Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942.
Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942.
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DOI：
10.3389/fmicb.2016.00475
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发表时间：
2016
影响因子：
5.2
通讯作者：
Price GD
中科院分区：
文献类型：
--
作者：
Benson PJ;Purcell-Meyerink D;Hocart CH;Truong TT;James GO;Rourke L;Djordjevic MA;Blackburn SI;Price GD
从光合作用固定的CO2生产碳商品的兴趣集中在蓝藻作为代谢工程和途径研究的目标。我们研究了在氮剥夺下，在模式蓝藻物种细长聚球藻PCC 7942中碳通量的重定向，以优化工业上理想的化合物丙酮酸的生产。在氮限制条件下，过量的碳自然地储存为多分支多糖，糖原，但是糖原合成的阻断，通过编码ADP-葡萄糖焦磷酸化酶（glgC）的基因中的敲除突变，导致有机酸，丙酮酸和2-酮戊二酸的积累，作为溢流排泄物进入细胞外培养基。ΔglgC菌株在缺氮48 h下首次分泌丙酮酸。此外，通过将培养物pH增加至pH 10，可以显著提高丙酮酸的排泄，这表明涉及未知的底物/质子同向转运体用于输出。ΔglgC突变体还被工程化以表达葡萄糖和蔗糖的外源转运蛋白，然后在外源有机碳供应下光混合营养生长，如在N-剥夺期间添加5 mM葡萄糖或蔗糖。在这些条件下，我们观察到细胞外丙酮酸排泄增加四倍时，加入葡萄糖，和一个较小的增加与加入蔗糖。虽然丙酮酸排泄的大小与ΔglgC菌株的碳酸氢盐依赖性光合O2释放能力或光强度无关，但是，在N-剥夺之前的发酵剂培养物的密度与最终的胞外丙酮酸浓度之间观察到正相关。有助于增强丙酮酸排泄的因素进行了讨论，以及考虑丙酮酸排泄的碳源是否可能来自光合作用CO2固定或现有碳储存的再动员。
Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized production of the industrially desirable compound, pyruvate. Under nitrogen limited conditions, excess carbon is naturally stored as the multi-branched polysaccharide, glycogen, but a block in glycogen synthesis, via knockout mutation in the gene encoding ADP-glucose pyrophosphorylase (glgC), results in the accumulation of the organic acids, pyruvate and 2-oxoglutarate, as overflow excretions into the extracellular media. The ΔglgC strain, under 48 h of N-deprivation was shown to excrete pyruvate for the first time in this strain. Additionally, by increasing culture pH, to pH 10, it was possible to substantially elevate excretion of pyruvate, suggesting the involvement of an unknown substrate/proton symporter for export. The ΔglgC mutant was also engineered to express foreign transporters for glucose and sucrose, and then grown photomixotrophically with exogenous organic carbon supply, as added 5 mM glucose or sucrose during N- deprivation. Under these conditions we observed a fourfold increase in extracellular pyruvate excretion when glucose was added, and a smaller increase with added sucrose. Although the magnitude of pyruvate excretion did not correlate with the capacity of the ΔglgC strain for bicarbonate-dependent photosynthetic O2 evolution, or with light intensity, there was, however, a positive correlation observed between the density of the starter culture prior to N-deprivation and the final extracellular pyruvate concentration. The factors that contribute to enhancement of pyruvate excretion are discussed, as well as consideration of whether the source of carbon for pyruvate excretion might be derived from photosynthetic CO2 fixation or from remobilisation of existing carbon stores.