High ectoine production by an engineered Halomonas hydrothermalis Y2 in a reduced salinity medium
High ectoine production by an engineered Halomonas hydrothermalis Y2 in a reduced salinity medium
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工程改造的热液盐单胞菌 Y2 在低盐度培养基中高四氢嘧啶产量
DOI：
10.1186/s12934-019-1230-x
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发表时间：
2019-10
影响因子：
6.4
通讯作者：
Yang Chunyu
中科院分区：
文献类型：
--
作者：
Zhao Qi;Yang Chunyu
背景
四氢-2-甲基-4-嘧啶羧酸（四氢嘧啶）作为一种亲和吸附溶质，在许多领域显示出巨大的应用潜力。但由于其产能低、介质含盐量高等问题，严重阻碍了其广泛应用.
结果
在四氢嘧啶分泌菌株水热盐单胞菌Y2的基因组中鉴定了整个四氢嘧啶代谢，包括四氢嘧啶合成途径和催化剂途径。通过对编码四氢嘧啶羟化酶（EctD）和（或）四氢嘧啶水解酶（DoeA）的基因进行框内缺失，破坏了四氢嘧啶的利用途径，从而显著提高了四氢嘧啶的产量。在500 mL烧瓶中使用含有100 g L − 1 NaCl的优化培养基，Y2/Δ ectD/Δ doeA的双突变体在培养30 h后合成3.13 g L − 1四氢嘧啶。这远远高于野生型菌株的产量（1.91 g L − 1），也超过了Y2/Δ ectD的产量（2.21 g L − 1）。Y2/Δ ectD/Δ doeA对四氢嘧啶的富集作用明显增强，提示Doe途径在四氢嘧啶催化过程中起重要作用。此外，为了降低发酵培养基的盐度，克服废水处理的困难，以菌株Y2/Δ ectD/Δ doeA为基础，构建了缺失关键Na +/H+逆向转运蛋白Mrp和（或）NhaD2的突变株。结果，Mrp缺陷型菌株在含有较低浓度NaCl的培养基中可以合成等量的四氢嘧啶（约7 g L-1或500 mg（g DCW）-1）。在60 g L − 1 NaCl胁迫的补料分批发酵过程中，Mrp缺陷型菌株最多积累10.5 g L − 1四氢嘧啶，比产量为765 mg（g DCW）− 1，谷氨酸产量为0.21 g g − 1。
结论
Y2/Δ ectD/Δ doeA对四氢嘧啶合成的显著促进作用提示Doe途径在四氢嘧啶合成中起着重要作用。此外，Mrp缺陷型菌株降低的盐度要求意味着许多相容性溶质生物合成的可行方案，即，通过沉默一些Na +/H+反向转运蛋白在其嗜盐生产，从而降低介质盐度。
Background
As an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications.
Results
The entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L−1 NaCl in a 500-mL flask, the double mutant of Y2/ΔectD/ΔdoeA synthesized 3.13 g L−1 ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L−1), and also exceeds the production of Y2/ΔectD (2.21 g L−1). The remarkably enhanced accumulation of ectoine by Y2/ΔectD/ΔdoeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na+/H+ antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/ΔectD/ΔdoeA. As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L−1 or 500 mg (g DCW) −1) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L−1 NaCl stress, a maximum 10.5 g L−1 ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW)−1 and a yield of 0.21 g g−1 monosodium glutamate.
Conclusion
The remarkably enhanced production of ectoine by Y2/ΔectD/ΔdoeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na+/H+ antiporters in their halophilic producers and thus lowering the medium salinity.
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影响因子：
5.1
作者：
Schwibbert K;Marin-Sanguino A;Bagyan I;Heidrich G;Lentzen G;Seitz H;Rampp M;Schuster SC;Klenk HP;Pfeiffer F;Oesterhelt D;Kunte HJ
通讯作者：
Kunte HJ
影响因子：
2.1
作者：
K. Göller;Alexandra Ofer;E. Galinski
通讯作者：
K. Göller;Alexandra Ofer;E. Galinski
影响因子：
5.3
作者：
通讯作者：
--
影响因子：
4.1
作者：
Qi Zhao;Yiwei Meng;Shannan Li;Pei-jun Lv;P. Xu;Chunyu Yang
通讯作者：
Qi Zhao;Yiwei Meng;Shannan Li;Pei-jun Lv;P. Xu;Chunyu Yang
影响因子：
4.1
作者：
Chen, Ruifeng;Zhu, Lijun;Qian, Junqing
通讯作者：
Qian, Junqing