Platteeuw C, Hugenholtz J, Starrenburg M, van Alen-Boerrigter I, de Vos W M
Department of Biophysical Chemistry, NIZO, Ede, The Netherlands.
Appl Environ Microbiol. 1995 Nov;61(11):3967-71. doi: 10.1128/aem.61.11.3967-3971.1995.
The als gene for alpha-acetolactate synthase of Lactococcus lactis MG1363 was cloned on a multicopy plasmid under the control of the inducible L. lactis lacA promoter. More than a hundredfold overproduction of alpha-acetolactate synthase was obtained in L. lactis under inducing conditions as compared with that of the host strain, which contained a single chromosomal copy of the als gene. The effect of alpha-acetolactate synthase overproduction on the formation of end products in various L. lactis strains was studied under different fermentation conditions. Under aerobic conditions and with an initial pH of 6.0, overexpression of the als gene resulted in significant acetoin production that amounted to more than one-third of the pyruvate converted. However, the effect of the alpha-acetolactate synthase overproduction was even more pronounced in the lactate dehydrogenase-deficient strain L. lactis NZ2700. Anaerobic cultivation of this strain resulted in a doubling of the butanediol formation of up to 40% of the converted pyruvate. When cultivated aerobically at an initial pH of 6.8, overexpression of the als gene in L. lactis NZ2700 resulted in the conversion of more than 60% of the pyruvate into acetoin, while no butanediol was formed. Moreover, at an initial pH of 6.0, similar amounts of acetoin were obtained, but in addition approximately 20% of the pyruvate was converted into butanediol. These metabolic engineering studies indicate that more than 80% of the lactose can be converted via the activity of the overproduced alpha-acetolactate synthase in L. lactis.
乳酸乳球菌MG1363的α-乙酰乳酸合酶的als基因被克隆到一个多拷贝质粒上，该质粒受可诱导的乳酸乳球菌lacA启动子控制。与含有als基因单拷贝染色体的宿主菌株相比，在诱导条件下乳酸乳球菌中α-乙酰乳酸合酶的过量表达超过了一百倍。在不同发酵条件下，研究了α-乙酰乳酸合酶过量表达对各种乳酸乳球菌菌株终产物形成的影响。在好氧条件下且初始pH为6.0时，als基因的过表达导致大量乙偶姻生成，其产量超过转化丙酮酸的三分之一。然而，在缺乏乳酸脱氢酶的乳酸乳球菌NZ2700菌株中，α-乙酰乳酸合酶过量表达的影响更为明显。该菌株的厌氧培养导致丁二醇生成量增加一倍，达到转化丙酮酸的40%。当在初始pH为6.8的条件下好氧培养时，乳酸乳球菌NZ2700中als基因的过表达导致超过60%的丙酮酸转化为乙偶姻，而没有生成丁二醇。此外，在初始pH为6.0时，获得了相似量的乙偶姻，但除此之外，约20%的丙酮酸转化为丁二醇。这些代谢工程研究表明，通过过量表达的α-乙酰乳酸合酶在乳酸乳球菌中的活性，超过80%的乳糖可以被转化。
