Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Apdo, Postal 510-3, Cuernavaca, Morelos 62250, Mexico.
Microb Cell Fact. 2013 Sep 30;12:86. doi: 10.1186/1475-2859-12-86.
During the last two decades many efforts have been directed towards obtaining efficient microbial processes for the production of shikimic acid (SA); however, feeding high amounts of substrate to increase the titer of this compound has invariably rendered low conversion yields, leaving room for improvement of the producing strains. In this work we report an alternative platform to overproduce SA in a laboratory-evolved Escherichia coli strain, based on plasmid-driven constitutive expression of six genes selected from the pentose phosphate and aromatic amino acid pathways, artificially arranged as an operon. Production strains also carried inactivated genes coding for phosphotransferase system components (ptsHIcrr), shikimate kinases I and II (aroK and aroL), pyruvate kinase I (pykF) and the lactose operon repressor (lacI).
The strong and constitutive expression of the constructed operon permitted SA production from the beginning of the cultures, as evidenced in 1 L batch-mode fermentors starting with high concentrations of glucose and yeast extract. Inactivation of the pykF gene improved SA production under the evaluated conditions by increasing the titer, yield and productivity of this metabolite compared to the isogenic pykF+ strain. The best producing strain accumulated up to 43 g/L of SA in 30 h and relatively low concentrations of acetate and aromatic byproducts were detected, with SA accounting for 80% of the produced aromatic compounds. These results were consistent with high expression levels of the glycolytic pathway and synthetic operon genes from the beginning of fermentations, as revealed by transcriptomic analysis. Despite the consumption of 100 g/L of glucose, the yields on glucose of SA and of total aromatic compounds were about 50% and 60% of the theoretical maximum, respectively. The obtained yields and specific production and consumption rates proved to be constant with three different substrate concentrations.
The developed production system allowed continuous SA accumulation until glucose exhaustion and eliminated the requirement for culture inducers. The obtained SA titers and yields represent the highest reported values for a high-substrate batch process, postulating the strategy described in this report as an interesting alternative to the traditionally employed fed-batch processes for SA production.
在过去的二十年中，人们做出了许多努力，旨在开发高效的微生物工艺来生产莽草酸（SA）；然而，为了提高这种化合物的含量，人们往往需要大量地添加底物，这导致转化率很低，因此仍有改进生产菌株的空间。在这项工作中，我们报告了一种替代平台，可以在经过实验室进化的大肠杆菌菌株中过量生产 SA，该平台基于从戊糖磷酸和芳香族氨基酸途径中选择的六个基因的质粒驱动组成型表达，这些基因被人工排列成一个操纵子。生产菌株还携带了失活的基因，这些基因编码磷酸转移酶系统成分（ptsHIcrr）、莽草酸激酶 I 和 II（aroK 和 aroL）、丙酮酸激酶 I（pykF）和乳糖操纵子抑制剂（lacI）。
构建的操纵子的强组成型表达允许从培养物开始就生产 SA，这在从高浓度葡萄糖和酵母提取物开始的 1 L 分批式发酵罐中得到了证明。在评估条件下，pykF 基因的失活通过提高该代谢物的含量、产率和生产力，与异源型 pykF+ 菌株相比，改善了 SA 的生产。最佳生产菌株在 30 小时内积累了高达 43 g/L 的 SA，并且检测到相对较低浓度的乙酸和芳香族副产物，其中 SA 占产生的芳香族化合物的 80%。这些结果与发酵开始时糖酵解途径和合成操纵子基因的高表达水平一致，这是通过转录组分析揭示的。尽管消耗了 100 g/L 的葡萄糖，但 SA 和总芳香族化合物的葡萄糖得率分别约为理论最大值的 50%和 60%。获得的产率和特定的生产和消耗速率在三种不同的底物浓度下是恒定的。
所开发的生产系统允许连续积累 SA，直到葡萄糖耗尽，并消除了对培养物诱导剂的需求。所获得的 SA 含量和产率代表了高底物分批工艺的最高报道值，这表明本报告中描述的策略是一种有前途的替代传统分批补料工艺的方法，用于 SA 生产。
