The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production.

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A nonautonomous element of 624 bp, called RetroCl1 (Retroelement Colletotrichum lindemuthianum 1), was identified in the plant pathogenic fungus Colletotrichum lindemuthianum. RetroCl1 contains terminal direct repeats (223 bp) that are surrounded by CTAGT sequences. It has a short internal domain of 178 bp and shows characteristics of terminal-repeat retrotransposon in miniature (TRIM) family. We used RetroCl1 sequence to develop molecular markers for the Colletotrichum genus. IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) markers were used to analyze the genetic diversity of C. lindemuthianum. Fifty-four isolates belonging to different races were used. A total of 45 loci were amplified. The Nei index showed significant differences among the populations divided according to race, indicating that they are structured according to pathotype. No clear correlation between IRAP and REMAP markers with pathogenic characterization was found. C. lindemuthianum has high genetic diversity, and the analysis of molecular variance showed that 51% of variability is found among the populations of different races. The markers were also tested in different Colletotrichum species. In every case, multiple bands were amplified, indicating that these markers can be successfully used in different species belonging to the Colletotrichum genus.

The lignocellulosic biomass, comprised mainly of cellulose, hemicellulose, and lignin, is a strong competitor for petroleum to obtain fuels and other products because of its renewable nature, low cost, and non-competitiveness with food production when obtained from agricultural waste. Due to its recalcitrance, lignocellulosic material requires an arsenal of enzymes for its deconstruction and the consequent release of fermentable sugars. In this context, enzymes currently classified as auxiliary activity 9 (AA9/formerly GH61) and 10 (AA10/formerly CBM 33) or lytic polysaccharide monooxygenases (LPMO) have emerged as cellulase boosting enzymes. AA9 and AA10 are the new paradigm for deconstruction of lignocellulosic biomass by enhancing the activity and decreasing the loading of classical enzymes to the reaction and, consequently, reducing costs of the hydrolysis step in the second-generation ethanol production chain. In view of that disclosed above, the goal of this work is to review experimental data that supports the relevance of AA9 and AA10 for the biomass deconstruction field.

Background Lytic polysaccharide monooxygenases (LPMOs) opened a new horizon for biomass deconstruction. They use a redox mechanism not yet fully understood and the range of substrates initially envisaged to be the crystalline polysaccharides is steadily expanding to non-crystalline ones. Results The enzyme Kp LPMO10A from the actinomycete Kitasatospora papulosa was cloned and overexpressed in Escherichia coli cells in the functional form with native N-terminal. The enzyme can release oxidized species from chitin (C1-type oxidation) and cellulose (C1/C4-type oxidation) similarly to other AA10 members from clade II (subclade A). Interestingly, Kp LPMO10A also cleaves isolated xylan (not complexed with cellulose, C4-type oxidation), a rare activity among LPMOs not described yet for the AA10 family. The synergistic effect of Kp LPMO10A with Celluclast ® and an endo-β-1,4-xylanase also supports this finding. The crystallographic elucidation of Kp LPMO10A at 1.6 Å resolution along with extensive structural analyses did not indicate any evident difference with other characterized AA10 LPMOs at the catalytic interface, tempting us to suggest that these enzymes might also be active on xylan or that the ability to attack both crystalline and non-crystalline substrates involves yet obscure mechanisms of substrate recognition and binding. Conclusions This work expands the spectrum of substrates recognized by AA10 family, opening a new perspective for the understanding of the synergistic effect of these enzymes with canonical glycoside hydrolases to deconstruct ligno(hemi)cellulosic biomass. Electronic supplementary material The online version of this article (10.1186/s13068-019-1449-0) contains supplementary material, which is available to authorized users.