Elleke Fenna Bosma

Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit

The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering.

Sphingomyelin synthase-related protein SMSr is a suppressor of ceramide-induced mitochondrial apoptosis

ABSTRACT Cells synthesize ceramides in the endoplasmic reticulum (ER) as precursors for sphingolipids to form an impermeable plasma membrane. As ceramides are engaged in apoptotic pathways, cells would need to monitor their levels closely to avoid killing themselves during sphingolipid biosynthesis. How this is accomplished remains to be established. Here we identify SMSr (SAMD8), an ER-resident ceramide phosphoethanolamine (CPE) synthase, as a suppressor of ceramide-mediated cell death. Disruption of SMSr catalytic activity causes a rise in ER ceramides and their mislocalization to mitochondria, triggering a mitochondrial pathway of apoptosis. Blocking de novo ceramide synthesis, stimulating ceramide export from the ER or targeting a bacterial ceramidase to mitochondria rescues SMSr-deficient cells from apoptosis. We also show that SMSr-catalyzed CPE production, although essential, is not sufficient to suppress ceramide-induced cell death and that SMSr-mediated ceramide homeostasis requires the N-terminal sterile &agr;-motif, or SAM domain, of the enzyme. These results define ER ceramides as bona fide transducers of mitochondrial apoptosis and indicate a primary role of SMSr in monitoring ER ceramide levels to prevent inappropriate cell death during sphingolipid biosynthesis.

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

ABSTRACT Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C5 and C6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 103 colonies per μg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C5 and C6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals.

Sustainable Production of Bio-Based Chemicals by Extremophiles

To improve microbial fermentation as an efficient way to sustainably produce green chemicals from renewable resources, novel production organisms are being explored. Extremophiles, in general, and moderate thermophiles in particular, offer important advantages over well-known mesophilic biotechnology hosts, such as Escherichia coli and Saccharomyces cerevisiae. These advantages include robust performance at temperatures that allow high substrate and product solubility, decreased contamination risk and growth conditions that match with the optimal conditions for enzymes used in saccharification processes, allowing efficient simultaneous saccharification and fermentation. In this review we will evaluate the use of extremophiles for the production of bio-based chemicals, with the main focus on the potential of these organisms for efficient production of bulk products such as platform chemicals. Examples include the application of thermophilic Bacillus and Clostridium species for organic acid production. Furthermore, we will discuss the development of genetic tools for biotechnologically-relevant extremophiles, as this is crucial for optimizing their specific production capacities, as well as for developing them as industrial platform organisms

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.CRISPR-Cas9 genome engineering tools have found wide application in a range of species, however they are unsuitable for applications at elevated temperatures. Here the authors characterise ThermoCas9 from which is functional from 20°C to 70°C.

Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9

Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45–55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.

Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains

BackgroundMicrobial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals. Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks and a process temperature matching that of commercial hydrolytic enzymes, enabling simultaneous saccharification and fermentation at higher efficiencies and with less enzymes. However, genetic tools for biotechnologically relevant thermophiles are still in their infancy. In this study we developed a markerless gene deletion method for the thermophile Bacillus smithii and we report the first metabolic engineering of this species as a potential platform organism.ResultsClean deletions of the ldhL gene were made in two B. smithii strains (DSM 4216T and compost isolate ET 138) by homologous recombination. Whereas both wild-type strains produced mainly l-lactate, deletion of the ldhL gene blocked l-lactate production and caused impaired anaerobic growth and acid production. To facilitate the mutagenesis process, we established a counter-selection system for efficient plasmid removal based on lacZ-mediated X-gal toxicity. This counter-selection system was applied to construct a sporulation-deficient B. smithii ΔldhL ΔsigF mutant strain. Next, we demonstrated that the system can be used repetitively by creating B. smithii triple mutant strain ET 138 ΔldhL ΔsigF ΔpdhA, from which also the gene encoding the α-subunit of the E1 component of the pyruvate dehydrogenase complex is deleted. This triple mutant strain produced no acetate and is auxotrophic for acetate, indicating that pyruvate dehydrogenase is the major route from pyruvate to acetyl-CoA.ConclusionsIn this study, we developed a markerless gene deletion method including a counter-selection system for thermophilic B. smithii, constituting the first report of metabolic engineering in this species. The described markerless gene deletion system paves the way for more extensive metabolic engineering of B. smithii. This enables the development of this species into a platform organism and provides tools for studying its metabolism, which appears to be different from its close relatives such as B. coagulans and other bacilli.

Hijacking CRISPR-Cas for high-throughput bacterial metabolic engineering: advances and prospects

High engineering efficiencies are required for industrial strain development. Due to its user-friendliness and its stringency, CRISPR-Cas-based technologies have strongly increased genome engineering efficiencies in bacteria. This has enabled more rapid metabolic engineering of both the model host Escherichia coli and non-model organisms like Clostridia, Bacilli, Streptomycetes and cyanobacteria, opening new possibilities to use these organisms as improved cell factories. The discovery of novel Cas9-like systems from diverse microbial environments will extend the repertoire of applications and broaden the range of organisms in which it can be used to create novel production hosts. This review analyses the current status of prokaryotic metabolic engineering towards the production of biotechnologically relevant products, based on the exploitation of different CRISPR-related DNA/RNA endonuclease variants.

Lactobacilli and pediococci as versatile cell factories - Evaluation of strain properties and genetic tools

This review discusses opportunities and bottlenecks for cell factory development of Lactic Acid Bacteria (LAB), with an emphasis on lactobacilli and pediococci, their metabolism and genetic tools. In order to enable economically feasible bio-based production of chemicals and fuels in a biorefinery, the choice of product, substrate and production organism is important. Currently, the most frequently used production hosts include Escherichia coli and Saccharomyces cerevisiae, but promising examples are available of alternative hosts such as LAB. Particularly lactobacilli and pediococci can offer benefits such as thermotolerance, an extended substrate range and increased tolerance to stresses such as low pH or high alcohol concentrations. This review will evaluate the properties and metabolism of these organisms, and provide an overview of their current biotechnological applications and metabolic engineering. We substantiate the review by including experimental results from screening various lactobacilli and pediococci for transformability, growth temperature range and ability to grow under biotechnologically relevant stress conditions. Since availability of efficient genetic engineering tools is a crucial prerequisite for industrial strain development, genetic tool development is extensively discussed. A range of genetic tools exist for Lactococcus lactis, but for other species of LAB like lactobacilli and pediococci such tools are less well developed. Whereas lactobacilli and pediococci have a long history of use in food and beverage fermentation, their use as platform organisms for production purposes is rather new. By harnessing their properties such as thermotolerance and stress resistance, and by using emerging high-throughput genetic tools, these organisms are very promising as versatile cell factories for biorefinery applications.