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

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

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. It is worth to mention that synthetic bioengineering could be applied for any microorgan‐ ism, since it is a rational design of metabolic pathways.

While most biological routes being studied for the processing of lignocellulosic biomass fo‐ cused on the separate production of hydrolytic enzymes, in a process that usually comprises several steps, another approach is suggested to achieve this goal. This approach, termed consolidated bioprocessing (CBP) involves the production of cellulolytic enzymes, hydroly‐ sis of biomass, and fermentation of resulting sugars in a single stage via microorganisms or a consortium [157]. CBP appears to offer very large costs reduction if microorganisms can be developed that possess the required combination of substrate utilization and product forma‐ tion properties [158]. In a 2006 report in biomass conversion to biofuels, the U.S. Department of Energy endorsed the view that CBP technology is "the ultimate low-cost configuration for cellulose hydrolysis and fermentation" (DOE Joint Task Force, 2006; energy.gov). Currently, CBP technology is developing fast, especially due to partnerships with venture capital in‐ vestors and researchers. The main challenge of CBP is to generate engineered microorgan‐ isms able to produce the saccharolytic enzymes and converting the sugars released by those enzymes into the desired end-products. In addition, CBP microorganisms need to be able to perform these tasks rapidly and efficiently under challenging, industrial processes. A suc‐ cessful microbial platform for production of bioethanol from microalgae is currently availa‐ ble, and demonstrates an application of the CBP [159]. A DNA fragment encoding enzymes for alginate transport and metabolism from *Vibrio splendidus*, abundant and ubiquitous ma‐ rine bacteria, was introduced in the genome of *Escherichia coli*, a well-characterized microor‐ ganism. This microbial platform was able to simultaneously degrade, uptake and metabolize alginate, an abundant polysaccharide present in microalgae. When further engi‐ neered for ethanol synthesis, this platform enabled bioethanol production with satisfactory yield directly from microalgae via a consolidated bioprocess [159].

The approach required for generation of CBP microorganisms involves the knowledge of many topics discussed in this chapter, concerning to fundamental principles of microbial cellulose utilization and its regulation. Moreover, the principles of synthetic bioengineering discussed above can be applied to the development of new strains for CBP technology, and therefore the generation of new microbial platforms able to uptake and metabolize com‐ pletely the lignocellulosic biomass.
