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.

Surface Enhanced Raman Scattering for Quantification of p-Coumaric Acid Produced by Escherichia coli.

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.

Quantification of a bacterial secondary metabolite by SERS combined with SLM extraction for bioprocess monitoring.

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.

Enhanced protein and biochemical production using CRISPRi-based growth switches.

Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2.2-fold. Single-cell dynamic tracking in microfluidic systems was used to confirm functionality of the growth switches. Decoupling of growth from production of biochemicals was demonstrated for mevalonate, a precursor for isoprenoid compounds. Mass yield of mevalonate was increased by 41%, and production was maintained for more than 45h after activation of the pyrF-based growth switch. The developed methods represent a promising approach for increasing production yield and titer for proteins and biochemicals.

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. 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 [OD600]) 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 OD600 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.

Towards in vivo regulon kinetics: PurR activation by 5-phosphoribosyl-α-1-pyrophosphate during purine depletion in Lactococcus lactis.

Short-term adaptation to changing environments relies on regulatory elements translating shifting metabolite concentrations into a specifically optimized transcriptome. So far the focus of analyses has been divided between regulatory elements identified in vivo and kinetic studies of small molecules interacting with the regulatory elements in vitro. Here we describe how in vivo regulon kinetics can describe a regulon through the effects of the metabolite controlling it, exemplified by temporal purine exhaustion in Lactococcus lactis. We deduced a causal relation between the pathway precursor 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and individual mRNA levels, whereby unambiguous and homogeneous relations could be obtained for PurR regulated genes, thus linking a specific regulon to a specific metabolite. As PurR activates gene expression upon binding of PRPP, the pur mRNA curves reflect the in vivo kinetics of PurR PRPP binding and activation. The method singled out the xpt-pbuX operon as kinetically distinct, which was found to be caused by a guanine riboswitch whose regulation was overlaying the PurR regulation. Importantly, genes could be clustered according to regulatory mechanism and long-term consequences could be distinguished from transient changes--many of which would not be seen in a long-term adaptation to a new environment. The strategy outlined here can be adapted to analyse the individual effects of members from larger metabolomes in virtually any organism, for elucidating regulatory networks in vivo.

Optics miniaturization strategy for demanding Raman spectroscopy applications.

Raman spectroscopy provides non-destructive, label-free quantitative studies of chemical compositions at the microscale as used on NASA's Perseverance rover on Mars. Such capabilities come at the cost of high requirements for instrumentation. Here we present a centimeter-scale miniaturization of a Raman spectrometer using cheap non-stabilized laser diodes, densely packed optics, and non-cooled small sensors. The performance is comparable with expensive bulky research-grade Raman systems. It has excellent sensitivity, low power consumption, perfect wavenumber, intensity calibration, and 7 cm-1 resolution within the 400-4000 cm-1 range using a built-in reference. High performance and versatility are demonstrated in use cases including quantification of methanol in beverages, in-vivo Raman measurements of human skin, fermentation monitoring, chemical Raman mapping at sub-micrometer resolution, quantitative SERS mapping of the anti-cancer drug methotrexate and in-vitro bacteria identification. We foresee that the miniaturization will allow realization of super-compact Raman spectrometers for integration in smartphones and medical devices, democratizing Raman technology.

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.

The PurR regulon in Lactococcus lactis - transcriptional regulation of the purine nucleotide metabolism and translational machinery.

Purine nucleotides are either synthesized de novo from 5-phosphoribosyl-1-pyrophosphate (PRPP) or salvaged from the environment. In Lactococcus lactis, transcription of the de novo synthesis operons, purCSQLF and purDEK, has genetically been shown to be activated by the PurR protein when bound to a conserved PurBox motif present on the DNA at a fixed distance from the promoter -10 element. PurR contains a PRPP-binding site, and activation occurs when the intracellular PRPP pool is high as a consequence of low exogenous purine nucleotide pools. By an iterative approach of bioinformatics searches and motif optimization, 21 PurR-regulated genes were identified and used in a redefinition of the PurBox consensus sequence. In the process a new motif, the double-PurBox, which is present in a number of promoters and contains two partly overlapping PurBox motifs, was established. Transcriptional fusions were used to analyse wild-type promoters and promoters with inactivating PurBox mutations to confirm the relevance of the PurBox motifs as PurR-binding sites. The promoters of several operons were shown to be devoid of any -35 sequence, and found to be completely dependent on PurR-mediated activation. This suggests that binding of the PurR protein to the PurBox takes over the role of the -35 sequence. The study has expanded the PurR regulon to include promoters in nucleotide metabolism, C(1) compound metabolism, phosphonate transport, pyrophosphatase activity, (p)ppGpp metabolism, and translation-related functions. Of special interest is the presence of PurBox motifs in rrn promoters, suggesting a novel connection between nucleotide availability and the translational machinery.

A simplified method for rapid quantification of intracellular nucleoside triphosphates by one-dimensional thin-layer chromatography.

Quantification of nucleotides is an important part of metabolomics but has been hampered by the lack of fast, sensitive, and reliable methods. We present a less time-consuming, more sensitive, and more precise method for the quantitative determination of nucleoside triphosphates (NTPs), 5-ribosyl-1-pyrophosphate (PRPP), and inorganic pyrophosphate (PP(i)) in cell extracts. The method uses one-dimensional thin-layer chromatography (TLC) and radiolabeled biological samples. Nucleotides are resolved at the level of ionic charge in an optimized acidic ammonium formate and chloride solvent, permitting quantification of NTPs. The method is significantly simpler and faster than both current two-dimensional methods and high-performance liquid chromatography (HPLC)-based procedures, allowing a higher throughput while common sources of inaccuracies and technical problems are avoided. For determination of PP(i), treatment with inorganic pyrophosphatase (PPase) of the radiolabeled phosphate is employed for removal of contaminating pyrophosphate. Biological examples performed in triplicates showed standard deviations of approximately 10% of the mean for the determined concentrations of NTPs.

Two nucleoside transporters in Lactococcus lactis with different substrate specificities.

In an alternative to biosynthesis of nucleotides, most organisms are capable of exploiting exogenous nucleotide sources. In order to do so, the nucleotide precursors must pass the membrane, which requires the presence of transporters. Normally, phosphorylated compounds are not subject to transport, and the utilization of nucleotides is dependent on exogenous phosphatases. The composition of transporters with specificity for purine and pyrimidine nucleosides and nucleobases is subject to variation. The ability of Lactococcus lactis to transport different nucleosides across the cell membrane was characterized at both genetic and physiological level, using mutagenesis and by measuring the growth and uptake of nucleosides in the different mutants supplemented with different nucleosides. Two high affinity transporters were identified: BmpA-NupABC was shown to be an ABC transporter with the ability to actively transport all common nucleosides, whereas UriP was shown to be responsible for the uptake of only uridine and deoxyuridine. Interestingly, the four genes encoding the ABC transporter were found at different positions on the chromosome. The bmpA gene was separated from the nupABC operon by 60 kb. Moreover, bmpA was subject to regulation by purine availability, whereas the nupABC operon was constitutively expressed.

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.

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.

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.

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A.

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5'-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5'-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5'-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5'-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.

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.

Genetic toolbox for controlled expression of functional proteins in Geobacillus spp.

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.

CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae.

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.