**21. Conclusions and future perspectives**

instance, in *A. nidulans*, the xylanolytic transcriptional activator XlnR is repressed by glucose via CreA, the transcriptional factor responsible for CCR in this specie. The comprehension of such sophisticated regulatory network is essential for genetic engineering of new strains

In order to degrade efficiently plant biomass, a microorganism should possess characteristics that make the process economically viable. For cellulosic ethanol production, for instance, an efficient microorganism should produce high yields of the desired product, must have a broad substrate range and high ethanol tolerance and it has to be tolerant to the inhibitors present in lignocellulosic hydrolysates. Therefore, engineering microbial strains for improvement of ef‐ fectiveness in industrial applications is not a simple task. Concerning to bioethanol produc‐ tion, the most promising organism for genetic bioengineering is the yeast *Saccharomices cerevisiae*. This microorganism has a great capacity for cell-recycle fermentation, and it is toler‐ ant against various stresses, such as high temperature, low pH and many inhibitors [150]. Moreover, *S. cerevisiae* is a well-characterized model organism, with a diverse array of research

However, the metabolism of *S. cerevisiae* is very complex and punctual genetic modifications in the yeast may lead to unpredicted modifications in the whole metabolism, with undesired effects for industrial applications, such as generation of toxic by-products. In general, meta‐ bolic engineering of *S. cerevisiae* is performed on a trial-and error basis, with various modifi‐ cations being tested at the same time. In this way, the most promising approaches are implemented to increase target production [153]. Due to this challenge, a novel and rational strategy has recently emerged for a system-wide modification of metabolism. The novel ap‐ proach, termed synthetic bioengineering, is essential for creating effective yeast cell factories [152 - 154]. The first step in the synthetic bioengineering consists of an optimized metabolic pathway designed by computational simulation. Based on this metabolic profile, a list of tar‐ get genes to be inserted or deleted is determined. Next, a customized microbial cell factory is assembled using advanced gene manipulation techniques. After the detection of metabolic problems concerning the prototype strains by using transcriptomics and metabolomics, fur‐ ther improvement of the assembled strains are performed [153]. Currently, many research‐ ers have been using synthetic bioengineering approaches to improve microbial strains for industrial applications. For instance, efforts have been done in order to produce *S. cerevisiae* strains able to use xylose as carbon source, once this sugar is the second most abundant in lignocellulosic biomass. The yeast is not able to ferment xylose, but some groups already produced *S. cerevisiae* strains with improved capacity of xylose fermentation [30, 155 - 156]. A detailed description of synthetic bioengineering and its applications could be found at [153]. In this review, the authors describe engineered microbial strains producing higher al‐ cohols such as 1-butanol and isobutanol and strains overproducing glutathione, for instance.

**20. Improving microbial strains for degradation of lignocellulosic**

230 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

tools and resources, which facilitates metabolic engineering [151, 152].

able to produce a wide range of lignocellulolytic enzymes.

**biomass**

A large quantity of lignocellulosic residues is accumulating over the world, mainly due to the expansion of industrial processes, but other sources such as wood, grass, agricultural, forestry and urban solid wastes contribute to accumulation of lignocellulosic material. These residues constitute a renewable resource from which many useful biological and chemical products can be derived. The natural ability of fungi and other microorganisms to degrade lignocellulosic biomass, due to highly efficient enzymatic systems, is very attractive for the development of new strategies concerning industrial processes. Paper manufacture, com‐ posting, human and animal feeding, economically important chemical compounds and bio‐ mass fuel production are among some industrial applications derived from microbial lignocellulosic degradation.

**Author details**

**References**

lish.

Wagner Rodrigo de Souza\*

Address all correspondence to: wagnerusp@gmail.com

ISI:000246554000023. English.

PMID: 16102600.

506-77.

Institute of Biology, State University of Campinas (UNICAMP), Brasil

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Global climate change and future energy demands initiate a race in order to achieve sustain‐ able fuels derived from biomass residues. Conversion of sugars to ethanol is already cur‐ rently done at very low cost from sugarcane in Brazil, and from corn, in United States. However, the challenge is how to obtain the biofuel from the wastes generated from the mills producing ethanol. Residues such as sugarcane bagasse and corncobs contain large amounts of lignocellulosic material and therefore can be transformed into biofuels. A major advantage of using residues to produce biofuels is to reduce the competition between fuels and food. In this context, hydrolytic enzymes such as cellulases contribute for the large cost of cellulosic ethanol nowadays. The great bottleneck to achieve cellulosic biofuels is the plant biomass recalcitrance, and overcome such barrier is the key for the development of feasible industrial processes for biofuels production. For instance, it was recently demon‐ strated that *Aspergillus niger* growing on steam-exploded sugarcane bagasse was able to pro‐ duce and secrete a high number of (hemi)cellulases [98]. The challenge is to adapt the process of steam explosion of a waste residue, such as sugarcane bagasse, with its hydroly‐ sis by the fungi. The steam-explosion is a pretreatment that can decrease biomass recalci‐ trance, allowing the fungi to penetrate deeply within the biomass.

The comprehension of the machinery behind the enzymatic systems of fungi able to degrade plant cell wall polysaccharides favors the use of the microorganisms in industrial applications. Currently, through advanced molecular techniques, it is possible to engineer new microbial strains by insertion or deletion of genes involved in important metabolic pathways responsible for biomass degradation. The useful host cells to develop the synthetic bioengineering should have versatile genetic tools, resources and suitability for bio-refinery processes, such as stress tolerance. Therefore, a strain development in future requires insertion, deletion and expression controls of multiple genes and it is a difficult task to achieve. However, integrated advanced techniques could be able to overcome these challenges, including computational simulation of metabolic pathways, genome synthesis, directed evolution and minimum genome factory. The synthesis of the whole genome has already been done [160, 162] and, as discussed in reference [153], in a near future the synthesis of very large fragments of DNA will make it possible to de‐ sign a whole yeast artificial chromosome (YAC) encoding a number of genes. According to these authors, the *de novo* synthesis of YAC should be a breakthrough methodology for the fu‐ ture synthetic bioengineering, and cloning of individual genes and a construction of plasmid vectors would be obsolete. A rational design of metabolic pathways along with customized de‐ sign of genes with optimized expression may be obtained, making it possible to produce a whole sequence of the artificial chromosome [153, 163].

As said by Lee Lynd, a pioneering researcher in the field of biomass: "the first step toward realiz‐ ing currently improbable futures is to show that they are possible". These technologies descri‐ bed above are currently available for scientific community and, along with advances in industrial processes, endorse the possibility to take energy from plant biomass using microor‐ ganisms. Thus, the Humanity has never been so close to use new and sustainable ways of energy.
