Engineering the Metabolic Landscape of Microorganisms for Lignocellulosic Conversion.

Bacteria and yeast are being intensively used to produce biofuels and high-added-value products by using plant biomass derivatives as substrates. The number of microorganisms available for industrial processes is increasing thanks to biotechnological improvements to enhance their productivity and yield through microbial metabolic engineering and laboratory evolution. This is allowing the traditional industrial processes for biofuel production, which included multiple steps, to be improved through the consolidation of single-step processes, reducing the time of the global process, and increasing the yield and operational conditions in terms of the desired products. Engineered microorganisms are now capable of using feedstocks that they were unable to process before their modification, opening broader possibilities for establishing new markets in places where biomass is available. This review discusses metabolic engineering approaches that have been used to improve the microbial processing of biomass to convert the plant feedstock into fuels. Metabolically engineered microorganisms (MEMs) such as bacteria, yeasts, and microalgae are described, highlighting their performance and the biotechnological tools that were used to modify them. Finally, some examples of patents related to the MEMs are mentioned in order to contextualize their current industrial use.

Submergence Stress Alters the Expression of Clock Genes and Configures New Zeniths and Expression of Outputs in Brachypodium distachyon.

Plant networks of oscillating genes coordinate internal processes with external cues, contributing to increased fitness. We hypothesized that the response to submergence stress may dynamically change during different times of the day. In this work, we determined the transcriptome (RNA sequencing) of the model monocotyledonous plant, Brachypodium distachyon, during a day of submergence stress, low light, and normal growth. Two ecotypes of differential tolerance, Bd21 (sensitive) and Bd21-3 (tolerant), were included. We submerged 15-day-old plants under a long-day diurnal cycle (16 h light/8 h dark) and collected samples after 8 h of submergence at ZT0 (dawn), ZT8 (midday), ZT16 (dusk), ZT20 (midnight), and ZT24 (dawn). Rhythmic processes were enriched both with up- and down-regulated genes, and clustering highlighted that the morning and daytime oscillator components (PRRs) show peak expression in the night, and a decrease in the amplitude of the clock genes (GI, LHY, RVE) was observed. Outputs included photosynthesis-related genes losing their known rhythmic expression. Up-regulated genes included oscillating suppressors of growth, hormone-related genes with new late zeniths (e.g., JAZ1, ZEP), and mitochondrial and carbohydrate signaling genes with shifted zeniths. The results highlighted genes up-regulated in the tolerant ecotype such as METALLOTHIONEIN3 and ATPase INHIBITOR FACTOR. Finally, we show by luciferase assays that Arabidopsis thaliana clock genes are also altered by submergence changing their amplitude and phase. This study can guide the research of chronocultural strategies and diurnal-associated tolerance mechanisms.

AmyJ33, a truncated amylase with improved catalytic properties.

Biochemical and kinetic properties are of special interest for the specific applications of α-amylases in industrial sectors such as textile industries, detergents, biofuels and food among others. Therefore, protein engineering is currently directed towards a continuous demand to improve the properties of amylases and thus meet the specific characteristics for various industrial sectors. In the present work, modular protein engineering was performed to improve the biochemical and kinetic properties of AmyJ33r an α-amylase isolated from Bacillus siamensis JJC33M consisting of five domains, A, B, C, D and E (SBD) (Montor-Antonio et al. in 3 Biotech 7:336, 2017. https://doi.org/10.1007/s13205-017-0954-8 ). AmyJ33r is not active on native starch, only showing activity on gelatinized starch. At the C-terminal, AmyJ33r has a starch binding domain (SBD, domain E) belonging to the CBM26 family. In this study, four truncated versions were constructed and expressed in E. coli (AmyJ33-AB, AmyJ33-ABC, AmyJ33-ABCD, and SBD) to determine the role of the A, B, C, D, and E domains in the biochemical behavior of AmyJ33r on starch. Biochemical and kinetic characterization of the truncated versions showed that domain C is essential for catalysis; domain D improved enzyme activity at alkaline pH values, is also involved negatively in thermostability at 40, 50, and 60 °C and its presence favored the production of maltooligosaccharides with a higher degree of polymerization (DP4). E domain have interaction with raw starch, also the deletion of E domain (SBD) favors the affinity for the substrate while the deletion of D domain increased enzyme kcat at the time of product release. In conclusion, AmyJ33-ABC has better kinetic parameters than AmyJ33-ABCD and AmyJ33r, but is less stable than these two enzymes.

Differential Expression of Maize and Teosinte microRNAs under Submergence, Drought, and Alternated Stress.

Submergence and drought stresses are the main constraints to crop production worldwide. MicroRNAs (miRNAs) are known to play a major role in plant response to various stresses. In this study, we analyzed the expression of maize and teosinte miRNAs by high-throughput sequencing of small RNA libraries in maize and its ancestor teosinte (Zea mays ssp. parviglumis), under submergence, drought, and alternated stress. We found that the expression patterns of 67 miRNA sequences representing 23 miRNA families in maize and other plants were regulated by submergence or drought. miR159a, miR166b, miR167c, and miR169c were downregulated by submergence in both plants but more severely in maize. miR156k and miR164e were upregulated by drought in teosinte but downregulated in maize. Small RNA profiling of teosinte subject to alternate treatments with drought and submergence revealed that submergence as the first stress attenuated the response to drought, while drought being the first stress did not alter the response to submergence. The miRNAs identified herein, and their potential targets, indicate that control of development, growth, and response to oxidative stress could be crucial for adaptation and that there exists evolutionary divergence between these two subspecies in miRNA response to abiotic stresses.

Submergence and Waterlogging Stress in Plants: A Review Highlighting Research Opportunities and Understudied Aspects.

Soil flooding creates composite and complex stress in plants known as either submergence or waterlogging stress depending on the depth of the water table. In nature, these stresses are important factors dictating the species composition of the ecosystem. On agricultural land, they cause economic damage associated with long-term social consequences. The understanding of the plant molecular responses to these two stresses has benefited from research studying individual components of the stress, in particular low-oxygen stress. To a lesser extent, other associated stresses and plant responses have been incorporated into the molecular framework, such as ion and ROS signaling, pathogen susceptibility, and organ-specific expression and development. In this review, we aim to highlight known or suspected components of submergence/waterlogging stress that have not yet been thoroughly studied at the molecular level in this context, such as miRNA and retrotransposon expression, the influence of light/dark cycles, protein isoforms, root architecture, sugar sensing and signaling, post-stress molecular events, heavy-metal and salinity stress, and mRNA dynamics (splicing, sequestering, and ribosome loading). Finally, we explore biotechnological strategies that have applied this molecular knowledge to develop cultivars resistant to flooding or to offer alternative uses of flooding-prone soils, like bioethanol and biomass production.

Biotechnological Strategies to Improve Plant Biomass Quality for Bioethanol Production.

The transition from an economy dependent on nonrenewable energy sources to one with higher diversity of renewables will not be a simple process. It requires an important research effort to adapt to the dynamics of the changing energy market, sort costly processes, and avoid overlapping with social interest markets such as food and livestock production. In this review, we analyze the desirable traits of raw plant materials for the bioethanol industry and the molecular biotechnology strategies employed to improve them, in either plants already under use (as maize) or proposed species (large grass families). The fundamentals of these applications can be found in the mechanisms by which plants have evolved different pathways to manage carbon resources for reproduction or survival in unexpected conditions. Here, we review the means by which this information can be used to manipulate these mechanisms for commercial uses, including saccharification improvement of starch and cellulose, decrease in cell wall recalcitrance through lignin modification, and increase in plant biomass.

qPCR and HRM-based diagnosis of SNPs on growth differentiation factor 9 (GDF9), a gene associated with sheep (Ovis aries) prolificacy.

Prolificacy is a desirable trait for genetic improvement of sheep flocks, since it holds the potential to improve productivity. Animals carrying single-nucleotide polymorphisms (SNPs) in genes associated with this trait can be identified and employed to increase prolificacy in flocks. In this study, we report a diagnostic method based on quantitative PCR and high-resolution melting curves to detect different SNPs in the prolificacy-associated gene growth differentiation factor 9 (GDF9). The diagnostic method was validated using artificial sequences representing known SNPs in GDF9, then applied to a real flock comprising four breeds and admixed animals (n = 306). Five different SNPs were identified in this flock, as was a low or null frequency of occurrence of SNPs positively associated with prolificacy. This indicates a need to implement a breeding strategy for recovering or reintroducing such SNPs. Our method provides a genotyping strategy for identifying individuals with SNPs of interest for prolificacy, which will help producers plan a breeding strategy for this trait. This method can be adapted and expanded for the diagnosis of other traits of interest.

Transcriptomic analysis of submergence-tolerant and sensitive Brachypodium distachyon ecotypes reveals oxidative stress as a major tolerance factor.

When excessive amounts of water accumulate around roots and aerial parts of plants, submergence stress occurs. To find the integrated mechanisms of tolerance, we used ecotypes of the monocot model plant Brachypodium distachyon to screen for genetic material with contrasting submergence tolerance. For this purpose, we used a set of previously studied drought sensitive/tolerant ecotypes and the knowledge that drought tolerance is positively associated with submergence stress. We decided to contrast aerial tissue transcriptomes of the ecotype Bd21 14-day-old plants as sensitive and ecotype Bd2-3 as tolerant after 2 days of stress under a long-day photoperiod. Gene ontology and the grouping of transcripts indicated that tolerant Bd2-3 differentially down-regulated NITRATE REDUCTASE and ALTERNATIVE OXIDASE under stress and constitutively up-regulated HAEMOGLOBIN, when compared with the sensitive ecotype, Bd21. These results suggested the removal of nitric oxide, a gaseous phytohormone and concomitant reactive oxygen species as a relevant tolerance determinant. Other mechanisms more active in tolerant Bd2-3 were the pathogen response, glyoxylate and tricarboxylic acid cycle integration, and acetate metabolism. This data set could be employed to design further studies on the basic science of plant tolerance to submergence stress and its biotechnological application in the development of submergence-tolerant crops.

Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor.

Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200-300% more glucose, adult vegetative plants yielded 40-90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production.

Heavy metal detoxification in eukaryotic microalgae.

Microalgae are aquatic organisms possessing molecular mechanisms that allow them to discriminate non-essential heavy metals from those essential ones for their growth. The different detoxification processes executed by algae are reviewed with special emphasis on those involving the peptides metallothioneins, mainly the post transcriptionally synthesized class III metallothioneins or phytochelatins. Also, the features that make microalgae suitable organisms technologies specially to treat water that is heavily polluted with metals is discussed.

Phenotypic plasticity in Scenedesmus incrassatulus (Chlorophyceae) in response to heavy metals stress.

The microalgae genus Scenedesmus is commonly found in freshwater bodies, wastewater facilities and water polluted with heavy metals. Phenotypic plasticity in Scenedesmus has been documented in response to a wide variety of conditions; however, heavy metals have not been comprehensively documented as phenotypic plasticity inducers. In this study, we report the phenotypic plasticity of Scenedesmus incrassatulus (a non-spiny, four-cell coenobium forming species) in response to EC(50) value of copper, cadmium and hexavalent chromium. S. incrassatulus was grown in batch cultures in the presence of each metal. Chlorophyll-a content, cell size, parameters derived from the schematic energy-flux model for photosystem II, and morphotype expressions were recorded. Divalent cation metals induced unicellular forms, and hexavalent chromium produced out-of-shape coenobia corresponding to various stages of autospore formation. The changes induced by divalent metals were interpreted as phenotypic plasticity, because they were always associated to population doublings and were reversible when toxicant pressure was removed (only for Cu). Copper was the best inductor of unicellular forms and also affected significantly all the photosynthetic parameters measured. The developed morphotypes could confer ecological advantages to S. incrassatulus in metal stressed environments.

Avances en el diseño conceptual de fotobiorreactores para el cultivo de microalgas / Advances in conceptual design of photobioreactores for microalgal culture

Durante la década pasada se han usado múltiples diseños de fotobiorreactores para el cultivo de organismos fotoautróficos microscópicos como microalgas y cianobacterias. Los avances en el diseño de estos sistemas han permitido mejorar notablemente la densidad celular, la productividad y por ende la economía de los cultivos para distintos fines. En este trabajo se revisan diferentes aspectos del diseño de fotobiorreactores, tanto aquellos que implican el aprovechamiento adecuado de la energía luminosa (ciclos luz-oscuridad, trayectoria de la luz y geometría de fotobiorreactores) como los basados en conceptos fisiológicos (fotoinhibición por oxígeno, cultivos de alta densidad celular, ultra alta densidad celular, heterotrofía y mixotrofía). Se presentan las principales aplicaciones de los diferentes diseños, en la producción de compuestos de alto valor agregado y su uso potencial en la biotecnología ambiental