Application of Multiple Strategies To Debottleneck the Biosynthesis of Longifolene by Engineered Saccharomyces cerevisiae.

Longifolene as an important sesquiterpene had enormous biological benefits. However, the low productivity of longifolene relying on chemical catalysis and plant extraction limited its wide application. Herein, the longifolene biosynthetic pathway was introduced into Saccharomyces cerevisiae, and multiple genetic strategies were applied to debottleneck the synthesis of longifolene, including the regulation of the rate-limiting enzymes, the elimination of the competitive pathways, the screening of the molecular chaperone to improve synthase activity, and the enhancement of the precursor supply. After combinationally applying these optimum strategies, the production of longifolene reached 27.30 mg/L in shake flasks and 1249 mg/L in fed-batch fermentation, respectively, which was the highest yield of longifolene reported thus far. It was demonstrated that the strategies applied in our work were effective in promoting the biosynthesis of longifolene, which not only laid a significant foundation for its industrial production but also provided a platform for the synthesis of other terpenoids.

Mooring Stone-Like Arg114 Pulls Diverse Bulged Peptides: First Insight into African Swine Fever Virus-Derived T Cell Epitopes Presented by Swine Major Histocompatibility Complex Class I.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), which is a devastating pig disease threatening the global pork industry. However, currently, no commercial vaccines are available. During the pig immune response, major histocompatibility complex class I (MHC-I) molecules select viral peptide epitopes and present them to host cytotoxic T lymphocytes, thereby playing critical roles in eliminating viral infections. Here, we screened peptides derived from ASFV and determined the molecular basis of ASFV-derived peptides presented by the swine leukocyte antigen 1*0101 (SLA-1*0101). We found that peptide binding in SLA-1*0101 differs from the traditional mammalian binding patterns. Unlike the typical B and F pockets used by the common MHC-I molecule, SLA-1*0101 uses the D and F pockets as major peptide anchor pockets. Furthermore, the conformationally stable Arg114 residue located in the peptide-binding groove (PBG) was highly selective for the peptides. Arg114 draws negatively charged residues at positions P5 to P7 of the peptides, which led to multiple bulged conformations of different peptides binding to SLA-1*0101 and creating diversity for T cell receptor (TCR) docking. Thus, the solid Arg114 residue acts as a "mooring stone" and pulls the peptides into the PBG of SLA-1*0101. Notably, the T cell recognition and activation of p72-derived peptides were verified by SLA-1*0101 tetramer-based flow cytometry in peripheral blood mononuclear cells (PBMCs) of the donor pigs. These results refresh our understanding of MHC-I molecular anchor peptides and provide new insights into vaccine development for the prevention and control of ASF. IMPORTANCE The spread of African swine fever virus (ASFV) has caused enormous losses to the pork industry worldwide. Here, a series of ASFV-derived peptides were identified, which could bind to swine leukocyte antigen 1*0101 (SLA-1*0101), a prevalent SLA allele among Yorkshire pigs. The crystal structure of four ASFV-derived peptides and one foot-and-mouth disease virus (FMDV)-derived peptide complexed with SLA-1*0101 revealed an unusual peptide anchoring mode of SLA-1*0101 with D and F pockets as anchoring pockets. Negatively charged residues are preferred within the middle portion of SLA-1*0101-binding peptides. Notably, we determined an unexpected role of Arg114 of SLA-1*0101 as a "mooring stone" which pulls the peptide anchoring into the PBG in diverse "M"- or "n"-shaped conformation. Furthermore, T cells from donor pigs could activate through the recognition of ASFV-derived peptides. Our study sheds light on the uncommon presentation of ASFV peptides by swine MHC-I and benefits the development of ASF vaccines.

Diversified Anchoring Features the Peptide Presentation of DLA-88*50801: First Structural Insight into Domestic Dog MHC Class I.

Canines represent a crucial animal model for studying human diseases and organ transplantation, as well as the evolution of domestic animals. MHCs, with a central role in cellular immunity, are commonly used in the study of dog population genetics and genome evolution. However, the molecular basis for the peptide presentation of dog MHC remains largely unknown. In this study, peptide presentation by canine MHC class I DLA-88*50801 was structurally determined, revealing diversified anchoring modes of the binding peptides. Flexible and large pockets composed of both hydrophobic and hydrophilic residues can accommodate pathogen-derived peptides with diverse anchor residues, as confirmed by thermostability measurements. Furthermore, DLA-88*50801 contains an unusual α2 helix with a large coil in the TCR contact region. These results further our understanding of canine T cell immunity through peptide presentation of MHC class I and shed light on the molecular basis for vaccine development for canine infectious diseases, for example, canine distemper virus.