Zymomonas mobilis is a promising organism for biofuel production as it can produce ethanol from glucose at high rates. However, Z. mobilis does not natively ferment C5 sugars such as xylose. While it has been engineered to do so, the engineered strains do not metabolize these sugars at high rates. Previous research has identified some of the bottlenecks associated with xylose metabolism in Z. mobilis. In this work, we investigated transport as a possible bottleneck. In particular, we hypothesized that the slow uptake of xylose through the promiscuous Glf transporter may limit the efficiency of xylose metabolism in Z. mobilis. To test this hypothesis, we expressed XylE, the low-affinity xylose transporter from Escherichia coli, in a xylose-utilizing strain of Z. mobilis. Our results show that the expression of this pentose-specific transporter improves the rate of xylose utilization in Z. mobilis; however, this enhancement is seen only at high xylose concentrations. In addition, we also found that overexpression of the promiscuous Z. mobilis transporter Glf yielded similar results, suggesting that the transport bottleneck is not due to the specificity, but rather the capacity for sugar uptake.

Zymomonas mobilis is capable of producing ethanol at high rates and titers from glucose. This bacterium has previously been engineered to consume the pentose sugars xylose and arabinose, but the rate of consumption of these sugars is low. Recent research has utilized adaptive evolution to isolate strains of Z. mobilis capable of rapidly fermenting xylose. In this study, we also used adaptive evolution to isolate strains of Z. mobilis capable of rapidly fermenting xylose and arabinose. To determine the bottlenecks in pentose metabolism, we then used high-throughput sequencing to pinpoint the genetic changes responsible for the phenotypes of the adapted strains. We found that the transport of both xylose and arabinose through the native sugar transporter, Glf, limits pentose fermentations in Z. mobilis. We also found that mutations in the AddB protein increase plasmid stability and can reduce cellular aggregation in these strains. Consistent with previous research, we found that reduced xylitol production improves xylose fermentations in Z. mobilis. We also found that increased transketolase activity and reduced glyceraldehyde-3-phosphate dehydrogenase activity improve arabinose fermentations in Z. mobilis. Biotechnol.

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