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Lignocellulosic biomass can be hydrolyzed into two major sugars of glucose and xylose, and thus the strategy for the efficient consumption of both sugars is highly desirable. NADPH is the essential molecule for the production of industrially important value-added chemicals, and thus its availability is quite important. Escherichia coli mutant lacking the pgi gene encoding phosphoglucose isomerase (Pgi) has been preferentially used to overproduce the NADPH. However, there exists a disadvantage that the cell growth rate becomes low for the mutant grown on glucose. This limits the efficient NADPH production, and therefore, it is quite important to investigate how addition of different carbon source such as xylose (other than glucose) effectively improves the NADPH production. In this study, we have developed a kinetic model to propose an efficient NADPH production system using E. coli pgi-knockout mutant with a mixture of glucose and xylose. The proposed system adds xylose to glucose medium to recover the suppressed growth of the pgi mutant, and determines the xylose content to maximize the NADPH productivity. Finally, we have designed a mevalonate (MVA) production system by implementing ArcA overexpression into the pgi-knockout mutant using a mixture of glucose and xylose. In addition to NADPH overproduction, the accumulation of acetyl-CoA (AcCoA) is necessary for the efficient MVA production. In the present study, therefore, we considered to overexpress ArcA, where ArcA overexpression suppresses the TCA cycle, causing the overflow of AcCoA, a precursor of MVA. We predicted the xylose content that maximizes the MVA production. This approach demonstrates the possibility of a great progress in the computer-aided rational design of the microbial cell factories for useful metabolite production.

An integrated analysis of the cell growth characteristics, enzyme activities, intracellular metabolite concentrations was made to investigate the metabolic regulation of pgi gene knockout Escherichia coli based on batch culture and continuous culture which was performed at the dilution rate of 0.2h-1. The enzymatic study identified that pathways of pentose phosphate, ED pathway and glyoxylate shunt were all active in pgi mutant. The glycolysis enzymes i.e glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphatase, pyruvate kinase, triose phosphate isomerase were down regulated implying that the inactivation of pgi gene reduced the carbon flux through glycolytic pathway. Meanwhile, the pentose phosphate pathway was active as a major route for intermediary carbohydrate metabolism instead of glycolysis. The pentose phosphate pathway generates most of the major reducing co-factor NADPH as shown by the increased of NADPH/NADP+ ratio in the mutant when compared with the parent strain. The fermentative enzymes such as acetate kinase and lactate dehydrogenase were down regulated in the mutant. Knockout of pgi gene results in the significant increase in the intracellular concentration of glucose-6-phosphate and decrease in the concentration of oxaloacetate. The slow growth rate of the mutant was assumed to be affected by the accumulation of glucose-6-phosphate and imbalance of NADPH reoxidation.

Since most bio-production processes are conducted in a batch or fed-batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied 13C-metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild-type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild-type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose-6-phosphate dehydrogenase reaction was the rate-limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild-type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass can be hydrolyzed into two major sugars of glucose and xylose, and thus the strategy for the efficient consumption of both sugars is highly desirable. NADPH is the essential molecule for the production of industrially important value-added chemicals, and thus its availability is quite important. Escherichia coli mutant lacking the pgi gene encoding phosphoglucose isomerase (Pgi) has been preferentially used to overproduce the NADPH. However, there exists a disadvantage that the cell growth rate becomes low for the mutant grown on glucose. This limits the efficient NADPH production, and therefore, it is quite important to investigate how addition of different carbon source such as xylose (other than glucose) effectively improves the NADPH production. In this study, we have developed a kinetic model to propose an efficient NADPH production system using E. coli pgi -knockout mutant with a mixture of glucose and xylose. The proposed system adds xylose to glucose medium to recover the suppressed growth of the pgi mutant, and determines the xylose content to maximize the NADPH productivity. Finally, we have designed a mevalonate (MVA) production system by implementing ArcA overexpression into the pgi -knockout mutant using a mixture of glucose and xylose. In addition to NADPH overproduction, the accumulation of acetyl-CoA (AcCoA) is necessary for the efficient MVA production. In the present study, therefore, we considered to overexpress ArcA, where ArcA overexpression suppresses the TCA cycle, causing the overflow of AcCoA, a precursor of MVA. We predicted the xylose content that maximizes the MVA production. This approach demonstrates the possibility of a great progress in the computer-aided rational design of the microbial cell factories for useful metabolite production., (Copyright © 2020 Matsuoka and Kurata.)