| 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.Lignocellulose,one of the most abundant biomasses,can be enzymatically hydrolyzed to sugars like glucose,xylose,and arabinose.At least 200 million tons of corn stover,a lignocellulosic biomass,are produced in China each year,which may make it a low cost and widelyavailable resource for 2,3-BD production.However,the 2,3-BD yields and productivitiesfrom lignocellulose are generally low.At the same time,inulin,is a polydisperse fructan consisting mainly ofβ-(2,1)-D-fructosyl-fructose links terminated by a sucrose residue.The worldwide production of inulin is currently estimated to be about 350,000 tons.Although its total quantity is not comparable to that of lignocellulose biomass,inulin could be easily processed by using currently available technologies,and it may even be easier to process than lgnocellulose.Thus,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,the screening GRAS and efficient strains,enzyme isolated and engineering bacteria construction for production of optical(2S,3S)-2,3-BDwere carried out.Enterobacter cloacaestrain SDM was systematically and metabolically engineered to construct an efficient biocatalyst for production for the fuel and enantiopure bio-chemical—(2R,3R)-2,3-BD.First,various genes encoding(2R,3R)-2,3,butanediol dehydrogenases were expressed in abdh mutant of E.cloacae strain SDM under the Pb promoter.Then,carbon catabolite repression in strain SDM waseliminated via inactivation of ptsG,followed by overexpression of galP,and the resultant strain utilized glucose and xylose simultaneously.To improve the efficiency of(2R,3R)-2,3-BD production,we knocked out ldh,frdA,and adh,thereby enhancing the yield of(2R,3R)-2,3-BD by 16.5%in a 500-mL Erlenmeyer flask.Finally,using fed-batch fermentation in a 5-L bioreactor,we robustlysynthesized 152.0 g/L(2R,3R)-2,3-BD(purity>97.5%)within 44 h with a specific productivity of 3.5 g/[L·h]and a yield of 97.7%from a mixture of glucose and xylose,two major carbohydrate components in lignocellulosic hydrolysates.In addition,when a lignocellulosic hydrolysate was used as the substrate,119.4 g/L(2R,3R)-2,3-BD(purity>96.0%)was synthesized within 51 h with a productivity of 2.3 g/[L·h]and a yieldof 95.0%.These results show that the highest records have been acquired for enantiopure(2R,3R)-2,3-BD production by the smartlyengineeredE.cloacae strain from lignocellulose-derived sugars.In addition to producing the 2,3-BD,our highly systematic approach might also be used in the production of other important chemicals by using lignocellulose-derived sugars.Currently the higher yield of 2,3-butanediol production strains are not biosafety strains that have been reported in the papers.This paper used a Bacillus licheniformis MW3 which can efficiently use six-carbon sugar and five-carbon sugars to produce 2,3-butanediol.In order to utilize B.licheniformis MW3 effectively,we first determined the key enzymes in the metabolism of 2,3-butanediol—butanediol dehydrogenase and glycerol dehydrogenase.By multiple sequence alignment of the proteins,we also determined its possible role.Followed by purifing of the proteins,and substrate spectrum analysising,researching stereospecific catalytic activity,activity staining of different substrates to determine the butanediol dehydrogenase is mainly used in catalytic meso-2,3-butanediol,and glycerol dehydrogenase catalytic(2R,3R)-2,3-butanediol.Then we established that knocking out 2,3-butanediol dehydrogenase gene bdh to produce(2R,3R)-2,3-butanediol and knocking out glycerol dehydrogenase gene gdh to produce meso-2,3-butadiol.The fed-batch fermentation in 5-L fermentor by B.licheniformis MW3 △bdh,125.9 g/L(2R,3R)-2,3-BD was synthesized within 51 h with a productivity of 2.47 g/[L·h]and purity of 96.4%.In the 50-L fed-batch fermentation of B.licheniformis MW3 △gdh,the product of 2,3-butanediol at 35 h reached the maximum 92.77 g/L.2,3-butanediol productivity of 2.65 g/[L · h],a yield of 99.75%,and the final product purity meso-2,3-butanediol was 99.57%.With the expansion of production scale,purity has improved on the contrary,which is favorable industrial scale applications for us. |
