Centre of Biological Engineering, University of Minho, Braga, Portugal.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Oeiras, Portugal.
Microb Cell Fact. 2018 Nov 16;17(1):178. doi: 10.1186/s12934-018-1023-7.
Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075-4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production.
To drive MG production, the pathway for the synthesis of GDP-mannose, one of the MG biosynthetic precursors, was overexpressed in S. cerevisiae along with the MG biosynthetic pathway. MG production was evaluated under different cultivation modes, i.e., flask bottle, batch, and continuous mode with different dilution rates. The genes encoding mannose-6-phosphate isomerase (PMI40) and GDP-mannose pyrophosphorylase (PSA1) were introduced into strain MG01, hosting a plasmid encoding the MG biosynthetic machinery. The resulting engineered strain (MG02) showed around a twofold increase in the activity of PMI40 and PSA1 in comparison to the wild-type. In batch mode, strain MG02 accumulated 15.86 mg g , representing a 2.2-fold increase relative to the reference strain (MG01). In continuous culture, at a dilution rate of 0.15 h, there was a 1.5-fold improvement in productivity.
In the present study, the yield and productivity of MG were increased by overexpression of the GDP-mannose pathway and optimization of the mode of cultivation. A maximum of 15.86 mg g was achieved in batch cultivation and maximal productivity of 1.79 mg g h in continuous mode. Additionally, a positive correlation between MG productivity and growth rate/dilution rate was established, although this correlation is not observed for MG yield.
甘露糖甘油酯（MG）是适应热环境的海洋微生物中最广泛分布的兼容溶质之一。这种离子溶质具有出色的保护蛋白质免受热变性的能力，因此提出了大量的生物技术和临床应用。然而，目前高昂的生产成本对大规模应用造成了严重限制。所有已知的微生物生产者都是通过两步途径从 GDP-甘露糖和 3-磷酸甘油酸合成 MG 的，其中甘露糖-3-磷酸甘油酸是中间代谢物。在早期的一项工作中，该途径在酿酒酵母中表达，目的是确认基因功能（Empadinhas 等人，J Bacteriol 186:4075-4084, 2004），但 MG 积累水平较低。因此，鉴于该化合物具有潜在的生物技术价值，我们决定进一步努力将酿酒酵母转化为 MG 生产的高效细胞工厂。
为了促进 MG 的生产，在酿酒酵母中过量表达了 GDP-甘露糖合成途径的合成途径，这是 MG 生物合成前体之一，同时还表达了 MG 生物合成途径。在不同的培养模式下评估了 MG 生产，即在摇瓶、分批和不同稀释率的连续模式下。引入了编码甘露糖-6-磷酸异构酶（PMI40）和 GDP-甘露糖焦磷酸化酶（PSA1）的基因到携带 MG 生物合成机制质粒的菌株 MG01 中。与野生型相比，工程菌株（MG02）的 PMI40 和 PSA1 活性增加了约两倍。在分批培养模式下，菌株 MG02 积累了 15.86 mg g-1，比参考菌株（MG01）增加了 2.2 倍。在连续培养中，在稀释率为 0.15 h-1 时，生产力提高了 1.5 倍。
在本研究中，通过过表达 GDP-甘露糖途径和优化培养方式，提高了 MG 的产量和生产力。在分批培养中最高达到 15.86 mg g-1，在连续培养中最大生产力为 1.79 mg g-1 h-1。此外，建立了 MG 生产力与生长速率/稀释速率之间的正相关关系，尽管这种相关性不适用于 MG 产量。
