As A Chassis Bacterium Based On Engineering Klebsiella Pneumoniae CRISPR And Base Editing Tools

Due to the growing concern with climate change and the depletion of fossil fuels,traditional fossil fuels are partially being replaced by sustainable energy sources such as biodiesel and bioethanol.As a major byproduct of biodiesel industry,glycerol was adequately produced in the past two decades and has become an available and cost-effective raw material.Among many microbes using glycerol as carbon source,Klebsiella pneumoniae exhibits extraordinary capability to metabolize glycerol and thus grows rapidly.In particular,K.pneumoniae can naturally utilize glycerol to synthesize bulk chemicals such as3-hydroxypropionic acid（3-HP）,1,3-propanediol（1,3-PDO）and2,3-butanediol.As one of the main metabolites of K.pneumoniae,3-HP is an important C3 platform compound that can be converted into various important chemicals with high market demand.For biosynthesis of 3-HP,K.pneumoniae is currently the most promising host cell for producing3-HP due to only two-step catalysis from substrate glycerol.However,due to the fact that K.pneumoniae is a non-model bacterium with limited genetic tools and opportunistic pathogenicity,it cannot be directly recruited in large-scale industrial production.To modify K.pneumoniae into a chassis cell,this study focuses on development tools for translation regulation,cofactor-dependent pathway intensification,base editing,genome-wide mutation as well as biocontainment.Below are the main research contents.1.To inhibit or activate protein translation,CRISPR/dCas13d system was first developed in K.pneumoniae.Briefly,the enhanced green fluorescent protein（egfp）was used as a reporter,and a translation inhibition system targeting ribosome binding site（RBS）was designed and constructed in K.pneumoniae.Results showed that the engineeredCRISPR/Cas13d andCRISPR/dCas13d systems gave rise to 30.2%and30.4%reduction in fluorescence intensity,respectively.Afterward,dCas13d was fused with translation initiation factor IF3（inf C）,and sg RNA was designed to target the 27 bp upstream of the m RNA translation initiation site of egfp gene.Because of the engineered translation activation system,the fluorescence intensity of the recombinant strain was increased by up to 30.3%.When this translation activation system was employed for the expression of aldehyde dehydrogenase（Puu C）,the enzyme activity was increased by 21.2%and the 3-HP production was increased by 4%.2.For glycerol-based biosynthesis of 3-HP in K.pneumoniae, aldehyde dehydrogenase converts 3-hydroxypropionaldehyde（3-HPA）to3-HP.However,this catalysis reaction consumes a lot of cofactor nicotinamide adenine dinucleotide（NAD⁺）,which in turn constrains 3-HP formation.To address this issue,a NAD⁺regeneration pathway based on niacin supplement was constructed to supply NAD⁺and improve 3-HP production in K.pneumoniae.To begin with,a tac promoter-driven NAD⁺synthesis pathway was engineered in K.pneumoniae,and the strain only overexpressing nicotinic acid phosphoribosyltransferase（Pnc B）showed14.24%increase in the production of NAD⁺relative to the stain harboring an empty vector.After optimization of niacin concentration,supplementation of 30 mg/L niacin in shake-flask fermentation resulted in the production of 0.55 g/L 3-HP,which was 2.75 times that of the control.In a 5-L bioreactor,replenishment of niacin led to 36.43%increase of3-HP production.3.To modify K.pneumoniae for metabolic engineering purposes and minimize its toxicity,a panel of base editors were constructed.Firstly,two cytosine base editors Pm CDA1-dCas9 and Pm CDA1-n Cas9 were constructed based on CRISPR-dCas9 and cytosine deaminase Pm CDA1,which could convert cytosine（C）to thymine（T）.Interestingly,the mutator Pm CDA1-n Cas9 showed much higher editing efficiency than Pm CDA1-dCas9.Later,a stop codon was introduced by targeting the urease gene using the cytosine base editor,which successfully inactivated the toxic protein urease.In addition,tad A-n Cas9,an adenosine base editor relying on adenosine deaminase tad A-8e,was constructed,which was able to efficiently convert adenine（A）to guanine（G）.Lastly,a dual-base editor tadual-n Cas9 based on tadual（a variant of tad A-8e）was constructed,which could simultaneously convert C to T and A to G.4.To improve the tolerance of K.pneumoniae to 3-HP,multiple genome-wide mutators were constructed for in vivo continuous evolution of the genome.Firstly,Pm CDA1 was individually fused with theαsubunit of K.pneumoniae RNA polymerase（RNAPα）,σ70（rpo D）,σ32（rpo H）andσ38（rpo S）,leading to a total of four cytosine global mutators:Pm CDA1-RNAPα,Pm CDA1-rpo D,Pm CDA1-rpo H and Pm CDA1-rpo S.Then,two adenosine global mutators,tad A-RNAPαand tad A-rpo S,were constructed.After 70 cycles of in vivo continuous evolution using the aforementioned six global mutators,all 6 mutant libraries showed better overall growth than the initial strain under different 3-HP concentrations.Among the mutators,Pm CDA1-rpo H and Pm CDA1-rpo S showed best evolutionary effects,as they allowed bacterial growth at 190 m M of 3-HP,which is 20 m M increase compared to the initial strain.When used for in vivo continuous evolution,the global mutator Pm CDA1-RNAPαperformed best in improving bacterial tolerance to 3-HP,and the strain was able to tolerate 220 m M 3-HP,which is 40 m M increase compared to wild-type strain.In addition,the inactivation of the 3-HP transport negative regulatory protein Yiep by Pm CDA1-n Cas9 also improved the tolerance of K.pneumoniae to 3-HP.5.To prevent the engineered strain from contaminating environment,two biocontainment systems were constructed based on CRISPR/Cas9 kill switch and organophosphorus auxotrophy.To this end,Cas9 was used to digest genome and vector.To construct such a chassis cell,the Cas9 expression cassette in vector was successfully integrated into K.pneumoniae genome.Results showed that the expressedCas9could effectively digest vector and genome in the presence of sg RNA.In addition,λ-Red homologous recombination technology was harnessed to knock out the pit A,pst B,pst A,pst C,pst S,phn C,phn D and phn E genes participating in the phosphate and phosphite transport system of K.pneumoniae.As expected,the auxotrophy K.pneumoniae strain could not grow in inorganic phosphorus(PO₃^3-or PO₄^3-)medium within 15 h,indicating the reduced adaption of the engineered strain to harsh external environment.In LB medium,the mutant strain demonstrated only 25%decrease of biomass compared to wild-type strain in 24 h,indicating that the knockout of genes does not remarkably halt its growth in specific laboratory environments.