Advanced one-pot deconstruction and valorization of lignocellulosic biomass into triacetic acid lactone using Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides is capable of co-utilization of complex carbon sources and robust growth from lignocellulosic hydrolysates. This oleaginous yeast is therefore an attractive host for heterologous production of valuable bioproducts at high titers from low-cost, deconstructed biomass in an economically and environmentally sustainable manner. Here we demonstrate this by engineering R. toruloides to produce the polyketide triacetic acid lactone (TAL) directly from unfiltered hydrolysate deconstructed from biomass with minimal unit process operations. RESULTS: Introduction of the 2-pyrone synthase gene into R. toruloides enabled the organism to produce 2.4 g/L TAL from simple media or 2.0 g/L from hydrolysate produced from sorghum biomass. Both of these titers are on par with titers from other better-studied microbial hosts after they had been heavily engineered. We next demonstrate that filtered hydrolysates produced from ensiled sorghum are superior to those derived from dried sorghum for TAL production, likely due to the substantial organic acids produced during ensiling. We also demonstrate that the organic acids found in ensiled biomass can be used for direct synthesis of ionic liquids within the biomass pretreatment process, enabling consolidation of unit operations of in-situ ionic liquid synthesis, pretreatment, saccharification, and fermentation into a one-pot, separations-free process. Finally, we demonstrate this consolidation in a 2 L bioreactor using unfiltered hydrolysate, producing 3.9 g/L TAL. CONCLUSION: Many steps involved in deconstructing biomass into fermentable substrate can be combined into a distinct operation, and directly fed to cultures of engineered R. toruloides cultures for subsequent valorization into gram per liter titers of TAL in a cost-effective manner.

Lepidopteran mevalonate pathway optimization in Escherichia coli efficiently produces isoprenol analogs for next-generation biofuels.

Terpenes constitute the largest class of natural products with over 55,000 compounds with versatile applications including drugs and biofuels. Introducing structural modifications to terpenes through metabolic engineering is an efficient and sustainable way to improve their properties. Here, we report the optimization of the lepidopteran mevalonate (LMVA) pathway towards the efficient production of isopentenyl pyrophosphate (IPP) analogs as terpene precursors. First, we linked the LMVA pathway to NudB, a promiscuous phosphatase, resulting in the production of the six-carbon analog of 3-methyl-3-buten-1-ol (isoprenol), 3-ethyl-3-buten-1-ol (C6-isoprenol). Using C6-isoprenol as the final product, we then engineered the LMVA pathway by redirecting its upstream portion from a thiolase-dependent pathway to a beta-oxidation pathway. The beta-oxidation LMVA pathway transforms valeric acid, a platform chemical that can be produced from biomass, into C6-isoprenol at a titer of 110.3 mg/L, improved from 5.5 mg/L by the thiolase LMVA pathway, which used propionic acid as a feedstock. Knockout of the E. coli endogenous thiolase genes further improved the C6-isoprenol titer to 390 mg/L, implying efficient production of homo isopentenyl pyrophosphate (HIPP). The beta-oxidation LMVA-NudB pathway also converts butanoic acid and hexanoic acid into isoprenol and isoprenol's seven-carbon analog, 3-propyl-3-buten-1-ol (C7-isoprenol), respectively, suggesting the beta-oxidation LMVA pathway produces IPP and C7-IPP from the corresponding fatty acids. Fuel property tests revealed the longer chain isoprenol analogs have lower water solubilities, similar or higher energy densities, and comparable research octane number (RON) boosting effects to isopentenols. This work not only optimizes the LMVA pathway, setting the basis for homoterpene biosynthesis to expand terpene chemical space, but provides an efficient pathway to produce isoprenol analogs as next-generation biofuels from sustainable feedstocks.

Chemoinformatic-Guided Engineering of Polyketide Synthases.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKS module to produce a saturated ß-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates  into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structure similarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced by the host Streptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

Investigation of Bar-seq as a method to study population dynamics of Saccharomyces cerevisiae deletion library during bioreactor cultivation.

BACKGROUND: Despite the latest advancements in metabolic engineering for genome editing and characterization of host performance, the successful development of robust cell factories used for industrial bioprocesses and accurate prediction of the behavior of microbial systems, especially when shifting from laboratory-scale to industrial conditions, remains challenging. To increase the probability of success of a scale-up process, data obtained from thoroughly performed studies mirroring cellular responses to typical large-scale stimuli may be used to derive crucial information to better understand potential implications of large-scale cultivation on strain performance. This study assesses the feasibility to employ a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime and aims to understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance. RESULTS: We find that mutant population diversity is maintained through multiple seed trains, enabling large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes tested in this study, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that all encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Ecological analysis of beta diversity between all samples revealed significant population divergence over time and showed feed specific consequences of population structure. Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant stresses. Our observations indicate that, for this yeast deletion collection, the selection of the feeding scheme which affects the accumulation of the fermentative by-product ethanol impacts the diversity of the mutant pool to a higher degree as compared to the pH of the culture broth. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses. CONCLUSIONS: Our results demonstrate the feasibility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions and to understand critical stages of a scale-up process where variability emerges, and selection pressure gets imposed. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks.

BACKGROUND: In an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass. RESULTS: We developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A. CONCLUSION: Our findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.

Perfil Clínico-Epidemiológico e Sobrevida dos Casos de Retinoblastoma em um Hospital Referência em Oncologia do Estado de Goiás / Clinical-Epidemiological Profile and Survival of the Cases of Retinoblastoma in a Reference Hospital in Oncology in the State of Goiás / Perfil Clínico-Epidemiológico y Sobrevida de los Casos de Retinoblastoma en un Hospital de Referencia en Oncología del Estado de Goiás

Introdução: O retinoblastoma é a malignidade primária intraocular mais comum na infância, é raro e corresponde de 2% a 4% dos tumores malignos pediátricos. Objetivo: Descrever o perfil clínico-epidemiológico e a sobrevida dos casos de retinoblastoma em um hospital de referência em oncologia do Estado de Goiás, entre 2008 a 2014. Método: Estudo observacional analítico do tipo transversal, construído com base na análise de prontuários de pacientes diagnosticados com retinoblastoma entre 2008 e 2014. Realizaram-se análises por estatística descritiva e teste de associação qui-quadrado. Adotou-se o nível de significância de 5%. A sobrevida foi avaliada por meio do método de Kaplan-Meier. Resultados: Foram atendidos 55 pacientes com retinoblastoma, permitindo identificar o predomínio do sexo feminino (54,5%); na faixa etária de 1 a 4 anos (27,3%); com etnia parda (50,9%). As características clínicas mais prevalentes foram: acometimento intraocular (74,5%); unilateral (65,5%); sem histórico familiar (56,4%); e com sinal clínico de leucocoria (80%). A maioria não apresentou metástases ao diagnóstico (87,3%), sendo o principal tratamento a enucleação unilateral (72,7%). Verificou-se associação quanto à evolução clínica do paciente em relação à localização extraocular (p = 0,001), presença de metástase (p = 0,001) e estádio IV de Chantada et al. (p = 0,001). Pacientes classificados como E foram submetidos a maior número de enucleações (olho direito ­ p = 0,05 e olho esquerdo ­ p = 0,001). A sobrevida global em cinco anos foi de 72,7%. Conclusão: Tais achados são relevantes para o planejamento de ações de prevenção, pois o diagnóstico precoce é um dos principais aliados na determinação da cura e na preservação da visão.

Introduction: Retinoblastoma is the most common intraocular primary malignancy in childhood, it is rare and accounts for 2% to 4% of pediatric malignant tumors. Objective: To describe the clinical-epidemiological profile and survival of cases of retinoblastoma in a reference hospital in oncology in the state of Goiás, between 2008 and 2014. Method: Crosssectional analytical observational study built from the analysis of medical records of patients diagnosed with retinoblastoma between 2008 and 2014. Analyzes were performed using descriptive statistics and chi-square association test. A significance level of 5% was adopted. Survival was assessed using the Kaplan-Meier method. Results: 55 patients with retinoblastoma were treated, with predominance of females (54.5%), in the age group of 1 to 4 years (27.3%) and of brown ethnicity (50.9%). The most prevalent clinical characteristics were intraocular involvement (74.5%), unilateral (65.5%), with no family history (56.4%) and with clinical signs of leukocoria (80%). Most of them did not present metastases at diagnosis (87.3%), the main treatment being unilateral enucleation (72.7%). Association between the clinical evolution of the patient and extraocular location (p = 0.001) was found, presence of metastasis (p = 0.001), and stage IV classified by Chantada et al. (p = 0.001). Patients classified as E were submitted to higher volume of enucleation (right eye ­ p = 0.05 and left eye ­ p = 0.001). The 5-year overall survival was 72.7%. Conclusion: These findings are relevant for planning preventive actions, as early diagnosis is one of the main allies in determining the cure and preserving vision

Introducción: El retinoblastoma es la neoplasia maligna primaria intraocular más frecuente en la infancia, es raro y corresponde del 2% al 4% de los tumores malignos pediátricos. Objetivo: Describir el perfil clínicoepidemiológico y la sobrevida de los casos de retinoblastoma en un hospital de referencia en oncología en el Estado de Goiás, entre 2008 y 2014. Método: Estudio observacional analítico transversal, construido a partir del análisis de las historias clínicas de los pacientes diagnosticados con retinoblastoma entre 2008 y 2014. Los análisis se realizaron mediante estadística descriptiva y prueba de asociación ji cuadrada. Se adoptó un nivel de significancia del 5%. La sobrevida se evaluó mediante el método de Kaplan-Meier. Resultados: Fueron tratados 55 pacientes con retinoblastoma, lo que permitió identificar un predominio femenino (54,5%); en el grupo de edad de 1 a 4 años (27,3%); con etnia parda (50,9%). Las características clínicas más prevalentes fueron: afectación intraocular (74,5%); unilateral (65,5%); y sin antecedentes familiares (56,4%) y con signos clínicos de leucocoria (80%). La mayoría no presentaba metástasis al diagnóstico (87,3%); siendo el principal tratamiento la enucleación unilateral (72,7%). Hubo asociación entre la evolución clínica del paciente y la localización extraocular (p = 0,001), presencia de metástasis (p = 0,001) y estadio IV de Chantada et al. (p = 0,001). Los pacientes clasificados como E tenían más ojos enucleados (ojo derecho ­ p = 0,05 y ojo izquierdo ­ p = 0,001). La sobrevida global a los 5 años fue del 72,7%. Conclusión: Estos hallazgos son relevantes para la planificación de acciones preventivas, ya que el diagnóstico precoz es uno de los principales aliados para determinar la cura y preservar la visión.

Production of ethanol 3G from Kappaphycus alvarezii: evaluation of different process strategies.

This study evaluated the potential of Kappaphycus alvarezii as feedstock for ethanol production, i.e. ethanol 3G. First, aquatic biomass was subjected to a diluted acid pretreatment. This acid pretreatment generated two streams--a galactose-containing liquid fraction and a cellulose-containing solid fraction, which were investigated to determine their fermentability with the following strategies: a single-stream process (simultaneous saccharification and co-fermentation (SSCF) of both fractions altogether), which achieved 64.3 g L(-1) of ethanol, and a two-stream process (fractions were fermented separately), which resulted in 38 g L(-1) of ethanol from the liquid fraction and 53.0 g L(-1) from the simultaneous saccharification and fermentation (SSF) of the solid fraction. Based on the average fermentable carbohydrate concentration, it was possible to obtain 105 L of ethanol per ton of dry seaweed. These preliminaries results indicate that the use of the macro-algae K. alvarezii has a good potential feedstock for bioethanol production.

Cellulase production by Penicillium funiculosum and its application in the hydrolysis of sugar cane bagasse for second generation ethanol production by fed batch operation.

This study aimed to produce a cellulase blend and to evaluate its application in a simultaneous saccharification and fermentation (SSF) process for second generation ethanol production from sugar cane bagasse. The sugar cane bagasse was subjected to pretreatments (diluted acid and alkaline), as for disorganizing the ligocellulosic complex, and making the cellulose component more amenable to enzymatic hydrolysis. The residual solid fraction was named sugar cane bagasse partially delignified cellulignin (PDC), and was used for enzyme production and ethanol fermentation. The enzyme production was performed in a bioreactor with two inoculum concentrations (5 and 10% v/v). The fermentation inoculated with higher inoculum size reduced the time for maximum enzyme production (from 72 to 48). The enzyme extract was concentrated using tangential ultrafiltration in hollow fiber membranes, and the produced cellulase blend was evaluated for its stability at 37 °C, operation temperature of the simultaneous SSF process, and at 50 °C, optimum temperature of cellulase blend activity. The cellulolytic preparation was stable for at least 300 h at both 37 °C and 50 °C. The ethanol production was carried out by PDC fed-batch SSF process, using the onsite cellulase blend. The feeding strategy circumvented the classic problems of diffusion limitations by diminishing the presence of a high solid:liquid ratio at any time, resulting in high ethanol concentration at the end of the process (100 g/L), which corresponded to a fermentation efficiency of 78% of the maximum obtainable theoretically. The experimental results led to the ratio of 380 L of ethanol per ton of sugar cane bagasse PDC.

Potential of giant reed (Arundo donax L. ) for second generation ethanol production

Background The production of second generation ethanol from lignocellulosic biomasses that have not had their potential fully explored as feedstock is of great importance. Arundo donax is one these biomasses. It is a promising grassy plant to be used as a renewable resource for the production of fuels and chemicals, because of its fast growth rate, ability to grow in different soil types and climatic conditions. The present study evaluated its use as feedstock for the production of second generation ethanol. Results Initially its chemical characterization was carried out, and a protocol for fractioning the biomass through diluted acid pretreatment followed by alkaline pretreatment was developed, providing a solid fraction which was undergone to enzymatic hydrolysis reaching 42 g/L of glucose, obtained in 30 h of enzymatic hydrolysis. This partially delignified material was subjected to a simultaneous saccharification and fermentation (SSF) process, resulting in an ethanol concentration of 39 g/L at 70 h. Conclusions The fermentability of the pretreated biomass was performed successfully through the conception of simultaneous saccharification and fermentation resulting in approximately 75 L of ethanol per ton of cellulose.