Beyond restrictions : Towards biotechnological exploitation of thermophilic clostridia

Abstract

In the past years, most chemicals and energy were produced from fossil-based resources. The continuous dependency on these depleting fossil resources have negatively impacted the environment with the emission of greenhouse gases and harmful materials. The replacement of fossil-based technologies to sustainable production methods such as those using microbes and fermentation has become a key research area. Non‐model thermophilic clostridia with native ability to ferment lignocellulose biomass, make them potential production organisms for industrial applications. However, the limitation of genetic accessibility and genetic tools for clostridial manipulation pose a major barrier for exploiting them as platform organisms. This thesis explores the genetic accessibility and tool development for two thermophilic clostridia, the ethanol-producing Hungateiclostridium thermocellum and the succinate- producing Pseudoclostridium thermosuccinogenes. In addition, we studied aspects such as xylose consumption, sporulation and CRISPRi-mediated silencing of central metabolism to improve strain engineering. For industrial strain development high engineering efficiencies are desired. Due to the user-friendliness and stringency, CRISPR-Cas-based technologies have strongly enhanced strain engineering efforts in bacteria. This has enabled more rapid metabolic engineering of both the model and non-model organisms, opening new possibilities to use them 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 hosts to create novel platform organisms to produce biotechnologically relevant products. H. thermocellum DSM1313 has biotechnological potential as a whole-cell biocatalyst for ethanol production using lignocellulosic renewable sources. The full exploitation of H. thermocellum has been limited with the lack of high-throughput genome engineering tools. A thermophilic bacterial CRISPR- Cas9‐based system, that has been recently developed in our research group, was applied in the organism as a transcriptional gene repression tool. The ThermoCas9-based CRISPR interference (CRISPRi) was utilized to modulate the central metabolic lactate dehydrogenase (ldh) and phosphotransacetylase (pta) genes in H. thermocellum. The effects of gene repression were studied based on transcriptional expression and product formation. Single-guide RNA (sgRNA) under the control of native intergenic 16S/23S rRNA promoter from H. thermocellum directing the ThermodCas9 to the promoter region of both pta and ldh silencing transformants reduced expression up to 67% and 62%, correspondingly. This resulted in 24% and 17% decrease in lactate and acetate production, respectively. Hence, these data established for the first time, employing CRISPRi-mediated gene repression of metabolic genes in H. thermocellum can be used for remodelling of metabolic pathways without the requisite for genetic engineering. P. thermosuccinogenes DSM5809 is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a production organism. However, exploitation of P. thermosuccinogenes has been constrained partly due to the genetic inaccessibility and lack of genetic tools. In this study, we established the genetic accessibility of the organism by overcoming restriction barriers with in vivo methylation of plasmid DNA when transformed into an engineered E. coli HST04 strain expressing four native methylation systems of the thermophile. Transformation efficiencies of 102 CFU/µg plasmid DNA were achieved. The protocol was used to introduce  a  ThermodCas9-based   CRISPRi  tool  targeting  the  gene  encoding   malic   enzyme   in P. thermosuccinogenes, which resulted in a 75% downregulation of its expression. Additionally, the silencing of malic enzyme had an impact on the strain’s fermentation profile. This is the first example of genetic engineering in P. thermosuccinogenes, opening new possibilities for metabolic engineering of this bacterium. Apart from the development of genetic tools, this thesis also deals with heterologous expression of genes from P. thermosuccinogenes into H. thermocellum. The latter has been of interest for consolidated bioprocessing (CBP), because of its capability to ferment cellulose without any pre-treatment or additional enzymes. H. thermocellum has excellent cellulolytic activity for C6 substrates, but has the inability to grow on C5 substrates such as xylose. A GTP-dependent plasmid-based xylose gene cluster from P. thermosuccinogenes was introduced into H. thermocellum. The expression of the xylose utilization genes allowed for growth on xylose. Transcriptional analysis showed upregulation of the xylulokinase (xylB) and transcriptional regulator (xylR) genes on xylose compared to cellobiose. Unpredictably, introduction of the plasmid-based xylose gene cluster into H. thermocellum also impacted the cellobiose fermentation profile. Ethanol and acetate production was improved by 24% and 19%, respectively, for the xylose-plasmid bearing cells compared to the empty vector. To conclude, introducing the P. thermosuccinogenes xylose gene cluster is a useful step towards CBP with H. thermocellum to produce fuels and high value chemicals. Single-cell analysis of microbial population heterogeneity is a fast-growing research area in industrial biotechnology, environmental biology and pathogenesis due to its potential to identify and quantify the impact of subpopulations on microbial performance. Though several tools have been established, determination of population heterogeneity in anaerobic bacteria, especially spore-forming clostridia species has been studied thoroughly. We applied single cell analysis techniques such as flow cytometry (FCM) and fluorescence-assisted cell sorting (FACS) on the spore-forming succinate producer P. thermosuccinogenes. By combining FCM and FACS with fluorescent staining, we discriminated and enriched all sporulation-related morphologies of the thermophile. To evaluate the presence of metabolically active vegetative cells, a blend of the dyes propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) tested best. Side scatter (SSC-H) in combination with metabolic indicator cFDA dye provided the best separation of sporulation populations. Based on this protocol, we successfully determined sporulation dynamics of P. thermosuccinogenes by distinguishing between spores, forespores, dark and bright phase endospores, and vegetative cells populations. Henceforth, this methodology can be applied to further study population heterogeneity and its impact on fermentation performance in the clostridia. In conclusion, this PhD thesis focuses on the two non-model thermophilic organisms as bacterial cell factories. Taking the succinate producer P. thermosuccinogenes as a model, we described strategies to overcome restriction barriers by in vivo methylation of plasmid DNA to have a genetically accessible strain. As a proof-of-concept, CRISPRi silencing tool was developed for both the thermophilic clostridia and applied to impact their transcriptional expression and fermentation profiles. Lignocellulolytic capacities of H. thermocellum were   improved by the introduction of xylose genes from P. thermosuccinogenes. Besides,  tools    were developed to study sporulation dynamics of P. thermosuccinogenes for future optimization of industrial fermentations.