Pathway-Consensus Approach to Metabolic Network Reconstruction for Pseudomonas putida KT2440 by Systematic Comparison of Published Models

Over 100 genome-scale metabolic networks (GSMNs) have been published in recent years and widely used for phenotype prediction and pathway design. However, GSMNs for a specific organism reconstructed by different research groups usually produce inconsistent simulation results, which makes it difficult to use the GSMNs for precise optimal pathway design. Therefore, it is necessary to compare and identify the discrepancies among networks and build a consensus metabolic network for an organism. Here we proposed a process for systematic comparison of metabolic networks at pathway level. We compared four published GSMNs of Pseudomonas putida KT2440 and identified the discrepancies leading to inconsistent pathway calculation results. The mistakes in the models were corrected based on information from literature so that all the calculated synthesis and uptake pathways were the same. Subsequently we built a pathway-consensus model and then further updated it with the latest genome annotation information to obtain modelPpuQY1140 for P. putida KT2440, which includes 1140 genes, 1171 reactions and 1104 metabolites. We found that even small errors in a GSMN could have great impacts on the calculated optimal pathways and thus may lead to incorrect pathway design strategies. Careful investigation of the calculated pathways during the metabolic network reconstruction process is essential for building proper GSMNs for pathway design

High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases

Biogenesis of medium-chain-length polyhydroxyalkanoates

26 p.-3 fig.-2 tab.Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biotechnologically useful natural products found in many bacteria. This biopolymer functions as a carbon and energy storage reservoir in cells but has physical and mechanical properties that make it a promising bioplastic with applications ranging from adhesives to medical implants. Therefore, there is much interest in understanding the biology of mcl-PHA synthesis and metabolism. Increased knowledge of PHA biology serves as a foundation for the bioengineering of PHA and its eventual use as a biologically derived product. This chapter covers the state of knowledge on mcl-PHA, including its synthesis and its central role in cellular metabolism. Moreover, this chapter discusses methods for bioengineering mcl-PHA production in bacteria as well as synthetic biology methods for its study and production in the natural mcl-PHA producer, Pseudomonas putida.Research on polymer biotechnology in the laboratory of M. Auxiliadora Prieto is supported by funding from the European Union’s Horizon 2020 research and innovation program under grant agreements number 633962 and 679050. We also acknowledge support from the Community of Madrid (P2013/MIT2807) and the Spanish Ministry of Economy (BIO201344878R, BIO2014-61515-EXP).Peer reviewedPostprin

Advanced glycation end products (AGEs) in diabetic complications

Hyperglycemic condition in diabetes accelerates formation of advanced glycation end products (AGEs) that are formed as a result of series of reaction between reducing sugars and proteins. Accumulation of AGEs has been implicated in development of insulin resistance as well as in the pathogenesis of diabetic complications. The principal mechanism by which AGEs render harmful effects is through interaction with cell bound receptors. Certain receptors like AGE-R1 are involved in degradation of AGEs, while certain other receptors like receptor for AGE (RAGE) bring about counter effects exacerbating the situation. Accumulation of diverse AGEs, synergistically down regulate AGE-R1 while up regulate RAGE causing vicious cycle leading to enhanced formation and further accumulation of AGEs. In this article we discuss the formation of heterogeneous AGEs, importance of detection and quantification of AGEs, biological degradation of AGEs via different receptors, AGE-RAGE and its role in proinflammatory signaling, AGE mediated diabetic vascular complications such as nephropathy, retinopathy, neuropathy, cardiovascular and cerebrovascular diseases and finally the biological inhibition of AGEs is discussed along with chemical inhibitors for AGEs and natural products in AGE inhibition as a measure for the prevention of diabetic complications