Kirby James, Nishimoto Minobu, Chow Ruthie W N, Baidoo Edward E K, Wang George, Martin Joel, Schackwitz Wendy, Chan Rossana, Fortman Jeffrey L, Keasling Jay D
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA Joint BioEnergy Institute, Emeryville, California, USA.
California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA.
Appl Environ Microbiol. 2015 Jan;81(1):130-8. doi: 10.1128/AEM.02920-14. Epub 2014 Oct 17.
Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.
大多数细菌物种以及植物质体中的萜类合成是通过1-脱氧-D-木酮糖5-磷酸（DXP）途径进行的。该途径的第一步涉及DXP合酶（Dxs）将丙酮酸和3-磷酸甘油醛缩合，其中六分之一的碳以二氧化碳的形式损失。从磷酸戊糖到DXP的一种假设的新途径（nDXP）可以使从C5糖到萜类的途径更直接，并且还可以规避控制Dxs的调节机制，但目前还没有已知的酶能够将糖转化为其1-脱氧等价物。利用在以木糖为唯一碳源生长的大肠杆菌中对dxs缺失进行互补的筛选，我们发现了两个候选nDXP基因。通过野生型大肠杆菌yajO基因（注释为推定的木糖还原酶）的过表达或通过天然ribB基因中的各种突变实现了互补。用纯化的YajO和突变的RibB蛋白进行的体外分析表明，在这两种情况下，DXP都是由5-磷酸核酮糖（Ru5P）合成的。我们通过在大肠杆菌中改造DXP途径以生产倍半萜红没药烯（一种候选生物柴油），证明了这些基因在微生物萜类生物合成中的实用性。为了进一步提高从Ru5P进入该途径的通量，nDXP酶被表达为与DXP还原酶（Dxr）融合，Dxr是DXP途径中的第二个酶。Dxr-RibB（G / 108S）融合蛋白的表达使红没药烯滴度提高了4倍以上，并减轻了细胞内DXP的积累。
