Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa Cuernavaca, Apdo. Post. 510-3, 62210, Cuernavaca, Morelos, Mexico.
Departament d'Engiyeria Química, Biològica i Ambiental, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallés), Barcelona, Spain.
Microb Cell Fact. 2018 Jan 22;17(1):10. doi: 10.1186/s12934-018-0860-8.
Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P) ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions.
The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP ratio, showing that at the lowest OTR (2.4 mmol L h), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L h). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L h. At the highest OTR condition, a decrease in the NADPH/NADP ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH).
We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.
氮单胞菌是一种能产生海藻酸盐和聚羟基丁酸酯（P3HB）的细菌；然而，NAD(P)H/NAD(P) 比值在这些生物聚合物生物合成途径中的代谢通量的作用尚不清楚。本研究的目的是在氧限制和非氧限制条件下，评估 NAD(P)H/NAD(P) 比值和参与海藻酸盐和 P3HB 生物合成的代谢通量。
结果表明，氧气供应的变化对代谢通量和细胞内 NADPH/NADP 比值有重要影响，表明在最低 OTR（2.4 mmol L h）下，三羧酸（TCA）循环的通量降低了 27.6 倍，而在没有氧限制的培养物（OTR = 14.6 mmol L h）中，P3HB 生物合成的通量增加了 6.6 倍。这与 phbB 的转录水平和 P3HB 生物合成的增加一致。此外，在没有氧限制的条件下，与 2.4 mmol L h 的条件相比，碳摄取率（增加两倍）以及通过戊糖磷酸（PP）途径的通量（增加 4.8 倍）增加。在最高 OTR 条件下，观察到 NADPH/NADP 比值降低了三倍，这可能是由于在固氮条件下氮酶的呼吸保护导致呼吸速率增加所致，与解偶联呼吸链基因（ndhII 和 cydA）的高表达以及氮酶复合物（nifH）的基因表达诱导相关。
我们已经证明，氧气供应的变化会影响细胞的内部氧化还原状态和碳代谢通量。这也对 TCA 循环和 PP 途径以及海藻酸盐和 P3HB 生物合成通量产生强烈影响。
