Adaptation on xylose improves glucose–xylose co-utilization and ethanol production in a carbon catabolite repression (CCR) compromised ethanologenic strain

Abstract

Background Sugar hydrolysates from lignocellulosic biomass are majorly composed of glucose and xylose that can be fermented to biofuels. Bacteria, despite having the natural ability to consume xylose are unable to consume it in presence of glucose due to a carbon catabolite repression (CCR) mechanism. This leads to overall reduced productivity as well as incomplete xylose utilization due to ethanol build-up from glucose utilization. In our effort to develop a strain for simultaneous fermentation of glucose and xylose into ethanol, we deleted ptsG in ethanologenic E. coli SSK42 to make it deficient in CCR and performed adaptive laboratory evolution to achieve accelerated growth rate, sugar consumption and ethanol production. Finally, we performed proteomics study to identify changes that might have been responsible for the observed improved phenotype of the evolved strain. Results The parental strain of SSK42, i.e., wild-type E. coli B, did not co-utilize glucose and xylose as expected. After deleting the ptsG gene encoding the EIIBCGlc subunit of PTS system, glucose consumption is severely affected in wild-type E. coli B. However, the ethanologenic, SSK42 strain, which was evolved in our earlier study on both glucose and xylose, didn’t show such a drastic effect of EIIBCGlc deletion, instead consumed glucose first, followed by xylose without delay for switching from one sugar to another. To improve growth on xylose and co-utilization capabilities, the ptsG deleted SSK42 was evolved on xylose. The strain evolved for 78 generations, strain SCD78, displayed significant co-utilization of glucose and xylose sugars. At the bioreactor level, the strain SCD78 produced 3-times the ethanol titer of the parent strain with significant glucose–xylose co-utilization. The rate of glucose and xylose consumption also increased 3.4-fold and 3-fold, respectively. Proteome data indicates significant upregulation of TCA cycle proteins, respiration-related proteins, and some transporters, which may have a role in increasing the total sugar consumption and co-utilization of sugars. Conclusion Through adaptive evolution, we have obtained a strain that has a significant glucose–xylose co-utilization phenotype with 3-fold higher total sugar consumption rate and ethanol production rate compared to the unevolved strain. This study also points out that adaptation on xylose is enough to impart glucose–xylose co-utilization property in CCR compromised ethanologenic strain SSK42.

Metabolic engineering for 4-aminophenylalanine production from lignocellulosic biomass by recombinant Escherichia coli

“…22 Second-generation feedstocks of lignocellulosic biomass predominantly contain glucose followed by xylose with trace amounts of other monosaccharides such as arabinose and mannose; 10 diluted acid-pretreated sorghum bagasse contains 60% and 7% glucose and xylose as dry biomass, respectively, 12 and adaptive mutant of E. coli can simultaneously utilize both xylose and glucose along with arabinose. 32 These results suggest that the metabolic engineering of E. coli for the co-utilization of hexose and pentose could improve 4APhe in further studies.…”

Synergistic co‐utilization of biomass‐derived sugars enhances aromatic amino acid production by engineered Escherichia coli

“…Beyond aromatic amino acids, aromatic biochemicals in general are appealing bioproduction targets due to their potential to replace many conventional, bulk petrochemicals (Straathof & Bampouli, 2017). And, since aromatic biochemicals are almost exclusively derived from intermediates/products for the shikimic acid pathway (Báez‐Viveros et al, 2004; Liu et al, 2021; Yakandawala et al, 2008), xylose (co‐)catabolism is particularly intriguing from the perspective of enhancing product titers/yields (Chiang et al, 2013; Dev et al, 2022; Huccetogullari et al, 2019; Li & Frost, 1999; Vargas‐Tah et al, 2015; Wang et al, 2016). This is due at least in part to the increased availability of erythrose‐4‐phosphate (E4P) as flux is increased through the pentose phosphate (PP) pathway.…”

Composition of Lignocellulose Hydrolysate in Different Biorefinery Strategies: Nutrients and Inhibitors

“…In the CBP system, since all steps occur in the same bioreactor, the efficient consumption of the sugar mixture or the biomass hydrolysate is particularly critical. For instance, the hydrolysis of hemicelluloses produces xyloses, arabinoses, galactoses, and rhamnoses, but many host microorganisms in CBP cannot consume these sugars, leading to a carbon catabolite repression [192]. In such cases, CBP hosts are required to be modified by genetic engineering or metabolic engineering [32,193,194].…”