control logic module:​​

T7 promoter​​, ​​cold-inducible cspA promoter​​, blue-light and araBAD promoter were introduced in the PHA synthesis, to achieve precisely timed control over PHA synthesis and granule assembly. The ​​T7 promoter​​ was utilized to drive strong, IPTG-inducd expression of the core PHA synthesis operon (phaCAB). The ​​cold-inducible cspA promoter​​, the ​​optogenetic blue-light-responsive system​​, and the ​​L-arabinose-inducible araBAD promoter​​ were employed to separately regulate the expression of size-modulating proteins (PhaP, ADF3).

Summary:

In this collection, these modules form a complete and interacting system that achieves the function of controlling PHA synthesis and size regulation. We successfully constructed and verified this system. Functional experiments demonstrated that through different induction strategies, we observed efficient PHA production and significant changes in PHA particle size, thereby confirming the function of t his system in size regulation. All these parts comply with the RCF assembly standards. We have summarized them into a collection (UUID: b943558d-af2c-4ae2-8b16-4987c1f02b2f) and provided detailed documentation.

Furthermore, this collection provides a case reference and a powerful tool for the synthetic biology field to programmatically regulate the size of intracellular particles. These component sets are designed in a modular manner, and in the future, users can replace or upgrade specific modules as needed (for example, by using different promoters or different surface proteins), with strong scalability, which can inspire and facilitate more diverse biomanufacturing projects.

Table 1 Part collection of regulation module in this project.

No	Part number	Part name	Diagram	Part type I	Part type II
1	BBa_25DSR6K3	phaC		Basic part	New part
2	BBa_25Y9LCWS	phaA		Basic part	New part
3	BBa_25XUQQJP	phaB		Basic part	New part
4	BBa_25F3MLCY	phaP		Basic part	New part
5	BBa K5137008	ADF3		Basic part	Existing part
6	BBa_2584689S	​​maltose-binding protein (MBP)		Basic part	New part
7	BBa_K5410014	T7 promoter		Basic part	Existing part
8	BBa_K5410015	T7 terminator		Basic part	Existing part
9	BBa_K5432027	araBAD promoter		Basic part	Existing part
10	BBa_K3782007	cspA promoter		Basic part	Existing part
11	BBa_K3447133	light-on induced system		Composite part	Existing part
12	BBa_25UO52AB	pRSFDuet-1 plasmid backbone		Basic part	New part
13	BBa_25JWK4NZ	pBAD-Myc-HisA plasmid backbone		Basic part	New part

Table 2 Part collection of plasmids in this project.

No	Part number	Part name	Diagram	Part type I	Part type II
1	BBa_25N6960P	pRSFDuet-1-phaC		Composite part	New part
2	BBa_25R8QPIY	pRSFDuet-1-phaC-phaA-phaB		Composite part	New part
3	BBa_25FPQIWY	pRSFDuet-1-phaC-PcspA-phaP		Composite part	New part
4	BBa_25Z31U0T	pRSFDuet-1-phaC-phaA-phaB-PcspA-phaP		Composite part	New part
5	BBa_2546M5NK	pRSFDuet-1-phaC-Plight-phaP		Composite part	New part
6	BBa_25UMLEA5	pRSFDuet-1-phaC-phaA-phaB-Plight-phaP		Composite part	New part
7	BBa_25PLR114	pBAD-ADF3-MBP		Composite part	New part