Developing Genome Engineering Tools for Engineering Non-model Microorganisms for Biochemical Production and Sustainable Agriculture Applications

Abstract

Nowadays, the energy crisis is getting worse in the world, and the environmental pollution resulting from the consumption of fossil fuels cannot be underestimated. Therefore, the discovery and optimization of renewable and clean fuel alternatives are currently sought after. Biofuels and biochemicals, derived from renewable biomass, represent a crucial and viable solution in alleviating the crisis. N-butanol (hereafter referred to as butanol) is a valuable biofuel with numerous advantages over ethanol, the commonly used gasoline blendstock. It also serves as a valuable chemical feedstock with many applications across various industries. Additionally, hexanoate and hexanol are valuable fuels or fuel precursors that can be used as aviation fuels and chemical feedstocks in industries such as pharmaceuticals and cosmetics. Non-pathogenic clostridia possess natural C4 pathways for butanol production and can be engineered to produce hexanoate and hexanol. The goal of this study was to genetically engineer Clostridium hosts to produce these biochemicals. However, as Gram-positive anaerobic bacteria, these Clostridium species are generally challenging to engineer. Although CRISPR-Cas9-based genome engineering tools were developed previously, the engineering efficiency is still not high. Therefore, more efficient tools for genome engineering are needed. In this study, the CRISPR-AsCas12f1 system, which significantly reduces size of the plasmid carrying the CRISPR system compared to CRISPR-Cas9, was successfully developed for engineering C. beijerinckii, a prominent host for biobutanol production, with a higher genome editing efficiency. Remarkably, the resultant mutant with Cbei_1741 deleted with the CRISPR-AsCas12f1 system could produce 40% more butanol compared to the wild type strain. Characterization of the cell membrane indicated that the deletion of Cbei_1741, which encodes 1-acyl-sn-glycerol-3-phosphate acyltransferase, blocked the CDP-diacylglycerol production pathway. This blockage saved Acyl-CoA and reducing power, which in turn slightly enhanced butanol tolerance and butanol production in the engineered mutant strain. A good genome editing tool should have a broad functionality. So, the CRISPR-AsCas12f1 based genome engineering system was further developed in C. tyrobutyricum, another strain which was much more difficult to genetically engineer based on our experiences. As an initial step, the results of in vitro and in vivo codon optimized AsCas12f1 cleavage activity assay demonstrated that the optimal CRISPR-AsCas12f1 system was functional in C. tyrobutyricum. This genome editing system realizes one-step screening of mutant strains with an efficiency of up to 100%. Simultaneously, with the support of the plasmid curing system under the regulation of the theophylline-dependent riboswitch inducible gene expression system, the comprehensive genome engineering tool could significantly shorten the cycle of knocking out a certain gene in C. tyrobutyricum. The engineered C. tyrobutyricum was developed by using the optimal CRISPR-AsCas12f1 system to be able to produce the hexenoate. The mutant with highest hexanoate production in this study was constructed by overexpressed thiolase from Ruminococcaceae bacterium CPB6 (ThlCPB6), which is a key gene for producing C6-acyl-CoA intermediate by condensing the acetyl-CoA and butyryl-CoA, and replaced the native Cat1 (butyryl-CoA:acetate CoA-transferase) with Cat from CPB6, resulting in hexanoate production to 4.4 g/L. In addition, the ThlCPB6 was introduced into butanol producing C. tyrobutyricum strain to produce hexanol, allowing the titer of 91 mg/L. As a part of this dissertation work, the genome engineering technology has been further applied in plant-growth promoting microorganisms towards agricultural sustainability. Recently, due to soil drought caused by environmental issues or climate changes, food production has dropped significantly; however, this stands in stark contrast to the increasing demand for high food production driven by population growth in the world. Recently, ACC (1-aminocyclopropane-1-carboxylate) was demonstrated to improve drought tolerance of plants. Here, one Plant Growth Promoting Rhizobacteria (PGPR) with ACC deaminase activity was isolated from the root of peanut, and the sequence of the ACC deaminase encoded gene was determined. Subsequently, a sacB-based genome editing tool was developed for the isolated PGPR from peanut root, Bradyrhizobium sp. strain 9. With the SacB as the counter-selection marker, the gene encoding the ACC deaminase was successfully deleted with higher efficiency. The results of ACC deaminase activity assays and Sanger sequencing further illustrate the efficacy of the SacB-based genome editing tool as an effective method for genome editing in the PGPR. Furthermore, a 5X ACC deaminase mutant strain was developed to further enhance the ACC deaminase activity. Finally, the plant tests of successful nodulations for these two mutant strains indicate that engineered PGPR could nodulate the root of peanuts without affecting peanut growth.