Instituto de Investigaciones en Biodiversidad y Biotecnología, Consejo Nacional de Investigaciones Científicas y Técnicas, Mar del Plata, Buenos Aires, Argentina; Fundación para Investigaciones Biológicas Aplicadas, Argentina.
Instituto de Investigaciones en Biodiversidad y Biotecnología, Consejo Nacional de Investigaciones Científicas y Técnicas, Mar del Plata, Buenos Aires, Argentina; Fundación para Investigaciones Biológicas Aplicadas, Argentina.
Metab Eng. 2017 Mar;40:59-68. doi: 10.1016/j.ymben.2017.01.002. Epub 2017 Jan 9.
The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N and CO from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N from the air into crops.
一些细菌和古菌进行的生物固氮作用是替代合成氮肥最有吸引力的方法之一。然而，除了共生的根瘤菌-豆科植物系统外，朝着更广泛地实现这一目标的进展一直很缓慢。在这项研究中，我们操纵了需氧细菌固氮菌（Azotobacter vinelandii）中固氮和同化作用的内源性调节。用外源诱导启动子替代谷氨酰胺合成酶的天然启动子，产生了条件致死突变株，在没有诱导剂的情况下无法进行固氮生长。这种突变表型可以在携带 nifL 基因缺失的双突变菌株中恢复，导致 nif 基因的组成型表达和铵的产量增加。在 GS 非诱导条件下，单突变株和双突变株都持续向生长培养基中释放出非常高水平的铵（>20mM）。与单突变株相比，双突变株在更宽的诱导剂浓度范围内生长并分泌高水平的铵。诱导突变细胞可以用不同水平的谷氨酰胺合成酶装载，这导致随后细胞外铵积累的模式不同。将工程细菌接种到微藻培养物中，除了空气中的氮和二氧化碳外，没有其他碳和氮源，结果导致微藻大量增殖，而添加诱导剂后则抑制了微藻的增殖。单突变株和双突变株也促进了黄瓜植物在没有添加氮肥的情况下的生长，而在亲本菌株中，这种特性只是微不足道的。这项研究提供了一个简单的合成遗传回路，可能会激发对优化接种剂的工程设计，从而有效地将空气中的氮输送到作物中。
