Promoters play an essential role in the regulation of gene expression for fine-tuning genetic circuits and metabolic pathways in Saccharomyces cerevisiae (S. cerevisiae). However, native promoters in S. cerevisiae have several limitations which hinder their applications in metabolic engineering. These limitations include an inadequate number of well-characterized promoters, poor dynamic range, and insufficient orthogonality to endogenous regulations. Therefore, it is necessary to perform promoter engineering to create synthetic promoters with better properties. Here, we review recent advances related to promoter architecture, promoter engineering and synthetic promoter applications in S. cerevisiae. We also provide a perspective of future directions in this field with an emphasis on the recent advances of machine learning based promoter designs.

for the timely accumulation of anthocyanins in sweetpotato leaves. These results may also provide clues for similar studies of juvenile red fading in other plant species.

Background Saccharomyces cerevisiae is an important synthetic biology chassis for microbial production of valuable molecules. Promoter engineering has been frequently applied to generate more synthetic promoters with a variety of defined characteristics in order to achieve a well-regulated genetic network for high production efficiency. Galactose-inducible (GAL) expression systems, composed of GAL promoters and multiple GAL regulators, have been widely used for protein overexpression and pathway construction in S. cerevisiae. However, the function of each element in synthetic promoters and how they interact with GAL regulators are not well known. Results Here, a library of synthetic GAL promoters demonstrate that upstream activating sequences (UASs) and core promoters have a synergistic relationship that determines the performance of each promoter under different carbon sources. We found that the strengths of synthetic GAL promoters could be fine-tuned by manipulating the sequence, number, and substitution of UASs. Core promoter replacement generated synthetic promoters with a twofold strength improvement compared with the GAL1 promoter under multiple different carbon sources in a strain with GAL1 and GAL80 engineering. These results represent an expansion of the classic GAL expression system with an increased dynamic range and a good tolerance of different carbon sources. Conclusions In this study, the effect of each element on synthetic GAL promoters has been evaluated and a series of well-controlled synthetic promoters are constructed. By studying the interaction of synthetic promoters and GAL regulators, synthetic promoters with an increased dynamic range under different carbon sources are created.

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Saccharomyces cerevisiae has been widely used as a microbial cell factory to produce recombinant proteins. Therefore, enhancing the protein production efficiency of yeast cell factories to expand the market demand for protein products is necessary. Recombinant proteins are often retained in the secretory pathway because of the limited protein transport performed by vesicle trafficking. Cell polarization describes the asymmetric organization of the plasma membrane cytoskeleton and organelles and tightly regulates vesicle trafficking for protein transport. Engineering vesicle trafficking has broadly been studied by the overexpression or deletion of key genes involved but not by modifying cell polarization. Here, we used α−amylase as a reporter protein, and its secretion and surface−display were first improved by promoter optimization. To study the effect of engineering cell polarization on protein production, fourteen genes related to cell polarization were overexpressed. BUD1, CDC42, AXL1, and BUD10 overexpression increased the activity of surface−displayed α−amylase, and BUD1, BUD3, BUD4, BUD7, and BUD10 overexpression enhanced secreted α−amylase activity. Furthermore, BUD1 overexpression increased the surface−displayed and secreted α−amylase expression by 56% and 49%, respectively. We also observed that the combinatorial modification and regulation of gene expression improved α-amylase production in a dose−dependent manner. BUD1 and CDC42 co−overexpression increased the α−amylase surface display by 100%, and two genomic copies of BUD1 improved α−amylase secretion by 92%. Furthermore, these modifications were used to improve the surface display and secretion of the recombinant β−glucosidase protein. Our study affords a novel insight for improving the surface display and secretion of recombinant proteins.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles that deliver bioactive proteins, nucleic acids, lipids, and other small molecules from donor to recipient cells. They have attracted significant interest recently due to their important roles in regulating plant-microbe interaction. During microbial infection, plant EVs play a prominent role in defense by delivering small regulatory RNA into pathogens, resulting in the silencing of pathogen virulence genes. Pathogens also deliver small RNAs into plant cells to silence host immunity genes. Recent evidence indicates that microbial EVs may be involved in pathogenesis and host immunity modulation by transporting RNAs and other biomolecules. However, the biogenesis and function of microbial EVs in plant-microbe interaction remain ill-defined. In this review, we discuss various aspects of microbial EVs, with a particular focus on current methods for EV isolation, composition, biogenesis, and their roles in plant-microbe interaction. We also discussed the potential role of microbial EVs in cross-kingdom RNA trafficking from pathogens to plants, as it is a highly likely possibility to explore in the future.

Leaves of sweetpotato (Ipomoea batatas L.) are promising healthy leafy vegetable. Juvenile red fading (JRF) leaves of sweetpotato, with anthocyanins in young leaves, are good candidates for developing functional vegetables. Here, metabolic profiling and possible antioxidants were analyzed for five leaf stages of the sweetpotato cultivar “Chuanshan Zi”. The contents of anthocyanins, total phenolics, and flavonoids all declined during leaf maturation, corresponding to declining antioxidant activities. By widely targeted metabolomics, we characterized 449 metabolites belonging to 23 classes. A total of 193 secondary metabolites were identified, including 82 simple phenols, 85 flavonoids, 18 alkaloids, and eight terpenes. Analysis of the metabolic data indicates that the antioxidant capacity of sweetpotato leaves is the combined result of anthocyanins and many other colorless compounds. Increased levels of “chlorogenic acid methyl ester”, a compromised form of chlorogenic acid, significantly correlated with the declined antioxidant abilities. Besides anthocyanins, some significant metabolites contributing to the high antioxidant property of the sweetpotato leaves were highlighted, including chlorogenic acids, isorhamnetin glycosides, trans-4-hydroxycinnamic acid methyl ester, 4-methoxycinnamic acid, esculetin, caffeate, and trigonelline. This study provides metabolic data for the utilization of sweetpotato leaves as food sources, and sheds light on the metabolomic change for JRF leaves of other plants.

Galactose-inducible (GAL) promoters have been widely used in metabolic engineering in Saccharomyces cerevisiae for production of valuable products. Endogenous GAL promoters and GAL transcription factors have often been engineered to improve GAL promoter activities. Heterologous GAL promoters and GAL activator (Gal4p-like transcriptional activators), although existing in other yeasts or fungi, have not been well explored. In this study, we comprehensively characterized the activation effects of Gal4p activators from different yeasts or fungi on a variant of GAL promoters. Overexpressing endogenous Gal4p driven by PHHF1 increased the activities of native PGAL1 and heterologous P SkGAL2 by 131.20% and 72.45%, respectively. Furthermore, eight transcriptional activators from different organisms were characterized and most of them exhibited functions that were consistent with ScGal4p. Expression of KlLac9p from Kluyveromyces lactis further increased the activity of P ScGAL1 and P SkGAL2 by 41.56% and 100.63%, respectively, compared to ScGal4p expression, and was able to evade Gal80p inhibition. This optimized GAL expression system can be used to increase the production of β-carotene by 9.02-fold in S. cerevisiae. Our study demonstrated that a combination of heterologous transcriptional activators and GAL promoters provided novel insights into the optimization of the GAL expression system.