An automated workflow for multi-omics screening of microbial model organisms.

Multi-omics datasets are becoming of key importance to drive discovery in fundamental research as much as generating knowledge for applied biotechnology. However, the construction of such large datasets is usually time-consuming and expensive. Automation might enable to overcome these issues by streamlining workflows from sample generation to data analysis.

Laboratory evolution reveals general and specific tolerance mechanisms for commodity chemicals.

Although strain tolerance to high product concentrations is a barrier to the economically viable biomanufacturing of industrial chemicals, chemical tolerance mechanisms are often unknown. To reveal tolerance mechanisms, an automated platform was utilized to evolve Escherichia coli to grow optimally in the presence of 11 industrial chemicals (1,2-propanediol, 2,3-butanediol, glutarate, adipate, putrescine, hexamethylenediamine, butanol, isobutyrate, coumarate, octanoate, hexanoate), reaching tolerance at concentrations 60%-400% higher than initial toxic levels. Sequencing genomes of 223 isolates from 89 populations, reverse engineering, and cross-compound tolerance profiling were employed to uncover tolerance mechanisms.

High-Resolution Scanning of Optimal Biosensor Reporter Promoters in Yeast.

Small-molecule binding allosteric transcription factors (aTFs) derived from bacteria enable real-time monitoring of metabolite abundances, high-throughput screening of genetic designs, and dynamic control of metabolism. Yet, engineering of reporter promoter designs of prokaryotic aTF biosensors in eukaryotic cells is complex. Here we investigate the impact of aTF binding site positions at single-nucleotide resolution in >300 reporter promoter designs in From this we identify biosensor output landscapes with transient and distinct aTF binding site position effects for aTF repressors and activators, respectively.

Glucose-Dependent Promoters for Dynamic Regulation of Metabolic Pathways.

For an industrial fermentation process, it can be advantageous to decouple cell growth from product formation. This decoupling would allow for the rapid accumulation of biomass without inhibition from product formation, after which the fermentation can be switched to a mode where cells would grow minimally and primarily act as catalysts to convert substrate into desired product. The switch in fermentation mode should preferably be accomplished without the addition of expensive inducers.

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data.

Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity.

Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.

Background: In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs.

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes.

Glucose-based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae.

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis.

Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli.

Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer's disease in mice and to prevent complications associated with diabetes type I.

CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae.

Background: One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae.

Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence.

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine.

Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the β-alanine biosynthetic route as the most economically attractive according to the metabolic modeling.

Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance.

Biologically produced 3-hydroxypropionic acid (3 HP) is a potential source for sustainable acrylates and can also find direct use as monomer in the production of biodegradable polymers. For industrial-scale production there is a need for robust cell factories tolerant to high concentration of 3 HP, preferably at low pH. Through adaptive laboratory evolution we selected S.

Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli.

This study describes the construction of two flavonoid biosensors, which can be applied for metabolic engineering of Escherichia coli strains. The biosensors are based on transcriptional regulators combined with autofluorescent proteins. The transcriptional activator FdeR from Herbaspirillum seropedicae SmR1 responds to naringenin, while the repressor QdoR from Bacillus subtilis is inactivated by quercetin and kaempferol.

EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae.

Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system.

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode.

Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals.

Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications.

Background: The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering.

Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes.

The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways.

Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae.

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S.

Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiae.

The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Over-expression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous over-expression of tHMG1 and repression of ERG9 did not further improve cubebol production.

Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering.

A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function.

In silico strategy to rationally engineer metabolite production: A case study for threonine in Escherichia coli.

Genetic engineering of metabolic pathways is a standard strategy to increase the production of metabolites of economic interest. However, such flux increases could very likely lead to undesirable changes in metabolite concentrations, producing deleterious perturbations on other cellular processes. These negative effects could be avoided by implementing a balanced increase of enzyme concentrations according to the Universal Method [Kacser and Acerenza (1993) Eur J Biochem 216:361-367].

Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae.

A eukaryotic mevalonate pathway transferred and expressed in Escherichia coli, and a mammalian hydrocortisone biosynthetic pathway rebuilt in Saccharomyces cerevisiae are examples showing that transferring metabolic pathways from one organism to another can have a powerful impact on cell properties. In this study, we reconstructed the E. coli isoprenoid biosynthetic pathway in S.