University of Nottingham, Bioprocess, Environmental and Chemical Technologies Research Group, Nottingham, UK.
Ingenza Ltd, Edinburgh, UK.
Microbiology (Reading). 2018 Mar;164(3):287-298. doi: 10.1099/mic.0.000611. Epub 2018 Feb 5.
Bio-production of fuels and chemicals from lignocellulosic C5 sugars usually requires the use of the pentose phosphate pathway (PPP) to produce pyruvate. Unfortunately, the oxidation of pyruvate to acetyl-coenzyme A results in the loss of 33 % of the carbon as CO2, to the detriment of sustainability and process economics. To improve atom efficiency, we engineered Escherichia coli to utilize d-xylose constitutively using the Weimberg pathway, to allow direct production of 2-oxoglutarate without CO2 loss. After confirming enzyme expression in vitro, the pathway expression was optimized in vivo using a combinatorial approach, by screening a range of constitutive promoters whilst systematically varying the gene order. A PPP-deficient (ΔxylAB), 2-oxoglutarate auxotroph (Δicd) was used as the host strain, so that growth on d-xylose depended on the expression of the Weimberg pathway, and variants expressing Caulobacter crescentus xylXAB could be selected on minimal agar plates. The strains were isolated and high-throughput measurement of the growth rates on d-xylose was used to identify the fastest growing variant. This strain contained the pL promoter, with C. crescentus xylA at the first position in the synthetic operon, and grew at 42 % of the rate on d-xylose compared to wild-type E. coli using the PPP. Remarkably, the biomass yield was improved by 53.5 % compared with the wild-type upon restoration of icd activity. Therefore, the strain grows efficiently and constitutively on d-xylose, and offers great potential for use as a new host strain to engineer carbon-efficient production of fuels and chemicals via the Weimberg pathway.
从木质纤维素 C5 糖生物生产燃料和化学品通常需要利用戊糖磷酸途径 (PPP) 来生产丙酮酸。不幸的是，丙酮酸氧化为乙酰辅酶 A 会导致 33%的碳以 CO2 的形式损失，从而不利于可持续性和工艺经济性。为了提高原子效率，我们通过工程改造大肠杆菌，利用 Weimberg 途径组成型利用 d-木糖，从而可以直接生产 2-氧代戊二酸而不会损失 CO2。在体外确认酶表达后，我们通过组合方法在体内优化了途径表达，通过筛选一系列组成型启动子，同时系统地改变基因顺序。使用 PPP 缺陷型（ΔxylAB）、2-氧代戊二酸营养缺陷型（Δicd）作为宿主菌株，因此 d-木糖上的生长取决于 Weimberg 途径的表达，并且可以在最小琼脂平板上选择表达 Caulobacter crescentus xylXAB 的变体。分离出这些菌株，并通过高通量测量 d-木糖上的生长速率来鉴定生长最快的变体。该菌株含有 pL 启动子，C. crescentus xylA 位于合成操纵子的第一个位置，与使用 PPP 的野生型大肠杆菌相比，在 d-木糖上的生长速度提高了 42%。值得注意的是，与野生型相比，当 icd 活性恢复时，生物质产量提高了 53.5%。因此，该菌株能够高效且组成型地在 d-木糖上生长，为通过 Weimberg 途径工程化高效利用碳生产燃料和化学品提供了巨大潜力。
