Exploration of the metabolic flexibility of glycogen accumulating organisms through metatranscriptome analysis and metabolic characterization

Glycogen accumulating organisms(GAOs) are closely related to the deterioration of enhanced biological phosphorus removal systems. However, the metabolic mechanisms that drive GAOs remain unclear. Here, the two-thirds supernatant of a reactor were decanted following the anaerobic period to enrich GAOs. Long-term monitoring demonstrated that the system was stable and exhibited typical characteristics of GAOs metabolism. Acetate was completely consumed after 60 min of the anaerobic phase. The level of glycogen decreased from 0.20 to 0.14 g/gSS during the anaerobic phase, whereas the level of glycogen significantly increased to 0.21g/gSS at the end of the aerobic period. Moreover, there was almost no phosphate release and absorption in the complete periods, thus confirming the successful construction of a GAOs enrichment system. Microbial community analysis demonstrated that Ca. Contendobacter was among the core functional genera and showed the highest activity among all of the communities. Furthermore, our study is the first to identify the involvement of the ethyl-malonyl-CoA pathway in the synthesis of polyhydroxyvalerate via croR, ccr, ecm, mcd, mch and mcl genes. The Embden-Meyerhof-Parnas(EMP) pathway was preferentially used via glgP. Furthermore, the glyoxylate cycle was the main source of ATP under anaerobic conditions, whereas the tricarboxylic acid cycle provided ATP under aerobic conditions. aceA and mdh appeared to be major modulators of the glyoxylate pathway for controlling energy flow. Collectively, our findings not only revealed the crucial metabolic mechanisms in a GAOs enrichment system but also provided insights into the potential application of Ca. Contendobacter for wastewater treatment.

Using high-throughput data and dynamic flux balance modeling techniques to identify points of constraint in xylose utilization in Saccharomyces cerevisiae

Background Several enzymes and cofactors have been identified as contributing to the slow utilization of xylose by xylose-fermenting strains of Saccharomyces cerevisiae.However,there has been no consensus on which of these possible bottle-necks are the most important to address.A previous strain characterization study from our lab suggested that insufficient NAD+limits fermentation and may be the most important bottleneck affecting utilization of xylose for the production of ethanol.The development and validation of a genome scale dynamic flux balance model would help to verify the existence and extent of this and other metabolic bottlenecks and suggest solutions to guide future strain development thereby minimiz-ing bottleneck impact on process economics.Results A dynamic flux balance model was developed to identify bottlenecks in several strains of S.cerevisiae,both with wild-type pentose phosphate pathway expression and with the pathway over expressed.ZWF1 was found to be limiting in the oxidative portion of the pentose phosphate pathway under oxygen replete conditions.This pathway is used to regenerate NADPH.Under oxygen limiting conditions,respiration of xylose was limited by the lack of oxygen as a terminal electron acceptor.Ethanol production was also limited under these conditions due to the inability to balance NAD+/NADH.The model suggests the use of the anaplerotic glyoxylate pathway to improve NAD+/NADH balance,increasing ethanol produc-tion by 50%while producing succinate as a coproduct at upwards of 20 g/l.Conclusion In the production of high value chemicals from biomass,the use of the respiratory metabolism is a waste of feedstock carbon.Bottlenecks previously identified in the oxidative pentose phosphate pathway are currently only relevant under oxygen-replete conditions and cannot impact the partitioning of carbon between the respiratory and fermentative pathways.Focusing future efforts on the non-respiratory balancing of NAD+/NADH,perhaps through the glyoxylate pathway,would improve the economics of ethanol production both directly and through coproduct formation.