Engineering Product Forming Ability into Biomass Degraders

Several species of cellulolytic fungi, such as Trichoderma reesei, naturally pro­duce a large repertoire of saccharolytic enzymes to digest lignocellulose effi­ciently, assimilate all lignocellulosic sugars, and convert these sugars into ethanol, showing that they naturally possess all pathways for conversion of lignocellulose into bioethanol (Chambergo et al. 2002; Lynd et al. 2002). It has been shown that a biorefinery consuming thousands of tons of biomass per day will require many tons of cellulase preparation to operate assuming that enzymes with far greater specific activity are not identified (Xu et al. 2009). Currently, only fungi naturally produce the required amounts of cellulase and some strains of T. reesei produce more than 100 g/L cellulase (Cherry and Fidantsef 2003). Thus, advantages of T. reesei as a CBP organism are: (1) the production of sufficient quantities of

Table 8.3 Characteristics of an ideal CBP organism

1. Ability to ferment all hexoses and pentoses present in lignocellulose

2. High product yield, titer, and productivity

3. High product and inhibitor tolerance

4. General robustness for industrial processes, cellulase production in toxic environment

5. Tolerance to low pH and high temperature

6. Amenability to DNA manipulation

7. High levels of heterologous protein production and secretion (if cellulolytic ability must be

engineered)

8. Concurrent fermentation of sugars (hexose and pentose co-utilization)

9. GRAS status

10. Recyclability in successive processes

11. Minimum nutrient supplementation requirement

Van Zyl et al. (2007) cellulases at reasonable cost, (2) several strains are established commercially, and (3) it can utilize all lignocellulose sugars for production of ethanol. Challenges to overcome before T. reesei can be considered as a CBP organism include the observations that ethanol yield, rate of production and tolerance are low, and that mixing during fermentation may require more energy owing to the filamentous cell morphology. Preliminary studies showed that T. reesei could produce cellulases when grown aerobically on cellulose that continued to degrade cellulose to sugars and ferment these sugars to ethanol when cultures were rendered anaerobic (Xu et al. 2009). However, acetic acid was produced as a major byproduct. The major limitation for efficient ethanol production by T. reesei does not lie in the absence of the relevant genes and pathways but are more likely related to the low expression of these genes or the activity of the enzymes encoded. Approaches to solving these problems are to enhance the expression of the relevant genes at the transcriptional level and/or to introduce heterologous genes that encode enzymes with higher activities and to knockout genes responsible for the production of byproducts. Recently, the laccase gene lacA from Trametes sp. AH28-2 was heterologously expressed in T. reesei under control of a constitutive promoter (Zhang et al. 2012). Transformants were identified that were able to secrete the recombinant laccase. Reducing sugar yields obtained from saccharification of corn residue by crude enzyme extracts prepared from the transformants increased by 31.3-71.6 %, respectively, compared to the host strain.

Another filamentous fungus, Fusarium oxysporum, also produces the enzymes required to break down cellulose and hemicellulose while simultaneously fer­menting the released sugars to ethanol albeit at relatively low yields (Anasontzis et al. 2011; Panagiotou et al. 2005). In SSF of a cellulosic substrate a F. oxysporum wild-type strain was able to grow in aerobic conditions and produced ethanol with a yield of 0.35 g/g cellulose under anaerobic conditions. The strain was also shown to effectively produce a complete system of hydrolytic enzymes when grown on various agro-industrial lignocellulose by-products, such as dry citrus peels, corn cob, and brewer’s spent grain with concomitant ethanol production (Anasontzis et al. 2011; Xiros et al. 2008). It was hypothesized that homologous overexpres­sion of cellulases and hemicellulases under constitutive control, could provide a higher breakdown rate of the biomass and thus increase the supply of sugars to the ethanol production pathway. To this end, the endoxylanase two of F. oxysporum, was overproduced under control of the constitutive Aspergillus nidulans gpdA promoter (Anasontzis et al. 2011). The fermentative performance of the trans­formants were evaluated and compared to that of the wild type in simple CBP systems using corn cob or wheat bran as sole carbon sources. Transformants produced approximately 60 % more ethanol compared to the wild type on corn cob and wheat bran likely due to the * 2-2.5-fold higher extracellular xylanase activities in the fermentation broths of the transformants.

High-temperature conversion process conditions potentially provide a significant energy saving since reactors would not have to be cooled to mesophilic conditions before inoculation and then reheated for distillation (Xu et al. 2010). Furthermore, it has been shown that a 10 °C increase in temperature approximately doubles enzymatic reaction rates, decreasing the amount of enzyme required (Ibrahim and El-diwany 2007). In addition, the use of reaction and fermentation temperatures in excess of 60 °C minimizes the risk of contamination. Since cellulose hydrolysis and sugar release is in most cases the rate-limiting step in a typical CBP process, high — temperature hydrolysis will be therefore be advantageous. Thermophilic bacteria capable of cellulose hydrolysis and ethanol production show great potential as CBP organisms (Xu et al. 2010). The cellulosome producing thermophilic Gram-positive anaerobic bacterium C. thermocellum is regarded as a potential CBP organism as it is very efficient at hydrolyzing crystalline cellulose (Lynd et al. 2002). While growth of wild-type strains is inhibited in the presence of ethanol concentrations above 2 % (v/v) strains have been evolved that remained viable at ethanol concentrations of up to 8 % (v/v) (Xu et al. 2010). This group also investigated the effect of some of these inhibitors on cellulosome activity of C. thermocellum. It was shown that that some organic acids actually promoted cellulolytic activity and that the C. thermocellum cellulosome could tolerate certain concentrations of furfural (up to 5 mM), p-hydroxybenzoic acid (up to 50 mM) and catechol (up to 1 mM). The C. thermo — cellum cellulosomes were also able to tolerate higher ethanol concentrations and temperatures than the T. reesei enzymes used commercially.

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