Wen Rex C, Shen Claire R
Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, 30013 Taiwan.
Biotechnol Biofuels. 2016 Dec 19;9:267. doi: 10.1186/s13068-016-0680-1. eCollection 2016.
As a natural fermentation product secreted by species, bio-based 1-butanol has attracted great attention for its potential as alternative fuel and chemical feedstock. Feasibility of microbial 1-butanol production has also been demonstrated in various recombinant hosts.
In this work, we constructed a self-regulated 1-butanol production system in by borrowing its endogenous fermentation regulatory elements (FRE) to automatically drive the 1-butanol biosynthetic genes in response to its natural fermentation need. Four different cassette of 5' upstream transcription and translation regulatory regions controlling the expression of the major fermentative genes , , , and were cloned individually to drive the 1-butanol pathway genes distributed among three plasmids, resulting in 64 combinations that were tested for 1-butanol production efficiency. Fermentation of 1-butanol was triggered by anaerobicity in all cases. In the growth-decoupled production screening, only combinations with formate dehydrogenase (Fdh) overexpressed under FRE demonstrated higher titer of 1-butanol anaerobically. In vitro assay revealed that 1-butanol productivity was directly correlated with Fdh activity under such condition. Switching cells to oxygen-limiting condition prior to significant accumulation of biomass appeared to be crucial for the induction of enzyme synthesis and the efficiency of 1-butanol fermentation. With the selection pressure of anaerobic NADH balance, the engineered strain demonstrated stable production of 1-butanol anaerobically without the addition of inducer or antibiotics, reaching a titer of 10 g/L in 24 h and a yield of 0.25 g/g glucose under high-density fermentation.
Here, we successfully engineered a self-regulated 1-butanol fermentation system in based on the natural regulation of fermentation reactions. This work also demonstrated the effectiveness of selection pressure based on redox balance anaerobically. Results obtained from this study may help enhance the industrial relevance of 1-butanol synthesis using and solidifies the possibility of strain improvement by directed evolution.
作为一种由特定物种分泌的天然发酵产物，生物基1-丁醇因其作为替代燃料和化学原料的潜力而备受关注。在各种重组宿主中也已证明了微生物生产1-丁醇的可行性。
在本研究中，我们通过借用其内源发酵调控元件（FRE）构建了一个自我调控的1-丁醇生产系统，以根据其天然发酵需求自动驱动1-丁醇生物合成基因。分别克隆了控制主要发酵基因、、、和表达的4种不同的5'上游转录和翻译调控区域盒，以驱动分布在三个质粒中的1-丁醇途径基因，产生了64种组合，并对其1-丁醇生产效率进行了测试。在所有情况下，1-丁醇发酵均由厌氧触发。在生长解耦生产筛选中，只有在FRE下甲酸脱氢酶（Fdh）过表达的组合在厌氧条件下表现出更高的1-丁醇滴度。体外试验表明，在这种条件下丁醇生产率与Fdh活性直接相关。在生物量显著积累之前将细胞切换到限氧条件似乎对酶合成的诱导和1-丁醇发酵效率至关重要。在厌氧NADH平衡的选择压力下，工程菌株在不添加诱导剂或抗生素的情况下厌氧稳定生产1-丁醇，在高密度发酵下24小时内达到10 g/L的滴度，葡萄糖产率为0.25 g/g。
在此，我们基于发酵反应的天然调控成功构建了一个自我调控的1-丁醇发酵系统。这项工作还证明了基于厌氧氧化还原平衡的选择压力的有效性。本研究获得的结果可能有助于提高利用生产I-丁醇的工业相关性，并巩固通过定向进化改进菌株的可能性。
