Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides

This is the final version of the article. Available from Frontiers Media via the DOI in this record.Rhodotorula (Rhodosporidium) toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70% of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3, and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides.This work was supported by Biotechnology and Biological Sciences Research Council doctoral training partnership award BB/J014400/1 to AJ

Draft genome sequences of Achromobacter piechaudii GCS2, Agrobacterium sp. Strain SUL3, Microbacterium sp. Strain GCS4, Shinella sp. Strain GWS1, and Shinella sp. Strain SUS2 isolated from Consortium with the Hydrocarbon-Producing Alga Botryococcus braunii.

This is the final version of the article. Available from American Society for Microbiology via the DOI in this record.A variety of bacteria associate with the hydrocarbon-producing microalga Botryococcus braunii, some of which may influence its growth. We report here the genome sequences for Achromobacter piechaudii GCS2, Agrobacterium sp. strain SUL3, Microbacterium sp. strain GCS4, and Shinella sp. strains GWS1 and SUS2, isolated from a laboratory culture of B. braunii, race B, strain Guadeloupe.K.J.J. was funded by a Ph.D. studentship from the Biotechnology and Biologaical Sciences Research Council (BBSRC) LoLa award BB/ K003240/2. The Exeter Sequencing Service was supported by the Wellcome Trust Institutional Strategic Support Fund (WT097835MF), a Wellcome Trust Multi User Equipment Award (WT101650MA), and a BBSRC LoLa award (BB/K003240/2). We gratefully acknowledge the Exeter Sequencing Service and computational core facilities at the University of Exete

Design of Experiments Methodology to Build a Multifactorial Statistical Model Describing the Metabolic Interactions of Alcohol Dehydrogenase Isozymes in the Ethanol Biosynthetic Pathway of the Yeast Saccharomyces cerevisiae

This is the author accepted manuscript. The final version is available from the American Chemical Society via the DOI in this recordMultifactorial approaches can quickly and efficiently model complex, interacting natural or engineered biological systems in a way that traditional one-factor-at-a-time experimentation can fail to do. We applied a Design of Experiments (DOE) approach to model ethanol biosynthesis in yeast, which is well-understood and genetically tractable, yet complex. Six alcohol dehydrogenase (ADH) isozymes catalyze ethanol synthesis, differing in their transcriptional and post-translational regulation, subcellular localization, and enzyme kinetics. We generated a combinatorial library of all ADH gene deletions and measured the impact of gene deletion(s) and environmental context on ethanol production of a subset of this library. The data were used to build a statistical model that described known behaviors of ADH isozymes and identified novel interactions. Importantly, the model described features of ADH metabolic behavior without explicit a priori knowledge. The method is therefore highly suited to understanding and optimizing metabolic pathways in less well-understood systems.We wish to thank Dr. Alex Johns for helpful discussions. S.R.B. would also like to thank Shell Biodomain for funding for this PhD research project

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli.

This is the final version of the article. Available from National Academy of Sciences via the DOI in this record.Data deposition: The synthetic nucleotide sequences reported in this paper have been deposited in GenBank database (accession nos. JQ901708, JQ901709, and JQ901710).Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.This work was supported by a grant from Shell Research Ltd. and a Biotechnology and Biological Sciences Research Council (BBSRC) Industry Interchange Partnership grant (to J.L.)

Genome sequence of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609

This is the final version of the article. Available from the publisher via the DOI in this record.Most eukaryotic oleaginous species are yeasts and among them the basidiomycete red yeast, Rhodotorula (Rhodosporidium) toruloides (Pucciniomycotina) is known to produce high quantities of lipids when grown in nitrogen-limiting media, and has potential for biodiesel production. The genome of the CGMCC 2.1609 strain of this oleaginous red yeast was sequenced using a hybrid of Roche 454 and Illumina technology generating 13 × coverage. The de novo assembly was carried out using MIRA and scaffolded using MAQ and BAMBUS. The sequencing and assembly resulted in 365 scaffolds with total genome size of 33.4 Mb. The complete genome sequence of this strain was deposited in GenBank and the accession number is LKER00000000. The annotation is available on Figshare (doi:10.6084/m9.figshare.4754251).This research was funded by grants from Shell Global Solutions (UK). We gratefully acknowledge Liverpool Advanced Genomics Facility and Exeter Sequencing Service and computational core facilities at the University of Exeter supported by Wellcome Trust Institutional Strategic Support Fund (WT097835MF) and Wellcome Trust Multi User Equipment Award (WT101650MA)

Redundant Mechanisms Prevent Mitotic Entry Following Replication Arrest in the Absence of Cdc25 Hyper-Phosphorylation in Fission Yeast

Following replication arrest the Cdc25 phosphatase is phosphorylated and inhibited by Cds1. It has previously been reported that expressing Cdc25 where 9 putative amino-terminal Cds1 phosphorylation sites have been substituted to alanine results in bypass of the DNA replication checkpoint. However, these results were acquired by expression of the phosphorylation mutant using a multicopy expression vector in a genetic background where the DNA replication checkpoint is intact. In order to clarify these results we constructed a Cdc25(9A)-GFP native promoter integrant and examined its effect on the replication checkpoint at endogenous expression levels. In this strain the replication checkpoint operates normally, conditional on the presence of the Mik1 kinase. In response to replication arrest the Cdc25(9A)-GFP protein is degraded, suggesting the presence of a backup mechanism to eliminate the phosphatase when it cannot be inhibited through phosphorylation

Phosphorylation of the MBF Repressor Yox1p by the DNA Replication Checkpoint Keeps the G1/S Cell-Cycle Transcriptional Program Active

Background: In fission yeast Schizosaccharomyces pombe G1/S cell-cycle regulated transcription depends upon MBF. A negative feedback loop involving Nrm1p and Yox1p bound to MBF leads to transcriptional repression as cells exit G1 phase. However, activation of the DNA replication checkpoint response during S phase results in persistent expression of MBF-dependent genes.Methodology/Principal Findings: This report shows that Yox1p binding to MBF is Nrm1-dependent and that Yox1p and Nrm1p require each other to bind and repress MBF targets. In response to DNA replication stress both Yox1p and Nrm1p dissociate from MBF at promoters leading to de-repression of MBF targets. Inactivation of Yox1p is an essential part of the checkpoint response. Cds1p (human Chk2p) checkpoint protein kinase-dependent phosphorylation of Yox1p promotes its dissociation from the MBF transcription factor. We establish that phosphorylation of Yox1p at Ser114, Thr115 is required for maximal checkpoint-dependent activation of the G1/S cell-cycle transcriptional program.Conclusions/Significance: This study shows that checkpoint-dependent phosphorylation of Yox1p at Ser114, Thr115 results in de-repression of the MBF transcriptional program. The remodeling of the cell cycle transcriptional program by the DNA replication checkpoint is likely to comprise an important mechanism for the avoidance of genomic instability

Control of DNA synthesis genes in fission yeast by the cell-cycle gene <i>cdc10</i>⁺

In the budding yeast Saccharomyces cerevisiae, cell-cycle control over DNA synthesis occurs partly through the coordinate expression in late G1 phase of many, if not all, of the genes required for DNA synthesis. A cis-acting hexamer element ACGCGT (an MluI restriction site) is responsible for coordinating transcriptional regulation of these genes at the G1/S phase boundary and we have identified a binding activity, DSC1, that recognizes these sequences in a cell-cycle-dependent manner. In the distantly related fission yeast Schizosaccharomyces pombe, only one of the known DNA synthesis genes, &lt;i&gt;cdc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt;, which encodes a subunit of ribonucleotide reductase, is periodically expressed in late G1 (ref. 6). The promoter region of &lt;i&gt;cdc22&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; has two MluI sites and five related sequences, suggesting that similar controls over DNA synthesis genes could occur in fission yeast. We report here a binding activity in fission yeast that is very similar to DSC1 in budding yeast. We also show that the fission yeast &lt;i&gt;cdc10&lt;/i&gt;&lt;sup&gt;+&lt;/sup&gt; gene product, which is required for Start and entry into S phase, is a component of this binding activity