Engineering

Introduction

  In petroleum, residual naphthalene poses potential environmental risks due to its persistence and toxicity. To address this issue, our research team initiated a project focused on identifying a gene capable of degrading naphthalene, aiming to develop an efficient biological degradation solution.

  After an extensive review of relevant literature, databases, and prior research findings, we identified a potential candidate: the nahC gene, which is widely reported to be associated with naphthalene degradation pathways in certain microorganisms. To further study its function, we planned to clone this gene into the pET-30a vector—a commonly used expression vector in prokaryotic systems, known for its high copy number and compatibility with Escherichia coli (E. coli) hosts. Despite dedicated efforts over several days, including optimizing ligation conditions, adjusting transformation protocols (such as heat shock time and temperature), and verifying the integrity of the recombinant vector via gel electrophoresis, we encountered repeated failures in successfully transforming the nahC-containing pET-30a into E. coli. This setback prompted us to reassess our strategy and explore alternative approaches.

  We then shifted our focus to a novel concept: instead of relying on intracellular expression of the naphthalene-degrading enzyme, could we engineer the enzyme to be expressed on the cell membrane surface of E. coli? This design would allow the enzyme to directly interact with naphthalene in the extracellular environment, potentially enhancing degradation efficiency by eliminating the need for substrate uptake into the cell. To achieve membrane surface expression, we researched and identified inteins—short peptide segments with unique self-splicing and protein ligation capabilities, which can covalently link target proteins to membrane-anchored proteins. Theoretically, fusing the naphthalene-degrading enzyme gene with an intein gene and a membrane anchor gene could enable the enzyme to be displayed on the E. coli cell surface. However, this approach was ultimately abandoned due to two critical challenges: first, the lack of detailed structural information about the naphthalene-degrading enzyme, which made it impossible to predict optimal fusion sites; second, the high complexity of protein conformation—even small changes in the enzyme’s amino acid sequence (from fusion with inteins) could disrupt its active site, rendering it non-functional.

  Given the difficulties with direct enzyme expression, we decided to first optimize the separation and bioavailability of naphthalene and phenanthrene (another common polycyclic aromatic hydrocarbon, PAH, in petroleum). Through further research, we discovered that rhlAB—a gene cluster encoding enzymes responsible for synthesizing rhamnolipids—could serve as a key auxiliary component. Rhamnolipids are biosurfactants that can reduce the surface tension of aqueous solutions, enhance the solubility and desorption of hydrophobic PAHs (like naphthalene) from environmental matrices (e.g., soil or oil), and thus improve their bioavailability for microbial degradation.

  To harness the function of rhlAB, we cloned this gene cluster into the pET-28a vector (a sister vector to pET-30a, featuring a kanamycin resistance gene and a His-tag for protein purification) and used the T7 promoter—a strong, inducible promoter that drives high-level gene expression in E. coli strains containing the T7 RNA polymerase gene (e.g., BL21(DE3)). After optimizing induction conditions (including IPTG concentration and induction time), we successfully achieved high-level expression of rhlAB in E. coli, confirmed by detecting rhamnolipid production via thin-layer chromatography (TLC) and measuring its surfactant activity.

  Next, we sought to expand our PAH degradation capacity to phenanthrene, a more complex PAH than naphthalene. We selected the phnC gene from the phn gene cluster (phn cluster), which is documented to play a crucial role in the upstream steps of phenanthrene degradation. Theoretically, PhnC (the protein encoded by phnC) acts as a transporter or enzyme that initiates the conversion of phenanthrene into catechol—a central intermediate in many aromatic compound degradation pathways, which can be further metabolized by E. coli to produce energy and biomass.

  We cloned the phnC gene into a suitable expression vector, transformed it into E. coli, and verified the successful construction of the phnC-engineered strain via PCR and DNA sequencing. To maximize PAH degradation efficiency, we then combined this phnC-containing strain with the previously constructed rhlAB-expressing strain in a co-culture system. In this system, the rhlAB strain produces rhamnolipids to enhance the desorption and solubility of naphthalene and phenanthrene, while the phnC strain converts phenanthrene into catechol for subsequent degradation.

  To evaluate the performance of this co-culture system, we conducted soil-based degradation experiments. Soil samples contaminated with naphthalene and phenanthrene were treated with the co-culture, and after a set incubation period, we used a UV-visible spectrophotometer to measure the residual PAH concentrations (based on the characteristic absorption peaks of naphthalene and phenanthrene in the UV range). The results clearly showed a significant reduction in the total PAH content in the treated soil compared to the untreated control group, confirming the effectiveness of our engineered strain co-culture in degrading petroleum-derived PAHs. This research provides a promising biological strategy for the remediation of PAH-contaminated environments.