Genetic Engineering Of Actinobacillus Succinogenes For Succinic Acid Production

Succinic acid is an important C4 platform compound,which has a wide application prospect in food,medicine,surfactants,green solvents,biodegradable plastics and other fields.Actinobacillus succinogenes take succinic acid as the main metabolite and is considered as one of the most promising succinic acid producing bacteria.However,due to the need to add pH neutralizer MgCO₃ in the process of fermentation acid production,the difficulty of engineering operation and the opportunity of bacterial contamination are increased.At the same time,the shortage of genetic manipulation tools makes it difficult to obtain strains with excellent performance,thus hindering the industrial application of A.succinogenes.In order to solve the above problems,this paper studied the effect of exogenous acid resistant system on improving succinic acid production of strains by genome sequencing and acid resistance mechanism analysis.In this study,a series of genome editing methods based on CRISPR/Cas were established,and the A.succinogenes engineering strain was constructed,in order to achieve the transformation of A.succinogenes and improve the fermentation ability of the strain to produce succinic acid.The main research contents are as follows:（1）Through genome sequencing and analysis of A.succinogenes ZK,the acid-resistant mechanism of A.succinogenes includes:F₀F₁-ATPase,repair protein Rce F,Rad C,Rce O,DNA mismatch repair protein Mut S,Dna K,and AP endonuclease for macromolecular repair or protection,confirming that the absence of glutamate decarboxylate（Gad）acid-resistant system,which is more common in bacteria.By introducing Gad system into A.succinogenes,recombinant strains gadB-SW and gadBC-SW were constructed,and it was found that the survival rate of the cells was improved at pH4.6,but the growth was affected at neutral pH.Under the condition of shake-flask fermentation with less MgCO₃,the yield of succinic acid from gadBC-SW was 8.4%higher than that from wild type.The concentration of succinic acid in gadBC-SW reached 47 g/L and 31 g/L,respectively,when the pH 6.5 and pH 6.0 were controlled by Na₂CO₃ in a 3-L fermenter for 34 h.（2）The gene knockout method of Actinobacillus succinogenes was studied by using the gene encoding acetokinase（ackA）as the target gene.As for CRISPR/Cas gene knockout system of A.succinogenes:Western Blotting results showed that Cpf1 from Francisella tularensis andCas9 from Streptococcus pyogenes after codon optimization could be successfully expressed in A.succinogenes;With e GFP as the reporter protein,7 promoters with different expression intensity were identified:pck A>J23119>kasop>gadph>Km>erm E;Different CRISPR/Cas9 gene knockout plasmids and CRISPR/Cpf1 gene knockout plasmids were constructed,and it was speculated that optimizing the expression levels of Cas protein and g RNA and screening for auxiliary recombinases might be the key.A homologous recombinant gene knockout system was established.Using plasmid p CVD442 as knockout vector,the knockout strainΔackA was obtained by conjugation,double homologous recombination,resistance screening and sucrose back-screening.（3）The A.succinogenes gene interference system（CRISPRi）was constructed.Using pLGZ922 as the skeleton vector,dCas9 was obtained by site-specific mutation of Cas9,and CRISPRi based on CRISPR/Cas9 was constructed,but the inhibition effect on the target gene ackA was not obvious.dCpf1 was obtained by site-specific mutation of Cpf1,and CRISPRi based on CRISPR/Cpf1 was constructed,which had inhibitory effect on ackA target gene,among which dCpf1（E1006A）CRISPRi could inhibit ackA by more than 75%.Compared with ackA knockout strain,the growth OD of down-regulated ackA expression strain was improved,and the concentration of acetic acid,was significantly decreased in glucose or xylose medium.The strain with down-regulated ackA expression was fermented in 3-L fermenter for 60 h,which produced 57.06 g/L succinic acid,2.44 g/L acetic acid and 0.79 g/g sugar and acid conversion rate.（4）The base editors based on CRISPR/Cas were developed.Cytosine base editor（CBE）and adenine base editor（ABE）of CRISPR/Cas9 and CRISPR/Cpf1 were designed and constructed respectively,and it was found that only ABE and CBE based on CRISPR/Cas9could achieve C to T and A to G conversion in A.succinogenes.By extending linker and introducing 2 copies of UGI to optimize CBE,nCas9（D10A）max is obtained,editing efficiency is increased to 50%,editing window is C2-12 of PAM upstream.By fusing the amino terminal of Cas9 with the mutant of E.coli adenosine deaminase（Tad A-8e）,yielding nCas9（D10A）-ABE.The editing window was A4-8 of PAM upstream,and the editing efficiency reached 100%.In addition,the guanine base editor（Td-GABE）was constructed by introducing N46L mutation into Tad A-8e,which realized the conversion of G to T at G4 site of PAM upstream,the editing efficiency was 11%.Td-CBE and Td-CBEmax are constructed,and the editing efficiency is100%at the C5 site of PAM upstream,and the accurate conversion from C to T is realized.At the same time,it was found that nCas9（D10A）-ABE could edit at least 6 sites at the same time,and the editing efficiency could reach 100%.Td-CBE and Td-CBEmax can edit two sites at the same time,and the editing efficiency of the first site is 100%,and the editing efficiency of the second site is 10%.（5）Genetic engineering of A.succinogenes for SA production.According to succinic acid metabolism of A.succinogenes,it was found that the expression of phosphoenolpyruvate carboxylase PEPC derived from Corynebacterium acetoacidophilum can increase the fixation of CO₂ and promote the formation of SA.The production of acetic acid can be eliminated by knocking out ackA gene of C3 pathway,and the expression formate dehydrogenase from Candida boidinii can eliminate formic acid.By silencing the hypothetical C4 transporter genes Asuc＿0715 and Asuc＿0716,the production of succinic acid was reduced by about 16.8%,and were related to the secretion of succinic acid by measuring the intracellular and extracellular SA concentrations during fermentation.The silencing of glucose transporter gene Asuc＿0914increased the yield of succinic acid,and was associated with glucose uptake by measuring intracellular and extracellular glucose concentration during fermentation.Asuc＿0914 knockout strain produced 39.26 g/L succinic acid and 0.94 g/g sugar and acid conversion rate by shake-flask fermentation.The concentration of succinic acid reached 71.92 g/L and the conversion rate of sugar and acid was 1.03 g/g after fed-batch fermentation 60 h in 3-L fermenter,which showed the prospect of industrial application.