Isolation And Engineered Bacillus Strains For Production Of 2,3-butanediol

2,3-Butanediol(2,3-BD),a fuel and platform bio-chemical,is used as a starting material for the manufacture of bulk chemicals such as methyl ethyl ketone and 1,3-butadiene.2,3-BD can be produced by chemical or biotechnological methods.Due to the gradual exhaustion of crude oil reserves,interest in the biotechnological production of 2,3-BD has increased greatly in recent years.Currently,microbes,including Klebsiellapneumonia,K.oxytoca and Serratiamarcescensare regarded as the most efficient 2,3-BD producer.However,these strains are all categorized by the World Health Organization(WHO)as risk group 2 species(pathogenic)andthus not desirable in industrial-scaleproduction of 2,3-BD.Some generally regarded as safe(GRAS)organisms including Bacilluslicheniformis,Paenibacillus polymyxa(formerly B.polymyxa),and B.amyloliquefaciens have been isolated for 2,3-BD production.However,the concentration of 2,3-BD obtained by these strains from xlyose are low.At the same time,most of the reported strains for 2,3-BD production haveoptimal fermentation temperature of 30～40℃.Due to themoderate temperature conditions,these fermentationscan be easily contaminated.At the same time,inulin,is a polydisperse fructan consisting mainly of β-(2,1)-D-fructosyl-fructose links terminated by a sucrose residue.Recently,inulin has recently received much attention as a renewable feedstock for the production of fructose and chemicals,such as ethanol and lactic acid.However,there have been few studies on 2,3-BD productionfrom inulin as substrate.To solve these problems in 2,3-BD production,in this study,isolation of GRAS and efficient strains,expressionof inulinase and construction of engineered bacteriafor production of 2,3-BDwere performed.In the secord part of this work,a thermophilic strain LR was newly isolated for(2R,3R)-2,3-butanediol((2R,3R)-2,3-BD)production.Strain LR was identified as Bacillus amyloliquefaciensby 16S rDNA and other physiological and biochemical experiments.In a fed-batch fermentation,56.0 g/L of(2R,3R)2,3-BD was obtainedfrom glucose.This is a new record about(2R,3R)-2,3-BD production by a thermophilicB.amyloliquefaciensstrain.In the third part of this work,engineered B.subtilis strains were constructed to produce 2,3-BD and acetoin from inulin.First,theRbdhgene of B.subtilis168was deleted.The resulting strain could mainly produce acetoin.Then,an inuA gene encoding an inulinase from Paenibacillus polymyxawas overexpressing under the P43promotor.To obtained an engineeredstrain producing meso-2,3-BD from inulin,a Mbdh gene ecoding a meso-2,3-BDH from B.licheniformiswas co-expressing with the inuA.We synthesized 44.0 g/L meso-2,3-BD(purity>97%)from inulin within 60 h ina fed-batch fermentation.