Abstract: Aspartate kinase（AK）and aspartate semi-aldehyde dehydrogenase（ASD）encoded by gene lysC and gene asdA are two key rate-limiting enzymes playing vital roles in L-threonine synthesis pathway,and AK was co-inhibited by lysine and threonine. Selecting Corynebacterium glutamicum T11 as the original strain,the lysC-asdA gene co-expression cassette were constructed,the key rate-limiting enzyme gene lysC was mutated by site-directed mutation at Ala279Th,and the anti-feedback inhibition mutant gene,named lysC^r-asdA,was obtained,and was inserted into the shuttle expression vector pZ8-1 containing the strong promoter tac,thus the tandem expression plasmid pZ8-1-lysC^r-asdA was constructed successfully and transformed into the starting strain,then,the engineering strain T11/pZ8-1-lysC^r-asdA was obtained. The production of L-threonine by shaking flask was 7.18 g/L,which was 27.8% higher than that by the original strain. The results of further batch fermentation in 30 L fermenter showed that the yield of L-threonine reached 65.5 g/L for 60 h and the conversion rate of sugar and acid reached 39.5 g/L,increasing by 29.5% and 33.9% higher than by the original strain. It provides a strong basis for further constructing Corynebacterium glutamicum engineering strain highly yielding threonine.

Key words: L-threonine, Corynebacterium glutamicum, aspartate kinase, aspartate semi-aldehyde dehydrogenase, co-expession