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

Abstract

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.

Improving Furfural Tolerance of Escherichia coli by Integrating Adaptive Laboratory Evolution with CRISPR-Enabled Trackable Genome Engineering (CREATE)

“…Compared with the reported E. coli strains that only tolerated about 3 g/L furfural, ,, 4.7 g/L is a breakthrough in the performance of furfural tolerance, representing the highest value reported for E. coli thus far.…”

“…The specific growth rate of the pfkA overexpression strain (CG01) reached 61.9 and 66.8% of the KQ52 strain in the presence of 1 and 2 g/L furfural, respectively. It is generally known that the pentose phosphate pathway is one of the main sources of NADPH and glycolysis has a vital function to provide ATP for biomass production . Therefore, the upregulation of glycolysis and the pentose phosphate pathway could be an important source of additional ATP and NADPH to maintain the biomass yield under furfural stress.…”

“…Notably, its concentration can reach up to 5 g/L, , while the growth of E. coli is significantly inhibited by 1 g/L furfural in a minimal medium . The furfural influence is multifaceted, including effects of membrane integrity, hindering the synthesis of proteins and RNA, inhibiting biological activity, decreasing NADH and ATP concentrations, and directly restricting intracellular central carbon metabolism. , In spite of the significant effort, it is still difficult to develop a strain tolerant to furfural applying rational engineering strategies because of poor understanding knowledge of the underlying molecular mechanisms .…”

Unraveling the mechanism of furfural tolerance in engineered Pseudomonas putida by genomics

“…It was reported to inhibit the glycolytic and fermentative enzymes (Wang et al, 2018 ). Furfural has been shown to severely affect intracellular redox metabolism, induce the accumulation of reactive oxygen species, and cause damage to cell organelles (Jilani et al, 2020 ; Li et al, 2022 ). To reduce the toxicity level of furfural, different strategies for engineering microbial tolerance against furan aldehydes have been reported (Nieves et al, 2015 ).…”

Improved furfural tolerance in Escherichia coli mediated by heterologous NADH-dependent benzyl alcohol dehydrogenases

“…Correspondingly, for E. coli , many of the successful strategies for engineering of furfural resistance have focused on addressing the depletion of NADPH. These strategies include the identification and deletion of the furfural-active reductases that use NADPH [ 22 ], the expression of a transhydrogenase capable of replenishing the pool of NADPH [ 23 ], the rerouting of carbon flux through the pentose phosphate pathway [ 24 , 25 ], and the expression of reductases that are able to reduce furfural without using NADPH [ 26 , 27 ].…”