Saccharomyces cerevisiae is an excellent microbial cell factory for producing valuable recombinant proteins because of its fast growth rate, robustness, biosafety, ease of operability via mature genomic modification technologies, and the presence of a conserved post-translational modification pathway among eukaryotic organisms. However, meeting industrial and market requirements with the current low microbial production of recombinant proteins can be challenging. To address this issue, numerous efforts have been made to enhance the ability of yeast cell factories to efficiently produce proteins. In this review, we provide an overview of recent advances in S. cerevisiae engineering to improve recombinant protein production. This review focuses on the strategies that enhance protein production by regulating transcription through promoter engineering, codon optimization, and expression system optimization. Additionally, we describe modifications to the secretory pathway, including engineered protein translocation, protein folding, glycosylation modification, and vesicle trafficking. Furthermore, we discuss global metabolic pathway optimization and other relevant strategies, such as the disruption of protein degradation, cell wall engineering, and random mutagenesis. Finally, we provide an outlook on the developmental trends in this field, offering insights into future directions for improving recombinant protein production in S. cerevisiae.

The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals.

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.

Background: Vanillin is one of the important phenolic inhibitors inSaccharomyces cerevisiaefor bioconversion of lignocellulosic materials and has been reported to inhibit the translation process in cells. In our previous studies, it was confirmed that the deletion of the transcription factor geneYRR1enhanced vanillin resistance by promoting some translation-related processes at the transcription level. In this work, we investigated the effects of proteomic changes upon induction of vanillin stress and deletion ofYRR1to provide unique perspectives from a transcriptome analysis for comprehending the mechanisms ofYRR1deletion in the protective response of yeast to vanillin.Results: In wild-type cells, vanillin reduced two dozens of ribosomal proteins contents while upregulated proteins involved in glycolysis, oxidative phosphorylation, and the pentose phosphate pathway in cells. The ratios of NADPH/NADP⁺and NADH/NAD⁺were increased when cells responded to vanillin stress. The differentially expressed proteins perturbed byYRR1deletion were much more abundant than and showed no overlaps with transcriptome changes, indicating that Yrr1 affects the synthesis of certain proteins. Forty-eight of 112 upregulated proteins were involved in the stress response, translational and transcriptional regulation.YRR1deletion increased the expression ofHAA1-encoding transcriptional activator,TMA17-encoding proteasome assembly chaperone andMBF1-encoding coactivator at the protein level, as confirmed by ELISA. Cultivation data showed that the overexpression ofHAA1andTMA17enhanced resistance to vanillin inS. cerevisiae.Conclusions: Cells conserve energy by decreasing the content of ribosomal proteins, producing more energy and NAD(P)H for survival in response to vanillin stress. Yrr1 improved vanillin resistance by increasing the protein quantities of Haa1, Tma17 and Mbf1. These results showed the response ofS. cerevisiaeto vanillin and howYRR1deletion increases vanillin resistance at the protein level. These findings may advance our knowledge of howYRR1deletion protects yeast from vanillin stress and offer novel targets for genetic engineering of designing inhibitor-resistant ethanologenic yeast strains.

As a potential alternative to fossil-based fuels, cellulosic ethanol has attracted much attention due to its great benefit to energy sustainability and environmental friendliness. However, at present, the industrial competitiveness of cellulosic ethanol production is still insufficient compared with fossil-based fuels because of the higher costs. Expanding the range of lignocellulosic biomass may be a promising measure to promote the economical production of cellulosic ethanol. Corn fiber, a byproduct from the corn deep-processing, is an attractive feedstock for cellulosic ethanol production because of its rich carbohydrate content (generally exceeding 65% of dry weight), almost no transportation cost, and low lignin content allow it to be easily handled. This study first optimized the hydrolysis conditions, including the pretreatment and enzymolysis process based on dilute sulfuric acid, to achieve a high sugar yield. Then, the corn fiber hydrolysates obtained under different hydrolysis conditions were suitably fermented by different C5/C6 co-fermentation Saccharomyces cerevisiae, indicating that the hydrolysate at high solid loading (20%) needs to detoxification to a certain extent but not low solid loading (10%) to achieve high ethanol yield. Finally, the fermentation of the 20% solid loading hydrolysates with resin detoxification was performed in a 50 L bioreactor, achieving the sugar (glucose and xylose) metabolic rate of 2.24 g L ⁻¹ h ⁻¹ and ethanol yield of 92% of the theoretical value, which are the highest reported levels to date. This study provided a potential process route for cellulosic ethanol production from corn fiber from the perspective of the suitability between the upstream hydrolysis process and the downstream fermentation strain.