In this cycle, we utilized PHA synthesis genes derived from Ralstonia eutropha, which exhibit high compatibility with heterologous hosts such as E. coli (Table 1).

Table 1 The PHA synthesis-related genes used in this project.

Heterologous expression of phaC in E. coli enables the polymerization of short-chain-length (C3–C5) hydroxyacyl-CoA substrates into biodegradable polyesters [2]. However, although PhaC alone is capable of polymerizing PHA precursors, its activity is limited by the host’s innate ability to supply the essential monomer, (R)-3-hydroxyacyl-CoA, resulting in a substantial metabolic burden on the host cells. To address this limitation, we evaluated the ​​complete gene cluster—phaC, phaA, and phaB​​—which facilitates autonomous precursor generation and polymerization at the same time. This approach was employed to assess whether it mitigates host metabolic burden and enhances PHA yield.

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 construction strategy 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. The principle of this method is that Nile red specifically binds to intracellular PHA granules, and the fluorescence intensity is proportional to the PHA concentration. To obtain accurate quantitative results, we first prepared a PHA standard curve. The results showed a strong linear relationship between RFU and PHA concentration (y = 190.45x - 12.606) with an excellent coefficient of determination (R² = 0.9959) (Fig. 6A). This indicated that the standard curve was highly reliable and suitable for accurate quantification of PHA in subsequent experimental samples.

As shown in Fig. 6B-C, 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) PHA standard curve. (B) Nile Red staining of the bacterial cells. (C) PHA quantitative results.

4. PHA characterization

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 showed 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.

The function of the phaC gene alone is constrained by the host's inherent capacity to supply the required monomer, (R)-3-hydroxyacyl-CoA. In contrast, the complete phaCAB gene cluster provides a self-sufficient pathway: ​​PhaA​​ (β-ketothiolase) condenses acetyl-CoA to form acetoacetyl-CoA, and ​​PhaB​​ (acetoacetyl-CoA reductase) subsequently reduces it to the monomer. This enables efficient, autonomous PHA synthesis directly from central metabolites like acetyl-CoA. This comprehensive system significantly reduces the host's metabolic burden.

Fluorescence microscopy and TEM analyses confirmed that the engineered strain harboring the ​​complete phaCAB operon​​ produced ​​a substantial amount of intracellular PHA granules​​. However, the resulting granules exhibited ​​significant heterogeneity in size and morphology​​, which is undesirable for industrial applications requiring uniformity and reproducibility.

To address this limitation, we proceeded to the next DBTL cycles​​ by introducing a ​​hydrophobin-based size regulation module​​. This module aims to achieve precise control over granule assembly, enabling the production of ​​highly uniform and tunable PHA particles​​ suitable for scalable manufacturing and specific industrial requirements.

In this module, we integrated a size-regulation system into the PHA synthesis framework using PhaP (GenBank: CAJ92517.1), a native PHA granule-associated protein, modulates granule size via surface charge-mediated dispersion [2]. This strategy was designed to control the size of PHA particles, which is of importance for applications that require the fabrication of uniform microspheres. Furthermore, we introduced the cold-shock promoter cspA to regulate the transcription of the phaP gene, enabling precise temporal induction of phaP expression under low-temperature conditions (about 15°C).

To investigate temperature-responsive size control of PHA granules, we constructed two recombinant plasmids: ​​pRSFDuet-1-phaC-P_cspA-phaP (control group) and ​​pRSFDuet-1-phaCAB-P_cspA-phaP​​ (experimental group). Two recombinant plasmids were constructed via restriction enzyme-based ligation. The PHA synthesis module (phaC or phaCAB) and the phaP module (P_cspA-phaP​​) were individually digested with XbaI and HindIII, then ligated into the pRSFDuet-1 backbone, yielding two recombinant plasmids, respectively (Fig. 9).

Fig. 9 Plasmids construction strategy of (A) pRSFDuet-1-P_cspA-phaC and (B) pRSFDuet-1-P_cspA-phaCAB.

Restriction digestion analysis confirmed the successful digestion of all plasmids (Fig. 10). For the control plasmid, digestion of the lac-phaC fragment yielded a band of 3324 bp, while the phaP-kan module produced a 3049 bp fragment. For the phaCAB operon-containing plasmid, the lac-phaCAB fragment showed the expected larger size of 5413 bp, with the phaP-kan fragment maintaining consistency at 3019 bp. All fragments matched their predicted sizes.

Fig. 10 The agarose gel electrophoresis of plasmid digestion.

Colony PCR and sequencing analysis successfully validated both plasmid constructs. The pRSFDuet-1-phaC-P_cspA-phaP showed the expected 3017 bp band, while pRSFDuet-1-phaCAB-P_cspA-phaP displayed the correct 4338 bp fragment (Fig. 11). DNA sequencing confirmed accurate insertion of all genetic elements with no mutations, verifying proper construction of both recombinant plasmids for subsequent functional studies.

Fig. 11 The colony PCR and sequencing results.

1. Growth curve test

The verified plasmids were transformed into E. coli BL21(DE3), and positive clones were confirmed by colony PCR. Following inoculation and culture expansion, IPTG induction was applied for 12 h to initiate PhaC or PhaCAB expression. The medium was then replaced with inducer-free fresh medium, and the temperature was shifted to 15 °C to induce PhaP expression from 12 to 36 h. Growth curves revealed that the ​​strain carrying the phaCAB operon​​ exhibited significantly better growth than the ​​strain with phaC alone​​, reaching a final OD₆₀₀ of 2.070 compared to 2.668 at 36 h (Fig. 12). This suggested that the complete operon alleviated metabolic burden through autonomous precursor supply. However, ​​both strains showed growth limitation after the temperature downshift to 15 °C​​, indicating that low-temperature induction imposed a general stress on cellular metabolism, despite the functional advantage of the phaCAB system.



Fig. 12 The Growth curve of the strain.

2. Protein expression

The SDS-PAGE results clearly indicated that both constructs successfully expressed target proteins upon dual induction (IPTG and low temperature). In the phaC-P_cspA-phaP strain (left), induced sample shows a slight PhaC expression (~66 kDa). However, compared to phaC-P_cspA-phaP strain, it showed a significant reduction in PhaC expression. In the phaCAB-P_cspA-phaP strain (right), induced sample shows strong expression of PhaB (~26 kDa) and PhaP (~20 kDa), but significantly diminished PhaA (~40 kDa) (Fig. 13). We hypothesized that low temperature induction may impair translation efficiency of certain proteins like PhaA, potentially due to codon usage bias or mRNA stability issues under cold stress. Additionally, metabolic burden from simultaneous expression might also trigger partial protein degradation.



Fig. 13 The SDS-PAGE results of protein expression in the whole bacteria.

3. PHA concentration determination

Quantification of PHA production revealed significantly different yields between the two strains (Fig. 14). The ​​phaCAB-P_cspA-phaP​​ strain consistently demonstrated higher PHA accumulation, reaching 0.987 mg/mL at 36 hours. In contrast, the ​​phaC-P_cspA-phaP​​ strain showed markedly lower production, peaking at only 0.240 mg/mL. However, after low-temperature induction, the accumulation amount of PHA in the bacterial cells was significantly less than that in the cells without P_cspA-phaP (phaCAB: 1.925 mg/mL).



Fig. 14 PHA quantitative results. (A) Nile Red staining of the bacterial cells. (B) PHA quantitative results.

4. PHA characterization

For the ​​phaCAB-P_cspA-phaP​​​​ strain, the negative control group showed minimal fluorescence, while the sample induced by IPTG and low temperature exhibited slightly bright red fluorescent spots. This indicated that a small amount of PHA particles were formed under the induction conditions, which is consistent with the previous quantitative results (Fig. 15).



Fig. 15 Results of bacterial staining observed under a fluorescence microscope.

TEM images showed that compared with the strain expressing phaCAB, the strain co-expressing phaCAB and phaP has significantly smaller, more uniform-sized and more dispersed particles (Figure 16). This morphological change confirmed that PhaP can effectively regulate the size and organization of PHA particles, which is consistent with its role as a particle size regulator. However, it is possible that due to the use of a cold-induced cspA promoter for PhaP protein expression after PHA accumulation, the number of intracellular PHA particles decreased significantly. 



Fig. 16 The results of the PHA morphology under TEM.

Compared with the control group that was not induced by cold, the group that used the cold-induced cspA promoter to regulate phaP significantly inhibited cell growth and reduced the production of PHA. This might be due to the fact that the low-temperature induction (at 15°C) disrupted metabolic activities, restricted biomass formation, and reduced the efficiency of PHA biosynthesis. This suggested that the cold-induction promoter may not be suitable for the accumulation of PHA.

To address the limitations of cold-inducible systems, we adopted a ​​blue-light-inducible module​​ BBa_K3447133 (from iGEM20_Jilin_China) for precise regulation of phaP transcription. The phaP gene was inserted downstream of this optogenetic system, which activates phaP expression ​​only under blue light illumination​​ while remaining repressed in darkness. This strategy aims to achieve tighter control of granule size modulation while avoiding the metabolic drawbacks associated with low-temperature induction.

As illustrated in Fig. 17, the PHA synthesis module (phaC or phaCAB operon), the blue-light induction module (BBa_K3447133), and the phaP gene were individually amplified by PCR. These fragments were subsequently assembled into the pRSFDuet-1 vector via homologous recombination, yielding the final plasmids: pRSFDuet-1-phaC-P_light-phaP and pRSFDuet-1-phaCAB-P_light-phaP.

Fig. 17 Plasmids construction strategy of (A) pRSFDuet-1-phaC-P_light-phaP and (B) pRSFDuet-1-phaCAB-P_light-phaP.

The PCR amplification of the fragments was successfully verified by agarose gel electrophoresis (Fig. 18). Four distinct bands were observed at 3005 bp, 3665 bp, 5746 bp, and 3191 bp, corresponding to the expected sizes of the amplification products. The clear band separation and correct molecular weights confirmed the specific amplification of the target fragment, providing the essential component for subsequent plasmid construction.

Fig. 18 The agarose gel electrophoresis of DNA amplification

Colony PCR analysis confirmed the correct construction of both recombinant plasmids. For pRSFDuet-1-phaC-P_light-phaP, a specific band of 2347 bp was amplified, while pRSFDuet-1-phaCAB-P_light-phaP showed the expected 4569 bp fragment. Furthermore, the sequencing results showed that the plasmids were correct (Fig. 19). 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 for the following test.

Fig. 19 The colony PCR and sequencing results.

1. Growth curve test

The growth kinetics of the two recombinant strains were monitored 36 hours. The phaCAB-P_light-phaP strain exhibited significantly better growth, reaching a final OD₆₀₀ of 6.528, compared to 5.611 for the phaC-P_light-phaP strain (Fig. 20). This substantial difference in biomass accumulation demonstrated that the complete phaCAB operon provided a clear growth advantage under the dual-induction conditions (IPTG and blue light).



Fig. 20 The Growth curve of the strains.

2. Protein expression

Protein expression result was analyzed by SDS-PAGE. For both constructs, induced samples (lane 2) showed clear expression of target proteins: PhaC (66.1 kDa) was present in both, while additional bands corresponding to PhaA (40.6 kDa) and PhaB (26.4 kDa) were specifically detected in the phaCAB-containing strain. Uninduced controls (lane 1) showed minimal background expression (Fig. 21).



Fig. 21 The SDS-PAGE results of protein expression in the whole bacteria.

3. PHA concentration determination

PHA production was quantified over 36 hours of induction. The phaCAB-P_light-phaP strain produced significantly higher PHA yields, reaching 1.568 mg/mL, compared to 0.430 mg/mL for the phaC-P_light-phaP strain. The visual pink coloration in the experimental tubes correlated with the quantitative data, demonstrating the superior performance of the complete phaCAB operon in PHA biosynthesis under optogenetic control (Fig. 22).



Fig. 22 PHA quantitative results. (A) Nile Red staining of the bacterial cells. (B) PHA quantitative results.

4. PHA characterization

For the ​​phaCAB-P_light-phaP​​ strain, the negative control exhibited minimal fluorescence, while the sample induced by ​​IPTG and blue light​​ showed abundant bright red fluorescent spots (Fig. 23). This indicates successful formation of PHA granules under optogenetic induction, consistent with quantitative yield measurements. 



Fig. 23 Results of bacterial staining observed under a fluorescence microscope.

The transmission electron microscope images showed that under blue light induction, the strain expressing both phaCAB and phaP exhibits significantly higher density of PHA particles than the strain induced by cold-shock promoter. These particles are evenly distributed throughout the cytoplasm and have a diameter of 289.9 ± 64.44 nm (Fig. 24). The results indicate that blue light induction effectively activates the synthesis of PhaP and promotes the formation of a greater number and more evenly distributed particles.



Fig. 24 The results of the PHA morphology under TEM.

The implementation of the ​​blue-light-inducible module​​ for regulating phaP transcription demonstrated significant advantages over the cold-inducible cspA promoter system. Strains subjected to ​​blue-light induction​​ maintained ​​normal growth profiles​​ without the metabolic burden associated with low-temperature stress, enabling unimpaired cell proliferation. Crucially, ​​PHA accumulation remained robust​​, with quantitative analyses revealing ​​markedly higher PHA yields​​ compared to cold-induced cultures. Fluorescence microscopy and TEM imaging confirmed the presence of abundant, well-formed PHA granules in the blue-light-induced strains, indicating efficient phaP-mediated granule assembly under optogenetic control. The blue-light module presents a more suitable and efficient regulatory tool for scalable PHA biomanufacturing.

Furthermore, we found that after introducing the PhaP protein, the size of PHA particles would decrease and become more uniform. This is mainly due to its dual mechanism of action. The PhaP molecules would bind to the hydrophobic surface of the newly formed PHA core, effectively reducing the interfacial tension and preventing small particles from merging into larger irregular clumps. At the same time, on the particle surface, PhaP produced physical and electrostatic three-dimensional hindering effects, which could limit the occurrence of disordered fusion and promote the formation of uniformly sized independent particles [3]. Therefore, PhaP can facilitate the formation of a large number of small and uniform particles, rather than fewer but larger aggregates.

To achieve a more diverse range of PHA particle sizes, we introduced an additional hydrophobin module: the spider silk protein ​​ADF3 (GenBank: AAC47010.1)​, sourced from Araneus diadematus. The ​​β-sheet domain of ADF3​​ specifically adsorbs onto the surface of PHA granules through hydrophobic interactions, effectively regulating the degree of interparticle aggregation [4]. To precisely control its expression, we employed an ​​L-arabinose-inducible promoter​​ to drive the transcription of the ADF3 gene. This strategic combination allows for tunable modulation of PHA granule size and providing a broader spectrum of material properties for downstream applications.

The spider silk protein ADF3 exhibits ​​significantly stronger hydrophobicity​​ than the PhaP, enabling it to ​​promote robust intergranular aggregation​​ of PHA particles. To overcome the high risk of ​​insoluble inclusion body formation​​ during heterologous expression in E. coli, we fused ADF3 with the ​​maltose-binding protein (MBP)​​ tag, a highly effective solubility enhancer [5]. This ​​ADF3-MBP fusion​​ was then cloned into the ​​pBAD vector​​ downstream of the ​​araBAD promoter​​, allowing precise, arabinose-inducible expression. The final construct was assembled via ​​homologous recombination​​, ensuring seamless integration and controlled expression (Fig. 25).

Fig. 25 Plasmids construction strategy of pBAD-ADF3-MBP

As shown in Fig. 26, agarose gel electrophoresis confirmed the successful amplification of the target DNA fragments. Both the ​​ADF3-MBP​​ insert and the ​​pBAD​​ vector backbone exhibited clear bands at approximately ​​3061 bp and 4053 bp​​, matching the expected length.

Fig. 26 The agarose gel electrophoresis of DNA amplification

Colony PCR and sequencing analysis (Fig. 27) confirmed the successful construction of the pBAD-ADF3-MBP plasmid. A specific band of approximately 3286 bp was amplified, corresponding to the expected size of the ADF3-MBP fusion fragment. DNA sequencing further validated the correct insertion of all genetic elements, confirming the proper assembly of the recombinant plasmid.

Fig. 27 The colony PCR and sequencing results.

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. The strains possess a complete system, including the PHA synthesis module and the size regulation module (P_araBAD-ADF3).

Fig. 28 Plasmid co-transfection strategy.

Colony PCR analysis confirmed the successful co-transformation of both plasmid combinations. For the pBAD-ADF3-MBP and pRSFDuet-1-phaC combination, specific bands corresponding to ADF3-MBP (3286 bp) and phaC (2249 bp) were detected. Similarly, the pBAD-ADF3-MBP and pRSFDuet-1-phaCAB combination showed clear bands for ADF3-MBP (3286 bp) and phaCAB (4338 bp). All bands matched the expected sizes, validating the simultaneous presence of both plasmids in the engineered strains (Fig. 29).

Fig. 29 The colony PCR and sequencing results.

1. Growth curve test

​​ The growth curves of the two recombinant strains were monitored over 36 hours (Fig. 30). The ​​phaCAB + ADF3-MBP​​ strain demonstrated significantly better growth, reaching a final OD₆₀₀ of 4.432, compared to 3.436 for the ​​phaC + ADF3-MBP​​ strain. This consistent superiority at all time points indicates that the complete phaCAB operon provided a growth advantage, even when co-expressed with the ADF3-MBP fusion protein.



Fig. 30 The Growth curve of the strains.

2. Protein expression

SDS-PAGE analysis confirmed successful protein expression under dual induction (IPTG + L-arabinose). In the ​​phaCAB + ADF3-MBP​​ system, induced samples (lane 2) showed clear bands for ADF3-MBP (100 kDa), PhaC (66.1 kDa), PhaA (40.6 kDa), and PhaB (26.4 kDa). The ​​phaC + ADF3-MBP​​ system exhibited bands only for ADF3-MBP and PhaC (Fig. 31).



Fig. 31 The SDS-PAGE results of protein expression in the whole bacteria.

3. PHA concentration determination

​​ Quantitative analysis of PHA production demonstrated significantly higher yields from the ​​phaCAB + ADF3-MBP​​ strain compared to ​​phaC + ADF3-MBP​​. The complete operon system achieved a PHA concentration of 1.875 mg/mL at 36 hours, representing a 2.3-fold increase over the PhaC strain (0.801 mg/mL). This substantial enhancement, visually confirmed by more intense pink coloration (Fig. 32), validated the superior efficiency of the autonomous phaCAB pathway for PHA biosynthesis even when co-expressed with the ADF3 size-regulation module.



Fig. 32 PHA quantitative results. (A) Nile Red staining of the bacterial cells. (B) PHA quantitative results.

4. PHA characterization

For the ​​phaCAB + ADF3-MBP​​ strain, the negative control showed minimal fluorescence, while the sample induced by ​​IPTG and L-Arabinose​​ exhibited abundant bright red fluorescent spots (Fig. 33). This indicates successful formation of PHA granules under dual induction conditions, confirming the synergistic effect of ADF3-MBP fusion protein on PHA accumulation. 



Fig. 33 Results of bacterial staining observed under a fluorescence microscope.

TEM images revealed distinct granule morphologies between the two regulatory systems. Strain expressing ​​PhaP​​ contained numerous small, uniformly dispersed granules(289.9 ± 64.44 nm). In contrast, ​​ADF3-MBP​​ expressing strain exhibited significantly larger and more densely packed granules (923.2 ± 147.3 nm) (Fig. 34). This size difference demonstrates ADF3's superior capability in promoting PHA granule fusion compared to the PhaP.



Fig. 34 The results of the PHA morphology under TEM.

The ​​spider silk protein ADF3​​ promotes the formation of ​​larger and more uniform PHA granules​​ through its ​​amphiphilic molecular structure​​. The ​​hydrophobic β-sheet domains​​ of ADF3 strongly adsorb onto the hydrophobic surface of nascent PHA granules via hydrophobic interactions, effectively ​​reducing interfacial tension​​ and facilitating fusion of smaller granules [4]. Simultaneously, the ​​exposed hydrophilic regions​​ provide steric hindrance and electrostatic repulsion, preventing excessive aggregation and ensuring uniform size distribution. This dual mechanism—​​hydrophobic-driven assembly​​ and ​​charge-mediated stabilization​​—enables precise control over granule growth, resulting in a homogeneous population of enlarged PHA particles ideal for industrial applications.