2.2.1 Selecting Appropriate Designer Enzymes

One of the key features in the present invention is the creation of a designer ethanol — production pathway to tame and work with the natural photosynthetic mechanisms to achieve the desirable synthesis of ethanol directly from CO2 and H2O. The natural photosynthetic mechanisms (illustrated in Fig. 1) include (1) the process of photo­synthetic water splitting and proton gradient-coupled electron transport through the
thylakoid membrane of the chloroplast, which produces the reducing power (NADPH) and energy (ATP), and (2) the Calvin cycle, which reduces CO2 by consumption of the reducing power (NADPH) and energy (ATP).

In accordance with the present invention, a series of enzymes are used to create a designer ethanol-production pathway that takes an intermediate product of the Calvin cycle and converts the intermediate product into ethanol. A “designer etha­nol-production-pathway enzyme” is hereby defined as an enzyme that serves as a catalyst for at least one of the steps in a designer ethanol-production pathway. The intermediate products of the Calvin cycle are shown in Fig. 4a. According to the present invention, a number of intermediate products of the Calvin cycle can be utilized to create designer ethanol-production pathway(s); and the enzymes required for a designer ethanol-production pathway are selected depending on from which intermediate product of the Calvin cycle the designer ethanol-production pathway branches off.

In one example, a designer pathway is created that takes glyceraldehydes-3- phosphate and converts it into ethanol by using, for example, a set of enzymes consisting of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase, as shown in Fig. 4b. In this designer pathway, for conversion of one molecule of glyceraldehyde-3-phosphate to ethanol, an NADH molecule is generated from NAD+ at the step from glyceraldehyde-3-phosphate to 1,3-diphos — phoglycerate catalyzed by glyceraldehyde-3-phosphate dehydrogenase; meanwhile an NADH molecule is converted to NAD+ at the terminal step catalyzed by alcohol dehydrogenase to reduce acetaldehyde to ethanol. Consequently, in this designer pathway (Fig. 4b), the number of NADH molecules consumed is balanced with the number of NADH molecules generated. Therefore, this designer ethanol-production pathway can operate continuously.

In another example, as shown in Fig. 4c, a designer pathway is created that takes the intermediate product, 3-phosphoglycerate, and converts it into ethanol by using, for example, a set of enzymes consisting of phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase. It can be seen that the last five enzymes of the designer pathway shown in Fig. 4b are identical with those utilized in the designer pathway shown in Fig. 4c. In other words, the designer enzymes depicted in Fig. 4b permit ethanol production from both the point of 3-phosphoglycerate and the point glyceraldehydes 3-phosphate in the Calvin cycle. These two pathways (Fig. 4b. c), however, have different characteristics. Unlike the glyceraldehyde-3-phosphate-branched ethanol-production pathway (Fig. 4b), the 3-phosphoglycerate-branched pathway which consists of the activities of only five enzymes as shown in Fig. 4c could not itself generate any NADH for use in the terminal step to reduce acetaldehyde to ethanol. That is, if (or when) an alcohol dehydrogenase that strictly uses only NADH but not NADPH is employed, it would require a supply of NADH for the 3-phosphoglycerate-branched pathway to operate. Consequently, in order for the 3-phosphoglycerate-branched ethanol — production pathway (Fig. 4c) to operate, it is important to use an alcohol dehydro­genase that can use NADPH which can be supplied by the photo-driven electron

this designer ethanol-production pathway (Fig. 4c). Alternatively, when an alcohol dehydrogenase that can use only NADH is employed, it is prefer­ably here to use an additional embodiment that can confer an NADPH/NADH conversion mechanism (to supply NADH by converting NADPH to NADH, see more detail later in the text) in the designer organism’s chloroplast to facilitate photosynthetic production of ethanol through the 3-phosphoglycerate-branched designer pathway.

In still another example, a designer pathway is created that takes fructose-1,6- diphosphate and converts it into ethanol by using, for example, a set of enzymes consisting of aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyru­vate kinase, pyruvate decarboxylase, and alcohol dehydrogenase, as shown in Fig. 4d, with aldolase and triose phosphate isomerase being the only two additional enzymes relative to the designer pathway depicted in Fig. 4b. The addition of yet one more enzyme in the designer organism, phosphofructose kinase, permits the creation of another designer pathway which branches off from the point of fructose — 6-phosphate for the production of ethanol (Fig. 4e) . Like the glyceraldehyde-3- phosphate-branched ethanol-production pathway (Fig. 4b), both the fructose-1, 6-diphosphate-branched pathway (Fig. 4d) and the fructose-6-phosphate-branched pathway (Fig. 4e) can themselves generate NADH for use in their terminal step to reduce acetaldehyde to ethanol. In each of these designer ethanol-production pathways, the numbers of NADH molecules consumed are balanced with the numbers of NADH molecules generated. Therefore, these designer ethanol-production pathways can operate continuously.

Table 1 lists examples of the enzymes including those identified above for con­struction of the designer ethanol-production pathways. Throughout this specification, when reference is made to an enzyme, such as, for example, any of the enzymes listed in Table 1, it include their isozymes, functional analogs, designer modified enzymes, and combinations thereof. These enzymes can be selected for use in con­struction of the designer ethanol-production pathways. The “isozymes or functional analogs” refer to certain enzymes that have the same catalytic function but may or may not have exactly the same protein structures. For example, in Saccharomyces bayanus, there are four different genes (accession numbers: AY216992, AY216993, AY216994, and AY216995) encoding four alcohol dehydrogenases. These alcohol dehydrogenases essentially have the same function as an alcohol dehydrogenase, although there are some variations in their protein sequences. Therefore, the isozymes or functional analogs can also be selected and/or modified for use in con­struction of the designer ethanol-production pathway. The most essential feature of an enzyme is its active site that catalyzes the enzymatic reaction. Therefore, certain enzyme-protein fragment(s) or subunit(s) that contains such an active catalytic site may also be selected for use in this invention. For various reasons, some of the natu­ral enzymes contain not only the essential catalytic structure but also other structure components that may or may not be desirable for a given application. With techniques

Chlamydomonas reinhardtii cytoplasm; Aspergillus fumigatus; Coccidioides immitis; Leishmania braziliensis; Ajellomyces capsulatus; Monocercomonoicles sp.; Aspergillus clavatus; Arabidopsis thaliana; Zea mays C. reinhardtii cytoplasm; A. thaliana’, Leishmania Mexicana; Lodderomyces elongisporus; Babesia bovis; Sclerotinia sclerotiorum; Pichia guilliennon- clii; Spirotrichonympha leidyi; Otyza sativa; T. pyrifonnis; Leuconostoc mesenteroides; Davidiella tassiana; Aspergillus oryzjae; Schizosaccharomyces pombe; Brassica napus; Z. mays C. reinhardtii cytoplasm; A. thaliana; Saccharomyces cerevisiae; B. bovis; S. sclerotiorum; Trichomonas vaginalis; P. guillietmondii; Pichia stipitis; L. elongisporus; C. immitis; T. pyrifonnis; Glycine max (soybean)

C. reinhardtii cytoplasm; P. stipitis; L. elongisporus; A. thaliana; Lycoris aurea; Chaetomium globosum; Citrus sinensis; Petunia x hybrida; Candida glabrata; Saccharomyces kluyveri; Z. mays; Rhizopus oryzae; Lotus comiculatus; Zymomonas mobiles; Lachancea kluyveri; O. sativa C. reinhardtii mitochondria; Kluyveromyces lactis; Kluyveromyces marxianus; S. cerevisiae; Saccharomyces bayanus; P. stipitis; Entamoeba histolytica; T. vaginalis; L. braziliensis; Botryotinia fuckeliana; A. fumigatus; Dianthus caiyophyllus; Saccharomyces pastorianus; L. kluyveri

JGI Chlre2 protein ID 161689, GenBank: AF268078; XMJ747847; XM_749597; XM 001248115; XM_001569263; XM_001539892; DQ665859; XM_001270940; NM_117020; M80912

GenBank: X66412, P31683; AK222035;DQ221745; XM_001528071; XM_001611873; XMOO1594215; XM_001483612; AB221057; EF122486, U09450; DQ845796; AB088633; U82438; D64113; U13799; AY307449; U17973

of bioinformatics-assisted molecular design, it is possible to select the essential catalytic structure(s) for use in construction of a designer DNA construct encoding a desirable designer enzyme. Therefore, in one of the various embodiments, a designer enzyme gene is created by artificial synthesis of a DNA construct according to bioinformatics-assisted molecular sequence design. With the computer-assisted synthetic biology approach, any DNA sequence (thus its protein structure) of a designer enzyme may be selectively modified to achieve more desirable results by design. Therefore, the terms “designer modified sequences” and “designer modified enzymes” are hereby defined as the DNA sequences and the enzyme proteins that are modified with bioinformatics-assisted molecular design. For example, when a DNA construct for a designer chloroplast-targeted enzyme is designed from the sequence of a mitochondrial enzyme, it is a preferred practice to modify some of the protein structures, for example, by selectively cutting out certain structure component(s) such as its mitochondrial transit-peptide sequence that is not suitable for the given application, and/or by adding certain peptide structures such as an exogenous chloroplast transit-peptide sequence (e. g., a 135-bp Rubisco small — subunit transit peptide (RbcS2)) that is needed to confer the ability in the chloroplast — targeted insertion of the designer protein. Therefore, one of the various embodiments flexibly employs the enzymes, their isozymes, functional analogs, designer modified enzymes, and/or the combinations thereof in construction of the designer ethanol — production pathway(s).

As shown in Table 1, many genes of the enzymes identified above have been cloned and/or sequenced from various organisms. Both genomic DNA and/or mRNA sequence data can be used in designing and synthesizing the designer DNA constructs for transformation of a host alga, plant, plant tissue or cells to create a designer organism for photobiological ethanol production (Fig. 5). However, because of possible variations often associated with various source organisms and cellular compartments with respect to a specific host organism and its chloroplast environment where the ethanol-production pathway(s) is designed to work with the Calvin cycle, certain molecular engineering artwork in DNA construct design including codon-usage optimization and sequence modification is often necessary for a designer DNA construct (Fig. 6) to work well. For example, if the source sequences are from cytosolic enzymes (sequences), a functional chloroplast-target — ing sequence must be added to provide the capability for a designer unclear gene- encoded enzyme to insert into a host chloroplast to confer its function for a designer ethanol-production pathway. Furthermore, to provide the switchability for a designer ethanol-production pathway, it is also important to include a functional inducible promoter sequence such as the promoter of a hydrogenase (Hydl) or nitrate reductase (Nial) gene in certain designer DNA construct(s) as illustrated in Fig. 6a to control the expression of the designer gene(s). In addition, as mentioned before, certain functional derivatives or fragments of these enzymes (sequences), chloroplast-tar — geting transit peptide sequences, and inducible promoter sequences can also be selected for use in full, in part or in combinations thereof, to create the designer organisms according to various embodiments of this invention. The arts in creating and using the designer organisms are further described herein below.

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