The process of recycling poly(ethylene terephthalate) (PET) remains a major challenge due to the enzymatic degradation of high-crystallinity PET (hcPET). Recently, a bacterial PET-degrading enzyme, PETase, was found to have the ability to degrade the hcPET, but with low enzymatic activity. Here we present an engineered whole-cell biocatalyst to simulate both the adsorption and degradation steps in the enzymatic degradation process of PETase to achieve the efficient degradation of hcPET. Our data shows that the adhesive unit hydrophobin and degradation unit PETase are functionally displayed on the surface of yeast cells. The turnover rate of the whole-cell biocatalyst toward hcPET (crystallinity of 45%) dramatically increases approximately 328.8-fold compared with that of purified PETase at 30 °C. In addition, molecular dynamics simulations explain how the enhanced adhesion can promote the enzymatic degradation of PET. This study demonstrates engineering the whole-cell catalyst is an efficient strategy for biodegradation of PET.

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP5_1–486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP5_1–486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function. Significance Guanylate-binding proteins (GBPs) belong to interferon-inducible GTPases and mediate a broad spectrum of innate immune responses against various pathogens. Their protective functions require oligomerization induced by nucleotide binding and/or catalysis, but the actual molecular mechanisms are still elusive. Here, we report the crystal structures of human GBP5 (hGBP5) in both its nucleotide-free state and nucleotide-bound state, as well as nucleotide-free human GBP2 (hGBP2). We show that hGBP5 forms a closed face-to-face dimer upon GTP loading. This closed conformation is crucial to its anti–HIV-1 activity. Furthermore, with hGBP2 structure and SAXS validation, we propose a plausible working model for GTP-induced assembly of GBPs. Our findings lay the foundation to better understand the molecular mechanisms of GBPs and their immune functions.

Significance: Mosquito-borne diseases cause more than 700,000 deaths annually. Wolbachia pipientis , an intracellular bacterium infecting many insect species, can manipulate host reproduction through cytoplasmic incompatibility (CI) and is being used in several promising strategies for controlling mosquito populations and reducing the spread of mosquito-vectored viruses. Although the key factors involved in CI (called CI factors or Cifs) have been identified, the molecular mechanisms of CI induction and rescue remain elusive. We present comprehensive structural, biochemical, and functional studies on two major types of CI systems and demonstrate the importance of Cif complex formation for rescuing CI. Our findings lay the foundation for understanding the molecular mechanisms of CI and the engineering of Cifs to control agricultural pests and arthropod-borne human pathogens.

Cytoplasmic incompatibility (CI) results when Wolbachia bacteria-infected male insects mate with uninfected females, leading to embryonic lethality. “Rescue” of viability occurs if the female harbors the same Wolbachia strain. CI is caused by linked pairs of Wolbachia genes called CI factors (CifA and CifB). The co-evolution of CifA-CifB pairs may account in part for the incompatibility patterns documented in insects infected with different Wolbachia strains, but the molecular mechanisms remain elusive. Here, we use X-ray crystallography and AlphaFold to analyze the CI factors from Wolbachia strain wMel called CidA^wMel and CidB^wMel. Substituting CidA^wMel interface residues with those from CidA^wPip (from strain wPip) enables the mutant protein to bind CidB^wPip and rescue CidB^wPip-induced yeast growth defects, supporting the importance of CifA-CifB interaction in CI rescue. Sequence divergence in CidA^wPip and CidB^wPip proteins affects their pairwise interactions, which may help explain the complex incompatibility patterns of mosquitoes infected with different wPip strains.