Engineering Of Saccharomyces Cerevisiae Research Articles

Large-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae

Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae.

Targeting Mosquitoes through Generation of an Insecticidal RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae.

The global deployment of RNAi yeast insecticides involves transitioning from the use of laboratory yeast strains to more robust strains that are suitable for scaled fermentation. In this investigation, the RNA-guided Cas-CLOVER system was used in combination with Piggybac transposase to produce robust Saccharomyces cerevisiae strains with multiple integrated copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. This enabled the constitutive high-level expression of an insecticidal shRNA corresponding to a target sequence that is conserved in mosquito Shaker genes, but which is not found in non-target organisms. Top-expressing Cas-CLOVER strains performed well in insecticide trials conducted on Aedes, Culex, and Anopheles larvae and adult mosquitoes, which died following consumption of the yeast. Scaled fermentation facilitated the kilogram-scale production of the yeast, which was subsequently heat-killed and dried. These studies indicate that RNAi yeast insecticide production can be scaled, an advancement that may one day facilitate the global distribution of this new mosquito control intervention.

Highly Efficient Biosynthesis of γ-Bisabolene with a New Sesquiterpene Synthase AcTPS5 by Dual Cytoplasmic-Peroxisomal Engineering in Saccharomyces cerevisiae

γ-bisabolene is a monocyclic sesquiterpene with various biological activities; it has also been approved as a food additive. Additionally, the hydrogenated form of bisabolene is considered as a potential alternative to D2 diesel. Saccharomyces cerevisiae has the ability to produce a large amount of acetyl-CoA in both cytosol and peroxisomes, which serves as a precursor in terpene biosynthesis. In this study, AcTPS5 was identified as a new γ-bisabolene synthase. By expressing AcTPS5 and the mevalonate pathway in peroxisomes, γ-bisabolene titer was achieved at 125.0 mg/L. Deleting the peroxisome autophagy gene atg36 further improved γ-bisabolene production to 216.9 mg/L. The implementation of dual cytoplasmic–peroxisomal engineering further boosted γ-bisabolene production to 296.4 mg/L. Finally, through increasing the acetyl-CoA supply and down-regulating the expression of ERG9, γ-bisabolene production was achieved at 584.14 mg/L in shake-flask fermentation and 2.69 g/L in fed-batch fermentation, which is the highest reported production of γ-bisabolene to date. The strategy presented in this study provides an efficient approach for terpene production in S. cerevisiae.

Manipulation of IME4 expression, a global regulation strategy for metabolic engineering in Saccharomyces cerevisiae.

Metabolic engineering has been widely used for production of natural medicinal molecules. However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of metabolic network. N6-Methyladenosine (m6A) modification of RNA plays critical roles in regulation of gene expression. Herein, we identify 1470 putatively m6A peaks within 1151 genes from the haploid Saccharomyces cerevisiae strain. Among them, the transcript levels of 94 genes falling into the pathways which are frequently optimized for chemical production, are remarkably altered upon overexpression of IME4 (the yeast m6A methyltransferase). In particular, IME4 overexpression elevates the mRNA levels of the methylated genes in the glycolysis, acetyl-CoA synthesis and shikimate/aromatic amino acid synthesis modules. Furthermore, ACS1 and ADH2, two key genes responsible for acetyl-CoA synthesis, are induced by IME4 overexpression in a transcription factor-mediated manner. Finally, we show IME4 overexpression can significantly increase the titers of isoprenoids and aromatic compounds. Manipulation of m6A therefore adds a new layer of metabolic regulatory machinery and may be broadly used in bioproduction of various medicinal molecules of terpenoid and phenol classes.

Engineering an Efficient Expression Using Heterologous GAL Promoters and Transcriptional Activators in Saccharomyces cerevisiae.

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 PSkGAL2 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 PScGAL1 and PSkGAL2 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.

Evolutionary and reverse engineering in Saccharomyces cerevisiae reveals a Pdr1p mutation-dependent mechanism for 2-phenylethanol tolerance

Background2-Phenylethanol (2-PE), a higher alcohol with a rose-like odor, inhibits growth of the producer strains. However, the limited knowledge regarding 2-PE tolerance mechanisms renders our current knowledge base insufficient to inform rational design.ResultsTo improve the growth phenotype of Saccharomyces cerevisiae under a high 2-PE concentration, adaptive laboratory evolution (ALE) was used to generate an evolved 19–2 strain. Under 2-PE stress, its OD600 and growth rate increased by 86% and 22% than that of the parental strain, respectively. Through whole genome sequencing and reverse engineering, transcription factor Pdr1p mutation (C862R) was revealed as one of the main causes for increased 2-PE tolerance. Under 2-PE stress condition, Pdr1p mutation increased unsaturated fatty acid/saturated fatty acid ratio by 42%, and decreased cell membrane damage by 81%. Using STRING website, we identified Pdr1p interacted with some proteins, which were associated with intracellular ergosterol content, reactive oxygen species (ROS), and the ATP-binding cassette transporter. Also, the results of transcriptional analysis of genes encoded these proteins confirmed that Pdr1p mutation induced the expression of these genes. Compared with those of the reference strain, the ergosterol content of the PDR1_862 strain increased by 72%–101%, and the intracellular ROS concentration decreased by 38% under 2-PE stress. Furthermore, the Pdr1p mutation also increased the production of 2-PE (11% higher).ConclusionsIn the present work, we have demonstrated the use of ALE as a powerful tool to improve yeast tolerance to 2-PE. Based on the reverse engineering, transcriptional and physiological analysis, we concluded that Pdr1p mutation significantly enhanced the 2-PE tolerance of yeast by regulating the fatty acid proportion, intracellular ergosterol and ROS. It provides new insights on Pdr1p mediated 2-PE tolerance, which could help in the design of more robust yeasts for natural 2-PE synthesis.

Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species.

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of Saccharomyces cerevisiae has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for S. cerevisiae are discussed in terms of their contributions to the required standardization efforts. In addition, the toolkits designed for emerging nonconventional yeast species including Yarrowia lipolytica, Komagataella phaffii, and Kluyveromyces marxianus are also reviewed. Without a doubt, the characterized DNA parts combined with the standardized assembly strategies highlighted in these toolkits have greatly contributed to the rapid development of many metabolic engineering and diagnostics applications among others. Despite the growing capacity in deploying synthetic biology for common yeast genome engineering works, the yeast community has a long journey to go to exploit it in more sophisticated and delicate applications like bioautomation.

Glutathione production by Saccharomyces cerevisiae: current state and perspectives.

Glutathione (L-γ-glutamyl-cysteinyl-glycine, GSH) is a tripeptide synthesized through consecutive enzymatic reactions. Among its several metabolic functions in cells, the main one is the potential to act as an endogenous antioxidant agent. GSH has been the focus of numerous studies not only due to its role in the redox status of biological systems but also due to its biotechnological characteristics. GSH is usually obtained by fermentation and shows a variety of applications by the pharmaceutical and food industry. Therefore, the search for new strategies to improve the production of GSH during fermentation is crucial. This mini review brings together recent papers regarding the principal parameters of the biotechnological production of GSH by Saccharomyces cerevisiae. In this context, aspects, such as the medium composition (amino acids, alternative raw materials) and the use of technological approaches (control of osmotic and pressure conditions, magnetic field (MF) application, fed-batch process) were considered, along with genetic engineering knowledge, trends, and challenges in viable GSH production. KEY POINTS: • Saccharomyces cerevisiae has shown potential for glutathione production. • Improved technological approaches increases glutathione production. • Genetic engineering in Saccharomyces cerevisiae improves glutathione production.

Engineering eukaryote-like regulatory circuits to expand artificial control mechanisms for metabolic engineering in Saccharomyces cerevisiae

Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast (Saccharomyces cerevisiae) suffers from several drawbacks; specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae. Trans- and cis- modules, including eukaryotic trans-activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L−1 in flask cultivation. These negative/positive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications.

Cloning, Transformation and Expression of Human IFN-γ by Genetic Engineering in Saccharomyces cerevisiae

Human interferon-gamma (hIFN-γ) is a Glycoprotein pertinence to a distinct group of interferon, called type II interferons, which have an immunological function to respond to antigenic stimuli such as bacteria, viruses, fungi, and any infections by a microorganism. The hIFN-γ is produced by natural killer T (NKT) and natural killer (NK) cells during the immune response as part of the innate immune response and by Th1 CD4 and CD8 cytotoxic T lymphocytes (CTL) effector T cells upon the development of antigen-specific immunity. Recombinant hIFN-γ has been produced in different expression systems comprising prokaryotic, insect, fungal (yeasts), protozoan, mammalian cells, and plant. In the present study, pET28a plasmid was used in this research for preparation to insert and pYES2 plasmid for cloning and expression of human IFN-γ gene in yeast. The trademark of hIFN-γ has been produced in Escherichia coli is termed ACTIMMUNE®, but the human interferon- gamma was produced in the prokaryotic expression system is unglycosylated form. This result increases in cost due to increased purification, as well as short-life in the bloodstream, but it is biologically active. This study aimed to use Saccharomyces cerevisiae 4741 strain as a eukaryotic system for expression of hIFN-γ cDNA instead of a prokaryotic system in glycosylation patterns as modified translation. But the results are not satisfactory as the produce of yields.

Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase.

Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved kcat/Km values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest.

Corrigendum to: Metabolic engineering of Saccharomyces cerevisiae for production of ß-carotene from hydrophobic substrates

Corrigendum to: Metabolic engineering of Saccharomyces cerevisiae for production of ß-carotene from hydrophobic substrates

Metabolic engineering of Saccharomyces cerevisiae for high-level production of gastrodin from glucose

BackgroundThe natural phenolic glycoside gastrodin is the major bioactive ingredient in the well-known Chinese herb Tianma and is widely used as a neuroprotective medicine in the clinic. Microbial production from sustainable resources is a promising method to replace plant extraction and chemical synthesis which were currently used in industrial gastrodin production. Saccharomyces cerevisiae is considered as an attractive host to produce natural plant products used in the food and pharmaceutical fields. In this work, we intended to explore the potential of S. cerevisiae as the host for high-level production of gastrodin from glucose.ResultsHere, we first identified the plant-derived glucosyltransferase AsUGT to convert 4-hydroxybenzyl alcohol to gastrodin with high catalytic efficiency in yeast. Then, we engineered de novo production of gastrodin by overexpressing codon-optimized AsUGTsyn, the carboxylic acid reductase gene CARsyn from Nocardia species, the phosphopantetheinyl transferase gene PPTcg-1syn from Corynebacterium glutamicum, the chorismate pyruvate-lyase gene UbiCsyn from Escherichia coli, and the mutant ARO4K229L. Finally, we achieved an improved product titer by a chromosomal multiple-copy integration strategy and enhancement of metabolic flux toward the aglycon 4-hydroxybenzyl alcohol. The best optimized strain produced 2.1 g/L gastrodin in mineral medium with glucose as the sole carbon source by flask fermentation, which was 175 times higher than that of the original gastrodin-producing strain.ConclusionsThe de novo high-level production of gastrodin was first achieved. Instead of chemical synthesis or plants extraction, our work provides an alternative strategy for the industrial production of gastrodin by microbial fermentation from a sustainable resource.

Combinatorial Metabolic Engineering in Saccharomyces cerevisiae for the Enhanced Production of the FPP-Derived Sesquiterpene Germacrene.

Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.

Promoter Architecture and Promoter Engineering in Saccharomyces cerevisiae.

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.

Advances in RNAi-Assisted Strain Engineering in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a widely used eukaryotic model and microbial cell factory. RNA interference (RNAi) is a conserved regulatory mechanism among eukaryotes but absent from S. cerevisiae. Recent reconstitution of RNAi machinery in S. cerevisiae enables the use of this powerful tool for strain engineering. Here we first discuss the introduction of heterologous RNAi pathways in S. cerevisiae, and the design of various expression cassettes of RNAi precursor reagents for tunable, dynamic, and genome-wide regulation. We then summarize notable examples of RNAi-assisted functional genomics and metabolic engineering studies in S. cerevisiae. We conclude with the future challenges and opportunities of RNAi-based approaches, as well as the potential of other regulatory RNAs in advancing yeast engineering.

Multidimensional Metabolic Engineering for Constructing Efficient Cell Factories

Microbial production of medium-chain fatty acids (MCFAs) is challenging, as they are rare metabolites and toxic to cells. Recently, Zhu et al. performed multidimensional engineering in Saccharomyces cerevisiae, including engineering fatty acid synthases and improving cellular tolerance to MCFAs, to build an efficient MCFA cell factory.

Evolutionary engineering in Saccharomyces cerevisiae reveals a TRK1-dependent potassium influx mechanism for propionic acid tolerance

BackgroundPropionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries. Microbial PA production through engineering yeast as a cell factory is a potentially sustainable alternative to replace petroleum refining. However, PA inhibits yeast growth at concentrations well below the titers typically required for a commercial bioprocess.ResultsAdaptive laboratory evolution (ALE) with PA concentrations ranging from 15 to 45 mM enabled the isolation of yeast strains with more than threefold improved tolerance to PA. Through whole genome sequencing and CRISPR–Cas9-mediated reverse engineering, unique mutations in TRK1, which encodes a high-affinity potassium transporter, were revealed as the cause of increased propionic acid tolerance. Potassium supplementation growth assays showed that mutated TRK1 alleles and extracellular potassium supplementation not only conferred tolerance to PA stress but also to multiple organic acids.ConclusionOur study has demonstrated the use of ALE as a powerful tool to improve yeast tolerance to PA. Potassium transport and maintenance is not only critical in yeast tolerance to PA but also boosts tolerance to multiple organic acids. These results demonstrate high-affinity potassium transport as a new principle for improving organic acid tolerance in strain engineering.

A new strategy for seamless gene editing and marker recycling in Saccharomyces cerevisiae using lethal effect of Cwp1

Technologies development for seamless gene editing and marker recycling has allowed frequent genomic engineering in Saccharomyces cerevisiae for desired laboratory strains and cell factory. Alternative new approaches are still required for complicated scenarios. In this study, we report that inducible overexpression of cell wall protein 1 (Cwp1) by galactose addition confers yeast cells a robust growth inhibition. Direct repeats flanking the Gal‐CWP1:selectable marker cassette allow for its homology recombination excision and counter selection upon galactose addition, therefore enable seamless gene editing and marker recycling. We used this strategy and efficiently generated scarless Ade8 deletion mutants. Our results highlight the utility of lethal effect of Cwp1 overexpression a new counter selection strategy and a simple and efficient method for seamless gene editing and marker recycling in S. cerevisiae and potentially other fungi.

Advancing Metabolic Engineering of Saccharomyces cerevisiae Using the CRISPR/Cas System.

Thanks to its ease of use, modularity, and scalability, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been increasingly used in the design and engineering of Saccharomyces cerevisiae, one of the most popular hosts for industrial biotechnology. This review summarizes the recent development of this disruptive technology for metabolic engineering applications, including CRISPR-mediated gene knock-out and knock-in as well as transcriptional activation and interference. More importantly, multi-functional CRISPR systems that combine both gain- and loss-of-function modulations for combinatorial metabolic engineering are highlighted.