Use of carbohydrate-directed enzymes for the potential exploitation of sugarcane bagasse to obtain value-added biotechnological products.

Microorganisms, such as fungi and bacteria, are crucial players in the production of enzymatic cocktails for biomass hydrolysis or the bioconversion of plant biomass into products with industrial relevance. The biotechnology industry can exploit lignocellulosic biomass for the production of high-value chemicals. The generation of biotechnological products from lignocellulosic feedstock presents several bottlenecks, including low efficiency of enzymatic hydrolysis, high cost of enzymes, and limitations on microbe metabolic performance. Genetic engineering offers a route for developing improved microbial strains for biotechnological applications in high-value product biosynthesis. Sugarcane bagasse, for example, is an agro-industrial waste that is abundantly produced in sugar and first-generation processing plants. Here, we review the potential conversion of its feedstock into relevant industrial products via microbial production and discuss the advances that have been made in improving strains for biotechnological applications.

The fermentation efficiency exhibited by Saccharomyces cerevisiae on Sugarcane bagasse hydrolysate, by analyzing the effects of pre-treatment and detoxification / A eficiência da fermentação exibida por Saccharomyces cerevisiae no hidrolisado de bagaço de cana-de-açúcar, analisando os efeitos do pré-tratamento e destoxicação

In this study, the possibility of increasing fermentation efficiency of Saccharomyces cerevisiae on sugarcane bagasse (a type of lignocellulosic waste) was analyzed. Sugarcane bagasse was subjected to hydrothermal and acidic pre-treatment. Next, the enzymatic hydrolysis of raw biomass and each pre treated biomass was performed using CellicCtec® enzymatic complex to obtain sugarcane hydrolysate, hydrothermal hydrolysate and acidic hydrolysate. Next, these were fermented by S. cerevisiae to check if the by-products of enzymatic hydrolysis, furfural and acetic acid had an inhibitory effect on fermentation efficiency. Next, each pre-treated biomass was subjected to detoxification involving activated charcoal. Each detoxified biomass was tested for fermentation efficiency. The lignocellulosic composition for sugarcane hydrolysate, hydrothermal hydrolysate and acidic hydrolysate, varied significantly, and were found to be, for cellulose 36.7%, 27.7% and 63.7% respectively; for hemicellulose 22.2%, 4.4% and 12% respectively; and for lignin 21.2%, 27.7% and 28.7% respectively. The presence of furfural and acetic acid had a strong influence on the fermentation efficiency of S. cerevisiae, and affected the consumption of sugars in each biomass by more than 90%. Further, we found that the detoxification process increased fermentation efficiency by 12.7% for the hydrothermal hydrolysate while for the acidic hydrolysate no significant difference was observed. This study showed that fermentation with greater efficiency is viable through the combined use of hydrothermal pre-treatment and detoxification. This combination of methods also causes less pollution as compared with the method involving acid pre-treatment due to the reduced number of effluents produced.(AU)

Nesse trabalho avaliou-se a possibilidade de se aumentar a eficiência de fermentação de um hidrolisado de bagaço de cana submetido aos pré-tratamentos hidrotérmico (195 ºC, usando 200 rpm por 10 min) e ácido (0,5% (v/v) de ácido sulfúrico a 121ºC por 15 min) (carga de sólidos de 10% m/v). A hidrólise enzimática do material pré-tratado foi realizada utilizado o complexo enzimático CellicCtec® (60 FPU/gbiomassa seca, tampão citrato a 50 mM e pH 4,8) a 50ºC usando 150 rpm por 72h. Antes do processo de detoxificação, realizou-se um teste com a espécie de Saccharomyces cerevisiae para verificar se os compostos furfural (1 e 4g.L-1) e ácido acético (1 e 5% v/v) exerciam significativa inibição na espécie testada. O processo de detoxificação avaliou a concentração de carvão ativado (1, 3 e 5% m/v) e o tempo do processo (30, 45 e 60 min) a 30 ºC, 150 rpm por 24 h. A composição lignocelulosica da biomassa in natura e pré-tratada (hidrotérmico e ácido) foi para celulose (36,7, 27,7 e 63,7%), hemicelulose (22,2, 4,4 e 12%) e lignina (21,2, 27,7 e 28,7%), respectivamente e com rendimento mássico em torno de 60%. A presença de furfural e ácido acético exibiu forte influência na espécie considerada, chegando a prejudicar em mais de 90% o consumo de açúcares no meio. O processo de destoxificação aumentou 13% a eficiência de fermentação para o hidrolisado obtido hidrotermicamente, enquanto que para o ácido não houve diferença significativa. Obtendo assim uma fermentação com maior eficiência, tecnicamente viável e menos poluente.(AU)

Gene Editing Technologies for Sugarcane Improvement: Opportunities and Limitations.

Plant-based biofuels present a promising alternative to depleting non-renewable fuel resources. One of the benefits of biofuel is reduced environmental impact, including reduction in greenhouse gas emission which causes climate change. Sugarcane is one of the most important bioenergy crops. Sugarcane juice is used to produce table sugar and first-generation biofuel (e.g., bioethanol). Sugarcane bagasse is also a potential material for second-generation cellulosic biofuel production. Researchers worldwide are striving to improve sugarcane biomass yield and quality by a variety of means including biotechnological tools. This paper reviews the use of sugarcane as a feedstock for biofuel production, and gene manipulation tools and approaches, including RNAi and genome-editing tools, such as TALENs and CRISPR-Cas9, for improving its quality. The specific focus here is on CRISPR system because it is low cost, simple in design and versatile compared to other genome-editing tools. The advance of CRISPR-Cas9 technology has transformed plant research with its ability to precisely delete, insert or replace genes in recent years. Lignin is the primary material responsible for biomass recalcitrance in biofuel production. The use of genome editing technology to modify lignin composition and distribution in sugarcane cell wall has been realized. The current and potential applications of genome editing technology for sugarcane improvement are discussed. The advantages and limitations of utilizing RNAi and TALEN techniques in sugarcane improvement are discussed as well.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.