Accumulation of d -Glucose from Pentoses by Metabolically Engineered Escherichia coli

Abstract

T he pentose phosphate (PP) pathway interconverts phosphosugars having 3 to 7 carbon atoms principally by the action of the reversible enzymes transketolase and transaldolase. During the consumption of hexoses such as D-glucose or D-fructose, entry of carbon into this pathway provides many microorganisms, including Escherichia coli, the means to generate the reduced cofactor NADPH and to synthesize specific building-block compounds derived from intermediates of this pathway (e.g., phenylalanine, histidine, and ribose). For microorganisms having the requisite kinases and sugar transport mechanisms, the PP pathway also provides convenient entry points for the catabolism of many other sugars, including D-xylose and L-arabinose (49).We have previously studied D-xylose and L-arabinose metabolism in E. coli that lacks the ability to metabolize D-glucose due to knockouts in the ptsG, manZ, and glk genes (1-3). Recently, small but consistent amounts (about 50 mg/liter) of D-glucose were observed as the accumulated end product when E. coli ptsG manZ glk was grown on 5 g/liter of either pentose in a defined medium (unpublished data). How might D-glucose be derived from these pentoses?Both D-xylose and L-arabinose are converted to the common intermediate D-xylulose-5-phosphate (D-xylulose-5P) (Fig. 1), which via the PP pathway partitions to 67% D-fructose-6P and 33% D-glyceraldehyde-3P without the involvement of ATP:(1) During growth of cells having a complete glycolytic pathway, the 2 mol of D-fructose-6P formed via equation 1 readily generates 4 mol of D-glyceraldehyde-3P. For D-glucose to accumulate from pentoses in cells prevented from metabolizing D-glucose, we reasoned that some D-fructose-6P generated from these pentoses (i.e., by equation 1) is converted "back" to D-glucose and that once formed, the D-glucose is unable to reenter metabolism in the triple knockout strain. We furthermore hypothesized that even more D-glucose would accumulate from pentoses in cells that were further constrained from metabolizing D-fructose-6P or D-glucose-6P.Because D-fructose-6P conversion to D-glyceraldehyde-3P is ubiquitous in wild-type organisms, D-glucose is not typically considered a product of D-xylose or L-arabinose metabolism, and the conversion of these pentoses to readily available D-glucose would in itself not seem to be an economically viable process. However, if the yields and rates were sufficiently large, the accumulation of hexoses directly from pentoses might advance the use of lignocellulosic hydrolysates with organisms, such as Saccharomyces cerevisiae, which metabolize D-glucose readily but are natively unable to consume pentoses. Moreover, conversion of 5-carbon saccharides into 6-carbon saccharides derived from D-fructose-6P offers a unique platform both to build carbon length and potentially to generate compounds in industrially relevant organisms such as E. coli that might not be possible under typical conditions in which products of D-fructose-6P do not accumulate.The objectives of this study were to examine ...

Phosphatases and phosphate affect the formation of glucose from pentoses in Escherichia coli

“…Previous studies with agp knockouts coding glucose‐1‐phosphatase demonstrated that glucose‐6P, and not glucose‐1P, is the primary phosphorylated source of glucose in E. coli ptsG manZ glk pfkA zwf , a result which is consistent with glucose‐6P formation being favored in the equilibrium with glucose‐1P mediated by phosphoglucomutase .…”

“…Escherichia coli MEC143 has deletions in the genes which provide for glucose conversion to glucose‐6P ( ptsG , manZ , glk ), and in key genes involved in the metabolism of glucose‐6P and fructose‐6P ( zwf , pfkA ). MEC143 has been established to accumulate glucose by the dephosphorylation of glucose‐6P when grown on either xylose or l ‐arabinose . Mass glucose yields of 0.260 g glucose/g xylose and 0.316 g glucose/g l ‐arabinose were observed in shake flask studies using 5 g/L of either pentose (Fig.…”

“…Metabolically engineered Escherichia coli MEC143 converts pentoses such as arabinose and xylose into glucose . The transformation of five‐carbon sugars to six‐carbon glucose occurs because of the reversibility of transketolase and transaldolase, key enzymes of the pentose phosphate pathway (Fig.…”

“…The quantity of ATP required for this conversion depends on the mechanism the cells use for pentose transport . Escherichia coli MEC143 contains deletions in several genes involved in the initial catabolism of glucose (phosphotransferase enzymes capable of glucose uptake, ptsG and manZ genes, and glucokinase, glk ), and in glucose‐6P metabolism (6P‐fructokinase I, pfkA , and glucose‐6P 1‐dehydrogenase, zwf ).…”

“…into central metabolism by the Embden–Meyerhof–Parnas and pentose phosphate pathways. After an equilibrium between fructose‐6P and glucose‐6P by phosphoglucose isomerase ( pgi gene), the final step in the conversion of pentoses via fructose‐6P to glucose is the dephosphorylation of glucose‐6P : …”