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Strategies for investigating and optimizing the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations., Heterologous expression of recombinant proteins often results in misfolding, aggregation and degradation. Here, we show an in vivo dual-biosensor system that simultaneously assesses protein translation and protein folding, thereby enabling rapid screening of expression strains as well as mutant libraries.

Inducible expression systems can be applied to control the expression of proteins or biochemical pathways in cell factories. However, several of the established systems require the addition of expensive inducers, making them unfeasible for large‐scale production. Here, we establish a genome integrated trp‐T7 expression system where tryptophan can be used to control the induction of a gene or a metabolic pathway. We show that the initiation of gene expression from low‐ and high‐copy vectors can be tuned by varying the initial concentration of tryptophan or yeast extract, and that expression is tightly regulated and homogenous when compared with the commonly used lac‐T7 system. Finally, we apply the trp‐T7 expression system for the production of l‐serine, where we reach titers of 26 g/L in fed‐batch fermentation., Inducible expression of biochemicals and proteins is widely used. Here, a tryptophan‐controlled, tightly regulated and tuneable T7‐based induction system is described and shown to be compatible with fed‐batch fermentation.

Background Sucrose is an attractive industrial carbon source due to its abundance and the fact that it can be cheaply generated from sources such as sugarcane. However, only a few characterized Escherichia coli strains are able to metabolize sucrose, and those that can are typically slow growing or pathogenic strains. Methods To generate a platform strain capable of efficiently utilizing sucrose with a high growth rate, adaptive laboratory evolution (ALE) was utilized to evolve engineered E. coli K-12 MG1655 strains containing the sucrose utilizing csc genes (cscB, cscK, cscA) alongside the native sucrose consuming E. coli W. Results Evolved K-12 clones displayed an increase in growth and sucrose uptake rates of 1.72- and 1.40-fold on sugarcane juice as compared to the original engineered strains, respectively, while E. coli W clones showed a 1.4-fold increase in sucrose uptake rate without a significant increase in growth rate. Whole genome sequencing of evolved clones and populations revealed that two genetic regions were frequently mutated in the K-12 strains; the global transcription regulatory genes rpoB and rpoC, and the metabolic region related to a pyrimidine biosynthetic deficiency in K-12 attributed to pyrE expression. These two mutated regions have been characterized to confer a similar benefit when glucose is the main carbon source, and reverse engineering revealed the same causal advantages on M9 sucrose. Additionally, the most prevalent mutation found in the evolved E. coli W lineages was the inactivation of the cscR gene, the transcriptional repression of sucrose uptake genes. Conclusion The generated K-12 and W platform strains, and the specific sets of mutations that enable their phenotypes, are available as valuable tools for sucrose-based industrial bioproduction in the facile E. coli chassis. Electronic supplementary material The online version of this article (10.1186/s12934-019-1165-2) contains supplementary material, which is available to authorized users.

Development of microsystems, which enable "sample-to-answer" detection from real samples, is often challenging. We present the first integration of supported liquid membrane extraction combined with electrochemical detection on a centrifugal fluidic platform. The developed lab-on-a-disc (LoD) system enabled the separation, enrichment, and subsequent electrochemical detection of the target analyte from a complex sample mixture. As a case study, we quantified the amount of a dietary supplement and pharmaceutical precursor, p-coumaric acid, from bacterial growth media at different time points during production. The assay, extraction, and detection, performed on the LoD device, proved to be a low cost and environmentally friendly approach, requiring only a few tens of microliters of organic solvent and enabled detection in a 3 μL volume. In addition, the data obtained from the centrifugal platform showed a good correlation with data obtained from the high performance liquid chromatography analysis.

Growth decoupling can be used to optimize production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference (CRISPRi) to increase production of a single domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways, and found that repression of 11 pathway genes led to increased GFP production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.

Growth decoupling can be used to optimize microbial production of biobased compounds by inhibiting excess biomass formation and redirect carbon flux from growth to product formation. However, identifying suitable genetic targets through rational design is challenging. Here, we conduct a genome-wide CRISPRi screen to discover growth switches suitable for decoupling growth and production. Using an sgRNA library covering 12 238 loci in the

Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single‐domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression‐optimized sdAb. Proteomics analysis of the sdAb‐producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome‐associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake‐flask fermentation where the product yield was improved 2.6‐fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best‐performing pyrG growth decoupling strain., Decoupling growth and production is a promising approach to improve cell factory performance. In this study, Landberg et al. inhibit the expression of selected genes in the nucleotide metabolism to stop cell growth and increase the production of a single‐domain antibody. Using proteomics, they show that the growth inhibited cells maintain their capacity for protein synthesis and do not enter stationary phase.

Publisher's version (útgefin grein)., Background: Lactobacillus reuteri is a heterofermentative Lactic Acid Bacterium (LAB) that is commonly used for food fermentations and probiotic purposes. Due to its robust properties, it is also increasingly considered for use as a cell factory. It produces several industrially important compounds such as 1,3-propanediol and reuterin natively, but for cell factory purposes, developing improved strategies for engineering and fermentation optimization is crucial. Genome-scale metabolic models can be highly beneficial in guiding rational metabolic engineering. Reconstructing a reliable and a quantitatively accurate metabolic model requires extensive manual curation and incorporation of experimental data. Results: A genome-scale metabolic model of L. reuteri JCM 1112T was reconstructed and the resulting model, Lreuteri_530, was validated and tested with experimental data. Several knowledge gaps in the metabolism were identified and resolved during this process, including presence/absence of glycolytic genes. Flux distribution between the two glycolytic pathways, the phosphoketolase and Embden-Meyerhof-Parnas pathways, varies considerably between LAB species and strains. As these pathways result in different energy yields, it is important to include strain-specific utilization of these pathways in the model. We determined experimentally that the Embden-Meyerhof-Parnas pathway carried at most 7% of the total glycolytic flux. Predicted growth rates from Lreuteri_530 were in good agreement with experimentally determined values. To further validate the prediction accuracy of Lreuteri_530, the predicted effects of glycerol addition and adhE gene knock-out, which results in impaired ethanol production, were compared to in vivo data. Examination of both growth rates and uptake- and secretion rates of the main metabolites in central metabolism demonstrated that the model was able to accurately predict the experimentally observed effects. Lastly, the potential of L. reuteri as a cell factory was investigated, resulting in a number of general metabolic engineering strategies. Conclusion: We have constructed a manually curated genome-scale metabolic model of L. reuteri JCM 1112T that has been experimentally parameterized and validated and can accurately predict metabolic behavior of this important platform cell factory., This study was supported by the Marine Biotechnology ERA-NET Thermo-Factories project grant number 5178–00003B; the Technology Development fund in Iceland grant number 159004-0612; The Novo Nordisk Foundation in Denmark; and the European Union’s Horizon 2020 research and innovation programme under grant agreement No 686070 (DD-DeCaF).

The number of newly developed genetic variants of microbial cell factories for production of biochemicals has been rapidly growing in recent years, leading to an increased need for new screening techniques. We developed a method based on surface-enhanced Raman scattering (SERS) coupled with liquid-liquid extraction (LLE) for quantification of p-coumaric acid (pHCA) in the supernatant of genetically engineered Escherichia coli (E. coli) cultures. pHCA was measured in a dynamic range from 1 μM up to 50 μM on highly uniform SERS substrates based on leaning gold-capped nanopillars, which showed an in-wafer signal variation of only 11.7%. LLE using dichloromethane as organic phase was combined with the detection in order to increase selectivity and sensitivity by decreasing the effect of interfering compounds from the analytes of interest. The difference in pHCA production yield between three genetically engineered E. coli strains was successfully evaluated using SERS and confirmed with high-performance liquid chromatography. As this novel approach has potential to be automated and parallelized, it can be considered for high-throughput screening in metabolic engineering.

Microbial cell factories have been engineered to produce a variety of biochemicals, ranging from biofuels, food addtives to pharmaceuticals. However, for most compounds, the production yield is far from reaching economical targets. Accumulation of excess biomass contributes to decreasing production yields, and a method for limiting biomass formation while allowing for continued production of biochemicals is therefore desirable. In this study, we investigated eight different culturing setups aiming at inhibiting biomass formation of Escherichia coli, based on nutrient limitations or the addition of growth inhibitors. The ability to control cell growth and the production of biochemicals, exemplified by mevalonate and tyrosine, was characterized. An increased mass yield of both mevalonate and tyrosine was achieved by limiting phosphate, sulfate or magnesium in the media. Sulfate limitation, in particular, resulted in an increase in mass yield of mevalonate and tyrosine by 80% and 50%, respectively. By tracking production and biomass concentrations, it was observed that the production was maintained for more than 10 h after inhibition of cell growth, despite cell maintenance requirements. The outlined method serves as promising approach for increasing production yield of a range of different biochemicals.

Acetogens are microbes which produce acetate as a fermentation by-product. They have diverse phylogeny but a metabolic feature in common called the Woods-Ljungdahl Pathway (WLP), which confers the ability to fix carbon dioxide via a non-photosynthetic route. Electrons for this process are derived from diverse substrates including molecular hydrogen and carbon monoxide. The ability of acetogens to utilise components of syngas (H2, CO, CO2) make them an attractive target for metabolic engineering for industrially relevant products. We have previously reported the construction of a genome-scale metabolic model of the model acetogen Acetobacterium woodii using a sequenced and annotated genome of strain DSM1030. The model consists of 836 metabolites, 909 reactions and 84 transporters and can account for growth on diverse substrates reported in the literature. We identified the reactions used to catabolise fifteen single substrates and 121 substrate pair combinations, and used this to construct a sub-model representing a core set of energy producing catabolic pathways. We then introduced heterologous reactions to allow for the production of chemical of interest. Elementary modes analysis of this extended sub-model was applied to further decompose it into unique sets of the smallest functioning sub-networks. With CO2 and H2 as substrates, we find routes for the production of several chemicals where small amounts of excess ATP are produced simultaneously. Repeated analysis with alternative renewable feedstocks such as methanol and formate, indicate a wider potential in producing compounds of interest while also maintaining energy generation and co-factor conservation.

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs., Single molecule real-time DNA sequencing allows genome-wide identification of DNA methylation patterns. Here, Jensen et al. present a high-throughput method that allows rapid coupling of DNA methylation patterns with their corresponding methyltransferase genes in bacteria.

Given the commercial importance of the compounds produced by genetically modified organisms, there is a need for screening methods which facilitate the evaluation of newly developed strains, especially during the phase of proof-of-concept development. We report a time-efficient analysis method for the screening of bacterial strains, which enables the detection of two structurally similar secondary bacterial metabolites. By combining liquid-liquid extraction and surface-enhanced Raman scattering we were able to quantify p-coumaric acid and cinnamic acid, produced by genetically modified E. coli from tyrosine and phenylalanine, respectively. With the simple sample pre-treatment method, and by applying a partial least squares data analysis method, we simultaneously detected the analytes from four E. coli strains cultured in the presence or absence of tyrosine and phenylalanine.

L-serine is a widely used amino acid that has been proposed as a potential building block biochemical. The high theoretical yield from glucose makes a fermentation based production attractive. In order to achieve this goal, serine degradation to pyruvate and glycine in E. coli MG1655 was prevented by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very high serine production yield (0.45 g/g glucose) during small-scale batch fermentation in minimal medium. Serine, however, was found to be highly toxic even at low concentrations to this strain, which lead to slow growth and production during fed batch fermentation, resulting in a serine production of 8.3 g/L. The production strain was therefore evolved by random mutagenesis to achieve increased tolerance towards serine. Additionally, overexpression of eamA, a cysteine/homoserine transporter was demonstrated to increase serine tolerance from 1.6 g/L to 25 g/L. During fed batch fermentation, the resulting strain lead to the serine production titer of 11.7 g/L with yield of 0.43 g/g glucose, which is the highest yield reported so far for any organism.

A standard protocol for analysis of microbial factories requires the screening of several populations in order to find the best performing ones. Standard analytical methods usually include high performance liquid chromatography (HPLC), thin layer chromatography (TLC) or spectrophotometry, which are expensive and time-consuming processes. Surface Enhanced Raman Spectroscopy (SERS), instead, is a highly sensitive spectroscopic technique for specific, fast and real-time sensing of biological samples. Here we demonstrate the use of SERS to discriminate between two different bacterial populations based on detection of p-coumaric acid (pHCA) in cell supernatant. SERS active substrates, based on leaning gold-capped silicon nanopillars, were used for detection. They were successfully used to detect culture medium spiked with pHCA, and the effect of medium dilution was studied. For analysis of biological production of pHCA, triplicate cultures of E. coli strains expressing a pHCA-forming enzyme (P) as well as of a non-producing strain (C) were grown. Then, supernatant samples were collected and their pHCA content was measured using SERS and HPLC for comparison. The intensity of the pHCA Raman mode at 1169 cm-1 (CH-rocking motion) showed different trends for P and C strains, similar to the results obtained using the HPLC method. Results illustrate that SERS can be used for quick and semi- quantitative discrimination of pHCA concentrations in cell supernatant medium.

This mini-review provides an overview of traditional, emerging, and future applications of lactic acid bacteria (LAB) and discusses how genome editing tools can be used to overcome current challenges in all these applications. It also describes currently available tools and how these can be further developed, and takes current legislation into account. Genome editing tools are necessary for the construction of strains for new applications and products, but can also play a crucial role in traditional ones, such as food and probiotics, as a research tool for understanding mechanistic insights and discovering new properties. Traditionally, recombinant DNA techniques for LAB have strongly focused on being food-grade, but they lack throughput and the number of genetically tractable strains is still rather limited. Further tool development in this direction will enable rapid construction of multiple mutants or mutant libraries on a genomic level in a wide variety of LAB strains. We also propose an iterative Design-Build-Test-Learn workflow cycle for LAB cell factory development based on systems biology, with “cell factory” expanding beyond its traditional meaning of production strains and making use of high-throughput genome editing tools to advance LAB understanding, applications and strain development.

Biological production and application of a range of organic compounds is hindered by their limited solubility and toxicity. This work describes a process for functionalization of phenolic compounds that increases solubility and decreases toxicity. We achieve this by screening a wide range of sulfotransferases for their activity towards a range of compounds, including the antioxidant resveratrol. We demonstrate how to engineer cell factories for efficiently creating sulfate esters of phenolic compounds through the use of sulfotransferases and by optimization of sulfate uptake and sulfate nucleotide pathways leading to the 3′-phosphoadenosine 5′-phosphosulfate precursor (PAPS). As an example we produce the antifouling agent zosteric acid, which is the sulfate ester of p-coumaric acid, reaching a titer of 5 g L−1 in fed-batch fermentation. The described approach enables production of sulfate esters that are expected to provide new properties and functionalities to a wide range of application areas., Toxicity and limited solubility inhibits the biological production of many organic compounds. Here the authors metabolically engineer sulfate uptake and activation in order to produce sulfate esters of phenolic compounds, such as zosteric acid, thereby addressing these issues.

In vitro selection of aptamers that recognize small organic molecules has proven difficult, in part due to the challenge of immobilizing small molecules on solid supports for SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This study describes the implementation of RNA Capture-SELEX, a selection strategy that uses an RNA library to yield ligand-responsive RNA aptamers targeting small organic molecules in solution. To demonstrate the power of this method we selected several aptamers with specificity towards either the natural sweetener rebaudioside A or the food-coloring agent carminic acid. In addition, Bio-layer interferometry is used to screen clonal libraries of aptamer candidates and is used to interrogate aptamer affinity. The RNA-based Capture-SELEX strategy described here simplifies selection of RNA aptamers against small molecules by avoiding ligand immobilization, while also allowing selection against multiple candidate targets in a single experiment. This makes RNA Capture-SELEX particularly attractive for accelerated development of RNA aptamers targeting small metabolites for incorporation into synthetic riboswitches and for analytical biosensors.

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 towards eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant towards three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found towards p-coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome-wide approach, Tn-seq. Libraries containing a large number of miniTn5-Km transposon insertion mutants were grown in the presence and absence of p-coumaric acid, and the enrichment or depletion of mutants was quantified by high-throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance towards p-coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance towards p-coumaric acid is related to transport and membrane integrity. This article is protected by copyright. All rights reserved.

The use of gas fermentation for production of chemicals and fuels with lower environmental impact is a technology that is gaining increasing attention. Over 38 Gt of CO2 is annually being emitted from industrial processes, thereby contributing significantly to the concentration of greenhouse gases in the atmosphere. Together with the gasification of biomass and different waste streams, these gases have the potential for being utilized for production of chemicals through fermentation processes. Acetogens are among the most studied organisms capable of utilizing waste gases. Although engineering of heterologous production of higher value compounds has been successful for a number of acetogens, the processes are challenging due to the redox balance and the lack of efficient engineering tools. In this review, we address the availability of different gaseous feedstock and gasification processes, and we focus on the advantages of alternative fermentation scenarios, including thermophilic production strains, multi-stage fermentations, mixed cultures, as well as mixotrophy. Such processes have the potential to significantly broaden the product portfolio, increase the product concentrations and yields, while enabling the exploitation of alternative and mixed feedstocks. The reviewed processes also have the potential to address challenges associated with product inhibition and may contribute to reducing the costs of downstream processing. Given the widespread availability of gases, such processes will likely significantly impact the transition towards a more sustainable society.

During the last few decades, great advances have been reached in high-throughput design and building of genetically engineered microbial strains, leading to a need for fast and reliable screening methods. We developed and optimized a microfluidic supported liquid membrane (SLM) extraction device and combined it with surface enhanced Raman scattering (SERS) sensing for the screening of a biological process, namely for the quantification of a bacterial secondary metabolite, p-coumaric acid (pHCA), produced by Escherichia coli. The microfluidic device proved to be robust and reusable, enabling efficient removal of interfering compounds from the real samples, reaching more than 13-fold up-concentration of the donor at 10 μL min−1 flow rate. With this method, we quantified pHCA directly from the bacterial supernatant, distinguishing between various culture conditions based on the pHCA production yield. The obtained data showed good correlation with HPLC analysis.

Species of genus Geobacillus are thermophilic bacteria and play an ever increasing role as hosts for biotechnological applications both in academia and industry. Here we screened a number of Geobacillus strains to determine which industrially relevant carbon sources they can utilize. One of the strains, G. thermoglucosidasius C56-YS93, was then chosen to develop a toolbox for controlled gene expression over a wide range of levels. It includes a library of semi-synthetic constitutive promoters (76-fold difference in expression levels) and an inducible promoter from the xylA gene. A library of synthetic in silico designed ribosome binding sites was also created for further tuning of translation. The PxylA was further used to successfully express native and heterologous xylanases in G. thermoglucosidasius. This toolbox enables fine-tuning of gene expression in Geobacillus species for metabolic engineering approaches in production of biochemicals and heterologous proteins.

Pseudomonas putida KT2440 has gained increasing interest as a host for the production of biochemicals. Because of the lack of a systematic characterization of inducible promoters in this strain, we generated ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning. A set of ProUSER-reporter vectors was further created to characterize different inducible promoters. The PrhaB and Pm promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of p-coumaric acid, P. putida was engineered to prevent degradation of tyrosine and p-coumaric acid. Pm and PrhaB were used to control the expression of a tyrosine ammonia lyase or AroG* and TyrA* involved in tyrosine production, respectively. Pathway expression was optimized by modulating inductions, resulting in small-scale p-coumaric acid production of 1.2 mM, the highest achieved in Pseudomonads under comparable conditions. With broad-host-range compatibility, the ProUSER vectors will serve as useful tools for optimizing gene expression in a variety of bacteria.

Background Economically viable biobased production of bulk chemicals and biofuels typically requires high product titers. During microbial bioconversion this often leads to product toxicity, and tolerance is therefore a critical element in the engineering of production strains. Results Here, a systems biology approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress. Twelve chemicals with significant production potential were selected, consisting of organic solvent-like chemicals (butanol, hydroxy-γ-butyrolactone, 1,4-butanediol, furfural), organic acids (acetate, itaconic acid, levulinic acid, succinic acid), amino acids (serine, threonine) and membrane-intercalating chemicals (decanoic acid, geraniol). The transcriptional response towards these chemicals revealed large overlaps of transcription changes within and between chemical groups, with functions such as energy metabolism, stress response, membrane modification, transporters and iron metabolism being affected. Regulon enrichment analysis identified key regulators likely mediating the transcriptional response, including CRP, RpoS, OmpR, ArcA, Fur and GadX. These regulators, the genes within their regulons and the above mentioned cellular functions therefore constitute potential targets for increasing E. coli chemical tolerance. Fitness determination of genome-wide transposon mutants (Tn-seq) subjected to the same chemical stress identified 294 enriched and 336 depleted mutants and experimental validation revealed up to 60 % increase in mutant growth rates. Mutants enriched in several conditions contained, among others, insertions in genes of the Mar-Sox-Rob regulon as well as transcription and translation related gene functions. Conclusions The combination of the transcriptional response and mutant screening provides general targets that can increase tolerance towards not only single, but multiple chemicals. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0577-5) contains supplementary material, which is available to authorized users.

Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. 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. Results Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes. Conclusions The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0288-3) contains supplementary material, which is available to authorized users.

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. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p -coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus , a bacterial-type enzyme from the amoeba Dictyostelium discoideum , and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD 600 ]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p -coumaric acid in Escherichia coli (producing 440 μM p -coumaric acid OD 600 unit −1 in the medium) and in Lactococcus lactis . The enzymes were also efficient in Saccharomyces cerevisiae , where p -coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.

Chitin, an insoluble polymer of GlcNAc, is an abundant source of carbon, nitrogen, and energy for marine microorganisms. Microarray expression profiling and mutational studies of Vibrio cholerae growing on a natural chitin surface, or with the soluble chitin oligosaccharides (GlcNAc) 2–6 , GlcNAc, or the glucosamine dimer (GlcN) 2 identified three sets of differentially regulated genes. We show that ( i ) ChiS, a sensor histidine kinase, regulates expression of the (GlcNAc) 2–6 gene set, including a (GlcNAc) 2 catabolic operon, two extracellular chitinases, a chitoporin, and a PilA-containing type IV pilus, designated ChiRP ( chi tin- r egulated p ilus) that confers a significant growth advantage to V. cholerae on a chitin surface; ( ii ) GlcNAc causes the coordinate expression of genes involved with chitin chemotaxis and adherence and with the transport and assimilation of GlcNAc; ( iii ) (GlcN) 2 induces genes required for the transport and catabolism of nonacetylated chitin residues; and ( iv ) the constitutively expressed MSHA pilus facilitates adhesion to the chitin surface independent of surface chemistry. Collectively, these results provide a global portrait of a complex, multistage V. cholerae program for the efficient utilization of chitin.

Polyphosphate- and polyhydroxyalkanoate (PHA)-accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods. Results of 16S rDNA clone library and fluorescence in situ hybridization (FISH) with rRNA-targeted, group-specific oligonucleotide probes indicated that the microbial community consisted mostly of the alpha- (9.5% of total cells), beta- (41.3%) and gamma- (6.8%) subclasses of the class Proteobacteria, Flexibacter-Cytophaga (4.5%) and the Gram-positive high G+C (HGC) group (17.9%). With individual phylogenetic groups or subgroups, members of Candidatus Accumulibacter phosphatis in the beta-2 subclass, a novel HGC group closely related to Tetrasphaera spp., and a novel gamma-proteobacterial group were the predominant populations. Furthermore, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups. Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA. In addition, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA. No cellular inclusions were detected in most members of the alpha-Proteobacteria and the Cytophaga-Flavobacterium group. The diversified functional traits observed suggested that different substrate metabolisms were used by predominant phylogenetic groups in EBPR processes.

A flow cell-grown model consortium consisting of two organisms, Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1), was studied. These bacteria have the potential to interact metabolically because Pseudomonas sp. B13(FR1) can metabolize chlorobenzoate produced by Burkholderia sp. LB400 when grown on chlorobiphenyl. The expected metabolic interactions in the consortium were demonstrated by high performance liquid chromatography (HPLC) analysis. The spatial structure of the consortium was studied by fluorescent in situ rRNA hybridization and scanning confocal laser microscopy. When the consortium was fed with medium containing a low concentration of chlorobiphenyl, microcolonies consisting of associated Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1) bacteria were formed, and separate Pseudomonas sp. B13(FR1) microcolonies were evidently not formed. When the consortium was fed citrate, which can be metabolized by both species, the two species formed separate microcolonies. The structure development In the consortium was studied online using a gfp-tagged Pseudomonas sp. B13(FR1) derivative. After a shift In carbon source from citrate to a low concentration of chlorobiphenyl, movement of the Pseudomonas sp. B13(FR1) bacteria led to a change in the spatial structure of the consortium from the unassociated form towards the associated form within a few days. Experiments Involving a gfp-based Pseudomonas sp. B13(FR1) growth activity reporter strain Indicated that chlorobenzoate supporting growth of Pseudomonas sp. B13(FR1) is located close to the Burkholderia sp. LB400 microcolonies in chlorobiphenyl-grown consortia.

Locked nucleic acid (LNA) is a new class of bicyclic high affinity DNA analogs. LNA-containing oligonucleotides confer significantly increased affinity against their complementary DNA targets, increased mismatch discrimination (delta Tm) and allow full control of the melting point of the hybridization reaction. LNA chemistry is completely compatible with the traditional DNA phosphoramidite chemistry and therefore LNA-DNA mixmer oligonucleotides can be designed with complete freedom for optimal performance. These properties render LNA oligonucleotides very well suited for SNP genotyping and have enabled several approaches for enzyme-independent SNP genotyping based on allele-specific hybridization. In addition, allele-specific PCR assays relying on enzymatically-enhanced discrimination can be improved using LNA-modified oligonucleotides. The use of LNA transforms enzyme-independent genotyping approaches into experimentally simple, robust and cost-effective assays, which are highly suited for genotyping in clinical and industrial settings.