Mechanism Analysis Of High Butanol Yield Mediated By Gain-of-oxygen Tolerance Mutation And SLDS Fermentation By Clostridium Beijerinckii

Butanol has been becoming a new type of renewable fuel because of its excellent properties.At the same time,butanol is also an important platform compound,extractant and vehicle brake agent.Bio-butanol is mainly produced from anaerobic fermentation of acetone butanol ethanol?ABE fermentation?by Clostridium.Compared with butanol fro m petrochemical industry,however,bio-butanol production has high cost,low efficiency and complicated solvent separation.Therefore,the core improving market competitiveness of bio-butanol is to solve the cost problem of ABE fermentation.Breakthroughs can be made in the following aspects:1)increasing the strain tolerance to oxygen;2)improving the yield and production efficiency of solvent);3)reducing the solvent toxicity on cells;4)Looking for cheap and non-grain fermentation materials;5)improving the strain stability.Here,Clostridium beijerinckii NCIMB 8052 was selected to intensively study based on strain tolerance to oxygen,improving yield and production efficiency as well as looking for cheap and non-grain fermentation materials,aiming at reducing production cost and analyzing the mechanism of high yield.The main research results are as follows:?1?Successfully constructing a gain-of-oxygen tolerance mutant?Cbei₁336 mutant?in C.beijerinckii,which showed a significantly increased yield and productivity of butanol and acetone.Cbei₁336 mutant,an oxygen tolerant facultative anaerobic mutant,was obtained by Group II intron knockout of PerR inhibitor gene?Cbei₁336?involved in regulation of peroxide reduction in C.beijerinckii solventogenic degradation strains?C.beijerinckii DS?.This mutant not only grew well in aerobic environments,but also the biomass was increased by 8.11±0.11?OD600??3.67±0.35 times?,the acetone and butanol concentrations were increased by 4.7±0.40 g/L?32.78±0.02 times?and 10.01±0.57g/L?57.23±0.01 times?,respectively,and the total solvent production productivity was increased from 0.07±0.01 g/L/h?C.beijerinckii DS?to 0.19±0.01 g/L/h?Cbei₁336mutant?compared with C.beijerinckii DS,the productivity was increased 2.71±0.01 times.?2?Molecular mechanisms of oxygen tolerance and high butanol and acetone yield and productivity in Cbei₁336 mutant were further analysis by transcriptome.Transcriptome analysis was used to compare the gene expression changes in C.beijerinckii DS and Cbei₁336 mutant after 12 h,24h and 36 h fermentation.The results showed that there were 2893,2597 and 3036 differentially expressed genes at 3 time points,respectively,of which 975 and 222 were jointly up-regulated and down-regulated at 3 time points,respectively.Gene Ontology?GO?analysis showed that the proportion of genes involved in the metabolic process were the highest,accounting for 26.69%and 29.28%,respectively.Further differentially expressed genes analysis showed that genes encoding key enzymes involved in metabolic pathway of ABE fermentation,including mannitol transporter subunit IIB 6-phosphofructokinase,glyceraldehyde-3-phosphate dehydro-genase,pyruvate kinase,2-oxoacid ferredoxin oxidoreductase subunit beta,acetyl-Co A acetyltransferase,acetoacetate decarboxylase and NADPH-BDH were significantly up-regulated,while genes encoding phosphate encoding acetyltransferase?PTA?and acetate kinase were significantly down-regulated in the Cbei₁336 mutant.In addition,genes involved in oxido-reduction,such as Ferredoxin,flavodoxin,rubrerythrin and radical SAM protein;genes associated with cell detoxification,such as glutaredoxin multicopper oxidase?MCO?and Superoxide dismutase?SOD?;genes related toiron-dicitrate ABC transporter ATP-binding protein,iron transporter?FeoB?and manganese transporter;genes involved in spore formation,such as acid-soluble spore protein?SASP?and spore coat protein gene were significantly up-regulated in Cbei₁336mutant at 3 time points,suggesting that the process involved in glucose consumption and solvent production,cell detoxification,oxido-reduction,metal ion transport as well as sporulation may play an important role in oxygen resistant mechanism and high solvent yield and productivity in Cbei₁336 mutant.?3?The practice research found that scarification liquid of DZW starch?SLDS?was a kind of cheap and non-grain raw material for ABE fermentation by C.beijerinckii,which could significantly increase yield and productivity of butanol and acetone.SLDS contains a variety of nutrients including carbohydrates?glucose?,amino acids,nucleic acids,organic acids,vitamins and flavonoids,which could promote the solvent product of ABE fermentation by C.beijerinckii.Compared with the traditional P₂ medium,ABE fermentation by SLDS effectively improved the biomass,glucose consumption and consumption rate,yield and productivity of acetone and butanol,as well as shortens fermentation period about 24 h.This was associated with up-regulated expression of five metabolic enzyme genes involved in glucose consumption and acetone and butanol production,including glucose phosphotransferase system?Cbei₀242?,pyruvate kinase?Cbei₀485?,acetoacetate decarboxylase?Cbei₃835?and NADPH-dependent butanol dehydrogenase?Cbei₂181and Cbei₂421?.SLDS is a byproduct and industrial waste of diosgenin industry.Its arbitrary emissions will lead to nutrients loss and environmental pollution.Therefore,SLDS recycling can not only reduce the cost of ABE fermentation,but also reduce environmental pollution and waste of resources.