1. phaC (BBa_25DSR6K3)

Name: phaC

Base Pairs: 1770 bp

Origin:

Recombinant PHA synthase derived from Ralstonia eutropha, with codon optimization for high-yield expression in E. coli.

Properties:

Encoding β-keto hydrolase, condensing two acetyl coenzyme A molecules to form acetoacetyl coenzyme A and releasing a HS-CoA molecule^[1].

Usage and Biology:

By expressing the phaC gene in engineered bacteria, it enables the large-scale production of biodegradable PHA bioplastics as sustainable alternatives to conventional petroleum-based plastics, addressing issues of plastic pollution. During catalysis, a cysteine residue in the active site of phaC synthase forms a covalent bond with the growing polymer chain, creating an elongating "enzyme-polymer" complex. This mechanism allows the efficient synthesis of high-molecular-weight, insoluble PHA granules from water-soluble monomer substrates within cells.

2. phaA (BBa_25Y9LCWS)

Name: phaA

Base Pairs: 1182 bp

Origin:

Derived from Ralstonia eutropha, with codon optimization for high-yield expression in E. coli.

Properties:

Encoding acetoacetyl coenzyme A reductase, uses NADPH as a reducing agent to reduce acetoacetyl coenzyme A to (R)-3-hydroxybutyryl coenzyme A^[1].

Usage and Biology:

The primary application of phaA lies in metabolic engineering for bioplastic production. It is co-expressed with other PHA synthesis genes (phaB, phaC) in microbial chassis to construct efficient pathways for converting renewable carbon sources into biodegradable PHA polymers. Under unbalanced nutrient conditions, phaA diverts acetyl-CoA from the central TCA cycle toward PHA synthesis. This enzymatic step controls the precursor supply for the entire PHA biosynthesis pathway, making phaA activity a critical determinant of both PHA yield and polymer composition in production strains.

3. phaB (BBa_25XUQQJP)

Name: phaB

Base Pairs: 741 bp

Origin:

Derived from Ralstonia eutropha, with codon optimization for high-yield expression in E. coli.

Properties:

Encoding PHA synthase, polymerizes the (R)-3-hydroxybutyryl coenzyme A monomers into a PHA polymer^[1].

Usage and Biology:

phaB is essential for engineered bioproduction of PHAs. It is co-expressed with phaA and phaC in microbial factories to establish a complete pathway for converting sugars or lipids into biodegradable plastics. phaB serves a critical role in redox balance and precursor supply. By consuming NADPH, it links PHA biosynthesis to the cellular redox state. The enzyme's stereospecificity ensures the production of the (R)-enantiomer of hydroxyacyl-CoA, which is the exclusive substrate for the polymerization reaction catalyzed by PHA synthase (phaC), ultimately determining the crystallinity and material properties of the resulting biopolymer.

To achieve PHA synthesis in E. coli, we designed two expression plasmids corresponding to the two PHA synthesis modules. We selected the ​​pRSFDuet​​ vector as the plasmid backbone to facilitate subsequent transcription and expression of multiple genes. This plasmid contains a strong inducible ​​T7 promoter​​, which enables highly efficient transcription of the target genes under its regulation. As illustrated in Fig. 1, the plasmid construction strategy involved amplifying the target genes (phaC and phaCAB) and the plasmid backbone separately, followed by assembly using homologous recombination to obtain the recombinant constructs.

Fig. 1 Plasmids diagram of (A) pRSFDuet-1-phaC and (B) pRSFDuet-1-phaCAB.

The agarose gel electrophoresis confirmed successful amplification of all target DNA fragments. The phaC gene (2249 bp) was amplified using the pUC-phaC plasmid as template, while the phaCAB operon (4338 bp) was amplified from the pUC-phaCAB template. Additionally, the pRSFDuet-1 vector backbone (3646 bp) was amplified from its respective template. All samples showed sharp bands at their expected molecular weights, indicating high amplification specificity and efficiency. These results verified the successful preparation of DNA fragments for subsequent cloning steps (Fig. 2).

Fig. 2 The agarose gel electrophoresis of DNA amplification.

After obtaining the DNA fragments, they are assembled through homologous recombination. After transferring the recombinant product into E. coli DH5α, we conducted colony PCR identification. For the colonies transformed with pRSFDuet-1-phaC, a specific PCR product (2249 bp) amplified, which was consistent with the expected size of the phaC gene. Similarly, the colonies containing pRSFDuet-1-phaC-phaA-phaB showed a clear band at approximately 4338 bp, which matched the expected size of the phaCAB gene, confirming the success of the recombinant plasmid construction. The further sequencing results confirmed we successfully obtain the correct plasmids.

Fig. 3 The colony PCR and sequencing results.

1. Growth curve test

Following the verification of plasmid sequences, the recombinant plasmids were successfully transformed into E. coli BL21(DE3). Positive clones, confirmed by colony PCR, were inoculated and cultured overnight before being subjected to IPTG induction for the expression of either phaC or the complete phaCAB operon. To assess the impact of these two genetic modules on bacterial growth, the OD₆₀₀ was monitored over a 36-hour period.

The growth curve results indicated a clear physiological difference between the two strains (Fig.4). The strain harboring the complete ​​phaCAB operon​​ exhibited a significantly faster growth rate and reached a higher final biomass (OD₆₀₀ = 5.989 at 36 hours) compared to the strain expressing only ​​phaC​​ (OD₆₀₀ = 4.805 at 36 hours). This suggested that the autonomous precursor supply enabled by the full phaCAB operon alleviated the metabolic burden on the host cells. In contrast, the phaC-only strain, which relies on the host's native metabolism to generate PHA precursors, experienced a greater growth burden, resulting in a slower growth rate and a lower maximum cell density.



Fig. 4 The Growth curve of the strain containing phaC or phaCAB

2. Protein expression

SDS-PAGE analysis confirmed the IPTG-induced expression of PHA synthesis enzymes in recombinant E. coli strains (Fig. 5). In the ​​strain (phaC)​​, a distinct band of approximately 66.1 kDa, corresponding to PhaC synthase, was observed only in the IPTG-induced lane, with negligible expression in the uninduced control. In the ​​strain (phaCAB)​​, induction with IPTG resulted in clear expression of all three pathway enzymes: PhaC (66.1 kDa), PhaA (40.6 kDa), and PhaB (26.4 kDa). Notably, ​​​​differential basal leakage expression was detected in the uninduced samples​​: PhaA showed high leakiness while PhaB demonstrated slight background expression. This variation in basal expression levels suggests differences in transcriptional regulation efficiency between these genes within the operon. 



Fig. 5 The SDS-PAGE results of protein expression in the whole bacteria.

3. PHA concentration determination

Next, we quantitatively analyzed the PHA produced by E. coli using the Nile red staining method. As shown in Fig. 6A-B, the phaCAB strain showed significantly higher PHA accumulation, reaching 2.014 mg/mL at 12 h and maintaining high levels thereafter, while the phaC strain exhibited minimal production (0.1-0.2 mg/mL). Visual observation confirmed these results, with phaCAB cultures turning distinctly pink compared to the light purple of phaC and negative controls. These findings clearly demonstrated the superior efficiency of the complete phaCAB operon over phaC alone for PHA production under these induction conditions.



Fig. 6 PHA quantitative results. (A) Nile Red staining of the bacterial cells. (B)PHA quantitative results.

Before and after induction, we performed Nile red staining on the bacterial cells and observed them using a fluorescence microscope (Fig. 7). For the phaC strain, the images showed that the group induced by IPTG only exhibited weak fluorescence, indicating that the PHA content was relatively low. For the phaCAB strain, the induced group contained a large number of bright red fluorescent spots, indicating that the PHA particles were successfully formed, which was consistent with the previous PHA quantitative results.

Fig. 7 Results of bacterial staining observed under a fluorescence microscope.

The TEM images show that in the strain expressing phaCAB, there are numerous PHA particles of various sizes (460.5 ± 213.1 nm ), while this phenomenon is absent in the negative control group (Fig. 8). These particles have the typical PHA morphology, which confirms the efficient accumulation of the polymer.

Fig. 8 The results of the PHA morphology under TEM.

1. phaP (BBa_25F3MLCY)

Name: phaP

Base Pairs: 579 bp

Origin:

Recombinant gene derived from Ralstonia eutropha, with codon optimization for high-yield expression in E. coli.

Properties:

phaP encodes a small protein called phasin, which is a structural protein that non-covalently binds to the surface of growing polyhydroxyalkanoate (PHA) granules. Phasins play a crucial role in regulating the number, size, and stability of these intracellular storage granules [2].

Usage and Biology:

phaP is primarily used in metabolic engineering to enhance PHA production. Its co-expression with the PHA synthesis enzymes (phaA, phaB, phaC) in production strains increases PHA yield and modifies granule morphology for easier extraction. The primary function of phaP is to emulsify and stabilize the hydrophobic PHA polymer within the aqueous cytoplasm. By coating the granule surface, it prevents coalescence and provides a functional interface for metabolic enzymes. Furthermore, phaP helps to mitigate the metabolic burden of polymer accumulation by regulating granule size and number, thereby maintaining cell viability during high-level PHA synthesis.

2. Maltose-binding protein (MBP) (BBa_2584689S)

Name: MBP

Base Pairs: 1101 bp

Origin:

The MBP was derived from the E. coli K12 strain and was commercially synthesized.

Properties:

MBP is a periplasmic protein that serves as a component of the bacterial maltose transport system. MBP exhibits specific affinity for maltose and longer maltodextrin polymers. Its notable characteristic is the conformational change it undergoes upon ligand binding, which facilitates the transport process [3].

Usage and Biology:

MBP is widely utilized as a fusion partner to improve the solubility and proper folding of recombinant proteins expressed in E. coli. The MBP-tagged proteins can be purified using affinity chromatography on cross-linked amylose resins. MBP functions in the bacterial ABC transport system where it binds maltose in the periplasm and delivers it to the inner membrane transport complex. The protein's ability to enhance solubility of fusion partners stems from its large, stable structure and chaperone-like properties that prevent aggregation of nascent polypeptide chains during expression.

In order to establish a PHA particle synthesis system based on the regulation of phaP hydrophobic protein, the gene encoding the hydrophobic protein ​​PhaP​​ was cloned into the pRSFDuet-1-phaC/phaCAB backbone to ensure coordinated regulation and expression balance with the synthesis genes. We also introduced the cold-shock promoter cspA or ​​blue-light-inducible module BBa_K3447133 to regulate the transcription of the phaP gene, enabling precise temporal induction of phaP expression under low-temperature conditions (Fig. 9).

Fig. 9 Plasmids diagram of the PHA synthesis system regulated by phaP.
(A) phaP induced by ​​blue-light-inducible module. (B) phaP induced by cold-shock promoter cspA.

For the incorporation of the ​​spider silk protein ADF3​, we adopted a ​​dual-plasmid co-transformation approach​​. The ADF3 gene was constructed into a compatible plasmid pBAD, allowing it to be co-transformed with the pRSFDuet-1 plasmid construct into the same E. coli host (Fig. 10). This strategy enables the assembly of a multi-modular system where PHA synthesis. To precisely control its expression, we employed an ​​L-arabinose-inducible promoter​​ to drive the transcription of the ADF3 gene.

Fig. 10 Plasmid co-transfection strategy.

For pRSFDuet-1-phaC/phaCAB-P_cspA-phaP, the phaC or phaCAB gene and the P_cspA-phaP​​ module were individually digested with XbaI and HindIII, then ligated into the pRSFDuet-1 backbone. All samples showed sharp bands at their expected molecular weights (Fig.11). After obtaining the DNA fragments, they are assembled through enzyme ligation. After transferring the recombinant product into E. coli DH5α, we conducted colony PCR identification. The further sequencing results confirmed that all extracted plasmids contained the correct sequences without mutations, indicating that we successfully obtained these recombinant plasmids (Fig. 11B-C). Finally, the recombinant plasmids were successfully transformed into E. coli BL21(DE3). Positive clones, confirmed by colony PCR, were inoculated and cultured overnight for the following test.

Fig. 11 Plasmid construction results of pRSFDuet-1-phaC/phaCAB-P_cspA-phaP.
(A) Enzyme digestion results. (B-C) Colony PCR and sequencing results.

For pRSFDuet-1-phaC/phaCAB-P_light-phaP, the agarose gel electrophoresis confirmed successful amplification of all target DNA fragments: phaP-Kan (3005 bp), Lac-phaC (3665 bp), Lac-phaCAB (5746 bp), and Plight (3191 bp) (Fig. 12A). After obtaining the DNA fragments, they are assembled through homologous recombination. After transferring the recombinant product into E. coli DH5α, we conducted colony PCR identification. The further sequencing results confirmed that all extracted plasmids contained the correct sequences without mutations, indicating that we successfully obtained these recombinant plasmids (Fig. 12B-C). Finally, the recombinant plasmids were successfully transformed into E. coli BL21(DE3). Positive clones, confirmed by colony PCR, were inoculated and cultured overnight for the following test.

Fig. 12 Plasmid construction results of pRSFDuet-1-phaC/phaCAB-P_light-phaP.
(A) DNA amplification results. (B-C) Colony PCR and sequencing results.

For the pBAD-ADF3-MBP plasmid, we first amplified the DNA fragments and the agarose gel electrophoresis results showed that all fragments were successfully amplified (Fig. 13A). After obtaining the DNA fragments, they are assembled through homologous recombination. The colony PCR and sequencing analysis to confirm the successful construction of it (Fig. 13B). The sequence-verified pBAD-ADF3-MBP plasmid was subsequently transferred into E. coli BL21(DE3). Then, it was prepared into competent cells and separately introduced with pRSFDuet-1-phaC or pRSFDuet-1-phaCAB plasmids, thereby generating two strains (Fig. 13C). The strains possess a complete system, including the PHA synthesis module and the size regulation module (P_araBAD-ADF3).

Fig. 13 Plasmid construction results of pBAD-ADF3-MBP.

(A) DNA amplification results. (B) Colony PCR and sequencing results. (C-D) Co-transformation of pBAD-ADF3-MBP and (C) pRSFDuet-1-phaC, and (D) pRSFDuet-1-phaCAB.

The results from the fluorescence microscope showed that the effects of different gene constructs and induction conditions on PHA accumulation were significant. For the regulatory effect of induction conditions: the fluorescence intensity was lower under low-temperature induction (phaCAB-P_cspA-phaP​​), which might be related to the inhibition of cell metabolism and enzyme activity by low temperature; while the blue light induction system (phaCAB-P_Light-phaP​​) showed significantly enhanced fluorescence, indicating that photogenetic control does not affect the yield of PHA (Fig. 17A-B). For the dual plasmid system (phaCAB + ADF3-MBP​​), it displayed clear fluorescence under dual induction with IPTG and arabinose, confirming the compatibility of the size regulation module (ADF3) with the core synthesis pathway, and not significantly interfering with the yield of PHA (Fig. 17C).

Fig. 17 Fluorescent microscopy detection of PHA staining.

We used TEM to observe the size and shape of PHA. The PHA particles appeared bright white due to their low electron scattering property, while the background, enriched with heavy metal dyes (sodium peroxodiphosphate, phosphotungstic acid), was dark, creating a sharp contrast. As shown in Fig. 18A-B, compared with the sole expression of phaCAB, the strains co-expressing phaP have significantly smaller particle sizes (289.9 ± 64.44 nm) and a uniform distribution, indicating that PhaP achieves size fine-tuning by inhibiting particle fusion. The bacteria induced by the cold-inducible promoter to synthesize phaP showed fewer PHA particles, which is consistent with the previous PHA content determination results compared to the blue light-induced group. The strain expressing ADF3-MBP had larger particle sizes (923.2 ± 147.3 nm), indicating that ADF3 promotes particle fusion through hydrophobic interactions, forming larger and more uniform PHA particles (Fig. 18).

Fig. 18 The results of the PHA morphology under TEM.

Reference:

[1] Cataldi P, Steiner P, Raine T, et al. Multifunctional Biocomposites Based on Polyhydroxyalkanoate and Graphene/Carbon Nanofiber Hybrids for Electrical and Thermal Applications. Acs Applied Polymer Materials, 2020, 2(8): 3525-3534.

[2] Seo M C, Shin H D, Lee Y H. Functional role of granule-associated genes, phaP and phaR, in poly-β-hydroxybutyrate biosynthesis in recombinant E-coli harboring phbCAB operon. Biotechnology Letters, 2003, 25(15): 1243-1249.

[3] Srinivasan U, Bell J A. A convenient method for affinity purification of maltose binding protein fusions. Journal of Biotechnology, 1998, 62(3): 163-167.