Metabolic Engineering Of Bacillus Licheniformis For Efficient Production Of Acetoin And Lactate

In the 21st century,the demand for energy is growing repldly,which has lead to the decline of oil resources availability and global environmental issues.In recent years,due to the continuous escalation of global energy and environmental problems,green and sustainable production methods have gradually emerged.Among them,microbial fermentation plays an important role.Compared with other production methods,microbial fermentation is superior in several aspects:cheaper raw materials,lower operating cost,higher product purity,and less pollution.Acetoin(AC)and lactic acid are widely used in food and chemical synthesis.Their production methods have attracted great attention.To produce acetoin and lactic acid from biomass through microbial fermentation is of great importanee in reducing the dependence on fossil energy and building the amicable type of environmental development pattern.Acetoin is widely used in cigarettes,cosmetics,detergents,chemical synthesis and other fields.The two active chemical groups(hydroxyl group and ketone group)in acetoin are chemically reactive.Hence,acetoin can be used as a precursor for the synthesis of many value-added compounds,for example,tetramethylpyrazine.In addition,acetoin can be used as a chelating agent to stabilize alkoxide.Lactic acid can be used as food additive to improve the taste and flavor.In the medicine industry,it can be used to promote the body's absorption of medicine because of its hydrophilic properties in dissolving proteins and other difficult-dissolved medicine.The principal application of lactic acid is the synthesis of poly lactic acid(PLA).PLA exhibits excellent properties in biodegradability and biocompatibility.In chapter 2,metabolic engineering of Bacillus licheniformis MW3 was conducted to produce acetoin from glucose.Pyruvate is produced through glycolysis,and then converted into acetoin by?-acetolactate synthetase and ?-acetolaetate decarboxylase.Since acetoin is the precursor of 2,3-butanediol,only a small amount of acetoin is accumulated during the fermentation process of Bacillus licheniformis MW3.The previous studies in our laboratory has shown that the gene budC encodes 2,3-butanediol dehydrogenase which catalyzes the conversion of acetoin into(2S,3S)-2,3-butanediol and meso-2,3-butanediol and the gene gdh encodes glycerol dehydrogenase which catalyzes the conversion of acetoin into(2R,3R)-2,3-butanediol.In this context,the key genes budC and gdh in the process of forming the 2,3-butanediol were knocked out to produce acetoin in high yield.Besides,spores were formed during the fermentation process of B.licheniformis MW3,which might reduce the production efficiency and affect acetoin production.Consequently,the key factor spo0A mediating spore formation was knocked out to construct the genetically engineered strain B.licheniformis MW3(?budC ?gdh ?spo0A).During fed-batch fermentation,61.77 g/L acetoin was produced with a yield 0.38 g/g,and the productivity was 1.72 g/[L·h].The results domentrated here might expand the application scope of B.licheniformis for acetoin production.In chapter 3,the constructed strain B.licheniformis MW3(?budC?gdh?spo0A)was used as the original strain for artificial evolution.High concentrations of acetoin was used as the evolutionary pressure.The strain B.licheniformis MW3(?budC ?gdh?spo0A)was capable of producing acetoin efficiently,however,further formation of acetoin was restricted which might result from the tolerance of acetoin of the strain.Artificial evolution has been widely used to re-design the microorganism fermentation processes.In this study,an evolvedstrain was achieved after continuous cultivation of six months.Interestingly,it was able to efficiently produce acetoin instead of acetoin.Compared with the original strain,,the enzyme activity of L-lactate dehydrogenase in the evolved strian was increased from 0.06 U/mg to 0.74 U/mg.Moreover,gene alsR mediating acetoin synthesis was knocked out to reduce the production of acetoin..After transformation,the target strain MW3(?budC?gdh?spo0A?slsR)was obtained.During the fed-batch fermentation process,the finally constructed strain MW3(?budC ?gdh ?spo0A ?alsR)was able to produce 156.53 g/L L-lactate at a yield of 0.91 g/g.The results domentrated here might serve as an alternative method for the lactic acid production.