Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway.
Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway.
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DOI：
10.1186/1475-2859-11-120
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发表时间：
2012-09-06
影响因子：
6.4
通讯作者：
Ohtake H
中科院分区：
文献类型：
--
作者：
Ye X;Honda K;Sakai T;Okano K;Omasa T;Hirota R;Kuroda A;Ohtake H
生物技术与化学制造的结合已被认为是建立可持续社会的关键技术。然而，生物催化化学转化的实际应用往往受到限制，因为它们的复杂性涉及产品产率的不可预测性和发酵过程中难以控制的问题。克服这些限制的可能策略之一是取消使用活微生物，只使用参与代谢途径的酶。使用在高温下产生嗜热酶的重组中介菌可以使天然蛋白质变性并消除不希望发生的副反应；因此，可以容易地制备高选择性和稳定的生物催化模块。通过将这些模块合理地组合在一起，可以设计和构建专门用于化学制造的人工合成路径。利用9株高产嗜热嗜热菌7种糖酵解酶、非辅因子非依赖性磷酸甘油变位酶或柯达达卡耐热球菌非磷酸化甘油醛-3-磷酸脱氢酶的9株重组大肠杆菌，构建了一条平衡消耗和再生ATP和ADP的嵌合Embden-Meyerhof(EM)途径。通过将该途径与Thermus苹果酸/乳酸脱氢酶偶联，从葡萄糖中产生化学计量比的乳酸，总的ATP周转率为31。在这项研究中，开发了一种新颖而简单的技术，可以灵活地设计定制的代谢途径。这一概念已经通过一条不形成三磷酸腺苷的嵌合EM途径得到了验证。我们将这项技术命名为“合成代谢工程”。我们的技术原则上适用于所有嗜热酶，只要它们能在宿主中功能性表达，因此可能适用于按需生产任何化学品或材料的生物催化制造。
The integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed. A chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31. In this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.
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