Fungal Metabolic Engineering for Biofuel Production

Mycology: Current and Future Developments, 2015, Vol. 1, 3-22

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CHAPTER 5

Fungal Metabolic Engineering for Biofuel Production Daniel P. Kiesenhofer, Astrid R. Mach-Aigner, Robert L. Mach* Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria Abstract: The search for alternatives to conventional fuels becomes more and more important. Second generation biofuels might be a solution to provide us with renewable energy. There are several alternatives to the fossil fuels, such as ethanol, isobutanol, sesquiterpenoids and fatty acid ethyl esters (FAEEs). Saccharomyces cerevisiae is a potent and versatile cell factory, which is able to produce these substances. Except for ethanol the so far achieved yields need to be improved. S. cerevisiae has to be equipped with the proper tools to degrade lignocellulose. This can be achieved by different strategies: secretion or cell surface display of lignocellulose-degrading enzymes, synthesis of cellulosomes or intracellular cellodextrin hydrolysis. Many filamentous fungi possess the ability to efficiently degrade lignocellulose, but they do not produce larger quantities of ethanol due to intolerance towards the product and anaerobic conditions. Therefore, the goal of production of second generation biofuels could also be achieved by enabling them to produce and tolerate higher amounts of ethanol.

Keywords: Biofuel, Consolidated bioprocessing, Ethanol, Filamentous fungi, Lignocellulose, Metabolic engineering, Saccharomyces cerevisiae.

INTRODUCTION The substitution of fossil fuels through ethanol and other alternative, renewable biofuels becomes more and more important. After the first generation biofuels faced criticism due to the use of putative food products, the second generation tries to circumvent this issue by using, otherwise idle, waste products from feed and pulp industries. In order to produce biofuels from renewable biopolymers adequate microorganisms have to be chosen to synthesize alternative fuels. Saccharomyces cerevisiae has been used in the food industry for centuries and the knowledge about this model organism is large. But this potentially ethanol producing organism has one major disadvantage: it cannot degrade lignocellulose and therefore, the ethanol fermentation depends on the addition of sugar, which contradicts the aim of second generation biofuels. To equip S. cerevisiae with the proper tools to utilize such a complex carbon source for ethanol production it has to be genetically and metabolically engineered. On the other side of the spectrum of possible ethanol producers are filamentous fungi, which are able to degrade and utilize Corresponding author Robert L. Mach: Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria Tel.: +43158801166502. E-Mail: [email protected] *

Roberto Nascimento Silva (Ed) All rights reserved-© 2015 Bentham Science Publishers

4 Mycology: Current and Future Developments, Vol. 1

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lignocellulose but lack the ability to produce and tolerate higher quantities of ethanol. So, in their case is also need for improvement, which is also facilitated via genetic and metabolic engineering methods. Looking at biofuels there is a variety of substances, which can substitute the conventional ones: ethanol, isobutanol, sesquiterpenoids or fatty acid ethyl esters (FAEEs). Exemplary chemical structures are depicted in (Fig. 1). Besides ethanol, these products can be produced in different S. cerevisiae strains. In their case, the necessity for new or altered pathways in yeast and improving the production arises.

Fig. (1). Examples for sesquiterpenoid biofuels: (A) α-bisabolene, (B) α-farnesene; examples for FAEEs: (C) ethyl-oleate, (D) ethyl-decanoate

Before engineering a production organism, we have to address a series of questions [1, 2]. On the one hand, there are product-specific aspects which have to be considered: Is the desired product toxic for the host? Is it necessary to manipulate the host in order to increase its robustness towards the properties of the process (e.g. temperature-tolerance)? On the other hand, there are host-specific issues, which have to be addressed: Has the pathway to be altered for increased productivity? Are other pathways interfering with product formation? Has the expression level of enzymes to be adjusted? Is the redox balance of essential cofactors maintained? In this book chapter we will discuss the potential of S. cerevisiae for the production of several different biofuels and strategies how to give this host the ability to utilize lignocellulose. Secondly, we will focus on the ability of filamentous fungi to produce ethanol from lignocellulose and its prospects for future biofuel applications. Table 1 provides a summary of the presented biofuel producing organisms and their biofuel yields.

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Mycology: Current and Future Developments, Vol. 1 5

SACCHAROMYCES CEREVISIAE Biofuels Ethanol S. cerevisiae is the most important and well known producer of ethanol in first generation biofuels. Industrial strains are able to produce up to 160 g ethanol per litre culture broth. A major by-product during ethanol fermentation is glycerol. It is an electron sink for reduced cofactors and important for osmotic balance of yeast cells. Deletion of gpd1 and gpd2, which encode for cytosolic NADH-dependent glycerol-3-phosphate dehydrogenases, lead to a reduction or complete abolishment of glycerol formation [3], but double deletion strains cannot grow anaerobically. Furthermore, a Δgpd2 strain showed an increase of ethanol synthesis with the drawbacks of decreased productivity and growth [4]. A modification of the double knock-out genotype by introduction of an acetylating NAD+-dependent acetaldehyde dehydrogenase from Escherichia coli resulted in a strain that could only grow anaerobically when acetate is present and produced ethanol from acetate [5]. To counter the alteration of osmotic balance and the energy pool (reduced NADH and ATP) by deletion of gpd1, Guo and colleagues introduced a nonphosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase from Bacilluscereus and facilitated overexpression of the two trehalose synthase genes tps1 and tps2 [26]. These alterations were successful and resulted in a productive yeast strain, which produced more ethanol and less glycerol than the parental strain. Another positive achievement is the increased thermotolerance mediated by the overexpression of tps1, which is beneficial for simultaneous saccharification and fermentation processes. Concerning the utilization of pentoses in S. cerevisiae, heterologously expressed xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and overexpression of endogenous xylulokinase are required [27]. Table 1. Exemplary representation of ethanol producers, their substrates, and ethanol yields.

Microorganism

Substrate concentration

and

YE1 [g/l]

YSC2 [g/g]

Reference

Isobutanol S. cerevisiae GEVO3694

35.7 g/l D-glucose

12.3

0.46

[6]

20 g/l D-glucose

0.52

0.03

[7]

FAEE S. cerevisiae YPH499 Sesquiterpenoids S. cerevisiae pYAMG044/AURGG101

50 g/l D-galactose3

0.145 I farnesol 0.098 neridol

[8]


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(Table ) contd.....

Substrate concentration

Microorganism S.cerevisiae

and

18 g/l D-galactose + 2 g/l D-glucose

YSC2 [g/g]

YE1 [g/l] 0.994 I bisabolene

Reference [9]

Ethanol S. cerevisiae MNII/cocI'BEC34

20 g/l PASCa

S. cerevisiae 598.E1

100 g/l PCSC

20.5

Fusarium oxysporum F3

40 g/l D-glucose

17.5

0.42

[12]

F. oxysporum Fx13

40 g/l wheat straw

8.83

0.22

[13]

Neurospora crassa DSM1129

alkali-treated BG

5.6

0.074

[14]

N. crassa DSM1129

80 g/l pretreated SoB

27.6

0.345

[15]

Trichoderma reesei HJ48

50 g/l D-glucose

9.7

0.25

[16]

50 g/l SuB

3.1

0.1

Aspergillus niger NRRL 326

50 g/l D-glucose

5.7

Rhizopus oryzae NRRL 1501

100 g/l corn fiber cellulose

15.5

Aspergillus parasiticus TJW35.21

60 g/l sucrose

638

Trametes hirsuta

20 g/l wheat bran

4.3

Trametes versicolor KT9427

20 g/l wheat bran

5

20 g/l rice straw

4.8

Fusarium verticillioides

40 g/l pretreated SuB

4.6

0.31

Acremonium zeae

40 g/l pretreated SuB

3.9

0.31

Phlebia sp. MG-60

20 g/l UHKP

8.4

0.42

5

7.6 b

0.51

c d7

e

91 g/l UHKP

[17]

[18] 0.44

Flammulina velutipes Fv-1

150 g/l SB cellulose

Paecilomyces variotii ATHUM 8891

15 g/l D-xylose/D-glucose

[21]

[22] [23]

0.36 10.5

20 g/l BG

[19] [20]

37.3 9

[10] [11]

6

[24] [25]

0.06

ethanol yield, ethanol yield per gram of consumed substrate, exogenous fatty acids were added, 4 engineered to display lignocellulose degrading proteins, 5 engineered to secrete lignocellulose degrading proteins, 6 original data states 2.6% ethanol, conversion into g/l by the authors, 7 exogenous cellulase and β glucosidase added to culture broth, 8 original data states 8% ethanol, conversion into g/l by the authors, 9 exogenous cellulase added to culture broth a phosphoric acid swollen cellulose, b pretreated corn stover cellulose, c brewer’s spent grain, d sorghum bagasse, e sugarcane bagasse 1

2

3

A prerequisite for second generation biofuels is the ability of a potential ethanol producer to metabolize mixed sugars into ethanol because lignocellulose consists of three components: cellulose, hemicellulose, and lignin. Cellulose is finally degraded to Dglucose, hemicellulose is mostly depolymerized to D-xylose or L-arabinose. One possibility to enable S. cerevisiae to increase its metabolization of pentoses is the employment of isomerase based pathways, the overexpression of non-oxidative pentose-



phosphate pathway enzymes, and increasing the efficiency of pentose transporters [28 30]. Mixed sugar fermentation using S. cerevisiae exhibits one major problem: D-glucose is the favoured source of energy, while pentoses are just consumed for ethanol synthesis when D-glucose is depleted from the medium [31], because D-glucose represses the activity of pentose transporters. Subtil and colleagues tried to tackle this issue by searching for pentose transporters, which are not regulated by D-glucose, but their effort was not successful [28]. In contrast, Bellissimi and colleagues observed co-consumption of D-glucose and D-xylose in fed-batch experiments. As a consequence the duration of the process was extended and acetic acid was accumulated [32]. Another approach to establish co-fermentation of hexoses and pentoses was a step-by-step strain evolution experiment. In a first step, the S. cerevisiae strain was grown in media containing Dglucose, D-xylose, and L-arabinose, the second medium did not contain D-glucose anymore, and the last medium used for growth solely contained L-arabinose as carbon source. These repeated batch cultivations led to a strain, which was able to speed up the process of ethanol fermentation from different sugars [33].

Isobutanol Isobutanol is synthesized via the Ehrlich pathway in S. cerevisiae. α-ketoisovalerate, which can be a product of valine degradation, is shuttled from the mitochondria into the cytosol, where it is decarboxylated by the ketoisovalerate decarboxylase (kivD) to isobutyraldehyde and reduced by the alcohol dehydrogenase (adh) to isobutanol [34]. In order to improve isobutanol synthesis via the endogenous pathway, Kondo and colleagues used a S. cerevisiae strain lacking the pyruvate decarboxylase gene pdc1, expressing kivD from Lactococcus lactis, and over-expressing endogenous adh6 and acetaloacetate synthase ilv2. The deletion of pdc1 reduces acetaldehyde formation from pyruvate and therefore, increases the pool of substrate available for isobutanol synthesis [35]. Another approach to increase the yield is to relocate the pathway from the mitochondria into the cytosol: ilv2, the ketoacid reductoisomerase ilv5, and the dihydroxyacid dehydrogenase ilv3 were stripped of their mitochondrial localization signal. Additionally, the expression of kivD from L. lactis increased the isobutanol yield [36]. To avoid a possible competition between mitochondrial and cytosolic pathways, Brat and colleagues deleted ilv2 and used a different kivD gene. This was done additionally to the approach mentioned before and resulted in an isobutanol concentration of 0.63 g/l [37]. An extensive experimental design by Lies and colleagues resulted in isobutanol yield of 12.3 g/l and led to a patent [6]. Based on the knowledge from several other patents [38 - 40] they created a high producing strain of S. cerevisiae, using Bacillus subtilis alsS as the cytosolic acetolactate dehydrogenase [38, 39], combined with the expression of E. coli ilvC, L. lactis ilvD, kivD [38], and adhA. These genes were introduced into a yeast strain with a Δald6, Δtma29, Δgpd1, Δgpd2, Δpdc1/5/6 [40 - 42] genotype [6]. Altogether, isobutanol synthesis in S. cerevisiae is difficult and results in low titers [43].


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But recently some corporations began to file patents and claim to reach yields, which surpass the ones reached in E. coli.

FAEE FAEEs possess high energy density and exhibit low host toxicity [44]. Examples for the structure formulas are provided in (Fig. 1C and D). The synthesis of FAEEs is facilitated by condensation of acyl-CoA subunits and ethanol by wax ester synthases/acyl-CoA diacylglycerol acyltransferases. The application of this mechanism for the purpose of biofuel synthesis was shown by Li and colleagues as well as Runguphan and colleagues [45, 46]. The highest titers using this mechanism were reported to be 0.016 g/l [47]. Another approach to increase FAEE synthesis is the overexpression of acetyl-CoA carboxylase acc1. Acc1 is negatively regulated via phosphorylation by the protein kinase Snf1. The mutation of Acc1 abolishes phosphorylation and led to a higher yield of FAEEs. This increase was more pronounced than the change in an Δsnf1 S. cerevisiae strain, which might be due to the versatile function of Snf1 [47]. The obstruction of other acyl-CoA consuming pathways like β-oxidation or sterylesters/triacylglycerols biosynthesis is also a good strategy to increase FAEE production. The side-effect of this approach is a higher pool of free fatty acids, which might be used to synthesize other fatty acid-derived molecules [48]. One of the highest FAEE production in S. cerevisiae was reported to be 0.52 g/l, which was reached by using glycerol as carbon source, an upregulation of ethanol biosynthesis, a down-regulation of glycerol export, and exogenous fatty acids [7].

Sesquiterpenoids Sesquiterpenoids consist of three isoprenoid subunits (examples for structure formulas are given in Fig. 1A and B) and might also be interesting as biofuels: for instance hydrogenated farnesenes (e.g. farnesol), which could substitute jet fuel, or bisabolane, hydrogenated bisabolene, which can be used as diesel substitute. Precursors of isopronoids are isopentenyl diphosphate and dimethylallyl diphosphate, which are products of the mevalonate (MVA) or the 2-methylerythritol 4-phopsphate (MEP) pathways, respectively. The first step towards isoprenoid synthesis is to enlarge the pool of acetyl-CoA, the precursor of the MVA pathway. This can be achieved by up-regulating aldehyde dehydrogenase ald6, and heterologous expression of Salmonella entericaacs1 to prevent D-glucose repression of the pyruvate to acetyl-CoA path [49]. Up-regulation of endogenous alcohol dehydrogenase adh2 can improve this strategy further [50]. To prevent synthesis of farnesyl diphosphate (FPP)-derived steroid hormones, the repression of the squalene synthase erg9 leads to a higher production of FPP sesquiterpenoids [51, 52]. Overexpression of the HMG1-CoA reductase-encoding gene hmg1 increased the production of farnesol and other isoprenoids. A truncated version of the protein led to a feedback-insensitive enzyme with slightly increased productivity of farnesol [8]. The



knock-out of squalene synthase represents another step towards increased farnesol production [53]. There is an alternative way to synthesize farnesol in S. cerevisiae by dephosphorylation of FPP. This is a two-step process facilitated by alkaline phosphatase Pho8p and lipid phosphatases Dpp1p and Lpp1p [54]. Overexpression of a truncated Hmg1, the FPP synthase Erg20, and the global transcriptional regulator Upc2-1, as well as heterologous expression of the Abies grandyis bisabolene synthase, and simultaneous repression of erg9 led to one of the highest bisabolene titers of 0.994 ± 0.241 g/l on complex D-galactose/D-glucose medium [9]. Overexpression of all genes involved in the MVA pathway, plus three copies of truncated hmg1 resulted in high levels of amorphadiene, a precursor of an antimalarial drug. The yield reached 37 g/l in fed-batch culture using ethanol as carbon source [55]. These strategies might be used and combined to increase yields of sesquiterpenoids as alternative biofuels.

ENGINEERING STRATEGIES FOR CONSOLIDATED BIOPROCESSING Consolidated bioprocessing (CBP) is closely associated with second generation biofuels. It describes a strategy to produce ethanol from lignocellulose using only one host. This implies that this microorganism has the ability to degrade lignocellulose and synthesize ethanol by itself. The goal can be reached by enabling an ethanol producer to express lignocellulolytic enzymes or vice versa, equip lignocellulose-degrading microorganisms with the ability to produce ethanol. Mostly, CBP is mentioned in connection with S. cerevisiae and the introduction of lignocellulolytic machinery into this host. Lignocellulose consists of three components: cellulose, hemicellulose, and lignin. CBP focuses on the degradation and utilization of the first two polymers. Generally spoken, cellobiohydrolases cleave the polymer releasing cello-oligosaccharides from its reducing and non-reducing end whereas endoglucanases induce nicks in cellulose exposing new reducing and non-reducing ends. Finally, β-glucosidases cleave released oligomers including cellubiose to D-glucose. The degradation of hemicellulose is carried out by endo-1,4-β-xylanases, which cleave xylan resulting in xylooligosaccharides. These are further degraded by β-xylosidase resulting in D-xylose. So far, there are four different strategies to enable S. cerevisiae to degrade lignocellulose: (1) secretion of lignocellulose-degrading enzymes; (2) membrane anchoring of these lignocellulosedegrading enzymes, called cell surface display; (3) import of break-down products (e.g. cellodextrin) and subsequent intracellular utilization; (4) synthesis of cellulosomes with bacterial origin. The selection of adequate genes to reach the goal of CBP has to take into account all possible problems of heterologous protein expression: efficient protein expression and correct processing (correct folding and modification like glycosylation), the protein localization, and the final enzyme activity.


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Secretion of Lignocellulose-Degrading Enzymes Introduction of the Trichoderma reesei endoglucanaseEGLI and the Saccharomycopsis fibuligera β-glucosidaseBgl1 into S. cerevisiae resulted in a strain able to grow on phosphoric swollen cellulose (PASC) and to produce ethanol [56]. In another experiment a S. cerevisiae strain, which expressed and secreted EGLII of T. reesei and BGLI from Acremonium aculeatus, was able to produce ethanol from pre-treated corn stover cellulose. The final ethanol concentration reached 2.6% (v/v) after 96 hours [11]. This CBP strategy has to deal with two issues, on the one hand, the host has to have a certain secretory capacity to export the necessary amount of lignocellulose-degrading enzymes, and on the other hand, the host has to continually express proteins. This stress can result in impaired growth rates of the CBP yeast. Another aspect is the extracellular hydrolysis of lignocellulose resulting in the abundance of D-glucose. As mentioned before, Dglucose will influence the uptake and gene regulation of the host organism.

Cell Surface Display of Lignocellulose-Degrading Enzymes Protein fusion is an efficient way to anchor heterologous proteins in the cell membrane. Therefore, a fusion with α-agglutinin or flocculin Flo1p is sufficient to successfully display heterologously expressed proteins on the surface of yeast cells [57]. Engineered S. cerevisiae displayed cellobiohydrolase CBHII and endoglucanase EGLII from T. reesei, and β-glucosidaseBgl1 from Acremonium aculeatus on its cell surface. This strain was able to produce 2.9 g ethanol per litre from 10 g/l PASC [58]. Higher copy number of the genes only slightly increased the ethanol concentration to 3.1 g/l from 20 g/l PASC [10]. A S. cerevisiae strain, able to convert D-xylose to ethanol (vide supra page 7), was engineered to present xylanase XYNII from T. reesei, β-xylosidase XylA from Aspergillus oryzae, and Bgl1 from Acremonium cellulolyticus on its cell surface. This approach was successful, producing 8.2 g ethanol per litre from hemicellulosic hydrolysate from rice straw [59]. Cellular surface display has some important advantages in comparison to secretion of enzymes: (1) there is no need for constant protein synthesis due to secretion because enzymes are anchored in the membrane and can be recycled; (2) synergistic effects between different enzymes can be employed due to potential colocalization; (3) released D-glucose is transported immediately into the host and has less impact on enzyme repression or as substrate for contaminants.

Import of Oligosaccharides The CBP tries to transfer the release of D-glucose (from the oligomer) from the culture broth into the cytosol. Degradation products of cellulose are imported into yeast cells, where they are completely hydrolysed to D-glucose. Intracellular D-glucose does not interfere with pentose transporters, therefore, pentoses, which originate from extracellular hemicellulose degradation, can still be taken up and utilized. The advantages behind this



approach are similar to the cell surface display, there is no need for continuous protein synthesis and secretion, and the amount of D-glucose in the culture broth is kept at a minimum. Ha and colleagues heterologously expressed a cellodextrin transporter from N. crassa in S. cerevisiae. Hence, they were successful in establishing a cultivation process, which co-utilized cellobiose and D-xylose. The only limitation they encountered is that the transporter works as importer and exporter of cellodextrin decreasing the efficiency of oligosaccharide uptake and therefore, the ethanol productivity [60].

FILAMENTOUS FUNGI Fusarium Oxysporum F. oxysporum is able to reach 80% of the theoretical yield of ethanol from D-glucose under anaerobic conditions [12] and up to 8.2 g ethanol per litre. Using brewer’s spent grain (BG) as substrate, Xiros and colleagues were able to produce 109 g ethanol from 1 kg substrate, which correlates with 60% of the theoretical yield. They also found that the hydrolysis of BG is the bottleneck in this experiment [61]. The overexpression of an endoxylanase and the use of wheat bran as substrate increased the production of ethanol about 60% [13]. Introduction of alternative transaldolase genes from S. cerevisiae or Pichia stipites increased the ethanol production by 28% and 11%, respectively [62, 63]. An important influence on ethanol synthesis was identified by Ali and colleagues: a transporter for pentose and hexose sugars, termed hxt. The abundance of Hxt is highest when D-glucose concentration is around 10 mM. Silencing of hxt led to a decrease of the ethanol yield ranging from -15% to -40%, while biomass formation was not influenced. Overexpression of hxt enhances the D-glucose, D-galactose, and D-xylose uptake rate, the conversion rate, and the ethanol yield. The same group reported on an increase of the theoretical yield from 70.1% in the wild-type to 78.5% in the overexpression strain on alkali-treated straw cultivated for 96 hours under oxygen-limiting conditions. The same observation was made for D-glucose as carbon source: while the wild-type reached 71.8%, the overexpression reached over 80% of the theoretical yield after 144 hours. A comparison between wild-type and overexpression strain on untreated wheat straw was also performed during this study. The theoretical ethanol yield increased from 23.8% in the wild-type to 33.8% in the overexpression strain [64]. These findings are important since the uptake and metabolization of D-xylose is impaired in the presence of D-glucose [65]. Overexpression of homologous phosphoglucomutase and transaldolase led to ethanol concentrations of 20 g/l from D-glucose [66]. Partial removal of ethanol in a stepwise manner from the culture broth led to final ethanol concentrations of 38.4 g/l (Paschos et al., unpublished results, mentioned in [66]).

Neurospora Crassa N. crassa is able to produce up to 2% ethanol endogenously by aerobic fermentation of


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D-glucose [67]. 80 – 90% of the ethanol is produced through the Embden-Meyerhof pathway, the rest originates from the hexose monophosphate pathway [68]. The rate of D-glucose utilization exceeds the rate of the TCA cycle, therefore, ethanol fermentation is preferred over acetate formation to regenerate NAD+ [69]. N. crassa also has different preferences towards sugars concerning their utilization. D-mannose is fully converted to ethanol within two days, D-galactose conversion reaches 75% and takes up to six days [70]. D-xylose and L-arabinose are yielding 65% and 44% of theoretical ethanol yield, respectively [71]. This difference is peculiar because both sugars are utilized via the pentose phosphate pathway and its intermediate xylulose-5-phosphate, but this finding was not further investigated by the group. In comparative experiments N. crassa strains NCIM 870 and NCIM 1021 were grown aerobically for two days followed by eight days of non-aerated fermentation. The ethanol yields were 1.8% and 1.1% (v/v), respectively, on medium containing D-glucose [72]. Under oxygen-limited conditions Zhang and colleagues were able to obtain ethanol yields of 6.7 g/l or 66% of theoretical yield from D-xylose [71]. When alkali pre-treated BG was used as carbon source, N. crassa could produce 74 g ethanol per kg of dry BG (or 5.6 g/l) corresponding to 36% of theoretical yield under micro-aerobic conditions [14]. The use of dilute-acid pretreated sorghum bagasse (SoB) and the addition of 6 U of filter paperase per g of substrate for ethanol production by N. crassa yielded 345 g ethanol per kg (or 27.6 g/l) [15].

Trichoderma Reesei In the presence of D-glucose genes coding for enzymes involved in the TCA cycle are not or just moderately repressed in T. reesei. As a consequence, it is more likely that pyruvate is reduced by pyruvate dehydrogenase and introduced into the TCA cycle as acetyl-CoA than that it is decarboxylized by pyruvate decarboxylase and fermented to ethanol [69]. Another issue is the repression of genes of the glycolytic pathway under anaerobic conditions. In this case the metabolic flux is interrupted for ATP production [73]. T. reesei can produce ethanol from cellulose by initial aerobic growth followed by cultivation under anaerobic conditions. This might be the strategy of choice for ethanol production in T. reesei, because the genes encoding cellobiohydrolase CBHI, endoglucanase EGLI, β-glucosidase BGLII, and β-galactosidase BGA1 are downregulated in chemostat cultures under anaerobic conditions [74]. Nonetheless, productivity and yield are very low due to T. reesei’s sensitivity towards ethanol and anaerobic condition [75]. Huang and colleagues were able to create a T. reesei strain by genome shuffling and subsequent selection for ethanol production. This strain was able to produce 9.7 g ethanol per litre under aerobic conditions and 4.8 g ethanol per litre under anaerobic conditions from D-glucose. When unprocessed sugarcane bagasse was used as carbon source, the strain produced an ethanol concentration of 3.1 g/l after 120 hours [16].



OTHER POTENTIAL PRODUCTION ORGANISMS Aspergillus spp. Aspergillus terreus is able to ferment a series of hexoses and pentoses to ethanol, the highest yield of 2.46% (v/v) originates from D-glucose [76]. A. nidulans can produce ethanol under anaerobic conditions, under which gene expression of genes involved in glycolysis and TCA cycle genes are up-regulated, and the expression of genes of the KOG categories ‘transcription’, ‘replication, recombination, and repair’, and ‘intracellular trafficking, secretion, and vesicular transport’ are down-regulated [77]. One by-product of these fermentations is usually lactate. Some engineering approaches include the introduction of a pdc gene from Zymomonas mobilis into A. nidulans [17] or the knockout of velvet gene veA in A. parasiticus which led to three to four times higher levels of ethanol [18].

Rhizopus spp. It has been reported that Rhizopus oryzae is generally able to produce ethanol in the presence of high amounts of lactic acid, which is at the same time a by-product of ethanol production. In 1985, it was reported that Rhizopus koji reached 50% of the productivity of a yeast system on D-glucose [78]. Millati and colleagues reported that D-glucose and Dxylose are the best substrates for ethanol production by Rhizopus spp. [79]. Büyükkileci and colleagues found that the highest yield of ethanol (37.2 ± 3.1 g/l) and lowest byproduct formation was achieved, when the spore concentration of the inoculum of R. oryzae was low (10-6 spores/ml) [80]. As mentioned, a major drawback of using R. oryzae is lactic acid formation. By silencing the lactate dehydrogenase, the accumulation of lactic acid was decreased by 85% and the ethanol yield was increased by 15.4% [81]. In a simultaneous saccharification and fermentation experiment using Rhizophus sp. W-08 and yeast the ethanol yield reached 21% (v/v) [82].

Trametes spp. Trametes hirsute is able to produce ethanol from D-glucose, D-mannose, D-xylose, maltose, and cellobiose under anaerobic conditions. Furthermore, it can utilize starch, wheat bran, and rice straw without the need of pretreatment. The ethanol yields from these three complex carbon sources reach 9.1, 4.3, and 3.0 g/l, respectively [19]. Trametes versicolor displays similar properties as T. hirsute, being able to convert the same sugars and complex carbon sources without pretreatment to ethanol. The maximum concentrations exceed those of T. hirsute slightly [20].

Other Fusarium verticillioides is able to utilize mixtures of D-glucose and D-xylose, and can


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produce up to 4.6 g ethanol per litre from 40 g/l pretreated sugarcane bagasse. The same is true for Acremonium zeae, which is able to produce ethanol concentrations of 3.9 g/l from the same carbon source as mentioned above [21]. Hypersaline-tolerant, white-rot fungus Phlebia sp. MG-60 is able to utilize D-glucose, Dmannose, D-galactose, D-fructose, D-xylose, cellobiose, and maltose for ethanol synthesis [22]. The process was performed in semi-aerobic conditions and the fungus produced ethanol from unbleached hardwood kraft pulp without additional enzymes. The highest yields were 37.3 g ethanol per litre after 360 hours [23]. The basidiomycete Flammulina velutipes Fv-1 displays good ethanol conversion rates for D-glucose, D-mannose, D-fructose, sucrose, maltose, and cellobiose, but the ethanol yield from pentoses is very low. This fungus expresses β-glucosidase, cellbiohydrolase, and cellulase, but no endo-β-1,4-glucanase during ethanol synthesis. Therefore, the highest ethanol yields (0.36 g ethanol per g cellulose, 69.6% theoretical yield) were reached with the addition of 9 mg commercial cellulase per g cellulose, but this process took 22 days [24]. Additionally, F. velutipes Fv-1 produces antitumor polysaccharides and immunosuppressive proteins [83]. Paecilomyces variotii shows higher ethanol yields in presence of D-xylose than Dglucose. The concentration of ethanol reaches 13.9 g/l and 91% of theoretical yield from D-xylose. A mixture of those two sugars led to an ethanol concentration of 10.5 g/l resulting from the consumption of both sugars [25].

CONCLUDING REMARKS S. cerevisiae is an important organism for ethanol production. Its potential for the production of other biofuels like isobutanol or sesquiterpenoids is still a matter of research. Due to the increased importance of second generation biofuels the establishment of yeast as a CBP organism is in progress. This is demonstrated by the range of different approaches that are examined by many research groups around the world. But the achievable yields are too small to be industrially applied at the moment. All three presented CBP strategies (cell surface display, enzyme secretion, and import of oligosaccharides) have their prospects and advantages. It is difficult to predict, which strategy is going to prevail. On the other hand, the potential of filamentous fungi as ethanol producers is limited because they are mostly obligate aerobe organisms, enzymes crucial for glycolysis are repressed in the absence of oxygen, and many species display only low ethanol tolerance [73]. Even their prospects as CBP organisms are questionable due to the aforementioned reasons. It might be more difficult to introduce ethanol tolerance into a host than the ability to degrade lignocellulose into an already well established ethanol producer because the number of genes involved in tolerance towards ethanol is much higher and



still a matter of research.

CONFLICT OF INTEREST None Declare.

DISCLAIMER This published chapter may be subject to further revision/updates until the complete eBook is finally published.

ACKNOWLEDGEMENTS This work was supported by two grants from the Austrian Science Fund (FWF): P24851 and V232 given to ARMA, as well as by the TOP-Anschubfinanzierung (“ABC”) of Vienna University of Technology.

ABBREVIATIONS BG

= Brewer’s spent grain

CBP

= Consolidated bioprocessing

FAEE = Fatty acid ethyl ester FPP

= Farnesyl diphosphate

MEP

= Methylerythritol 4-phosphate

MVA

= Mevalonate

PASC = Phosphoric acid swollen cellulose SoB

= Sorghum bagasse

GENE AND PROTEIN GLOSSARY acc1

= Gene coding for acetyl-CoA carboxylase from S. cerevisiae

acs1

= Gene coding for from S. enterica

adh, adh2, adh6; adhA

= Alcohol dehydrogenases from S. cerevisiae and L. lactis

ald6

= Gene coding for aldehyde dehydrogenase from S. cerevisiae

alsS

= Gene coding for cytosolic acetolactate dehydrogenase from B. subtilis

bga1

= Gene coding for β-galactosidase from T. reesei

bgl1; bgl2

= Genes coding for β-glucosidases from S. fibuligera and T. reesei, respectively

BGLI, Bgl1

= β-glucosidase from A. aculeatus (BGLI) and A. cellulolyticus (Bgl1) respectively

16 Mycology: Current and Future Developments, Vol. 1 cbh1

Kiesenhofer et al.

= Gene coding for cellobiohydrolase I (CBHI) from T. reesei

Dpp1p, = Lipid phosphatase from S. cerevisiae Lpp1p egl1, egl2

= Genes coding for endoglucanase I (EGLI) and endoglucanase II (EGLII) from T. reesei

Erg20

= FPP synthase from S. cerevisiae

erg9

= Gene coding for squalene synthase from S. cerevisiae

gpd1, gpd2

= Gene coding for cytosolic NADH-dependent glycerol-3-phosphate dehydrogenases from S. cerevisiae

Hmg1

= HMG1-CoA reductase from S. cerevisiae

hxt

= Gene coding for a sugar transporter from F. oxysporum

ilv2

= Gene coding for acetaloacetate synthase from S. cerevisiae

ilv3; ilvD

= Gene coding for dihydroxyacid dehydrogenase from S. cerevisiae and L. lactis, respectively

ilv5; ilvC

= Gene coding for ketoacid reductoisomerase from S. cerevisiae and E. coli, respectively

kivD

= Gene coding for ketoisovalerate decarboxylase from S. cerevisiae and L. lactis

pdc1

= Gene coding for pyruvate decarboxylase from S. cerevisiae

Pho8p

= Alkaline phosphatase from S. cerevisiae

snf1

= Gene coding for protein kinase from S. cerevisiae

tps1, tps2

= Genes coding for trehalose synthases from S. cerevisiae Upc2-1- transcriptional regulator in S. cerevisiae

veA

= Velvet gene from A. parasiticus

XylA

= β-xylosidase from A. oryzae

XYNII

= Xylanase II from T. reesei

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