| Acidithiobacillus caldus is an ancient sulfur-oxidizing bacteria widely distributed in the acid mine drainage environment.It relies on the oxidation of reduced inorganic sulfur compounds（RISCs）in ores to obtain the energy needed for survival.However,the continuous production of sulfuric acid inevitably causes significant environmental pressure on its survival.The acid resistance of A.caldus directly determines its ability to occupy an ecological niche in acid mine drainage ecosystems.However,due to the unique nature of its habitat,coupled with the lack of effective genetic manipulation tools,our current understanding of its acid-resistance mechanisms remains limited.On the other hand,industrial microorganisms used for organic acids production often face challenges such as inhibited cell growth and reduced production intensity due to the accumulation of acidic metabolites.The current conventional approaches to address this issue is to add alkaline reagents.However,this approach is accompanied by drawbacks such as increased osmotic stress in the fermentation system and increased downstream separation and purification costs.In the face of these challenges,this study started from the screening and identification of the extreme acidophile A.caldus CCTCC AB 2019256,and preliminarily elucidated its acid-resistant mechanism from multiple levels,and the acid-resistant components were explored for the engineering modifications of industrial fermentation microorganisms.Finally,the engineered strains with improved succinic acid tolerance and yield were obtained.The main research contents and results are as follows:（1）Screening and physiochemical characterization of the extremely acidophilus A.caldus.Using the method of enrichment culture and serial dilution,an extreme acidophilic sulfur-oxidizing bacteria strain was isolated from an acid mine drainage sample collected from the Zijin Copper Mine in Fujian Province,China.The strain was identified as A.caldus based on the 16S r RNA gene sequence analysis,and it was deposited in the China Center for Type Culture Collection（CCTCC AB 2019256）.This bacterium can maintain an intracellular p H close to neutral（6.57-6.96）under extreme acidic conditions.Under constant p H culture conditions（p H2.0,p H 1.5 and p H 1.0）,the final biomass,elemental sulfur consumption,maximum specific growth rate and maximum elemental sulfur specific consumption rate all decreased with the increase of acid stress intensity.RT-qPCR analysis showed that compared to the p H 2.0treatment condition,the transcription levels of two cyclopropane fatty acid synthase-encoding genes（cfa1,cfa2）and the global post-transcriptional regulator Hfq-encoding gene（hfq）were up-regulated by 1.92-fold,1.79-fold,and 1.69-fold,respectively,under p H 1.5 treatment condition.Under p H 1.0 treatment condition,the transcription levels of these genes were up-regulated by 2.43-fold,2.26-fold,and 1.94-fold,respectively.（2）Analysis of the biological function of AcHfq involved in bacterial resistance to environmental stress.Functional complementation experiments demonstrated that the recombinant strainΔhfq/Achfq constructed by CRISPR/Cas9-mediated chromosome integration fully or partially restored the phenotypic defects caused by hfq gene deletion in Escherichia coli,including impaired growth performance,abnormal cell morphology,impaired swarming motility,decreased stress resistance,decreased levels of intracellular ATP and free amino acids,and attenuated biofilm formation.Of particular note,the intracellular ATP level and biofilm production of the recombinant strainΔhfq/Achfq were increased by 12.2%and7.0%,respectively,compared to theΔhfq mutant.Comparative transcriptomic analysis revealed that AcHfq globally regulates various cellular processes such as metabolism,environmental signal processing,and motility in recombinant E.coli even under heterologous expression.（3）Analysis of the impact of AcCfaS-based cell membrane remodeling on bacterial acid resistance.The analysis of cell membrane fatty acid profile showed that the cyclopropane fatty acid Cy-19:0 had a relatively high abundance（24.8%-28.3%）in the cell membrane fatty acid composition of A.caldus CCTCC AB 2019256 under gradient acid stress treatment conditions.Overexpression of Accfa2 significantly enhanced the resistance of E.coli to moderate acid stress,that is,after incubation for 20 h in E minimal medium at p H 5.0,the OD600 of the recombinant strain MG102 increased by 20.9%compared with the control strain.Meanwhile,its intracellular levels of glutamic acid,arginine,aspartic acid,methionine and alanine were1.54,1.42,1.85,1.20 and 1.05 times higher than those of the control strain,respectively.In addition,overexpression of Accfa2 decreased the fluidity and permeability of E.coli cell membrane as well as the hydrophobicity of cell surface.（4）AcCfaS2-mediated membrane engineering strategy improves acid resistance of E.coli.Integrated expression of the Accfa2 gene in the E.coli genome improved the resistance of the recombinant strain to severe acid stress.That is,the OD600 of the recombinant strain M1-93-Accfa2 increased by 23.4%and 25.3%compared with the control strain after incubation in the E minimal medium at p H 4.8 for 10 h and 14 h,respectively.Analysis of the cell membrane fatty acid profile showed that the proportions of two cyclopropane fatty acids,Cy-17:0 and Cy-19:0,in the cell membrane composition reached 6.27%and 6.21%,respectively,which were1.26 and 5.26 times higher than those of the control strain.On the other hand,the proportions of unsaturated fatty acids C16:1w9c and C18:1w9c decreased by 1.31 and 5.81 times,respectively.Correspondingly,the permeability and fluidity of the cell membrane decreased significantly.HPLC analysis showed that the contents of glutamic acid,arginine,methionine and aspartic acid in M1-93-Accfa2 cells after acidification were 2.59,2.04,22.07 and 2.65 times that of the control strain,respectively.The membrane engineering strategy mediated by AcCfaS2 not only helps to improve the resistance of engineered strains to succinic acid stress,but also has a certain effect on the increase of succinic acid production.Specifically,after incubation in E minimal medium containing 40 m M succinic acid for 16 h,the OD600 of the recombinant strain M1-93-Accfa2 increased by 27.4%compared to the control strain.After 72h of flask fermentation,the succinic acid production of the strain M1-93-Accfa2-p CDF-tac-ppc reached 8.49 g·L-1,which was 10.3%higher than that of the control strain. |
