Metabolic Regulation Of The Synthesis Of2,3-butanediol In Klebsiella Pneumoniae

2,3-Butanediol (2,3-BD) is an important chemical and liquid fuel which is widelyused in varied fields such as fuel, chemical industry, food industry, aerospace.Klebsiella pneumoniae is a potentially useful producer in the industrial production of2,3-BD because of its wide substrate spectrum, high efficiency, and culturaladaptability. Many by-products are also produced by K. pneumoniae, such as ethanol,lactate, and acetate, which decrease the2,3-BD yield and increase the cost of productdownstream separation. Only a few studies have applied metabolic engineering in K.pneumoniae. So, the metabolic regulation study of the metabolic pathway of2,3-BDand main byproduct synthesis is necessary, which will provide important theoreticalsupport to improve the2,3-BD production. Thesis includes the following aspects:(1) Mutants with overexpression of-acetolactate synthase (ALS),-acetolactatedecarboxylase (ALDC) and acetoin reductase (AR) either individually or incombination were constructed to improve2,3-BD production in K. pneumoniae. Themutant K. pneumoniae strain (KG-rs) that overexpressed both ALS and AR showedan improved2,3-BD yield. The2,3-BD production of KG-rs in a batch fermentationwith glucose as the carbon source was12%higher than that of the parental strain. Theincreased of NADH and enzyme activity in the KG-rs strain maybe the reasons for the2,3-BD yield improvement. When cultured in the media with5different carbonsources (glucose, galactose, fructose, sucrose and lactose), the mutant exhibitedhigher2,3-BD productivity and production than the parental strain in all the testedcarbon sources except for lactose.(2) K. pneumoniae△ldhA、△adhE and△pta strain with lactate dehydrogenase(LDH), acetaldehyde dehydrogenase (ADH), and phosphotransacetylase (PTA)deletion individually was constructed.2,3-BD production dramatically improved withthe inactivation of adhE and pta, but not with the inactivation of ldhA. Theinactivation of ldhA resulted in faster cell growth speed and shorter fermentation timethan the wild type. Batch fermentation demonstrated that the Δpta and ΔadhE strainsresulted in high2,3-BD production rates, which were33.65%and19.58%higher thanthat of the parent strain, respectively. And the cell growth speed, enzyme activity,concentration of NADH and NAD+, and metabolic flux were measured to clarify the reasons for the differences of fermentation performance in the recombinant strains△ldhA、△adhE and△pta.(3) The△ldhA、△adhE and△pta strain exhibited higher2,3-BD productivity andproduction than the parental strain in glucose, galactose or fructose medium, butexcept for lactose medium. The major cause of inefficient lactose consumption wasthe low activity of lactose permease and β-galactosidase.