C4二元羧酸广泛用于食品、医药和化学等行业,市场潜在需求量巨大。酿酒酵母被认为是发酵生产C4二元羧酸的潜在最适微生物,却产生大量的乙醇,导致了碳流的损失。通过敲除硫胺素合成途径中的调控基因THI2,阻断了硫胺素的合成,使得副产物乙醇产量从5.27±0.23 g/L下降到0.53±0.12 g/L,但影响了葡萄糖消耗和菌体的生长。在此基础上,通过外源添加0.04μmol/L的硫胺素二磷酸,促进了葡萄糖的消耗和菌体生长;进一步通过外源添加1 000μg/L的NAD+,使得葡萄糖的消耗量和菌体的生长分别提高了48.6%和47.2%,而乙醇产量仅增加了0.56 g/L。通过调控辅因子水平(硫胺素和NAD+)可以有效减少副产物乙醇的积累,为解决利用酿酒酵母生产C4二元羧酸中副产物乙醇积累这一普遍性问题提供了一个新的策略。

Four-carbon dicarboxylic acids are widely used in food,pharmaceutical and chemical industries.Thus there is a huge market potential for then.S.cerevisiae is regarded as the best candidate microorganism for producing four-carbon dicarboxylic acids.Unfortunately,there is strong tendency for ethanol formation under fully aerobic conditions when sugar is present in access in batch culture,which leads to the loss of carbon flow.To reduce ethanol formation,the positive regulate gene THI2 in thiamine synthetic pathway was deleted,and the ethanol titer decreased from 5.27 ± 0.23 g /L to 0.53 ± 0.12 g /L.However,this strategy led to a week growth and glucose consumption.Then cell growth and glucose consumption were improved but still weak when 0.04 μmol /L ThDP was added.To further improve cell growth and glucose consumption,the effect of NAD+on FMME-002△THI2 was explored.Results show that glucose consumption and cell growth were improved by 48.6% and 47.2%,respectively,while ethanol titer only increased 0.56 g /L when 1 000 μg /L NAD+was added.In brief,ethanol formation was reduced efficiently by regulating the level of cofactors thiamine and NAD+,which provideed a novel strategy to solve this universal problem in the field of production of four-carbon dicarboxylic acids from S.cerevisiae.