Fu Jilagamazhi, Sharma Parveen, Spicer Vic, Krokhin Oleg V, Zhang Xiangli, Fristensky Brian, Cicek Nazim, Sparling Richard, Levin David B
Department of Biosystem Engineering, University of Manitoba, Winnipeg, Manitoba, Canada.
Department of Internal Medicine & Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, Winnipeg, Manitoba, Canada.
PLoS One. 2015 Nov 6;10(11):e0142322. doi: 10.1371/journal.pone.0142322. eCollection 2015.
Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA). The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. 'Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the 'Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA). Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in determining the monomer composition of mcl-PHA polymers. Understanding the relationships between genome content, gene and gene product expression, and how these factors influence polymer synthesis, will aid in optimization of mcl-PHA production by P. putida LS46 using biodiesel waste streams.
在允许或不允许合成中链长度聚羟基脂肪酸酯（mcl-PHA）的条件下，比较了以生物柴油衍生的废甘油或废游离脂肪酸作为唯一碳源培养的恶臭假单胞菌LS46的转录组和蛋白质组。本研究的目的是阐明影响生物聚合物合成激活、细胞内积累和单体组成的机制，并确定这些机制是否在生理上特定于用于恶臭假单胞菌LS46生长的碳源。恶臭假单胞菌LS46的活性mcl-PHA合成与关键mcl-PHA生物合成基因和/或基因产物的高表达水平相关，这些基因和产物包括单体供应蛋白、PHA合酶和颗粒相关蛋白。组学数据表明，当在两种废碳源上培养时，这些基因的表达在处于不同生理状态的恶臭假单胞菌LS46细胞中受不同的遗传机制调控。恶臭假单胞菌LS46的最佳聚合物产量主要受废甘油上mcl-PHA合成过程中甘油代谢效率较低的限制。将组学数据映射到mcl-PHA生物合成途径揭示了基因表达的显著差异，主要涉及：1）甘油运输；2）回收还原当量并产生关键mcl-PHA生物合成途径中间体（如NADH/NADPH、乙酰辅酶A）的酶促反应。在以废游离脂肪酸培养的培养物的指数期观察到mcl-PHAs的活性合成，并且这与脂肪酸β-氧化途径相关。β-氧化途径中一个可能调节脂肪酸β-氧化中间体水平从而调节碳流向mcl-PHA生物合成的假定硫酯酶被高度上调。最后，数据表明所选脂肪酸代谢和mcl-PHA单体供应酶的表达差异可能在决定mcl-PHA聚合物的单体组成中起作用。了解基因组内容、基因和基因产物表达之间的关系，以及这些因素如何影响聚合物合成，将有助于优化恶臭假单胞菌LS46利用生物柴油废物流生产mcl-PHA的过程。
