Cell surface display of recombinant proteins has become a powerful tool for biotechnology and biomedical applications. As a model eukaryotic microorganism, Saccharomyces cerevisiae is an ideal candidate for surface display of heterologous proteins. However, the frequently used commercial yeast surface display system, the a-agglutinin anchor system, often results in low display efficiency. We initially reconstructed the a-agglutinin system by replacing two anchor proteins with one anchor protein. By directly fusing the target protein to the N-terminus of Aga1p and inserting a flexible linker, the display efficiency almost doubled, and the activity of reporter protein α-galactosidase increased by 39%. We also developed new surface display systems. Six glycosylphosphatidylinositol (GPI) anchored cell wall proteins were selected to construct the display systems. Among them, Dan4p and Sed1p showed higher display efficiency than the a-agglutinin anchor system. Linkers were also inserted to eliminate the effects of GPI fusion on the activity of the target protein. We further used the newly developed Aga1p, Dan4p systems and Sed1p system to display exoglucanase and a relatively large protein β-glucosidase, and found that Aga1p and Dan4p were more suitable for immobilizing large proteins. Our study developed novel efficient yeast surface display systems, that will be attractive tools for biotechnological and biomedical applications

Bacillus amyloliquefaciens NCPSJ7 showed potential fungicidal activities for the effective control of fungal infection. From the PCR test, the key genes (srfAA, sfp, fenD, bmyB, ituD, and ituC) were detected in B. amyloliquefaciens NCPSJ7. These genes were closely related to the lipopeptides (LPs) synthesis. Next, three LPs families were identified with liquid chromatography–mass spectrometry (LC/MS), including iturin A, fengycin A, and surfactin. After purification with C18, the main active antifungal compound was proven to be C14-iturin A by ESI-HRMS, which has significant activities against fungi. These results proved that C14-iturin A played an important role in inhibiting the growth of fungi for B. amyloliquefaciens NCPSJ7. Furthermore, the isolated LP could inhibit mycelial growth and conidia germination at 30 μg/mL. SEM allowed us to observe that mycelial morphology and conidia germination were also affected. The mycelial ultrastructure TEM observations showed that the external electron-dense outer layer cell wall, which mainly consisted of glycoproteins, was affected. Furthermore, swollen mitochondria, enriched glycogen, and increased vacuoles were also found. LP also affected the intact wall and membranes, leading to their increased permeability, which was proved by propidium iodide (PI) staining and conductivity measurements. Meanwhile, the ergosterol, which has an affinity for iturin A, also increased. These results indicated that LP caused fungal dysfunction and membrane permeability increase, leading to fungal inhibition. Identifying and studying LPs is important in exploring the fungicidal activities of B. amyloliquefaciens, which promotes the use of B. amyloliquefaciens NCPSJ7 as a potential candidate for biocontrol.

Cellulase expression via extracellular secretion or surface display in Saccharomyces cerevisiae is one of the most frequently used strategies for a consolidated bioprocess (CBP) of cellulosic ethanol production. However, the inefficiency of the yeast secretory pathway often results in low production of heterologous proteins, which largely limits cellulase secretion or display. In this study, the components of the vesicle trafficking from the endoplasmic reticulum (ER) to the Golgi and from the Golgi to the plasma membrane, involved in vesicle budding, tethering and fusion, were over-expressed in Clostridium thermocellum endoglucanase (CelA)- and Sacchromycopsis fibuligera β-glucosidase (BGL1)-secreting or -displaying strains. Engineering the targeted components in the ER to Golgi vesicle trafficking, including Sec12p, Sec13p, Erv25p and Bos1p, enhanced the extracellular activity of CelA. However, only Sec13p over-expression increased BGL1 secretion. By contrast, over-expression of the components in the Golgi to plasma membrane vesicle trafficking, including Sso1p, Snc2p, Sec1p, Exo70p, Ypt32p and Sec4p, showed better performance in increasing BGL1 secretion compared to CelA secretion, and the over-expression of these components all increased BGL1 extracellular activity. These results revealed that various cellulases showed different limitations in protein transport, and engineering vesicle trafficking has protein-specific effects. Importantly, we found that engineering the above vesicle trafficking components, particularly from the ER to the Golgi, also improved the display efficiency of CelA and BGL1 when a-agglutinin was used as surface display system. Further analyses illustrated that the display efficiency of a-agglutinin was increased by engineering vesicle trafficking, and the trend was consistent with displayed CelA and BGL1. These results indicated that fusion with a-agglutinin may affect the proteins’ properties and alter the rate-limiting step in the vesicle trafficking. We have demonstrated, for the first time, engineering vesicle trafficking from the ER to the Golgi and from the Golgi to the plasma membrane can enhance the protein display efficiency. We also found that different heterologous proteins had specific limitations in vesicle trafficking pathway and that engineering the vesicle trafficking resulted in a protein-specific effect. These results provide a new strategy to improve the extracellular secretion and surface display of cellulases in S. cerevisiae.

Mulberry leaf tea (MT) is a popular Chinese food with nutrition and medicinal functions. Solid-state fermentation with Eurotium cristatum of MT (FMT) can improve their quality. Differences in chromaticity, taste properties, and flavor characteristics were analyzed to evaluate the improvements of the sensory quality of FMT. After fermentation, the color of the tea infusion changed. The E-tongue evaluation results showed a significant decrease in unpleasant taste properties such as sourness, bitterness, astringency, and aftertaste-bitterness, while umami and saltiness taste properties were enhanced post-fermentation. Aroma-active compounds in MT and FMT were identified and characterized. A total of 25 key aroma-active compounds were screened in MT, and 2-pentylfuran showed the highest relative odor activity value (ROAV). A total of 26 key aroma-active compounds were identified in FMT, and the newly formed compound 1-octen-3-one showed the highest ROAV, which contributed to FMT’s unique mushroom, herbal, and earthy flavor attributes. 1-octen-3-one, (E)-2-nonenal, trimethyl-pyrazine, 2-pentylfuran, and heptanal were screened as the potential markers that contributed to flavor differences between MT and FMT. E. cristatum fermentation significantly altered the sensory properties and flavor compounds of MT. This study provides valuable insights into the sensory qualities of MT and FMT, offering a theoretical basis for the development of FMT products.

Myrcene is a high-value monoterpene extensively applied in the fragrance, flavor, and agricultural industries, yet its efficient microbial production remains challenging due to pathway competition and limited metabolic flux. Compartmentalization offers a unique strategy to spatially organize heterologous metabolic pathways in Saccharomyces cerevisiae, enabling improved pathway efficiency through physical separation from competing cytosolic metabolism. In this study, we engineered the S. cerevisiae nucleus as a synthetic metabolic compartment for myrcene biosynthesis. Screening of myrcene synthases identified two highly active enzymes from Snapdragon Oc15 and Picea abies that function efficiently in S. cerevisiae. Myrcene production was detected only when myrcene synthase and the engineered GPP synthase mERG20p were co-localized to the nucleus, whereas cytosolic expression failed to yield detectable myrcene under their co-expression. Reconstruction of the complete mevalonate (MVA) pathway in the nucleus further increased myrcene titers. By identifying and optimizing rate-limiting steps, we substantially enhanced metabolic flux toward myrcene, achieving a final titer of 23.4 mg L⁻¹ in flask-shaking fermentation. This work demonstrates the feasibility of repurposing the yeast nucleus for myrcene efficient biosynthesis and provide a new strategy for further improving microbial production of myrcene.