Ethanol production by Escherichia coli from detoxified lignocellulosic teak wood hydrolysates with high concentration of phenolic compounds

Teak wood residues were subjected to thermochemical pretreatment, enzymatic saccharification, and detoxification to obtain syrups with a high concentration of fermentable sugars for ethanol production with the ethanologenic Escherichia coli strain MS04. Teak is a hardwood, and thus a robust deconstructive pretreatment was applied followed by enzymatic saccharification. The resulting syrup contained 60 g L−1 glucose, 18 g L−1 xylose, 6 g L−1 acetate, less than 0.1 g L−1 of total furans, and 12 g L−1 of soluble phenolic compounds (SPC). This concentration of SPC is toxic to E. coli, and thus two detoxification strategies were assayed: 1) treatment with Coriolopsis gallica laccase followed by addition of activated carbon and 2) overliming with Ca(OH)2. These reduced the phenolic compounds by 40 and 76%, respectively. The detoxified syrups were centrifuged and fermented with E. coli MS04. Cultivation with the over-limed hydrolysate showed a 60% higher volumetric productivity (0.45 gETOH L−1 h−1). The bioethanol/sugars yield was over 90% in both strategies.

Overexpression of an oxidoreductase YghA confers tolerance against furfural in ethanologenic Escherichia coli strain SSK42

Furfural is a common furan inhibitor formed due to dehydration of pentose sugar like xylose and acts as an inhibitor of microbial metabolism. Overexpression of NADH specific FucO and deletion of NADPH specific YqhD had been a successful strategy in the past in conferring tolerance against furfural in E. coli , which highlight the importance of oxidoreductases in conferring tolerance against furfural. In a screen consisting of various oxidoreductases, dehydrogenases, and reductases, we identified yghA gene as an overexpression target to confer tolerance against furfural. YghA preferably used NADH as a cofactor and had apparent K m value of 0.03 mM against furfural. In presence of 1 g L −1 furfural and 10% xylose (wt/vol), yghA overexpression in an ethanologenic E. coli strain SSK42 resulted in a 5.3-fold increase in ethanol titers as compared to the control strain with an efficiency of ∼97%. YghA also exhibited activity against the lesser toxic inhibitor 5-hydroxymethyl furfural that is formed due to dehydration of hexose sugars and thus is a formidable target for overexpression in ethanologenic strain for fermentation of sugars in biomass hydrolysate. IMPORTANCE Lignocellulosic biomass represents an inexhaustible source of carbon for second-generation biofuels. Thermo-acidic pretreatment of biomass is performed to loosen the lignocellulosic fibers and make the carbon bioavailable for microbial metabolism. The pretreatment process also results in the formation of inhibitors that inhibit microbial metabolism and increase production costs. Furfural is a potent furan inhibitor that increases the toxicity of other inhibitors present in the hydrolysate. Thus it is desirable to engineer furfural tolerance in E. coli for efficient fermentation of hydrolysate sugars.

Structure and mechanism of LGK: insights into the bioconversion of levoglucosan

Lignocellulosic biomass is an abundant source of carbohydrates that can be used for the production of biofuels. Additional processing of biomass-derived sugars arises from the fact that the most abundant sugar product from biomass pyrolysis is the unusual anhydro-ring containing sugar, levoglucosan (LG). LG does not fall within the native substrate range of commonly used biocatalysts such as Escherichia coli and Saccharomyces cerevisiae. However, LG can be further converted to glucose-6-phosphate through the activity of the sugar kinase known as levoglucosan kinase (LGK), which has been isolated from a variety of fungal sources. Integration of recombinant LGK from Lipomyces starkeyi into an engineered ethanologenic Escherichia coli strain has been shown to allow for the utilization of LG as the sole carbon source for ethanol production [1]. However, challenges associated with effective utilization of LG include a high Km of LGK for LG, and the relatively low activity of LGK at physiological pH. In addition to the practical applications of LGK for biofuel production, the enzyme is an exceptional target for structural and mechanistic studies since it appears to possess dual hydrolase and kinase functionality. In order to gain a better understanding of the structure and mechanism of LGK, we have crystallized and determined several high-resolution X-ray structures of Lipomyces starkeyi LGK bound to reaction substrates and products. We have also recently collected low-resolution neutron diffraction for an LGK crystal, and further optimization of LGK crystals is currently underway to improve crystal size and diffraction. Neutron diffraction will reveal the protonation states of key residues in the active site of LGK and provide highly detailed information about hydrogen bonds, including water-bonding interactions. The rational design of new LGK constructs will be used to improve applications of this enzyme towards levoglucosan derived biofuel production.

Polyamine Transporters and Polyamines Increase Furfural Tolerance during Xylose Fermentation with Ethanologenic Escherichia coli Strain LY180

ABSTRACTExpression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenicEscherichia coliLY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression ofyqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion ofpotEin EMFR9 lowered furfural tolerance to that of the parent. Deletion ofpotEandpuuPin LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural.