Effects of acetoacetyl-CoA synthase expression on production of farnesene in Saccharomyces cerevisiae

Efficient production of sesquiterpenes in Saccharomyces cerevisiae requires a high flux through the mevalonate pathway. To achieve this, the supply of acetyl-CoA plays a crucial role, partially because nine moles of acetyl-CoA are necessary to produce one mole of farnesyl diphosphate, but also to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA and, therefore, represents a potential target to increase the flux through the mevalonate pathway. This study investigates the effect of acetoacetyl-CoA synthase on growth as well as the production of farnesene and compares different homologs regarding their efficiency. While plasmid-based expression of nphT7 did not improve final farnesene titers, the construction of an alternative pathway, which exclusively relies on the malonyl-CoA bypass, was detrimental for growth and farnesene production. The presented results indicate that the overall functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain CL190. © 2017, The Author(s)

Emergence of superconductivity in the canonical heavy-electron metal YbRh2Si2

We report magnetic and calorimetric measurements down to T = 1 mK on the canonical heavy-electron metal YbRh2Si2. The data reveal the development of nuclear antiferromagnetic order slightly above 2 mK. The latter weakens the primary electronic antiferromagnetism, thereby paving the way for heavy-electron superconductivity below Tc = 2 mK. Our results demonstrate that superconductivity driven by quantum criticality is a general phenomenon.Comment: 39 pages including Supplementary Materials. Version before copy-edited by the journa

Expanded metabolite coverage of Saccharomyces cerevisiae extract through improved chloroform/methanol extraction and tert-butyldimethylsilyl derivatization

AbstractWe present an improved extraction and derivatization protocol for GC–MS analysis of amino/non-amino acids in Saccharomyces cerevisiae. Yeast cells were extracted with chloroform: aqueous-methanol (1:1, v/v) and the resulting non-polar and polar extracts combined and dried for derivatization. Polar and non-polar metabolites were derivatized using tert-butyldimethylsilyl (t-BDMS) dissolved in acetonitrile. Using microwave treatment of the samples, the derivatization process could be completed within 2 h (from >20 h of the conventional method), providing fully derivatized metabolites that contain multiple derivatizable organic functional groups. This results in a single derivative from one metabolite, leading to increased accuracy and precision for identification and quantification of the method. Analysis of combined fractions allowed the method to expand the coverage of detected metabolites from polar metabolites i.e. amino acids, organic acids and non-polar metabolites i.e. fatty alcohols and long-chain fatty acids which are normally non detectable. The recoveries of the extraction method was found at 88 ± 4%, RSD, N = 3 using anthranilic acid as an internal standard. The method promises to be a very useful tool in various aspects of biotechnological applications i.e. development of cell factories, metabolomics profiling, metabolite identification, 13C-labeled flux analysis or semi-quantitative analysis of metabolites in yeast samples

Engineering Yeast Metabolism for Production of Sesquiterpenes

Sesquiterpenes belong to the large and diverse group of isoprenoids, which are ubiquitous in the plant kingdom and have various applications in the chemical industry as fragrances, pharmaceuticals and as substitutes for petroleum-based gasoline, diesel and jet fuels. In this project, production of sesquiterpenes was studied in Saccharomyces cerevisiae using farnesene as an example with the objective to gain new insights into the synthesis of these compounds and to evaluate different metabolic engineering strategies.Farnesyl diphosphate (FPP) represents the universal precursor for all sesquiterpenes and different strategies were addressed to increase production of this intermediate and to allow for its efficient conversion to farnesene. As FPP is provided by the mevalonate pathway, we aimed at increasing the flux through the pathway by incorporation of malonyl-CoA using a recently identified acetoacetyl-CoA synthase from Streptomyces sp. strain CL190. While the enzyme had detrimental effects on growth as well as on product formation, it was able to compensate for the loss of the essential, endogenous acetoacetyl-CoA thiolase. Additionally, a homologous enzyme with superior efficiency could be identified. Secondly, as FPP is required as substrate for different pathways and represents a metabolic branch point, a novel tool for enzyme co-localization was developed to divert more flux towards farnesene. The system utilizes scaffolds based on affibodies and could improve product yields by more than twofold. Furthermore, the role of terpene synthases on the production of farnesene was elucidated by comparing farnesene synthases with different plant origins, i.e. Malus domestica, Citrus junos and Artemisia annua. While the selected candidates produced similar amounts of farnesene (up to 170 mg/L), these enzymes appeared to be superior in comparison to other sesquiterpene synthases, i.e. santalene synthase. Lastly, the response to increased product formation was investigated using transcriptome and metabolome analysis, which highlighted changes across various metabolic pathways and identified pantothenic acid as a potential target for future engineering strategies. In conclusion, the study evaluates different metabolic engineering strategies for production of sesquiterpenes in S. cerevisiae and provides new insights into the cellular response during their production. Additionally, utilization of affibodies as a novel tool in metabolic engineering to increase chemical production in S. cerevisiae was highlighted

Engineering Yeast Metabolism for Production of Sesquiterpenes

Sesquiterpenes belong to the large and diverse group of isoprenoids, which are ubiquitous in the plant kingdom and have various applications in the chemical industry as fragrances, pharmaceuticals and as substitutes for petroleum-based gasoline, diesel and jet fuels. In this project, production of sesquiterpenes was studied in Saccharomyces cerevisiae using farnesene as an example with the objective to gain new insights into the synthesis of these compounds and to evaluate different metabolic engineering strategies.Farnesyl diphosphate (FPP) represents the universal precursor for all sesquiterpenes and different strategies were addressed to increase production of this intermediate and to allow for its efficient conversion to farnesene. As FPP is provided by the mevalonate pathway, we aimed at increasing the flux through the pathway by incorporation of malonyl-CoA using a recently identified acetoacetyl-CoA synthase from Streptomyces sp. strain CL190. While the enzyme had detrimental effects on growth as well as on product formation, it was able to compensate for the loss of the essential, endogenous acetoacetyl-CoA thiolase. Additionally, a homologous enzyme with superior efficiency could be identified. Secondly, as FPP is required as substrate for different pathways and represents a metabolic branch point, a novel tool for enzyme co-localization was developed to divert more flux towards farnesene. The system utilizes scaffolds based on affibodies and could improve product yields by more than twofold. Furthermore, the role of terpene synthases on the production of farnesene was elucidated by comparing farnesene synthases with different plant origins, i.e. Malus domestica, Citrus junos and Artemisia annua. While the selected candidates produced similar amounts of farnesene (up to 170 mg/L), these enzymes appeared to be superior in comparison to other sesquiterpene synthases, i.e. santalene synthase. Lastly, the response to increased product formation was investigated using transcriptome and metabolome analysis, which highlighted changes across various metabolic pathways and identified pantothenic acid as a potential target for future engineering strategies. In conclusion, the study evaluates different metabolic engineering strategies for production of sesquiterpenes in S. cerevisiae and provides new insights into the cellular response during their production. Additionally, utilization of affibodies as a novel tool in metabolic engineering to increase chemical production in S. cerevisiae was highlighted

Developing language learning environment in biology and physics class

Förmågorna som eleverna ska utveckla på grundskolan inom de naturvetenskapliga ämnena bygger till en stor del på deras språkliga förmågor. Detta kan vara en enorm utmaning speciellt för elever med utländsk bakgrund som utgör 26% av alla elever i dagsläget. Mycket forskning har bedrivits beträffande hur andraspråksinlärning fungerar och främjas. Däremot är studier som undersöker såväl utfallet som effekten av ett språkutvecklande arbetssätt för andraspråksinlärare oftast inte tillgängliga. Detta utvecklingsarbete undersöker hur språkutvecklande NO-undervisning, inom biologi och fysik, kan utformas utan att ämnesinnehållet förenklas eller förminskas. Studien genomfördes på två skolor där majoriteten av eleverna har svenska som andraspråk. Eleverna svarade på enkätfrågor om hur de upplevt sitt lärande under lektionerna. Lektionerna hämtade mycket inspiration och metoder från Pauline Gibbons böcker om språkutvecklande arbete i skola. Även om studiens omfång är begränsat visar enkätens utvärdering tillsammans med våra observationer att eleverna har upplevt metoderna som gynnsamma för deras språk- och ämneskunskaper

Improved quantification of farnesene during microbial production from Saccharomyces cerevisiae in two-liquid-phase fermentations

Organic solvents are widely used in microbial fermentations to reduce gas stripping effects and capture hydrophobic or toxic compounds. Reliable quantification of biochemical products in these overlays is highly challenging and practically difficult. Here, we present a significant improvement of identification and quantification methods for farnesene produced by Saccharomyces cerevisiae in two-liquid-phase fermentations using GC-MS and GC-FID. By increasing the polarity of the stationary phase introducing a ZB-50 column (50%-phenyl-50%-dimethylsiloxane) peak intensity could be increased and solvent carryover could be minimized. Direct quantification of farnesene in dodecane was achieved by GC-FID whereas GC-MS demonstrated to be an excellent technique for identification of known and unknown metabolites. The GC-FID is a suitable technique for direct quantification of farnesene in complex matrices as shown by the good calibration curve (R2>0.998, N=5) within the tested concentration range of 1-50 µg/mL and the reproducibility of the intensity (intraday; <10% RSD at each concentration; N=5). The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.24 and 0.80 µg/mL, respectively. Furthermore, the FID method proved to be highly stable with regard to the intensity of the calibration (N=6) when the measurements were performed across 250 samples that were derived from a dodecane overlay

Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.

Enzyme fusions have been widely used as a tool in metabolic engineering to increase pathway efficiency by reducing substrate loss and accumulation of toxic intermediates. Alternatively, enzymes can be colocalized through attachment to a synthetic scaffold via noncovalent interactions. Here we describe the use of affibodies for enzyme tagging and scaffolding. The scaffolding is based on the recognition of affibodies to their anti-idiotypic partners in vivo, and was first employed for colocalization of farnesyl diphosphate synthase and farnesene synthase in S. cerevisiae. Different parameters were modulated to improve the system, and the enzyme:scaffold ratio was most critical for its functionality. Ultimately, the yield of farnesene on glucose YSFar could be improved by 135% in fed-batch cultivations using a 2-site affibody scaffold. The scaffolding strategy was then extended to a three-enzyme polyhydroxybutyrate (PHB) pathway, heterologously expressed in E. coli. Within a narrow range of enzyme and scaffold induction, the affibody tagging and scaffolding increased PHB production 7-fold. This work demonstrates how the versatile affibody can be used for metabolic engineering purposes