**6. Evolutionary adaptation and genetic engineering of thermophiles**

The thermophilic anaerobes mentioned in the previous sections make logical targets for genetic improvement due to their ability to produce ethanol from a wide range of substrates as evidenced by acceptable yields on lignocellulosic biomass. There are two general strategies for enhancing characteristics for ethanol production by wild type microorganisms: evolutionary adaptation or genetically modify the organisms. Early work often used classical methods such as the selection of clones and nonspecific mutagenesis to improve ethanol production [70]. These methods are time-consuming, and genetic modification is not without drawbacks as modified strains can exhibit poor growth and unexpected shifts in end-product formation. More recent work has focused more on modern techniques in molecular biology discussed herein.

#### **6.1. Evolutionary adaptation**

One of the major drawbacks of using thermophiles for the production of ethanol is their low substrate and ethanol tolerance. The use of classical evolutionary adaptation methods, such as non-specific mutagenesis and artificial selection, to enhance specific traits of microorganisms for industrial bioethanol production have been applied to thermophilic anaerobes on a limited basis. Examples of adaptation methods on three new mutant strains of *Thermoanaerobacter ethanolicus* were obtained by selection of pyruvate and iron deprivation [51] leading to enhanced ethanol tolerance (10% v/v) at substrate concentrations above 10 g/L. *Clostridium thermocellum* showed increased ethanol tolerance (up to 5% v/v) by stepwise increasing and transferring cultures to increased ethanol concentrations [71]. *Thermoanaerobacter pentosaceus* has been gradually adapted to higher substrate concentrations and demonstrated higher ethanol tolerance and substrate utilization [72]. Thus, evolutionary adaptation, may still be used for evolving of wild type strains and further improving GM strains to meet requirements for tolerance to high ethanol titers, improve substrate utilization, and potentially resistance to inhibitory compounds generated during biomass pretreatment such as 5-HMF and fufuraldehyde.

#### **6.2. Genetic engineering**

Early experiments on ethanol production from lignocellulose included as the ethanol-producing organisms *Thermoanaerobacter ethanolicus* and *Clostridium thermocellum* with hemicellulose from birch- and beechwood as a feedstock [66]. *Clostridium thermocellum* produced between 7.2 mM ethanol /g and 8.0 mM/g from avicel and Whatman paper, respectively. Studies of ethanol production from paddy straw, sorghum stover and corn stubs, pretreated with alkali showed similar results [68]. However, these results were obtained with relatively low substrate loadings (8.0 g/L) but later studies showed that increased substrate loadings lowered the ethanol yields considerable [69]. The highest ethanol yields reported from lignocellulose are by *Thermoanaerobacter* BG1L1 grown on corn stover and wheat straw [42, 43] that were pretreated with acid or wet oxidation, or 9.2 mM/g for biomass hydrolysates. *Thermoanaerobacterium* strain AK17 showed ethanol yields of 2.0 (paper) mM/g, 2.9 (grass) mM/g and 5.8 (cellulose) mM/g biomass [23]. Optimization experiments showed an increase in ethanol yields on grass and cellulose up to 4.0 and 8.6 mM·g−1, respectively. The main culture factors increasing ethanol yields was obtained by lowering of the substrate concentration from 7.5 to 2.5 g/L [39]. Recent investigations on two *Thermoanaerobacter* strains, AK5 and J1, showed promising results from various types of hydrolysates made from chemically and

enzymatically pretreated lignocellulosic biomass [17, 38] (**Table 1**).

herein.

**6.1. Evolutionary adaptation**

108 Fuel Ethanol Production from Sugarcane

**6. Evolutionary adaptation and genetic engineering of thermophiles**

The thermophilic anaerobes mentioned in the previous sections make logical targets for genetic improvement due to their ability to produce ethanol from a wide range of substrates as evidenced by acceptable yields on lignocellulosic biomass. There are two general strategies for enhancing characteristics for ethanol production by wild type microorganisms: evolutionary adaptation or genetically modify the organisms. Early work often used classical methods such as the selection of clones and nonspecific mutagenesis to improve ethanol production [70]. These methods are time-consuming, and genetic modification is not without drawbacks as modified strains can exhibit poor growth and unexpected shifts in end-product formation. More recent work has focused more on modern techniques in molecular biology discussed

One of the major drawbacks of using thermophiles for the production of ethanol is their low substrate and ethanol tolerance. The use of classical evolutionary adaptation methods, such as non-specific mutagenesis and artificial selection, to enhance specific traits of microorganisms for industrial bioethanol production have been applied to thermophilic anaerobes on a limited basis. Examples of adaptation methods on three new mutant strains of *Thermoanaerobacter ethanolicus* were obtained by selection of pyruvate and iron deprivation [51] leading to enhanced ethanol tolerance (10% v/v) at substrate concentrations above 10 g/L. *Clostridium thermocellum* showed increased ethanol tolerance (up to 5% v/v) by stepwise increasing and transferring cultures to increased ethanol concentrations [71]. *Thermoanaerobacter pentosaceus* has been gradually adapted Despite other promising features, one of the main drawback of most wild type thermophiles is their production of mixed end-products resulting in lower ethanol yields and the fact that highly ethanologenic organisms are not natively cellulolytic and *vice versa*. Two main strategies have been used to metabolically engineer thermophilic organisms for consolidated bioprocessing (CBP). The first strategy is to increase the ethanol yields of cellulase-producing organisms while the other is to express enzymes for biomass deconstruction in highly ethanologenic microorganisms [73, 74]. The first approach involves increasing ethanol yields by redirecting the flow of carbon and electrons which involves eliminating other potential fermentation products. Obvious targets include knocking out acetate and lactate pathways. The second approach involves addition of cellulolytic genes to the genome of a good ethanol producing bacterium.

The first thermophilic bacterium to be genetically modified to increase ethanol yields was *Thermoanaerobacterium saccharolyticum* in 2004 [75]. Since then, several other ethanologenic thermophiles have been genetically modified to increase ethanol titers and minimize the formation of other end-products (**Table 2**).

Deletion of genes involved to the production of various end-products to increase ethanol production capacity is the most obvious way to increase ethanol titers. This has been done by knocking out lactate dehydrogenase in *Thermoanaerobacterium saccharolyticum* [73, 82], *Thermoanaerobacter mathranii* [79], *Clostridium thermocellum* [83] and *Geobacillus thermoglucosidasius* [78].

Wild type *Clostridium thermocellum* produces a mixture of ethanol, acetate, lactate, hydrogen, and carbon dioxide [84] from cellulose and other substrates. The first successful transformation of the species was performed in 2006 [85], later on leading to the development of genetic systems to knock out the *pta* gene and thus acetate formation [85]. However, growth of the resultant strain was abnormal although cellulase active remained intact. Later work on *C. thermocellum* showed improved ethanol yields in an adapted strain (Δ*hpt*, Δ*ldh*, Δ*pta*) lacking acetate and lactate pathways and was successfully used in co-culture with *Thermoanaerobacterium saccharolyticum* [85].

Early work on *Thermoanaerobacterium saccharolyticum* were performed by using electroporation and shuttle vectors [86], but later on this strain has been further modified by inserting a cellobiohydrolase gene from *Clostridium thermocellum* into its genome [77]. Also a *ldh* gene knock out was done using *Thermoanaerobacterium saccharolyticum* [75] and then a double knock out of both *ldh* and *ak* [73]. The knocking out of acetate production led to less available energy,


thus less cell biomass and increased ethanol yields, both from glucose and xylose. Another double knock out of *Thermoanaerobacterium saccharolyticum* focused on the electron transfer system of the bacterium [74]. The *hfs* gene cluster, which codes for hydrogenase, and the *ldh* gene were knocked out resulting in a considerable increase in ethanol (44%) production as

*Thermoanaerobacter mathranii* has been modified and used in several investigations. The first mutant generated was BG1L1 by knocking out *ldh* resulting in a more than two-fold increase in ethanol production as compared with the wild type [87]. This strain showed good ethanol yields from undetoxified pretreated corn stover and wheat straw [42, 43]. Further manipulation of this strain involves overexpression of NAD(P)H-dependent alcohol dehydrogenase, resulting in the strain BG1E1. Clearly, this enzyme is of great importance for ethanol production and its overexpression resulted in higher ethanol yields [79]. The electron balance for sugar degradation was additionally focused upon with this strain when mannitol, which is more reduced than glucose and xylose, was used as a substrate [87] and this resulted in higher ethanol yields. The BG1G1 strain of *Thermoanaerobacter mathranii* was developed which

yields by 40% greater than the type strain. Additionally, the strain utilized the highly reduced

Recently, the highly ethanologenic strain *Thermoanaerobacter* BG1 "Pentocrobe 411" was genetically engineered by knocking out lactate dehydrogenase, phosphotransacetylase, and acetate kinase [80]. Pentocrobe 411 achieved very high ethanol titers (1.84 to 1.92 mol ethanol/ mol hexose equivalent) nearing the maximum theoretical yield from hexoses and pentoses on

Thermophilic bacteria within the genus of *Geobacillus* have also attracted increased interest due to their ethanol production capacity. *Geobacillus* strains are facultative anaerobes and can ferment various sugars to pyruvate by pyruvate dehydrogenase to acetyl-Coenzyme A) [78]. Under aerobic conditions, however, pyruvate formate lyase is used and a variety of end-products are formed. A research group led by Cripps manipulated *Geobacillus thermoglucosidasius*, producing variant with upregulated pyruvate dehydrogenase expression under anaerobic conditions in a strain lacking lactate dehydrogenase activity [78]. Several mutants were developed (TM89; *ldh* knockout; TM180; *ldh* knockout and upregulated *pdh*; TM242; *ldh*, upregulated *pdh* and *pfl*). The TM180 strain produced 1.45 mol ethanol/mol hexose (the wild type produced 0.39 mol ethanol/mol hexose and TM89 produced 0.94 mol ethanol/mol hexose). The triple mutant TM242 produced 1.65 mol ethanol/mol hexose. This mutant also showed good yields on xylose (1.33 mol ethanol/mol xylose) and good productivity rates. *Geobacillus thermoglucosidasius* has recently been genetically modified by expressing pyruvate decarboxylase from *Gluconobacter oxydans* [88]. Ethanol yields obtained were as high as 1.37 mol ethanol/

A natural target for the strategy of converting a cellulolytic organism into a good ethanol producer would be members of the genus of *Caldicellulosiruptor* which has several cellulolytic members although none are good ethanol producers. Recent work with *Caldicellulosiruptor bescii*, a naturally cellulolytic organism, has produced ethanol producing strains [89–93].


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compared with the wild type.

included the insertion of a NAD<sup>+</sup>

mol glucose.

glycerol and co-metabolism of glycerol and sugars.

various pretreated biomass in continuous culture.

*ack*—acetate kinase; *GldA*—glycerol dehydrogenase A; *hfs*—hydrogenase; *hpt*—hypoxanthine phosphoribosyl transferase; *pdh*—pyruvate decarboxylase; *pyrF*—orotidine-5-phoshate decarboxylase; *pfl*—pyruvate formate lyase; and *ure*—urease.

**Table 2.** Ethanol yields of genetically engineered thermophilic bacteria from different substrates and fermentation conditions.

thus less cell biomass and increased ethanol yields, both from glucose and xylose. Another double knock out of *Thermoanaerobacterium saccharolyticum* focused on the electron transfer system of the bacterium [74]. The *hfs* gene cluster, which codes for hydrogenase, and the *ldh* gene were knocked out resulting in a considerable increase in ethanol (44%) production as compared with the wild type.

**Strain Genotype Substrate Mode Ethanol yields** 

*T. saccharolyticum* TD1 Δ*ldh* Xylolse (5.0 g/L) Batch 0.98 [77] *T. saccharolyticum* ALK2 Δ*pta*, Δ*ack*, Δ*ldh* Cellobiose (70.0 g/L) Con ND [73] *T. saccharolyticium* HK07 Δ*ldh* , Δ*hfs* Cellobiose (1.8 g/L) Batch 0.86 [74] *T. saccharolyticium* M0355 Δ*ldh* , Δ*ack*, Δ*pta* Cellobiose (50.0 g/L) Batch 1.73 [74] *T. saccharolyticum* M1051 Δ*ldh* , Δ*ack* Δ*pta*, *ure* Cellobiose (27.5 g/L) Batch 1.73 [74]

Δ*pta*::gapDHp-cat

Δ*pta*::gapDHp-cat

(evolved)

*T. mathranii* BG1L1 Δ*ldh* Wheat straw

*T. mathranii* BG1G1 Δ*ldh*, *GldA* Glucose + glycerol

*T. mathranii* BG1G1 Δ*ldh*, *GldA* Xylose + glycerol

*T. mathranii* BG1G1 Δ*ldh*, *GldA* Xylose + glycerol

Δ*hpt*, Δ*ldh*, Δ*pta* (evolved) and Δ*pta*, Δ*ack*, Δ*ldh*

*C. thermocellum* Δ*pyrF*,

110 Fuel Ethanol Production from Sugarcane

*C. thermocellum* Δ*pyr*F,

*C. thermocellum* Δ*hpt*, Δ*ldh*, Δ*pta*

*C. thermocellum* adhE\*(EA)

*C. thermocellum*/*T. saccharolyticum*

*G. thermoglucosidasius*

*G. thermoglucosidasius*

*G. thermoglucosidasius*

*Thermoanarobacter* Pentocrobe 411

*ure*—urease.

conditions.

TM242

TM242

TM242

Δldh

**(mol/mol)**

Con 1.53–1.67 [65]

Batch 1.68 [79]

Batch 1.57 [79]

Con 1.53 [79]

Cellobiose (5.0 g/L) Batch 0.59 [76]

Avicel (5.0 g/L) Batch 0.71 [76]

Avicel (19.5 g/L) Batch 1.08 [77]

Avicel (19.5 g/L) Batch 1.26 [77]

Δ*hpt*, Δ*ldh* Cellobiose (5.0 g/L) Batch 0.37 [77]

Δ*ldh*-, *pdh* up, *pfl*B- Glucose (34.0 g/L) Batch 1.73 [78]

Δ*ldh*-, *pdh* up, Δ*pflB*- Glucose (34.0 g/L) Batch 1.84 [78]

Δ*ldh*-, Δ*pdh* up, Δ*pflB*- Xylose (29.0 g/L) Batch 1.37 [78]

Δ*ldh*, Δ*ack*, Δ*pta* Wheat straw (65 g/L) Con 1.84 [80]

(30-120 g/L)

(5.0 g/L)

(5.0 g/L)

*C. bescii* JWCB018 Δ*ldh*- Celo (10 g/L) Batch 0 [81] *C. bescii* JWCB032 Δ*ldh*-, *adhE*+ Celo (10 g/L) Batch 0.66 [81] *C. bescii* JWCB049 Δ*pyrFA*, Δ*ldh*- Celo (10 g/L) Batch 0.54 [81] *C. bescii* JWCB054 Δ*pyrFA*, Δ*ldh*- Celo (10 g/L) Batch 0.28 [81]

(12.8 and 7.2 g/L)

*ack*—acetate kinase; *GldA*—glycerol dehydrogenase A; *hfs*—hydrogenase; *hpt*—hypoxanthine phosphoribosyl transferase; *pdh*—pyruvate decarboxylase; *pyrF*—orotidine-5-phoshate decarboxylase; *pfl*—pyruvate formate lyase; and

**Table 2.** Ethanol yields of genetically engineered thermophilic bacteria from different substrates and fermentation

**References**

*Thermoanaerobacter mathranii* has been modified and used in several investigations. The first mutant generated was BG1L1 by knocking out *ldh* resulting in a more than two-fold increase in ethanol production as compared with the wild type [87]. This strain showed good ethanol yields from undetoxified pretreated corn stover and wheat straw [42, 43]. Further manipulation of this strain involves overexpression of NAD(P)H-dependent alcohol dehydrogenase, resulting in the strain BG1E1. Clearly, this enzyme is of great importance for ethanol production and its overexpression resulted in higher ethanol yields [79]. The electron balance for sugar degradation was additionally focused upon with this strain when mannitol, which is more reduced than glucose and xylose, was used as a substrate [87] and this resulted in higher ethanol yields. The BG1G1 strain of *Thermoanaerobacter mathranii* was developed which included the insertion of a NAD<sup>+</sup> -dependent glycerol dehydrogenase which increased ethanol yields by 40% greater than the type strain. Additionally, the strain utilized the highly reduced glycerol and co-metabolism of glycerol and sugars.

Recently, the highly ethanologenic strain *Thermoanaerobacter* BG1 "Pentocrobe 411" was genetically engineered by knocking out lactate dehydrogenase, phosphotransacetylase, and acetate kinase [80]. Pentocrobe 411 achieved very high ethanol titers (1.84 to 1.92 mol ethanol/ mol hexose equivalent) nearing the maximum theoretical yield from hexoses and pentoses on various pretreated biomass in continuous culture.

Thermophilic bacteria within the genus of *Geobacillus* have also attracted increased interest due to their ethanol production capacity. *Geobacillus* strains are facultative anaerobes and can ferment various sugars to pyruvate by pyruvate dehydrogenase to acetyl-Coenzyme A) [78]. Under aerobic conditions, however, pyruvate formate lyase is used and a variety of end-products are formed. A research group led by Cripps manipulated *Geobacillus thermoglucosidasius*, producing variant with upregulated pyruvate dehydrogenase expression under anaerobic conditions in a strain lacking lactate dehydrogenase activity [78]. Several mutants were developed (TM89; *ldh* knockout; TM180; *ldh* knockout and upregulated *pdh*; TM242; *ldh*, upregulated *pdh* and *pfl*). The TM180 strain produced 1.45 mol ethanol/mol hexose (the wild type produced 0.39 mol ethanol/mol hexose and TM89 produced 0.94 mol ethanol/mol hexose). The triple mutant TM242 produced 1.65 mol ethanol/mol hexose. This mutant also showed good yields on xylose (1.33 mol ethanol/mol xylose) and good productivity rates. *Geobacillus thermoglucosidasius* has recently been genetically modified by expressing pyruvate decarboxylase from *Gluconobacter oxydans* [88]. Ethanol yields obtained were as high as 1.37 mol ethanol/ mol glucose.

A natural target for the strategy of converting a cellulolytic organism into a good ethanol producer would be members of the genus of *Caldicellulosiruptor* which has several cellulolytic members although none are good ethanol producers. Recent work with *Caldicellulosiruptor bescii*, a naturally cellulolytic organism, has produced ethanol producing strains [89–93]. The type strains of *C. bescii* typically yield a mixture of lactic and acetic acid in addition to hydrogen and CO2 as end-products although other strains within the genus of *Caldicellulosiruptor* have been noted to produce low ethanol titers. Work by Cha [89] deleted the gene coding for lactate dehydrogenase by introducing a non-replicating plasmid via marker replacement. The resultant knockout strain did demonstrate increased biomass yield as well as acetate and hydrogen production with no lactate production when grown on cellobiose and lactose as well as switch grass hydrolysates. Subsequent work by Chung [81] inserted a NADHdependent *adhE* gene (from *Clostridium thermocellum*) into the *ldh* mutant (JWCB018) resulting in strain *C. bescii* JWCB032. The resultant *ldh*<sup>−</sup> *adhE*<sup>+</sup> strain yielded less acetate (4.3 mM) but produced 14.8 mM of ethanol from 29.2 mM cellobiose or 12.7% of the theoretical yield. It should be noted that this strain only used a small portion (4.4 mM of 29.2 mM cellobiose) provided and not produce ethanol above 65°C. Work by Cha [89] and Chung [93] introduced the alcohol dehydrogenase genes (*adhB* and *adhE*) from *Thermoanaerobacter pseudoethanolicus* into the *ldh* deficient strain. The two resultant strains yielded ethanol at temperatures greater than 65°C although titers were lower than the aforementioned strain JWCB032 (*ldh*<sup>−</sup> *adhE+* ). The *C. thermocellum* strain with *adhB* only produced 1.4 mM ethanol on avicel and 0.4 mM on switch grass while a strain with *adhE* gave 2.3 and 1.6 mM of ethanol on avicel and switch grass, respectively. One of the reasons for suggested for the low ethanol titers is the availability of cofactors and it should be noted that *T. pseudoethanolicus* ADHs utilize NADPH while the gene products from *C. thermocellum* use NADH as a source of reducing potential. Additional work is therefore needed to more carefully mimic the complex NAD(P)H system of multiple ADHs in *Thermoanaerobacter pseduoethanolicus*.

**Author details**

**References**

Sean Michael Scully and Johann Orlygsson\*

Biotechnology. 2009;**27**:398-405

2006;**17**:320-326

thermophilic bacteria. Energies. 2015;**8**:1-30

Molecular Biology Reviews. 2005;**69**:124-154

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review. Bioresource Technology. 2010;**101**:4980-4991

Microbiology and Molecular Biology Reviews. 2000;**64**:34-50

stable enzymes in biorefining. Microbial Cell Factories. 2007;**6**:9

\*Address all correspondence to: jorlygs@unak.is

Faculty of Natural Resource Sciences, University of Akureyri, Akureyri, Iceland

different feedstocks. Bioresource Technology. 2008;**99**:5270-5295

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Overall, efforts to engineer thermophilic anaerobes to increase ethanol titers has resulted in modest gains in yields while minimizing or eliminating the formation of unwanted end products. Future targets for genetic manipulation might include the inclusion of the cellulolytic machinery of *C. thermocellum* into highly ethanologenic *Thermoanaerobacter* and *Thermoanaerobacterium* strains.
