Levander Fredrik, Svensson Malin, Rådström Peter
Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund Institute of Technology, Lund University, SE-221 00 Lund, Sweden.
Appl Environ Microbiol. 2002 Feb;68(2):784-90. doi: 10.1128/AEM.68.2.784-790.2002.
It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal(+)) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal(+) strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal(+) strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal(+) strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.
通过改变影响前体核苷酸糖产生的中心代谢中酶的水平，有可能提高乳酸菌胞外多糖（EPSs）的低产量。为了验证这一假设，我们鉴定并克隆了嗜热链球菌LY03中编码UDP葡萄糖焦磷酸化酶（GalU）的galU基因。该基因的同源过表达导致GalU活性增加了10倍，但以乳糖为碳源时对EPS产量没有任何影响。然而，当galU与编码磷酸葡萄糖变位酶（PGM）的pgmA一起过表达时，EPS产量从乳糖的0.17 g/mol碳增加到0.31 g/mol碳。还发现一种具有增加的Leloir酶活性的半乳糖发酵LY03突变体（Gal(+)）的EPS产量（0.24 g/mol碳）高于亲本菌株。通过在该菌株中过表达galU，EPS产量进一步提高到0.27 g/mol碳。然而，当在Gal(+)菌株中敲除pgmA时，获得了最高的EPS产量，即0.36 g/mol碳。对培养物中细胞内代谢物水平的测量表明，Gal(+)菌株的葡萄糖1-磷酸水平明显高于其他菌株，而缺乏PGM活性的菌株的葡萄糖1-磷酸水平比其他Gal(+)菌株高三倍。这些结果表明，通过改变中心碳水化合物代谢中酶的水平来提高EPS产量是可能的。
