Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
J Biotechnol. 2017 Nov 20;262:40-46. doi: 10.1016/j.jbiotec.2017.09.021. Epub 2017 Sep 28.
l-Malate, an important chemical building block, has been widely applied in the food, pharmaceutical, and bio-based materials industries. In previous work, we engineered Aspergillus oryzae by rewiring the reductive tricarboxylic acid pathway to produce l-malate from glucose. To decrease the production cost, here, we further engineered A. oryzae to efficiently produce l-malate directly from corn starch with simultaneous liquefaction-saccharification and fermentation. First, a promoter PN5 was constructed by quintuple tandem of the 97-bp fragment containing the cis-element of the glucoamylase gene promoter (PglaA), and with the promoter PN5, the transcriptional level of glaA gene increased by 25-45%. Then, by co-overexpression of glaA, a-amylase (amyB) and a-glucosidase (agdA) genes with the promoter PN5, the l-malate titer increased from 55.5g/L to 72.0g/L with 100g/L corn starch in shake flask. In addition, to reduce the concentration of byproducts succinate and fumarate, a fumarase from Saccharomyces cerevisiae, which facilitated the transformation of fumarate to l-malate, was overexpressed. As a result, the concentration of succinate and fumarate decreased from 12.6 and 1.29g/L to 7.8 and 0.59g/L, and the l-malate titer increased from 72.0g/L to 78.5g/L. Finally, we found that the addition of glucose at the initial fermentation stage facilitated the cell growth and l-malate synthesis, and the l-malate titer further increased to 82.3g/L by co-fermentation of 30g/L glucose and 70g/L corn starch, with a productivity of 1.18g/L/h and a yield of 0.82g/g total carbon sources.
L-苹果酸是一种重要的化学基础物质,广泛应用于食品、制药和生物基材料等行业。在之前的工作中,我们通过重新布线还原性三羧酸途径,利用葡萄糖生产 L-苹果酸,从而对米曲霉进行了工程改造。为了降低生产成本,我们进一步对米曲霉进行了工程改造,使其能够直接利用玉米淀粉进行高效生产 L-苹果酸,同时实现液化-糖化和发酵。首先,构建了一个由包含葡糖淀粉酶基因启动子(PglaA)顺式元件的 97bp 片段五重串联而成的启动子 PN5,并利用该启动子,glaA 基因的转录水平提高了 25-45%。然后,通过共表达 glaA、α-淀粉酶(amyB)和α-葡萄糖苷酶(agdA)基因,并利用启动子 PN5,摇瓶中 100g/L 玉米淀粉的 L-苹果酸产量从 55.5g/L 提高到 72.0g/L。此外,为了降低副产物琥珀酸和富马酸的浓度,过表达了来自酿酒酵母的延胡索酸酶,该酶促进了富马酸向 L-苹果酸的转化。结果,琥珀酸和富马酸的浓度从 12.6 和 1.29g/L 降低到 7.8 和 0.59g/L,L-苹果酸的产量从 72.0g/L 提高到 78.5g/L。最后,我们发现初始发酵阶段添加葡萄糖有利于细胞生长和 L-苹果酸合成,通过共发酵 30g/L 葡萄糖和 70g/L 玉米淀粉,L-苹果酸产量进一步提高到 82.3g/L,生产强度为 1.18g/L/h,总碳源得率为 0.82g/g。