以法尼烯为评价效应物,研究了缺损乙醇合成途径、甘油合成途径、胞质乙酰辅酶A转运途径和法尼基焦磷酸消耗支路关键基因对酿酒酵母WHE4菌株合成法尼烯的影响。通过CRISPR-cas9基因编辑技术,获得8株关键基因缺损菌株。结果表明,与WHE4菌株相比,缺损乙醇脱氢酶基因ADH3-6对乙醇和法尼烯产量没有影响;单独缺损甘油三磷酸脱氢酶基因GPD1和GPD2使甘油积累量分别降低了15%和34%,缺损半乳糖激酶基因GAL1、GAL7、GAL10下调了甲羟戊酸途径所有基因转录水平,它们的缺损均不能提高菌株的法尼烯产量;缺损香叶基香叶基焦磷酸合酶基因BTS1和二酰基甘油二磷酸磷酸酶基因DPP1,法尼烯产量提高了29%,在5 L发酵罐补料分批发酵,菌株WHE4-33(WHE4 Δbts1, Δdpp1)的法尼烯产量达到1 578.91 mg/L。该研究对甲羟戊酸途径上游和下游关键节点基因进行了缺损影响法尼烯合成研究,为构建酿酒酵母萜类化合物高效平台提供了参考价值。
王均华
,
付闻文
,
李由然
,
朱惠霖
,
徐沙
,
石贵阳
,
张梁
,
丁重阳
,
顾正华
. 酿酒酵母关键节点基因缺损对法尼烯合成的影响[J]. 食品与发酵工业, 2022
, 48(2)
: 1
-7
.
DOI: 10.13995/j.cnki.11-1802/ts.027491
In this study, the effects of deleting key genes of ethanol and glycerol biosynthesis pathway and branches of cytosolic Acetyl-CoA and farnesyl diphosphate on farnesene production was studied in Saccharomyces cerevisiae WHE4. With the utilization of CRISPR-cas9, eight engineered strains were obtained and cultivation was carried out at lab-scale fermentation. Compared with that of strain WHE4, results showed that the deletion of ethanol dehydrogenase (ADH3-6) had no effect on ethanol and farnesene production. The deletion of glycerol-3-phosphatase dehydrogenase (GPD1 or GPD2) caused the decline of glycerol production (decreased by 15% and 34%), the deletion of galactokinase (GAL1, GAL7 and GAL10) caused decreased transcriptional levels of the mevalonate pathway genes, and the deletion of them caused the decline of farnesene production. The farnesene production was increased by 29% with the deletion of geranylgeranyl diphosphate synthase (BTS1) and diacylglycerol pyrophosphate phosphatase (DPP1) genes. And the highest farnesene production of strain WHE4-33 (WHE4 Δbts1, Δdpp1) reached 1 578.91 mg/L at a 5 L bioreactor by fed-batch fermentation. In conclusion, the influence of deleting key genes of upper and downstream pathways of the mevalonate pathway on farnesene production was studied, and this would provide guidance for constructing effective terpenoid-producing platform in S. cerevisiae.
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