As a generally-recognized-as-safe microorganism, Saccharomyces cerevisiae is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in S. cerevisiae by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, S. cerevisiae has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in S. cerevisiae are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.
Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism* ; Golgi Apparatus/metabolism* ; Metabolic Engineering ; Vacuoles/metabolism*

Sweet potato is an important food crop that can also be used as an industrial raw material. Sucrose is the main form of long-distance carbohydrate transport in plants, and sucrose transporter (SUT) regulates the transmembrane transport and distribution of sucrose during plant growth and metabolism. Moreover, SUT plays a key role in phloem mediated source-to-sink sucrose transport and physiological activities, supplying sucrose for the sink tissues. In this study, the full-length cDNA sequences of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were obtained by rapid amplification of cDNA ends (RACE) cloning according to the transcripts of the two SUT coding genes which were differentially expressed in sweet potato storage roots with different starch properties. Phylogenetic analysis was performed to clarify the classification of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆. The subcellular localization of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ was determined by transient expression in Nicotiana benthamiana. The function of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sucrose and hexose absorption and transport was identified using yeast functional complementarity system. The expression pattern of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sweet potato organs were analyzed by real-time fluorescence quantitative PCR (RT-qPCR). Arabidopsis plants heterologous expressing IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ genes were obtained using floral dip method. The differences in starch and sugar contents between transgenic and wild-type Arabidopsis were compared. The results showed IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ encoded SUT proteins with a length of 505 and 521 amino acids, respectively, and both proteins belonged to the SUT1 subfamily. IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were located in the cell membrane and were able to transport sucrose, glucose and fructose in the yeast system. In addition, IbSUT₆₂₇₈₈ was also able to transport mannose. The expression of IbSUT₆₂₇₈₈ was higher in leaves, lateral branches and main stems, and the expression of IbSUT₈₁₆₁₆ was higher in lateral branches, stems and storage roots. After IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were heterologously expressed in Arabidopsis, the plants grew normally, but the biomass increased. The heterologous expression of IbSUT₆₂₇₈₈ increased the soluble sugar content, leaf size and 1 000-seed weight of Arabidopsis plants. Heterologous expression of IbSUT₈₁₆₁₆ increased starch accumulation in leaves and root tips and 1 000-seed weight of seeds, but decreased soluble sugar content. The results obtained in this study showed that IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ might be important genes regulating sucrose and sugar content traits in sweet potato. They might carry out physiological functions on cell membrane, such as transmembrane transport of sucrose, sucrose into and out of sink tissue, as well as transport and unloading of sucrose into phloem. The changes in traits result from their heterologous expression in Arabidopsis indicates their potential in improving the yield of other plants or crops. The results obtained in this study provide important information for revealing the functions of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in starch and glucose metabolism and formation mechanism of important quality traits in sweet potato.
Ipomoea batatas/metabolism* ; Arabidopsis/metabolism* ; Sucrose/metabolism* ; Saccharomyces cerevisiae/metabolism* ; DNA, Complementary ; Phylogeny ; Plants, Genetically Modified/genetics* ; Membrane Transport Proteins/metabolism* ; Starch/metabolism* ; Plant Proteins/metabolism* ; Gene Expression Regulation, Plant

The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His₆-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His₆-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.
Animals ; Antifungal Agents/pharmacology* ; Escherichia coli/metabolism* ; Proteins/metabolism* ; Protease Inhibitors/chemistry* ; Bombyx/chemistry* ; Saccharomyces cerevisiae/metabolism* ; Peptide Hydrolases

CRISPR/Cas9 has been widely used in engineering Saccharomyces cerevisiae for gene insertion, replacement and deletion due to its simplicity and high efficiency. The selectable markers of CRISPR/Cas9 systems are particularly useful for genome editing and Cas9-plasmids removing in yeast. In our previous research, GAL80 gene has been deleted by the plasmid pML104-mediated CRISPR/Cas9 system in an engineered yeast, in order to eliminate the requirement of galactose supplementation for induction. The maximum artemisinic acid production by engineered S. cerevisiae 1211-2 (740 mg/L) was comparable to that of the parental strain 1211 without galactose induction. Unfortunately, S. cerevisiae 1211-2 was inefficient in the utilization of the carbon source ethanol in the subsequent 50 L pilot fermentation experiment. The artemisinic acid yield in the engineered S. cerevisiae 1211-2 was only 20%-25% compared with that of S. cerevisiae 1211. The mutation of the selection marker URA3 was supposed to affect the growth and artemisinic acid production. A ura3 mutant was successfully restored by a recombinant plasmid pML104-KanMx4-u along with a 90 bp donor DNA, resulting in S. cerevisiae 1211-3. This mutant could grow normally in a fed-batch fermentor with mixed glucose and ethanol feeding, and the final artemisinic acid yield (> 20 g/L) was comparable to that of the parental strain S. cerevisiae 1211. In this study, an engineered yeast strain producing artemisinic acid without galactose induction was obtained. More importantly, it was the first report showing that the auxotrophic marker URA3 significantly affected artemisinic acid production in a pilot-scale fermentation with ethanol feeding, which provides a reference for the production of other natural products in yeast chassis.
Artemisinins ; Fermentation ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

In order to improve the salt tolerance of banana NHX genes, we cloned a MaNHX5 gene from Musa acuminata L. AAA group and predicted the key salt-tolerant amino acid sites and mutant protein structure changes of MaNHX5 by using bioinformatics tools. The 276-position serine (S) of MaNHX5 protein was successfully mutated to aspartic acid (D) by site-directed mutagenesis, and the AXT3 salt-sensitive mutant yeast was used for a functional complementation test. The results showed that after the mutated MaNHX5 gene was transferred to AXT3 salt-sensitive mutant yeast, the salt tolerance of the mutant yeast was significantly improved under 200 mmol/L NaCl treatment. It is hypothesized that Ser276 of MaNHX5 protein plays an important role in the transport of Na⁺ across the tonoplast.
Amino Acids/metabolism* ; Gene Expression Regulation, Plant ; Musa/metabolism* ; Plant Proteins/metabolism* ; Plants, Genetically Modified ; Saccharomyces cerevisiae/metabolism*

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.
Fermentation ; Geranyltranstransferase/genetics* ; Monoterpenes/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

Perilla (Perilla frutescens L.) is an important edible-medicinal oil crop, with its seed containing 46%-58% oil. Of perilla seed oil, α-linolenic acid (C18:3) accounts for more than 60%. Lysophosphatidic acid acyltransferase (LPAT) is one of the key enzymes responsible for triacylglycerol assembly in plant seeds, controlling the metabolic flow from lysophosphatidic acid to phosphatidic acid. In this study, the LPAT2 gene from the developing seeds of perilla was cloned and designated as PfLPAT2. The expression profile of PfLPAT2 gene was examined in various tissues and different seed development stages of perilla (10, 20, 30, and 40 days after flowering, DAF) by quantitative real-time PCR (qRT-PCR). In order to detect the subcellular localization of PfLPAT2 protein, a fusion expression vector containing PfLPAT2 and GFP was constructed and transformed into Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. In order to explore the enzymatic activity and biological function of PfLPAT2 protein, an E. coli expression vector, a yeast expression vector and a constitutive plant overexpression vector were constructed and transformed into an E. coli mutant SM2-1, a wild-type Saccharomyces cerevisiae strain INVSc1, and a common tobacco (Nicotiana tabacum, variety: Sumsun NN, SNN), respectively. The results showed that the PfLPAT2 open reading frame (ORF) sequence was 1 155 bp in length, encoding 384 amino acid residues. Functional structure domain prediction showed that PfLPAT2 protein has a typical conserved domain of lysophosphatidic acid acyltransferase. qRT-PCR analysis indicated that PfLPAT2 gene was expressed in all tissues tested, with the peak level in seed of 20 DAF of perilla. Subcellular localization prediction showed that PfLPAT2 protein is localized in cytoplasm. Functional complementation assay of PfLPAT2 in E. coli LPAAT mutant (SM2-1) showed that PfLPAT2 could restore the lipid biosynthesis of SM2-1 cell membrane and possess LPAT enzyme activity. The total oil content in the PfLPAT2 transgenic yeast was significantly increased, and the content of each fatty acid component changed compared with that of the non-transgenic control strain. Particularly, oleic acid (C18:1) in the transgenic yeast significantly increased, indicating that PfLPAT2 has a higher substrate preference for C18:1. Importantly, total fatty acid content in the transgenic tobacco leaves increased by about 0.42 times compared to that of the controls, with the C18:1 content doubled. The increased total oil content and the altered fatty acid composition in transgenic tobacco lines demonstrated that the heterologous expression of PfLPAT2 could promote host oil biosynthesis and the accumulation of health-promoting fatty acids (C18:1 and C18:3). This study will provide a theoretical basis and genetic elements for in-depth analysis of the molecular regulation mechanism of perilla oil, especially the synthesis of unsaturated fatty acids, which is beneficial to the genetic improvement of oil quality of oil crops.
Acyltransferases ; Cloning, Molecular ; Escherichia coli/metabolism* ; Fatty Acids ; Perilla frutescens/metabolism* ; Plant Oils ; Plant Proteins/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Seeds/chemistry* ; Tobacco/genetics*

Ubiquitination is one of the reversible protein post-translational modifications, in which ubiquitin molecules bind to the target protein in a cascade reaction of ubiquitin activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases. The deubiquitinating enzymes (DUBs) remove ubiquitin residues from the substrates, which play key roles in the formation of mature ubiquitin, the removal and trimming of ubiquitin chains, as well as the recycling of free ubiquitin chains. Ubp14, a member of the ubiquitin specific proteases family in Saccharomyces cerevisiae, is mainly responsible for the recycling of intracellular free ubiquitin chains. To investigate its global biological function, a ubp14∆ mutant was constructed by homologous recombination technique. The growth rate of ubp14∆ mutant was lower than that of the wild-type (WT) strain. Using stable isotope labeling by amino acids in cell culture (SILAC) combined with deep coverage proteomics analysis, the differentially expressed proteins of ubp14∆ mutant relative to the wild-type strain were systematically analyzed. A total of 3 685 proteins were identified in this study, and 109 differentially expressed proteins were filtered out by statistical analysis. Gene ontology analysis found that differentially expressed proteins caused by Ubp14 loss were mainly involved in amino acid metabolism, REDOX, heat shock stress and etc, which shed light on the broad biological function of this DUB. This study provides highly reliable proteomic data for further exploring the biological functions of the deubiquitination enzyme Ubp14, and further understanding the relationship between the free ubiquitin homeostasis and biological process regulation.
Saccharomyces cerevisiae/metabolism* ; Proteomics ; Endopeptidases/metabolism* ; Ubiquitin/metabolism* ; Ubiquitination ; Proteins/metabolism* ; Deubiquitinating Enzymes/metabolism* ; Biological Phenomena

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.
Acetic Acid ; DNA-Binding Proteins/metabolism* ; Fermentation ; Leucine ; RNA, Transfer/genetics* ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism* ; Transcription Factors

Lager yeast is the most popular yeast strain used for beer production in China. The flocculation of yeast plays an important role in cell separation at the end of fermentation. Therefore, appropriately enhancing the flocculation capability of the lager yeast without affecting its fermentation performance would be desirable for beer industry. Our previous study showed that the defect of gene RIM21 might contribute to the enhanced flocculation capability of a lager yeast G03. To further investigate the role of the RIM21 gene in flocculation of strain G03, this study constructed a RIM21-deleted mutant strain G03-RIM21Δ through homologous recombination. Deletion of RIM21 improved the flocculation capability of strain G03 during wort fermentation at 11 °C without changing its fermentation performance significantly. The expression of FLO5, Lg-FLO1 and some other genes involved in cell wall integrity pathway were up-regulated in strain G03-RIM21Δ. In addition, the disruption of RIM21 enhanced resistance of yeast cells to cell wall inhibitors. These results provide a basis for elucidating the flocculation mechanism of lager yeast under low-temperature fermentation conditions.
Beer ; Fermentation ; Flocculation ; Receptors, Cell Surface ; Saccharomyces/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*