Department of Bioengineering, University of California, Berkeley, CA, 94720, USA.
Appl Microbiol Biotechnol. 2014 Feb;98(4):1567-81. doi: 10.1007/s00253-013-5361-4. Epub 2013 Nov 21.
The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene). RBSs of various strengths, selected based on their theoretical strengths, were cloned 5' of the genes encoding mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and amorphadiene synthase. Operons containing one copy of each gene and all combinations of RBSs were constructed and tested for their impact on growth, amorphadiene production, enzyme level, and accumulation of select pathway intermediates. Pathways with one or more inefficiently translated enzymes led to the accumulation of pathway intermediates, slow growth, and low product titers. Choosing the most appropriate RBS combination and carbon source, we were able to reduce the accumulation of toxic metabolic intermediates, improve growth, and improve the production of amorphadiene approximately fivefold. This work demonstrates that balancing flux through a heterologous pathway and maintaining steady growth are key determinants in optimizing isoprenoid production in microbial hosts.
编码甲羟戊酸（MVA）基法呢基焦磷酸（FPP）生物合成途径的基因编码在两个操纵子中，并在大肠杆菌中表达，以增加倍半萜的产量。几个途径基因的翻译效率低下造成了瓶颈，并导致了几种途径中间体的积累，即甲羟戊酸和 FPP，以及倍半萜产物法呢烯的产量不理想。由于选择核糖体结合位点（RBS）来优化翻译效率存在困难，因此采用组合方法来选择最适合 MVA 途径下半部分（从甲羟戊酸到法呢烯）基因的 RBS。根据其理论强度选择各种强度的 RBS，克隆到编码甲羟戊酸激酶、磷酸甲羟戊酸激酶、甲羟戊酸二磷酸脱羧酶和法呢烯合酶的基因 5'端。包含每个基因一个拷贝和所有 RBS 组合的操纵子被构建并测试其对生长、法呢烯产量、酶水平和选择途径中间体积累的影响。含有一个或多个翻译效率低下的酶的途径导致途径中间体的积累、生长缓慢和低产物滴度。选择最合适的 RBS 组合和碳源，我们能够减少有毒代谢中间体的积累，提高生长速度，并将法呢烯的产量提高约五倍。这项工作表明，平衡异源途径中的通量和保持稳定生长是优化微生物宿主中异戊二烯生产的关键决定因素。
