Butyl Acetate And Isobutanol Production By Recombinant Clostridium

With the development of modern industry and agriculture,the demand for energy and bulk chemicals is increasing rapidly.However,this trend is accompanied with environmental problems caused by the petrochemical industry and potential energy depletion.To address these potential threats,there is a call for the wide use of clean,renewable green energy and sustainable production of chemicals.Like C.acetobutylicum,C.beijerinckii and C.diolis are also a solvent-producing Clostridium.Their metabolic pathway is mainly to convert the sugar to pyruvate through glycolysis or pentose phosphate pathway and further generate acetyl-Co A,then acetyl-Co A will be transformed into ethanol and butanol through intermediate metabolism pathway.Acetone,acetic acid,and butyric acid are also common metabolites.Because the metabolic pathways of solventogenic Clostridium have been thoroughly studied and its genetic manipulation technology is relatively mature,it has the potential to be modified as the chassis cell for the production of alcohol,acid,ester and other chemicals.First,we over-expressed the alcohol acyltransferase gene（ATF1）to construct a butyl acetate-producing recombinant C.beijerinckii strain and a recombinant C.diolis strain.Both strains successfully produced 1.49 g/L and 1.37 g/L butyl acetate directly from glucose in 72hours,respectively.We found that both of the solvent-producing Clostridia showed intolerance to butyl acetate.After a series of tolerance tests,we found that in-situ extraction of butyl acetate by adding an extractant into the fermentation broth can greatly reduce the toxic effect so that show good growth of the strain.Then,we selected C.beijerinckii with excellent butyl acetate production capacity as the initial strain,and examined the effect of extraction rates of butyl acetate by several extractants such as n-hexane,n-decane,n-dodecane,and n-hexadecane on the growth of the strain,and the production of the product.The results showed that the extraction efficiency of n-hexane was89.3%.The other three extraction results were almost the same,at about 82%.The results also showed that the four extractants are non-toxic to C.beijerinckii.The fermentation level of the strain with dioxane was improved,and the butyl acetate production was increased by 216.6%.With the dodecane level during in-situ extraction fermentation further studied and considering the economic relationship between the amount of dodecane and the amount of butyl acetate produced,the usage of dodecane extractant was decided to be 10%（v/v）.In addition,we examined the effects of adding acetic acid,butyric acid,and calcium carbonate to the substrate.The results showed that the effects of additions of acetic acid and calcium carbonate was more pronounced in promotion of butyl acetate production.In 48 hours,the concentration of butyl acetate produced by the recombinant Clostridium baileyi was 5.56g/L after optimization which was 24 hours faster than the result without optimization.The yield of butyl acetate was increased by 2.74 times and the productivity was increased by 0.97 times.In this study,for the first time,efficient one-step butyl acetate production was achieved in Clostridium,suggesting a certain industrial prospect.Moreover,we constructed a recombinant C.beijerinckii strain and a recombinant C.diolis strain that contained both the fluoren-keto acid decarboxylase gene（kiv D）and the aldehyde dehydrogenase gene（yqhD）.The intermediate metabolite hydrazone-keto acid can be converted into branched-chain alcohols by kiv D and yqhD.The Recombinant C.beijerinckii strain produced 43 mg/L isobutanol and 3.34 g/L butanol in 72 h,and recombinant C.diolis strain produced 41 mg/L isobutanol and 3.20 g/L butanol in 72 h.This means that we have achieved the co-production of butanol and isobutanol for the first time.We optimized the butanol-isobutanol co-fermentation in recombinant C.beijerinckii.The effects of the addition of Ca CO₃,tryptone,valine and isobutyric acid on the yield of butanol and isobutanol were investigated.We found that when 5 g/L,1 g/L,5 g/L,and 1 g/L of Ca CO₃,tryptone,valine,and isobutyric acid respectively,were added to the fermentation medium,the recombinant C.beijerinckii exhibited best.Under the optimal fermentation conditions,the recombinant C.beijerinckii starin consumed 45.21 g/L of glucose to produce 194 mg/L of isobutanol,which is 3.5-fold higher than that obtained before optimization.Simultaneously,7.16 g/L of butanol was produced within 72 h.In summary,this study has conducted a systematic study on the one-step synthesis of butyl acetate by recombinant Clostridium,and for the first time realized the simultaneous synthesis of butanol and isobutanol by Clostridium,which has an important role in promoting the exploration of new products for Clostridium synthesis.