An atlas of rational genetic engineering strategies for improved xylose metabolism in Saccharomyces cerevisiae.

Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.

Kluyveromyces marxianus: a potential biocatalyst of renewable chemicals and lignocellulosic ethanol production.

Kluyveromyces marxianus is an ascomycetous yeast which has shown promising results in cellulosic ethanol and renewable chemicals production. It can survive on a variety of carbon sources under industrially favorable conditions due to its fast growth rate, thermotolerance, and acid tolerance. K. marxianus, is generally regarded as a safe (GRAS) microorganism, is widely recognized as a powerhouse for the production of heterologous proteins and is accepted by the US Food and Drug Administration (USFDA) for its pharmaceutical and food applications. Since lignocellulosic hydrolysates are comprised of diverse monomeric sugars, oligosaccharides and potential metabolism inhibiting compounds, this microorganism can play a pivotal role as it can grow on lignocellulosic hydrolysates coping with vegetal cell wall derived inhibitors. Furthermore, advancements in synthetic biology, for example CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, will enable development of an engineered yeast for the production of biochemicals and biopharmaceuticals having a myriad of industrial applications. Genetic engineering companies such as Cargill, Ginkgo Bioworks, DuPont, Global Yeast, Genomatica, and several others are actively working to develop designer yeasts. Given the important traits and properties of K. marxianus, these companies may find it to be a suitable biocatalyst for renewable chemicals and fuel production on the large scale. This paper reviews the recent progress made with K. marxianus biotechnology for sustainable production of ethanol, and other products utilizing lignocellulosic sugars.

Biochemical conversion of sugarcane bagasse into the alcohol fuel mixture of isopropanol-butanol-ethanol (IBE): Is it economically competitive with cellulosic ethanol?

This work presents a techno-economic analysis of the production of isopropanol, butanol, and ethanol (IBE) from sugarcane bagasse using clostridia and compares IBE with cellulosic ethanol for the minimum selling price (MSP) and sustainability aspects. The MSPs of the fuels are similar (15 USD/GJ) provided that glucose and xylose are effectively utilized in both processes, and the IBE process is equipped with a genetically-modified Clostridium species with enhanced IBE yield and a highly productive continuous bioreactor with integrated product recovery. Notably, these technologies can reduce the size (from 23 × 3785-m3 to 3 × 3027-m3 fermentation tanks) and the wastewater footprint (from 50 to 10 m3/m3 IBE) of the IBE plant. Furthermore, given that the production of either fuel results in a similar increase in the value created by the sugarcane biorefinery and its energy efficiency, the alcohol mixture produced by clostridia is a promising alternative to the less energy-dense ethanol fuel.

A new approach to obtain cellulose nanocrystals and ethanol from eucalyptus cellulose pulp via the biochemical pathway.

The feasibility of integration of cellulosic ethanol production with the manufacture of cellulose nanofibers (CNF) and cellulose nanocrystals (CNC) was evaluated using eucalyptus cellulose pulp as feedstock and employing the biochemical route alone. For the enzymatic hydrolysis step, experimental central composite design (CCD) methodology was used as a tool to evaluate the effects of solids loading (SL) and enzymatic loading (EL) on glucose release and cellulose conversion. Glucose concentrations from 45 to 125 g/L were obtained after 24 h, with cellulose conversions from 35 to 96%. Validation of the statistical model was performed at SL of 20% and EL of 10 mg protein/g, which was defined by the desirability function as the optimum condition. The sugars released were used for the production of ethanol by Saccharomyces cerevisiae, resulting in 62.1 g/L ethanol after 8 h (yield of 95.5%). For all the CCD experimental conditions, the residual solids presented CNF characteristics. Moreover, the use of a new strategy with temperature reduction from 50 to 35°C after 24 h of enzymatic hydrolysis enabled CNC to be obtained after 144 h. The CNC showed a crystallinity index of 83%, length of 260 nm, diameter of 15 nm, and aspect ratio (L/D) of 15. These characteristics are suitable for many applications, such as reinforcement in polymeric materials and other lower volume higher value bio-based products. The findings indicate the viability of obtaining ethanol and CNC using the biochemical route exclusively, potentially contributing to the future implementation of forest biorefineries. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1085-1095, 2017.

Development of a low-cost cellulase production process using Trichoderma reesei for Brazilian biorefineries.

BACKGROUND: During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery. RESULTS: Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous ß-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw. CONCLUSIONS: In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei. With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases.

Production of cellulosic ethanol from sugarcane bagasse by steam explosion: Effect of extractives content, acid catalysis and different fermentation technologies.

The production of cellulosic ethanol was carried out using samples of native (NCB) and ethanol-extracted (EECB) sugarcane bagasse. Autohydrolysis (AH) exhibited the best glucose recovery from both samples, compared to the use of both H3PO4 and H2SO4 catalysis at the same pretreatment time and temperature. All water-insoluble steam-exploded materials (SEB-WI) resulted in high glucose yields by enzymatic hydrolysis. SHF (separate hydrolysis and fermentation) gave ethanol yields higher than those obtained by SSF (simultaneous hydrolysis and fermentation) and pSSF (pre-hydrolysis followed by SSF). For instance, AH gave 25, 18 and 16 g L(-1) of ethanol by SHF, SSF and pSSF, respectively. However, when the total processing time was taken into account, pSSF provided the best overall ethanol volumetric productivity of 0.58 g L(-1) h(-1). Also, the removal of ethanol-extractable materials from cane bagasse had no influence on the cellulosic ethanol production of SEB-WI, regardless of the fermentation strategy used for conversion.

Ethanol production from sugars obtained during enzymatic hydrolysis of elephant grass (Pennisetum purpureum, Schum.) pretreated by steam explosion.

In this work, steam explosion was used a pretreatment method to improve the conversion of elephant grass (Pennisetum purpureum) to cellulosic ethanol. This way, enzymatic hydrolysis of vaccum-drained and water-washed steam-treated substrates was carried out with Penicillium echinulatum enzymes while Saccharomyces cerevisiae CAT-1 was used for fermentation. After 48 h of hydrolysis, the highest yield of reducing sugars was obtained from vaccum-drained steam-treated substrates that were produced after 10 min at 200 °C (863.42 ± 62.52 mg/g). However, the highest glucose yield was derived from water-washed steam-treated substrates that were produced after 10 min at 190 °C (248.34 ± 6.27 mg/g) and 200 °C (246.00 ± 9.60 mg/g). Nevertheless, the highest ethanol production was obtained from water-washed steam-treated substrates that were produced after 6 min at 200 °C. These data revealed that water washing is a critical step for ethanol production from steam-treated elephant grass and that pretreatment generates a great deal of water soluble inhibitory compounds for hydrolysis and fermentation, which were partly characterized as part of this study.

Alcoholic fermentation of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the presence of inhibitory compounds and seawater.

Production of cellulosic ethanol and holocellulosic ethanol from vegetable or microbial biomass starts with a hydrolysate containing compounds which may produce negative effects in the enzymatic hydrolysis and fermentation stages due to the need of pretreatment of the materials. In this way, the simultaneous presence of hydroxymethylfurfural (HMF), furfural, acetic acid, levulinic acid, and formic acid in different concentrations was tested in the fermentation using Saccharomyces cerevisiae, Pichia stipitis, and Zymomonas mobilis. The substitution of freshwater by seawater in the culture medium was also analyzed. Thus, inhibitory effects were stronger in the fermentation using P. stipitis, followed by Z. mobilis and S. cerevisiae. Formic acid and acetic acid presented more significant effects among the inhibitory compounds, followed by HMF, furfural and levulinic acid. Fermentation performed in culture medium with seawater showed promising results, especially in the ethanol yield using S. cerevisiae (0.50 g ethanol/g glucose) and Z. mobilis (0.49 g ethanol/g glucose). Whereas the production of cellulosic ethanol and holocellulosic ethanol are in early stages of development on an industrial scale, and that the availability and use of freshwater may cause socio-environmental problems for expansion of ethanol production, the use of seawater appears as an alternative to mitigate this problem.

Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis.

In the near future, grasses must provide most of the biomass for the production of renewable fuels. However, grass cell walls are characterized by a large quantity of hydroxycinnamic acids such as ferulic and p-coumaric acids, which are thought to reduce the biomass saccharification. Ferulic acid (FA) binds to lignin, polysaccharides and structural proteins of grass cell walls cross-linking these components. A controlled reduction of FA level or of FA cross-linkages in plants of industrial interest can improve the production of cellulosic ethanol. Here, we review the biosynthesis and roles of FA in cell wall architecture and in grass biomass recalcitrance to enzyme hydrolysis.

Family 1 carbohydrate binding-modules enhance saccharification rates.

Cellulose degrading enzymes usually have a two-domain structure consisting of a catalytic domain and a non-catalytic carbohydrate-binding module. Although it is well known the importance of those modules in cell wall degrading process, their function is not yet fully understood. Here, we analyze the cellulose-hydrolysis activity enhancement promoted by the cellobiohydrolase I carbohydrate-binding module from Trichoderma harzianum. It was cloned, expressed, purified and used in combination with either a commercial cellulase preparation, T. reesei cellobiohydrolase I or its separate catalytic domain to hydrolyze filter paper. In all cases the amount of glucose released was increased, reaching up to 30% gain when the carbohydrate-binding module was added to the reaction. We also show that this effect seems to be mediated by a decrease in the recalcitrance of the cellulosic substrate. This effect was observed both for crystalline cellulose samples which underwent incubation with the CBM prior to application of cellulases and for the ones incubated simultaneously. Our studies demonstrate that family 1 carbohydrate-binding modules are able to potentiate the enzymatic degradation of the polysaccharides and their application might contribute to diminishing the currently prohibitive costs of the lignocellulose saccharification process.