**3. Key aspects of the engineered ethanol cassette in** *Synechocystis*

To metabolically engineer *Synechocystis* as a cell factory a 'cassette' of genes and sequences are needed. A key ingredient of a functional ethanol cassette, suitable for expression in *Synechocystis*, is the functional expression of a pyruvate decarboxylase gene encoding the enzyme pyruvate decarboxylase (PDC). The PDC produced converts the metabolic intermediate pyruvate to acetaldehyde, which is in turn converted to ethanol by engineered alcohol dehydrogenase (ADH) or by the native *Synechocystis* ADH.

and perhaps also on other host enzymes that use ThDP as a co-factor. Thus this may affect competitiveness of engineered strains and in the long term, engineering a thiamine trans-

Metabolic Engineering of the Model Photoautotrophic Cyanobacterium *Synechocystis*…

http://dx.doi.org/10.5772/intechopen.77271

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While there is a potential choice of PDCs to use, in practice most work so far has been carried out on the *Zymomonas mobilis* PDC. This enzyme is a homo tetramer of 240 kDa [29] and has an optimum pH of 6.0 [30]. Given the pH optimum for growth of *Synechocystis* is ~pH 8, full enzymatic function or co-factor binding [31] may be somewhat compromised by the pH difference between the enzyme optimum and the host pH optimum which may suggest looking

In most reports on engineered ethanol cassettes the source of ADH has been *Zymomonas mobilis*. Two ADH isozymes are known to be present within the genome of *Zymomonas mobilis* - ADH I and ADH II (EC 1.1.1.1) [32]. For metabolic engineering of ethanol production the Fe2+ containing ADHII encoded by the *adh*B gene of *Zymomonas* has been utilized [5]. This enzyme has a pH optimum of pH 8.5 (as opposed to pH 6.5 for ADHI) and a cofactor requirement for Nicotinamide adenine dinucleotide (NADH) for the reduction of acetaldehyde to ethanol [33]. Unusually *Synechocystis* also encodes its own native ADH gene via the *adh*A gene (slr1192). This is a medium chain alcohol dehydrogenase, which catalyzes the reversible oxidation of alcohols to aldehydes or ketones [34]. The *Synechocystis* ADH encodes a 140 kDa zinc dependent enzyme with broad alcohol dehydrogenase activity and which interestingly is Nicotinamide adenine dinucleotide phosphate (NADPH) dependent as opposed to the *Zymomonas* activity, which is NADH dependent [34]. Indeed *Synechocystis* has been reported to possess multiple *adh* genes but does not contain an native *pdc* gene [35] suggesting that the native ADH may play an as yet unknown function in the cyanobacterium. Indeed the *Synechocystis adh* gene has been substituted for the *Zymomonas* gene [9] and functions very well. Recently we have reported [36] metabolic engineered cassettes with a copy of the *Zymomonas mobilis adh* gene and the native *Synechocystis adh* gene with increased ethanol producing activity. This may occur because the two activities rely on different co-factors NADH (*Zymomonas*) and NADP (*Synechocystis*) which

porter may be needed in *Synechocystis* production strains to overcome such issues.

**4.** *Zymomonas mobilis* **and** *Synechocystis* **alcohol dehydrogenase** 

may spread the co-factor requirement and availability within the cell [36].

**5. Construction of functional ethanol cassettes in** *Synechocystis*

In general, terms the construction of an ethanol cassette follows the basic components as reported [4, 5]. The *Zymomonas mobilis pdc* gene is amplified and fused with the *Zymomonas mobilis adh*B gene under the control of an inducible promoter. The light inducible P*psbA2* promoter is often utilized but other promoters have also been evaluated [37]. There is then the need for a strong selection of the cassette encoded generally by an antibiotic resistance determinant such as kanamycin or zeocin [36]. Homology sequences are needed at both ends of the

at other potential PDC candidates.

**(ADH)**

Pyruvate decarboxylase (PDC, EC 4.1.1.1) carries out the decarboxylation of pyruvate to acetaldehyde in alcohol fermentations and requires thiamine diphosphate/pyrophosphate (ThDP) and the divalent cation Mg2+ as cofactors. Several other enzymes in various metabolic pathways also require these cofactors to function and it is believed that each of them use a similar mechanism of action. PDC can be found in fungi, plants and yeast and is not present in humans [21]. PDC genes have been observed and characterized from only a small number of bacterial species as it appears to be rather rare amongst prokaryotes. These include *Zymomonas mobilis*, [22], *Zymobacter palmae* [23], *Acetobacter pasteurianus* [24], *Gluconacetobacter diazotrophicus* [25], *Thermococcus quaymacensis* [26], *Geobacillus thermoglucosidasius* [27] and *Sarcina ventriculi* [28]. Although the *Zymomonas mobilis* PDC is the most extensively utilized in ethanol production there is much potential to utilize some of the other bacterial PDC's on the basis of pH optimum or lower Km (see **Table 2**). With model organisms, such as *Synechocystis,* using a PDC with a lower Km may increase the flux from pyruvate and couple the product acetaldehyde better with ADH resulting in higher ethanol yields. There is thus some scope for improvement of the ethanol cassette given that some of the newly characterized PDCs have better kinetics than the original *Zymomonas* PDC. All known PDC's have specific co-factor requirements and co-factor availability is an issue when expressing engineered cassettes. While there may be little problem with Mg2+ supply, the availability of ThDP will be limited as the host organism must synthesis it (as *Synechocystis* does not possess a thiamine transporter) [18]. Equally ThDP will be required for other cellular metabolic reactions and its availability will be squeezed by added engineered PDC. Hence if metabolic engineering were to result in high level expression of heterologous PDC, the limited availability of ThDP would pose limitations on its function


**Table 2.** Properties of known bacterial PDCs [23, 25, 27].

and perhaps also on other host enzymes that use ThDP as a co-factor. Thus this may affect competitiveness of engineered strains and in the long term, engineering a thiamine transporter may be needed in *Synechocystis* production strains to overcome such issues.

enzyme pyruvate decarboxylase (PDC). The PDC produced converts the metabolic intermediate pyruvate to acetaldehyde, which is in turn converted to ethanol by engineered alcohol

Pyruvate decarboxylase (PDC, EC 4.1.1.1) carries out the decarboxylation of pyruvate to acetaldehyde in alcohol fermentations and requires thiamine diphosphate/pyrophosphate (ThDP) and the divalent cation Mg2+ as cofactors. Several other enzymes in various metabolic pathways also require these cofactors to function and it is believed that each of them use a similar mechanism of action. PDC can be found in fungi, plants and yeast and is not present in humans [21]. PDC genes have been observed and characterized from only a small number of bacterial species as it appears to be rather rare amongst prokaryotes. These include *Zymomonas mobilis*, [22], *Zymobacter palmae* [23], *Acetobacter pasteurianus* [24], *Gluconacetobacter diazotrophicus* [25], *Thermococcus quaymacensis* [26], *Geobacillus thermoglucosidasius* [27] and *Sarcina ventriculi* [28]. Although the *Zymomonas mobilis* PDC is the most extensively utilized in ethanol production there is much potential to utilize some of the other bacterial PDC's on the basis of pH optimum or lower Km (see **Table 2**). With model organisms, such as *Synechocystis,* using a PDC with a lower Km may increase the flux from pyruvate and couple the product acetaldehyde better with ADH resulting in higher ethanol yields. There is thus some scope for improvement of the ethanol cassette given that some of the newly characterized PDCs have better kinetics than the original *Zymomonas* PDC. All known PDC's have specific co-factor requirements and co-factor availability is an issue when expressing engineered cassettes. While there may be little problem with Mg2+ supply, the availability of ThDP will be limited as the host organism must synthesis it (as *Synechocystis* does not possess a thiamine transporter) [18]. Equally ThDP will be required for other cellular metabolic reactions and its availability will be squeezed by added engineered PDC. Hence if metabolic engineering were to result in high level expression of heterologous PDC, the limited availability of ThDP would pose limitations on its function

**Bacterial host and enzyme kM (mM) pyruvate Optimum pH Optimum temperature (°C)**

5.0–5.5 45–50

7.0 55

3.5–6.5 65

6.0–6.5 60

6.3–6.7 N/A

4.5–5.0 53

0.6 pH 6.0 1.2 pH 7.0

0.71 pH 7.0

5.1 pH 7.0

0.94 pH 7.0

4.0 pH 7.0

2.8 pH 7.0

*Gluconacetobacter diazotrophicus* 0.06 pH 5.0

*Zymobacter palmae* 0.24 pH 6.0

*Acetobacter pasteurianus* 0.39 pH 5.0

*Zymomonas mobilis* 0.43 pH 6.0

*Sarcina ventriculi* 5.7 pH 6.5

*Gluconobacter oxydans* 0.12 pH 5.0

**Table 2.** Properties of known bacterial PDCs [23, 25, 27].

dehydrogenase (ADH) or by the native *Synechocystis* ADH.

202 Fuel Ethanol Production from Sugarcane

While there is a potential choice of PDCs to use, in practice most work so far has been carried out on the *Zymomonas mobilis* PDC. This enzyme is a homo tetramer of 240 kDa [29] and has an optimum pH of 6.0 [30]. Given the pH optimum for growth of *Synechocystis* is ~pH 8, full enzymatic function or co-factor binding [31] may be somewhat compromised by the pH difference between the enzyme optimum and the host pH optimum which may suggest looking at other potential PDC candidates.
