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

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.

Deletion of pgi gene in E. coli increases tolerance to furfural and 5-hydroxymethyl furfural in media containing glucose-xylose mixture.

BACKGROUND: Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF. In present study, deletion of pgi gene as a strategy to augment carbon flow through pentose phosphate pathway (PPP) was studied in ethanologenic Escherichia coli strain SSK101 to impart tolerance towards either furfural or 5-HMFor both inhibitors together. RESULTS: A key gene of EMP pathway, pgi, was deleted in an ethanologenic E. coli strain SSK42 to yield strain SSK101. In presence of 1 g/L furfural in minimal AM1 media, the rate of biomass formation for strain SSK101 was up to 1.9-fold higher as compared to parent SSK42 strain, and it was able to clear furfural in half the time. Tolerance to inhibitor was associated with glucose as carbon source and not xylose, and the tolerance advantage of SSK101 was neutralized in LB media. Bioreactor studies were performed under binary stress of furfural and 5-HMF (1 g/L each) and different glucose concentrations in a glucose-xylose mixture with final sugar concentration of 5.5%, mimicking major components of dilute acid treated biomass hydrolysate. In the mixture having 6 g/L and 12 g/L glucose, SSK101 strain produced ~ 18 g/L and 20 g/L ethanol, respectively. Interestingly, the maximum ethanol productivity was better at lower glucose load with 0.46 g/(L.h) between 96 and 120 h, as compared to higher glucose load where it was 0.33 g/(L.h) between 144 and 168 h. Importantly, parent strain SSK42 did not exhibit significant metabolic activity under similar conditions of inhibitor load and sugar concentration. CONCLUSIONS: E. coli strain SSK101 with pgi deletion had enhanced tolerance against both furfural and 5-HMF, which was associated with presence of glucose in media. Strain SSK101 also had improved fermentation characteristics under both hyperosmotic as well as binary stress of furfural and 5-HMF in media containing glucose-xylose mixture.

Improvement in ethanol productivity of engineered E. coli strain SSY13 in defined medium via adaptive evolution.

E. coli has the ability to ferment both C5 and C6 sugars and produce mixture of acids along with small amount of ethanol. In our previous study, we reported the construction of an ethanologenic E. coli strain by modulating flux through the endogenous pathways. In the current study, we made further changes in the strain to make the overall process industry friendly; the changes being (1) removal of plasmid, (2) use of low-cost defined medium, and (3) improvement in consumption rate of both C5 and C6 sugars. We first constructed a plasmid-free strain SSY13 and passaged it on AM1-xylose minimal medium plate for 150 days. Further passaging was done for 56 days in liquid AM1 medium containing either glucose or xylose on alternate days. We observed an increase in specific growth rate and carbon utilization rate with increase in passage numbers until 42 days for both glucose and xylose. The 42nd day passaged strain SSK42 fermented 113 g/L xylose in AM1 minimal medium and produced 51.1 g/L ethanol in 72 h at 89% of maximum theoretical yield with ethanol productivity of 1.4 g/L/h during 24-48 h of fermentation. The ethanol titer, yield and productivity were 49, 40 and 36% higher, respectively, for SSK42 as compared to unevolved SSY13 strain.