乳酸是一种单羟基酸,因其羟基结合的位置不同分为D-乳酸和L-乳酸,在食品、医药和生物材料中具有广泛的应用。随着对可降解塑料的需求增加,利用生物法生产乳酸的研究也越来越受到重视。该文以二倍体热带假丝酵母(Candida tropicalis)为出发菌株,探究了丙酮酸脱羧酶(pyruvate decarboxylase, PDC)基因缺失以及D-乳酸脱氢酶(D-lactic acid dehydrogenase, D-LDH)和2-羟基酸脱氢酶(2-hydroxyacid dehydrogenase, 2-HAD)基因的过表达对细胞生长和D-乳酸合成的影响。利用同源重组的方法敲除pdc1和pdc2基因以及整合d-ldh和2-had基因,成功构建了pdc1基因敲除菌株AK01、pdc2基因敲除菌株AK02、pdc1/pdc2基因敲除菌株AK03、2-had基因过表达菌株AK04、AK06和d-ldh基因过表达菌株AK05、AK07。结果发现,pdc1基因敲除的菌株AK01和AK03出现了一定的生长抑制且显著降低了菌株乙醇的产量;表达异源d-ldh的菌株AK05和AK07也出现了不同程度的生长抑制。和出发菌株相比,敲除pdc1基因同时整合异源d-ldh基因的菌株AK05能较好的生产D-乳酸,摇瓶发酵水平达到6.1 g/L,是出发菌株的29倍。
Lactic acid is a kind of hydroxy acid, which can be divided into D-lactic acid and L-lactic acid due to its combination site of hydroxyl. It is widely applied in food, medicine and biomaterial industries. As the growing demand for biodegradable plastics, researches on producing lactic acid by biotechnology have drawn wide attentions. This study investigated the impact of pdc gene knock out and gene overexpression of LDH and 2-HAD on cell growth and D-lactic acid production, based on the diploid Candida tropicalis. pdc1 gene knock out strain AK01, pdc2 gene knock out strain AK02, pdc1/pdc2 gene knock out strain AK03, 2-had gene overexpression strain AK04, AK06 and d-ldh gene overexpression strain AK05, AK07 were obtained by homologous recombination. The results showed that there was growth inhibition and notably ethanol reduction in pdc1 gene knock out strain AK01 and AK03. The growth inhibition was also found in d-ldh gene overexpression strain AK05 and AK07. pdc1 gene knock out and d-ldh gene overexpression strain AK05 produced 6.1 g/L D- lactic acid, which was 28 times more than that produced by the parental strain.
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