scholarly journals Engineering of Saccharomyces cerevisiae for 24-Methylene-Cholesterol Production

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1710
Author(s):  
Jiao Yang ◽  
Changfu Li ◽  
Yansheng Zhang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oryza Sativa ◽  
Xenopus Laevis ◽  
Chemical Synthesis ◽  
Expression Cassette ◽  
Yeast Genome ◽  
Anticancer Activities ◽  
Common Precursor ◽  
Physalis Angulata

24-Methylene-cholesterol is a necessary substrate for the biosynthesis of physalin and withanolide, which show promising anticancer activities. It is difficult and costly to prepare 24-methylene-cholesterol via total chemical synthesis. In this study, we engineered the biosynthesis of 24-methylene-cholesterol in Saccharomyces cerevisiae by disrupting the two enzymes (i.e., ERG4 and ERG5) in the yeast’s native ergosterol pathway, with ERG5 being replaced with the DHCR7 (7-dehydrocholesterol reductase) enzyme. Three versions of DHCR7 originating from different organisms—including the DHCR7 from Physalis angulata (PhDHCR7) newly discovered in this study, as well as the previously reported OsDHCR7 from Oryza sativa and XlDHCR7 from Xenopus laevis—were assessed for their ability to produce 24-methylene-cholesterol. XlDHCR7 showed the best performance, producing 178 mg/L of 24-methylene-cholesterol via flask-shake cultivation. The yield could be increased up to 225 mg/L, when one additional copy of the XlDHCR7 expression cassette was integrated into the yeast genome. The 24-methylene-cholesterol-producing strain obtained in this study could serve as a platform for characterizing the downstream enzymes involved in the biosynthesis of physalin or withanolide, given that 24-methylene-cholesterol is a common precursor of these chemicals.

scholarly journals Developing a Yeast Platform Strain for an Enhanced Taxadiene Biosynthesis by CRISPR/Cas9

Metabolites ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 147
Author(s):  
Joseph C. Utomo ◽  
Fabio C. Chaves ◽  
Philippe Bauchart ◽  
Vincent J. J. Martin ◽  
Dae-Kyun Ro
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Anticancer Drug ◽  
Biosynthetic Pathway ◽  
Budding Yeast ◽  
Yeast Genome ◽  
Yeast Saccharomyces Cerevisiae ◽  
Geranylgeranyl Pyrophosphate ◽  
Common Precursor ◽  
The Future ◽  
Terpenoid Pathway

Paclitaxel is an important diterpenoid commonly used as an anticancer drug. Although the paclitaxel biosynthetic pathway has been mostly revealed, some steps remain to be elucidated. The difficulties in plant transformations and the scarcity of the precursor of paclitaxel, (+)-taxa-4(5), 11(12)-diene (taxadiene), have hindered the full comprehension of paclitaxel biochemistry and, therefore, its production by biotechnological approaches. One solution is to use the budding yeast, Saccharomyces cerevisiae, as a platform to elucidate the paclitaxel biosynthesis. As taxadiene is a diterpenoid, its common precursor, geranylgeranyl pyrophosphate (GGPP), needs to be increased in yeast. In this study, we screened various GGPP synthases (GGPPS) to find the most suitable GGPPS for taxadiene production in yeast. We also optimized the taxadiene production by increasing the flux toward the terpenoid pathway. Finally, to remove selection markers, we integrated the required genes using a CRISPR/Cas9 system in the yeast genome. Our result showed that a titer of 2.02 ± 0.40 mg/L (plasmid) and 0.41 ± 0.06 mg/L (integrated) can be achieved using these strategies. This platform strain can be used to readily test the gene candidates for microbial paclitaxel biosynthesis in the future.

Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

2019 ◽  
Author(s):  
Thomas Siemon ◽  
Zhangqian Wang ◽  
Guangkai Bian ◽  
Tobias Seitz ◽  
Ziling Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Chemical Synthesis ◽  
Building Block ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Economical Method ◽  
Englerin A ◽  
Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

2019 ◽  
Author(s):  
Thomas Siemon ◽  
Zhangqian Wang ◽  
Guangkai Bian ◽  
Tobias Seitz ◽  
Ziling Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Chemical Synthesis ◽  
Building Block ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Economical Method ◽  
Englerin A ◽  
Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

scholarly journals Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

Genetics ◽  
1991 ◽  
Vol 127 (4) ◽  
pp. 681-698 ◽  
Author(s):  
A J Link ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Physical Map ◽  
Single Copy ◽  
Restriction Endonucleases ◽  
Yeast Genome ◽  
Hybridization Probes ◽  
Copy Dna ◽  
Single Copy Dna

Abstract A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

scholarly journals Upstream – News in Genomics

2002 ◽  
Vol 3 (3) ◽  
pp. 221-225
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ovarian Cancer ◽  
Oryza Sativa ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Proteomic Analysis ◽  
Large Scale ◽  
Protein Complexes ◽  
The Other ◽  
Blood Samples

In recent months a bumper crop of genomes has been completed, including the fission yeast (Schizosaccharomyces pombe) and rice (Oryza sativa). Two large-scale studies ofSaccharomyces cerevisiaeprotein complexes provided a picture of the eukaryotic proteome as a network of complexes. Amongst the other stories of interest was a demonstration that proteomic analysis of blood samples can be used to detect ovarian cancer, perhaps even as early as stage I.

scholarly journals Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2429-2435 ◽  
Author(s):  
D M Donovan ◽  
N J Pearson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Relative Rate ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Protein Product ◽  
Yeast Genome ◽  
Base Pairs ◽  
Beta Galactosidase

The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae.

scholarly journals CRISPR-Cas9: A Powerful Tool to Efficiently Engineer Saccharomyces cerevisiae

Life ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 13
Author(s):  
João Rainha ◽  
Joana L. Rodrigues ◽  
Lígia R. Rodrigues
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Genome ◽  
Specific Method ◽  
Complex Processes ◽  
Biological Studies ◽  
Precise Tool ◽  
Long Time ◽  
Gene Knockouts ◽  
Heterologous Pathway ◽  
A Current

Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and specific method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artificial heterologous pathway integration, transcriptional regulation, or tolerance engineering.

Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (11) ◽  
pp. 2887-2893
Author(s):  
M Neitz ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Restriction Fragment ◽  
Dna Sequences ◽  
Yeast Genome ◽  
Ura3 Gene ◽  
Dna Restriction ◽  
Meiotic Analyses ◽  
The Right ◽  
In Cis

A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis. DNA sequence analysis revealed that CEN14 possesses sequences (elements I, II, and III) that are characteristic of other yeast centromeres. Mitotic and meiotic analyses indicated that the CEN14 function resides on a 259-base-pair (bp) RsaI-EcoRV restriction fragment, containing sequences that extend only 27 bp to the right of the element I to III region. In conjunction with previous findings on CEN3 and CEN11, these results indicate that the specific DNA sequences required in cis for yeast centromere function are contained within a region about 150 bp in length.

Saccharomyces cerevisiae contains two functional citrate synthase genes

1986 ◽  
Vol 6 (6) ◽  
pp. 1936-1942
Author(s):  
K S Kim ◽  
M S Rosenkrantz ◽  
L Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Nuclear Gene ◽  
Tricarboxylic Acid ◽  
Yeast Genome ◽  
Gene Encoding ◽  
Acid Cycle ◽  
Nonfermentable Carbon Source

The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed.

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