Christian Bille Jendresen

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.

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

ABSTRACT 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.

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.

Bistability in a metabolic network underpins the de novo evolution of colony switching in Pseudomonas fluorescens

Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation of a number of underlying molecular mechanisms. However, little is known about how phenotypic switches arise and function in their early evolutionary stages. The first opportunity to provide empirical insight was delivered by an experiment in which populations of the bacterium Pseudomonas fluorescens SBW25 evolved, de novo, the ability to switch between two colony phenotypes. Here we unravel the molecular mechanism behind colony switching, revealing how a single nucleotide change in a gene enmeshed in central metabolism (carB) generates such a striking phenotype. We show that colony switching is underpinned by ON/OFF expression of capsules consisting of a colanic acid-like polymer. We use molecular genetics, biochemical analyses, and experimental evolution to establish that capsule switching results from perturbation of the pyrimidine biosynthetic pathway. Of central importance is a bifurcation point at which uracil triphosphate is partitioned towards either nucleotide metabolism or polymer production. This bifurcation marks a cell-fate decision point whereby cells with relatively high pyrimidine levels favour nucleotide metabolism (capsule OFF), while cells with lower pyrimidine levels divert resources towards polymer biosynthesis (capsule ON). This decision point is present and functional in the wild-type strain. Finally, we present a simple mathematical model demonstrating that the molecular components of the decision point are capable of producing switching. Despite its simple mutational cause, the connection between genotype and phenotype is complex and multidimensional, offering a rare glimpse of how noise in regulatory networks can provide opportunity for evolution.

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.

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.

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.

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.

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.

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.