Construction Of Succinate-producing Recombinant Ethanologenic Zymomonas Mobilis And Its Application In Fermentation

Zymomonas mobilis has become a preferred host in cellulosic ethanol fermentation or other biomass-based building block chemicals production for its special ED metabolic pathway and higher efficiency of sugar utilization. In order to product succinic acid from biomass by Z. mobilis, construction of succinic acid anabolic pathways, assessment of engineered strains by metabolism, physiological and biochemical, adaptive evolution and genome re-sequencing were performed in this study. The main conclusions are as followed:(1) Based on analysis of Z. mobilis ZM4 ED pathway, four key genes, lactate dehydrogenase(ZMO1237, ldhA), formate acetyltransferase(ZMO1570, pfl), pyruvate decarboxylase(ZMO1360, pdc) and alcohol dehydrogenase(ZMO1596, adhB)of Z. mobilis ZM4 in ED metabolic pathway were be deleted by using homologous recombination, four recombinant strains were be successfully constructed, and named as Z. mobilis ZM1237, ZM1570, ZM1360 and ZM1596, respectively.(2) The profiling of cell growth between Z. mobilis ZM4 and its mutants in RM or high glucose medium culture showed that wild type strain grew better than engineered strains. However, all strains showed the same trend when cultured to stationary phase. Metabolite analysis showed that engineered strains could produce more succinic acid than wild type, especially for mutant strain ZM1360. Higher yield of 2.8 g/L succinic acid was achieved, and the productivity was 0.1 g/L//h. Meanwhile, LDH, PDC and ADH activity of Z. mobilis ZM1237, ZM1360, ZM1596 showed decreasing of 19.5%, 35.85% and 69.51% lower than the wild type strain, separately. The results of real-time PCR showed that expression level of ldhA, pdc and adhB significantly declined as expected. Actually, gene expression level of ldhA in ZM1237 decreased 8.8%, pdc in ZM1360 decreased 82%, and adhB in ZM1596 wasn’t detected. Taken together, we successfully constructed engineered Z. mobilis strain for succinic acid production by blocking of competition of succinic acid anabolic pathways, and the strategy could changed ethanol metabolic flux.(3) For improving of Z. mobilis tolerant to some inhibitors resulted from lignocellulosic hydrolyaste, strategy of adaptive laboratory evolution(ALE) was used for development of furfural or acetic acid tolerant strains. After three round evolution under corresponding stress conditions, different evolved mutants(Z. mobilis ZMA7-2 and Z. mobilis ZMF3-3) were successfully obtained via ALE method. Z. mobilis ZMA7-2(stored at CGMCC No. 9987) was tolerant of 7 g/L acetic acid, and ZMF3-3(stored at CGMCC No. 9986) was tolerant of 3 g/L furfural. After cultured with 18 h, the cell density of ZMA7-2 under 7 g/l acetic-acid showed 2.64 times than wild type strain; the cell density of ZMF3-3 under 3 g/l furfural showed 2.24 times than Z. mobilis ZM4. Although ZMA7-2 had showed higher growth than Z. mobilis ZM4, there was no significantly difference in the yield of ethanol under acetic-acid stress condition. Interestingly, the efficiency of producing ethanol by ZMA7-2 was significantly higher than the wild strain. It was difficult to grow for Z. mobilis ZM4 under the stress of 3 g/L furfural. Therefore, the ethanol yield of Z. mobilis ZM4 was significantly decreased by 89.8% under furfural stress condition when compared to normal condition. On the other hand, ZMF3-3 had higher ethanol yield than Z. mobilis ZM4 under 3 g/l furfural stress condition, which showed 94.84% theoretical ethanol yield. RAPD results showed that there were amount of SNPs in evolved strains. Assay of the key enzymes activity indicated that furfural had more negative effect on activity of PDC and ADH than acetic-acid, especially for ADH.(4) Whole comparative genome sequencing(CGS) of the evolved strains suggested that ZMA7-2 had seventy nine SNPs in genome locus, and ZMF3-3 had eighty two SNPs. On the other hand, both mutant strains shared seventy one SNPs, ZMA7-2 have eight unique SNPs, and eleven unique SNPs were also found in strain ZMF3-3. Similarly, structure variation(SV), such as insertion or deletion occurred in the genome locus of the evolved strains due to environmental stress. These results provide some genetic basis of phenotype evolution under stress conditions by ALE strategy.(5) Two mutants ZMA7-2△pdc and ZMA7-2△adhB were also successfully obtained by using Z. mobilis ZMA7-2 and ZMF3-3 as novel host.