Abstract:
Although a high titre of malic acid is achieved by filamentous fungi, by-product succinic acid accumulation leads to a low yield of malic acid and is unfavourable for downstream processing. Herein, we conducted a series of metabolic rewiring strategies in a previously constructed Myceliophthora thermophila to successfully improve malate production and abolish succinic acid accumulation. First, a pyruvate carboxylase CgPYC variant with increased activity was obtained using a high-throughput system and introduced to improve malic acid synthesis. Subsequently, shifting metabolic flux to malate synthesis from mitochondrial metabolism by deleing mitochondrial carriers of pyruvate and malate, led to a 53.7% reduction in succinic acid accumulation. The acceleration of importing cytosolic succinic acid into the mitochondria for consumption further decreased succinic acid formation by 53.3%, to 2.12 g/L. Finally, the importer of succinic acid was discovered and used to eliminate by-product accumulation. In total, malic acid production was increased by 26.5%, relative to the start strain JG424, to 85.23 g/L and 89.02 g/L on glucose and Avicel, respectively, in the flasks. In a 5-L fermenter, the titre of malic acid reached 182.7 g/L using glucose and 115.8 g/L using raw corncob, without any by-product accumulation. This study would accelerate the industrial production of biobased malic acid from renewable plant biomass.
摘要:
尽管丝状真菌可以获得高滴度的苹果酸,副产物琥珀酸的积累导致苹果酸的低收率,并且不利于下游加工。在这里,我们在先前构建的嗜热菌中进行了一系列代谢重新布线策略,以成功地改善苹果酸的产生并消除琥珀酸的积累。首先,使用高通量系统获得具有增加的活性的丙酮酸羧化酶CgPYC变体,并引入该变体以改善苹果酸合成。随后,通过缺失丙酮酸和苹果酸的线粒体载体,将代谢通量从线粒体代谢转移到苹果酸合成,导致琥珀酸积累减少53.7%。向线粒体输入胞质琥珀酸用于消费的加速进一步减少了53.3%的琥珀酸形成,至2.12g/L最后,琥珀酸的进口商被发现并用于消除副产品积累。总的来说,苹果酸产量增加了26.5%,相对于起始菌株JG424,在葡萄糖和Avicel上为85.23g/L和89.02g/L,分别,在烧瓶中。在一个5升发酵罐里,使用葡萄糖,苹果酸的滴度达到182.7g/L,使用生玉米芯达到115.8g/L,没有任何副产品积累。这项研究将加速从可再生植物生物质中工业生产生物基苹果酸。