Modulating metabolism through synthetic biology: Opportunities for two-stage fermentation.

Bio-based production of fuels, chemicals and materials is needed to replace current fossil fuel based production. However, bio-based production processes are very costly, so the process needs to be as efficient as possible. Developments in synthetic biology tools has made it possible to dynamically modulate cellular metabolism during a fermentation. This can be used towards two-stage fermentations, where the process is separated into a growth and a production phase, leading to more efficient feedstock utilization and thus potentially lower costs. This article reviews the current status and some recent results in application of synthetic biology tools towards two-stage fermentations, and compares this approach to pre-existing ones, such as nutrient limitation and addition of toxins/inhibitors.

Biocatalysis with In-Situ Product Removal Improves p-Coumaric Acid Production.

Natural and pure p-coumaric acid has valuable applications, and it can be produced via bioprocessing. However, fermentation processes have so far been unable to provide sufficient production metrics, while a biocatalytic process decoupling growth and production historically showed much promise. This biocatalytic process is revisited in order to tackle product inhibition of the key enzyme tyrosine ammonia lyase. In situ product removal is proposed as a possible solution, and a polymer/salt aqueous two-phase system is identified as a suitable system for extraction of p-coumaric acid from an alkaline solution, with a partition coefficient of up to 13. However, a 10 % salt solution was found to reduce tyrosine ammonia lyase activity by 19 %, leading to the need for a more dilute system. The cloud points of two aqueous two-phase systems at 40 °C and pH 10 were found to be 3.8 % salt and 9.5 % polymer, and a 5 % potassium phosphate and 12.5 % poly(ethylene glycol-ran-propylene glycol) mW~2500 system was selected for in situ product removal. An immobilized tyrosine ammonia lyase biocatalyst in this aqueous two-phase system produced up to 33 g/L p-coumaric acid within 24 hours, a 1.9-fold improvement compared to biocatalysis without in situ product removal.

Probing efficient microbial CO2 utilisation through metabolic and process modelling.

Acetogenic gas fermentation is increasingly studied as a promising technology to upcycle carbon-rich waste gasses. Currently the product range is limited, and production yields, rates and titres for a number of interesting products do not allow for economically viable processes. By pairing process modelling and host-agnostic metabolic modelling, we compare fermentation conditions and various products to optimise the processes. The models were then used in a simulation of an industrial-scale bubble column reactor. We find that increased temperatures favour gas transfer rates, particularly for the valuable and limiting H2 , while furthermore predicting an optimal feed composition of 9:1 mol H2 to mol CO2 . Metabolically, the increased non-growth associated maintenance requirements of thermophiles favours the formation of catabolic products. To assess the expansion of the product portfolio beyond acetate, both a product volatility analysis and a metabolic pathway model were implemented. In-situ recovery of volatile products is shown to be within range for acetone but challenging due to the extensive evaporation of water, while the direct production of more valuable compounds by acetogens is metabolically unfavourable compared to acetate and ethanol. We discuss alternative approaches to overcome these challenges to utilise acetogenic CO2 fixation to produce a wider range of carbon negative chemicals.

Characterization of tyrosine ammonia lyases from Flavobacterium johnsonian and Herpetosiphon aurantiacus.

p-Coumaric acid (pCA) can be produced via bioprocessing and is a promising chemical precursor to making organic thin film transistors. However, the required tyrosine ammonia lyase (TAL) enzyme generally has a low specific activity and suffers from competitive product inhibition. Here we characterized the purified TAL variants from Flavobacterium johnsoniae and Herpetosiphon aurantiacus in terms of their susceptibility to product inhibition and their activity and stability across pH and temperature via initial rate experiments. FjTAL was found to be more active than previously described and to have a relatively weak affinity for pCA, but modeling revealed that product inhibition would still be problematic at industrially relevant product concentrations, due to the low solubility of the substrate tyrosine. The activity of both variants increased with temperature when tested up to 45°C, but HaTAL1 was more stable at elevated temperature. FjTAL is a promising biocatalyst for pCA production, but enzyme or bioprocess engineering are required to stabilize FjTAL and reduce product inhibition.

An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator.

Parageobacillus thermoglucosidasius is a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential of P. thermoglucosidasius, more efficient tools for genetic engineering are required. The present study describes an improved shuttle vector, which speeds up recombination-based genomic modification by incorporating a thermostable sfGFP variant into the vector backbone. This additional selection marker allows for easier identification of recombinants, thereby removing the need for several culturing steps. The novel GFP-based shuttle is therefore capable of facilitating faster metabolic engineering of P. thermoglucosidasius through genomic deletion, integration, or exchange. To demonstrate the efficiency of the new system, the GFP-based vector was utilised for deletion of the spo0A gene in P. thermoglucosidasius DSM2542. This gene is known to be a key regulator of sporulation in Bacillus subtilis, and it was therefore hypothesised that the deletion of spo0A in P. thermoglucosiadius would produce an analogous sporulation-inhibited phenotype. Subsequent analyses of cell morphology and culture heat resistance suggests that the P. thermoglucosidasius ∆spo0A strain is sporulation-deficient. This strain may be an excellent starting point for future cell factory engineering of P. thermoglucosidasius, as the formation of endospores is normally not a desired trait in large-scale production.

Folding of heterologous proteins in bacterial cell factories: Cellular mechanisms and engineering strategies.

The expression of correctly folded and functional heterologous proteins is important in many biotechnological production processes, whether it is enzymes, biopharmaceuticals or biosynthetic pathways for production of sustainable chemicals. For industrial applications, bacterial platform organisms, such as E. coli, are still broadly used due to the availability of tools and proven suitability at industrial scale. However, expression of heterologous proteins in these organisms can result in protein aggregation and low amounts of functional protein. This review provides an overview of the cellular mechanisms that can influence protein folding and expression, such as co-translational folding and assembly, chaperone binding, as well as protein quality control, across different model organisms. The knowledge of these mechanisms is then linked to different experimental methods that have been applied in order to improve functional heterologous protein folding, such as codon optimization, fusion tagging, chaperone co-production, as well as strain and protein engineering strategies.

High-throughput colorimetric assays optimized for detection of ketones and aldehydes produced by microbial cell factories.

Randomized strain and pathway engineering are critical to improving microbial cell factory performance, calling for the development of high-throughput screening and selection systems. To facilitate this effort, we have developed two 96-well plate format colorimetric assays for reliable quantification of various ketones and aldehydes from culture supernatants, based on either a vanillin-acetone reaction or the 2,4-dinitrophenylhydrazine (2,4-DNPH) reagent. The vanillin-acetone assay enabled accurate and selective measurement of acetone titers up to 2 g l-1 in a minimal culture medium. The 2,4-DNPH-based assay can be used for a wide range of aldehydes and ketones, shown here through the optimization of conditions for 15 different compounds. Both assays were implemented to improve acetone production from different substrates by an engineered Escherichia coli strain. The fast and user-friendly colorimetric assays proposed here open the potential for iterative rounds of (automated) strain and pathway engineering and screening, facilitating the efforts towards further boosting production titers of industrially relevant ketones and aldehydes.

The ProUSER2.0 Toolbox: Genetic Parts and Highly Customizable Plasmids for Synthetic Biology in Bacillus subtilis.

Versatile DNA assembly standards and compatible, well-characterized part libraries are essential tools for creating effective designs in synthetic biology. However, to date, vector standards for Gram-positive hosts have limited flexibility. As a result, users often revert to PCR-based methods for building the desired genetic constructs. These methods are inherently prone to introducing mutations, which is problematic considering vector backbone parts are often left unsequenced in cloning workflows. To circumvent this, we present the ProUSER2.0 toolbox: a standardized vector platform for building both integrative and replicative shuttle vectors forBacillus subtilis. The ProUSER2.0 vectors consist of a ProUSER cassette for easy and efficient insertion of cargo sequences and six exchangeable modules. Furthermore, the standard is semicompatible with several previously developed standards, allowing the user to utilize the parts developed for these. To provide parts for the toolbox, seven novel integration sites and six promoters were thoroughly characterized in B. subtilis. Finally, the capacity of the ProUSER2.0 system was demonstrated through the construction of signal peptide libraries for two industrially relevant proteins. Altogether, the ProUSER2.0 toolbox is a powerful and flexible framework for use in B. subtilis.

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli.

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.

Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli.

Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in E. coli to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ mol-1 more stable than the wild-type. In one case, the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ mol-1 more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

Genome-scale metabolic modeling of P. thermoglucosidasius NCIMB 11955 reveals metabolic bottlenecks in anaerobic metabolism.

Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published 13C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform.

CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli.

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.

Genome-Wide CRISPRi-Based Identification of Targets for Decoupling Growth from Production.

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 Escherichia coli genome, we screen for targets that inhibit growth while allowing for continued protein production. In total, we identify 1332 sgRNAs that simultaneously decrease growth and maintain or increase accumulation of GFP. The top target sibB/ibsB shows more than 5-fold increase in GFP accumulation and 45% decrease in biomass formation. Overall, our genome-wide CRISPRi screen provides key targets for growth decoupling, and the approach can be applied to improve biobased production in other microorganisms.

An autoinducible trp-T7 expression system for production of proteins and biochemicals in Escherichia coli.

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.

Production of zosteric acid and other sulfated phenolic biochemicals in microbial cell factories.

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.

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

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.

Generation of an E. coli platform strain for improved sucrose utilization using adaptive laboratory evolution.

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.

Simultaneous quantification of multiple bacterial metabolites using surface-enhanced Raman scattering.

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.

Extraction, Enrichment, and in situ Electrochemical Detection on Lab-on-a-Disc: Monitoring the Production of a Bacterial Secondary Metabolite.

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.

Injection molded lab-on-a-disc platform for screening of genetically modified E. coli using liquid-liquid extraction and surface enhanced Raman scattering.

We present the development of an automated centrifugal microfluidic platform with integrated sample pre-treatment (filtration and liquid-liquid extraction) and detection (SERS-based sensing). The platform consists of eight calibration and four assay modules, fabricated with polypropylene using injection molding and bonded with ultrasonic welding. The platform was used for detection of a secondary bacterial metabolite (p-coumaric acid) from bacterial supernatant. The obtained extraction efficiency was comparable to values obtained in batch experiments and the SERS-based sensing showed a good correlation with HPLC analysis.