scholarly journals The Plasma Membrane at the Cornerstone Between Flexibility and Adaptability: Implications for Saccharomyces cerevisiae as a Cell Factory

2021 ◽  
Vol 12 ◽  
Author(s):  
Luís Ferraz ◽  
Michael Sauer ◽  
Maria João Sousa ◽  
Paola Branduardi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Cell Factory ◽  
Membrane Composition ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
A Cell ◽  
Biotechnological Processes ◽  
Membrane Engineering ◽  
The Impact

In the last decade, microbial-based biotechnological processes are paving the way toward sustainability as they implemented the use of renewable feedstocks. Nonetheless, the viability and competitiveness of these processes are often limited due to harsh conditions such as: the presence of feedstock-derived inhibitors including weak acids, non-uniform nature of the substrates, osmotic pressure, high temperature, extreme pH. These factors are detrimental for microbial cell factories as a whole, but more specifically the impact on the cell’s membrane is often overlooked. The plasma membrane is a complex system involved in major biological processes, including establishing and maintaining transmembrane gradients, controlling uptake and secretion, intercellular and intracellular communication, cell to cell recognition and cell’s physical protection. Therefore, when designing strategies for the development of versatile, robust and efficient cell factories ready to tackle the harshness of industrial processes while delivering high values of yield, titer and productivity, the plasma membrane has to be considered. Plasma membrane composition comprises diverse macromolecules and it is not constant, as cells adapt it according to the surrounding environment. Remarkably, membrane-specific traits are emerging properties of the system and therefore it is not trivial to predict which membrane composition is advantageous under certain conditions. This review includes an overview of membrane engineering strategies applied to Saccharomyces cerevisiae to enhance its fitness under industrially relevant conditions as well as strategies to increase microbial production of the metabolites of interest.

scholarly journals Recent Advances on Feasible Strategies for Monoterpenoid Production in Saccharomyces cerevisiae

2020 ◽  
Vol 8 ◽  
Author(s):  
Qiyu Gao ◽  
Luan Wang ◽  
Maosen Zhang ◽  
Yongjun Wei ◽  
Wei Lin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plant Biomass ◽  
Extraction Methods ◽  
Active Components ◽  
Plant Essential Oils ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Plant Extraction ◽  
Low Efficiency ◽  
Metabolic Activities

Terpenoids are a large diverse group of natural products which play important roles in plant metabolic activities. Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs. Some monoterpenoids are widely used in medicine, cosmetics and other industries, and they are mainly obtained by plant biomass extraction methods. These plant extraction methods have some problems, such as low efficiency, unstable quality, and high cost. Moreover, the monoterpenoid production from plant cannot satisfy the growing monoterpenoids demand. The development of metabolic engineering, protein engineering and synthetic biology provides an opportunity to produce large amounts of monoterpenoids eco-friendly using microbial cell factories. This mini-review covers current monoterpenoids production using Saccharomyces cerevisiae. The monoterpenoids biosynthetic pathways, engineering of key monoterpenoids biosynthetic enzymes, and current monoterpenoids production using S. cerevisiae were summarized. In the future, metabolically engineered S. cerevisiae may provide one possible green and sustainable strategy for monoterpenoids supply.

scholarly journals Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

2021 ◽  
Author(s):  
Eline Postma ◽  
Else-Jasmijn Hassing ◽  
Venda Mangkusaputra ◽  
Jordi Geelhoed ◽  
Pilar de la Torre ◽  
...  
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Metabolic Networks ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Microbial Cell Factories ◽  
Cell Factories

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

scholarly journals Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae.

1994 ◽  
Vol 5 (11) ◽  
pp. 1185-1198 ◽  
Author(s):  
C Berkower ◽  
D Loayza ◽  
S Michaelis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Sufficient Information ◽  
Plasma Membrane Atpase ◽  
Amino Acid Permease ◽  
Membrane Atpase ◽  
A Cell ◽  
Pattern Characteristic ◽  
General Amino Acid Permease ◽  
Punctate Pattern

STE6, a member of the ATP binding cassette (ABC) transporter superfamily, is a membrane protein required for the export of the a-factor mating pheromone in Saccharomyces cerevisiae. To initiate a study of the intracellular trafficking of STE6, we have examined its half-life and localization. We report here that STE6 is metabolically unstable in a wild-type strain, and that this instability is blocked in a pep4 mutant, suggesting that degradation of STE6 occurs in the vacuole and is dependent upon vacuolar proteases. In agreement with a model whereby STE6 is routed to the vacuole via endocytosis from the plasma membrane, we show that degradation of STE6 is substantially reduced at nonpermissive temperature in mutants defective in delivery of proteins to the plasma membrane (sec6) or in endocytosis (end3 and end4). Whereas STE6 appears to undergo constitutive internalization from the plasma membrane, as do the pheromone receptors STE2 and STE3, we show that two other proteins, the plasma membrane ATPase (PMA1) and the general amino acid permease (GAP1), are significantly more stable than STE6, indicating that rapid turnover in the vacuole is not a fate common to all plasma membrane proteins in yeast. Investigation of STE6 partial molecules (half- and quarter-molecules) indicates that both halves of STE6 contain sufficient information to mediate internalization. Examination of STE6 localization by indirect immunofluorescence indicates that STE6 is found in a punctate, possibly vesicular, intracellular pattern, distinct from the rim-staining pattern characteristic of PMA1. The punctate pattern is consistent with the view that most of the STE6 molecules present in a cell at any given moment could be en route either to or from the plasma membrane. In a pep4 mutant, STE6 is concentrated in the vacuole, providing further evidence that the vacuole is the site of STE6 degradation, while in an end4 mutant STE6 exhibits rim-staining, indicating that it can accumulate in the plasma membrane when internalization is blocked. Taken together, the results presented here suggest that STE6 first travels to the plasma membrane and subsequently undergoes endocytosis and degradation in the vacuole, with perhaps only a transient residence at the plasma membrane; an alternative model, in which STE6 circumvents the plasma membrane, is also discussed.

scholarly journals Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Pingping Wang ◽  
Wei Wei ◽  
Wei Ye ◽  
Xiaodong Li ◽  
Wenfang Zhao ◽  
...  
Keyword(s):  
Yeast Cell ◽  
Batch Fermentation ◽  
Cell Factory ◽  
Ginsenoside Rh2 ◽  
Fed Batch ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Fed Batch Fermentation ◽  
Shake Flasks ◽  
Produce Plant

AbstractSynthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3–OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones.

scholarly journals A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin.

1994 ◽  
Vol 14 (7) ◽  
pp. 4825-4833 ◽  
Author(s):  
C F Lu ◽  
J Kurjan ◽  
P N Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Soluble Form ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Experimental Support ◽  
Cell Biol ◽  
A Cell

Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall.

scholarly journals Navigating the Valley of Death: Perceptions of Industry and Academia on Production Platforms and Opportunities

2020 ◽  
Author(s):  
Linde FC Kampers ◽  
Enrique Asin Garcia ◽  
Peter J Schaap ◽  
Annemarie Wagemakers ◽  
Vitor AP Martins dos Santos
Keyword(s):  
Industrial Applications ◽  
Third Party ◽  
Cell Factory ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Start Up ◽  
Industrial Performance ◽  
Research Choices ◽  
Depth Interviews ◽  
Valley Of Death

AbstractRational lifestyle engineering using computational methods and synthetic biology has made it possible to genetically improve industrial performance of microbial cell factories for the production of a range of biobased chemicals. However, only an estimated 1 in 5,000 to 10,000 innovations make it through the Valley of Death to market implementation.To gain in-depth insights into the views of industry and academia on key bottlenecks and opportunities to reach market implementation, a qualitative and exploratory study was performed by conducting 12 in depth interviews with 8 industrial and 4 academic participants. The characteristics that any cell factory must have were schematically listed, and commonly recognised opportunities were identified.We found that academics are limited by only technical factors in their research, while industry is restricted in their research choices and flexibility by a series of technical, sector dependent and social factors. This leads to a misalignment of interest of academics and funding industrial partners, often resulting in miscommunication. Although both are of the opinion that academia must perform curiosity-driven research to find innovative solutions, there is a certain pressure to aim for short-term industrial applications. All these factors add up to the Valley of Death; the gap between development and market implementation.A third party, in the form of start-up companies, could be the answer to bridging the Valley of Death.

scholarly journals A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Eline D. Postma ◽  
Sofia Dashko ◽  
Lars van Breemen ◽  
Shannara K. Taylor Parkins ◽  
Marcel van den Broek ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Engineering ◽  
De Novo ◽  
Transcriptional Unit ◽  
Dna Assembly ◽  
Cellular Processes ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Expression Platform

ABSTRACTThe construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes Synthetic Chromosomes (SynChs) as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular SynChs of 50 and 100 Kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. SynChs made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding SynChs were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.

scholarly journals Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

2020 ◽  
Author(s):  
Quentin M. Dudley ◽  
Ashty S. Karim ◽  
Connor J. Nash ◽  
Michael C. Jewett
Keyword(s):  
Protein Synthesis ◽  
Metabolic Engineering ◽  
Biosynthetic Enzymes ◽  
List Type ◽  
Free Protein ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
The Impact ◽  
Cell Free Protein Synthesis

AbstractMetabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.TOC FigureHighlightsApplied the iPROBE framework to build the nine-enzyme pathway to produce limoneneAssessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performanceIteratively optimized the cell-free production of limonene by exploring more than 580 unique reactionsExtended pathway to biofuel precursors pinene and bisabolene

scholarly journals A targeted next generation sequencing approach to develop robust, genotype-specific mutation profiles and uncover novel variants in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Natalie A. Lamb ◽  
Jonathan Bard ◽  
Michael J. Buck ◽  
Jennifer A. Surtees
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Stability ◽  
Underlying Mechanism ◽  
Position Effects ◽  
Replication Fidelity ◽  
Specific Mutation ◽  
Targeted Next Generation Sequencing ◽  
A Cell ◽  
Mutation Spectra ◽  
The Impact

ABSTRACTDistinct mutation signatures arise from environmental exposures and/or from defects in metabolic pathways that promote genome stability. The presence of a particular mutation signature in a cell or a tumor can therefore predict the underlying mechanism of mutagenesis, which, in practice, may be clinically important. These insults to the genome often alter dNTP pools, which itself impacts replication fidelity. Therefore, the impact of altered dNTP pools should be considered when making mechanistic predictions based on mutation signatures. We developed a targeted deep-sequencing approach on the CAN1 gene in Saccharomyces cerevisiae to define information-rich mutational profiles associated with distinct rnr1 backgrounds that alter replication fidelity by elevating dNTP levels.. The mutation spectra of rnr1Y285F and rnr1Y285A alleles were characterized previously; our analysis was consistent with this prior work but the sequencing depth achieved in our study allowed a significantly more robust and nuanced computational analysis of the variants observed, generating profiles that integrated information about mutation spectra, position effects, and sequence context. This approach revealed novel, genotype-specific mutation profiles in the presence of even modest changes in dNTP pools. Furthermore, we identified broader sequence contexts and specific nucleotide motifs that influenced variant profiles in different rnr1 backgrounds, which allowed us to make specific mechanistic predictions about the impact of altered dNTP pools on replication fidelity.

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