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"Stuart, David T."
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Evaluating the effects of synthetic POM cycles and NAD+ kinase expression on fatty alcohol production in Saccharomyces cerevisiae
Efficient regeneration of NADPH can be a limiting factor for anabolic processes in engineered microbial cells. We tested the ability of four distinct Pyruvate-Oxaloacetate-Malate “POM” cycles composed of Saccharomyces cerevisiae pyruvate carboxylase ( PYC1 or PYC2 ), malate dehydrogenase ( ‘MDH1 or ‘MDH2 ), and malic enzyme ( sMAE1 ) to improve NADPH regeneration. Only the PYC1 , ‘MDH2 , sMAE1 combination increased the titer of fatty alcohols produced by engineered S. cerevisiae indicating that not all combinations of POM cycle enzymes could drive this pathway. Metabolomic analysis revealed that introduction of the POM cycle altered the concentration of intermediates in amino acid biosynthetic pathways and the trichloroacetic acid cycle suggesting that the POM cycle had wider effects than previously anticipated. Overexpression of the endogenous NAD + kinases UTR1 , YEF1 , and a cytosolic version of POS5 were also tested. Only expression of POS5c resulted a significant increase in fatty alcohol titer. In these minimally engineered strains, combined overexpression of the PYC1 , ‘ MDH2 , sMAE1 POM cycle and POS5c did not further increase titers. These findings indicate that more extensive metabolomic and proteomic investigations are required to identify combinations of enzymes that will yield an optimal increase in NADPH to meet anabolic demands without imposing excessive metabolic burden or disrupting pathways that might compromise bioproduct synthesis.
Journal Article
Lipomyces starkeyi: an emerging cell factory for production of lipids, oleochemicals and biotechnology applications
Oils and oleochemicals produced by microbial cells offer an attractive alternative to petroleum and food-crop derived oils for the production of transport fuel and oleochemicals. An emerging candidate for industrial single cell oil production is the oleaginous yeast Lipomyces starkeyi. This yeast is capable of accumulating storage lipids to concentrations greater than 60% of the dry cell weight. From the perspective of industrial biotechnology L. starkeyi is an excellent chassis for single-cell oil and oleochemical production as it can use a wide variety of carbon and nitrogen sources as feedstock. The strain has been used to produce lipids from hexose and pentose sugars derived from cellulosic hydrolysates as well as crude glycerol and even sewage sludge. L. starkeyi also produces glucanhydrolases that have a variety of industrial applications and displays potential to be employed for bioremediation. Despite its excellent properties for biotechnology applications, adoption of L. starkeyi as an industrial chassis has been hindered by the difficulty of genetically manipulating the strain. This review will highlight the industrial potential of L. starkeyi as a chassis for the production of lipids, oleochemicals and other biochemicals. Additionally, we consider progress and challenges in engineering this organism for industrial applications.
Journal Article
Moesziomyces antarcticus MMF1 Has a Role in the Secretion of Mannosylerythritol Lipids
Mannosyl erythritol lipids (MELs) are glycolipid biosurfactants produced by Ustilaginomycete yeasts. The MEL biosynthetic pathway has been characterized in Ustilago maydis where a putative transporter encoded by MMF1 is required for the secretion of the glycolipid surfactant to the extracellular space. The anamorphic yeast Moesziomyces antarcticus is a prolific producer of MELs, but the mechanism of MEL secretion is less well characterized than in U. maydis. Homologous recombination was employed to generate a disruption of the MMF1 gene in M. antarcticus JCM10317. This mutation did not prevent the intracellular accumulation of MEL species but did result in significantly reduced secretion of the conventional MEL-A, MEL-B and MEL-C species detectable by thin-layer chromatography. However, the mutant strain did secrete a glycolipid species that is distinct from conventional MEL-A/B/C and similar to a glycolipid secreted by MMF1 mutant strains of U. maydis and Pseudozyma tsukubaensis. Despite the defect in MEL secretion displayed by the M. antarcticus strain harbouring a disrupted MMF1 gene, these cells did not display a significant defect in growth or cell morphology. The findings of this investigation provide evidence that M. antarcticus MMF1 encodes a transporter required for the secretion of MELs but not required for MEL synthesis or cell growth.
Journal Article
Biosynthesis of Fatty Alcohols in Engineered Microbial Cell Factories: Advances and Limitations
Concerns about climate change and environmental destruction have led to interest in technologies that can replace fossil fuels and petrochemicals with compounds derived from sustainable sources that have lower environmental impact. Fatty alcohols produced by chemical synthesis from ethylene or by chemical conversion of plant oils have a large range of industrial applications. These chemicals can be synthesized through biological routes but their free forms are produced in trace amounts naturally. This review focuses on how genetic engineering of endogenous fatty acid metabolism and heterologous expression of fatty alcohol producing enzymes have come together resulting in the current state of the field for production of fatty alcohols by microbial cell factories. We provide an overview of endogenous fatty acid synthesis, enzymatic methods of conversion to fatty alcohols and review the research to date on microbial fatty alcohol production. The primary focus is on work performed in the model microorganisms, Escherichia coli and Saccharomyces cerevisiae but advances made with cyanobacteria and oleaginous yeasts are also considered. The limitations to production of fatty alcohols by microbial cell factories are detailed along with consideration to potential research directions that may aid in achieving viable commercial scale production of fatty alcohols from renewable feedstock.
Journal Article
Bioengineered Probiotics: Synthetic Biology Can Provide Live Cell Therapeutics for the Treatment of Foodborne Diseases
The rising prevalence of antibiotic resistant microbial pathogens presents an ominous health and economic challenge to modern society. The discovery and large-scale development of antibiotic drugs in previous decades was transformational, providing cheap, effective treatment for what would previously have been a lethal infection. As microbial strains resistant to many or even all antibiotic drug treatments have evolved, there is an urgent need for new drugs or antimicrobial treatments to control these pathogens. The ability to sequence and mine the genomes of an increasing number of microbial strains from previously unexplored environments has the potential to identify new natural product antibiotic biosynthesis pathways. This coupled with the power of synthetic biology to generate new production chassis, biosensors and “weaponized” live cell therapeutics may provide new means to combat the rapidly evolving threat of drug resistant microbial pathogens. This review focuses on the application of synthetic biology to construct probiotic strains that have been endowed with functionalities allowing them to identify, compete with and in some cases kill microbial pathogens as well as stimulate host immunity. Weaponized probiotics may have the greatest potential for use against pathogens that infect the gastrointestinal tract: Vibrio cholerae , Staphylococcus aureus , Clostridium perfringens and Clostridioides difficile . The potential benefits of engineered probiotics are highlighted along with the challenges that must still be met before these intriguing and exciting new therapeutic tools can be widely deployed.
Journal Article
Co-Production of Isobutanol and Ethanol from Prairie Grain Starch Using Engineered Saccharomyces cerevisiae
Isobutanol is an important and valuable platform chemical and an appealing biofuel that is compatible with contemporary combustion engines and existing fuel distribution infrastructure. The present study aimed to compare the potential of triticale, wheat and barley starch as feedstock for isobutanol production using an engineered strain of Saccharomyces cerevisiae. A simultaneous saccharification and fermentation (SSF) approach showed that all three starches were viable feedstock for co-production of isobutanol and ethanol and could produce titres similar to that produced using purified sugar as feedstock. A fed-batch process using triticale starch yielded 0.006 g isobutanol and 0.28 g ethanol/g starch. Additionally, it is demonstrated that Fusarium graminearum infected grain starch contaminated with mycotoxin can be used as an effective feedstock for isobutanol and ethanol co-production. These findings demonstrate the potential for triticale as a purpose grown energy crop and show that mycotoxin-contaminated grain starch can be used as feedstock for isobutanol biosynthesis, thus adding value to a grain that would otherwise be of limited use.
Journal Article
Optimization of C16 and C18 fatty alcohol production by an engineered strain of Lipomyces starkeyi
The oleaginous yeast
Lipomyces starkeyi
was engineered for the production of long-chain fatty alcohols by expressing a fatty acyl-CoA reductase,
mFAR1,
from
Mus musculus
. The optimal conditions for production of fatty alcohols by this strain were investigated. Increased carbon-to-nitrogen ratios led to efficient C16 and C18 fatty alcohol production from glucose, xylose and glycerol. Batch cultivation resulted in a titer of 1.7 g/L fatty alcohol from glucose which represents a yield of 28 mg of fatty alcohols per gram of glucose. This relatively high level of production with minimal genetic modification indicates that
L. starkeyi
may be an excellent host for the bioconversion of carbon-rich waste streams, particularly lignocellulosic waste, to C16 and C18 fatty alcohols.
Journal Article
Dual Phosphorylation of Cdk1 Coordinates Cell Proliferation with Key Developmental Processes in Drosophila
Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Here, we expressed wild-type and phospho-acceptor mutant Cdk1 proteins to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2-phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the premitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1–Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2-phase cells are ready for mitosis.
Journal Article
Structural elucidation of a PRP8 core domain from the heart of the spliceosome
The spliceosome consists of five RNAs and more than 100 associated proteins. One of these, PRP8, is both one of the largest and most highly conserved spliceosomal proteins. Previous genetic and cross-linking data pointed to the importance of domain IV of PRP8 in spliceosome assembly and/or catalysis. Its structure has now been solved and found to contain an RNase H fold, suggestive of an RNA binding surface. The RNA binding data suggest that the PRP8 core recognizes, rather than a specific sequence, a structure resembling the four-helix junction proposed for the catalytically active U2/U6 snRNA interaction.
The spliceosome is a complex ribonucleoprotein (RNP) particle containing five RNAs and more than 100 associated proteins. One of these proteins, PRP8, has been shown to interact directly with the splice sites and branch region of precursor-mRNAs (pre-mRNAs) and spliceosomal RNAs associated with catalysis of the two steps of splicing. The 1.85-Å X-ray structure of the core of PRP8 domain IV, implicated in key spliceosomal interactions, reveals a bipartite structure that includes the presence of an RNase H fold linked to a five-helix assembly. Analysis of mutant yeast alleles and cross-linking results in the context of this structure, coupled with RNA binding studies, suggests that domain IV forms a surface that interacts directly with the RNA structures at the catalytic core of the spliceosome.
Journal Article