多形汉逊酵母细胞工厂实现甲醇生物转化合成3-羟基丙酸

Bioconversion of methanol to 3-hydroxypropionate by engineering Ogataea polymorpha

3-羟基丙酸(3-HP)是公认的高附加值化学品之一,可用于合成多种化学品,例如丙烯酰胺、丙烯酸、1,3-丙二醇以及可降解塑料.目前3-HP的生物合成过程均以糖为原料,且需求不断增长,亟需寻找不依赖耕地的替代原料.碳一资源来源广泛,是理想的生物制造原料.其中,甲醇可由煤、天然气制得,也可由CO_(2)加氢制备.多形汉逊酵母作为典型的甲基营养型酵母,能够以甲醇为唯一碳源和能源进行生长,并且能够耐受高温、高渗透压和低pH等条件,是非常优秀的工业菌株.近年来基因编辑技术的发展,大大加快了多形汉逊酵母代谢工程改造,有望构建高效细胞工厂,实现甲醇生物转化.本文以多形汉逊酵母为宿主,系统改造其细胞代谢实现甲醇高效转化为3-HP.由于汉逊酵母自身不能合成3-HP,因此需要表达来源于Chloroflexus aurantiacus的3-HP合成途径关键基因MCR,该途径以乙酰-CoA和丙二酰-CoA为前体,以还原力NADPH为辅因子.首先优化MCR表达,采用融合蛋白并结合甲醇诱导型启动子PAOX基因组整合MCR表达, 3-HP合成效率最高;其次,强化供应前体乙酰-CoA以及丙二酰-CoA,使3-HP产量提高26%;进一步改造氧化磷酸戊糖途径以增强辅因子NADPH的供应,使3-HP产量提高30%;最后,将增强前体和辅因子供应的有效策略结合,工程菌HP15相比出发菌HP07摇瓶发酵3-HP产量提高135%,达到1.45 g/L.通过摇瓶补料分批发酵, 3-HP产量达到7.10 g/L,得率达到0.14 g/g甲醇,为目前报道碳一资源合成3-HP的较高产量,表明多形汉逊酵母作为甲醇细胞工厂合成化学品具有很好的潜力.预期结合“液态阳光”CO_(2)制备甲醇技术以及甲醇生物转化过程,有望将CO_(2)制备成高附加值化学品,有助于实现碳中和.

Methanol bioconversion toward chemical production can be helpful for carbon neutrality. Here metabolic engineering an industrial methylotrophic yeast Ogataea polymorpha was conducted for overproduction of 3-hydroxypropionate(3-HP), an important platform chemical, which can be used for production of special chemicals including acrylamide, acrylic acid, 1,3-propanediol, as well as biodegradable plastics. We developed several metabolic engineering strategies to optimize the 3-HP biosynthetic pathway and enhance the supply of precursors acetyl-CoA and malonyl-CoA, and cofactor NADPH, which enabled the 3-HP production of 1.45 g/L under batch fermentation and 7.10 g/L under fed-batch fermentation at shake flask scale, with a yield of 0.14 g/g methanol. This was, to our knowledge, the highest 3-HP titer from methanol and even one-carbon sources. This study demonstrated the potential of O. polymorpha as a cell factory for chemical production from methanol.