Menkel E, Thierbach G, Eggeling L, Sahm H
Institut für Biotechnologie der Kernforschungsanlage Jülich, Federal Republic of Germany.
Appl Environ Microbiol. 1989 Mar;55(3):684-8. doi: 10.1128/aem.55.3.684-688.1989.
Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.
增加谷氨酸棒杆菌菌株中天冬氨酸的可利用性，以评估其对赖氨酸生产的影响。向具有反馈抗性天冬氨酸激酶的菌株中添加富马酸后，赖氨酸产量从20 mM增加到30 mM。这种增加伴随着苹果酸和琥珀酸的排泄。在该菌株中，富马酸通过富马酸水合酶、苹果酸脱氢酶和天冬氨酸氨基转移酶的活性转化为天冬氨酸。为了实现富马酸向天冬氨酸的直接转化，构建了含有大肠杆菌aspA +（天冬氨酸酶）基因的穿梭载体。将这些构建体导入原本缺乏天冬氨酸酶的谷氨酸棒杆菌中。使用质粒pZ1-9时，这导致天冬氨酸酶活性为0.3 U/mg（大肠杆菌中天冬氨酸酶活性的70%），使用质粒pCE1 delta时活性高达1.05 U/mg。在表达aspA +的菌株中，赖氨酸排泄进一步增加了20%。此外，在携带pCE1 delta的菌株中，排泄出高达27 mM的天冬氨酸。这表明谷氨酸棒杆菌中天冬氨酸到赖氨酸的反应序列存在未确定的限制。
