# Patent

## Title
Genetically modified cyanobacteria for the production of ethanol

## Abstract

The invention provides novel compositions of matter for the production of ethanol from carbon dioxide and water. Particularly, the invention provides photoautotrophic organisms having a first and second genetic modification, wherein the first genetic modification improves the ethanol production from organisms having the second genetic modification.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. provisional application 61/065,292, filed Feb. 8, 2008, which is incorporated by reference in its entirety. The present application is related to a PCT application titled “GENETICALLY MODIFIED PHOTOAUTOTROPHIC ETHANOL PRODUCING HOST CELLS, METHOD FOR PRODUCING THE HOST CELLS, CONSTRUCTS FOR THE TRANSFORMATION OF THE HOST CELLS, METHOD FOR TESTING A PHOTOAUTOTROPHIC STRAIN FOR A DESIRED GROWTH PROPERTY AND METHOD OF PRODUCING ETHANOL USING THE HOST CELLS,” filed on even the same day with the present application and claiming priority to U.S. provisional application 61/065,292, which PCT application is incorporated by reference in its entirety.


FIELD OF THE INVENTION
This invention is related to the field of direct production of ethanol from carbon dioxide and water using genetically modified cyanobacteria.
This application refers to a “Sequence Listing” listed below, which is provided as a text document. The document is entitled “12368060SeqList2.txt” (506,402 bytes, created Nov. 13, 2009) and is hereby incorporated by reference in its entirety herein.
BACKGROUND OF THE INVENTION
There is a current need to find alternate energy sources to substitute for the use of fossil fuels for transportation applications. Biologically produced ethanol has been proposed as an alternative to petroleum-derived liquid fuels. There are different ways to generate ethanol through biological means. Obtaining ethanol from grains and seeds has been criticized for contributing to rising food prices and leading to deforestation. The disadvantages of obtaining ethanol from grain and cellulosic sources are further explained in paragraph [0007] of published U.S. patent application 20090017512. The present invention is addressed to the direct production of ethanol from carbon dioxide and water using genetically-modified cyanobacteria and overcomes problems associated with grain or cellulosic sources of ethanol.
Further, the present invention discloses the capability to produce ethanol using desert lands and salt water and resolves problems associated with demand on food-producing land and water resources. Moreover, the ethanol productivity of the present invention is higher than for corn-based ethanol. The present invention has projected productivity of 6,000 gallons (22,700 liter) ethanol per acre compared to 370 gallons (1,400 liter) ethanol for corn ethanol. [Bryan Walsh, “Biofuels: the New Alchemy,” TIME magazine, http://www.time.com/time/specials/packages/article/0,28804,18 72110_1872133_1872143-1,00.html; see also Emily Waltz, “Biotech's Green Gold,” 27 Nature Biotechnology 15-18 (2009)].
The present invention improves upon work disclosed by Woods et al. in U.S. Pat. Nos. 6,306,639 and 6,699,696, which taught the genetic modification of Cyanobacteria by incorporating the genetic information encoding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh). Specifically, the coding sequences of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh) from the bacterium Zymomonas mobilis were cloned into the shuttle vector pCB4 and then used to transform the cyanobacterium Synechococcus sp. strain PCC 7942. The pdc and adh genes were expressed at high levels, and the transformed cyanobacterium synthesized ethanol, which diffused from the cells into the culture medium.
Methods to improve ethanol production in such ethanologenic organisms are needed to facilitate commercial implementation of this ethanol source. The ability to modify the genetics of specific species has been stated to be currently limiting progress. E. T. Johnson and C. Schmidt-Dannert, “Light Energy Conversion in Engineered Microorganisms,” Trends in Biotechnology, Volume 26, Issue 12, December 2008, Pages 682-689. The problem of genetic engineering is complicated. Some of the obstacles to achieving high yields of products are a result of the interdependence of metabolic networks, which are strongly influenced by the global levels of a handful of metabolites: ATP/ADP, NAD+/NADH, NADP+/NADPH, and acyl-CoAs. ( . . . ) The incorporation of new pathways for biofuel synthesis can destabilize the balance of these important metabolites, leading to the production of undesirable byproducts and a decrease in yield. Sung Kuk Lee, Howard Chou, Timothy S Ham, Taek Soon Lee, Jay D Keasling, Current Opinion in Biotechnology, Volume 19, Issue 6, December 2008, Pages 556-563. [Generally, see also Biochemistry. Fifth Edition. Berg J M, Tymoczko J L, and Stryer L. New York. W.H. Freeman and Company. 2002.]
One way to increase ethanol production in a microbial host cell is to down-regulate and/or disrupt genes encoding enzymes involved in metabolic pathways that may compete for substrates, intermediates, and/or co-factors that influence ethanol production. The present invention discloses how this can be successfully done to increase ethanol production.
SUMMARY OF THE INVENTION
The present invention discloses genetically-modified cyanobacteria with ethanol-production capabilities enhanced over the currently-reported art, and methods of making such cyanobacteria.
The genetically modified photoautotrophic ethanol producing host cell comprises at least two different genetic modifications, a first and a second genetic modification.
The first genetic modification changes the enzymatic activity or affinity of an endogenous host enzyme, resulting in a higher level of biosynthesis of acetyl-CoA, acetaldehyde, pyruvate or precursors thereof. The endogenous host enzyme is already present in an unmodified wild type host cell and its activity or affinity is changed by the first genetic modification in order to increase the level of biosynthesis of metabolic intermediates, which are also present in the wild type host cell and which can be used to form ethanol. Suitable endogenous host enzymes are selected from the group consisting of phosphoglycerate mutase, enolase, pyruvate kinase, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), malic enzyme, phosphoenolpyruvate [PEP]carboxylase, malic enzyme, and malate dehydrogenase. The enhanced level of metabolic intermediates can arise through an increase in the rate of synthesis of the metabolic intermediate or elimination or reduction of the rate of use of the metabolic intermediate by a competing reaction. Examples of the latter include knockout of alanine dehydrogenase, phosphotransacetylase, acetate kinase, lactate dehydrogenase, and a knock-down of pyruvate dehydrogenase activity, in which we are attempting to reduce the turnover and thus keep the metabolite pools higher.
The second genetic modification is in the form of at least one overexpressed enzyme, which can form ethanol, for example from the above-mentioned important metabolic intermediates. In a further embodiment the overexpressed enzyme for ethanol formation can catalyze the last step of ethanol formation leading to the final product ethanol. The overexpressed enzyme for ethanol formation can also catalyze the penultimate step of ethanol formation resulting in a metabolic intermediate, which can further be converted by another enzyme for ethanol formation into the final product ethanol. The second genetic modification can take the form of overexpression of pdc or the form of overexpression of adh and pdc.
Enhanced ethanol production has been obtained by identifying a variety of genes as targets for downregulation, which are set forth more fully in the examples below. The fermentative pathway is impacted in the double mutant Δack/Δpta and the carbohydrate storage pathway is impacted in the ΔglgA1/ΔglgA2 double mutant.
In one aspect the invention provides a method for the production of ethanol comprising the steps
a) providing an ethanologenic cyanobacterial host cell, having genetic modifications relative to the corresponding wild type cell comprising
i) changing the enzymatic activity or affinity of an endogenous host cell enzyme
ii) creating an ethanologenic biosynthetic pathway comprising an overexpressed first enzyme for the formation of ethanol,
b) growing the host cell of (a) under conditions whereby ethanol is produced; and
c) isolating the ethanol produced at step (b).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1, comprising 1A, 1B, 1C, 1D, 1E, and 1F illustrates some relevant metabolic pathways
FIG. 2 illustrates possible pathways leading to ethanol production.
FIG. 3 illustrates possible pathways leading to ethanol production
FIG. 4a presents the amino acid sequence of a glycogen synthase gene of Synechocystis sp. PCC 6803 that is encoded by the gene sll0945 (glgA1). (SEQ ID NO:1)
FIG. 4B presents the amino acid sequence of a second glycogen synthase of Synechocystis sp. PCC 6803 that is encoded by the gene sll1393 (glgA2). (SEQ ID NO:2)
FIG. 4C presents a schematic representation of restriction sites used in the cloning strategy for pUC 19-glgA1-Cm.
FIG. 4D is a schematic representation of gene organization for the plasmid pUC 19-glgA1-Cm.
FIG. 4E presents the nucleotide sequence of the construct pUC 19-glgA1-Cm. (SEQ ID NO:3)
FIG. 4F presents a schematic representation of restriction sites used in the cloning strategy for pUC 19-glgA2-Kan.
FIG. 4G is a schematic representation of gene organization for the plasmid pUC 19-glgA2-Kan.
FIG. 4H presents the nucleotide sequence of the construct pUC 19-glgA2-Kan. (SEQ ID NO:4)
FIG. 5A presents the amino acid sequence of the open reading frame sll1682, which encodes alanine dehydrogenase (EC 1.4.1.1) (Genbank No BAA16790) of Synechocystis sp. strain PCC6803. (SEQ ID NO:5)
FIG. 5B presents a schematic representation of gene organization for the plasmid pGEM-T/ald-KManti.
FIG. 5C presents the nucleotide sequence of the insert of construct pGEM-T/ald-KManti. (SEQ ID NO:6)
FIG. 6A presents the amino acid sequence of the open reading frame slr1176, which encodes ADP-glucose pyrophosphorylase (EC 2.7.7.27) (Genbank No BAA18822) of Synechocystis sp. strain PCC6803. (SEQ ID NO:7)
FIG. 6B presents a schematic representation of gene organization for the plasmid pGEM-T/glgC-KManti.
FIG. 6C presents the nucleotide sequence of the insert of construct pGEM-T/glgC-KManti. (SEQ ID NO:8)
FIG. 6D presents a schematic representation of gene organization for the plasmid pDrive/glgC-CMantisense.
FIG. 6E presents the nucleotide sequence of the insert of construct pDrive/glgC-CMantisense. (SEQ ID NO:9)
FIG. 7A presents the amino acid sequence of the open reading frame slr0301 that encodes pyruvate water dikinase/PEP synthase (EC 2.7.9.2) (Genbank No BAA10668) of Synechocystis sp. strain PCC6803. (SEQ ID NO:10)
FIG. 7B presents a schematic of gene organization for the plasmid pGEM-T/ppsA-anti.
FIG. 7C presents the nucleotide sequence of the insert of construct pGEM-T/ppsA-anti. (SEQ ID NO:11)
FIG. 8A presents the amino acid sequence of open reading frame slr 1556 that encodes a putative lactate dehydrogenase (EC 1.1.1.28), (annotated as 2-hydroxyaciddehydrogenase homolog) (GenBank No. P74586) of Synechocystis sp. strain PCC6803. (SEQ ID NO:12)
FIG. 8B presents a schematic representation of restriction sites used in the cloning strategy for pBlue ldh-Kan-a.
FIG. 8C presents a schematic for the gene organization of the plasmid pBlue ldh-Kan-a.
FIG. 8D presents the nucleotide sequence of the insert contained in the construct pBlue ldh-Kan-a. (SEQ ID NO:13)
FIG. 9A presents the amino acid sequence of the open reading frame sll 1299 that encodes a putative acetate kinase (EC 2.7.2.1) (Genbank No. P73162). (SEQ ID NO:14)
FIG. 9B presents a schematic representation of restriction sites used in the cloning strategy for pBlue-ack-Kan-b.
FIG. 9C presents a schematic for the gene organization of the plasmid pBlue-ack-Kan-b.
FIG. 9D presents the nucleotide sequence of the insert of construct pBlue-ack-Kan-b. (SEQ ID NO:15)
FIG. 10A presents the amino acid sequence of the open reading frame slr2132 that encodes a phosphoacetyltransacetylase (EC 2.3.1.8) (Genbank No. P73662) of Synechocystis sp. strain PCC6803. (SEQ ID NO:16)
FIG. 10B presents a schematic representation of restriction sites used in the cloning strategy for pUC pta-Cm.
FIG. 10C presents a schematic for the gene organization of the plasmid pUC pta-Cm.
FIG. 10D presents the nucleotide sequence of the insert of construct pUC pta-Cm. (SEQ ID NO:17)
FIG. 11A presents the amino acid sequence of open reading frame slr1830 that encodes poly(3-hydroxyalkanoate) synthase [EC:2.3.1.] (Genbank No BAA17430) of Synechocystis sp. strain PCC6803. (SEQ ID NO:18)
FIG. 11B presents a schematic representation of gene structure for the plasmid as pIC2OH/ΔphaC-KM.
FIG. 11C presents the nucleotide sequence of the insert of construct as pIC2OH/ΔphaC-KM. (SEQ ID NO:19)
FIG. 11D presents the amino acid sequence of ORF all4645 for PCC 7120 (SEQ ID NO:20).
FIG. 11E presents a schematic representation of restriction sites and gene organization for the PCC 7120 glgC knockout.
FIG. 11F presents the sequence of the insert of pRL271 agp (all4645)::C.K3-PpetE-pdc-adhII (SEQ ID NO:21).
FIG. 11G presents the amino acid sequence of Glucose-1-phosphate adenylyltransferase (ADP-glucose-pyrophosphorylase, agp, glgC), EC 2.7.7.27, of Anabaena variabilis ATCC29314 (SEQ ID NO:22).
FIG. 12A presents the amino acid sequence of open reading frame sll1721 that encodes the β-subunit of the E1 component of the pyruvate dehydrogenase (EC 1.2.4.1) (Genbank No BAA17445) of Synechocystis sp. strain PCC6803. (SEQ ID NO:23)
FIG. 12B presents a schematic of gene organization for the plasmid pSK9/pdhBanti.
FIG. 12C presents the nucleotide sequence of the insert for pSK9/pdhBanti. (SEQ ID NO:24)
FIG. 12D presents a schematic representation of gene organization for the plasmid pSK9/pdhB.
FIG. 12E presents the nucleotide sequence of the insert for the construct pSK9/pdhB. (SEQ ID NO:25)
FIG. 12F presents a schematic of gene organization for the plasmid pGEM-T/ΔpdhB-KMantisense.
FIG. 12G presents the nucleotide sequence of the insert of construct pGEM-T/ΔpdhB-KMantisense. (SEQ ID NO:26)
FIG. 13A presents a schematic representation of the cloning vector pGEM-T.
FIG. 13B presents the nucleotide sequence of vector pGEM-T. (SEQ ID NO:27)
FIG. 14A presents a schematic representation of the cloning vector pDrive.
FIG. 14B presents the nucleotide sequence of vector pDrive. (SEQ ID NO:28)
FIG. 15A presents a schematic representation of the cloning vector pBluescript II SK (+).
FIG. 15B presents the nucleotide sequence of the vector pBluescript II SK (+). (SEQ ID NO:29)
FIG. 16A presents a schematic representation of the cloning vector pUC 19.
FIG. 16B presents the nucleotide sequence of the vector pUC 19. (SEQ ID NO:30)
FIG. 17A presents a schematic representation of genes organized in the vector pSK9.
FIG. 17B presents the nucleotide sequence of the vector pSK9. (SEQ ID NO:31)
FIG. 18A presents the amino acid sequence of open reading frame slr0721 that encodes malic enzyme 1 (EC 1.1.1.38) (Genbank No P72661) of Synechocystis sp. strain PCC6803. (SEQ ID NO:32)
FIG. 18B presents a schematic of genes organized in the construct of Synechocystis sp. strain PCC6803.
FIG. 18C presents the nucleotide sequence of the insert of construct pSK9/me-long. (SEQ ID NO:33)
FIG. 19A presents the amino acid sequence of open reading frame sll0891 that encodes malate dehydrogenase (EC 1.1.1.37) (Genbank No Q55383) of Synechocystis sp. strain PCC6803. (SEQ ID NO:34)
FIG. 19B presents a schematic representation of gene organization for the construct pSK9-mdh.
FIG. 19C presents the nucleotide sequence of the insert of construct pSK9-mdh. (SEQ ID NO:35)
FIG. 19D presents a schematic representation of gene organization for the construct pSK9/me-mdh.
FIG. 19E presents the nucleotide sequence of the insert of construct pSK9/me-mdh. (SEQ ID NO:36)
FIG. 20A presents the amino acid sequence of open reading frame sll0587 that encodes a pyruvate kinase 1 (EC 2.7.1.40 (PK1)) (Genbank No Q55863) of Synechocystis sp. strain PCC6803. (SEQ ID NO:37)
FIG. 20B presents a schematic representation of gene organization for the construct pVZ321-pyk1.
FIG. 20C presents the nucleotide sequence of the insert of construct pVZ321-pyk1. (SEQ ID NO:38)
FIG. 20D presents a schematic representation of gene organization for the construct pVZ321 PpetJ pyk1.
FIG. 20E presents the nucleotide sequence of the insert found in construct pVZ321 PpetJ pyk1. (SEQ ID NO:39)
FIG. 21A presents the amino acid sequence of open reading frame sll1275 that encodes pyruvate kinase 2 (EC 2.7.1.40 (PK2)) (Genbank No P73534) of Synechocystis sp. strain PCC6803. (SEQ ID NO:40)
FIG. 21B presents a schematic representation of gene organization for the construct pVZ321pyk2.
FIG. 21C presents the nucleotide sequence of the insert of pVZ321pyk2. (SEQ ID NO:41)
FIG. 21D presents a schematic representation of gene organization for the construct. pVZ321 PpetJ pyk2.
FIG. 21E presents the nucleotide sequence for the insert of the construct pVZ321 PpetJ pyk2. (SEQ ID NO:42)
FIG. 22A presents a schematic representation of the gene organization for the p67 insert.
FIG. 22B presents the amino acid sequence of pyruvate kinase I (E. coli K12). (SEQ ID NO:43)
FIG. 22C presents the amino acid sequence of enolase (Zymomonas mobilis). (SEQ ID NO:44)
FIG. 22D presents the amino acid sequence of phosphoglycerate mutase (Zymomonas mobilis). (SEQ ID NO:45)
FIG. 22E presents the nucleotide sequence of the insert of plasmid #67. (SEQ ID NO:46)
FIG. 22F presents a schematic representation of gene organization for the construct pVZ321-p67.
FIG. 22G presents a schematic representation of gene organization for construct pVZ322-p67.
FIG. 23A presents the amino acid sequence of open reading frame slr0752 that encodes the enolase (eno, 2-phosphoglycerate dehydratase) (EC 4.2.1.11) (Genbank No. BAA18749) of Synechocystis sp. strain PCC6803. (SEQ ID NO:47)
FIG. 23B presents a schematic representation of gene organization for construct pVZ321 PpetJ eno.
FIG. 23C presents the nucleotide sequence of the insert of construct pVZ321-PpetJ-eno. (SEQ ID NO:48)
FIG. 24A presents the amino acid sequence of open reading frame slr1124 that encodes the phosphoglycerate mutase (pgm or gpmB) (EC 5.4.2.1) (Genbank No. BAA16651) of Synechocystis sp. strain PCC6803. (SEQ ID NO:49)
FIG. 24B presents a schematic representation of the gene organization of construct pVZ321-PpetJ-pgm.
FIG. 24C presents the nucleotide sequence of the insert of construct pVZ321-PpetJ-pgm. (SEQ ID NO:50)
FIG. 24D presents a schematic representation of gene organization for the construct pVZ322-PpetJ-pyk1-eno-pgm.
FIG. 24E presents a schematic representation of gene organization for the construct pVZ322-PpetJ-pyk2-eno-pgm.
FIG. 24F presents the nucleotide sequence of the insert of construct pVZ322-PpetJ-pyk1-eno-pgm. (SEQ ID NO:51)
24G presents the nucleotide sequence of the insert of construct pVZ322-PpetJ-pyk2-eno-pgm. (SEQ ID NO:52)
FIG. 25A presents the amino acid sequence for open reading frame slr0453 that encodes the probable phosphoketolase (phk), (EC 4.1.2.-) (Genbank No. P74690) of Synechocystis sp. strain PCC6803. (SEQ ID NO:53)
FIG. 25B presents a schematic representation of the gene organization for the construct pVZ322 PpetJ-phk.
FIG. 25C presents the nucleotide sequence of the insert of the construct pVZ322 PpetJ-phk. (SEQ ID NO:54)
FIG. 26A presents the amino acid sequence of open reading frame slr2132 that encodes a phosphoacetyltransacetylase (pta) (EC 2.3.1.8) (Genbank No. P73662) of Synechocystis sp. strain PCC6803. (SEQ ID NO:55)
FIG. 26B presents a schematic representation of gene organization in the construct pVZ322 PpetJ pta.
FIG. 26C presents the nucleotide sequence of the insert of construct pVZ322 PpetJ pta. (SEQ ID NO:56)
FIG. 26D presents a schematic representation of gene organization in construct pVZ322 PpetJ phk pta.
FIG. 26E presents the nucleotide sequence of the insert of construct pVZ322 PpetJ phk pta. (SEQ ID NO:57)
FIG. 27A presents the amino acid sequence of open reading frame slr0091 encodes a aldehyde dehydrogenase (aldh) (EC 1.2.1.3) (Genbank No. BAA10564) of Synechocystis sp. strain PCC6803. (SEQ ID NO:58)
FIG. 27B is a schematic representation of gene organization in construct pVZ 322 PpetJ aldh.
FIG. 27C presents the nucleotide sequence of construct pvc 322 PpetJ aldh. (SEQ ID NO:59)
FIG. 28A presents the amino acid sequence of open reading frame sll0920 that encodes phosphoenolpyruvate carboxylase (EC 4.1.1.31) (Genbank No. BAA18393) of Synechocystis sp. strain PCC6803. (SEQ ID NO:60)
FIG. 28B is a schematic representation of gene organization in pVZ321-PpetJ-ppc.
FIG. 28C presents the nucleotide sequence of the insert of construct pVZ321-PpetJ-ppc. (SEQ ID NO:61)
FIG. 28D presents the nucleotide sequence of primer SynRbc-BglII-fw (SEQ ID NO:62).
FIG. 28E presents the nucleotide sequence of primer SynRbc-PstI-rev (SEQ ID NO:63).
FIG. 28F presents the nucleotide sequence of primer SynRbc-SacI-fw (SEQ ID NO:64).
FIG. 28G presents the nucleotide sequence of the rbcLXS operon of Synechocystis PCC 6803 (SEQ ID NO:65).
FIG. 28H presents the amino acid sequence of the rbcL large subunit of Synechocystis PCC 6803 (SEQ ID NO:66).
FIG. 28I presents the amino acid sequence of the rbcX Rubisco chaperonin protein of Synechocystis PCC 6803 (SEQ ID NO:67).
FIG. 28J presents the amino acid sequence of the ribulose bisphosphate carboxylase small subunit (rbcS) of Synechocystis PCC 6803 (SEQ ID NO:68).
FIG. 28K is a schematic presentation of gene organization for plasmid pVZ321b-Prbc-SynRbcLXS.
FIG. 28L is a schematic presentation of gene organization for plasmid pVZ321b-PpetJ-PDC/SynRbcLXS.
FIG. 29A is a schematic representation of the structure of the vector pSK9.
FIG. 29B presents the nucleotide sequence of the vector pSK9. (SEQ ID NO:69)
FIG. 30A is a schematic representation of gene organization in construct pVZ321. (GenBank No. AF100176).
FIG. 30B presents the nucleotide sequence of the pVZ321 vector. (SEQ ID NO:70)
FIG. 31A is a schematic representation of gene organization for construct pVZ322.
FIG. 31B presents the nucleotide sequence of the pVZ322 vector (SEQ ID NO:71).
FIG. 32A is a schematic representation of gene organization of construct pIC PpetJ.
FIG. 32B presents the nucleotide sequence of the construct pIC PpetJ. (SEQ ID NO:72)
FIG. 32C is a graphic presentation demonstrating growth properties and extracellular pyruvate levels of the ΔglgA1/ΔglgA2 double mutant (M8) under nitrogen replete and nitrogen starved conditions.
FIG. 32D is a graphic presentation of pyruvate levels in wildtype and mutant (ΔglgA1/ΔglgA2) media/cells as determined enzymatically and by ion chromatography.
FIG. 32E is a graphic presentation of the conductimetric detection of pyruvate in methanol extracts (snapshot) of cultures of wildtype and a glycogen synthase deficient mutant after 24 h under N-deficient conditions.
FIG. 32F is a graphic depiction showing the that the pyruvate concentration in the growth medium is higher for the M8 mutant without Adh and Pdc enzymes than for the M8 mutant including both ethanol forming enzymes under the conditions of nitrogen starvation.
FIG. 32G is a graphic depiction of the ethanol concentration determined in the growth medium for the M8 mutant with the Adh and Pdc enzymes under the conditions of nitrogen starvation and without nitrogen starvation.
FIG. 32H is a graphic depiction of ethanol generation in glycogen deficient Synechocystis pVC mutants with ZmPDC and ZmADHII under the control of the iron-dependent isiA promoter.
FIG. 32I is a graphic presentation of ethanol production in wildtype, ack and ack/pta double mutant cells.
FIG. 32J is a graphic presentation of ethanol production in wildtype, ack and ack/pta double mutant cells when normalized for optical density.
FIG. 32K is a graphic presentation of demonstrating that pVZ321b-Prbc-SynRbcLXS grows as fast as the Synechocystis wild type and shows no phenotypical differences except for the chlorophyll content that is reduced by 20-30% compared to wild type.
FIG. 32L is a graphic presentation of demonstrating the growth parameter (OD at 750 nm and Chlorophyll content) of Synechocystis wild type and a mutant that over-express the endogenous RuBisCO operon.
FIG. 32M is a graphic presentation of ethanol production for the mutant Synechocystis PCC6803 harboring the pSK10-PisiA-PDC/ADHII plasmid and the mutant additionally containing the vector pVZ321b-Prbc-SynRbc.
FIG. 32N is a graphic presentation of ethanol production normalized to the OD750 for the mutant Synechocystis PCC6803 harboring the pSK10-PisiA-PDC/ADHII plasmid and the mutant additionally containing the vector pVZ321b-Prbc-SynRbc
FIG. 33A is a schematic representation of gene organization for the construct pVZ-PisiA-pdc/adh.
FIG. 33B is a schematic representation of gene organization for the construct pVZ-PntcA-pdc/adh.
FIG. 33C is a schematic representation of gene organization for the construct pVZ-PnblA-pdc/adh.
FIG. 33D presents the nucleotide sequence of the insert of the vector pCB4-LR(TF)pa that encodes Z. mobilis adhII and pdc genes. (SEQ ID NO:73)
FIG. 33E is a schematic representation of restriction sites present in the Z. mobilis adhII and pdc fragment.
FIG. 33F presents the amino acid sequence of Z. mobilis pdc protein. (SEQ ID NO:74)
FIG. 33G presents the amino acid sequence of the Z. mobilis adhII protein. (SEQ ID NO:75)
FIG. 34A presents the nucleotide sequence for the isiA promoter (Synechocystis sp. PCC6803) (isiA gene: sll0247), which is induced under iron starvation conditions. (SEQ ID NO:76)
FIG. 34B presents the nucleotide sequence for the nblA promoter (Synechocystis sp. PCC6803) (nblA gene: ssll10452), which is induced under nitrogen starvation conditions. (SEQ ID NO:77)
FIG. 34C presents the nucleotide sequence for the ntcA promoter (Synechocystis sp. PCC6803) (ntcA gene: sll1423), which is induced under nitrogen starvation. (SEQ ID NO:78)
FIG. 35A presents the nucleotide sequence of the cloning vector pVZ321b, a derivative of pVZ321. (SEQ ID NO:79)
FIG. 35B is a schematic representation of gene organization for the cloning vector pVZ321b.
FIG. 36A presents the nucleotide sequence for the petJ promoter (Synechocystis sp. PCC 6803) (petJ gene: sll1796) (encoding for cytochrome c553), which is induced under copper starvation conditions. (SEQ ID NO:80)
FIG. 36B is a schematic representation of gene organization for the construct pVZ321b-PpetJ-PDC-ADHII.
FIG. 36C presents the nucleotide sequence of the sigB promoter (Synechocystis sp. PCC 6803) (sigB gene: sll0306) (encoding for RNA polymerase group 2 sigma factor), which is induced after heat shock, in stationary growth phase/nitrogen starvation and darkness. (SEQ ID NO:81)
FIG. 36D is a schematic representation of gene organization for the construct pVZ321b-PsigB-PDC-ADHII.
FIG. 36E presents the nucleotide sequence of the htpG promoter (Synechocystis sp. PCC 6803) (htpG gene: sll0430) (encoding for heat shock protein 90, molecular chaperone), which is induced after heat shock. (SEQ ID NO:82)
FIG. 36F is a schematic representation of gene organization for the construct pVZ321b-PhtpG-PDC-ADHII.
FIG. 36G presents the nucleotide sequence of the lrtA promoter (Synechocystis sp. PCC 6803) (lrtA gene: sll0947) (encoding the light repressed protein A homolog), which is induced after light to dark transition. (SEQ ID NO:83)
FIG. 36H is a schematic representation of gene organization in the construct pVZ321b-PlrtA-PDC-ADHII.
FIG. 36I presents the nucleotide sequence of the psbA2 promoter (Synechocystis sp. PCC 6803) (psbA2 gene: slr1311) (encoding the photosystem II D1 protein), which is induced after dark to light transition. (SEQ ID NO:84)
FIG. 36J is a schematic representation of gene organization for the construct pVZ321b-PpsbA2-PDC-ADHII.
FIG. 36K presents the nucleotide sequence of the rbcL promoter (Synechocystis sp. PCC 6803) (rbcL gene: slr0009) (encoding the ribulose biphosphate carboxylase/oxygenase large subunit), which is a constitutive and strong promoter under continuous light conditions. (SEQ ID NO:85)
FIG. 36L is a schematic representation of gene organization for the construct pVZ321b-PrbcL-PDC-ADHII.
FIG. 36M presents the nucleotide sequence for the psaA promoter (Synechocystis sp. PCC6803) (psaA gene: slr1834) (encoding P700 apoprotein subunit Ia), which is induced under low white light and orange light, low expression level under high light and red light, and repressed in darkness. (SEQ ID NO:86)
FIG. 36N is a schematic representation of the gene organization of the construct pVZ321b-PpsaA-PDC-ADHII.
FIG. 36O presents the nucleotide sequence of the ggpS promoter (Synechocystis sp. PCC6803) (ggpS gene: sll1566) (encoding glucosylglycerolphosphate synthase), which is induced after salt stress. (SEQ ID NO:87)
FIG. 36P is a schematic representation of the gene organization of the construct pVZ321b-PggpS-PDC-ADHII.
FIG. 36Q presents the nucleotide sequence of the nirA promoter (Synechocystis sp. PCC6803) (nirA gene: slr0898) (encoding ferredoxin-nitrite reductase), which is induced after transition from ammonia to nitrate. (SEQ ID NO:88)
FIG. 36R is a schematic representation of the gene organization of the construct pVZ321c-PnirA-PDC-ADHII.
FIG. 36S presents the nucleotide sequence of the petE promoter (Anabaena sp. PCC7120) (petE gene: all0258) (encoding plastocyanin precursor), which is induced at elevated copper concentrations. (SEQ ID NO:89)
FIG. 36T is a schematic representation of gene organization for the construct pVZ321c-PpetE-PDC-ADHII.
FIG. 36U presents the nucleotide sequence of the hspA promoter (Synechocystis sp. PCC6803) (hspA gene: sll1514) 16.6 kDa small heat shock protein, molecular chaperone multi-stress responsive promoter (heat, cold, salt and oxidative stress). (SEQ ID NO:90)
FIG. 36V is a schematic representation of gene organization for the construct pVZ321c-PhspA-PDC-ADHII.
FIG. 36W presents the nucleotide sequence for the hliB promoter (Synechocystis sp. PCC6803) (hliB gene: ssr2595) high light-inducible polypeptide HliB, CAB/ELIP/HLIP superfamily) (multi-stress responsible promoter (heat, cold, salt and oxidative stress). (SEQ ID NO:91)
FIG. 36X is a schematic representation of gene organization of the construct pVZ321c-PhliB-PDC-ADHII.
FIG. 36Y presents the nucleotide sequence of the clpB1 promoter (Synechocystis sp. PCC6803) (clpB1 gene: slr1641) ATP-dependent Clp protease, Hsp 100, ATP-binding subunit ClpB multi-stress responsible promoter (heat, cold, salt and oxidative stress). (SEQ ID NO:92)
FIG. 36Z is a schematic representation of the gene organization for the construct pVZ321c-PclpB1-PDC-ADHII.
FIG. 37A presents the nucleotide sequence of the adhA gene from Zymomonas mobilis ZM4. (SEQ ID NO:93)
FIG. 37B presents the amino acid sequence for the ZmAdhI protein sequence (AAV89860). (SEQ ID NO:94)
FIG. 37C is a schematic presentation of the gene organization for construct pVZ321b-PisiA-PDC-ADHI.
FIG. 37D is a schematic presentation of the gene organization for construct pVZ321b-PntcA-PDC-ZmADHI.
FIG. 37E is a schematic presentation of the gene organization for construct pVZ321b-PnblA-PDC-ZmADHI.
FIG. 38A presents the nucleotide sequence of SynAdh, the adh gene (slr1192) of Synechocystis sp. PCC 6803. (SEQ ID NO:95)
FIG. 38B presents the amino acid sequence of SynAdh (protein sequence BAA18840) of Synechocystis sp. PCC 6803. (SEQ ID NO:96)
FIG. 38C is a schematic representation of the gene organization for construct pVZ321b-PisiA-PDC-SynADH.
FIG. 38D is a schematic representation of the gene organization for construct pVZ321b-PntcA-PDC-SynADH.
FIG. 38E is a schematic representation of the gene organization for construct pVZ321b-PnblA-PDC-SynADH.
FIG. 39A presents the nucleotide sequence of EcAdhE, the adhE gene from E. coli K12. (SEQ ID NO:97)
FIG. 39B presents the amino acid sequence of EcAdhE (protein sequence NP_415757). (SEQ ID NO:98)
FIG. 39C is a schematic representation of the gene organization for construct pVZ321b-PisiA-PDC-EcAdhE.
FIG. 39D is a schematic representation of the gene organization for construct pVZ321b-PntcA-PDC-EcAdhE.
FIG. 39E is a schematic representation of the gene organization for construct pVZ321b-PnblA-PDC-EcAdhE.
FIG. 40A presents the nucleotide sequence of ThAdhE, the adhE gene (tlr0227) from Thermosynechococcus elongatus BP-1. (SEQ ID NO:99)
FIG. 40B presents the amino acid sequence of ThAdhE (protein sequence BAC07780). (SEQ ID NO:100)
FIG. 40C is a schematic representation of the gene organization for the construct pVZ321b-PisiA-ThAdhE.
FIG. 40D is a schematic representation of the gene organization for the construct pVZ321b-PntcA-ThAdhE.
FIG. 40E is a schematic representation of the gene organization for the construct pVZ321b-PnblA-ThAdhE.
FIG. 41A presents the nucleotide sequence of ZpPdcpdc gene from Zymobacter palmae ATCC 51623 (SEQ ID NO:101)
FIG. 41B presents the amino acid sequence of ZpPdc (protein sequence AAM49566). (SEQ ID NO:102)
FIG. 42A presents the nucleotide sequence of pSK10 cloning vector (derivate of pSK9 [V. V. Zinchenko, Moscow, Russia; unpublished]). (SEQ ID NO:103)
FIG. 42B is a schematic representation of the gene organization for the plasmid pSK10.
FIG. 42C is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-ADHII.
FIG. 42D is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-ADHII.
FIG. 42E is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-ADHII.
FIG. 42F is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-ADHI.
FIG. 42G is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-ADHI.
FIG. 42H is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-ADHI.
FIG. 42I is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-SynADH.
FIG. 42J is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-SynADH.
FIG. 42K is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-SynADH.
FIG. 42L is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-EcAdhE.
FIG. 42M is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-EcAdhE.
FIG. 42N is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-EcAdhE.
FIG. 42O is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-ThAdhE.
FIG. 42P is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-ThAdhE.
FIG. 42Q is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-ThAdhE.
FIG. 42R presents the nucleotide sequence of the crhC promoter (Anabaena sp. PCC7120) (crhC gene: alr4718, RNA helicase crhC cold shock inducible) (SEQ ID NO:104).
FIG. 42S presents the nucleotide sequence of the petE promoter (Anabaena sp. PCC7120) petE gene: all0258, plastocyanin precursor (petE) induced by addition of Cu (SEQ ID NO:X). (SEQ ID NO:105)
FIG. 42T presents the gene organization of plasmid pRL1049-PpetE-PDC-ADHII.
FIG. 42U presents the nucleotide sequence of plasmid pRL1049-PpetE-PDC-ADHII (SEQ ID NO:106).
FIG. 42V depicts the gene organization of plasmid pRL593-PisiA-PDC-ADHII.
FIG. 42W presents the nucleotide sequence of plasmid pRL593-PisiA-PDC-ADHII (SEQ ID NO:107).
FIG. 42X is a graphic depiction of ethanol production rate in Anabaena PCC7120 harboring pRL593-PisiA-PDC-ADHII following induction by iron starvation was measured in BG11 medium (+N) and in medium lacking combined nitrogen (−N) in day (12 h)/night (12 h) cycle.
FIG. 42Y is a graphic depiction of ethanol production rate in Anabaena PCC7120 harboring pRL593-PisiA-PDC-ADHII following induction by iron starvation was measured in BG11 medium (+N) and in medium lacking combined nitrogen (−N) in day (12 h)/night (12 h) cycle, wherein values are normalized for optical density.
FIG. 43A is a photographic depiction of a Western Blot that was used to quantify the induction rate of the used promoters by determining the relative abundance of the Z. mobilis ADHII and PDC enzymes expressed in Synechocystis with and without nutrient starvation.
FIG. 43B is a photograph of a Western Blot that was used to determine the relative abundance of the Z. mobilis ADHII and PDC enzymes expressed in Synechocystis with and without nutrient starvation.
FIG. 44A is a graphic representation of ethanol production rates of genetically modified photoautotrophic host cells containing Zymomonas mobilis PDC and ADHII as a second genetic modification.
FIG. 44B is a graphic representation of ethanol production in Synechocystis pVZ mutants having ZmPdC and ZmADHII under the control of isiA, and iron-dependent promoter.
FIG. 44C is a graphic presentation of ethanol production in glycogen deficient Synechocystis pVZ mutants having ZmPdC and ZmADHII under the control of isiA, an iron-dependent promoter.
FIG. 44D is a graphic presentation of ethanol production in Synechocystis pVZ mutants having ZmPdC and SynADH under the control of rbcLS, a constitutive promoter.
FIG. 45 is a graphic presentation of ethanol production in Synechocystis expressing different 3 variants of E. coli AdhE compared to wild-type.
FIG. 46A is a graphic representation of growth over time for the captioned mutant strains.
FIG. 46B is a graphic representation of ethanol production over time (% v/v) for the captioned mutant strains.
FIG. 46C is a graphic representation of ethanol production per growth for the captioned mutant strains.
FIG. 46D is a graphic representation of measurements on outgas samples of Synechocystis mutants that express ZmPdc/ZmAdhI (dashed line), ZmPdc/ZmAdhII (solid line) and ZmPdc/SynAdh (dotted line) analysed by gas chromatography. The grey arrow indicates the acetaldehyde, and the black arrow indicates the ethanol peak.
FIG. 46E is a graphic depiction of acetaldehyde production after addition of ethanol in different concentrations. Wild type and ethanol producing transgenic cells are presented.
FIG. 46F is a graphic depiction of the pH-dependency of acetaldehyde reduction by crude extracts containing the Synechocystis Adh.
FIG. 46G is a graphic depiction summarizing the acetaldehyde reduction rates at different cosubstrate concentrations. Measurements were performed at pH 6.1
FIG. 46H is a graphic depiction of Lineweaver-Burk plots, which depict the reciprocal of the rate of acetaldehyde reduction versus the reciprocal of the concentration of NADH (squares) or NADPH (rhombi), respectively. Km and vmax values are discussed in the text.
46-I is a photographic depiction of SDS/PAGE analysis of recombinantly expressed SynADH showing that SynADH was enriched, but not purified to homogeneity.
FIG. 47A is a clade analysis of alcohol dehydrogenase enzymes.
FIG. 47B(1) is a table of several alcohol dehydrogenase genes from subclade A, indicating the originating organism, the accession number, gene information, and protein designation.
FIG. 47B(2) is a table of several alcohol dehydrogenase genes from subclade B, indicating the originating organism, the accession number, gene information, and protein designation.
FIG. 47B(3) is a table of several alcohol dehydrogenase genes from subclade C, indicating the originating organism, the accession number, gene information, and protein designation.
FIGS. 47C through 47X depict the amino acid sequences of several proteins described herein, which are also present in the sequence listing.
FIG. 48A is a graphic depiction of the OD750 growth properties of Synechocystis wild type and mutants that express Pdc/Adh enzyme and Pdc enzyme alone.
FIG. 48B is a graphic depiction of ethanol production for Synechocystis wild type and mutants that express Pdc/Adh enzyme and Pdc enzyme alone.
FIG. 48C is a tabular presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented graphically in FIG. 48D.
FIG. 48D is a graphical presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented in tabular form in FIG. 48C.
FIG. 48E is a tabular presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented graphically in FIG. 48F.
FIG. 48F is a graphical presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented in tabular form in FIG. 48E.
FIG. 48G is a tabular presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented graphically in FIG. 48H.
FIG. 48H is a graphical presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented in tabular form in FIG. 48G.
FIG. 48I is a tabular presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented graphically in FIG. 48J.
FIG. 48J is a graphical presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented in tabular form in FIG. 48I.
FIG. 49A is a tabular presentation of cyanobacterial promoters used to express ethanologenic enzymes in Synechocystis 6803.
FIG. 49B is a graphic depiction of growth properties of 6803 transformed with pVZ321b-PisiA-PDC/ADH as monitored by determining the OD750.
FIG. 49C is a graphic depiction of iron-induced ethanol production of 6803 transformed with pVZ321b-PisiA-PDC/ADH.
FIG. 49D is a graphic depiction of ethanol production of Synechocystis 6803 pVZ321b-PnblA-PDC/ADH that express Pdc/Adc enzymes under the control of the nitrogen dependent nblA-promoter.
FIG. 49E is a graphic depiction of the growth properties of cells with PnirA-PDC when nitrogen is provided by ammonia or nitrate.
FIG. 49F is a graphic depiction of ethanol production of cells with PnirA-PDC when nitrogen is provided by ammonia or nitrate.
FIG. 49G is a graphic depiction of ethanol production normalized for culture optical density of cells with PnirA-PDC when nitrogen is provided by ammonia or nitrate.
FIG. 49H is a graphic depiction of growth of Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH.
FIG. 49I is a graphic depiction of ethanol production of Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH.
FIG. 49J is a graphic depiction ethanol productivity per growth of Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH.
FIG. 49K is a graphic depiction of the growth of Synechocystis 6803 pVZ321b-PpetE-PDC/ADH.
FIG. 49L is a graphic depiction ethanol production of Synechocystis 6803 pVZ321b-PpetE-PDC/ADH.
FIG. 49M is a graphic depiction of ethanol production of Synechocystis 6803 pVZ321b-PcrhC-PDC/ADH.
FIG. 49N is a graphic depiction of growth properties of Synechocystis 6803 pVZ321b-PhspA-PDC, pVZ321b-PhtpG-PDC, pVZ321b-PhliB-PDC and pVZ321b-PclpB1-PDC.
FIG. 49O is a graphic depiction of ethanol production of Synechocystis 6803 pVZ321b-PhspA-PDC, pVZ321b-PhtpG-PDC, pVZ321b-PhliB-PDC and pVZ321b-PclpB1-PDC.
FIG. 49P is a graphic presentation of growth properties under different conditions of cells containing pVZ321b-PpetJ-PDC/SynADH.
FIG. 49Q is a graphic presentation of ethanol production under different growth conditions of cells containing pVZ321b-PpetJ-PDC/SynADH.
FIG. 49R is a graphic presentation of ethanol production per OD under different growth conditions of cells containing pVZ321b-PpetJ-PDC/SynADH.



DETAILED DESCRIPTION OF EMBODIMENTS
Definitions
As used herein, the term “genetically modified” refers to any change in the endogenous genome of a wild type cell or to the addition of non-endogenous genetic code to a wild type cell, e.g., the introduction of a heterologous gene. More specifically, such changes are made by the hand of man through the use of recombinant DNA technology or mutagenesis. The changes can involve protein coding sequences or non-protein coding sequences such as regulatory sequences as promoters or enhancers.
The term “nucleic acid” is intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. The gene can be endogenous to the host cell or can be recombinantly introduced into the host cell.
The phrase “operably linked” means that the nucleotide sequence of the nucleic acid molecule or gene of interest is linked to the regulatory sequence(s) in a manner which allows for expression (e.g., enhanced, increased, constitutive, basal, attenuated, decreased or repressed expression) of the nucleotide sequence and expression of a gene product encoded by the nucleotide sequence (e.g., when the recombinant nucleic acid molecule is included in a recombinant vector, as defined herein, and is introduced into a microorganism).
The term “recombinant nucleic acid molecule” includes a nucleic acid molecule (e.g., a DNA molecule) that has been altered, modified or engineered such that it differs in nucleotide sequence from the native or natural nucleic acid molecule from which the recombinant nucleic acid molecule was derived (e.g., by addition, deletion or substitution of one or more nucleotides). Advantageously, a recombinant nucleic acid molecule (e.g., a recombinant DNA molecule) includes an isolated nucleic acid molecule or gene of the present invention
The terms “host cell” and “recombinant host cell” are intended to include a cell suitable for genetic manipulation, e.g., which can incorporate heterologous polynucleotide sequences, e.g., which can be transfected. The cell can be a prokaryotic or a eukaryotic cell. The term is intended to include progeny of the cell originally transfected. In particular embodiments, the cell is a prokaryotic cell, e.g., a cyanobacterial cell. Particularly, the term recombinant host cell is intended to include a cell that has already been selected or engineered to have certain desirable properties and suitable for further modification using the compositions and methods of the invention.
The term “promoter” is intended to include a polynucleotide segment that can transcriptionally control a gene-of-interest, e.g., a pyruvate decarboxylase gene, that it does or does not transcriptionally control in nature. In one embodiment, the transcriptional control of a promoter results in an increase in expression of the gene-of-interest. In another embodiment, a promoter is placed 5′ to the gene-of-interest. A promoter can be used to replace the natural promoter, or can be used in addition to the natural promoter. A promoter can be endogenous with regard to the host cell in which it is used or it can be a heterologous polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used. Promoters of the invention may also be inducible, meaning that certain exogenous stimuli (e.g., nutrient starvation, heat shock, mechanical stress, light exposure, etc.).
The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value/range, it modifies that value/range by extending the boundaries above and below the numerical value(s) set forth. In general, the term “about” is used herein to modify a numerical value(s) above and below the stated value(s) by a variance of 20%.
As used herein, the phrase “increased activity” refers to any genetic modification resulting in increased levels of enzyme in a host cell. As known to one of ordinary skill in the art, enzyme activity may be increased by increasing the level of transcription, either by modifying promoter function or by increasing gene copy number, increasing translational efficiency of an enzyme messenger RNA, e.g., by modifying ribosomal binding, or by increasing the stability of a enzyme protein, which because the half-life of the protein is increased, will lead to more enzyme molecules in the cell. All of these represent non-limiting examples of increasing the activity of an enzyme. (mRNA Processing and Metabolism: Methods and Protocols, Edited by Daniel R. Schoenberg, Humana Press Inc., Totowa, N.J.; 2004; ISBN 1-59259-750-5; Prokaryotic Gene Expression (1999) Baumberg, S., Oxford University Press, ISBN 0199636036; The Structure and Function of Plastids (2006) Wise, R. R. and Hoober J. K., Springer, ISBN 140203217X; The Biomedical Engineering Handbook (2000) Bronzino, J. D., Springer, ISBN 354066808X).
Genetic knockdown includes techniques by which the expression of one or more of the organism's genes is reduced. In genetic knockout, the gene is made inoperative.
In one aspect the invention also provides nucleic acids, which are at least 60%, 70%, 80% 90% or 95% identical to the promoter nucleic acids disclosed therein and to the nucleic acids, which encode proteins, for example enzymes for ethanol formation or host cell enzymes involved in the conversion or formation of acetyl CoA, acetaldehyde or pyruvate or for formation of reserve compounds. The invention also provides amino acid sequences for enzymes for ethanol formation or host cell enzymes involved in the conversion or formation of acetyl-CoA, acetaldehyde or pyruvate or for formation of reserve compounds, which are at least 60%, 70%, 80% 90% or 95% identical to the amino acid sequences disclosed therein.
The percentage of identity of two nucleic acid sequences or two amino acid sequences can be determined using the algorithm of Thompson et al. (CLUSTALW, 1994 Nucleic Acid Research 22: 4673-4,680). A nucleotide sequence or an amino acid sequence can also be used as a so-called “query sequence” to perform against public nucleic acid or protein sequence databases in order, for example, to identify further unknown homologous promoters, which can also be used in embodiments of this invention. In addition, any nucleic acid sequences or protein sequences disclosed in this patent application can also be used as a “query sequence” in order to identify yet unknown sequences in public databases, which can encode for example new enzymes, which could be useful in this invention. Such searches can be performed using the algorithm of Karlin and Altschul (1999 Proceedings of the National Academy of Sciences U.S.A. 87: 2,264 to 2,268), modified as in Karlin and Altschul (1993 Proceedings of the National Academy of Sciences U.S.A. 90: 5,873 to 5,877). Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al. (1999 Journal of Molecular Biology 215: 403 to 410). Suitable parameters for these database searches with these programs are, for example, a score of 100 and a word length of 12 for BLAST nucleotide searches as performed with the NBLAST program. BLAST protein searches are performed with the XBLAST program with a score of 50 and a word length of 3. Where gaps exist between two sequences, gapped BLAST is utilized as described in Altschul et al. (1997 Nucleic Acid Research, 25: 3,389 to 3,402).
Database entry numbers given in the following are for the CyanoBase, the genome database for cyanobacteria (http://bacteria.kazusa.or.jp/cyanobase/index.html); Yazukazu et al. “CyanoBase, the genome database for Synechocystis sp. Strain PCC6803: status for the year 2000”, Nucleic Acid Research, 2000, Vol. 18, page 72.
Embodiments
It is one object of embodiments of the invention to provide a genetically modified host cell, which can be used for production of ethanol.
This object is reached by providing a genetically modified host cell according to base claim 1. Further embodiments of the genetically modified host cell, as well as constructs for producing the genetically modified host cells and a method for producing ethanol using the genetically modified host cells are subject matters of further claims.
Embodiment of genetic knockout and/or overexpression of metabolic pathway enzymes
In a first aspect the invention provides a genetically modified photoautotrophic, ethanol producing host cell comprising:

    
    
        at least one first genetic modification changing the enzymatic activity or affinity of an endogenous host cell enzyme,
        the first genetic modification resulting in an enhanced level of biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or precursors thereof compared to the respective wild type host cell,
        at least one second genetic modification different from the first genetic modification comprising an overexpressed enzyme for the formation of ethanol.
    
    


Acetaldehyde, pyruvate and acetyl-coA or their precursors are important metabolic intermediates for energy production in cells. In photoautotrophic cells, which use light, CO2, and water as a source of energy to produce carbohydrates via photosynthesis, acetaldehyde, pyruvate, acetyl-CoA and their precursors can be formed by conversion of organic molecules obtained via CO2 fixation in the Calvin-cycle, for example 3-phosphoglycerate. Pyruvate, acetyl-CoA and their precursors are important metabolic intermediates obtained e.g. by photosynthetic CO2 fixation in photoautotrophic cells. Acetaldehyde is a metabolic intermediate of the anoxygenic fermentation pathway in many photoautotrophic cells. Precursors of pyruvate and acetyl-CoA are organic compounds, which can be converted into these important metabolic intermediates via the enzymatic action of enzymes of the photoautotrophic cell. For example the organic compounds 2-phosphoglycerate, 3-phosphoglycerate or phosphoenolpyruvate can be converted into pyruvate by enzymes of the glycolytic pathway in photoautotrophic cells.
The genetically modified photoautotrophic ethanol producing host cell comprises at least two different genetic modifications, a first and a second genetic modification. The first genetic modification changes the enzymatic activity or affinity of an endogenous host enzyme, resulting in a higher level of biosynthesis of acetyl-CoA, acetaldehyde, pyruvate or precursors thereof. The endogenous host enzyme is already present in an unmodified wild type host cell and its activity or affinity is changed by the first genetic modification in order to increase the level of biosynthesis of metabolic intermediates, which are also present in the wild type host cell and which can be used to form ethanol.
Furthermore the genetically modified photoautotrophic ethanol producing host cell comprises a second genetic modification in the form of at least one overexpressed enzyme, which can form ethanol, for example from the above-mentioned important metabolic intermediates. In a further embodiment the overexpressed enzyme for ethanol formation can catalyze the last step of ethanol formation leading to the final product ethanol. The overexpressed enzyme for ethanol formation can also catalyze the penultimate step of ethanol formation resulting in a metabolic intermediate, which can further be converted by another enzyme for ethanol formation into the final product ethanol.
The enzyme for ethanol formation can, for example, be an endogenous enzyme already present in a wild type photoautotrophic host cell, which is not genetically modified. In this case the activity or affinity of the enzyme for ethanol formation can be enhanced by the second genetic modification, for example by genetic engineering or random mutagenesis. This can, for example, be done by genetically modifying the amino acid sequence of the enzyme by site directed or random mutagenesis of the gene encoding this endogenous enzyme, thereby enhancing its activity for formation of ethanol. Another possibility is to increase the number of gene copies encoding for the enzyme in the host cell or simply by enhancing the rate of transcription of the gene already present in the wild type cell to increase the abundance of its messenger RNA in the second genetic modification. This can be done for example by replacing or mutating the endogenous promoter controlling the transcription of the endogenous gene encoding the enzyme for ethanol formation.
Alternatively or additionally a heterologous enzyme for ethanol formation can be introduced into the host cell by the second genetic modification, if that enzyme is not present in an genetically unmodified wild type host cell. This can be done, for example, by introducing a construct, for example a DNA vector into the host cell including a heterologous gene encoding the overexpressed enzyme for ethanol formation. In the case that an endogenous enzyme for ethanol formation is already present in a photoautotrophic wild type host cell, the heterologous enzyme for ethanol formation can enhance the activity of the endogenous enzyme resulting in a higher rate of ethanol formation.
The enzymatic activity and the affinity of an enzyme for its substrate are important kinetic constants. The enzymatic activity is given by the parameter Vmax, which reflects the maximal velocity of an enzymatic reaction occurring at high substrate concentrations when the enzyme is saturated with its substrate. The affinity is given by the Michaelis-Menten constant Km which is the substrate concentration required for an enzyme to reach one-half its maximum velocity. In order to increase the enzymatic activity Vmax has to be increased, whereas for increasing the affinity Km has to be reduced. Regarding a further explanation of enzyme kinetics we refer to the chapter “enzyme kinetics” in the textbook “Biochemistry” by Donald Voet and Judith Voet (John Wiley & Sons, 1990, pages 335 to 340).
The higher level of biosynthesis of acetyl-CoA, acetaldehyde, pyruvate or precursors thereof results in a change of the flux of the acetyl-CoA, acetaldehyde, pyruvate or precursors thereof in the direction of the at least one overexpressed enzyme for ethanol formation so that formation of ethanol can be increased in comparison to a photoautotrophic ethanol producing host cell harboring only the second genetic modification, but lacking the first genetic modification.
Acetyl-CoA, acetaldehyde, pyruvate or precursors thereof are transient metabolic intermediates, which are often rapidly processed into other metabolites by the photoautotrophic host cell and therefore a change in the level of biosynthesis of these metabolic intermediates can be hard to detect in photoautotrophic host cells featuring the first genetic modification but lacking the second genetic modification.
A first genetic modification therefore results in a higher level of biosynthesis of acetyl-CoA, acetaldehyde, pyruvate or precursors thereof compared to the respective wild type host cell, if after introduction of the second genetic modification a high level of ethanol formation can be detected, even if a change in the level of biosynthesis of these metabolic intermediates could not be detected in the photoautotrophic host cell harboring the first genetic modification but lacking the second genetic modification.
The genetically modified photoautotrophic host cell can comprise more than one first genetic modification and also more than one second genetic modification. For example the first genetic modification can comprise at least two genetic modifications, one first genetic modification (a), which is a down-regulation or a knock out of gene expression of a metabolic enzyme and at least one further first genetic modification (b), which is an increase in metabolic enzyme activity and/or substrate affinity for a endogenous enzyme for formation of acetyl-CoA, pyruvate or acetaldehyde or precursors thereof.
In a further embodiment thereof, the total number of possible one first genetic modifications (a) is represented by N, wherein N is a number from 0 to 50, and N indicates the number of genetic modifications resulting in the down-regulation or knockout of metabolic enzyme activity and/or substrate affinity, and the number of further first genetic modifications (b) is represented by P, wherein P is a number from 0 to 50, resulting in an increase in metabolic enzyme activity and/or substrate affinity for a endogenous enzyme for formation of acetyl-CoA, pyruvate or acetaldehyde or precursors thereof. The numerical values for genetic modification (a) N and genetic modification (b) P are selected independently from one another as long as the sum of P+N is at least one. By way of non-limiting example, (a) N may have a numerical value of 1, indicating a single genetic modification, and (b) P may have a numerical value of 2, indicating two genetic modifications. Alternatively (a) N may have numerical value of 2, indicating two genetic modifications, and (b) P may have a numerical value of 1, indicating a single genetic modification. Thus, as will be understood to those skilled in the art, the invention provides herein for a wide variety of genetically modified, photoautotrophic ethanol producing host cells comprising a multitude of genetic modifications, the combination of which result in an enhanced level of biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or precursors thereof.
The genetically modified photoautotrophic host cell shows a high production of ethanol due to the fact that the ethanol forming enzyme is overexpressed due to the second genetic modification leading to a high enzymatic activity or activity for ethanol formation and that at the same time a higher level of biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or their precursors is formed in the cells compared to the respective wild type cells due to the first genetic modification. Acetaldehyde, pyruvate, acetyl-CoA or their precursors serve as substrates for the ethanol production. These metabolic intermediates can either be a direct substrate for the overexpressed enzyme for the formation of ethanol or for another second overexpressed enzyme for ethanol formation, which then catalyzes the formation of a substrate for the first overexpressed enzyme for ethanol formation.
In yet another embodiment of the genetically modified host cell

    
    
        the at least one endogenous host cell enzyme is selected from enzymes of the glycolysis pathway, Calvin-cycle, intermediate steps of metabolism, amino acid metabolism, the fermentation pathway and the citric acid cycle, wherein the activity of at least one of these enzymes is enhanced compared to the respective wild type host cell.
        Enzymes of intermediate steps of metabolism are enzymes which connect different metabolic pathways. For example the glycolysis and the citric acid cycle are connected via the enzyme malic enzyme converting malate into pyruvate.
    
    


In particular the endogenous host cell enzyme can be selected from only one of the above pathways or in the case that more than one endogenous host cell enzyme is mutated in a first genetic modification can be selected from any possible combination of the above pathways.
The Calvin-cycle is an important part of photosynthesis and includes the light-independent reactions, where CO2 is captured from the environment of the cell and converted into organic compounds, for example three-carbon compounds such as 3-phosphoglycerate. CO2 may also be captured by alternative routes into four-carbon compounds such as oxaloacetate. These processes are also referred to as C3-carbon fixation and C4-carbon fixation.
Photosynthetic CO2 fixation can lead to the production of carbon storage compounds such as reserve carbohydrates like glycogen, starch or sucrose.
The glycolysis pathway is normally the first step of carbohydrate catabolism in order to generate adenosine triphosphate (ATP) and reductants such as nicotinamide adenine dinucleotide (NADH). Glycolysis furthermore can produce pyruvate which is an important compound for the citric acid cycle that generates reductant for aerobic respiration and intermediates for biosynthesis. Furthermore, glycolysis serves to synthesize various 6- and 3-carbon intermediate compounds which can be used for other cellular processes such as amino acid biosynthesis.
Pyruvate produced via glycolysis is one of the major sources for the citric acid cycle, which is an important part of a metabolic pathway for the chemical conversion of carbo-hydrates, fat and proteins into carbon dioxide and water to generate energy for the host cell. Pyruvate can, for example, be fed into the citric acid cycle via acetyl-CoA (acetyl-CoA). Furthermore, pyruvate can also be metabolized to acetaldehyde via other enzymes. Therefore, enhancing the activity or affinity of at least one of the endogenous host cell enzymes of the Calvin-cycle or glycolysis pathway or the citric acid cycle in a first genetic modification can result in a higher level of biosynthesis of pyruvate, or acetyl-CoA or their precursors, respectively. This in turn can result in a higher ethanol production due to the fact that these metabolic intermediates can be ultimately converted to ethanol via the at least one overexpressed enzyme for the formation of ethanol provided by the second genetic modification.
In certain aspects and embodiments of the invention the enzymatic activity or affinity of any of these enzymes can be enhanced, for example, by increasing the activity or affinity of the enzymes present in the wild type host cell. Non-limiting examples contemplated by the invention include site directed mutagenesis or random mutagenesis and by increasing the amount of enzymes in the host cell. The latter is achieved, for example by introducing mutations in the promoter regions controlling the transcriptional activity of the genes encoding the enzymes or by introducing additional gene copies coding for these enzymes into the host cell.
In a further embodiment at least one enzyme of the glycolysis pathway, the citric acid cycle, the intermediate steps of metabolism, the amino acid metabolism, the fermentation pathway or the Calvin-cycle of the host cell is overexpressed. Overexpression of an enzyme already present in a wild type host cell is an effective method to enhance the enzymatic activity of enzymes in a cell. Overexpression can also be achieved by introducing a heterologous enzyme into the host cell, which exhibits the same enzymatic activity as the host cell enzyme, which should be overexpressed. For example if 3-phosphoglycerate mutase should be overexpressed in the cyanobacterium Synechocystis a plasmid comprising a heterologous gene encoding 3-phosphoglycerate mutase from Zymomonas mobilis can be introduced into the host cell. Another non-limiting example is the overexpression of pyruvate kinase from E. coli in Synechocystis, thereby raising the enzymatic activity of the endogenous host cell enzyme pyruvate kinase in Synechocystis. 
In the case that the enzymatic activity of malate dehydrogenase, an enzyme of the citric acid cycle and malic enzyme, an enzyme of the intermediate steps of metabolism is enhanced, malate dehydrogenase can stimulate the conversion of oxaloacetate to pyruvate via malate. Malate dehydrogenase catalyzes the conversion of oxaloacetate to malate using NADH:

Oxaloacetate+NADH+H+→malate+NAD+

Malic enzyme catalyzes the conversion of malate into pyruvate using NADP+:

malate+NADP+→pyruvate+CO2+NADPH

In C4-plants the released CO2 can be fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) and NADPH can be used for CO2-fixation in the Calvin-cycle. The enzymatic activity or affinity of RubisCO can be enhanced in a first genetic modification in order to increase the CO2-fixation and direct more carbon towards ethanol formation. This can be done for example by overexpressing only the small and the large subunits of RubisCO or a complete RubisCO operon also including a RubisCO Chaperonin in the photoautotrophic host cells such as prokaryotic cells. The RubisCO Chaperonin can assist in the folding of the RubisCO enzyme, which is a complex of eight large and eight small subunits in cyanobacteria and algae. The binding sites for the substrate ribulose 1,5-bisphosphate are located on the large subunits, whereas the small subunits have regulatory functions. RubisCO catalyzes bifunctional the initial step in the carbon dioxide assimilatory pathway and photorespiratory pathway in photosynthetic organisms. The enzyme catalyzes the carboxylation of ribulose-1,5-bisphosphate into two molecules of 3-phosphoglycerate (3-PGA) in the carbon dioxide assimilatory pathway, but also the oxygenation of ribulose-1,5-bisphosphate resulting in 3-PGA and 2-Phosphoglycolate (2-PG) in the photorespiratory pathway. In order to direct the carbon provided by the CO2-fixation into ethanol formation, the carbon dioxide assimilatory pathway has to be enhanced and the activity of the photorespiratory pathway has to be reduced. Some photoautotrophic cells such as cyanobacteria have mechanisms to actively uptake CO2 and HCO3− and to raise the CO2-concentration in the proximity of RubisCO (Badger M. R., and Price, G. D. (2003) J. Exp. Bot. 54, 609-622). This reduces the oxygenase activity of the enzyme. Nevertheless the cyanobacterial photosynthesis is not efficient enough to completely abolish the formation of 2-PG. Cyanobacteria produce significant amounts of 2-PG, particularly at elevated oxygen concentrations or after a change to low CO2-concentrations.
In order to enhance the carbon dioxide fixating activity of RubisCO random or side directed mutagenesis can be performed to achieve higher CO2 fixation according to some embodiments of the invention. Efforts to select RubisCO enzymes with improved activity using random mutagenesis were successful when the large subunit of RubisCO from Synechococcus PCC 7942 was mutagenized and co-expressed with the small subunit of RubisCO and phosphoribulokinase (prkA) in E. coli (Directed evolution of RubisCO hypermorphs through genetic selection in engineered E. coli, Parikh et al, Protein Engineering, Design & Selection vol. 19 no. 3 pp. 113-119, 2006). This strategy was also successful in the case of the similar enzymes from Synechococcus PCC 6301 in E. coli (Artificially evolved Synechococcus PCC 6301 RubisCO variants exhibit improvements in folding and catalytic efficiency, Greene et al., Biochem J. 404 (3): 517-24, 2007).
Another way of increasing the enzymatic activity of RubisCO according to the invention involves overexpressing heterologous RubisCO in order to increase the CO2 fixation as it was shown in case of the heterologous expression of RubisCO from Allochromatium vinosum in Synechococcus PCC 7942 (Expression of foreign type I ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) stimulates photosynthesis in cyanobacterium Synechococcus PCC 7942 cells, Iwaki et al, Photosynthesis Research 88: 287-297, 2006).
Overexpression of RubisCO in photoautotrophic host cells such as cyanobacteria also harboring at least one overexpressed enzyme for the formation of ethanol surprisingly not just results in an increased activity of RubisCO, but also leads to an increased biomass of the cells and a higher growth rate accompanied by a slight increase in the rate of ethanol production.
In addition the photorespiration activity of RubisCO can be reduced or eliminated by random or side directed mutagenesis.
Certain embodiments of the invention relate to the overexpression of at least one enzyme from the glycolysis pathway. Non-limiting examples are phosphoglycerate mutase, enolase and pyruvate kinase.
Phosphoglycerate mutase catalyzes the reversible reaction leading from 3-phosphoglycerate formed in the Calvin-cycle to 2-phosphoglycerate. 2-phosphoglycerate in turn can then, in a reversible reaction catalyzed by the enzyme enolase, be converted to phosphoenolpyruvate. Phosphoenolpyruvate can further be converted to pyruvate via the enzymatic action of pyruvate kinase. Therefore, enhancing the activity of any or all of these enzymes enhances the pyruvate pool in the host cell by enhancing the conversion of 3-phosphoglycerate formed in the Calvin-cycle to pyruvate. Pyruvate itself can then either be a direct substrate for the at least one overexpressed enzyme for ethanol formation or it can further be converted into another intermediate, which then can be further metabolized by the enzyme for ethanol formation in order to form high amounts of ethanol.
An enzyme of the fermentation pathway, which can be overexpressed is for example the acetaldehyde dehydrogenase enzyme, which can convert acetyl-CoA to acetaldehyde, thereby increasing the level of biosynthesis of acetaldehyde in the host cell. Alternatively other aldehyde dehydrogenases enzymes could be expressed in order to increase the level of biosynthesis of acetaldehyde in the host cell.
Enzymes of the intermediate steps of metabolism, which can be overexpressed are for example pyruvate dehydrogenase enzyme converting pyruvate into acetyl-CoA, increasing the level of biosynthesis of acetyl-CoA in the host cell. In addition or alternatively phosphotransacetylase converting acetyl-CoA to acetylphosphate can be overexpressed in the host cell, thereby increasing the level of biosynthesis of acetaldehyde in the host cell.
Another non-limiting example of an enzyme, whose activity or affinity can be increased is the enzyme PEP-carboxylase (phosphoenolpyruvate carboxylase). This enzyme catalyzes the addition of CO2 to phosphoenolpyruvate (PEP) to form the four-carbon compound oxaloacetate (OAA). This PEP-carboxylase catalyzed reaction is used for CO2 fixation and can enhance the photosynthetic activity leading to higher CO2 fixation, which can be used for ethanol formation.
In particular the enzymatic activity or affinity of PEP-carboxylase, malate dehydrogenase and malic enzyme can be enhanced concomitantly. This leads to a higher CO2 fixation and an enhanced level of biosynthesis of pyruvate. In addition the decarboxylation of malate to pyruvate catalyzed by the enzyme malic enzyme, enhances the CO2 partial pressure leading to an increased efficiency of the Calvin cycle. PEP-carboxylase is used for CO2 fixation in C4-plants and can also be found in cyanobacteria.
Furthermore it is possible to overexpress enzymes of the amino acid metabolism of the host cell, which for example convert certain amino acids into pyruvate leading to an enhanced biosynthesis of pyruvate in the host cell. For example serine can directly be converted to pyruvate in the cyanobacterium Synechocystis PCC 6803. The open reading frame slr 2072, which is annotated as ilvA (threonine dehydratase), EC 4.3.1.19, can catalyze the deamination of serine to pyruvate.
According to a further aspect of the invention the enzymatic activity or affinity of the enzyme phosphoketolase (EC 4.1.2.-, putative phosphoketolase in Synechocystis PCC 6803 slr 0453) is enhanced in a first genetic modification in order to increase the level of biosynthesis of precursor molecules for the generation of acetyl-CoA and acetaldehyde. Phosphoketolase catalyses the formation of acetyl phosphate and glyceraldehyde 3-phosphate, a precursor of 3-phosphoglycerate from xylulose-5-phosphate which is an intermediate of the Calvin cycle.
According to another embodiment of the invention in combination with enhancing the enzymatic activity or affinity of phosphoketolase enzyme, the polyhydroxybutyrate (PHB) pathway is knocked out in order to avoid PHB accumulation due to an increased level of acetyl-CoA biosynthesis (Control of Poly-β-Hydroxybutyrate Synthase Mediated by Acetyl Phosphate in Cyanobacteria, Miyake et al., Journal of Bacteriology, p. 5009-5013, 1997). Additionally AdhE can be overexpressed at the same time to convert the acetyl-CoA to ethanol.
Endogenous host enzymes of the glycolysis pathway, the Calvin-cycle, the intermediate steps of metabolism, the amino acid metabolism pathways, the fermentation pathways or the citric acid cycle, can be dependent upon a cofactor. The invention also provides an enhanced level of biosynthesis of this cofactor compared to the respective wild type host cell, thereby increasing the activity of these enzymes. Such an enhanced level of biosynthesis of this cofactor can be provided in a first genetic modification.
An enhanced level of the cofactor biosynthesis also results in an enhanced enzymatic activity or affinity of these above mentioned enzymes and therefore in an enhanced level of biosynthesis of pyruvate, acetyl-CoA, acetaldehyde or their precursors in the cell.
For example, alcohol dehydrogenase enzymes are often NAD+/NADH cofactor dependent enzymes. In this case, their enzymatic activity can be enhanced by raising the level of NADH biosynthesis in the host cell. This can, for example, be done by overexpressing NAD(P)+ transhydrogenases, which transfer reduction equivalents between NADP(H) to NAD(H). These NAD(P)+ transhydrogenases are oxidoreductases.
Furthermore the host cell can comprise a host NADH dehydrogenase converting NADH to NAD+ wherein the activity of the NADH dehydrogenase is reduced compared to the wild type host cell.
For example, point mutations can be introduced into the gene encoding the NADH dehydrogenase in order to reduce the activity or affinity of this enzyme or alternatively the gene encoding the NADH dehydrogenase can be knocked-out by inserting for example heterologous nucleic acid sequences into the gene, thereby disrupting it.
Alternatively, in order to enhance the enzymatic activity of an enzyme, which is NADP+/NADPH cofactor dependent as, for example the malic enzyme, the level of NADP+/NADPH in the host cell also can be increased.
In many of photoautotrophic cells the level of NAD+ plus NADH to NADP+ plus NADPH is around 1:10. Due to this high imbalance of NADH to NADPH, the conversion of an NADP+/NADH cofactor specific enzyme via site directed mutagenesis or random mutagenesis of the enzyme into an NADP+/NADPH dependent enzyme can increase its activity. The changing of the cofactor specificity of alcohol dehydrogenase via in vitro random mutagenesis is for example described in the publication “Alteration of Substrate Specificity of Zymomonas mobilis Alcohol Dehydrogenase-2 Using in Vitro Random Mutagenesis” (Protein Expression and Purification Volume 9, Issue 1, February 1997, Pages 83-90).
A further embodiment of the invention provides a genetically modified host cell

    
    
        wherein the at least one endogenous host cell enzyme is for the conversion of pyruvate or acetyl-CoA or for the formation of reserve compounds, wherein its activity or affinity is reduced.
    
    


Alternatively or in addition to enhancing the activity of enzymes forming pyruvate, acetaldehyde, acetyl-CoA or precursors thereof, the activity of the enzymes converting the above-mentioned important intermediate metabolic compounds into other compounds can be reduced by the way of the first genetic modification. The inventors found out that by reducing the activity of at least one of these enzymes the level of biosynthesis of pyruvate, acetyl-CoA, acetaldehyde or their precursors can be risen compared to a wild type host cell. In addition, the inventors made the observation that by reducing the activity of host enzymes forming reserve compounds, for example glycogen, more carbohydrates formed via photosynthesis in the photoautotrophic host cells are shuffled into the glycolysis pathway and the citric acid cycle, thereby enhancing the level of biosynthesis of pyruvate, acetaldehyde, acetyl-CoA or their precursors. Due to the fact that these metabolic intermediates are used by at least one overexpressed enzyme for the formation of ethanol, a higher ethanol production of such a genetically modified host cell can be observed.
The enzymatic activity of at least one of these enzymes can be reduced, for example by introducing point mutations into the genes encoding these enzymes, thereby reducing the activity of these enzymes. Alternatively or in addition, the promoter regions controlling the transcriptional activity of these genes can be mutated, resulting in a lower transcriptional activity and therefore a reduced level of protein translation in the genetically modified host cell.
A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide.
A “promoter” is an array of nucleic acid control sequences that direct transcription of an associated nucleic acid sequence, which may be a heterologous or endogenous nucleic acid sequence. A promoter includes nucleic acid sequences near the start site of transcription, such as a polymerase binding site for a RNA polymerase used for the synthesis of messenger RNA. The promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
Furthermore, it is possible that the host cell comprises disruptions in the host gene encoding at least one of the enzymes of the host cell converting pyruvate, acetyl-CoA, the precursors thereof or for forming reserve compounds. In this case, the enzymatic activity of the enzymes can be eliminated to a full extent due to the fact that the disrupted gene does not encode for a functional protein anymore.
The disruption of the gene can be furthermore caused by an insertion of a biocide resistance gene into the respective gene. This has the advantage that so-called “knockout mutants” containing the insertions in the respective genes can easily be selected by culturing the genetically modified host cells in selective medium containing the biocide to which the genetically modified host cell is resistant.
The term “biocide” refers to a chemical substance, which is able to inhibit the growth of cells or even kill cells, which are not resistant to this biocide. Biocides can include herbicides, algaecides and antibiotics, which can inhibit the growth of plants, algae or microorganisms such as bacteria, for example cyanobacteria.
Alternatively or in addition for disrupting the gene encoding one of the enzymes converting pyruvate, acetyl-CoA or acetaldehyde or forming reserve compounds, the enzymatic activity of one of these enzymes can also be reduced by using the antisense messenger RNA concept.
A wild type cell normally comprises at least one host gene encoding for the host enzyme or protein, wherein transcription of this gene results in a sense messenger RNA (mRNA), which codes for the functional protein and is translated into the protein via translation mediated by the ribosomes, ribonucleoprotein complexes present in cells. The messenger RNA is normally a single stranded RNA molecule encoding the amino acid sequence of the enzyme in the form of the genetic code. Specifically, the genetic code defines a mapping between tri-nucleotide sequences called codons in the messenger RNA and the amino acids of the amino acid sequence; every triplet of nucleotides in a nucleic acid sequence of the mRNA specifies a single amino acid. This messenger RNA molecule is normally called sense RNA. In order to reduce or even eliminate the enzymatic activity of the enzyme encoded by this gene a nucleic acid sequence can be introduced into the host cell, which upon transcription results in a RNA strand complementary to the sense messenger RNA strand, the so-called antisense RNA. This antisense RNA can then interact with the sense RNA, forming a double-stranded RNA species which cannot be translated by the ribosomes into a functional protein anymore. Depending on the ratio of the sense RNA to the antisense RNA in the host cell, the level of enzymatic activity of the enzyme can be reduced or even eliminated. Different antisense RNA approaches for the regulation of gene expression are described in the following publications:

    Dühring U, Axmann I M, Hess W R, Wilde A.
    “An internal antisense RNA regulates expression of the photosynthesis gene isiA” (Proc Natl Acad Sci USA. 2006 May 2; 103(18):7054-8).
    Udekwu K I, Darfeuille F, Vogel J, Reimegård J, Holmqvist E, Wagner E G.
    “Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA” (Genes Dev. 2005 Oct. 1; 19(19):2355-66)


Prime enzyme targets for down regulation of enzymatic activity or for elimination of enzymatic activity are ADP-glucose-pyrophosphorylase, glycogen synthase, alanine dehydrogenase, lactate dehydrogenase, pyruvate water dikinase, phosphotransacetylase, and acetate kinase as well as pyruvate dehydrogenase.
ADP-glucose-pyrophosphorylase catalyzes the conversion of glucose-1-phosphate into ADP-glucose, which is a precursor for the reserve polysaccharide glycogen in many photoautotrophic host cells. The enzyme glycogen synthase catalyzes the addition of further glucose monomers donated by ADP glucose to the ends of glycogen primers.
The inventors found out that by reducing or even eliminating the formation of reserve carbohydrates such as starch or glycogen, the level of biosynthesis of pyruvate, acetyl-CoA or acetaldehyde can be raised compared to the level of biosynthesis a wild type host cell. This finding was particularly true for the reduction of the enzymatic affinity and activity of glycogen synthase and ADP-glucose-pyrophosphorylase. A knock out of both enzymes in photoautotrophic host cells lacking at least one overexpressed enzyme for ethanol production as a second genetic modification resulted in a big increase of pyruvate secreted into the growth medium. Further introducing a second genetic modification into these photoautotrophic host cells resulted in an increased fraction of fixed carbon being diverted to ethanol production.
Alanine dehydrogenase catalyzes the reversible reductive amination of pyruvate to alanine using NADH as a reductant. A reduction of activity of alanine dehydrogenase can result in a higher level of pyruvate.
The enzyme lactate dehydrogenase catalyzes the inter-conversion of pyruvate to the fermentative end product lactate using NADH as a reductant. Reducing or inhibiting the enzymatic action of lactate dehydrogenase can result in an increase of the level of biosynthesis of pyruvate in the genetically modified host cell.
The enzyme pyruvate water dikinase catalyzes the ATP-dependent conversion of pyruvate, ATP and water to adenosine monophosphate (AMP), phosphoenolpyruvate and phosphate. Due to that a reduction of the enzymatic activity of pyruvate water dikinase can also result in an increased level of pyruvate in the host cell.
The enzyme phosphotransacetylase catalyzes the reversible transfer of an acetyl group from acetyl-CoA to a phosphate thereby forming acetylphosphate. A reduction of the enzymatic activity of this enzyme can also result in an increased level of acetyl-CoA as well as of its precursor pyruvate.
The enzyme acetate kinase catalyzes the conversion of acetylphosphate to the fermentative end product acetate whereas the phosphate group is transferred from acetylphosphate to adenosine diphosphate (ADP) so adenosine triphosphate (ATP) is formed. An inactivation or a reduction of the enzymatic activity of this enzyme can therefore result in a higher level of acetylphosphate and maybe acetyl-CoA in the cell.
Reducing the enzymatic activity or knocking out of the gene encoding phosphotransacetylase (PTA) can be important, since this enzyme is at the branch point of acetate generation via acetylphosphate. Acetylphosphate itself is an important intermediate, because it is needed for ADP regeneration to ATP and it stimulates the activity of polyhydroxybutyrate (PHB) synthase. Knock out of the PTA therefore can avoid loss of acetyl-CoA into the acetate branch and additionally can minimize PHB generation. Thus acetyl-CoA can be channeled to the ethanol generating branch.
The inventors found out that a reduction in the enzymatic affinity or activity of the enzymes of the complete acetate fermentation pathway, in particular phosphotransacetylase and acetate kinase can lead to an increase in the ethanol production rate without reducing the photosynthetic capacity of the photoautotrophic host cells. For example a knock out of both genes coding for phosphotransacetylase and acetate kinase can enhance the ethanol production rate compared to a photoautotrophic host cell harboring only at least one overexpressed enzyme for ethanol formation as a second genetic modification but lacking the first genetic modification, the knock out mutations of both enzymes.
On the other hand acetylphosphate is the natural precursor of fermentative EtOH synthesis via acetaldehyde and therefore overexpressing the phosphotransacetylase together with the acetaldehyde forming enzyme and knocking-out or reducing the enzymatic activity of the PHB synthase can also increase the level of biosynthesis of acetaldehyde in the genetically modified host cell.
In some bacterial cells both enzymes phosphotransacetylase and acetate kinase can also catalyze the reverse reaction from acetate to acetylphosphate and from acetylphosphate to acetyl-CoA. In the case that the level of biosynthesis of acetyl-CoA should be raised compared to the wild type cells the activity or affinity of both enzymes can be enhanced for example via overexpression in different first genetic modifications. Alternatively only acetate kinase can be overexpressed in a first genetic modification in the case that the second genetic modification comprises at least acetaldehyde dehydrogenase converting the acetylphosphate to acetaldehyde and further Adh, such as AdhI and/or AdhII converting the acetaldehyde into ethanol.
Another possible target enzyme for down-regulation to increase the level of biosynthesis of pyruvate is pyruvate dehydrogenase, which catalyzes the thiamine pyrophosphate (TPP) cofactor dependent decarboxylation of pyruvate resulting in acetyl-CoA, NADH and CO2.
With regard to the enzymes forming reserve compounds for the cell, the gene for glycogen synthase can be disrupted, for example by inserting a heterologous nucleic acid sequence encoding for a biocide resistance cassette into the gene. The inventors found out that such a knockout of both glycogen synthase genes glgA1 and glgA2 in the phototropic genetically modified host cell of the genera Synechocystis results in an enhanced pyruvate level of up to 50-fold compared to the unmodified wild type host cell.
In particular, the enzymes forming one of the following reserve compounds can be a prime target for a reduction of their enzymatic activity of even for knockout: Glycogen, polyhydroxyalkanoates like, for example poly-3-hydroxybutyrate or poly-4-hydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, polyhydroxy-octanoate, amylopectin, starch, cyanophycin and their copolymers, glucosyl glycerol and bacterial extracellular polymeric substances such as extracellular polysaccharides. Enzymes which are involved in the synthesis of these reserve compounds are for example beta-ketothiolase, acetoacetyl-CoA reductase, polyhydroxybutyrate synthase, glucosylglycerolphosphate synthase.
Polyhydroxybutyrate is synthesized from acetyl-CoA via three enzymatic reactions: 3-thiolase (EC 2.3.1.9) converts two acetyl-CoA molecules to an acetoacetyl-CoA molecule, NADPH-dependent acetoacetyl-CoA reductase (EC 1.1.1.36) converts acetoacetyl-CoA to D-3-hydroxybutyryl-CoA with NADPH oxidation, and the last enzyme, PHB synthase, catalyzes the linkage of the D-3-hydroxybutyryl moiety to an existing PHB molecule by an ester bond.
The biosynthetic pathway of glucosyl glycerol begins with ADP-glucose and glycerol-3-phosphate (G3P), which are used by the GG-phosphate synthase (GGPS), and proceeds via the intermediate GG-phosphate (GGP), which is dephosphorylated to GG by the GGphosphate phosphatase (GGPP).
Hydrolyzed EPSs (bacterial extracellular polymeric substances) showed the compositional involvement of four sugar moieties viz. mannose, glucose, xylose and ribose in varying combinations. Chemical analysis of EPS revealed a heteropolysaccharidic nature, with xylose, glucose, galactose, and mannose the main neutral sugars found.
In the case that a genetically modified host cell exhibits a reduced enzymatic activity for the formation of any of the above-mentioned reserve compounds, it is expected that the precursors for these reserve compounds are fed into the glycolysis pathway or the citric acid cycle, thereby resulting in an enhanced level of, pyruvate, acetyl-CoA, acetaldehyde or their precursors. This in turn can result in a higher ethanol production in the case that pyruvate, acetyl-CoA or acetaldehyde are used by the at least one overexpressed enzyme for ethanol formation in order to produce ethanol.
In yet a further embodiment of the host cell of the invention, the at least one overexpressed enzyme for the formation of ethanol is an alcohol dehydrogenase.
An alcohol dehydrogenase catalyzes the reduction of a substrate to ethanol. This reaction is normally dependent on the cofactor NADH. Alternatively there are alcohol dehydrogenases which are NADPH-dependent.
Furthermore, the alcohol dehydrogenase can be a thermophilic alcohol dehydrogenase. Thermophilic alcohol dehydrogenase can, for example, be obtained from a host cell which can normally grow well at temperatures above 45° C. Thermophilic alcohol dehydrogenases can be more stable and probably more active than alcohol dehydrogenases obtained from mesophilic host cells, which normally grow at temperatures below 45° C. One possible example for such a thermophilic alcohol dehydrogenase is the alcohol dehydrogenase AdhE obtained from the thermophilic cyanobacterium Thermosynechococcus sp. or from E. coli. 
One possible substrate for alcohol dehydrogenase can be acetyl-CoA, which for example can be directly converted to ethanol by the above-mentioned alcohol dehydrogenase AdhE from Thermosynechococcus or E. coli. Overexpressing such an alcohol dehydrogenase in a genetically modified host cell has the advantage that only one enzyme has to be overexpressed in order to enhance the level of ethanol production. In the case that the level of biosynthesis of acetyl-CoA of the host cell is increased due to overexpression of acetyl-coenzyme A forming enzymes and due to the reduction of enzymatic activity of acetyl-CoA converting enzymes, a high level of ethanol formation can result.
In addition the enzymatic activity or affinity of AdhE can be increased by introducing mutations, in particular point mutations into the protein via site directed or random mutagenesis. The AdhE is an iron-dependent, bifunctional enzyme containing a CoA-depending aldehyde dehydrogenase and an alcohol dehydrogenase activity. One characteristic of iron-dependent alcohol dehydrogenases (AdhII) is the sensitivity to oxygen. In the case of the AdhE from E. coli a mutant was described that shows in contrast to the wildtype also Adh activity under aerobic conditions. The site of the mutation was determined in the coding region at the codon position 568. The G to A nucleotide transition in this codon results in an amino acid exchange from glutamate to lysine (E568K). The E568K derivate of the E. coli AdhE is active both aerobically and anaerobically. This mutation is therefore a solution for the use of this oxygen-sensitive enzyme in an oxygen-producing photosynthetic host cell. [Holland-Staley et al., Aerobic activity of Escherichia coli alcohol dehydrogenase is determined by a single amino acid, J. Bacteriol. 2000 November; 182(21):6049-54].
In a further embodiment of the invention, a genetically modified host cell can be provided, which further comprises:

    
    
        pyruvate decarboxylase converting pyruvate to acetaldehyde, wherein

the alcohol dehydrogenase converts the acetaldehyde to ethanol.

    
    


In this case, the substrate for the alcohol dehydrogenase is provided by a further overexpressed enzyme, for example pyruvate decarboxylase, which is introduced into the host cell via a further second genetic modification. Due to the fact that the level of biosynthesis of pyruvate of the host cell is increased due to the above-mentioned modifications of the pyruvate forming and converting enzymatic activities by way of the first genetic modification, more acetaldehyde is formed via the enzymatic activity of pyruvate decarboxylase. Therefore there is an increased synthesis of acetaldehyde, which is then further converted by alcohol dehydrogenase to ethanol resulting in a higher intracellular or extracellular ethanol level in the host cell. The alcohol dehydrogenase, as well as the pyruvate decarboxylase can be obtained from alcohol-fermenting organisms such as Zymomonas mobilis, Zymobacter palmae or the yeast Saccharomyces cerevisiae. 
In another embodiment of the invention the genetically modified host cell comprises two second genetic modifications, one comprising alcohol dehydrogenases Adh converting acetaldehyde into ethanol and another second genetic modification comprising a CoA-dependent acetaldehyde dehydrogenase converting acetyl-CoA into acetaldehyde. One example of such an acetylating CoA-dependent acetaldehyde dehydrogenase is mhpF from E. coli. 
In yet a further embodiment of the invention the genetically modified host cell harbors a pyruvate decarboxylase enzyme as the only second genetic modification. Such a single second genetic modification is particularly advantageous in genetically modified host cells, which already have an endogenous alcohol dehydrogenase enzyme. The inventors surprisingly found that the activity of such an endogenous alcohol dehydrogenase enzyme can be high enough in order to convert all or almost all of the acetaldehyde formed by the overexpressed pyruvate decarboxylase enzyme into ethanol.
For example all cyanobacterial host cells harbor at least one endogenous alcohol dehydrogenase enzyme. A preferred example is the cyanobacterium Synechocystis in particular Synechocystis PCC6803 or nitrogen fixing cyanobacteria such as Nostoc/Anabaena spec. PCC7120 and Anabaena variabilis ATCC 29413.
The alcohol dehydrogenase can be a zinc-dependent dehydrogenase. In comparison to iron-dependent dehydrogenases, a zinc-dependent dehydrogenase is less oxygen-sensitive and therefore can exhibit a higher enzymatic activity in a photoautotrophic host cell compared to an iron-dependent alcohol dehydrogenase. For example, the alcohol dehydrogenase AdhI obtained from Zymomonas mobilis is a zinc-dependent alcohol dehydrogenase, which can convert acetaldehyde to ethanol by using NADH as a reductant. Alternatively a zinc-dependent alcohol dehydrogenase can be obtained from the cyanobacterium Synechocystis, which also depends on the cofactor NADH.
Alternatively or additionally the alcohol dehydrogenase can comprise AdhII for example from Zymomonas mobilis, which is a Fe2+ dependent alcohol dehydrogenase converting acetaldehyde into ethanol.
In one embodiment, the photoautotrophic ethanol producing host cell comprises at least three second genetic modifications, wherein the at least three overexpressed enzymes for ethanol production have at least three different substrate specificities.
In one embodiment thereof, the three substrate specificities are for the substrates pyruvate, acetaldehyde and acetyl-CoA. For example the three different overexpressed enzymes for ethanol formation can be AdhE converting acetyl-CoA to ethanol, Pdc converting pyruvate to acetaldehyde and AdhI or AdhII converting the acetaldehyde to ethanol. In another embodiment the three different overexpressed enzymes for ethanol formation can be a CoA-dependent acetaldehyde dehydrogenase converting acetyl-CoA to acetaldehyde and Pdc converting pyruvate to acetaldehyde and AdhI or AdhII converting the acetaldehyde to ethanol.
In a further embodiment thereof, the three substrate specificities are for the substrates pyruvate, acetaldehyde and acetylphosphate. In this case the three different overexpressed enzymes for ethanol formation can be acetaldehyde dehydrogenase converting acetylphosphate to acetaldehyde, Pdc converting pyruvate to acetaldehyde and AdhI or AdhII converting the acetaldehyde to ethanol.
In another embodiment, the photoautrophic ethanol producing host cell comprises at least four second genetic modifications, wherein the at least four overexpressed enzymes for ethanol production have at least four different substrate specificities. In one embodiment thereof, the four substrate specificities are for the substrates pyruvate, acetaldehyde and acetyl-CoA and acetylphosphate.
A further embodiment of the invention provides a genetically modified host cell further comprising:

    
    
        a host cell genome, wherein
        a gene encoding the at least overexpressed enzyme for the formation of ethanol is integrated into the host cell genome. The host cell genome can be arranged in at least one chromosome containing coding as well as non-coding sequences. The coding sequences of the genome encode all the proteins and nucleic acids present in a wild type host cell. The gene encoding the at least one overexpressed enzyme for the formation of ethanol can be integrated into the host cell genome, for example via homologous recombination. Integration of the gene coding for the at least one overexpressed enzyme for ethanol formation into the host cell genome can be advantageous for host cells, which exhibit a natural competence for homologous recombination, for example the cyanobacterium Synechocystis sp.
    
    


Yet another embodiment of the invention provides a genetically modified host cell further comprising:

    
    
        at least one host gene encoding the enzyme converting pyruvate or acetyl-CoA or acetaldehyde or forming reserve compounds,
        wherein a heterologous or endogenous gene encoding the at least one overexpressed enzyme for the formation of ethanol is integrated into that host gene thereby disrupting the host gene.
    
    


Such a genetically modified host cell can be produced in just one genetic engineering step, by simply inserting the heterologous or endogenous gene, encoding the at least one overexpressed enzyme for ethanol formation into the host genome into a gene encoding an enzyme converting pyruvate or Acetyl-CoA or forming reserve compounds. Such a procedure knocks out the gene for the enzyme with the undesired activity and at the same time provides a genetic modification introducing an ethanol producing enzyme into a host cell. These genetically modified host cells are therefore easier to obtain than other genetically modified host cells wherein the reduction of enzymatic activity of the enzymes converting pyruvate, acetyl-CoA or acetaldehyde and the introduction of a gene encoding the overexpressed enzyme for ethanol formation is done in two separate steps.
Furthermore, the gene encoding the heterologously or endogenously expressed enzyme can be under the transcriptional control of a promoter endogenous to the host cell. This have the advantage that no exogenous promoter has to be introduced into the host cell. In the case that an exogenous promoter is introduced into a genetically modified host cell a further heterologous gene encoding a transcription factor which recognizes the heterologous promoter, can be introduced into the host cell as well, which complicates the genetic engineering step. Therefore, the introduction of an endogenous promoter, which is also present in an genetically unmodified wild type host cell, has the advantage that this promoter is easily recognized by the genetically modified host cell without the need to introduce further genetic modifications. For example, an inducible promoter such as isiA, which can be induced under iron starvation and stationary growth phase conditions for the host cells can be introduced into Synechocystis PCC 6803 as an endogenous promoter. Further non-limiting examples for suitable promoters will be explained later on.
The gene encoding the heterologously or endogenously expressed enzyme for ethanol formation can also be under the transcriptional control of a heterologous promoter, which is not present in a wild type host cell. For example, heat inducible promoters such as the CI-PL promoter from the bacteriophage lambda can be used to control the transcription of genes.
According to another embodiment of the invention the gene encoding the heterologously or endogenously expressed enzyme for ethanol formation is under the transcriptional control of an inducible promoter.
Such a genetically modified host cell can accumulate large amounts of acetyl-CoA, pyruvate, acetaldehyde or their precursors in the uninduced state due to the above-mentioned modifications and can then, after induction of the promoter, produce high amounts of ethanol via the enzymatic action of the enzyme for ethanol formation, which is now induced. Ethanol can be harmful to the cell. Therefore, larger amounts of ethanol can be produced by first accumulating the substrate necessary for ethanol formation without producing ethanol (uninduced state of the host cell) and then after induction directly converting these substrates into large amounts of ethanol. Therefore inducible promoters can be a good genetic tool in order to decouple the accumulation of acetyl-CoA, pyruvate, acetaldehyde or their precursors in host cells from the ethanol production.
Inducible promoters can be induced for example by nutrient starvation of the host cell, by stationary phase growth of the host cell culture or by subjecting the host cell to stressful conditions.
These kind of promoters are useful, because a genetically modified host cell culture can grow and reach a certain density, thereby leading to a nutrient starvation of the host cell and also increasing the stress for the host cell culture in the case that the growth medium is not continuously supplemented with nutrients. In this case a genetically modified cell culture can accumulate for example acetyl-CoA, pyruvate or their precursors in the exponential growth phase in the non-induced state without producing ethanol, and upon having reached the stationary growth phase can convert these metabolic products into ethanol due to induction of the promoters. For example, the inducible promoters can be inducible by nitrogen starvation or by trace element starvation, such as iron or copper. Examples of such kinds of promoters are the ntcA promoter, the nblA promoter as well as the sigB promoter from Synechocystis, which are inducible by nitrogen starvation and the isiA promoter which is inducible upon iron starvation. The petJ promoter is inducible by copper starvation. In addition, the isiA or sigB promoter can be also inducible by stationary growth phase of the host cell culture. The sigB promoter can also be induced by subjecting the host cell culture to darkness. Further stressful conditions can be heat shock for induction (sigB promoter or the htpG promoter) and cold shock, which induces for example the crhC promoter. Heat shock can be induced, for example by raising the growth temperature of the host cell culture from 30° C. to 40° C. In contrast to that, a cold shock can be induced by reducing the growth temperature of the cell culture from 30° C. to 20° C. A further example of an inducible promoter is the nirA promoter, which can be induced by nitrate starvation or light.
Further relevant promoters are a promoter of a gene encoding light repressed protein A homolog (lrtA promoter), which can be induced by a transition from light to dark conditions. In addition the promoter of gene of P700 apoprotein subunit Ia (psaA promoter), which can be induced under low white light and orange light and repressed in darkness.
Alternatively the gene encoding the heterologously or endogenously expressed enzyme for ethanol formation can be under the transcriptional control of a constitutive promoter, which allows a certain level of transcription and therefore enzymatic activity of the overexpressed enzyme for ethanol formation during the whole period of cultivation even without induction. This can be advantageous in the case that the metabolic intermediate converted by the overexpressed enzyme for ethanol formation is harmful to the cell, as for example acetaldehyde. In this case the acetaldehyde is continuously converted to ethanol and is not present in the genetically modified host cell in high amounts.
A further embodiment of the invention provides a genetically modified photoautotrophic, ethanol producing host cell comprising:

    
    
        at least one first genetic modification changing the enzymatic activity or affinity of an endogenous host enzyme of the host cell,
        the first genetic modification resulting in a level of biosynthesis of a first metabolic intermediate for energy production of the host cell, which is enhanced compared to the level of biosynthesis in the respective wild type host cell,
        at least one second genetic modification different from the first genetic modification comprising an overexpressed first enzyme for the formation of ethanol from the first metabolic intermediate.
    
    


The first metabolic intermediate can be any metabolic intermediate involved in the energy production of the host cell or in the formation of reserve compounds in the claim, for example starch, glycogen or polyhydroxybutyrate. This first metabolic intermediate can, for example, be formed during the Calvin-cycle, the light-independent part of photosynthesis, the glycolysis, the fermentation pathway, the amino acid metabolism or the citric acid cycle. Some non-limiting examples for the first metabolic intermediate are pyruvate, acetyl-CoA or acetaldehyde.
Due to the fact that the level of biosynthesis of this first metabolic intermediate is enhanced compared to the wild type host cell and due to the fact that this first intermediate is used by the first enzyme for ethanol formation in order to produce ethanol, these genetically modified photoautotrophic host cells can produce a high amount of ethanol.
For example, the first metabolic intermediate can comprise acetyl-CoA and the at least one overexpressed first enzyme can comprise the alcohol dehydrogenase AdhE directly converting acetyl-CoA to ethanol. In this case only one overexpressed enzyme is necessary in order to produce a increased amount of ethanol.
It is also possible that the genetically modified host cell further comprises:

    
    
        at least one overexpressed second enzyme, converting the first metabolic intermediate into a second metabolic intermediate, wherein
        the at least one overexpressed first enzyme converts the second metabolic intermediate into ethanol.
    
    


In this case, the first enzyme uses another metabolic intermediate provided by a second overexpressed enzyme in order to produce ethanol.
For example, the first metabolic intermediate can comprise pyruvate and the second metabolic intermediate can comprise acetaldehyde and the at least one overexpressed second enzyme can comprise pyruvate decarboxylase converting pyruvate into acetaldehyde and the at least one overexpressed first enzyme can comprise alcohol dehydrogenase Adh, converting acetaldehyde into ethanol.
Some host cells, for example, cyanobacteria, normally do not have a pyruvate decarboxylase. Therefore, the transformation of cyanobacteria with a pyruvate decarboxylase and in addition the overexpression of an alcohol dehydrogenase which already can be present in the wild type cyanobacterial cell can result in increased amounts of ethanol.
Another embodiment of the invention provides a genetically modified host cell, which further comprises:

    
    
        at least one host enzyme for conversion of the first metabolic intermediate, wherein
        the activity of said host enzyme is reduced compared to the respective wild type host cell by genetic engineering.
    
    


As mentioned above, the activity of host enzymes can be reduced, for example by site directed mutagenesis or random mutagenesis of the gene encoding the host enzyme, which results in a protein with a lower activity. Alternatively or additionally the promoter sequences controlling the transcriptional activity of the genes encoding this host enzyme also can be genetically modified in order to reduce the transcriptional activity. Another example is to disrupt the gene encoding the host enzyme for conversion of the first metabolic intermediate with a heterologous nucleic acid sequence. The host enzyme, for example, can be any enzyme of the Calvin-cycle, the glycolysis pathway, the intermediate steps of metabolism, the amino acid metabolism or the citric acid cycle converting the first metabolic intermediate, which for example, can be pyruvate. In this case the host enzymes whose activity is reduced can, for example, be selected from a group consisting of pyruvate water dikinase, pyruvate dehydrogenase, phosphotransacetylase, acetate kinase, lactate dehydrogenase or alanine dehydrogenase.
In addition or alternatively the genetically modified host cell can further comprise:

    
    
        at least one host enzyme for forming the first metabolic intermediate, wherein
        the activity of said host enzyme is enhanced compared to the respective wild type host cell by genetic engineering.
    
    


In the case that the first metabolic intermediate is, for example, pyruvate the at least one host enzyme can be selected from the above-mentioned enzymes, which are: malate dehydrogenase, malic enzyme, pyruvate kinase, enolase, and phosphoglycerate mutase.
In the case that the first metabolic intermediate is, for example, acetyl-CoA the at least one host enzyme in addition to the above latter mentioned enzymes also can be selected from pyruvate dehydrogenase.
There are several methods for genetic engineering, which are useful in enhancing the enzymatic activity or affinity of an enzyme, for example introducing point mutations (site directed mutagenesis or random mutagenesis) into a gene encoding the host enzyme for forming the first metabolic intermediate in order to enhance the enzymatic activity of this enzyme. Furthermore, additional gene copies encoding the host enzyme can be introduced into the host cell therefore enhancing the amount of protein in the host cell. Alternatively or in addition, the promoter region controlling the transcriptional activity of the gene encoding the enzyme can be mutated in order to enhance the transcriptional activity of the gene. Overexpression can also be achieved by introducing a heterologous enzyme into the host cell, which exhibits the same enzymatic activity as the host cell enzyme, which should be overexpressed. For example if PGA mutase should be overexpressed in the cyanobacterium Synechocystis a plasmid comprising a heterologous gene encoding PGA mutase from Zymomonas mobilis can be introduced into the host cell. Another non-limiting example is the overexpression of pyruvate kinase from E. coli in Synechocystis, thereby raising the enzymatic activity of the endogenous host cell enzyme pyruvate kinase in Synechocystis. In addition homologous genes from other cyanobacterial sources such as Synechocystis can be overexpressed in photoautotrophic host cells. Non-limiting examples for overexpression are: PGA mutase genes slr1124, slr1945, sll0395 and slr1748 and the enolase homolog slr0752 from Synechocystis PCC 6803.
Yet another embodiment of the invention provides a construct for the transformation of a photoautotrophic host cell by disrupting a host gene sequence encoding a host enzyme in order to increase the biosynthetic level of pyruvate, acetyl-CoA, acetaldehyde or precursors thereof in the host cell comprising:

    
    
        a heterologous nucleic acid sequence comprising a promoter and a biocide resistance conferring gene under the transcriptional control of the promoter, wherein
        the heterologous nucleic sequence is flanked at its 5′ and 3′ end by nucleic acid sequences that bind to the host gene sequence encoding a host enzyme.
    
    


Such a construct can, for example, be used in order to knock out unwanted host enzymes which convert an important first metabolic intermediate into another metabolic compound. Due to the biocide resistance conferring gene, genetically modified host cells resulting from the transformation with such a construct can be selected by exposing the transformed host cells to a growth medium containing the biocide. The 5′ and 3′ flanking nucleic acid sequences are preferably homologous to the nucleic acid sequence of the host gene encoding the host enzyme for conversion of the first metabolic intermediate.
The term “binds to” is used herein to refer to the annealing or hydrogen bonding of one nucleic acid (polynucleotide) to another nucleic acid (polynucleotide) In a particularly preferred embodiment, binding occurs in vivo or within a cell between a heterologous nucleic acid sequence and a genomic or chromosomal nucleic acid sequence. This is particularly useful in promoting homologous recombination. In other circumstances, the term may refer to hybridization in a non-natural environment, particularly under stringent conditions in the laboratory. “Hybridization stringency” is a term well understood to those of ordinary skill in the art. A particular, non-limiting example of stringent (e.g. high stringency) hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) buffer at about 45 degrees Celsius, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65 degrees Celsius. Hybridization stringency may also be varied and used to identify and isolate nucleic acid sequences having different percent identity with the probe sequence.
In various embodiments of the invention, 5′ and 3′ flanking sequences of the invention are selected from a host cell enzyme gene sequence described herein. Moreover, in the Examples section provided herewith, the construction of various nucleic acid constructs is provided. As one of ordinary skill in the art would recognize, the invention is not limited to only those sequences disclosed herein because these examples provide ample teaching to select similar 5′ and 3′ sequences from host cell enzyme identified in sequence databases.
These sequences can, for example, have an identity at least 80%, 85%, 90%, 95% and 100% to the corresponding nucleic acid sequences of the host cell enzyme gene.
Another embodiment of the invention provides a construct for the transformation of a photoautotrophic host cell by disrupting a host cell gene sequence encoding a host cell enzyme in order to increase the biosynthetic level of pyruvate, acetyl-CoA, acetaldehyde or precursors thereof in the host cell, comprising:

    
    
        a heterologous nucleic acid sequence comprising a promoter and a first gene encoding at least one overexpressed first enzyme for the formation of ethanol from the first metabolic intermediate under the transcriptional control of the promoter, wherein
        the heterologous nucleic acid sequence is flanked at its 5′ and 3′ end by nucleic acid sequences that bind to said host gene.
    
    


Such a construct can, for example, be used in order to knock out a gene encoding a host enzyme for conversion of a first metabolic intermediate, which can be pyruvate, acetyl-CoA, acetaldehyde or precursors thereof and at the same time, introduce via genetic engineering a gene encoding a first enzyme for the formation of ethanol. Such a construct can therefore be used in order to enhance the level of a first metabolic intermediate in a genetically modified host cell and at the same time use this first metabolic intermediate as a substrate for ethanol production.
The 5′ and 3′ flanking nucleic acid sequences are preferably highly identical, more preferably completely identical, to the corresponding parts of the host cell gene encoding the host cell enzyme. Such a construct is integrated into the host genome of a host cell via homologous recombination.
Homologous recombination involves the alignment of similar sequences, preferably homologous nucleic acid sequences located in different nucleic acid strands, for example a recombinant integrative plasmid and the chromosome of a host cell. After a crossover between the aligned nucleic acid strands, the nucleic acid strands are broken and repaired in order to produce an exchange of nucleic acid material between the chromosome and the recombinant integrative plasmid. The process of homologous recombination naturally occurs in many host cells, for example cyanobacteria such as Synechocystis and can be utilized as a molecular biology technique for genetically engineering organisms and introducing genetic changes into the organisms. The 5′ and 3′ flanking nucleic acid sequences each can have a length of a few hundred base pairs, preferably at least around 500 base pairs or more, in order to enable homologous recombination. The length can be up to 1.5 kilobases or even 2 kilobases.
In various embodiments of the invention, the heterologous nucleic acid sequence further comprises a second gene encoding at least one overexpressed second enzyme converting the first metabolic intermediate into a second metabolic intermediate, wherein the at least one overexpressed first enzyme converts the second metabolic intermediate into ethanol.
In such a case the first metabolic intermediate can comprise pyruvate and the second metabolic intermediate can comprise acetaldehyde and the second gene can encode pyruvate decarboxylase converting pyruvate into acetaldehyde, and the first gene can encode alcohol dehydrogenase converting acetaldehyde into ethanol.
Alternatively, the first metabolic intermediate can comprise pyruvate and the second metabolic intermediate can, for example, comprise acetyl-CoA. In this case the first gene can encode pyruvate dehydrogenase, pyruvate formate lyase or pyruvate-ferredoxin-oxidoreductase which can convert pyruvate to acetyl-CoA. The second gene then can encode a coenzyme A dependent aldehyde dehydrogenase which can convert acetyl-CoA to acetaldehyde. In this case a third gene can be introduced into the construct which encodes alcohol dehydrogenase which can convert acetaldehyde to ethanol. Therefore, constructs according to certain embodiments of the inventions can comprise more than two or even more than three genes encoding more than two or three enzymes involved in ethanol formation.
Alternatively the first metabolic intermediate can comprise acetyl-CoA and the first gene can be alcohol dehydrogenase AdhE directly converting acetyl-CoA into ethanol. In this case one enzyme can be sufficient to trigger ethanol formation in a genetically modified host cell.
Furthermore a co-expression of the enzymes AdhE, Adh and Pdc in parallel is also able to convert acetyl-CoA into ethanol (e.g. in combination with a blocked or reduced acetate and lactate pathway) and to convert pyruvate into ethanol in parallel. This could avoid that pathways are shifted to acetyl-CoA in case of Pdc and Adh expression or to pyruvate in case of AdhE expression.
A further embodiment of the invention is directed to a genetically modified photoautotrophic, ethanol producing host cell comprising:

    
    
        a first genetic modification comprising at least one genetic modification of at least one host cell enzyme that is not pyruvate decarboxylase or alcohol dehydrogenase, wherein the first genetic modification results in an enhanced level of biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or precursors thereof compared to the respective wild type host cell, and
        a second genetic modification comprising at least one overexpressed enzyme for the formation of ethanol.
    
    


The subject matter of a further embodiment of the invention is a construct for the transformation of a host cell by disrupting a host gene encoding a host enzyme for conversion of a first metabolic intermediate for energy production of the host cell or forming reserve compounds, comprising:

    
    
        a heterologous nucleic acid sequence comprising an inducible promoter and a gene encoding the host enzyme for conversion of the first metabolic intermediate for energy production of the host cell or forming the reserve compounds under the transcriptional control of the inducible promoter, wherein
        the heterologous nucleic acid sequence is flanked at its 5′ and 3′ end by nucleic acid sequences which are able to bind to at least parts of said host gene.
    
    


As mentioned above, the 5′ and 3′ flanking nucleic acid sequences are necessary in order to ensure the insertion of this construct into the host cell genome, for example via homologous recombination. Such a construct can be useful in the case that the host enzyme for conversion of the first metabolic intermediate or for forming reserve compounds is a very crucial enzyme for the metabolism of the host cell so that it might not be possible to completely knock out this enzyme without killing the host cells during this process. Such a construct can be used in order to replace the uncontrollable wild type host gene by a copy of the gene which is under the control of an inducible promoter. Such a construct enables the controlling of the enzymatic activity of an important metabolic enzyme of the host cell without completely knocking out the enzymatic activity of this enzyme.
The host gene, for example, can encode glycogen synthase. Due to the fact that two copies are sometimes present in the genome of a host cell, two different constructs have to be designed in order to knock out both glycogen synthase coding genes.
The above-mentioned constructs can be part of a recombinant plasmid which further can comprise other genes, which for example encode biocide resistance conferring genes.
Subject matter of a further embodiment of the invention is a method for producing genetically modified host cells comprising the method steps:
A) Providing a wild type host cell showing a wild type level of biosynthesis of a first metabolic intermediate for energy production of the host cell,
B) enhancing the level of biosynthesis of the first metabolic intermediate in comparison to the wild type level by genetic engineering,
C) introducing a first heterologous or endogenous gene into the host cell, the first gene encoding at least one overexpressed first enzyme for the formation of ethanol from the first metabolic intermediate.
Such a method enhances in method step B) the level of biosynthesis of a useful first metabolic intermediate and then introduces in method step C) a gene into the host cell encoding a protein which can use the first metabolic intermediate for ethanol synthesis.
Alternatively first method step C) then method step B) can be carried out. Such a method, can be healthier for the cell due to the fact that the metabolic intermediate, which can be harmful would not accumulate in the cells, e.g. in case of acetaldehyde.
According to a further embodiment of the method of the invention in step C) a second heterologous or endogenous gene can be introduced into the host cell, the second heterologous or endogenous gene encoding at least one overexpressed second enzyme converting the first metabolic intermediate into a second metabolic intermediate, wherein the at least overexpressed first enzyme converts the second metabolic intermediate into ethanol.
As mentioned above, the first metabolic intermediate can comprise pyruvate and the second metabolic intermediate can comprise acetaldehyde so that the second gene can encode pyruvate decarboxylase converting pyruvate into acetaldehyde and the first gene can encode alcohol dehydrogenase converting acetaldehyde into ethanol.
Alternatively the first metabolic intermediate can comprise acetyl-CoA and the first gene can encode the alcohol dehydrogenase AdhE, which directly converts acetyl-CoA into ethanol.
In a further modification of the method of the invention in step A) a wild type host cell can be provided which further comprises a first host gene encoding at least one first host enzyme for conversion of the first metabolic intermediate or for forming reserve compounds, the first host gene is under the transcriptional control of a first host promoter.
Then in step B) the activity of the at least one first host enzyme can be reduced by genetic engineering.
In particular, in step B) the activity of the at least one host enzyme can be reduced by mutating either the first host promoter or the first host gene or by disrupting the first host gene by introducing a heterologous nucleic acid sequence into the first host gene.
According to a further embodiment of the method of the invention, in step A) a wild type host cell can be provided which further comprises a second host gene encoding at least one second host enzyme for forming the first metabolic intermediates or precursors thereof, the second host gene is under the transcriptional control of a second host promoter, and then in step B) the activity of the at least one second host enzyme is enhanced by genetic engineering. The activity of the at least one second host gene can be enhanced by mutating either the second host promoter or the second host gene or by overexpressing the second host enzyme.
A further embodiment of the invention provides a genetically modified photoautotrophic ethanol-producing host cell comprising:

    
    
        a heterologous or endogenous nucleic acid sequence comprising a promoter and a gene encoding at least one overexpressed enzyme for the formation of ethanol under the transcriptional control of the promoter, wherein
        the promoter can be induced by nutrient starvation, oxidative stress, light, darkness, heat shock, cold shock, salt stress, by a change of the nutrient source, by an increase in the concentration of one nutrient or stationary growth of the host cell.
    
    


In the following the inventions will be explained in more detail with reference to figures and certain embodiments:
FIGS. 1A to C depict general schemes of metabolic pathways in Cyanobacteria with marked enzymes for overexpression and down-regulation or knock-out for the increase of biosynthesis of different metabolic intermediates.
FIG. 2 shows a flow chart including some ethanologenic enzymes for ethanol production.
FIG. 1A shows some general metabolic pathways in cyano-bacteria as a non-limiting example. In particular the Calvin cycle as the light independent part of the photosynthesis is shown starting with the carbon dioxide fixation reaction catalyzed by the enzyme RubisCO. Further the glycolysis pathway, the pentose phosphate pathway and the citric acid cycle are shown. The general metabolic pathways depict boxed and circled enzymes, whose activity or affinity can be changed as part of at least one first genetic modification of an endogenous host enzyme of the cyanobacterial host cell. Boxed enzymes either have been overexpressed compared to the respective wild type cyanobacterial cells or are prime candidates for overexpression. Circled enzymes either have been knocked out or down regulated or are prime targets for knock-out or down-regulation. The main reason for the knock-out or overexpression is to enhance the level of pyruvate biosynthesis in the genetically modified cell by knocking-out or reducing the activity or affinity of enzymes consuming pyruvate or its metabolites and to enhance the enzymatic activity of enzymes producing pyruvate or its precursors such as phosphoenolpyruvate (PEP). The cyanobacterial host cell can comprise more than one first genetic modification. For example enzymes enhancing the level of pyruvate biosynthesis such as enolase or malic enzyme can be overexpressed and the activity or affinity of enzymes consuming pyruvate, such as lactate dehydrogenase or alanine dehydrogenase can be reduced or abolished by knock-out of the respective genes in one cyanobacterial host cell.
In addition two second genetic modifications resulting in an overexpression of enzymes for ethanol formation have been introduced into the metabolic cyanobacterial pathways shown in FIG. 1A. These enzymes are indicated by the thickly framed boxes denoted with the reference sign “A”. In particular these enzymes are alcohol dehydrogenase (abbreviated as Adh) and pyruvate decarboxylase (abbreviated as Pdc), which also have to be introduced into most cyanobacteria via genetic engineering.
FIG. 1B shows the same general metabolic pathways in cyano-bacteria as already presented in FIG. 1A for the case that the level of biosynthesis of acetyl-CoA is raised compared to a wildtype cyanobacterial cell. The enzymes, which are part of the first and second genetic modification are marked in the same way as in FIG. 1A. In addition the direct conversion of acetyl-CoA to ethanol catalyzed by the enzyme aldehyde-alcohol dehydrogenase AdhE, which has to be introduced into most cyanobacteria via a second genetic modification is denoted. AdhE is for example an endogenous enzyme in the cyanobacterium Thermosynechococcus or an heterologous enzyme from E. coli. In this case the expression of AdhE can be enhanced in a second genetic modification in Thermosynechococcus, for example by introducing additional gene copies into the cell or by mutating the promoter of the wildtype gene encoding AdhE in order to enhance transcription and translation. In the case of overexpression of AdhE the enzyme pyruvate dehydrogenase can be overexpressed (shown as a boxed enzyme). In addition to overexpression of AdhE it is still possible to overexpress Pdc and Adh simultaneously. Alternatively only AdhE can be overexpressed.
FIG. 1C gives an overview of metabolic enzymes in cyanobacteria, which can be overexpressed (boxed enzymes) or knocked out or downregulated (circled enzymes) in the case that the level of biosynthesis of acetaldehyde is to be increased in the cell. In this case the enzymes phosphotransacetylase and acetaldehyde dehydrogenase are overexpressed in comparison to the situation shown in FIG. 1B. The enzyme acetaldehyde dehydrogenase converting acetylphosphate to acetaldehyde is for example disclosed in the publication Stal (Stal, Moezelaar, “Fermentation in cyanobacteria”, FEMS Microbiology Reviews 21, (1997), pages 179-211). The enzymes, which are part of the first and second genetic modification are marked in the same way as in FIGS. 1A and 1B.
FIG. 1D depicts the exemplary metabolic pathway of other bacteria. In contrast to the metabolic pathways shown in the FIGS. 1A to 1C, the enzyme acetate kinase in addition also catalyzes the reaction in the other direction from acetate to acetylphosphate. In the case that the enzyme acetaldehyde dehydrogenase is overexpressed or its affinity or activity is enhanced in other ways described in this patent application, Overexpression of acetate kinase enzyme can enhance the level of biosynthesis of acetylphosphate, thereby enhancing ethanol formation by Adh. In addition the other ethanol forming enzyme AdhE can also be overexpressed.
FIG. 1E shows the same metabolic pathway as depicted in FIG. 1D with the exception that in addition to the acetate kinase enzyme the phosphotransacetylase enzyme also catalyzes the reverse reaction from acetylphosphate to acetyl-CoA. In this case phosphotransacetylase can be overexpressed in addition to acetate kinase enzyme in order to enhance the level of biosynthesis of acetyl-CoA in a first genetic modification. The second genetic modification comprises overexpression of AdhE, which converts the acetyl-CoA into ethanol. In addition the second genetic modification also can comprise overexpression of Adh and Pdc.
FIG. 1F shows some relevant metabolic pathways of cyanobacteria with different overexpressed enzymes for ethanol formation, which can be introduced into a photoautotrophic cyanobacterial host cell by second genetic modifications. In one aspect of the invention a CoA-dependent acetaldehyde dehydrogenase can be overexpressed in the host cell, which converts acetyl-CoA into acetaldehyde. The acetaldehyde can then further be converted to ethanol by a further enzyme for ethanol formation Adh, which can be AdhI enzyme or AdhII enzyme or a combination of both enzymes.
In addition or alternatively Pdc enzyme can be present in the host cell as a further overexpressed enzyme for ethanol formation introduced via a second genetic modification, which can convert pyruvate into acetaldehyde.
FIG. 2 shows in a more detailed way the last steps of ethanol synthesis in genetically modified cyanobacteria.
FIG. 3 depicts a further non-limiting representation of metabolic pathways of a cyanobacterium. In contrast to the FIGS. 1A to 1F a NAD dependent acetaldehyde dehydrogenase is shown, which can convert acetate into acetaldehyde, which then can be converted into ethanol by Adh enzyme.
Working Example of Genetic Knockout
In the following one embodiment of the invention, in particular a genetically modified host cell comprising a host enzyme forming reserve compounds, wherein the gene encoding this enzyme is disrupted by genetic engineering, is explained in more detail with reference to a working example. The host enzyme is glycogen synthase, which is encoded by two genes in the host cell Synechocystis sp. PCC 6803. In order to knock-out both genes a double knock-out mutant has to be generated.
Laboratory Protocols
Protocols for the Generation of a Glycogen Synthase Double Mutant of Synechocystis sp. PCC 6803
In the genome database of Synechocystis sp. PCC 6803 two genes encoding glycogen synthases are annotated (http://bacteria.kazusa.or.jp/cyano).
One glycogen synthase of Synechocystis sp. PCC 6803 is encoded by the gene sll0945 (glgA1), annotated as glycogen synthase 1 (GlgA1). The Accession number of the protein is P74521 (EC 2.4.1.21), its amino acid sequence is presented in FIG. 4A.
A second glycogen synthase of Synechocystis sp. PCC 6803 is encoded by the gene sll1393 (glgA2), annotated as glycogen (starch) synthase 2 (GlgA2). The Accession number of the protein is P72623 (EC 2.4.1.21), its amino acid sequence is presented in FIG. 4B.
Construction of DNA-Vectors (Knock-Out-Constructs) for the Two Glycogen Synthase Encoding Genes (glgA1 and glgA2) of Synechocystis sp. PCC 6803
In General:
DNA sequences encoding genes of interest are amplified by polymerase chain reaction (PCR) using specific primers. When the genomic sequence does not contain appropriate restriction sites for cloning, primers are designed containing restriction sites. Genomic DNA from Synechocystis sp. PCC 6803 are used as template. The amplified PCR fragments are digested with the appropriate restriction enzymes and ligated into a cloning vector.
An antibiotic resistance cassette is then inserted into selected sites of the cloned genes. Upstream and downstream on each site of the antibiotic resistance cassette at least 500 bps remain for homologous recombination.
Genetic engineering of constructs as well as PCRs, ligations into cloning vectors, insertions of antibiotic resistance cassettes and transformations into E. coli are done using standard procedures (state of the art) or according to the manufacturers instructions.
To generate a glycogen deficient mutant in Synechocystis sp. PCC 6803, constructs were created for inactivation both glycogen synthase genes. The resulting glycogen deficient mutant described below is named mutant M8.
For creating a knock-out construct to inactivate glgA1, a 1341 bp fragment containing the major part of the coding sequence from glycogen synthase 1 (sll0945) was amplified by PCR using the following primers:








#glgA-1fw:



5′-CGACGGTATGAAGCTTTTATTTG-3′,;
(SEQ ID NO: 130)






primer contains a HindIII restriction site for cloning (marked in bold letters).











#glgA-1rv:




5′-CCGGCGGAACGGTACCAAC-3′,,
(SEQ ID NO: 131)






primer contains a KpnI restriction site for cloning (marked in bold letters).

The PCR fragment was digested with HindIII and KpnI and cloned into plasmid pUC19 (Ac. No M77789). A single BstXI site present in the middle of glgA1 gene was used to insert a chloramphenicol resistance cassette (named Cm). The chloramphenicol resistance cassette, encoding a chloramphenicolacetyltransferase (cat) gene, was cut out of plasmid pACYC184 (Ac. No X06403) using BsaAI and BsaBI. The orientation of the antibiotic cassette was analyzed by digestion with HindIII and EcoRI; a restriction map is presented in FIG. 4C.
A knock-out-construct, named pUC-glgA1-Cm, has the structure presented in FIG. 4D, and the nucleotide sequence of the construct pUC-glgA1-Cm is presented in FIG. 4E.
For creating a knock-out construct to inactivate glgA2, a 1673 bp fragment containing the entire coding sequence from glycogen synthase 2 (sll1393) was amplified by PCR using the following primers:










#glgA-2fw:




5′-GGCCAGGGGAATTCTCCTCCAG-3′,
(SEQ ID NO: 132)






primer contains an EcoRI restriction site for cloning (marked in bold letters).









#glgA-2rv:



5′-GCGGATAATACTGAACGAAGCTTTG-3′,
(SEQ ID NO: 133)






primer contains a HindIII restriction site for cloning (marked in bold letters).

The PCR fragment was digested with EcoRI and HindIII and cloned into plasmid pUC19. A single HincII site present in the middle of glgA2 gene was used to insert a kanamycin resistance cassette (named Kan). The kanamycin resistance cassette, encoding an aminoglycoside 3′-phosphotransferase (aph) gene, was cut out of plasmid pUC4K (Ac. No X06404) using HincII. The orientation of antibiotic cassette was analyzed with the restriction enzyme HindIII. A restriction map of this clone is presented schematically in FIG. 4G.
The knock-out-construct used, named pUC-glgA2-Kan, has the structure presented in FIG. 4G and the nucleotide sequence presented in FIG. 4H.
Mutagenesis by transformation of the DNA-vectors (knock-out-constructs) using the natural competence of Synechocystis sp. PCC 6803 for DNA uptake and its system for homologous recombination.
The transformation was done in two steps. The first transformation knocks out gene sll0945 (glgA1) in the wild type of Synechocystis, and the corresponding mutant ΔglgA1 was selected. In a second step, gene sll1393 (glgA2) was knocked out in the ΔglgA1 mutant and the double mutant ΔglgA1/ΔglgA2 was selected.
General Transformation Protocol:


    
    
        Spin down 10 ml of exponentially growing culture of Synechocystis sp. at room temperature (RT) and remove the supernatant
        Resuspend the pellet in 0.5-1.0 ml of BG11 medium (media recipe:
        NaNO3: 1.5 g
        K2HPO4: 0.04 g
        MgSO4.7H2O: 0.075 g
        CaCl2.2H2O: 0.036 g
        Citric acid: 0.006 g
        Ferric ammonium citrate: 0.006 g
        EDTA (disodium salt): 0.001 g
        NaCO3: 0.02 g
        Trace metal mix A5_ 1.0 ml
        Agar (if needed): 10.0 g
        Distilled water: 1.0 L
        The pH should be 7.1 after sterilization
        Trace Metal Mix A5:
        H3BO3: 2.86 g
        MnCl2.4H2O: 1.81 g
        ZnSO4.7H2O: 0.222 g
        NaMoO4.2H2O: 0.39 g
        CuSO4.5H2O: 0.079 g
        Co(NO3)2.6H2O: 49.4 mg
        Distilled water: 1.0 L)
        Add 1-10 μg plasmid DNA (knock-out-construct carrying gene of interest and an antibiotic cassette for screening for homologous recombination)
        Incubate on a table top shaker for 5-6 hours in the light at RT
        Plate 500 it of a 1/100 dilution of the transformation mixture on a BG11 agar plate. Plate the remainder of the cell suspension on another plate. Include control plate (transformation mixture with water instead of plasmid DNA).
        Incubate 48 h in the light at room temperature (RT) when chloramphenicol is used for selection or over night when kanamycin is used for selection.
        Pipet 500 μl of the corresponding antibiotic in a suitable concentration under the agar for the selection of mutant clones (initial concentration for chloramphenicol: 1 μg/ml BG11 agar; initial concentration for kanamycin: 5 μg/ml)
        Incubate for approx. 2 weeks in the light at RT
        Transfer individual colonies to plates containing the corresponding antibiotic
        Thereafter, the concentrations of antibiotics were increased stepwise when the cells were transferred onto another agar plate or into liquid culture (for kanamycin from initially 5 to 15 μg/ml BG11, for chloramphenicol from initially 1 to 15 μg/ml BG11 medium) in order to get fully segregated (homozygous) mutants. Transfers were done every 2 weeks. In case of kanamycin, the concentration in the range from 50 to 150 μg/ml agar was increased gradually over the course of 4 weeks.

Cultivation of Cyanobacterial Wild Type and Mutant Strains

    
    


Wild type and mutant strains of Synechocystis PCC 6803 were grown as batch cultures in BG11 medium at 29° C. under continuous illumination with white light (intensity: 40 μE m−2 s−1) and aeration with air. For cultivation of mutants, the appropriate antibiotics were added to the medium (kanamycin 75 mg/l; chloramphenicol 15 mg/l).
Samples were analyzed briefly before the nitrogen step down (“+N”), directly after resuspension of the cells in BG11 medium lacking a nitrogen source (“−N”, 0 h) and after 3, 6 and 24 hours.
Generation of Knock-Out Mutants of Synechocystis sp. PCC 6803 Affecting the Following Genes:
a) alanine dehydrogenase (ald)
b) ADP-glucose pyrophosphorylase (glgC)
c) pyruvate water dikinase (ppsA)
d) lactate dehydrogenase (ldh)
e) acetate kinase (ack)
f) phosphoacetyltransacetylase (pta)
g) PHB knockout mutant (ΔphaC)
h) knockout mutant of ADP-glucose-pyrophosphorylase, agp, glgC in the filamentous, diazotrophic cyanobacteria Nostoc/Anabaena spec. PCC7120 and Anabaena variabilis ATCC 29413
Protocols for Generation of Knock-Down Mutants of Synechocystis sp. PCC 6803 Affecting the Following Gene:
a) pyruvate dehydrogenase (pdhB)
Description of the here Used Cloning Vectors
Protocols for the Generation of Knock-Out Mutants of Synechocystis sp. PCC 6803
Construction of DNA-Vectors for Generation of Knock-Out Mutants
In General:
DNA sequences encoding genes of interest were amplified by polymerase chain reaction (PCR) using specific primers. When the genomic sequence did not contain appropriate restriction sites for cloning, primers were designed containing restriction sites. Genomic DNA from Synechocystis sp. PCC 6803 was used as template. The amplified PCR fragments were digested with the appropriate restriction enzymes and ligated into a cloning vector.
An antibiotic resistance cassette was then inserted into selected sites of the cloned genes. Upstream and downstream on each site of the antibiotic resistance cassette at least 500 bps remained for homologous recombination. The following antibiotic resistance cartridges were used: kanamycin resistance cassette (named Kan) from pUC4K vector (Ac. No X06404) from the NCBI database under: http://www.ncbi.nlm.nih.gov/sites/entrez?term=X06404&cmd=Search&db=nuccore&QueryKey=1 encoding aminoglycoside 3′-phosphotransferase (aph) gene or chloramphenicol resistance cartridge (named Cm) from pACYC184 vector (Biolabs, Ac No. X06403) encoding chloramphenicolacyltransferase (cat) gene. Genetic engineering of constructs as well as PCRs, ligations into cloning vectors, insertions of antibiotic resistance cassettes and transformations into E. coli were done using standard procedures (state of the art) or according to the manufacturer instructions.
Sequences and structures of the used cloning and expression plasmids are described below (see 3).
Knock-outs were generated via homologous recombination of the wild type gene with the mutant genes. The method of transformation of the DNA-vectors (knock-out-constructs) using the natural competence of Synechocystis sp. PCC 6803 for DNA uptake was already described in detail for the generation of the glycogen deficient mutant.
a) Construction of a DNA-Vector for Generation of an Alanine Dehydrogenase Knock-Out Mutant (Δald)
The open reading frame (ORF) sll1682 encodes alanine dehydrogenase (EC 1.4.1.1), Ac. No BAA16790. The amino acid sequence of this protein is presented in FIG. 5A.
Two constructs were generated for knock-out of alanine dehydrogenase differing in orientation of the inserted kanamycin resistance cartridge (in sense and in antisense orientation to the ald ORF) using the following primers:










#Ald50.fw:




5′-GGCTGACCCCCAGTAGTGTA-3
(SEQ ID NO: 134)


 



#Ald1042.rv:




5′-ATTTTCCGGCTTGAACATTG-3′
(SEQ ID NO: 135)






A 993 bp ald PCR fragment was amplified by a BIOTAQTM DNA Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega) and restricted with SmaI (blunt ends; Fermentas). The kanamycin cartridge was remained by a restriction of the pUC4K vector with EcoRI (5′overhangs; Fermentas) and a following “fill in reaction” via the T4 DNA Polymerase (Promega). Plasmids were analyzed by restriction digest in order to select constructs with both orientations of the inserted kanamycin cartridge.
A construct designated as pGEM-T/Δald-antisense has the structure presented schematically in FIG. 5B.
The sequence of the insert for this construct (pGEM-T/Δald-antisense) is presented in FIG. 5C.
In the other construct, designated as pGEM-T/Δald-sense the kanamycin resistance cartridge is inserted in the other direction.
b) Construction of DNA-Vector for Generation of an ADP-Glucose Pyrophosphorylase Knock-Out Mutant (ΔglgC)
The open reading frame (ORF) slr1176 encodes ADP-glucose pyrophosphorylase (EC 2.7.7.27), Ac. No BAA18822. The amino acid sequence of this protein is presented in FIG. 6A.
Four constructs were generated for knock out of ADP-glucose pyrophosphorylase differing in the locus of insertion (EcoRI, BsaBI) and in orientation of the resistance (kanamycin-Km, chloramphenicol-Cm) cartridge (in sense and in antisense orientation to the glgC gene). Both insertion sites were tested because of a putative small non-coding RNA at the 5′-terminus of the glgC gene (in antisense orientation). Therefore, the insertion of the chloramphenicol cartridge at the BsaBI-site might affect the expression of the putative small non-coding RNA.
The following primers were used for PCR










#GglC5.fw:




5′-GTTGTTGGCAATCGAGAGGT-3′
(SEQ ID NO: 136)


 



#GlgCiR.rv:




5′-GTCTGCCGGTTTGAAACAAT-3′
(SEQ ID NO: 137)


 



BsaBI:




GATNNtNNATC
(SEQ ID NO: 138)


 



#GlgCiR.fw:




5′-ACCCCATCATCATACGAAGC-3′
(SEQ ID NO: 139)


 



#GlgC1233.rv:




5′-AGCCTCCTGGACATTTTCCT-3′
(SEQ ID NO: 140)






The first 1579 bp glgC PCR fragment was amplified by a BIOTAQTM DNA Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega) and restricted with EcoRI (5′overhangs; Fermentas). The kanamycin cartridge was remained by a restriction of the pUC4K vector with EcoRI (5′overhangs; Fermentas).
Plasmids were analyzed by restriction digest in order to select constructs with both orientations of the inserted kanamycin cartridge, respectively.
The construct pGEM-T/ΔglgC-KMantisense has the structure shown in FIG. 6B, and its insert the nucleotide sequence presented in FIG. 6C.
In the other construct, designated as pGEM-T/ΔglgC-KMsense the kanamycin resistance cartridge is inserted in the other direction.
The second 1453 bp glgC PCR fragment was amplified by a BIOTAQTM DNA Polymerase (BIOLINE), cloned into the pDrive vector (Qiagen) and restricted with BsaBI (blunt ends; Biolabs). The chloramphenicol cartridge was remained by restriction of the pACYC184 vector (Biolabs, Ac No. X06403) with BsaAI (blunt ends; Biolabs).
Plasmids were analyzed by restriction digest in order to select constructs with both orientations of the inserted chloramphenicol resistance (Cm) cartridge, respectively.
A construct designated as pDrive/ΔglgC-CMantisense was selected; its structure is presented schematically in FIG. 6D and the nucleotide sequence of the insert is presented in FIG. 6E.
In the other construct, designated as pDrive/ΔglgC-CMsense the chloramphenicol resistance cartridge is inserted in the other direction.
c) Construction of DNA-Vector for Generation of a Pyruvate Water Dikinase Knock-Out Mutant (ΔppsA)
The open reading frame (ORF) slr0301 encodes pyruvate water dikinase/PEP synthase (EC 2.7.9.2),
Ac. No BAA10668. This protein has the amino acid sequence that is presented in FIG. 7A.
Two constructs were generated for knock-out of pyruvate water dikinase differing in orientation of the inserted kanamycin resistance cartridge (in sense and in antisense orientation to the ppsA ORF) using the following primers:










#PpsA547.fw:




5′-TTCACTGACCGGGCTATTTC-3′
(SEQ ID NO: 141)


 



#PpsA2329.rv:




5′-CTTGGCCACAGATACCGATT-3′
(SEQ ID NO: 142)






A 1783 bp ppsA PCR fragment was amplified by a BIOTAQTM DNA Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega) and restricted with SmaI (blunt ends; Fermentas). The kanamycin cartridge was remained by a restriction of the pUC4K vector with EcoRI (5′ overhangs; Fermentas) and a following “fill in reaction” via the T4 DNA Polymerase (Promega). Plasmids were analyzed by restriction digest in order to select constructs with both orientations of the inserted kanamycin cartridge.
The construct used, designated as pGEM-T/ΔppsA-antisense, has the structure presented in FIG. 7B. The nucleotide sequence of it insert is presented in FIG. 7C.
In the other construct, designated as pGEM-T/ΔppsA-sense the kanamycin resistance cartridge is inserted in the other direction.
d) Construction of a DNA-Vector for Generation of a Lactate Dehydrogenase Knock-Out Mutant (Δldh)
The open reading frame (ORF) slr 1556 encodes a putative lactate dehydrogenase (EC 1.1.1.28), annotated as 2-hydroxyaciddehydrogenase homolog (P74586). This amino acid sequence for this protein is presented in FIG. 8A.
A 1931 bp fragment containing the entire coding sequence from lactate dehydrogenase (slr1556) was amplified by PCR using the following primer:








#lhd-1fw:



5′-GCGAACTACCCAACGCTGACCGG-3′
(SEQ ID NO: 143)


 


#ldh-2rv:



5′-GCATCAAGTGTTGGGGGATATCCCTG-3′,
(SEQ ID NO: 144)






primer contains a EcoRV restriction site (GATATC) for cloning (marked in bold letters).

The PCR fragment was digested with NheI/EcoRV (NheI site is present in the genomic sequence) and cloned into pBluescript SK+ vector using XbaI/EcoRV. The kanamycin resistance cassette was used from the DNA vector pUC4K and ligated into the BglII/BclI restriction sites of slr1556. A restriction map of this is presented in FIG. 8B.
The knock-out-construct used, named pBlue ldh-Kan-a, has the structure presented in FIG. 8C, and the nucleotide sequence for its insert is presented in FIG. 8D.
e) Construction of a DNA-Vector for Generation of an Acetate Kinase Knock-Out Mutant (Δack)
The open reading frame (ORF) sll 1299 encodes a putative acetate kinase (EC 2.7.2.1), Ac No. P73162. The amino acid sequence for this protein is presented in FIG. 9A.
A 2316 bp fragment containing the entire coding sequence from acetate kinase (sll1299) was amplified by PCR using the following primer:










#ack-1 fw:




5′-CCGGGACGTGACAGAACGGGTGG-3′
(SEQ ID NO: 145)


 



#ack-2 rv:




5′-GCGTTGGCGATCGCCGTCACTAG-3′
(SEQ ID NO: 146)






The PCR fragment was digested with SpeI (both sites are located in the genomic sequence) and cloned into pBluescript SK+ vector. The kanamycin resistance cassette was used from the DNA vector pUC4K and ligated into the HpaI restriction sites of slr1299. A restriction enzyme map of this region is presented in FIG. 9B.
The orientation of the kanamycin resistance cassette was either in the same direction as sll1299 (designed “a”) or in the opposite direction (designed “b”).
The knock-out-construct used, named pBlue ack-Kan-b, has the structure presented in FIG. 9C, and the nucleotide sequence of its insert is presented in FIG. 9D.
f) Construction of a DNA-Vector for Generation of a Phosphoacetyltransacetylase (Phosphoacyltransferase) Knock-Out Mutant (Δpta)
The open reading frame (ORF) slr2132 encodes a phosphoacetyltransacetylase (EC 2.3.1.8), Ac No. P73662. The amino acid sequence for this protein is presented in FIG. 10A.
A 2869 bp fragment containing the entire coding sequence from phosphoacetyl-transacetylase (slr2132) was amplified by PCR using the following primer:










#pta-1fw:




5′-GCCATTGTGGGGGTGGGTCAG-3′
(SEQ ID NO: 147)


 



#pta-2rv: 




5′-CAGTTTATGCCCCGCTACCGGG-3′
(SEQ ID NO: 148)






The PCR fragment was digested with MfeI/HindIII (both sites present in the genomic sequence) and cloned into pUC19 (EcoRI/HindIII) vector. The chloramphenicol resistance cassette was used from plasmid pACYC184 and ligated into the ClaI/PstI restriction sites of slr2132. A restriction map of this region is presented in FIG. 10B.
The knock-out-construct selected is named pUC pta-Cm. It's structure is presented schematically in FIG. 10C, and the nucleotide sequence of the insert for this clone is presented in FIG. 10D.
g) Construction of DNA-Vector for Generation of PHB Knockout Mutant (ΔphaC)
The open reading frame (ORF) slr1830 encodes poly(3-hydroxyalkanoate) synthase [EC:2.3.1.], Ac. No BAA17430. The amino acid sequence for this protein is presented in FIG. 11A.
One construct was generated for knock out of poly(3-hydroxyalkanoate) synthase by deletion/insertion (resistance cartridge: kanamycin) mutagenesis.








# phaC-25′HD —XbaI.fw:



5′-CCGATGtcTAGaTAATTCACCATC-3′
(SEQ ID NO: 149)


 


# phaC404_BamHI.rv:



5′-TCTAGGGggAtCCAACGATCG-3′
(SEQ ID NO: 150)


 


# phaC711_BamHI.fw:



5′-CCAGGGGATccTCTTAACCTAG-3′
(SEQ ID NO: 151)


 


# phaC1133′HD —ClaI.rv:



5′-TGTCGTatCGATAGCCAATGG-3′
(SEQ ID NO: 152)






Two PCR products (pos. 24 to pos. 404; pos. 711 to pos. 1133) of the phaC fragment were amplified by a BIOTAQTM DNA Polymerase (BIOLINE), ligated via BamHI sites and cloned into the pIC2OH vector. The kanamycin cartridge was remained by a restriction of the pUC vector (http://seq.yeastgenome.org/vectordb/vector_descrip/COMPLETE/PUC4K. SEQ.html) with BamHI (Fermentas). Plasmids were analyzed by restriction digest. Knockouts were generated via homologous recombination of the wild type gene with the mutant genes.
The construct selected is pIC2OH/ΔphaC-KM and has the structure presented schematically in FIG. 11B. The nucleotide sequence for the insert of this clone is presented in FIG. 11C.
h) Construction of DNA-Vectors for Generation of Knockout Mutants of ADP-Glucose-Pyrophosphorylase, agp (glgC) in the Filamentous, Diazotrophic Cyanobacteria Nostoc/Anabaena spec. PCC7120 and Anabaena variabilis ATCC 29413
In order to generate ethanol producing Anabaena strains, different constructs were created for conjugation into Anabaena PCC7120 and Anabaena variabilis ATCC29413. Constructs for genome integration of ethanologenic genes were created for both Anabaena strains. As integration site into the genome the glucose-1-phosphate adenylyltransferase gene (ADP-glucose-pyrophosphorylase, agp, glgC) was chosen. Thus, by integration of the ethanologenic genes simultaneously an agp knock-out mutant was created.
Glucose-1-phosphate adenylyltransferase (ADP-glucose-pyrophosphorylase, agp, glgC), EC 2.7.7.27, of Anabaena spec. PCC7120 is encoded by ORF all4645, Ac. No. P30521. The amino acid sequence of ORF all4645 is shown in FIG. 11D.
Constructs for conjugation into Anabaena PCC7120 were cloned as followed:
Two fragments representing the 5′ and 3′ part of the ADP-glucose-pyrophosphorylase (agp) gene, ORF all4645, were amplified by PCR using the following primers:










#agp1.1




5′-CATCCATCATGAGCTCTGTTAAC-3′
(SEQ ID NO: 153)



(SacI site inserted)



 



#agp2.1




5′-GTATCTCGAGCGATGCCTACAGG-3′
(SEQ ID NO: 154)



(XhoI site inserted)



 



#agp3.1




5′-CGCATTGGTTTCTAGATGGCGC-3′
(SEQ ID NO: 155)



(XbaI site inserted)



 



#agp4.1




5′-CGATAACTCTAGACGAGTCATTG-3′
(SEQ ID NO: 156)



(XbaI site inserted)







Inserted Restriction Sites in Primer Sequences are Marked in Bold Letters

As indicated in FIG. 11E, in between these agp fragments a C.K3 cassette (coding for kanamycin/neomycin resistance) was ligated into the XbaI site. [C.K3 cassette is described in Elhai, J. & Wolk, C. P. (1988) Gene, 68, 119-138.]
The entire “agp knock-out” fragment was cloned into suicide vector pRL271 (Ac. No. L05081). The pdc/adh genes, or only pdc, were cloned downstream of the inducible promoter PpetE and integrated into the “agp-C.K3” construct.
The following constructs have been generated:
pRL271 agp (all4645)::C.K3
pRL271 agp (all4645)::C.K3-PpetE-pdc-adhII
pRL271 agp (all4645)::C.K3-PpetE-pdc
The structures of the constructs are depicted in FIG. 11-2.
The sequence of the insert of pRL271 agp (all4645)::C.K3-PpetE-pdc-adhII is shown in FIG. 11F.
The same strategy was used to create constructs for expression in Anabaena variabilis ATCC29413. The nucleotide sequences of the agp genes from both strains are 97%, their protein sequences are 99.3% identical.
Glucose-1-phosphate adenylyltransferase (ADP-glucose-pyrophosphorylase, agp, glgC), EC 2.7.7.27, of Anabaena variabilis ATCC29314 is encoded by ORF Ava_2020, Ac. No. Q3MBJ4, and has the amino acid sequence as shown in FIG. 11G.
For PCR amplification of the genomic fragments of Anabaena variabilis the following primers were used:










#agp1.2




5′-GAGGCAATGAGCTCCACTGGACG-3′
(SEQ ID NO: 157)



(SacI site inserted)



 



#agp2.2




5′-CTGGCGTTCCACTCGAGCTTGG-3′
(SEQ ID NO: 158)



(XhoI site inserted)



 



#agp3.1




5′-CGCATTGGTTTCTAGATGGCGC-3′
(SEQ ID NO: 159)



(XbaI site inserted)



 



#agp4.2




5′-CGATAACTCTAGACGAGTCATCG-3′
(SEQ ID NO: 160)



(XbaI site inserted)







Inserted restriction sites in primer sequences are marked in bold letters.
Generation of the constructs was exactly as described for the constructs of Anabaena PCC7120.
The following constructs have been generated:
pRL271 agp::C.K3
pRL271 agp::C.K3-PpetE-pdc-adhII
pRL271 agp::C.K3-PpetE-pdc
All described plasmids were conjugated into Anabaena strains according the following method:
Conjugation of Nostoc spec. PCC7120/Anabaena variabilis 
Cargoplasmids
Cargoplasmids (pRL593, pRL1049 or pRL271) were transformed into competent E. coli HB101 (pRL528helperplasmid).
In Preparation for Conjugation
E. coli Cultures:


    
    
        inoculation of overnight cultures in LB with the appropriate antibiotics from
        
            Cargoplasmid in E. coli HB101 (pRL528helperplasmid)
            Helperstrain E. coli J53 (RP4)
        
        
        preparation of well growing culture (for each conjugation/plate 10 ml of HB101 (pRL528+cargo plasmid) and 10 ml of J53 (RP4) is needed): inoculate 0.25 ml overnight culture in 10 ml LB+antibiotic, grow for 2.5 h/37° C.
        spin down the well grown E. coli cultures in “Falcons” 10 min 4800 rpm.
        
            (for J53 culture: take 2 Falcons).
        
        
        “wash”/resuspend cells in equal volume of LB without antibiotics.
        for each conjugation spin 10 ml of resuspended HB101 (culture carrying pRL528+cargo plasmid) in 15 ml Falcon tube, remover supernatant
        add on the cell pellets 10 ml resuspended J53 (RP4) culture, spin down, remove supernatant and resuspend combined cells in 1 ml LB, transfer cells in Eppi tubes, resuspend again in 100 μl and incubate for 2 h at 30° C.

Cyanos

        determine the chlorophyll concentration of well grown Anabaena cultures
        for each conjugation, culture corresponding to about 10 μg Chlorophyll is needed.
        spin down the equivalent volume of Anabaena culture and resuspend to a volume corresponding to 10 μg Chlorophyll/100 μl BG11 medium.

Conjugation

        for each conjugation place one HATF filter on a plate (BG11)
        mix 100 μl E. coli suspension=100 μl Anabaena culture and plate on filter
        incubate plates at 30° C. overnight wrapped in paper
        next day remove paper
        after one day transfer filter on plates containing antibiotics.

Construction of DNA-Vectors for Generation of Knock-Down Mutants

a) Construction of a DNA-Vector for Generation of a Pyruvate Dehydrogenase (pdhB) Knock-Down Mutant

    
    


The open reading frame (ORF) sll1721 encodes the β-subunit of the E1 component of the pyruvate dehydrogenase, (EC 1.2.4.1), Ac. No BAA17445. This protein has the amino acid sequence presented in FIG. 12A.
Two strategies were considered for knock-down of the pyruvate dehydrogenase. A knock-down could be achieved by regulation of the expression of the adequate antisense RNA (i) or by insertion of a controllable wild type gene copy accompanied by a knock-out of the original wild type gene (ii). Therefore, four constructs were generated to knock-down the pyruvate dehydrogenase.
The PCR fragments for the expression of the adequate antisense RNA as well as for the controllable wild type gene copy were amplified by a High-Fidelity DNA Polymerase (PhusionTM; Finnzymes), adenylated (BIOTAQTM DNA Polymerase; BIOLINE), cloned into the pDrive vector (Qiagen) and restricted with ClaI/BglII (i) or NdeI/BglII1 (ii) (Fermentas). These fragments were cloned into the pSK9 1 BglII was used instead of ClaI because this inserted ClaI cleavage side was affected by Dam-methylation. The BglII cleavage side is part of the 3′ end of the amplified PCR product and do not affect the translation termination loop. vector, digested with ClaI/BglII (i) or NdeI/BglII (ii). The non-public pSK9 vector was generated in the lab of V. V. Zinchenko (Moscow, Russia). The gene is incorporated into a non-coding genome region via the integrated platform. The expression of the enzyme and the antisense RNA is under the control of the copper inducible promoter petJ. The termination of transcription is achieved either by the gene-specific terminator loop (ii) or by the oop-terminator of the lambda phage (i) (Toop is part of the reverse-Primer), both amplified by PCR reaction.









#PdhBantiClaI.fw:







(SEQ ID NO: 161)









5′-ATCGATATAATTTCCGGGTCGTAGCC-3′,






this primer contains a ClaI restriction site for cloning (marked in bold letters)

#PdhBantioopBglII.rv:








(SEQ ID NO: 162)


5′GATCTGGAATAAAAAACGCCCGGCGGCAACCGAGCGGCAGCCATTCGG


 


GATAATAA-3′,






this primer contains a BglII restriction site for cloning (marked in bold letters) and the oop terminator region of the lambda phage (underlined)








(SEQ ID NO: 163)









#PdhBNdeI.fw: 5′-CATATGGCTGAGACCCTACTGTTT-3′,






this primer contains a NdeI restriction site for cloning (marked in bold letters)








(SEQ ID NO: 164)


#PdhB1061ClaI.rv 5′-ATCGATCTTACAAGCTCCCGGACAAA-3′,






this primer contains a ClaI restriction site for cloning (marked in bold letters)

The 1142 bp pdhB PCR fragment for the knock-out of the original wild type gene was amplified by a BIOTAQTM DNA Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega) and restricted with Eco147I (blunt ends; Fermentas). The kanamycin cartridge was remained by a restriction of the pUC4K vector with EcoRI (5′ overhangs; Fermentas) and a following “fill in reaction” via the T4 DNA Polymerase (Promega) and ligated into the Eco147I site. Resulting plasmids were analyzed by restriction digest in order to select constructs with both orientations of the inserted kanamycin cartridge. Knock-outs were generated via homologous recombination of the wild type gene with the mutant genes. The following primers were used for PCR:







(SEQ ID NO: 165)









# PdhB.fw: 5′-AATCGACATCCACCCTTGTC-3′


 







(SEQ ID NO: 166)









# PdhB.rv: 5′-GCCTTAACTGCGTCCACAAT-3′






(i) Knock-Down by Regulation of the Expression of the Adequate Antisense RNA

The construct used, designated as pSK9/pdhBanti, has the structure presented in FIG. 12B, and the nucleotide sequence of its insert is presented in FIG. 12C.
(ii) Knock-Down by Insertion of a Controllable Wild Type Gene Copy Accompanied by a Knock-Out of the Original Wild Type Gene
The construct used, designated as pSK9/pdhB, has the structure presented in FIG. 12D, and the nucleotide sequence of the insert for this clone is presented in FIG. 12E.
The knock-out construct used, designated as pGEM-T/ΔpdhB-KMantisense, has the structure presented in FIG. 12F. The sequence for the insert in this clone is presented in FIG. 12G.
In the other construct, designated as pGEM-T/ΔpdhB-KMsense the kanamycin resistance cartridge is inserted in the other direction.
In the following the cloning vectors, which were used are described.
a) Cloning Vector pGEM®-T Structure and Sequence
PCR cloning vector pGEM®-T was from Promega corp., Madison Wis., USA. The structure of the plasmid is presented in FIG. 13A, and its nucleotide sequence is presented in FIG. 13B.
b) Cloning Vector pDrive Structure and Sequence
Cloning vector pDrive was from Qiagen, Hilden, Germany. The structure of this plasmid is presented in FIG. 14A and its nucleotide sequence in FIG. 14B.
c) Cloning Vector pBlueSK+ Structure and Sequence
Cloning vector pBluescript II® SK+ (Ac. No X52328) was from Stratagene, La Jolla, Calif., USA.
The structure of this plasmid is presented in FIG. 15A and, its nucleotide sequence is presented in FIG. 15B.
d) Cloning Vector pUC19 Structure and Sequence
Cloning vector pUC19 (Ac. No M77789) is presented schematically in FIG. 16A, and its nucleotide sequence is presented in FIG. 16B.
e) Plasmid pSK9 Structure and Sequence
The non-public pSK9 vector was generated in the lab of V. V. Zinchenko (Moscow, Russia). A schematic of pSK9 structure is presented in FIG. 17A, and its nucleotide sequence is presented in FIG. 17B.
Protocols for Generation of Synechocystis sp. PCC 6803 Mutants Overexpressing the Following Genes:
a) malic enzyme
b) malate dehydrogenase
c) malic enzyme and malate dehydrogenase
d) pyruvate kinase 1
e) pyruvate kinase 2
f) pyruvate kinase, enolase and phosphoglycerate mutase
g) enolase
h) phosphoglycerate mutase
i) pyruvate kinase (1 or 2)/enolase/phosphoglycerate mutase
j) phosphoketolase
k) phosphoacetyltransacetylase
l) phosphoketolase/phosphoacetyltransacetylase
m) acetaldehyde dehydrogenase
n) PEP carboxylase
o) ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)
Construction of DNA-Vectors for Overexpression
In General:
DNA sequences encoding genes of interest were amplified by polymerase chain reaction (PCR) using specific primers. When the genomic sequence did not contain appropriate restriction sites for cloning, primers were designed containing restriction sites. Genomic DNA from Synechocystis sp. PCC 6803 was used as template. The amplified PCR fragments were digested with the appropriate restriction enzymes and cloned into either a self replicating plasmid (pVZ series) or an integrative plasmid (pSK series). As promoters either the genomic 5′region of the specific gene itself was used or alternative an inducible promoter like PpetJ. (PpetJ, pVZ, pSK, for description see below mentioned adh/pdc constructs). An antibiotic resistance cassette for selection of positive clones is present on the appropriate plasmid. The structures and sequences of all used DNA-vectors are described below (see 2).
Genetic engineering of constructs as well as PCRs, ligations into cloning vectors, insertions of antibiotic resistance cassettes and transformations into E. coli were done using standard procedures (state of the art) or according to the manufacturer instructions.
All pVZ plasmids were transferred to Synechocystis sp. PCC 6803 bp conjugation. This method is described for the below mentioned adh/pdc constructs. The pSK constructs were transferred to Synechocystis sp. PCC 6803 by transformation. The method of transformation using the natural competence of Synechocystis sp. PCC 6803 for DNA uptake was already described in detail for the generation of the glycogen synthase mutant.
a) Construction of DNA-Vectors for Overexpression of Malic Enzyme
The open reading frame (ORF) slr0721 encodes malic enzyme 1 (EC 1.1.1.38), Ac. No P72661. The amino acid sequence for this protein is presented in FIG. 18A.
For overexpression of malic enzyme, the encoding me gene together with its gene-specific terminator region was PCR-amplified using the following primer:
Mae-NdeI.fw: 5′-CATATGGTTAGCCTCACCCCCAAT-3, (SEQ ID NO:167), primer contains a NdeI restriction site for cloning (marked in bold letters)
MeLongClaI.rv: 5′-ATCGATCGGGATGGCCTATTTATGG 3′ (SEQ ID NO:168), primer contains a ClaI restriction site for cloning (marked in bold letters)
The PCR fragment was amplified by a High-Fidelity DNA Polymerase (PhusionTM; Finnzymes), adenylated (BIOTAQTM DNA-Polymerase; BIOLINE), cloned into the pDrive vector (Qiagen) and restricted with NdeI/ClaI (Fermentas). This fragment was cloned into the pSK9 vector, digested with NdeI/ClaI. The gene is incorporated into a non-coding genome region of Synechocystis sp. PCC 6803 via the integrated platform. The expression of the enzyme is under control of the copper dependent promoter PpetJ.
The construct used, designated as pSK9/me-long, has the structure presented in FIG. 18B. The insert for this clone has the nucleotide sequence presented in FIG. 18C
b) Construction of DNA-Vector for Overexpression of Malate Dehydrogenase
An open reading frame (ORF) sll0891 encodes malate dehydrogenase (EC 1.1.1.37), Ac. No Q55383. The amino acid sequence for this protein is presented in FIG. 19A.
For overexpression of malate dehydrogenase a construct was generated including start-codon and the gene specific termination loop of the mdh gene using the following primers:







(SEQ ID NO: 169)


Mdh-NdeI.fw: 5′-CATATGAATATTTTGGAGTATGCTCC-3′,






primer contains a NdeI restriction site for cloning (marked in bold letters)








(SEQ ID NO: 170)









Mdh-ClaI.rv 5′-ATCGATAAGCCCTAACCTCGGTG-3′,






primer contains a ClaI restriction site for cloning (marked in bold letters)

The PCR fragment was amplified by a High-Fidelity DNA Polymerase (PhusionTM; Finnzymes), adenylated (BIOTAQTM DNA-Polymerase; BIOLINE), cloned into the pDrive vector (Qiagen) and restricted with NdeI/ClaI (Fermentas). This fragment was cloned into the pSK9 vector, digested with NdeI/ClaI. The expression of the enzyme is under the control of the copper dependent promoter PpetJ.
The construct used, designated as pSK9/mdh, has the structure presented in FIG. 19B; the nucleotide sequence for the insert of this clone is presented in 19C.
c) Construction of DNA-Vector for Co-Overexpression of Malic Enzyme and Malate Dehydrogenase
This construct was generated for co-overexpression of malic enzyme and malate dehydrogenase. These genes were amplified by PCR using primers including the start and stop-codon of the me gene (PCR fragment I) and including the ribosome binding site (RBS) and termination loop of the mdh gene (PCR fragment II). The co-expression of the enzymes is under the control of the copper dependent promoter PpetJ.
The following primers were used for amplification

    
    
        PCR Fragment I:
    
    









(SEQ ID NO: 171)









Mae-NdeI.fw: 5′-CATATGGTTAGCCTCACCCCCAAT-3′,






primer contains a ′NdeI restriction site for cloning (marked in bold letters)










MeShortClaI.rv:







(SEQ ID NO: 172)









5′-ATCGATACAATTCCCGATTAACTATTGACC-3′,






primer contains a ClaI restriction site for cloning (marked in bold letters)

    
    
        PCR fragment II:
    
    









(SEQ ID NO: 173)


MdhRBSClaI.fw: 5′-ATCGATTTTTCTCCACCATCAACACC-3′,






primer contains a ClaI restriction site for cloning (marked in bold letters)








(SEQ ID NO: 174)









MdhBglII.rv: 5′-AGATCTAAGCCCTAACCTCGGTG-3′,






primer contains a BglII restriction site for cloning (marked in bold letters)

The PCR fragments were amplified by a High-Fidelity DNA Polymerase (PhusionTM; Finnzymes), adenylated (BIOTAQTM DNA-Polymerase, BIOLINE), cloned into the pDrive vector (Qiagen) and restricted with NdeI/ClaI and ClaI/BglII (Fermentas), respectively. These fragments were cloned into the pSK9 vector, first digested with NdeI/ClaI for integration of malic enzyme and secondly with ClaI/BglII for integration of malate dehydrogenase.
The construct used, designated as pSK9/me-mdh, has the structure presented in FIG. 19D, and the nucleotide sequence of its insert is presented in FIG. 19E.
d) Construction of DNA-Vectors for Overexpression of Pyruvate Kinase 1
The open reading frame (ORF) sll0587 encodes a pyruvate kinase 1 (EC 2.7.1.40 (PK1)), Ac. No Q55863. The amino acid sequence of this protein is presented in FIG. 20A.
Two constructs were generated in order to overexpress pyruvate kinase 1. One, harboring the own pyruvate kinase promoter region, and another construct on which pyruvate kinase 1 is under control of the inducible promoter PpetJ.
For the construct with the genomic 5′-region of the pyruvate kinase gene itself serving as promoter, a 2376 bp fragment containing the entire coding sequence from pyruvate kinase 1 (sll 0587) plus 770 bp upstream of the gene (promoter region) and 320 bp downstream of the gene (terminator region) was amplified by PCR using the following primer:







(SEQ ID NO: 175)









#pykA-5fw: 5′-CCTGTTATTGGCCACGGGCAGTA-3′,


 







(SEQ ID NO: 176)









#pykA-2rv: 5′-GGTTTACCCTGGGCTCGAGAATTTAGG-3′,






primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

The PCR fragment was digested with MfeI/XhoI (MfeI site was present in the genomic sequence; MfeI shares compatible cohesive ends with EcoRI), subcloned into pIC20H (using EcoRI/XhoI), cut out of this plasmid with SalI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ321 (self replicating plasmid).
The construct used, named pVZ321-pyk1, has the structure presented in FIG. 20B, and its insert nucleotide sequence is presented in FIG. 20C.
For the construct on which pyruvate kinase 1 is under control of the inducible promoter PpetJ, a 1763 bp fragment containing the entire coding sequence from pyruvate kinase 1 (sll 0587) plus 320 bp downstream of the gene (terminator region) was amplified by PCR using the following primer:







(SEQ ID NO: 177)









#pykA-3fw: 5′-CCCGGTGAAGCATATGAGACCCCT-3′,






primer contains a NdeI restriction site (CATATG) for cloning (marked in bold letters). ATG in the restriction site represents the start codon of the gene.








(SEQ ID NO: 178)









#pykA-2rv: 5′-GGTTTACCCTGGGCTCGAGAATTTAGG-3′,






primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

The PCR fragment was digested with NdeI/XhoI, ligated to PpetJ (SalI/NdeI) and cloned into the E. coli-Synechocystis shuttle vector pVZ321 (self replicating plasmid).
The construct used, named pVZ321-PpetJ-pyk1, has the structure presented in FIG. 20D, and the nucleotide sequence of its insert is presented in FIG. 20E.
e) Construction of DNA-Vectors for Overexpression of Pyruvate Kinase 2
The open reading frame (ORF) sll1275 encodes pyruvate kinase 2 (EC 2.7.1.40 (PK2)), Ac. No P73534. The amino acid sequence for this protein is presented in FIG. 21A.
Two constructs were generated in order to overexpress pyruvate kinase 2. One, harboring the own pyruvate kinase promoter region, and another construct on which pyruvate kinase 2 is under control of the inducible promoter PpetJ.
For the construct with the genomic 5′ region of the pyk2 gene itself serving as promoter, a 2647 bp fragment containing the entire coding sequence from pyk 2 (sll 1275) plus 600 bp upstream of the gene (promoter region) and 280 bp downstream of the gene (terminator region) was amplified by PCR using the following primer:







(SEQ ID NO: 179)









#pykB-1fw: 5′-CCTAAATTCAGGTCGACCGGCAAAC-3′,






primer contains a SalI restriction site (GTCGAC) for cloning (marked in bold letters).











# pykB-2rv:




5′-CACCAACCAGGCTCGAGTGGG-3′,
(SEQ ID NO: 180)






primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

The PCR fragment was digested with SalI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ321 (self replicating plasmid).
The construct used, named pVZ321-pyk2, has the structure presented in FIG. 21B, and the nucleotide sequence of its insert is presented in FIG. 21C.
For the construct on which pyruvate kinase 2 is under control of the inducible promoter PpetJ, a 2057 bp fragment containing the entire coding sequence from pyruvate kinase 2 (sll 1275) plus 280 bp downstream of the gene (terminator region) was amplified by PCR using the following primer:








# pykB-3fw:



5′-CCTAATTTCAGCCCCATATGCAAACG-3′,
(SEQ ID NO: 181)






primer contains a NdeI restriction site (CATATG) for cloning (marked in bold letters). ATG in the restriction site represents the start codon of the gene.











# pykB-2rv:




5′-CACCAACCAGGCTCGAGTGGG-3′,
(SEQ ID NO: 182)






primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

The PCR fragment was digested with NdeI/XhoI, ligated to PpetJ (SalI/NdeI) and cloned into the E. coli-Synechocystis shuttle vector pVZ321 (self replicating plasmid).
The resulting construct, pVZ321-PpetJ-pyk2, has the structure presented in FIG. 21D, and the nucleotide sequence of its insert is presented in FIG. 21E.
f) Construction of DNA-Vector for Overexpression of Pyruvate Kinase, Enolase and Phosphoglycerate Mutase
A DNA-vector was constructed in order to express additional genes coding for pyruvate kinase, phosphoglycerate mutase and enolase. A DNA fragment encoding these genes was cut out of plasmid #67. This plasmid was constructed by Dr. John Coleman, University of Toronto, Toronto, Canada.
The insert of plasmid #67 has the structure presented in FIG. 22A.
The insert of plasmid #67 contains a 357 bases long cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) promoter (Prbc) from Synechococcus PCC 7942. Downstream of this promoter there are three inserted open reading frames, the first is pyruvate kinase 1 from E. coli, the second enolase and the third phosphoglycerate mutase both from Zymomonas mobilis. The pyruvate kinase region differs from E. coli K-12 pyruvate kinase 1 (Ac. No AAC74746) by 3 nucleotides and one amino acid. (G to D mutation, underlined in the sequence below). The enolase gene from Zymomonas mobilis (Ac. No YP_163343) is a 100% amino acid match. The nucleotide sequence differs by two synonymous substitutions in the enolase region. The phosphoglycerate mutase gene is one amino acid different from Zymomonas (Ac. No YP_162975), from G to D at 118th amino acid (underlined in the sequence below). A HindIII site links the E. coli pyruvate kinase and the Zymomonas enolase genes.
The amino acid sequences of the enzymes encoded by the described insert are presented in FIG. 22B for pyruvate kinase I (E. coli K12); in FIG. 22C for enolase (Zymomonas mobilis); and in FIG. 22D for phosphoglycerate mutase (Zymomonas mobilis).
The nucleotide sequence of the described insert of plasmid #67 is presented in FIG. 22E.
The insert of plasmid #67 was cut out the vector using restriction enzymes XmaI and SpeI and cloned into the E. coli-Synechocystis shuttle vector pVZ321 and pVZ322 (self replicating plasmids) (XmaI/XbaI); XbaI and SpeI share compatible cohesive ends.
Plasmid pVZ321-p67 has the structure presented in FIG. 22F, and plasmid pVZ322-p67 has the structure presented in FIG. 22G.
g) Construction of DNA-Vectors for Overexpression of Enolase
The open reading frame (ORF) slr0752 encodes the enolase (eno, 2-phosphoglycerate dehydratase) (EC 4.2.1.11), Ac. No BAA18749. The amino acid sequence for this protein is presented in FIG. 23A.
A construct was generated for overexpression of enolase under control of the inducible promoter PpetJ.
The construct includes the petJ promoter, the 1299 bp coding sequence for enolase (slr0752) and 214 bp downstream of the gene (terminator region). The enolase gene was amplified by PCR using the following primer:









# Eno-SacI-ATG







(SEQ ID NO: 183)









5′-TAGAGCTCTTAAGTAAAGTCCCCGCCACCAT-3′,


 



# Eno-XhoI-rev







(SEQ ID NO: 184)









5′-TACTCGAGGTCATTGCTTCCTTGGCTTAGAAC-3′,






Primers contain a SacI or XhoI restriction site, respectively, for cloning (marked in bold letters).
The PCR fragment was digested with SacI/XhoI and ligated downstream of the PpetJ promoter into pJet-PpetJ. The entire PpetJ-enolase fragment was cut out of this plasmid with SalI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ321 (self replicating plasmid).
The construct used, named pVZ321-PpetJ-eno, has the structure presented in 23B, and the nucleotide sequence of its insert is presented in 23C.
h) Construction of DNA-Vectors for Overexpression of Phosphoglycerate Mutase
The open reading frame (ORF) slr1124 encodes the phosphoglycerate mutase (pgm or gpmB) (EC 54.2.1), Ac. No BAA16651. The amino acid sequence for this protein is presented in FIG. 24A.
A construct was generated for overexpression of phosphoglycerate mutase under control of the inducible promoter PpetJ.
The construct includes the petJ promoter, the 1047 bp coding sequence for phosphoglycerate mutase (slr1124) and 143 bp downstream of the gene (terminator region). The phosphoglycerate mutase gene was amplified by PCR using the following primer:









# Pgm-SacI-ATG







(SEQ ID NO: 185)









5′-TAGAGCTCACCAAAGACGATGTGGCCCACCAA-3′


 



# Pgm-XhoI-rev







(SEQ ID NO: 186)









5′-TACTCGAGTATGACCCCGCTGTTGCAGTTC-3′






Primers contain a SacI or XhoI restriction site, respectively, for cloning (marked in bold letters).
The PCR fragment was digested with SacI/XhoI and ligated downstream of the PpetJ promoter into pJet-PpetJ. The entire PpetJ-phosphoglycerate mutase fragment was cut out of this plasmid with SalI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ321 (self replicating plasmid).
The construct used, named pVZ321-PpetJ-pgm, has the structure presented in FIG. 24B, the nucleotide sequence of its insert is presented in FIG. 24C.
i) Construction of DNA-Vectors for Co-Overexpression of Pyruvate Kinase 1 or 2, Enolase and Phosphoglycerate Mutase
Further plasmids were generated in order to overexpress the three glycolytic enzymes pyruvate kinase 1 or 2, enolase and phosphoglycerate mutase from one transcript.
One construct was generated for overexpression of pyruvate kinase 1 (ORF sll0587), enolase (ORF slr0752) and phosphoglycerate mutase (ORF slr1124); the second construct encodes pyruvate kinase 2 (ORF sll1275), enolase (ORF slr0752) and phosphoglycerate mutase (ORF slr1124). The protein sequences, EC and Accession numbers of the enzymes are already described herein.
In both constructs the overexpression of the three genes is under control of the inducible promoter PpetJ.
The glycolytic genes were amplified by PCR using the following primers:









pyruvate kinase 1 (pyk1):



# pykA-3fw







(SEQ ID NO: 187)









5′-CCCGGTGAAGCATATGAGACCCCT-3′


 



(NdeI-site inserted)



# Pyk1-SacI-rev







(SEQ ID NO: 188)









5′-TAGAGCTCTTAAGAAATACGGTGAATCTTG-3′


 



pyruvate kinase 2 (pyk2):



# pykB-3fw:







(SEQ ID NO: 189)









5′-CCTAATTTCAGCCCCATATGCAAACG-3′


 



(NdeI-site inserted)



# Pyk2-SacI-rev







(SEQ ID NO: 190)









5′-TAGAGCTCCCTATCCTTTGGACACC-3′


 



enolase (eno):



# Eno-SacI-fw







(SEQ ID NO: 191)









5′-TAGAGCTCGTGTTTGGAGCATTACACACCGATG-3′


 



# Eno-BglII-rev







(SEQ ID NO: 192)









5′-TAAGATCTTTTTAAGAATGTTTGGGACCCAG-3′


 



phospgoglycerate mutase (pgm):



# Pgm-BglII-fw







(SEQ ID NO: 193)









5′-TCAGATCTGCCCCTCTGGGAAAAAATGACCA-3′


 



# Pgm-XhoI-rev







(SEQ ID NO: 194)









5′-TACTCGAGTATGACCCCGCTGTTGCAGTTC-3′






All primers contain restriction sites for cloning (marked in bold letters).
PCR fragments were subcloned into PCR cloning plasmid pJet1.2 blunt. The genes were cut out of these plasmids with the appropriate restriction enzymes and ligated downstream of the PpetJ promoter into pIC-PpetJ as followed:







5′-XhoI-pIC-PpetJ-NdeI-3′


               5′-NdeI-pyk1-SacI-3′


                         5′-SacI-eno-BglII-3′


                                  5′-BglII-pgm-


                                  XhoI-3′






The same construct was generated using fragment 5′-NdeI-pyk2-SacI-3′ instead of 5′-NdeI-pyk1-SacI-3′.
The entire PpetJ-pyk1-eno-pgm or PpetJ-pyk2 eno-pgm fragments were cut out of the cloning plasmid with PstI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ322 (self replicating plasmid).
The construct named pVZ322-PpetJ-pyk1-eno-pgm has the structure presented in FIG. 24D and the construct pVZ322-PpetJ-pyk2-eno-pgm has the structure presented in FIG. 24E. The sequence of the insert of pVZ322-PpetJ-pyk1-eno-pgm is presented in the FIG. 24F and the sequence of the insert of pVZ322-PpetJ-pyk2-eno-pgm is presented in FIG. 24G.
j) Construction of DNA-Vector for Overexpression of Phosphoketolase
The open reading frame (ORF) slr0453 encodes the probable phosphoketolase (phk), (EC 4.1.2.-), Ac. No P74690. The amino acid sequence of the protein is presented in FIG. 25A.
A construct was generated for overexpression of phosphoketolase under control of the inducible promoter PpetJ.
The construct includes the petJ promoter, the 2418 bp coding sequence for phosphoketolase (slr0453) and 307 bp downstream of the gene (terminator region). The phosphoketolase gene was amplified by PCR using the following primer:







(SEQ ID NO: 195)








# phk1-NdeI
5′-GTGTCTCATATGGTTACATCCCCCTTTTCCCTT-3′


 







(SEQ ID NO: 196)








# phk2-XhoI
5′-CGAGCCCTGCTCGAGCAGGC-3′






Primers contain a NdeI or XhoI restriction site, respectively, for cloning (marked in bold letters).
The PCR fragment was digested with NdeI/XhoI and ligated downstream of the PpetJ promoter into pIC-PpetJ. The entire PpetJ-phosphoketolase fragment was cut out of this plasmid with PstI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ322 (self replicating plasmid).
The construct used, named pVZ321-PpetJ-phk, has the structure presented in FIG. 25B, and the nucleotide sequence of its insert is presented in FIG. 25C.
k) Construction of DNA-Vector for Overexpression of Phosphoacetyltransacetylase
The open reading frame (ORF) slr2132 encodes a phosphoacetyltransacetylase (pta), EC 2.3.1.8, Ac No. P73662. The amino acid sequence of this protein is presented in FIG. 26A.
A construct was generated for overexpression of phosphoacetyltransacetylase under control of the inducible promoter PpetJ.
The construct includes the petJ promoter, the 2094 bp coding sequence from ORF slr2132 and 258 bp downstream of the gene (terminator region). The phosphoacetyltransacetylase gene was amplified by PCR using the following primer:









# pta_pPETJ1-NdeI







(SEQ ID NO: 197)









5′-GTGCCTCATATGACGAGTTCCCTTTATTTAAGCAC-3′


 



# pta_pPETJ2-XhoI







(SEQ ID NO: 198)









5′-CGGTTGCTCGAGCATCTGGAACGGTTGGGTAAAT-3′






Primers contain a NdeI or XhoI restriction site, respectively, for cloning (marked in bold letters).
The PCR fragment was digested with NdeI/XhoI and ligated downstream of the PpetJ promoter into pIC-PpetJ. The entire PpetJ-phosphoacetyltransacetylase fragment was cut out of this plasmid with PstI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ322 (self replicating plasmid).
The construct used, named pVZ322-PpetJ-pta, has the structure presented in FIG. 26B, and the nucleotide sequence of the insert for construct pVZ322-PpetJ-pta is presented in FIG. 26C.
l) Construction of DNA-Vector for Co-Overexpression of Phosphoketolase and Phosphoacetyltransacetylase
One further construct was created in order to co-overexpress the phosphoketolase and phosphoacetyltransacetylase from one transcript. The protein sequences, EC and Accession numbers of the enzymes are already described above. The expression of the genes is under control of the inducible promoter PpetJ. The phosphoketolase and phosphoacetyltransacetylase genes were amplified by PCR using the following primers:







# phkl


(SEQ ID NO: 199)


5′-GTGTCTCATATGGTTACATCCCCCTTTTCCCTT-3′


 


# phk-BgIII-rev


(SEQ ID NO: 200)


5′-GGTCACAGATCTGTTGTCCCCCATGGCCTAGCTA-3′


 


phosphoacetyltransacetylase (pta)


# pta-BgIII-fw


(SEQ ID NO: 201)


5′-CCTTGCAGATCTGGATACGTTGAGGTTATTTAAATTATGA-3′


 


# pta_pPETJ2-XhoI


(SEQ ID NO: 202)


5′-CGGTTGCTCGAGCATCTGGAACGGTTGGGTAAAT-3′






All primers contain restriction sites for cloning (marked in bold letters).
PCR fragments were cut with the appropriate restriction enzymes and ligated downstream of the PpetJ promoter into pIC-PpetJ as followed:









5′-XhoI-pIC-PpetJ-NdeI-3′



               5′-NdeI-phk-BglII-3′



                        5′-BglII-pta-XhoI-3′






The entire PpetJ-phk-pta fragment was cut out of the cloning plasmid pIC20H with SmaI/NruI and ligated into SmaI site of the E. coli-Synechocystis shuttle vector pVZ322 (self replicating plasmid).
The construct named pVZ322-PpetJ-phk-pta has the structure presented in FIG. 26D, and the nucleotide sequence of the insert of pVZ322-PpetJ-phk-pta is presented in FIG. 26E.
m) Construction of DNA-Vector for Overexpression of Aldehyde Dehydrogenase
The open reading frame (ORF) slr0091 encodes a aldehyde dehydrogenase (aldh), EC 1.2.1.3, Ac No. BAA10564 Q55811. The amino acid sequence for the protein is presented in FIG. 27A.
A construct was generated for overexpression of aldehyde dehydrogenase under control of the inducible promoter PpetJ. The construct includes the petJ promoter, the 1369 bp aldehyde dehydrogenase fragment containing the entire coding sequence from ORF slr0091 and 205 bp downstream of the gene (terminator region). The aldehyde dehydrogenase (aldh) gene was amplified by PCR using the following primer:









# aldh1-NdeI-fw







(SEQ ID NO: 203)









5′-GTGCCTCATATGGAATACTGCTAAAACTGTTGTTGC-3′


 



# aldh2-XhoI-rev







(SEQ ID NO: 204)









5′-GATCTCCTCGAGGTAAAGAATCAGCATAGGTCTGG-3′






Primers contain a NdeI or XhoI restriction site, respectively, for cloning (marked in bold letters).
The PCR fragment was digested with NdeI/XhoI and ligated downstream of the PpetJ promoter into pIC-PpetJ. The entire PpetJ-aldehyde dehydrogenase fragment was cut out of this plasmid with PstI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ322 (self replicating plasmid).
The construct used, named pVZ322-PpetJ-aldh, has the structure presented in FIG. 27B, and the nucleotide sequence of the insert of construct pVZ322-PpetJ-aldh is presented in FIG. 27C.
n) Construction of DNA-Vectors for Overexpression of PEP Carboxylase
The open reading frame (ORF) sll0920 encodes the phosphoenolpyruvate carboxylase (EC 4.1.1.31), BAA18393. The amino acid sequence for this protein is presented in FIG. 28A.
One construct was generated for overexpression of phosphoenolpyruvate carboxylase under control of the inducible promoter PpetJ.
The construct includes the petJ promoter, the 3105 bp ppc-fragment containing the entire coding sequence from phosphoenolpyruvate carboxylase (sll 0920) and 59 bp downstream of the gene (terminator region) was amplified by PCR using the following primer:








# ppc.NdeI.fw:



5′-CTAGAGGTTCATATGAACTTGGC-3′,
(SEQ ID NO: 205)






this primer contains a NdeI restriction site (CATATG) for cloning (marked in bold letters)











# ppc.XhoI.rv:




5′-GTAAGCAGGCTCGAGGCAAG-3′,
(SEQ ID NO: 206)






this primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

The PCR fragment was digested with NdeI/XhoI, subcloned into K8 (using NdeI/XhoI), cut out of this plasmid with SalI/XhoI and ligated into the E. coli/Synechocystis shuttle vector pVZ321 (self replicating plasmid). The pVZ321 vector has the GenBank accession number AF100176.
The construct used, named pVZ321-PpetJ-ppc, has the structure presented in FIG. 28B, and the nucleotide sequence for the pVZ321-PpetJ-ppc insert is presented in FIG. 28C.
o) Construction of DNA-Vectors for Overexpression of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RubisCO)
Overexpression of the Synechocystis RuBisCO was reached by integration of a conjugative, self-replicating pVZ plasmid into Synechocystis containing either the rbcLXS operon alone or the rbcLXS operon as transcriptional fusion together with the pyruvate decarboxylase from Zymomonas mobilis. 
The entire rbc operon from Synechocystis sp. PCC6803 was amplified by PCR using the primer pairs:

    
    
        SynRbc-BglII-fw and SynRbc-PstI-rev for the over-expression from the rbcL-promoter, which are shown in FIGS. 28D and 28E, respectively.
        SynRbc-SacI-fw and SynRbc-PstI-rev for the over-expression as transcriptional fusion with the Pdc from Zymomonas mobilis. The sequence of SynRbc-SacI-fw is shown in FIG. 28F.
    
    


The database entry numbers for the CyanoBase, the genome database for cyanobacteria (http://bacteria.kazusa.or.jp/cyanobase/index.html) for the Synechocystis rbcL-rbcX-rbcS coding sequences are slr0009 for the ribulose bisphosphate carboxylase large subunit (rbcL), slr0011 for the possible Rubisco chaperonin (rbcX) and slr0012 for the ribulose bisphosphate carboxylase small subunit (rbcS). The DNA sequence coding for the rbcLXS operon is depicted in FIG. 28G. The protein sequence obtained by translation of the protein coding DNA sequence is depicted in FIG. 28H for the rbcL large subunit; the rbcX Rubisco chaperonin protein sequence is shown in FIG. 28I and the protein sequence of the ribulose bisphosphate carboxylase small subunit (rbcS) is shown in FIG. 28J.
Mutants were selected on streptomycin plates and grown in BG11 medium containing the appropriate antibiotics (kanamycin 100 mg/l; streptomycin 10 mg/l).
In Synechocystis sp. PCC6803 mutants were generated by transforming the cells with two different plasmids, pVZ321b-Prbc-SynRbcLXS (FIG. 28K) and pVZ321b-PpetJ-PDC/SynRbcLXS (FIG. 28L).
In the following the vectors, which were used are described.
a) Plasmid pSK9 Structure and Sequence
The non-public pSK9 vector was generated in the lab of V. V. Zinchenko (Moscow, Russia). The structure of this plasmid is schematically represented in FIG. 29A, and its nucleotide sequence is presented in FIG. 29B.
b) Self-Replicating Synechocystis Plasmid pVZ321 Structure and Sequence
The pVZ321 vector has the GenBank accession number AF100176. This vector is presented schematically in FIG. 30A, and the pVZ321 nucleotide sequence is presented in FIG. 30B.
c) Self-Replicating Synechocystis Plasmid pVZ322 Structure and Sequence
The pVZ322 vector has the GenBank accession number AF100175. FIG. 31A presents a schematic of its structure, and FIG. 31B presents its nucleotide sequence.
d) construction of the Cloning Vector pIC20H
For cloning procedures a plasmid was constructed harboring promoter PpetJ in the multi-cloning site of cloning vector pIC20H, Ac. No. L08912, (Marsh J. L., Erfle M., Wykes E. J.; “The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation”; Gene 32:481-485 (1984)). Promoter PpetJ was cut out of the herein described pSK9 plasmid with ClaI and KpnI and ligated into pIC20H (ClaI/KpnI), resulting in plasmid pIC-PeptJ.
The plasmid pIC-PpetJ has the structure presented schematically in FIG. 32A, and the nucleotide sequence of pIC PpetJ is presented in FIG. 32B.
Generation of Additional Knock-Out/Knock-Down Mutants of Synechocystis Sp. PCC 6803: Methods and Results
The following Knock-Out construct sequences have been conveniently described and provided herein: (a) alanine dehydrogenase (ald), (b) ADP-glucose pyrophosphorylase (glgC), (c) pyruvate water dikinase (ppsA), (d) lactate dehydrogenase (ldh), (e) acetate kinase (ack) and (f) phosphoacetyltransacetylase (pta). The following Knock-Down construct sequence is described and provided pyruvate dehydrogenase (pdhB). These constructs may be used singly or sequentially in order to provide one or more mutations.
Mutagenesis
Host cells are mutagenized by transformation of the DNA-vectors (knock-out-constructs) using the natural competence of Synechocystis sp. PCC 6803 for DNA uptake and its system for homologous recombination as previously described herein. The transformation may comprise one or more steps in order to create mutant cells having a single, double, triple, etc. knockout and/or knockdown mutations. Additionally, knockdown/knockout mutants may additionally be mutagenized by introducing one or more overexpressing DNA constructs as described herein. As noted previously herein, the concentration of the appropriate antibiotic(s) is increased stepwise when the cells are transferred onto another agar plate or into liquid culture (for kanamycin from initially 5 to 150 μg/ml BG11, for chloramphenicol from initially 1 to 15 μg/ml BG11 medium) in order to get fully segregated (homozygous) mutants. Transfers are done every 2 weeks. In case of kanamycin, the concentration in the range from 50 to 150 μg/ml agar is increased gradually over the course of 4 weeks.
Molecular Analysis of Mutant Host Cells
In order to clearly demonstrate that a targeted homologous recombination event occurred in the selected mutant(s) cell, a variety of methods well known to one of ordinary skill in the art may be utilized. A test for successful knockout mutagenesis will be done initially by PCR amplifying a DNA fragment from the inserted antibiotic resistance cassette into the gene that should be knocked out. In addition, knockout mutants as well as knock-down mutants will be also checked by the detection and non-detection respectively of the target enzyme mRNA level in the mutant and wild type cells by using different techniques known in the art, e.g. RT-PCR, Northern blot or RNase protection assays. These recombinant DNA/molecular biology methods are well known to one of ordinary skill in the art; For example see: Methods in Enzymology, Vol. 167, (L. Packer, A. N. Glazer, eds); For extraction of genomic DNA: Franche C, Damerval T. in Methods of Enzymology, Vol. 167 p. 803-808; for extraction of total RNA: David l. Lane, Katherine G. Field, Gary J. Olsen, and Norman R. Pace in Methods of Enzymology, Vol. 167 p. 138-144; for Extraction of plasmid DNA: Grant R. Lambert and Noel G. Carr, Rapid Small-Scale Plasmid Isolation by Several Methods from Filamentous Cyanobacteria, Arch Microbiol (1982) 133: 122-125; for Northern Blots: Axmann, I. M., Kensche, P., Vogel, J., Kohl, S., Herzel, H. & Hess, W. R. (2005) Genome Biol 6, R73; for RT-PCR: Emanuel C, von Groll U, Müller M, Börner T, Weihe A. Development- and tissue-specific expression of the RpoT gene family of Arabidopsis encoding mitochondrial and plastid RNA polymerases. Planta. 2006 April; 223(5):998-1009; for RNase protection assay: W. R. Hess, B. Hoch, P. Zeltz, T. Hübschmann, H. Kössel and T. Bürner. Plant Cell 6 (1994), pp. 1455-1465. Academic Press, Inc., 1988), which are incorporated herein by reference.
Also, sufficient nucleotide sequence information for all enzymes is provided herein or available from known nucleotide sequence databases for the selection of the appropriate probes/primers for these analyses. With Northern Blot analysis, the abundance and relative amount of a mRNA will be detected. The same would be the case using a RNase protection assay but with a much higher sensitivity. The abundance and also the absolute amount of a mRNA can be determined with a high sensitivity using the RT-PCR.
With the PCR analysis, one forward primer is derived from the genetic sequence of the targeted enzyme and one reverse primer is derived from the biocide gene sequence; the amplified hybrid DNA fragment will be characterized and analyzed for predicted size and/or nucleotide sequence content. Mutant(s) cells found not to be expressing wildtype mRNA and found to have the above noted characteristics will be selected for further analysis.
Characterization of Knock-Out/Knock-Down Mutants
Cultivation of Cyanobacterial Wild Type and Mutant Strains
For a knock-out or knock-down mutant(s) related to the formation of reserve compounds such as glycogen, e.g., mutants of further reserve metabolites syntheses as PHB or cyanophycin, wild type and mutant strains of Synechocystis PCC 6803 are grown as batch cultures in BG11 medium at 29° C. under continuous illumination with white light (intensity: 40 μE m−2 s−1) and aeration with air. For cultivation of mutants, the appropriate antibiotics are added to the medium (kanamycin 75 mg/l; chloramphenicol 15 mg/l). Samples are analyzed briefly before the nitrogen step down (“+N”), directly after resuspension of the cells in BG11 medium lacking a nitrogen source (“−N”, 0 h) and after 3, 6 and 24 hours.
All other knock-out or knock-down mutants will be grown under standard culture conditions known in the art.
As provided below, mutants and wild type cells will be characterized regarding their intra- and extracellular pyruvate content using optical enzymatic tests and their profile of all relevant metabolites respectively. (incl. 3-PGA, PEP, pyruvate, acetyl-CoA, glycogen, PHB, cyanophycin, malate, oxaloacetate, 2-oxoglutarate, acetate, lactate, etc.) using appropriate techniques for example, spectroscopic methods, chromatographic methods such ion chromatography or optical or enzymatic methods or combinations thereof. The analysis will always be done in comparison to the wild type.
Also the growth and pigmentation properties of mutant(s) will be compared to the wild type cell using standard protocols well known in the art.
The example presented here will provide a graphic depiction of growth properties for wild type and mutant cells as change in X vs. time, wherein X is ideally dry weight or biovolume. Alternatively, optical density, cell count and chlorophyll could be used as reference parameters. Alternatively, pigmentation could be quantified spectrophotometrically as another parameter.
Protocol for Characterization of Metabolic Mutants Containing at Least One First and/or One First and One Second Genetic Modification
Generation of knock-out and over-expression mutants with single, double, triple, etc. knock-out and/or knock-down and/or over-expression mutations as a first genetic modification and the molecular analysis of such mutant cells in general is already described above.
Characterization of Metabolic Mutants
Metabolic mutant strains having a first genetic modification were characterized regarding their growth properties and certain extra- and intracellular metabolites in comparison to wild type strains. In addition the afore described metabolic mutants were also transformed with PDC and ADH as a second genetic modification and were characterized regarding growth properties, extra- and intracellular metabolites and ethanol production rates in comparison to the appropriate reference strain(s) expressing PDC and ADH, but lacking the metabolic mutation (first genetic modification).
Cultivation of Cyanobacterial Wild Type and Mutant Strains
Wild type and mutant strains of Synechocystis PCC 6803 were grown as batch cultures in BG11 medium at 28-29° C. For cultivation of mutants the appropriate antibiotics were added to the medium (kanamycin 75 mg/l; chloramphenicol 10 mg/l; gentamycin 3 mg/l or streptomycin 10 mg/l). In order to avoid premature induction of gene expression in mutants having constructs with PpetJ or PisiA promoter, these mutants were grown in culture medium supplemented with excess copper or iron (5× Cu for PpetJ; 3× Fe for PisiA).
Prior to characterization experiments, pre-cultures were grown in BG11 medium (no excess of Cu or Fe) and aeration with 0.5% CO2 in air.
For characterization experiments, wild type and mutant strains were grown in BG11 medium. Mutants having constructs with PpetJ or PisiA (overexpression, knock-down mutants or mutants expressing PDC and ADH) were transferred to BG11 lacking Cu (PpetJ) or Fe (PisiA), respectively, in order to induce gene expression (described in detail for PDC/ADH expressing mutants).
The total culture volume in characterization experiments was 300 mL in a 500 mL Schott-Flask; the initial OD750 was 1. Cultures were aerated with 0.5% CO2 in air.
All mutants were characterized under constant light conditions (75-100 μE m-2 s-1). In fast growing cultures, the light intensity was increased during the growth experiment (75-100 μE m-2 s-1 up to OD5; then light intensity was increased to 200 μE m-2 s-1).
Knock-out mutants related to fermentative pathways such as lactate dehydrogenase, acetate kinase or phosphoacetyltransacetylase were additionally characterized under day/night conditions (12 h 100 μE m-2 s-1/12 h dark). Knock-out mutants related to the formation of reserve compounds such as glycogen or PHB were additionally examined after transferring the cells in BG11 medium lacking a nitrogen source (nitrogen starvation conditions) as previously described herein.
Principle of Ethanol Quantification:
Ethanol is oxidized by nicotinamide-adenine dinucleotide (NAD+) to acetaldehyde in a reaction, which is catalyzed by the enzyme alcohol dehydrogenase (ADH) (reaction 1). The acetaldehyde, which is formed in the reaction, is quantitatively oxidized to acetic acid by the enzyme aldehyde dehydrogenase (Al-DH) (reaction 2).




In reactions (1) and (2) reduced nicotinamide-adenine dinucleotide (NADH) is formed. The amount of NADH formed is proportionate to the amount of ethanol in the sample. NADH is easily quantified by means of its light absorbance. The absorbance is usually measured at 340 nm, Hg 365 nm or Hg 334 nm.
Procedure:
Preparation of solutions: Solution 1: 1.3 mg/ml NAD and 0.27 U aldehyde dehydrogenase in potassium diphosphate buffer, pH 9.0. Solution 2: Suspension of alcohol dehydrogenase (ADH) with approx. 4000 U/ml. Alternatively, the chemicals and solutions of the ethanol determination kit of Boehringer Mannheim/R-Biopharm (Cat. No. 10 176 290 035) can be used. Sample and solution 1 are mixed in a ratio of 3 ml solution 1 and 0.1 ml sample (if necessary the sample is diluted with water). After approx. 3 min the absorbance is measured (A1). The reaction is then started by the addition of ADH suspension (solution 2, 0.050 ml for 3 ml solution 1 and 0.1 ml sample). After completion of the reaction (approx. 5 to 10 min) the absorbance is measured again (A2). The absorption measurements can be performed using a photometer or a microplate reader. For plate reader measurements all volumes are downscaled.
From the measured absorbance difference ΔA=(A2−A1) the ethanol concentration in the sample is calculated with the equation:




  c
  =
  
    
      
        V
        ×
        MG
      
      
        ɛ
        ×
        d
        ×
        v
        ×
        2
        ×
        1000
      
    
    ×
    Δ
    ⁢
    
      
    
    ⁢
    A
  




c, ethanol concentration [g/L]; V, total volume [mL]; MG, molecular weight of ethanol (46.07 g/mol); e, extinction coefficient (6.3 L×mmol−1×cm−1 at 340 nm); d, light path [cm]; v, sample volume [mL]

LITERATURE


    Protocol of the kit Ethanol, UV method for the determination of ethanol in foodstuff and other materials, Cat. No. 10176290035, R-Biopharm AG, Darmstadt, Germany.
    H.-O. Beutler (1984) in: Methods in Enzymatic Analysis (Bergmeyer, H. U. ed.) 3rd ed. Vol. VI, pp. 598-606, Verlag Chemie, Weinheim, Germany.

Growth Properties



For characterization experiments, metabolic mutant and the appropriate reference strains were cultured as described. Growth was followed for about 14 days by measuring optical density (daily) and chlorophyll (every second day). Photosynthetic O2 production was determined several times during exponential growth phase using a Clark electrode as followed:
Measurement of Photosynthetic Oxygen Evolution
Cell are washed 2× with fresh growth medium by centrifugation (3000×g, 10 min, room temperature) and resuspension. The cells are finally resuspended in growth medium to a chlorophyll concentration of 10 to 15 μg chlorophyll/ml. Chlorophyll is measured as described by [N. Tandeau De Marsac and J. Houmard]. The cells are filled into the chamber of a Rank Brothers oxygen electrode (Digital Model 10, Rank Brothers, Cambridge, England) and sodium bicarbonate is added to a final concentration of 25 mM.
The excitation light for photosynthesis experiments is provided by a slide projector with a 150-watt lamp (Osram, Xenophot HLX Germany).
The oxygen concentration in the chamber is recorded continuously with chart recorder (REC 112, Amersham Pharmacia Biotech) connected to the electrode. The chamber of the oxygen electrode is maintained at 25° C. with a circulating, temperature-controlled water bath (RM6, Lauda Brinkmann). For the calibration of the electrode the signal difference of air-saturated water (100% saturation) and oxygen free water (zero point) is measured. Oxygen free water is obtained by adding sodium dithionite (approximately 1 mg/ml). The measured amplitude is equated with the solubility of oxygen in water at 25° C. and a pressure of 1 bar (8.11 mg oxygen/L).

    Literature: N. Tandeau De Marsac and J. Houmard in: Methods in Enzymology, Vol. 169, 318-328. L. Packer, ed., Academic Press, 1988

Determination of Ethanol Production



For characterization of mutants expressing PDC and ADH or only PDC or other ethanologenic enzymes as a second genetic modification, ethanol was measured daily during the growth experiment according to the afore described optical enzymatic method (“Ethanol UV method” test kit by Boehringer Mannheim/R-Biopharm, Darmstadt, Germany). Ethanol production of metabolic mutants expressing PDC and ADH were compared to the appropriate reference strain expressing PDC and ADH as a second genetic modification, but lacking the respective metabolic mutation, the first genetic modification.
The cells were cultured over a period of time of 14 days. These cell cultures were further characterized during their logarithmic growth phase at certain time points with regard to their ethanol production rate, their chlorophyll content and photosynthetic capacity (oxygen evolution in μmol O2/mg Chl*h). These three values were measured in a period of time of approximately 2 hours as described below. In the following these measurements are referred to as “short term measurements” or “short term experiments”.
Simultaneous Measurement of Photosynthetic Oxygen Evolution and Ethanol Production (Short Term Experiment)
For the comparison of ethanol production and photosynthesis, ethanol production rates and rates of photosynthetic oxygen evolution are measured simultaneous in a single assay.
Cells are washed 2× with fresh growth medium by centrifugation (3000×g, 10 min, room temperature) and resuspension. Cells are resuspended in growth medium to a chlorophyll concentration of 10 to 15 μg chlorophyll/mL. Chlorophyll is measured as described in [N. Tandeau De Marsac and J. Houmard in: Methods in Enzymology, Vol. 169, 318-328. L. Packer, ed., Academic Press, 1988]. 1.9 mL of the cells and 0.1 mL of 500 mM sodium bicarbonate are filled into the chamber of the oxygen electrode and the rate of the photosynthetic oxygen evolution is measured as described herein (Measurement of photosynthetic oxygen evolution). The oxygen concentration in the sample in the chamber is constantly measured and plotted by a flat bed reecorder [REC 112, Amersham Pharmacia Biotech] that runs with a constant speed (1 cm/min). When the light is switched on one observes an increase of the oxygen concentration. Within several minutes (2-5 min) the plotted curve becomes linear. The change of the oxygen concentration is observed for the next minutes to make sure, that the increase of the oxygen concentration remains linear. The amount of oxygen evolved as a result of photosynthesis per unit time is calculated. The chamber of the oxygen electrode is maintained at 25° C. with a circulating, temperature-controlled water bath (RM6, Lauda Brinkmann. For the calibration of the electrode the signal difference of air-saturated water (100% saturation) and oxygen free water (zero point) is measured. Oxygen free water is obtained by adding sodium dithionite (approximately 1 mg/ml). The measured amplitude is equated with the solubility of oxygen in water at 25° C. and a pressure of 1 bar (8.11 mg oxygen/L). [See also: N. Tandeau De Marsac and J. Houmard in: Methods in Enzymology, Vol. 169, 318-328. L. Packer, ed., Academic Press, 1988.]
After completion of this measurement illumination of the sample in the chamber is continued under unchanged conditions. Over a period of one hour samples of 0.15 ml are taken in defined intervals (in most cases every 10 minutes). Immediately after removal samples are centrifuged (14,000×g, 10 min, 4° C.) and the supernatant is stored on ice. After completion of the sampling, the ethanol concentration in the supernatants is measured as described herein. The ethanol concentration versus time is plotted. Using the linear equation the rate of the increase of the ethanol content in v/v in the assay per hour is calculated. The rate of ethanol production is usually given in the dimension μmol ethanol*h−1*mg chlorophyll−1, the chlorophyll content measured at the beginning of the experiment is then used.
Determination of Intra- and Extracellular Metabolites
Two different methods were used to determine the level of intracellular metabolites in particular pyruvate and acetaldehyde.
Protocol for Extraction of Intracellular Metabolites


    
    
        use 5 ml culture.
        Centrifuge for 10 min, 4500 rpm.
        Resuspend the pellet in 1 ml dd water.
        Centrifuge 5 min with 14000 rpm. Discard the supernatant.
        Resuspend the pellet in 1 ml double distilled water.
        Centrifuge 5 min, 14000 rpm, 4° C. Discard the complete supernatant.
    
    


Continue or store the pellet by −20° C. under Argon atmosphere.

    
    
        Add 600 ml of extraction buffer.
        
            Extraction buffer: 10:3:1—methanol:chloroform:
        
        
    
    


water

    
    
        Vortex briefly.
        Shake at 4 degrees for 10 min.
        Centrifuge 5 min with 14000 rpm.
        Transfer 500 μl to a new tube.
        Add 200 μl chloroform and 200 μl water.
        Centrifuge 5 min with 14000 rpm.
        Transfer 500 μl of the upper phase to a new tube and speed vac to dry.
        Resuspend the pellet in 100 μl double distilled water.
        Shake at 4 degrees for ≧20 min. Centrifuge 5 min with 14000 rpm.
        Transfer 95 μl to a vial for IC.

Extraction and Analysis of Extracellular Metabolites

Extraction of Metabolites Using a Retsch Mill:

    
    


The protocol for extraction of intracellular metabolites was designed by Dr. M. Gründel.
Protocol:
Cells (150 ml cell culture) are harvested by centrifugation and resuspended in 400 μl buffer (100 mM Tris/HCl, pH 7.5) to which 200 μl of glass beads (0.1 mm diameter) are added. Cell lysis is performed using a Retsch mill model MM 301 (treatment for 10 minutes, 4° C.). After removal of glass beads, remaining intact cells and cell debris was removed by centrifugation (10 minutes, 4° C.). The whole procedure is repeated once. Proteins in the combined supernatants are precipitated by deoxycholate/trichloroacetic acid treatment (Bensadoun and Weinstein. 1976. Anal. Biochem. 70:241-250) and removed by centrifugation. The supernatant, containing the soluble metabolites, is neutralized with 2 M K2CO3 and adjusted to a volume of 1.5 ml with 100 mM Tris/HCl buffer, pH 7.5. In order to determine the concentration of metabolites, aliquots of 100-500 μl are used in the optical tests.
Extraction of Metabolites Using Ice Cold Methanol (Snap Shot Extraction):
Literature Describing the Method:
According to R. P. Maharjan, T. Ferenci. 2002. Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. Anal. Biochem. 313:145-154.
This method allows for the immediate freezing of intracellular metabolite pools and the extraction of numerous intra- and extracellular metabolites at the same time.
Protocol:
Batches of cyanobacterial cultures are dropped into an equal volume of methanol, cooled by dry ice, and incubated on dry ice until completely frozen. After thawing in ice/water (10 min) the samples are centrifuged for 5 min (>=17.000×g, temperature as low as possible). The pellet is extracted a second time with cold 50% methanol (−20° C.). Supernatants are combined. Methanol is removed by evaporation at 35° C. under vacuum using a rotavapor apparatus. The remaining solution is lyophilized, the residue is resuspended in a minimal volume of water.
The efficiency of extraction of bacterial cells with cold methanol is similar to that with hot ethanol or hot methanol. But the method is very simple, rapid and changes in the stability and reactivity in metabolites are minimized.
When extracellular pyruvate and oxoglutarate are assayed, an extraction is not necessary since both metabolites are detectable directly in the media. Quantification of intracellular and extracellular pyruvate and oxoglutarate levels before and after nitrogen deprivation is done as previously described herein.
Pyruvate and phosphoenolpyruvate are quantified using an optic enzymatic test of Häusler et al. (2000), Anal. Biochem, 281:1-8. This method allows for the quantification of pyruvate and phosphoenolpyruvate in one test.
Protocol:
The quantifications are based on the reduction of pyruvate to lactate by lactate dehydrogenase (LDH) at the expense of NADH which is oxidized to NAD+. In the first step, pyruvate was assayed. After completion of this reaction, pyruvate kinase is added. Pyruvate kinase converts phosphoenolpyruvate to pyruvate and thus allows for determination of phosphoenolpyruvate.
To 450 μl master mix (9 μl 20 mM NADH, 12 μl 1 M MgCl2, 46 μl 1 M KCl, 12 μl 100 mM ADP, 360 μl 100 mM HEPES, 10 μl H2O) 520 μl sample (if necessary diluted with H2O) are added. Add 2 μl LDH to start the reaction. The oxidation of NADH is observed as decrease of absorbance at 340 nm. Either the difference of the absorbances at 340 nm minus 380 nm is measured by difference spectroscopy (turbid or colored samples; ε340-380=4.83 l×cm×mmol-1) or the absorbance at 340 nm is measured against water (ε340=6.28 l×cm×mmol-1). After complete reaction of pyruvate, 2 μl pyruvate kinase are added to the assay. NADH oxidation is measured as before. From the differences of the absorbances at the start and the end of the reactions, the amount of oxidized NADH (=amount of pyruvate, and phosphoenolpyruvate, respectively) is calculated.
Chemicals and Solutions:
1. Lactate dehydrogenase suspension from bovine heart (L-LDH, Sigma L2625-2.5 KU, suspension with 5629.5 U/ml), diluted 1:10
2. Pyruvate Kinase from rabbit muscle (PK, Serva 34085, suspension with 4000 U/ml), diluted 1:20
3. 100 mM HEPES/NaOH (pH 7.5)
4. 1 M MgCl2
5. 100 mM ADP
6. NADH (Sigma, N6005) 20 mM in H2O
7. 1 M KCl
Photometric Quantification of Pyruvate (and/or Lactate) in an Enzymatic Cycling System
Method:
According to E. Valero & F. Garcia-Carmona. 1996. Optimizing Enzymatic Cycling Assays: Spectrophotometric Determination of Low Levels of Pyruvate and L-Lactate. Anal. Biochem. 239:47-52
This method allows for the quantification of pyruvate (and/or lactate) with a 10-fold higher sensitivity than the pyruvate quantification method described before.
Protocol:
In a cyclic reaction pyruvate is reduced to lactate under consumption of NADH, the lactate is oxidized by lactate oxidase to pyruvate. The rate of NADH consumption, monitored spectrophotometrically at 340 nm is proportional to the amount of pyruvate (plus lactate if present) in the sample. For calibration curves, different amounts of pyruvate are added to the master mix (end volume 1000 μl) consisting of 50 mM TRIS-buffer, pH 7.5, 256 μM NADH, 1.8 μg lactate dehydrogenase and 60 μg lactate oxidase. The reaction is started by addition of lactate dehydrogenase and the time course of the reaction at 340 nm is followed for some minutes. Samples with unknown amounts of pyruvate and lactate are treated identically and quantified using the calibration curve. Detection limit is about 1 nmol pyruvate and/or lactate.
Chemicals and Solutions:
1. 50 mM TRIS/HCl (pH 7.5)
2. 20 mM NADH in H2O
3. 0.25 mg/ml lactate dehydrogenase in 50 mM TRIS/HCl (pH 7.5)
4. 2.6 mg/ml lactate oxidase in 50 mM TRIS/HCl (pH 7.5).
Spectrophotometric Quantification of 2-oxoglutarate Using an Enzymatic Test
Method:
The method used is an adaptation of a fluorimetric method (P. J. Senior. (1975). J. Bacteriol. 123:407-418) for spectrophotometry. The oxidation of NADH, followed by the absorption change at 340 nm, is proportional to the concentration of 2-oxoglutarate.
Protocol:
Cuvettes contained a final volume of 1000 μl: 100-500 μl sample; 10 μl ammonium sulfate; 10 μl NADH; 10 μl ADP; 10 μl glutamate dehydrogenase solution; TRIS buffer added to a final volume of 1000 μl. The reaction is started by the addition of glutamate dehydrogenase.
Chemicals and Solutions:
1. 1 M ammonium sulfate
2. 20 mM NADH
3. 0.1 M ADP
4. 2.6 enzyme units per ml glutamate dehydrogenase (from bovine liver; 104


    
    
        enzyme units per mg; Serva lot no. 22904)

5. 0.1 M TRIS/HCl pH 8.0

    
    


Acetaldehyde was quantified by a modification of the protocol of a kit for ethanol quantification (Ethanol kit, R-Biopharm AG). Acetaldehyde is converted by aldehyde dehydrogenase under formation of NADH, which is quantified by its absorption at 340 nm. The amount is proportionate to the acetaldehyde content of the sample.
All mutant strains were characterized regarding their profile of relevant intracellular metabolites using ion chromatography always in comparison to the wild type or appropriate reference strain, respectively.
Short description of the UV-method for the determination of acetic acid in foodstuff and other materials from Boehringer Mannheim/R-Biopharm, Darmstadt, Germany
Principle: Acetic acid (acetate) is converted to acetyl-CoA in the presence of the acetyl-CoA synthetase (ACS), adenosine-5′-triphosphate (ATP) and coenzyme A (CoA) (1).

Acetate+ATP+CoAACS acetyl-CoA+AMP+PP  (1)

Acetyl-CoA reacts with oxaloacetate to citrate in the presence of citrate synthase (CS) (2).

Acetyl-CoA+oxaloacetate+H2OCS citrate+CoA  (2)

The oxaloacetate required for reaction (2) is formed from L-malate and nicotinamide-adenine dinucleotide (NAD) in the presence of L-malate dehydrogenase (L-MDH) (3). In this reaction NAD is reduced to NADH.

L-malate+NAD+L-MDH oxaloacetate+NADH+H+  (3)

The determination is based on the formation of NADH measured by the increase in light absorbance at 340, 334 or 365 nm. Because of the equilibrium of the preceding indicator reaction, the amount of NADH formed is not linearly (directly) proportional to the acetic acid concentration (this fact is been taken into consideration in the calculation of acetic acid concentrations).
The above described methods for the quantification of acetate, pyruvate, acetaldehyde and 2-oxoglutarate can detect changes in the static steady state levels of these metabolic intermediates. As mentioned above the first genetic modification can result in a change of the metabolic flux of these metabolic intermediates, which is hard to detect by assays, which are able to detect the steady state level of a metabolite, but not the changes in the flux of the metabolite. In particular, these enzymatic assays might not properly show the changes in the metabolic activity of a photoautotrophic host cell, induced by the first genetic modification.
An overview of alternative assay methods, which can be used to detect the change in the metabolic activity of a photoautotrophic host cell of this invention is shown in the Review of Shimizu, “Metabolic Engineering-Integrating Methodologies of Molecular Breeding and Bioprocess Systems Engineering”, Journal of Bioscience and Bioengineering, Vol. 94, No. 6: 563-573 (2002), which is hereby incorporated by reference. These methods are more time-consuming and complex than the above described enzymatic assays and are for example metabolic flux analysis (MFA), cell capability analysis, metabolic control analysis (MCA) or 13C-NMR and gas chromatography. Mass spectroscopy (GCMS) measurements.
Wild type (WT) and mutant metabolite (pyruvate, acetaldehyde or acetyl-CoA or precursors thereof) measurements will be obtained as previously described herein and presented in the tables below.














Metabolite Intracellular
Metabolite Extracellular



level in mmol per liter
level in mmol per liter











OD750
wt
mutant
wt
mutant















1.0
+N
A
A + Δ
F
F + Δ



−N, 0 h
B
B + Δ
G
G + Δ



−N, 3.5 h
C
C + Δ
H
H + Δ



−N, 6 h
D
D + Δ
I
I + Δ



−N, 24 h
E
E + Δ
J
J + Δ





Data will be verified by repetitions.


A-J represent wild type values for the indicated conditions


Δ represents an increment relative to the wt measurement






The table shows an example for such an experiment. In other experiments the optical density (OD750) at the beginning of the experiment and the time points can be different














Metabolite Intracellular











level in mmol per liter
Metabolite Extracellular



(calculated per packed
level in mmol per liter


Time of
cell volume1)
culture volume











cultivation
wt
mutant
wt
Mutant





T1
A
A + Δ
E
E + Δ


T2
B
B + Δ
F
F + Δ


T3
C
C + Δ
G
G + Δ


T4
D
D + Δ
H
H + Δ





Data will be verified by repetitions.


A-H represent wild type values


Δ represents an increment relative to the wt measurement






Parameters such as OD750nm, Chlorophyll content, protein content and cell number will also be measured in standardizing and evaluating metabolite values at different time points.
In addition, measurements can be obtained for variations in culture conditions such as light intensity, growth in darkness and in day/night cycles respectively, CO2 supplementation and temperature. Also, further variations might concern the composition of the growth medium (e.g. concentration of nitrate, ammonium, phosphate, sulfate or microelements (e.g. Cu, Fe)). All these variations in culture conditions are known to one of ordinary skill in the art.
The data will be analyzed and presented graphically as previously described herein.
Analysis of Ethanol Production
In order to discover whether the enhanced level of biosynthesis of pyruvate, acetaldehyde or acetyl-CoA in the mutant(s) cells also leads to a higher production of ethanol, Synechocystis sp. PCC 6803, both wildtype as well as the mutant(s) cells are transformed with the plasmid pVZ containing the Zymomonas mobilis Pdc and AdhII enzymes or other plasmids encoding ethanologenic genes under the control of the iron dependent isiA promoter or other promoters.
Analysis of ethanol production is done as previously described herein. Synechocystis sp. PCC 6803 with and without Pdc and Adh and Synechocystis sp. PCC 6803 mutant(s) cells with and without Pdc and Adh will be compared. This example will present a graphic depiction of these results that clearly demonstrate that increased ethanol production is provided by the mutant(s) cells when compared to the wild type cell.
Generation of Overexpression Mutants of Synechocystis Sp. PCC 6803: Methods and Results
The following overexpression construct sequences have been conveniently described and provided herein: (a) malic enzyme, (b) malate dehydrogenase, (c) pyruvate kinase 1, (d) pyruvate kinase 2, and (e) pyruvate kinase, enolase and phospho-glycerate mutase. These constructs may be used singly or sequentially in order to provide one or more mutations. Also, constructs contain either the natural promoter for the enzyme gene of interest or an inducible promoter.
Mutagenesis
Host cells are mutagenized by transformation of the overexpression DNA-vectors using the natural competence of Synechocystis sp. PCC 6803 for DNA uptake. In case of integrative overexpression mutants, the system of Synechocystis sp. PCC 6803 for homologous recombination as previously described herein is used. In addition, self-replicating constructs may also be used. The transformation may comprise one or more steps in order to create mutant cells having a single, double, triple, etc. overexpression mutations. Additionally, one or more knockdown/knockout mutations (as described herein) may be introduced. As noted previously herein, the concentration of the appropriate antibiotic(s) is increased stepwise when the cells are transferred onto another agar plate or into liquid culture (for kanamycin from initially 5 to 150 μg/ml Bg11, for chloramphenicol from initially 1 to 15 μg/ml BG11 medium) in order to get fully segregated (homozygous) mutants. Transfers are done every 2 weeks. In case of kanamycin, the concentration in the range from 50 to 150 μg/ml agar is increased gradually over the course of 4 weeks.
Molecular Analysis of Mutant Host Cell
In order to establish that the selected mutant(s) cell is overexpressing the target enzyme, RNA will be extracted from wild type and mutant cells and will be examined by using different techniques known in the art, e.g. RT-PCR, Northern blot or RNase protection assays. These recombinant DNA/molecular biology methods are well known to one of ordinary skill in the art; For example see: Methods in Enzymology, Vol. 167, (L. Packer, A. N. Glazer, eds) Academic Press, Inc., 1988); For extraction of genomic DNA: Franche C, Damerval T. in Methods of Enzymology, Vol. 167 p. 803-808; for extraction of total RNA: David 1. Lane, Katherine G. Field, Gary J. Olsen, and Norman R. Pace in Methods of Enzymology, Vol. 167 p. 138-144; for Extraction of plasmid DNA: Grant R. Lambert and Noel G. Carr, Rapid Small-Scale Plasmid Isolation by Several Methods from Filamentous Cyanobacteria, Arch Microbiol (1982) 133: 122-125; for Northern Blots: Axmann, I. M., Kensche, P., Vogel, J., Kohl, S., Herzel, H. & Hess, W. R. (2005) Genome Biol 6, R73; for RT-PCR: Emanuel C, von Groll U, Müller M, Börner T, Weihe A. Development- and tissue-specific expression of the RpoT gene family of Arabidopsis encoding mitochondrial and plastid RNA polymerases. Planta. 2006 April; 223(5):998-1009; for RNase protection assay: W. R. Hess, B. Hoch, P. Zeltz, T. Hübschmann, H. Kössel and T. Börner. Plant Cell 6 (1994), pp. 1455-1465., which are incorporated herein by reference.
Also, sufficient nucleotide sequence information for all enzymes is provided herein or available from known nucleotide sequence databases for the selection of the appropriate probes/primers for these analyses. With Northern Blot analysis, the abundance and relative amount of a mRNA will be detected. The same would be the case using a RNase protection assay but with a much higher sensitivity. The abundance and also the absolute amount of a mRNA can be determined with a high sensitivity using the RT-PCR. Mutant(s) cells found to be overexpressing the target mRNA will be selected for further analysis.
Characterization of Overexpression Mutants
Cultivation of Cyanobacterial Wild Type and mutant Strains
wild type (WT) and mutant strains will be grown under standard culture conditions.
Nitrogen step-down conditions will be as previously described herein.
Conditions for the induction of inducible promoters is provided herein through the teachings of the specification and by way of reference to specific publications. See also D. A. Los, M. K. Ray and M. Murata, Differences in the control of the temperature-dependent expression of four genes for desaturases in Synechocystis sp. PCC 6803, Mol. Microbiol. 25 (1997), pp. 1167-1175.
As provided below, mutants and wild type cells will be characterized regarding their intra- and extracellular pyruvate content using optical enzymatic tests and their profile of all relevant metabolites respectively. (incl. 3-PGA, PEP, pyruvate, acetyl-CoA, glycogen, PHB, cyanophycin, malate, oxaloacetate, 2-oxoglutarate, acetate, lactate, etc.) using ion chromatography always in comparison to the wild type.
Also the growth and pigmentation properties of mutant(s) will be compared to the wild type cell using standard protocols well known in the art.
The example presented here will provide a graphic depiction of growth properties for wild type and mutant cells as change in X vs. time, wherein X is ideally dry weight or biovolume. Alternatively, optical density, cell count and chlorophyll could be used as reference parameters. Alternatively, pigmentation could be quantified spectrophotometrically as another parameter.
Wild type (WT) and mutant metabolite (pyruvate, acetaldehyde or acetyl-CoA or precursors thereof) measurements will be obtained as previously described herein and presented in the table below.














Metabolite Intracellular
Metabolite Extracellular



level in mmol per liter
level in mmol per liter











OD750
wt
mutant
wt
mutant















1.0
+N
A
A + Δ
F
F + Δ



−N, 0 h
B
B + Δ
G
G + Δ



−N, 3.5 h
C
C + Δ
H
H + Δ



−N, 6 h
D
D + Δ
I
I + Δ



−N, 24 h
E
E + Δ
J
J + Δ





Data will be verified by repetitions.


A-J represent wild type values for the indicated conditions


Δ represents an increment relative to the wt measurement






The table shows an example for such an experiment. In other experiments the optical density (OD750) at the beginning of the experiment and the time points can be different














Metabolite Intracellular











level in mmol per liter
Metabolite Extracellular



(calculated per packed
level in mmol per liter


Time of
cell volume1)
culture volume











cultivation
wt
mutant
wt
mutant





T1
A
A + Δ
E
E + Δ


T2
B
B + Δ
F
F + Δ


T3
C
C + Δ
G
G + Δ


T4
D
D + Δ
H
H + Δ





Data will be verified by repetitions.


A-H represent wild type values


Δ represents an increment relative to the wt measurement






Parameters such as OD750nm, Chlorophyll content, protein content and cell number will also be measured in standardizing and evaluating metabolite values at different time points.
In addition, measurements can be obtained for variations in culture conditions such as light intensity, growth in darkness and in day/night cycles respectively, CO2 supplementation and temperature. Also, further variations might concern the composition of the growth medium (e.g. concentration of nitrate, ammonium, phosphate, sulfate or microelements (e.g. Cu, Fe)). All these variations in culture conditions are known to one of ordinary skill in the art.
The data will be analyzed and presented graphically as previously described herein.
Analysis of Ethanol Production
In order to discover whether the enhanced level of biosynthesis of pyruvate, acetaldehyde or acetyl-CoA in the mutant(s) cells also leads to a higher production of ethanol, Synechocystis sp. PCC 6803, both wildtype as well as the mutant(s) cells are transformed with the plasmid pVZ containing the Zymomonas mobilis Pdc and AdhII enzymes or other plasmids encoding ethanologenic genes under the control of the iron dependent isiA promoter or other promoters.
Analysis of ethanol production is done as previously described herein. Synechocystis sp. PCC 6803 with and without Pdc and Adh and Synechocystis sp. PCC 6803 mutant(s) cells with and without Pdc and Adh will be compared. This example will present a graphic depiction of these results that clearly demonstrate that increased ethanol production is provided by the mutant(s) cells when compared to the wild type cell.
X. Experimental Data for Characterization of Metabolic Mutants Containing at Least One First or One First and One Second Genetic Modification
In the following available experimental data regarding pyruvate secretion are discussed for photoautotrophic cells harboring at least one first genetic modification. Furthermore ethanol production rate, if available, are also discussed for photoautotrophic cells containing in addition to the at least one first genetic modification at least one second genetic modification.
X.1 Metabolic Mutant Harbouring a Glycogen Synthase Double Knock Out Mutation as a First Genetic Modification
Characterization of the glycogen deficient glycogen synthase double knock out mutants of Synechocystis PCC 6803:
Nomenclature:














Enzyme:
Glycogen synthase 1
Glycogen (starch) synthase 2


EC no.:
EC 2.4.1.21
EC 2.4.1.21


Gene name:
glgA1
glgA2


Gene in
sll0945
sll1393









Synechocystis 

PCC 6803:

    Theoretical background: Diverting the production of storage reserves into an enhanced production of pyruvate/ethanol
    Genetic Manipulation: double knockout by insertion of a chloramphenicol cassette (ΔglgA1) and kanamycin cassette (ΔglgA2)
    
        M8-mutant: Cm, Km
        Complete segregation: yes

Characterization of the Mutants Harboring the Glycogen Synthase Double Knock Out Mutation as a the First Genetic Modification, but Lacking the Second Genetic Modification (Ethanologenic Enzymes).

Determination of Intracellular Glycogen Before and After a N Step Down

    
    


The procedure is an adaptation of the method described by Ernst et al. (A. Ernst, H. Kirschenloher, J. Diez, P. Boger. 1984. Arch. Microbiol. 140:120-125). Glycogen is isolated by alkaline hydrolysis of cells followed by precipitation of glycogen with ethanol. Isolated glycogen is digested with amylolytic enzymes to glucose, which is quantified in a standard optical test.
Protocol:

    
    
        Spin down 1-4 ml of Synechocystis culture before and after N step down resp. at RT and remove the supernatant
        Add 200 μl KOH (30% w/v) to the pellet and incubate 90 minutes at 95° C. in a heating block
        Add 600 μl cold ethanol (96%) and incubate 90 min on ice
        Spin down and discard the supernatant
        Wash once with ethanol (70%) and once with ethanol (96%)
        Dry the pellet in a vacuum centrifuge
        Dissolve the pellet in 45-90 μl acetate buffer
        5-10 μl enzyme mix (amyloglucosidase+alpha-amylase from Bacillus amyloliquefaciens, purchased from Roche) and incubate 90 min at 45° C.
        Use 10-40 μl of the resulting sample for the determination of glucose after manufacturer's instruction (Infinity glucose hexokinase liquid stable reagent for optical test at 340 nm; Cat No. TR15421 Thermo Electron Corporation)

Reaction:

    
    






Chemicals and Solutions:

1. aqueous solution of KOH (30% w/v)

2. ethanol 96% v/v

3. 100 mM acetate buffer, adjusted to pH 5.0 with NaOH

4. enzyme mixture of amylo glucosidase (26.7 mg/ml; Boehringer, lot 1490306) plus alpha-amylase (1.0 mg/ml; Boehringer, lot 84874220-34) in 100 mM acetate buffer pH 5.0)

Quantification of Intracellular and Extracellular Pyruvate and Oxoglutarate Levels Before and After Nitrogen Deprivation (“N Step Down”)

Explanation for “N Step Down”:

This means sedimentation of cyanobacterial cells by centrifugation, decantation of the nitrate-containing (+N) medium and resuspension of the culture in nitrate-free (−N) medium.
Cultivation Under Continuous Light (40 μE m−2 s−1), BG11, 29° C.:


    Growth properties: no difference between wild type (wt) and mutant (M8)
    
        (the growth of M8 is impaired under High Light conditions [130 μE m−2 s−1] and low inoculi [initial OD750<0.1])
    
    
    Pigmentation: no difference between wt and mutant
    Storage substances: no glycogen production by the mutants in contrast to the wt

Continuous Light (40 μEm−2 s−1), BG11 without Nitrogen (24 h, 48 h), 29° C.: (N Starvation)

    Growth properties: wt and mutant stopped growing. After passage to BG11 medium containing nitrogen, wt started to grow again whereas the mutant M8 gradually lost the ability to grow, depending on duration of nitrogen depletion.
    Pigmentation: After withdrawal of nitrogen, wt started to degrade phycobilisomes (measured as absorbance at 625 nm): yellow color; M8-mutant did not degrade phycobilisomes: still blue-green color; unchanged chlorophyll levels (absorbance at 681 nm) in both wt and mutant M8

Pyruvate Level:

















Intracellular




level in mmol per liter
Extracellular



(calculated per packed
level in mmol per liter



cell volume1)
culture volume











OD750
wt
M8
wt
M8















1.0
+N
0.8
0.8
0.007
0.018



−N, 0 h


nd
nd



−N, 3.5 h


0.005
0.038



−N, 6 h


0.004
0.08



−N, 24 h
0.9
1.6
0.007
0.470





Data were verified by repetitions.


nd, not detectable


The packed cell volume is less than 1% of the culture volume






Growth properties and extracellular pyruvate levels of the ΔglgA1/ΔglgA2 double mutant (M8) under nitrogen replete and nitrogen starved conditions are presented in FIG. 32C.
The glycogen deficient mutant M8 was grown up to an OD750 of 0.6. After a centrifugation step, the cells were washed twice with nitrogen deficient BG11 medium and transferred to medium with nitrogen (+N, control) and without nitrogen (−N), respectively. After 24 h incubation, nitrogen was added to the nitrogen deficient cultures (black arrow). The growth of the cultures was estimated by measurement of chlorophyll. Abbreviations: Chl, chlorophyll a; Pyr, pyruvate
Oxoglutarate Level:














Intracellular











level in mmol per liter
Extracellular


Hours after
(calculated per packed
level in mmol per liter


nitrogen
cell volume1)
culture volume











step down
wt
M8
wt
M8














0.5
0.036
0.038
nd
nd


2
0.17
0.22
nd
nd


5
0.18
0.26
nd
0.01


24
0.22
0.53
nd
0.14





1The packed cell volume is less than 1% of the culture volume


nd, not detectable






Light/Dark Cycle (16 h/8 h), BG11, 29° C.:

Growth properties: no difference between wt and mutants M1 and M8
Further mutant characterization of the glycogen deficient mutant M8 in comparison with the wild type strain of Synechocystis sp. PCC6803
Culture Conditions:


    Continuous light (150 μm−2 s−1), 28° C.:
    Aeration with air (no additional CO2 supplementation)
    Culturing in glass flasks with 5 cm diameter, 400 ml culture volume
    Media: BG11 buffered with TES buffer (Sigma-Aldrich Inc.) at pH 8

Storage Substances:

    No glycogen production by the mutants in contrast to the wild type.

Pyruvate Concentrations in the Media Determined by Using an Optical Enzymatic Test:























Pyruvate
Pyruvate
Pyruvate
Pyruvate





0 h after
3.5 h after
6 h after
24 h after



OD750
Chlorophyll
N step down
N step down
N step down
N step down






















WT
1.2
6.18 μg/ml
0 μM
5.1 μM
4.0 μM
 2.5 μM


M8
1.1
3.60 μg/ml
0 μM
 37 μM
 79 μM
473 μM


mutant









Pyruvate Concentrations in the Media Determined by Ion Chromatography:



















Pyruvate
Pyruvate





0 h after
24 h after



OD750
Chlorophyll
N step down
N step down




















WT
1.2
6.18 μg/ml
0 μM
13.4 μM


M8
1.1
3.60 μg/ml
0 μM
 511 μM


mutant









Pyruvate Concentrations in the Media Plus Cells (Snap Shot Extraction) Determined by Ion Chromatography:



















Pyruvate
Pyruvate





0 h after
24 h after



OD750
Chlorophyll
N step down
N step down




















WT
1.2
6.18 μg/ml
0 μM
6.12 μM


M8
1.1
3.60 μg/ml
0 μM
 523 μM


mutant









Wildtype and mutant were transferred into a medium without combined nitrogen and grown for 24 hours. Subsequently the amount of pyruvate in the culture medium was determined in with an optical enzymatic method and by ion chromatography. The sum of intra- and extracellular pyruvate was determined by ion chromatography after snapshot extraction
Shown is the conductimetric detection of pyruvate in methanol extracts (snapshot) of cultures of wildtype and a glycogen synthase deficient mutant after 24 h under N-deficient conditions. The area of the pyruvate peak corresponds to 523 pmoles.
Data results are presented graphically in FIGS. 32D and 32E.
Summary Pertaining to Ethanol Production:
The loss of the two functional glycogen synthases in Synechocystis PCC 6803 mutant M8 resulted in a two-times increased intracellular pyruvate level and an at least 10-times increased extracellular pyruvate level after nitrogen depletion (24 h). In dense cultures (OD750 1.0), the extracellular pyruvate level is actually increased up to 500 times. In the wild type, these concentrations remained unchanged and much lower. The enhanced pyruvate level is used for ethanol production.
Glycogen is made during the day and would therefore compete with ethanol production in the light. It is degraded during the night and may thus support ethanol production by a quasi continuous production.
Possible Advantages of Glycogen Deficiency:
Glycogen Synthesis Requires Energy (ATP):

    
    
        Photosynthesis→glucose phosphate
        glucose phosphate+ATP→ADP-glucose+pyrophosphate
        n ADP-glucose→glycogen+n ADP
    
    


During the night, glycogen will be degraded:

    
    
        glycogen+n phosphate→n glucose phosphate
        glucose phosphate→→pentose phosphate+CO2↑
        pentose phosphate→→pyruvate
        pyruvate→→ethanol+CO2↑

Conclusions:

        Ethanol production via glycogen requires more energy and releases 50% more CO2 than direct production.
        A further advantage may be that glycogen-deficient mutants degrade photosynthetic pigments at a much lower rate than the wild type under conditions of nitrogen deficiency. Thus, growth could be retarded during ethanol production by lowering nitrogen supply.
    
    


In order to find out whether the pyruvate produced by the glycogen synthase double knock out mutant in Synechocystis can be used for ethanol production, the glycogen synthase double knock out mutant cells (denoted as M8 in the below two graphs) were transformed with the plasmid pVZ321b-PnblA-pdc/adh containing the alcohol dehydrogenase and pyruvate decarboxylase genes under the transcriptional control of the nblA promoter inducible by nitrogen starvation (denoted as M8 PnblA in the below two graphs). The concentration of pyruvate in the growth medium was determined for the M8 mutant without the pVZ321b-PnblA-pdc/adh plasmid after having induced pyruvate secretion into the medium by nitrogen starvation (indicated by M8-N in the below graphs). In addition the concentration of pyruvate and ethanol in the growth medium was also determined for the M8 mutant including the pVZ321b-PnblA-pdc/adh plasmid after having induced pyruvate production by nitrogen starvation (indicated by M8 PnblA-N in the below graphs). For the reason of comparison the respective pyruvate concentrations are also shown for the uninduced cells (denoted with M8 PnblA+N and M8+N, respectively).
Both graphs depict on the Y-axis the concentrations of pyruvate and ethanol in μM normalized to the cell density measured at 750 nm (OD750nm). The x-axes denote the course of the experiments in hours.
As can be seen in FIG. 32F the graph shows the pyruvate concentrations. It can clearly be seen that the pyruvate concentration in the growth medium is higher for the M8 mutant without Adh and Pdc enzymes than for the M8 mutant including both ethanol forming enzymes under the conditions of nitrogen starvation. In the case that the cells are not subjected to nitrogen starvation pyruvate could not be detected in the growth medium.
FIG. 32G depicts the ethanol concentration determined in the growth medium for the M8 mutant with the Adh and Pdc enzymes under the conditions of nitrogen starvation and without nitrogen starvation. The graph shows that the ethanol concentration is higher for the M8 mutant under the conditions of nitrogen starvation than without nitrogen starvation. By comparing both graphs it can be observed that nearly all pyruvate produced by the M8 mutant can be converted into ethanol by the Adh and Pdc enzymes: The M8 mutant without the Adh and Pdc enzymes secretes high amounts of pyruvate into the growth medium, but the M8 including both enzymes only excretes small amounts of pyruvate but a high amount of ethanol into the growth medium.
Furthermore the glycogen deficient glycogen synthase double knock out mutants of Synechocystis PCC 6803 were transformed with the plasmid pVZ containing ZmPDC and ADHII under the control of the iron starvation inducible promoter isiA using the standard protocols described above. Ethanol production rates and the OD750nm were determined over the course of 15 days. Results are depicted graphically in FIG. 32H.
Further, short term measurements of ethanol production rates were carried out for the glycogen synthase double knock out mutant in Synechocystis PCC 6803 with and without a second genetic modification of at least one overexpressed enzyme for ethanol formation and these production rates were compared to the ethanol production rates of the corresponding Synechocystis cells only harboring the second genetic modification.










ΔglgA1/ΔglgA2 mutant















% of



μmol O2/
μmol EtOH/
μmol EtOH/
theoretical



mg Chl* h
mg Chl* h
μmol O2
fixed CO2















S. PCC6803
98.3
5.0
0.051
15.3


pVZ321b-PisiA-


PDC-ADHII


ΔglgA1/A2
34.8
5.4
0.200
60.1


pVZ321b-PisiA-


PDC-ADHII









The above table shows the ethanol production rates normalized either to the chlorophyll content, the maximal photosynthetic capacity as determined by the oxygen evolution and the percentage of theoretical fixed CO2 which is diverted to ethanol production for a Synechocystis strain without the glycogen synthase double knock out mutation, the first genetic modification (S. PCC6803 pVZ321b-PisiA-PDC-ADHII), and for Synechocystis strains having both the first and second genetic modification (ΔglgA1/A2 pVZ321b-PisiA-PDC-ADHII). The data show that the overall photosynthetic capacity of the cells harboring the double knock out mutation is reduced. The results also indicate that a higher percentage of carbon fixed via photosynthesis can be diverted to ethanol production via a reduction of the enzymatic affinity or activity of glycogen synthase for example by introducing a knock out mutation of both genes glgA1/glgA2 coding for glycogen synthase into cyanobacteria) cells such as Synechocystis. 
X.2 Metabolic Mutant Harbouring a Knock Out of ADP-Glucose-Pyrophosphorylase (ΔGLGC) as a First Genetic Modification
Construction of the DNA-vector pGEM-T/ΔglgC-KM, which was used for generation of ΔglgC mutant, was already described herein. The obtained ΔglgC mutant was partially segregated and was grown in BG11 medium containing 75 mg/l kanamycin. The segregation status was checked by southern blot analysis using a radio-labeled glgC probe. Approximately 80% of the wild-type gene copies were replaced by the introduced mutant gene copy.
The partially segregated mutant ΔglgC was examined in comparison to Synechocystis wild-type strain under constant light conditions as described herein.
Growth Characteristics Under Constant Light Conditions
The ΔglgC mutant is generally more sensitive to light at low concentrated inoculi than the wild type strain (Synechocystis PCC6803). During further batch culturing no significant differences were detected in cell growth and chlorophyll content between the mutant and the Synechocystis PCC6803 wild type. However, the photosynthetic capacity of the ΔglgC mutant was about 35% lower compared to the Synechocystis PCC6803 wild type. This finding is consistent with data reported by Miao et al., 2003 (Miao, X., Wu, Q., Wu, G. & Zhao, N. (2003) Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp.; Biotechnol Lett. 25, 391-396).
Like in the ΔglgA mutant described above, in the ΔglgC mutant the extracellular pyruvate level is strongly increased. Data from one representative experiment are shown in the following table:















4 days
7 days
9 days














OD750
pyruvate [mM]
OD750
pyruvate [mM]
OD750
pyruvate [mM]

















PCC6803 Wt
1.7
0.009
2.0
0.001
2.4
0.003


ΔglgC
1.1
0.087
2.0
0.093
2.2
0.199









In wild type cells glycogen synthesis is increased during nitrogen starvation. Therefore, in the ΔglgC mutant, that is not able to produce glycogen, an additional increase of the pyruvate level was achieved by a nitrogen step down.
After 9 days of culturing under standard conditions, the culture was split into two parts. With one half of the culture a nitrogen step down was performed (as described for the ΔglgA mutant) and cells were grown on BG11 lacking combined nitrogen (−N) for two days. The second half of the culture was grown in full BG11 medium (+N) as a control. Two days after the nitrogen step-down, the excretion of pyruvate into the medium was measured.














+N
−N












OD750
pyruvate [mM]
OD750
pyruvate [mM]















PCC 6803 Wt
1.7
0.012
1.2
0.010


ΔglgC
1.3
0.295
1.2
0.361









ADP Glucose Pyrophosphorylase (GlgC) Knock-Out Mutant Expressing PDC and ADH

The DNA-vector pGEM-T/ΔglgC-KM was transformed into the PDC-ADHII expressing mutant Synechocystis PCC6803 pSK10-PpetJ-PDC-ADHII. The obtained mutant ΔglgC pSK10-PpetJ-PDC-ADHII was fully segregated and was grown in BG11 medium containing 100 mg/l kanamycin and 10 mg/l streptomycin.
Ethanol production was induced by copper starvation and compared to that of Synechocystis wild-type pSK10-PpetJ-PDC-ADHII.
In short term experiments under optimal conditions (light, CO2) the overall as well as the relative (to photosynthetic activity) ethanol production rate of the ΔglgC pSK-PpetJ-PDC-ADHII mutant was higher compared to that of the reference strain S. PCC6803 pSK-PpetJ-PDC-ADHII. Therefore the short term experiments performed at the beginning of the log phase (day 5 and 6 during the growth experiment) indicate a higher potential for ethanol production for the ΔglgC pSK-PpetJ-PDC-ADHII mutant. (Data are the mean of 2 measurements)










ΔglgC mutant















% of



μmol O2/
μmol EtOH/
μmol EtOH/
theoretical



mg Chl* h
mg Chl* h
μmol O2
fixed CO2















S. PCC6803 pSK-
250
4
0.016
4.8


PpetJ-PDC-ADHII


ΔglgC pSK-PpetJ-
125
9
0.072
21.6


PDC-ADHII









Similar to the glycogen synthase double knock out mutation, these results indicate that by reducing the enzymatic affinity or activity of ADP-glucose-pyrophosphorylase for example by a knock out mutation of the gene encoding ADP-glucose-pyrophosphorylase a higher percentage of carbon fixed via photosynthesis can be redirected to ethanol production. In the case that the photoautotrophic host cells do not have a second genetic modification, a drastic increase of pyruvate secretion into the growth medium can be detected.
X.3 Metabolic Mutant Harbouring a Knock Out of Pyruvate Water Dikinase (ΔppsA) as a First Genetic Modification
Knock out of phosphoenolpyruvate synthase or pyruvate water-dikinase (PpsA) was accomplished by insertion of a kanamycin resistance cassette into gene slr0301. Construction of the DNA-vector pGEM-T/ΔppsA, which was used for generation of the ppsA knock-out mutant, was already described herein. The obtained ppsA knock-out mutant was fully segregated and cultivated in BG11 medium containing 75 mg/l kanamycin.
The mutant ΔppsA was characterized in comparison to the Synechocystis wild-type strain under constant light conditions as described herein.
No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between Synechocystis PCC6803 wild-type and the ΔppsA mutant. However, in several independent growth experiments the extracellular pyruvate level of the ΔppsA mutant was increased at the end of the log-phase. Data from one representative experiment are shown in the following table:















4 days
10 days
14 days














OD750
pyruvate [mM]
OD750
pyruvate [mM]
OD750
pyruvate [mM]

















PCC6803 Wt
2.0
0
12.8
0.009
13.3
0.010


ΔppsA
1.8
0.014
8
0.010
10.8
0.073









X.4 Metabolic Mutant Harbouring a Knock Out of Either Acetatekinase (ΔACK) or a Double Knock Out of Acetatekinase and Phosphoacetyltransacetylase (ΔACK/PTA) as a First Genetic Modification

The following knock-out mutants were generated: the single-mutants Δack and Δpta and the double mutant Δack/Δpta. Knock-out of acetatekinase (ack) was accomplished by replacement of a 0.65 kb fragment of slr1299 (ack gene) by a kanamycin resistance cassette. As described herein, plasmid pBlue-ack-Kan was used to generate the Δack mutant. Knock-out of phosphoacetyltrans-acetylase (pta) was accomplished by replacement of a 0.45 kb fragment of slr2132 (pta gene) by a chloramphenicol resistance cassette. The construction of plasmid pUC-pta-Cm, which was used for generation of Δpta mutant is described above. The double knock-out mutant Δack/Δpta was generated by transformation of pBlue-ack-Kan into the Δpta mutant.
All mutants were fully segregated. Mutants were grown in BG11 medium containing the appropriate antibiotics (kanamycin 75 mg/l; chloramphenicol 10 mg/l).
Mutants Δack, Δpta, Δack/pta and Synechocystis wild-type strains were examined under constant light conditions as described.
Results:
No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between the Synechocystis PCC6803 wild type and mutants Δack, Δpta and double mutant Δack/Δpta.
Excretion of pyruvate into the medium could be detected at the end of the log phase and was increased in the mutants compared to the wild type. Data from representative experiments are shown in the following tables. The optical density at 750 nm (OD750nm) and the concentration of pyruvate in the medium are given at two time points at the end of the log phase.
















10 days
14 days












OD750 nm
pyruvate [mM]
OD750 nm
pyruvate [mM]





PCC6803 wt
4.6
0.006
6.2
0.012


Δack
6.6
0.009
7.0
0.025


Δpta
6.7
0.010
6.3
0.019













10 days
12 days












OD750 nm
pyruvate [mM]
OD750 nm
pyruvate [mM]





PCC6803 wt
8
0.003
8
0.011


Δack/Δpta
6
0.004
7
0.026









Acetatekinase (ack) and Acetatekinase (ack)/Phosphoacetyltransacetylase (pta) Knock-Out Mutants Expressing PDC and ADH

The self-replicating plasmid pVZ321b-PpetJ-PDC-ADHII was conjugated into each of the mutants: Δack, and double mutant Δack/pta, resulting in mutants Δack pVZ321b-PpetJ-PDC-ADHII, and Δack/pta pVZ321b-PpetJ-PDC-ADHII. Mutants were grown in BG11 medium containing the appropriate antibiotics (kanamycin 75 mg/l; chloramphenicol 10 mg/l; streptomycin 10 mg/l). Ethanol production was induced by copper starvation under constant light and compared to Synechocystis wild-type harboring pVZ321b-PpetJ-PDC-ADHII as described above.
Results:
In several independent growth experiments, the double mutant Δack/pta, harboring pVZ321b-PpetJ-PDC-ADHII, exhibited significantly higher ethanol production rates compared to the reference strain S. PCC6803 pVZ321b-PpetJ-PDC-ADHII. In the single mutant Δack, harboring pVZ321b-PpetJ-PDC-ADHII, ethanol production was increased compared to the reference strain S. PCC6803 pVZ321b-PpetJ-PDC-ADHII. However, this effect was not apparent, when given relative to cell growth.
Data from one representative experiment are shown in the following table. FIGS. 32I and 32J depict a graphical presentation of these data.

















time [days]
0
6 d
11 d
13 d





















PCC6803 pVZ321b-
OD750 nm
1.2
2.5
3.2
3.9


PpetJ-PDC-ADHII
EtOH [%]
0.000
0.030
0.060
0.072


Δack/pta pVZ321b-
OD750 nm
1.2
2.3
2.6
2.7


PpetJ-PDC-ADHII
EtOH [%]
0.000
0.044
0.098
0.121


Δack pVZ321b-
OD750 nm
1.3
2.8
3.9
4.8


PpetJ-PDC-ADHII
EtOH [%]
0.000
0.034
0.082
0.094









The following table shows the ethanol concentration in the medium at the end of a growth experiment and the ethanol production rate relative to cell growth (given as the slope of ethanol production [%] per OD750nm and day.














EtOH [%] after 13
EtOH production



days of growth
rate [%/OD750 nm*d]


















PCC6803 pVZ321b-
0.072
0.001


PpetJ-PDC-ADHII


Δack/pta pVZ321b-
0.121
0.0039


PpetJ-PDC-ADHII


Δack pVZ321b-
0.094
0.001


PpetJ-PDC-ADHII









When mutants Δack pVZ321b-PpetJ-PDC-ADHII, and Δack/pta pVZ321b-PpetJ-PDC-ADHII and the reference strain S. PCC6803 pVZ321b-PpetJ-PDC-ADHII were grown under day/night cycle conditions, similar results were obtained. After induction of PDC and ADHII by copper starvation, strains Δack/pta pVZ321b-PpetJ-PDC-ADHII and Δack pVZ321b-PpetJ-PDC-ADHII showed higher ethanol production rates compared to the reference strain S. PCC6803 pVZ321b-PpetJ-PDC.
At three consecutive days during the logarithmic growth phase, photosynthetic capacity and ethanol production was measured in the oxygen electrode as described.
In these short-term measurements photosynthetic activity is measured under optimized conditions (saturating light and carbon supply). Results represent the maximal photosynthetic capacity of cells rather than the real photosynthetic activity during cultivation.
Following the reaction equation of photosynthesis 6 CO2+12H2O→C6H12O6+6O2+6H2O, the photosynthetic capacity [μmol O2/mg Chl*h] is equivalent to the maximal carbon fixation [μmol CO2/mg Chl*h]. Therefore the factor (μmol EtOH per/μmol O2) given in the following table puts EtOH production into perspective of carbon fixation/photosynthesis.
Values are the mean of three consecutive measurements.















PS capacity
EtOH production




[μmol O2/
[μmol EtOH/
μmol EtOH/



mg Chl* h]
mg Chl* h]
μmol O2



















PCC6803 pVZ321b-
221
3.6
0.016


PpetJ-PDC-ADHII


Δack/pta pVZ321b-
241
6.1
0.025


PpetJ-PDC-ADHII


Δack pVZ321b-
301
7.2
0.024


PpetJ-PDC-ADHII









Conclusions:

Ethanol production in the double mutant Δack/pta, harboring pVZ321b-PpetJ-PDC-ADHII, was significantly enhanced compared to the reference strain (wt) and also in comparison to the single mutant Δack pVZ321b-PpetJ-PDC-ADHII. For the single mutant Δack pVZ321b-PpetJ-PDC-ADHII, high ethanol production rates were obtained in short term experiments.
X.5 Metabolic Mutant Harbouring a Knock Down of Pyruvate Dehydrogenase E1 Component (Beta Subunit) (PdhBanti) as a First Genetic Modification
Knock-down of Pyruvate dehydrogenase (PdhB) was accomplished by regulated expression (PpetJ) of the corresponding antisense RNA (sll1721-pdhB). Construction of the DNA-vector pSK9/PpetJ-pdhBanti, which was used for the generation of a pdhB knock down mutant, was already described herein. The obtained pdhB knock-down mutant was fully segregated and was grown in BG11 medium containing 14 mg/l chloramphenicol. The mutant pdhBanti was characterized in comparison to the Synechocystis wild-type strain under constant light conditions as described herein. Expression of anti-sense RNA was induced by copper starvation as described for induction experiments with the promoter PpetJ. Expression of anti-sense RNA was verified by northern blot analysis.
Results:
No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between Synechocystis PCC6803 wild type and pdhBanti mutant. After induction of the petJ promoter, the level of extracellular pyruvate was slightly increased in the pdhBanti mutant compared to the wild-type. This effect was verified in three independent growth experiments, data from one representative experiment are shown.














7 days
9 days












OD750
pyruvate [mM]
OD750
pyruvate [mM]















PCC6803 wt
3.6
0
5.3
0.004


pdhBanti
3.9
0.004
6.1
0.015









X.6 Metabolic Mutant Harbouring an Overexpressed Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) as a First Genetic Modification

Mutant and Synechocystis wild-type strains were grown at 28° C., under constant light (70 μE m−2 s−1) and aerated with CO2-enriched air (0.5% CO2). The initial OD750 was about 1 in a total culture volume of 200 ml in a 250 ml Schott-flask. For comparison of the ethanol production ethanol producing mutants pVZ321b-PpetJ-PDC and pVZ321b-PpetJ-PDC/SynRbcLXS and wild type were cultivated in BG11 without copper in continuous light (75-200 μE m−2 s−1) and a culture volume of 300 ml in a 500 ml Schott-flask.
Methods:
The rate of oxygen evolution was measured with a Clark-type oxygen electrode (Rank Brothers, UK). Prior to the measurement cells were washed 2× and resuspended in BG-11 medium supplemented with 25 mM NaHCO3. Light intensity was saturating with approx. 500 μE/s*m2.
For preparation of cell extracts, cells were pelleted, washed two times with 20 mM HEPES/KOH, pH 7.5, 5 mM EDTA, 2 mM DTT, dissolved in this buffer and broken with a beadbeater (2×10 min). The supernatant of a centrifugation (15 min, 14000 rpm, 4° C., Micro 200R, Hettich) was used for the experiments. The protein content of cell extracts was measured with the method of Lowry.
RuBisCO activity was measured similar as described in Iwaki et al. (2006) Photosynth Res. 2006 June; 88(3):287-97. Epub 2006 May 12. Expression of foreign type I ribulose-1,5-bisphosphate carboxylase/oxygenase stimulates photosynthesis in cyanobacterium Synechococcus PCC7942 cells:
5 μl to 15 μl of cell extracts were mixed with 750 μl of 50 mM HEPES/KOH, pH 7.5, 20 mM MgCl2, 50 mM KHCO3, 0.15 mM NADH, 5 mM ATP, 2.5 mM Phosphocreatine, 1.5 μl carbonic anhydrase (10 U/μl in 50 mM HEPES, pH 7.5), 7.5 μl creatine kinase (0.5 U/μl) 3.75 μl of glyceraldehyde-3-phosphate dehydrogenase (12.5 mg/ml), phosphoglycerate kinase (suspension with 10 mg/ml). The assay was incubated at 30° C. for 10 min. Then the reaction was started by the addition of 7.5 μl of 250 mM ribulose-1,5-bisphosphate and the absorption of 340 nm was monitored.
Results and Conclusions:
The mutant with RuBisCO over-expression (6803 pVZ321b-Prbc-SynRbcLXS) grows as fast as the Synechocystis wild type and shows no phenotypical differences except for the chlorophyll content that is reduced by 20-30% compared to wild type (see FIG. 32K). Interestingly, at the same time the mutant produces significant more biomass observed by dry weight determination at several time points during the cultivation experiment (Tab. 1). At the end point the difference in dry weight accounts to about 30%. This means although both cultures are indistinguishable by the optical density the mutant seems to build up more biomass. Either the cells are larger in size or the cells are denser packed by biomass (eg. with carbohydrates like glycogen or fatty acids).
FIG. 32L shows the growth parameter (OD at 750 nm and Chlorophyll content) of Synechocystis wild type and a mutant that over-express the endogenous RuBisCO operon.







TABLE 1







Biomass (dry weight, mean value of triplicates) during the


(in FIG. 50-1A shown) cultivation experiment of Synechocystis


wild type cells and cells overexpressing RuBisCO.










Prbc-SynRbcLXS
WT 6803













time

Chl a
Dryweight

Chl a
Dryweight


[d]
OD750 nm
[mg/l]
[g/l]
OD750 nm
[mg/l]
[g/l]
















0
0.96
3.82
0.23
0.91
3.69
0.18


7
6.09
22.60
1.01
6.36
29.07
1.02


11
8.14
21.22
1.51
7.99
33.89
1.30


16
10.17
18.30
1.70
10.01
24.97
1.32









Measurements of the RuBisCO activity from the mutant with RuBisCO over-expression revealed an about 2-fold increase in the activity compared to the wild type (see Tab. 2). This was confirmed by semi-quantitative Western blot analyses, too (data not shown). Furthermore for this mutant and the wild type the oxygen evolution was determined. Based on the wild-type level a slight increase (about 15%) in the oxygen evolution was detectable for the cells overexpressing the Synechocystis RuBisCO.







TABLE 2







RuBisCO activity and photosynthetic oxygen evolution


of Synechocystis wild type and a mutant overexpressing


the endogenous RuBisCO operon.










RuBisCO activity
oxygen evolution



[μmol RBP/min * mg
[μmol O2/h *



protein]
mg chl]













PCC6803 wild type
0.23 (100%)
107.8 (100%)


pVZ321b-Prbc-
0.48 (209%)
124.6 (115%)


SynRbcLXS









In a further experiment the potential positive effect of the detected increased RubisCO activity for the ethanol production was analyzed. For this purpose growth and ethanol production of an integrative ethanol producing mutant (6803 pSK10-PisiA-PDC/ADHII) was compared to the isogenic, ethanologenic mutant containing moreover the RubisCO overexpressing plasmid (pVZ321b-Prbc-SynRbc).
FIGS. 32L, 32M and 32N, respectively show the OD750, the ethanol production and the ethanol production normalized to the OD750 for the mutant Synechocystis PCC6803 harboring the pSK10-PisiA-PDC/ADHII plasmid and the mutant additionally containing the vector pVZ321b-Prbc-SynRbc.
Both ethanologenic Synechocystis mutants exhibit a similar ethanol production rate of about 0.017% (v/v) per day for 14 days under continuous light illumination (see FIG. 32-4C). Over the whole time-scale the mutant with the RubisCO over-expression produces a bit more ethanol (about 8% compared to the reference). Also when the ethanol production is normalized to the cell density (OD at 750 nm as indicator for the growth) this difference in the ethanol production remains. This indicates that an elevated RubisCO activity can lead to an increased ethanol formation. The potential to direct additional carbon fixed via photosynthesis into ethanol production might be further improvable by optimization of the RubisCO expression level as well as by combination with other metabolic mutations, enhancing the level of substrates for the ethanologenic enzymes.
X.7 Metabolic Mutant Harbouring an Overexpressed Pyruvate Kinase 2 as a First Genetic Modification
Construction of the DNA-vector pVZ321-PpetJ-pyk2, which was used for the generation of a pyk2 overexpression mutant, was already described herein.
The obtained mutant Synechocystis PCC6803 pVZ321-PpetJ-pyk2 was cultivated in BG11 medium containing 14 mg/l chloramphenicol and characterized in comparison to the Synechocystis wild-type strain under constant light conditions as described herein. Expression of pyruvate kinase gene was induced by copper starvation.
Results:
No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between Synechocystis PCC6803 wild type and mutant PCC6803 PpetJ-pyk2.
After induction of the petJ promoter, the level of extracellular pyruvate was slightly increased in the PCC6803 PpetJ-pyk2 mutant compared to the wild-type.















6 days
9 days
14 days














OD750
pyruvate [mM]
OD750
pyruvate [mM]
OD750
pyruvate [mM]

















PCC6803 Wt
1.3
0.018
1.9
0.005
2.5
0.009


PpetJ-pyk2
0.8
0.016
1.3
0.051
1.9
0.064









Pyruvatekinase 2 Overexpression Mutant Expressing PDC and ADH

Pyruvate kinase 2 was also expressed from self-replicating plasmid pVZ321 under control of its endogeneous promoter Ppyk2 in the ethanol producing strain S. PCC6803 pSK-PisiA-PDC-ADHII. Generation of plasmid pVZ-Ppyk2-pyk2, which was conjugated into Synechocystis pSK-PisiA-PDC-ADHII, was already described herein.
The ethanol production rates and the oxygen evolution for the photosynthetic capacity of Synechocystis strains S. PCC6803 pSK-PisiA-PDC-ADHII harboring plasmid pVZ-Ppyk2-pyk2 and reference strain S. PCC6803 pSK-PisiA-PDC-ADHII were determined as mentioned above.










(data are mean of two measurements)











μmol O2/
μmol EtOH/
μmol EtOH/



mg Chl* h
mg Chl* h
μmol O2














S. PCC6803 pSK-
164.5
9.5
0.058


PisiA-PDC-ADHII


Ppyk2-pyk2 pSK-
134.3
10.0
0.074


PisiA-PDC-ADHII









X.8 Metabolic Mutant Photoautotrophic Cells Harbouring an Overexpressed Pyruvate Kinase (pyk) Enolase (eno) and Phosphoglycerate Mutase (Pgm) as First Genetic Modifications

Two mutants have been created for overexpression of the three glycolytic genes pyruvate kinase (pyk), enolase (eno) and phosphoglycerate mutase (pgm).
In one mutant expression of pyruvate kinase 1 (from E. coli), enolase and phosphoglycerate mutase (both from Zymomonas mobilis) is controlled by the ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) promoter (Prbc) from Synechococcus PCC7942. Construction of the DNA-vector pVZ321-p67, which was conjugated into Synechocystis PCC6803 to generate mutant PCC6803 Prbc-pyk-eno-pgm, was already described herein.
In the other mutant the expression of additional copies of the endogenous genes pyruvate kinase 2, enolase and phosphoglycerate mutase from Synechocystis PCC6803 is controlled by the PpetJ promoter. DNA-vector pVZ322-PpetJ-pyk2-eno-pgm, which was conjugated into Synechocystis PCC6803 to generate mutant PCC6803 PpetJ-pyk2-eno-pgm, was already described herein.
The obtained mutants PCC6803 pVZ321-Prbc-pyk-eno-pgm and PCC6803 pVZ322-PpetJ-pyk2-eno-pgm were cultivated in BG11 medium containing 14 mg/l chloramphenicol or 3 mg/l gentamycin, respectively, and characterized in comparison to the Synechocystis wild-type strain under constant light conditions as described herein.
Results:
No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between Synechocystis PCC6803 wild type and mutants PCC6803 Prbc-pyk-eno-pgm and PCC6803 PpetJ-pyk2-eno-pgm.
Excretion of pyruvate was increased in mutant PCC6803 Prbc-pyk-eno-pgm compared to wild-type, as shown in the following table:














10 days
14 days












OD750
pyruvate [mM]
OD750
pyruvate [mM]















PCC6803 (BG11)
4.6
0.006
6.2
0.012


PCC6803 Prbc-pyk-
3.0
0.017
6.1
0.032


eno-pgm









In mutant PCC6803 PpetJ-pyk2-eno-pgm the level of extracellular pyruvate was increased after induction of the glycolytic genes by copper starvation.














7 days
14 days












OD750
pyruvate [mM]
OD750
pyruvate [mM]















PCC6803
1.6
0
3
0.006


(BG11-Cu)


PCC6803 PpetJ-
3.1
0.013
3.7
0.024


pyk2-eno-pgm









Expression of Pyruvate Kinase, Enolase and Phospho-Glycerate Mutase in Synechocystis Strains Expressing Pdc Enzyme Alone as a Second Genetic Modification.

Plasmids pVZ321-p67 and pVZ322-PpetJ-pyk2-eno-pgm were each conjugated into the ethanol producing strain Synechocystis PCC6803 pSK10-PpetJ-pdc expressing only PDC. (Construct pSK10-PpetJ-pdc is a derivate of pSK10-PpetJ-pdc-adhII, from that the adhII gene was cut out with SacI and PstI.)
The resulting mutants were cultured in BG11 containing 10 mg/l streptomycin and 7 mg/l chloramphenicol or 2 mg/l gentamycin, respectively. Expression of pdc (and in mutant PpetJ-pyk2-eno-pgm also of the glycolytic genes) was induced by copper starvation (PpetJ).
In short term measurements both mutants expressing the glycolytic enzymes showed a better ethanol production rate (relative to photosynthetic activity) than the reference strains. Data in the following table are means of two consecutive measurements within one cultivation.















μmol O2/
μmol EtOH/
μmol EtOH/



mg Chl* h
mg Chl* h
μmol O2



















PCC6803 pSK-PpetJ-PDC
130
1.8
0.014


PCC6803 pSK-PpetJ-PDC
148
3.2
0.022


pVZ-Prbc-pyk-eno-pgm


PCC6803 pSK-PpetJ-PDC
197
2.5
0.012


PCC6803 pSK-PpetJ-PDC
104
2.8
0.028


pVZ-Ppet J-pyk2-eno-pgm









Conclusions

These data suggest that overexpression of the glycolytic enzymes pyruvate kinase, enolase and phosphoglycerate mutase leads to a higher flux from CO2 towards pyruvate which results in a higher ethanol production rate, relative to the photosynthetic capacity.
X.9 Metabolic Mutant Photoautotrophic Cells Harbouring an Overexpressed Malic Enzyme (Me) and Malate Dehydrogenase (Mdh) as First Genetic Modifications
Overexpression of malic enzyme (Me) and malate dehydrogenase (Mdh) were accomplished by regulated expression of the corresponding genes (slr0721-me; sll0891-mdh) via the PpetJ promoter. Construction of DNA-vectors pSK9/PpetJ-me and pSK9/PpetJ-me-mdh, which were used for generation of me- and me/mdh-overexpression mutants, was already described herein. The obtained overexpression mutants were fully segregated and were grown in BG11 medium containing 14 mg/l chloramphenicol. Mutants PpetJ-me and PpetJ-me/mdh were examined in comparison to the Synechocystis wild-type strain under constant light conditions as described herein. Expression of me and mdh genes was induced by copper starvation and successfully proven by northern blot analysis via a radio-labeled me- and mdh-probe, respectively (data not shown).
Results:
No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between Synechocystis PCC6803 wild type and PpetJ-me and PpetJ-me/mdh mutant, respectively.
An enhanced extracellular pyruvate level was detected in the medium of the PpetJ-me and the PpetJ-me/mdh mutants after induction by copper starvation. The following table shows the extracellular pyruvate concentrations measured 10 days after induction in comparison with values measured in medium from non-induced cells.














Not induced (BG11)
Induced (BG11 —Cu)



10 days
10 days












OD750
pyruvate [mM]
OD750
pyruvate [mM]















PCC6803 Wt
8.3
0.010
9.3
0.011


PpetJ-me
10.1
0.005
8.3
0.032


PpetJ-me-mdh
7.8
0.005
8.5
0.024









The higher extracellular pyruvate levels measured in the induced PpetJ-me and PpetJ-me/mdh mutants (compared to wildtype and non-induced cells) suggest, that overexpression of malic enzyme or malic enzyme in combination with malate dehydrogenase leads to a higher pyruvate level within the cyanobacterial cells.
X.10 Metabolic Mutant Cells of Nostoc/Anabaena PCC7120 and Anabaena Variabilis ATCC 29413 Harbouring a Knockout of the ADP-Glucose-Pyrophosphorylase as a First Genetic Modification
In the following the EtOH production in Anabaena PCC7120 transformed with the integrative PpetE-PDC-ADHII and PpetE-PDC constructs will be discussed.
In a first test experiment EtOH production in Anabaena PCC7120 with PpetE-pdc-adhII or PpetE-pdc inserted in ADP-glucose-pyrophosphorylase gene, agp, was measured of the following mutants: A.7120 Δagp (all4645)::C.K3-PpetE-pdc-adhII, named “PpetE-pdc-adhII” and A.7120 Δagp (all4645)::C.K3-PpetE-pdc, named “PpetE-pdc”. Mutant A.7120 Δagp (all4645)::C.K3, named Δagp, served as control.
Cultures of all mutants were grown at 28° C., under continuous light conditions (40 μE/m2 s1) in batches of 50 ml in 100 ml Erlenmeyer flasks with shaking. Precultures were grown in BG11 medium lacking copper sulfate (BG11−Cu), supplemented with neomycin (100 μg/ml). It should be noted here, that the petE promoter might not be fully repressed under this BG11−Cu conditions, as the glassware was not treated to remove trace amounts of copper from it. The petE promoter seems to be smoothly regulated in Anabaena PCC7120 [Buikema, W. J., and R. Haselkorn. 2001. Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions. PNAS USA 98:2729-2734], therefore trace amounts of copper coming from the glassware might be sufficient to induce expression.
Expression of the ethanologenic genes was induced by addition of 1× copper (0.32 μM CuSO4). This corresponds to the copper concentration present in BG11 medium.
As a measure of growth, chlorophyll was determined at several time points and ethanol was measured using the already described enzymatic method.







TABLE 1







Growth and ethanol production of Anabaena mutants expressing


ethanologenic genes under control of petE promoter.









time












0 (start)
5 days
9 days
14 days
















Chl
EtOH
Chl
EtOH
Chl
EtOH
Chl
EtOH



















“PpetE-pdc-
1
0.002
3
0.014
6
0.022
8
0.037


adhII”


“PpetE-pdc”
3
0.006
5
0.015
6
0.028
8
0.044


Δagp
8
0
16
0.0001
20
0.0001
25
0.0001


(control)





Chl, chlorophyll in [μg/ml] and EtOH [%].






Ethanol was produced by both integrative mutants, while in the control strain (mutant Δagp) no ethanol production was detected. The similar ethanol production rates obtained in mutants “PpetE-pdc-adhII” and “PpetE-pdc” clearly indicate that also in Anabaena PCC7120 expression of PDC alone is sufficient for ethanol production. Thus it appears that this strain constitutively expresses an endogenous ADH enzyme converting acetaldehyde into ethanol. Several open reading frames are annotated as alcohol dehydrogenases in Anabaena PCC7120 (http://bacteria.kazusa.or.jp/cyanobase/), however all genes show only little similarity (less than 30% identical amino acids) to SynADH.
Detailed Discussion of the Embodiments Involving Overexpressed Zn2+ Dependent Alcohol Dehydrogenase and Pdc and/or Adh Enzymes Under the Control of Various Inducible Promoters
In the following further embodiments of the invention related to for example an overexpressed Zn2+ dependent alcohol dehydrogenase, an overexpressed alcohol dehydrogenase, directly converting acetyl-CoA to ethanol, or promoters that can be induced by nutrient starvation, cold shock, heat shock, salt stress, light exposure or stationary growth of the host cell will be explained in more detail.

    Construction of selfreplicating (extrachromosomal) and chromosome-integrative vectors for the inducible overexpression of ethanologenic enzymes in cyanobacteria
    Construction of extrachromosomal pVZ-vectors for inducible overexpression of pyruvate decarboxylase (ZmPdc) and alcohol dehydrogenase (ZmAdhII) from Zymomonas mobilis 


The construction of the certain vectors including the different promoters were done by using the following general protocol:

    
    
        EcoRI/BamHI restriction of the pCB4-LR(TF)pa shuttle vector in order to cut off the pdc and adh genes. This shuttle vector was constructed by Dr. John Coleman, University of Toronto, Toronto, Canada.
        ligation of the pdc/adh containing EcoRI/BamHI fragment into the cloning vector pDrive (EcoRI/BamHI). The pDrive vector (Qiagen, Hilden, Germany, GenBank no.: DQ996013) was already described above.
        amplification of the isiA-, nblA- and ntcA-promoter using chromosomal DNA from Synechocystis sp. PCC 6803 and the following primers (all amplified promoters have a length of about 600 bp and include the ribosome binding site of the corresponding genes):
    
    









(SEQ ID NO: 207)








isiA-fw-SalI
5′-GTCGACCTTCCAGCACCACGTCAAC-3′


 







(SEQ ID NO: 208)








isiA-rev-EcoRI
5′-GAATTCACAGAATTGCCTCCTTAATTGAG-3′


 







(SEQ ID NO: 209)








nblA-fw-SalI
5′-ACGCGTCGACTTATGGTTGATTCGCATTG-3′


 







(SEQ ID NO: 210)








nblA-rev-EcoRI
5′-CGGAATTCATAGCTGTTGCCCTCCAAG-3′


 







(SEQ ID NO: 211)








ntcA-fw-SalI
5′-GTCGACAACGACGGAGGTTTAAGGG-3′


 







(SEQ ID NO: 212)








ntcA-rev-EcoRI
5′-GAATTCATGCACGTTCACGGTAATGG-3′






    
    
        All forward primer included the SalI restriction site, all reverse primer included a EcoRI restriction site for cloning (marked bold).
        ligation of the SalI/EcoRI cut promoter fragments into the pDrive-pdc/adh (SalI/EcoRI) generating the constructs pDrive-PisiA-pdc/adh, pDrive-PnblA-pdc/adh and pDrive-PntcA-pdc/adh
        SalI/PstI restriction of pDrive-PisiA-pdc/adh, pDrive-PnblA-pdc/adh and pDrive-PntcA-pdc/adh and ligation of the corresponding promoter-pdc/adh fusions into the self replicating broad-host range vector pVZ321b (SalI/PstI), a derivate of the pVZ321 (constructed by V. V. Zinchenko Moscow, Russia; described above) with an additional streptomycin resistance cassette/cartridge introduced into the XbaI site of pVZ321. The pVZ321b vector was constructed by Anne Karradt, Humboldt-Universitaet Berlin, Plant Biochemistry Department (Prof. Lockau) and was used as a cargo plasmid for conjugation.
        pVZ321 Gen Bank no.: AF100176.
        End products of the cloning procedure are the pVZ-vectors: FIG. 33A presents a schematic diagram of pVZ-PisiA-pdc/adh; FIG. 33B presents a schematic diagram of pVZ-PnblA-pdc/adh; and FIG. 33C presents a schematic diagram of pVZ-PntcA-pdc/.
    
    


FIG. 33D presents the nucleotide sequence of adhII and pdc genes from Zymomonas mobilis. The source of this polynucleotide is the shuttle vector pCB4-LR(TF)pa, a kind gift from John Coleman. FIG. 33E presents a schematic diagram of some restriction sites occurring within the adhII and pdc gene sequences. FIGS. 33F and 33G presents the amino acid sequence of ZmPdc and ZmAdhII, respectively. Various gene promoter elements were utilized to control constitutive and/or induced gene expression. Sequences for these elements are presented herein. As known to those skilled in the art, other genetic elements may serve the same purpose.
Remark: In all following nucleotide sequences of promoters restriction sites for clonings are marked (colored).
The isiA promoter (Synechocystis sp. PCC6803) element nucleotide sequence is presented in FIG. 34A. This genetic element induces gene expression under conditions of iron starvation.
The nblA promoter (Synechocystis sp. PCC6803) element nucleotide sequence is presented in FIG. 34B. This genetic element induces gene expression under conditions of nitrogen starvation.
The ntcA promoter (Synechocystis sp. PCC6803) element nucleotide sequence is presented in FIG. 34C. This genetic element induces gene expression under conditions of nitrogen starvation.
The pVZ321b cloning vector (derivate of pVZ321) was constructed by Anne Karradt, Humboldt-Universitaet Berlin, Plant Biochemistry Department (Prof. Lockau), Berlin. The nucleotide sequence for pVZ321b is presented in FIG. 35A, and the structure of this plasmid is presented schematically in FIG. 35B.
Introduction of further well suited inducible promoters into the existing pVZ-expression constructs (point 1).
In order to create expression constructs as described above (point 1) but under control of a different promoter, the promoter sequence was cut out by SalI/EcoRI digestion of the corresponding pVZ-Pxxx-pdc/adh construct (xxx for isiA, ntcA, nblA). The new promoter sequence containing the restriction sites SalI/EcoRI as described for the isiA-, nblA- and ntcA-promoter was ligated into the “promoter free” pVZ construct resulting in a pdc/adh expression construct under control of the new promoter.
Representative new promoters include, but are not limited to, the following:
(1) FIG. 36A depicts the nucleotide sequence of the petJ promoter (Synechocystis sp. PCC 6803) (petJ gene: sll1796 (encoding for cytochrome c553; induced expression under copper starvation);
REFERENCE


    J Biol Chem. 2004 Feb. 20; 279(8):7229-33. Epub 2003 December
    
        The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin.
        Durán R V, Hervas M, De La Rosa M A, Navarro J A.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36B.
(2) FIG. 36 C depicts the nucleotide sequence of the sigB promoter (Synechocystis sp. PCC 6803)
sigB gene: sll0306 (encoding for RNA polymerase group 2 sigma factor) induced expression after heat shock, in stationary growth phase/nitrogen starvation and darkness)
REFERENCES


    Arch Microbial. 2006 October; 186(4):273-86. Epub 2006 Jul. 26.
    
        The heat shock response in the cyanobacterium Synechocystis sp. Strain PCC 6803 and regulation of gene expression by HrcA and SigB.
        Singh A K, Summerfield T C, Li H, Sherman L A
    
    
    FEBS Lett. 2003 Nov. 20; 554(3):357-62.
    
        Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria.
        Imamura S, Asayama M, Takahashi H., Tanaka K, Takahashi H, Shirai M
    
    
    J Biol. Chem. 2006 Feb. 3; 281(5):2668-75. Epub 2005 Nov. 21.
    
        Growth phase-dependent activation of nitrogen-related genes by a control network of group 1 and group 2 sigma factors in a cyanobacterium.
        Imamura S, Tanaka K, Shirai M, Asayama M.
        A plasmid created with this promoter element is presented schematically in FIG. 36D.

(3) FIG. 36 E depicts the nucleotide sequence of the htpG promoter (Synechocystis sp. PCC 6803) htpG gene: sll0430: (encoding for heat shock protein 90, molecular chaperone) induced expression after heat shock

    
    


REFERENCE


    Plant Physiol. 1998 May; 117(1):225-34.
    
        Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002.
        Samartzidou H, Widger W R
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36F.
(4) FIG. 36 G shows the nucleotide sequence of the lrtA promoter (Synechocystis sp. PCC 6803) lrtA gene:sll0947 (encoding the light repressed protein A homolog induced expression after light to dark transition)
REFERENCE


    Plant Physiol. 1998 May; 117(1):225-34.
    
        Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002.
        Samartzidou H, Widger W R
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36H.
(5) the nucleotide sequence of the psbA2 promoter (Synechocystis sp. PCC 6803) (FIG. 36I) psbA2 gene: slr1311 (encoding the photosystem II D1 protein) induced expression after dark to light transition
REFERENCE


    Biochem Biophys Res Commun. 1999 Feb. 5; 255(1):47-53.
    
        Light-dependent and rhythmic psbA transcripts in homologous/heterologous cyanobacterial cells.
        Agrawal G K, Asayama M, Shirai M.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36J.
(6) FIG. 36K shows the nucleotide sequence of the rbcL promoter (Synechocystis sp. PCC 6803) rbcL gene: slr0009 (encoding the ribulose biphosphate carboxylase/oxygenase large subunit constitutive strong expression under continuous light conditions
REFERENCE


    Plant Mol. Biol. 1989 December; 13(6):693-700
    
        Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803.
        Mohamed A, Jansson C.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36L.
(7) FIG. 36M depicts the nucleotide sequence of the psaA promoter (Synechocystis sp. PCC6803); PsaA gene: slr1334 (encoding P700 apoprotein subunit Ia) induced expression under low white light and orange light, low expression level under high light and red light, repressed in darkness
REFERENCES


    Plant Cell Physiol. 2005 September; 46(9):1484-93. Epub 2005 Jun. 24.
    
        Regulation of photosystem I reaction center genes in Synechocystis sp. strain PCC 6803 during Light acclimation.
        Herranen M, Tyystjärvi T, Aro E M.
    
    
    Plant Cell Physiol. 2006 July; 47(7):878-90. Epub 2006 May 16.
    
        Characterization of high-light-responsive promoters of the psaAB genes in Synechocystis sp. PCC 6803.
        Muramatsu M, Hihara Y.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36N.
(8) FIG. 36O shows the nucleotide sequence of the ggpS promoter (Synechocystis sp. PCC6803); ggpS gene: sll1566 (encoding glucosylglycerolphosphate synthase) induced expression after salt stress
REFERENCE


    Plant Physiol. 2004 October; 136(2):3290-300. Epub 2004 Sep. 10.
    
        Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803.
        Malin K, Kanesaki Y, Los D A, Murata N, Suzuki Hagemann M.
    
    
    J Bacteriol. 2002 June; 184(11):2870-7.
    
        Salt-dependent expression of glucosylglycerol-phosphate synthase, involved in osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803.
        Marin K, Huckauf J, Fulda S, Hagemann M.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36P.
(9) FIG. 36Q depicts the nucleotide sequence of the nirA promoter (Synechocystis sp. PCC6803); nirA gene: slr0898 (encoding ferredoxin-nitrite reductase) induced expression after transition from ammonia to nitrate
REFERENCE


    Appl Environ Microbial. 2005 October; 71(10):5678-84.
    
        Application of the Synechococcus nirA promoter to establish an inducible expression system for engineering the Synechocystis tocopherol pathway.
        Qi Q, Hao M, Ng W O, Slater S C, Baszis S R, Weiss J D, Valentin H E.
    
    
    J. Bacterial. 1998 August; 180(16):4080-8
    
        cis-acting sequences required for NtcB-dependent, nitrite-responsive positive regulation of the nitrate assimilation operon in the cyanobacterium Synechococcus sp. strain PCC 7942.
        Maeda S, Kawaguchi Y, Ohe T A, Omata T.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36R.
(10) FIG. 36S depicts the nucleotide sequence of the petE promoter (Anabaena sp. PCC7120); petE gene: all0258 (encoding plastocyanin precursor) induced expression at elevated copper concentrations
REFERENCE


    Microbiology. 1994 May; 140 (Pt 5):1151-9.
    
        Cloning, sequencing and transcriptional studies of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120.
        Ghassemian M, Wong B, Ferreira F, Markley J L, Straus N A.
    
    
    Proc Natl Acad Sci USA. 2001 Feb. 27; 98(5):2729-34. Epub 2001 Feb. 20.
    
        Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions.
        Buikema W J, Haselkorn R.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36T
(11) FIG. 36U shows the nucleotide sequence of the hspA promoter (Synechocystis sp. PCC6803); hspA gene: sll154 16.6 kDa small heat shock protein, molecular chaperone multi-stress responsible promoter (heat, cold, salt and oxidative stress)
REFERENCE


    Curr Microbial. 2004 September; 49(3):192-8.
    
        Expression of the heat shock gene hsp16.6 and promoter analysis in the cyanobacterium, Synechocystis sp. PCC 6803.
        Fang F, Barnum S R.
    
    
    J Exp Bat. 2006; 57(7):1573-8. Epub 2006 Mar. 30.
    
        The heat shock response of Synechocystis sp. PCC 6803 analysed by transcriptomics and proteomics.
        Suzuki I, Simon W J, Slabas A R.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36V.
(12) FIG. 36W depicts the nucleotide sequence of the hliB promoter (Synechocystis sp. PCC6803); hliB gene: ssr2595: high light-inducible polypeptide HliB, CAB/ELIP/HLIP superfamily multi-stress responsible promoter (heat, cold, salt and oxidative stress)
REFERENCE


    J Biol Chem. 2001 Jan. 5; 276(1):306-14.
    
        The high light-inducible polypeptides in Synechocystis PCC6803. Expression and function in high light.
        He Q, Dolganov N, Bjorkman O, Grossman A R.
    
    
    Arch Microbial. 2007 April; 187(4):337-42. Epub 2007 Feb. 10.
    
        The response regulator RpaB binds the high light regulatory 1 sequence upstream of the high-light-inducible hliB gene from the cyanobacterium Synechocystis PCC 6803.
        Kappell A D, van Waasbe en L G.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36X
(13) FIG. 36Y shows the nucleotide sequence of the clpB1 promoter (Synechocystis sp. PCC6803); clpB1 gene: slr1641: ATP-dependent Clp protease, Hsp 100, ATP-binding subunit ClpB multi-stress responsible promoter (heat, cold, salt and oxidative stress)
REFERENCE


    Microbiology. 2004 May; 150 (Pt 5):1271-81.
    
        Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313.
        Mary I, Tu C J, Grossman A, Vaulot D.
    
    
    J Exp Bot. 2006; 57(7):1573-8. Epub 2006 Mar. 30.
    
        The heat shock response of Synechocystis sp. PCC 6803 analysed by transcriptomics and proteomics.
        Suzuki I, Simon W J, Slabas A R.
    
    


A plasmid created with this promoter element is presented schematically in FIG. 36Z.
Introduction of Alternative Ethanologenic Genes to ZmPdc and ZmAdhII into the Existing pVZ-Expression Constructs (point 1)
In order to create expression constructs as described above (point 1) but with different alcohol dehydrogenases, the adh encoding sequence was cut out by SacI/PstI digestion of the corresponding pVZ-Pxxx-pdc/adh construct (xxx for isiA, nblA, ntcA). The new adh sequence containing the restriction sites SacI/PstI (introduced by used primer) was ligated into the “adh free” pVZ construct resulting in a construct that expresses the ZmPdc together with new Adh.
Remark: In all following nt sequences of genes restriction sites are marked for clonings as well as translation starts (start codons) and translation stops (stop codons).
In this context, new alcohol dehydrogenases include the following:
(1) FIG. 37A presents the nucleotide sequence for ZmADHI (adhA gene from Zymomonas mobilis ZM4) and FIG. 37B presents the amino acid sequence for ZmAdhI AAV89860
FIG. 37C presents a schematic representation of the plasmid pVZ321b-PisiA-PDC-ZmADH1. FIG. 37D presents a schematic representation of the plasmid pVZ321b-PntcA-PDC-ZmAH1. FIG. 37E presents a schematic representation of the plasmid pVZ321b-PnblA-PDC-ZmADH1.
(2) The nucleotide sequence of SynAdh (adh gene (slr1192) Synechocystis sp. PCC 6803) is presented in FIG. 38A. The amino acid sequence of this protein (SynAdh protein sequence BAA18840) is presented in FIG. 38B.
FIG. 38C presents a schematic representation of the plasmid pVZ321b-PisiA-PDC-SynADH. FIG. 38D presents a schematic representation of the plasmid pVZ321b-PntcA-PDC-SynADH. FIG. 38E presents a schematic representation of the plasmid pVZ321b-PnblA-PDC*SynADH.
In order to create expression constructs as described above (point 1) but with AdhE-type alcohol dehydrogenases, the pdc/adh encoding sequence was cut out by EcoRI/BamHI and EcoRI/PstI digestion resp. of the corresponding pVZ-Pxxx-pdc/adh construct (xxx for isiA, ntcA, nblA). The adhE sequence of E. coli and Thermosynechococcus elongatus resp. containing the restriction sites EcoRI/BamHI and EcoRI/PstI resp. (introduced by used primer) were ligated into the “pdc/adh free” pVZ construct resulting in constructs that express the AdhE-type alcohol dehydrogenases.
(3) The nucleotide sequence for EcAdhE (adhE gene from E. coli K12) is presented in FIG. 39A. The amino acid sequence for this protein (EcAdhE protein sequence NP_415757) is presented in FIG. 39B.
FIG. 39C presents a schematic representation of the plasmid pVZ321b-PisiA-PDC-EcAdhE. FIG. 39D depicts a schematic representation of the plasmid pVZ321b-PntcA-PDC-EcAdhE. FIG. 39E presents a schematic representation of the plasmid pVZ321b-PnblA-PDC-EcAdhE.
(4) The nucleotide sequence for the ThAdhE gene (adhE gene (tlr0227) from Thermosynechococcus elongatus BP-1) is presented in FIG. 40A, and the amino acid sequence for this protein (ThAdhE protein sequence BAC07780) is presented in FIG. 40B.
FIG. 40C presents a schematic representation of the plasmid pVZ321b-PisiA-PDC-ThAdhE. FIG. 40D presents a schematic representation of the plasmid pVZ321b-PntcA-PDC-ThAdhE. FIG. 40E presents a schematic representation of the plasmid pVZ321b-PnblA-PDC-ThAdhE.
In order to create expression constructs as described above (point 1) but with an alternative pyruvate decarboxylase to the Zymomonas mobilis enzyme, the Pdc encoding sequence was cut out by EcoRI/SacI digestion of the corresponding pVZ-Pxxx-pdc/adh construct (xxx for isiA, ntcA, nblA). The pdc sequence from Zymobacter palmae containing the restriction sites EcoRI/SacI (introduced by used primer) was ligated into the “pdc free” pVZ construct resulting in a construct that express the Pdc from Zymobacter palmae together with the preexisting Adh.
FIG. 41A presents the nucleotide sequence for ZpPdc (pdc gene from Zymobacter palmae ATCC 51623), and the amino acid sequence for this protein (ZpPdc protein sequence AAM49566) is presented in FIG. 41B.
Construction of Chromosome Integrative pSK-Vectors
In order to create plasmids for stable chromosome integration in cyanobacteria the whole inserts from the described pVZ constructs (point 1 and 3) containing the promoter sequence and the coding region of the ethanologenic enzymes (Pdc and Adh) were cut out by SalI/PstI digestion. The resulting inserts were ligated into the pSK10, a derivate of the pSK9 (a kind gift of V. V. Zinchenko and described in Dühring et al., submitted 16th of December 2007, Plant Physiology) using the SalI/PstI restriction sites. In some cases other restriction sites were used, e.g. in case of pVZ321b-Pxxx-pdc-adhI the restriction sites XbaI/PstI were used, in case of pVZ321b-Pxxx-Ecdhe the restriction sites XbaI/BamHI were used.
FIG. 42A presents the nucleotide sequence of the pSK10 cloning vector (derivate of pSK9 [V. V. Zinchenko, Moscow, Russia; unpublished]). FIG. 42B presents a schematic representation of this plasmid.
Several pSK10 constructs with ZmPdc/ZmAdhII were obtained.
FIG. 42C presents a schematic diagram of pSK10-PisiA-PDC-ADHII.
FIG. 42D presents a schematic diagram of pSK10-PnblA-PDC-ADHII.
FIG. 42E presents a schematic diagram of pSK10-PntcA-PDC-ADHII.
Several pSK10 constructs with ZmPdc/ZmAdhI were obtained.
FIG. 42F presents a schematic diagram of pSK10-PisiA-PDC-ADHI.
FIG. 42G presents a schematic diagram of pSK10-PnblA-PDC-ADHI.
FIG. 42H presents a schematic diagram of pSK10-PntcA-PDC-ADHI.
Several pSK10 constructs with ZmPdc/SynAdh were obtained.
FIG. 42I presents a schematic diagram of pSK10-PisiA-PDC-SynADH.
FIG. 42J presents a schematic diagram of pSK10-PnblA-PDC-SynADH.
FIG. 42K presents a schematic diagram of pSK10-PntcA-PDC-SynADH.
Several pSK10 constructs with EcAdhE were obtained.
FIG. 42L presents a schematic diagram of pSK10-PisiA-PDC-EcAdhE.
FIG. 42M presents a schematic diagram of pSK10-PnblA-PDC-EcAdhE.
FIG. 42N presents a schematic diagram of pSK10-PntcA-PDC-EcAdhE.
Several pSK10 constructs with ThAdhE were obtained.
FIG. 42O presents a schematic diagram of pSK10-PisiA-PDC-ThAdhE.
FIG. 42P presents a schematic diagram of pSK10-PnblA-PDC-ThAdhE.
FIG. 42Q presents a schematic diagram of pSK10-PntcA-PDC-ThAdhE.
Expression of Pdc and Adh in the Filamentous; Diazotropic Cyanobacteria Nostoc/Anabaena Spec. PCC7120 and Anabaena variabilis ATCC 29413
In order to generate ethanol producing Anabaena strains, different constructs were created for conjugation into Anabaena PCC7120 and Anabaena variabilis ATCC29413.
Nostoc/Anabaena spec. PCC7120 and Anabaena variabilis ATCC 29413 were transformed using Self-replicating plasmids.
The ethanologenic genes were cloned into self-replicating plasmids for conjugation into Anabaena PCC7120. In these constructs different promoters were used to control expression of pdc and adhII.
pRL1049 Constructs
Genes encoding pdc and adhII from Zymomonas mobilis were cloned into the self-replicating plasmid pRL1049, which is known to replicate in Nostoc strains. Nucleotide and amino acid sequences of adhII and pdc genes from Zymomonas mobilis are already described herein.
The promoter-pdc-adhII fragment was cut out of the herein described pSK10-PpetJ-pdc-adhII plasmid with ClaI and BamHI and ligated into pRL1049. Promoter sequences were exchanged via EcoRI and SalI. Different promoters were used: promoters originating from PCC 6803: PisiA, PpetJ and PrbcL (nucleotide sequences are already described herein) and promoters originating from PCC 7120: PcrhC and PpetE.
Promoter sequences of PcrhC and PpetE are shown in FIGS. 42R and 42S, respectively (SalI and EcoRI restriction sites for cloning are marked in bold letters):
FIG. 42R depicts the crhC promoter (Anabaena sp. PCC7120) (crhC gene: alr4718, RNA helicase crhC cold shock inducible)
FIG. 42S shows the petE promoter (Anabaena sp. PCC7120) petE gene: all0258, plastocyanin precursor (petE) induced by addition of Cu
The structure of plasmid pRL1049-PpetE-PDC-ADHII is shown in FIG. 42T.
The sequence of the plasmid pRL1049-PpetE-PDC-ADHII is shown in FIG. 42U.
pRL593 Construct
In addition to pRL1049 the broad range plasmid pRL593 was used for expression of pdc and adhII in Anabaena PCC7120. The structure of plasmid pRL593-PisiA-PDC-ADHII is presented in FIG. 42V and the DNA sequence is depicted in FIG. 42W.
EtOH Production in Anabaena PCC7120 Harboring Self-Replicating Plasmid pRL593-PisiA-PDC-ADHII
EtOH production in Anabaena PCC7120 harboring pRL593-PisiA-PDC-ADHII following induction by iron starvation was measured in BG11 medium (+N) and in medium lacking combined nitrogen (−N) in day (12 h)/night (12 h) cycle. The results of this measurement is presented in FIGS. 42X and 42Y.
Ethanol production in medium +N appeared higher than under condition lacking combined nitrogen (−N); but this effect was not very pronounced when calculated per OD750 nm.
The best EtOH production rate in Anabaena PCC7120/pRL593-PisiA-pdc-adhII achieved was 0.0076% EtOH per day, constant for 19 days. This rate is lower compared to Synechocystis strains expressing pdc-adhII under control of PisiA, but continues for a longer time.
Characterization of Generated Ethanologenic Synechocystis cyanobacteria 
P.1 Experimental Data for Characterization of Genetically Modified Photoautotrophic Host Cells Containing at Least One Second Genetic Modification
Expression Levels of ZmPdc/ZmAdhII in Generated Synechocystis Cyanobacterial Mutants:
In order to quantify the induction rate of the used promoters, Pdc/AdhII protein levels in cultures with and without nutrient starvation were estimated by Western blot analysis.
In the case of the mutant with the isiA-promoter cultures were grown with and without addition of iron for about 48 hours. In the case of the mutants with the ntcA- and nblA-promoter cultures were grown with and without addition of nitrogen to the media. To get more comparable signals in the immunodetection, from the cultures under induced conditions, different dilutions of the prepared crude extracts were used.
Activities of ZmPdc/ZmAdhII in Cyanobacterial Mutants:
In order to compare the enzymatic activities of Pdc/AdhII with the estimated expression level, activities of Adh and Pdc were measured in crude extracts of the corresponding cultures.
In the case of the mutant with the isiA-promoter, cultures were grown with and without addition of iron for about 48 hours. The mutant with the ntcA-promoter was grown in standard BG11. Estimated activities were calculated on the corresponding protein concentration of the used crude extracts. It should be noted that Pdc activities were estimated in the presence of added thiamine pyrophosphate (cofactor for Pdc enzyme).
Results are presented in FIGS. 43A and 43B.
Ethanol Generation Rates in Cyanobacterial Mutants:
In general the inducible promoters used therein can be induced by medium exchange or by letting the cyanobacterial mutants grow into starvation conditions in the case of promoters which are inducible by nutrient starvation for example iron or copper starvation.
The use of inducible promoters for the over-expression of ethanologenic enzymes in cyanobacteria allow for switch on or switch off ethanol production on demand. Several promoters that are used for this purpose are inducible by the nutrient status, e.g. iron or copper availability. To reach these inducible conditions either a medium exchange or growth into these starvation conditions are possible.
Induction by Medium Exchange:
Mutants and Synechocystis wild-type strains were grown at 28° C., under constant light (50 μE m-2 s-1) either on a shaker (100 rpm) or in aerated culture vessels, bubbled with CO2-enriched air (0.5% CO2). The initial OD750 was between 2 and 3 in a total culture volume of 50 ml in Erlenmeyer flasks or 100 ml in the aerated culture vessels.
When an optical density of 2-3 was reached the culture was harvested by centrifugation and the supernatant was discarded. The cell pellet was washed with the new medium (e.g. without iron, without copper, without nitrate and thereafter resuspended in the respective medium for promoter induction. If iron starvation is needed (isiA-promoter) the ferric ammonium citrate in the BG11 was omitted, in the case of copper starvation (petJ-promoter) the trace metal mix used was prepared without addition of copper sulfate, for nitrogen starvation the sodium nitrate in the BG11 was omitted.
Induction by Letting the Cultures Grow into Starvation Conditions:
Promoter induction by growing into starvation is based on the consumption of nutrients due to the nutrient demand of a culture. After nutrients are consumed the culture enters the starvation condition which leads to the induction of the appropriate promoter. The duration to reach such a starvation condition can be influenced/limited by reduction of the amount of the respective nutrient in the BG11 medium, e.g. 1⁄3 of the Ferric ammonium citrate or copper sulfate concentration.
Furthermore, for repression of the nirA-promoter ammonia (0.265 g/l corresponds to 5 mM NH4Cl) was added to the BG11 medium, which already contains nitrate. The culture induces itself by consuming the ammonia as a preferred nitrogen source at first (nirA promoter not induced) and upon complete consumption of ammonia starts consuming the nitrate accompanied with induction of the nirA-promoter.
BG11 Media Recipe:
NaNO3: 1.5 g
K2HPO4: 0.04 g
MgSO4.7H2O: 0.075 g
CaCl2.2H2O: 0.036 g
Citric acid: 0.006 g
Ferric ammonium citrate: 0.006 g
EDTA (disodium salt): 0.001 g
NaCO3: 0.02 g
Trace metal mix A5 1.0 ml (see below)
Distilled water: 1.0 L
Trace Metal Mix A5:
H3BO3: 2.86 g
MnCl2.4H2O: 1.81 g
ZnSO4.7H2O: 0.222 g
NaMoO4.2H2O: 0.39 g
CuSO4.5H2O: 0.079 g
Co(NO3)2.6H2O: 49.4 mg
Distilled water: 1.0 L
P.8 Ethanol Production Rates of Genetically Modified Photoautotrophic Host Cells Containing Ethanologenic Enzymes Under the Transcriptional Control of Various Inducible Promoters
In this section several natural occurring promoters from Synechocystis were analyzed for their suitability to express the Pdc enzyme in Synechocystis. In Tab. 1 an overview of the chosen promoters with their characteristics is shown. For all these promoters corresponding mutants in Synechocystis PCC6803 were already created and characterized. This section reports only a summary of the best embodiments.
FIG. 49A shows a summary of the cyanobacterial promoters used to express ethanologenic enzymes in Synechocystis 6803. Characteristics were taken from the literature, mainly analyzed and described for the cyanobacterium Synechocystis 6803.
Mutant Generation:
From a preexisting pVZ plasmid (pVZ321b-PisiA-PDC/ADHII) containing Pdc/Adh genes from Zymomonas mobilis the respective promoter fragment (PisiA) was cut out by SalI/EcoRI digestion and subsequent ligation of a new promoter fragment into the residual plasmid leading to a new pVZ321b-Pxxx-PDC/ADHII derivate with exchanged promoter xxx. Mutants were selected on streptomycin plates and grown in BG11 medium containing the appropriate antibiotics (kanamycin 100 mg/l; streptomycin 10 mg/l).
Growth Conditions:
Cultures were grown in BG11 in continuous light (50-100 μE) either on a shaker in 100 ml Erlenmeyer flasks (100 rpm) or in bubbling flasks (200 ml) aerated with CO2-enriched air (0.5%). Depending on the current promoter BG11 without iron or copper was used as well as BG11 without nitrogen or supplemented with 5 mM NH4Cl. Pre-cultures were harvested by centrifugation, the supernatant discarded and the cell pellet resuspended in new medium with or without the specific nutrient, needed for the regarding promoter mutant.
The growth of the cultures was monitored by photo-densitometrical measurements at 750 nm. The ethanol production was determined in the culture supernatant by an optical enzymatic test (Boehringer Mannheim).
Results and Conclusions:
Transconjugants with the isiA-promoter are well growing and as pigmented in the same way as the wild type. Growth experiments reveal that the ethanol formation in the culture strongly depends on the availability of iron (FIG. 1). If iron is present the ethanol production is lower and time-shifted compared to the sub-culture without iron. As described in the literature iron starvation leads to very strong induction of the isiA-promoter. After transition of the cells to iron-free BG11 it needs usual 3-5 days until ethanol formation starts. Western blot analyses revealed that Pdc accumulates within 48 hours past iron depletion (up to 50-fold), but it strongly depends on the growth phase and the iron availability of the pre-culture. By supplementation the growth medium with additional iron (3× Fe) the ethanol production can be disabled for long time and starts very late with a low rate as depicted in FIG. 49C. FIG. 49B shoes the growth of the same culture monitored by determining the OD750. Thus, ethanol production in Synechocystis is excellent adjustable by using the iron depending isiA-promoter.
Until now best production rates were observed for the isiA-promoter. In continuous light about 0.02% (v/v) ethanol and in day/night cycle about 0.014% (v/v) ethanol was produced per day, respectively (for at least 10 days). Since longer iron deficiency limits the photosynthesis rate it is imaginable to use this promoter in a biphasic manner in which after a production period iron is added to regenerate the cells for the next production period. Furthermore auto-induction by stationary growth is a possibility for the application of the isiA-promoter, too.
Transconjugants with the nblA-promoter appear more slowly growing compared to transconjugants with the isiA-promoter and are also a bit more yellowish pigmented than the wild type. Growth experiments reveal that the ethanol formation in the culture depends on the availability of nitrogen as described in the literature for the nblA-promoter. If nitrogen is absent the ethanol production is significant higher compared to the control culture with nitrogen (FIG. 49D). Western blot analyses revealed a fast and strong induction of the Pdc expression after nitrogen starvation. Within 48 hours the Pdc accumulates up to 25-fold compared to control cells (with nitrogen). But the ethanol accumulation in the culture stops after 5-6 days (see FIG. 49D) most likely due to the nitrogen deficiency. Since Synechocystis is not able to fix nitrogen from the atmosphere, nitrogen deprivation leads to a reduction of photosynthesis because of the deficiency of amino acid biosynthesis in the absence of an utilizable nitrogen source. Within some days of nitrogen deprivation photosynthesis decreases significantly. But by using of nitrogen-fixing cyanobacterial species (e.g. Anabaena sp. PCC7120) the application of a nitrogen-dependent promoter like the nblA-promoter might be well suited.
FIG. 49D shows the ethanol production of Synechocystis 6803 pVZ321b-PnblA-PDC/ADH that express Pdc/Adc enzymes under the control of the nitrogen dependent nblA-promoter. Cultures were grown on a shaker in Erlenmeyer flasks in BG11 under continuous light. A pre-culture was divided into 2 sub-cultures (start OD750 nm=2), one with and the other without nitrate.
The next set of promoters consists of three promoters inducible by the nutrient status. Two of them, PpetJ and PpetE are inducible by the copper availability and the third one, PnirA, depending from the nitrogen source, ammonia or nitrate.
According to the literature the nirA-promoter is repressed if ammonia is present and turned on if nitrate is the sole nitrogen source. Furthermore this promoter is described as tight regulated and was already successful used for heterologous gene expression in Synechocystis PCC6803. Transconjugants with the nirA-promoter appear more yellowish compared to the wild type and grow very slowly, if grown on usual BG11 plates. This phenotype is common for strong ethanol producers and is not surprising since the sole nitrogen source of BG11 is nitrate, which switches the nirA-promoter on.
Growth experiments revealed that the ethanol accumulation depends from the nitrogen source (FIGS. 49F and 49G). Without supplementation of ammonia to the BG11, the culture grows more slowly as shown in FIG. 49E and produces at the same time more ethanol. If ammonia is present the ethanol production was significant lower. At the 8th day new ammonia was added to the culture to take care that enough ammonia is present for repression of the nirA-promoter. Due to this elevated ammonia availability the ethanol formation was transiently blocked whereas the reference culture (BG11 without ammonia) continues accumulating ethanol with a similar rate anymore. But already 5 days later most of the new supplemented ammonia is consumed by the cells and the promoter becomes activated and reaches ethanol production rates similar to the reference culture. If the produced ethanol in each sub-culture is normalized to the cell growth (optical density) a clear difference in the ethanol productivity is visible (FIGS. 49F and 49G). The reference culture without ammonia produces at least two times more ethanol per cell compared to the culture supplemented with ammonia.
FIGS. 49E to 49G depict the growth, ethanol production and productivity per growth of Synechocystis 6803 pVZ325PnirA-PDC. Cultures were grown in Erlenmeyer flasks with BG11 medium in continuous light. A pre-culture was divided into two sub-cultures (start OD750nm=3), one with and the other without ammonia supplementation. At the 8th day new ammonia (again 5 mM) was added to the subculture that already contained ammonia.
Thus, in general the nirA-promoter is applicable but in contrast to the literature no tight repression seems to be possible. If the leakiness of the nirA-promoter can be somehow reduced, it is imaginable that in the up-scaling process ammonia can be added to the BG11 to reach fast growth rates and reduced activity of the nirA-promoter. By consuming the ammonia over the time the culture induces itself, but can still grow by using the second nitrogen source, the nitrate that will stimulate the ethanol production. Thus, no medium exchange will be necessary.
Since copper is not essential for photosynthetic growth of Synechocystis (in contrast to iron) promoters of copper-responsible genes are very promising. Well described in the literature are the petJ- and the petE-promoter. The petJ-promoter is switched off if copper is present whereas the petE promoter is switched on. Both promoters have been already applied for heterologous expression in cyanobacteria, the petJ mainly in Synechocystis, whereas the petE was mainly used in Anabaena sp. PCC7120.
Transconjugants with the petJ-promoter show a reduced growth rate compared to wild type and appear also a bit yellowish. This is not surprising, since it is known that the limited copper availability in BG11 medium (0.3 μM) already activates the petJ-promoter to some extent. Growth experiments revealed that the ethanol formation in the culture with different concentrations of copper strongly depends on the availability of copper (see FIG. 49H to 49J). If copper is absent the ethanol production is significant higher compared to the control cultures with 0.3 μM (1×) or 1.5 μM copper (5×) but at the same time the culture without copper grows more slowly. Between 1× and 5× copper also a significant difference in growth and ethanol accumulation is detectable. If copper is added to the culture the growth rate is increased depending on the amount. A control experiment with the wild type was performed in which the growth was documented in dependence of the copper availability. Neither growth improvement nor retardation was detectable for the wild type by various copper concentrations (data not shown). Therefore the faster growth of the mutant at elevated copper concentration is not due to a growth stimulating effect of copper, it is a consequence of the lower ethanol production. The higher the ethanol production rate the lower the growth rate of corresponding mutants. If the ethanol accumulation is calculated per cell (ethanol per OD750nm) strong differences in the productivity were obvious depending on the copper availability (see FIG. 47-6C). Thus, it is possible to adjust the ethanol production and the growth rate by copper supplementation. The petJ-promoter seems to be therefore well suited. Till now best production rates for this promoter are 0.014% (v/v) ethanol per day in continuous light (for about 4 weeks) and about 0.007% (v/v) ethanol in day/night cycles (for about 3 weeks).
FIG. 49H to 49J show the growth, ethanol production and productivity per growth of Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH. Cultures were grown on a shaker in Erlenmeyer flasks in BG11 in continuous light. A pre-culture (1× copper) was divided into 3 sub-cultures (start OD750nm=3) and different concentrations of copper were added.
Since in contrast to Synechocystis PCC6803 for the nitrogen-fixing cyanobacterium Anabaena PCC7120 it was shown that the Anabaena petE-promoter responds to different copper concentrations. Therefore, instead of the Synechocystis promoter the petE-promoter from Anabaena PCC7120 was chosen for the over-expression of Pdc/Adh in Synechocystis. Transconjugants with the petE-promoter are well growing and as pigmented as the wild type when grown on copper-free BG11-plates. Growth experiments reveal that the ethanol formation in the culture depends on the availability of copper (FIG. 49L). If the copper concentration is elevated (5× copper corresponds 1.5 μM) the ethanol production is significant higher and the culture grows more slowly at the same time (compared to the reference culture in copper-free BG11). Thus, the petE-promoter from Anabaena works well for the over-expression of Pdc/Adh in Synechocystis. 
FIGS. 49K and 49L show the growth, ethanol production of Synechocystis 6803 pVZ321b-PpetE-PDC/ADH. Cultures were grown on a shaker in Erlenmeyer flasks with BG11 in continuous light. A pre-culture (1× copper) was divided into 2 sub-cultures (start OD750nm=3) with different concentrations of copper (without and 5× Cu).
The crhC-promoter (cold shock induced RNA helicase) was amplified from the genome of Anabaena PCC7120, since the chrC-gene from Synechocystis seems to be not regulated by the temperature or alternatively exhibit no induction by cold-shock. The Pdc enzyme expression level of the corresponding mutants is relatively low, also when induced by cold-chock. But at least a 3-fold increase in Pdc expression, verified by Western blot analysis, and also an elevated ethanol formation was detectable if the culture was grown at 20° C. (compared to reference culture at 28° C.). Although the crhC-promoter works in general and seems to be adjustable by temperature, this promoter allows only low expression level of ethanologenic enzymes in Synechocystis. However for Anabaena it was shown that the crhC-promoter works well. Therefore it might be possible that the crhC-promoter works more efficient by using other cyanobacterial species.
FIG. 49M shows the ethanol production of Synechocystis 6803 pVZ321b-PcrhC-PDC/ADH. Cultures were grown on a shaker in Erlenmeyer flasks in BG11 under continuous light conditions at 20° C. and 28° C.
Further multi-stress responsible promoters, the htpG-promoter (heat shock protein 90), the hspA-promoter (small heat shock protein A), the clpB1-promoter (clp protease, HSP100) and the hliB-promoter (high-light inducible protein B, HLIP) were analyzed in order to test their suitability for over-expression of ethanologenic ORFs in Synechocystis 6803.
All four mutants showed different degrees in growth retardation and yellow pigmentation if grown on a plate. Strongest yellow pigmentation and most slowly growth were observed for the mutants with the hspA-promoter, followed by the htpG, the hliB and the clpB1-promoter.
The growth experiment revealed that the mutant with the hspA-promoter was most productive till the 10th day concerning the ethanol formation, but grows more slowly compared to the three other mutants (FIGS. 49N and 49O). But after 10 days of cultivation the ethanol accumulation decreases compared to mutants with the htpG- and the hliB-promoter which show a comparable ethanol accumulation.
FIGS. 49N and 49O show the growth, ethanol production and productivity per growth of Synechocystis 6803 pVZ321b-PhspA-PDC, pVZ321b-PhtpG-PDC, pVZ321b-PhliB-PDC and pVZ321b-Pc1pB1-PDC. Cultures were grown in a culture vessel in BG11 in continuous light, bubbled with CO2 enriched air (0.5%).
If for these four mutants the ethanol production is normalized to the culture growth the first observation or rather the first assumption about the strength of each promoter (different degree of yellow pigmentation and growth retardation indicates) can be clearly confirmed. The hspA-promoter seems to be most active in this set of multi-stress responsible promoters. The htpG- and the hliB-promoter exhibit a quite similar expression level, but the expression level of hliB-promoter can be additional elevated by increasing the light intensity. The clpB1-promoter exhibit the lowest expression in this selection of promoters, probably too low for commercial application. Further tests are necessary to elucidate the full performance of these kind of promoters, since no stress conditions were tested which might increase the observed expression level additionally. It is noteworthy that cultivation of the mutant with the hspA-promoter revealed production rates of about 0.015% (v/v) ethanol per day in continuous light and about 0.01% (v/v) ethanol in day/night cycles (both for about 2 weeks) that is comparable to the maximal expression level of mutants with the isiA- and petJ-promoter.
Multi-stress inducible promoters are especially of interest because of their potential to respond to ethanol or side effects the ethanol production (probably indirect). In this case some kind of auto-induction or self-enhancement is imaginable, which might be advantageous, e.g. in combination with other promoters.
It can be summarized that the genome of Synechocystis contains several promoters useful for the ethanol production process. Well working examples are the isiA-, petJ- and the petE-promoter as well as the nirA-promoter, which are all adjustable by the nutrient status. Furthermore the hspA and the htpG as well as the hliB-promoter appear to be suited for the production process.

## Claims

1. A genetically modified cyanobacterial host cell which produces ethanol (C2H5OH) and oxygen (O2) comprising:
(a) a first genetic modification which comprises transforming the cyanobacterial host cell with a nucleic acid encoding a protein with ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity; and
(b) a second genetic modification which comprises transforming the cyanobacterial host cell with a nucleic acid encoding a pyruvate decarboxylase enzyme associated with the formation of ethanol.

2. The genetically modified cyanobacterial host cell of claim 1 wherein the production of C2H5OH per O2 produced with the first and second modifications is higher than without the first genetic modification but with the second modification.

3. The genetically modified cyanobacterial host cell of claim 1 wherein the protein with RubisCO activity comprises a large subunit, a small subunit and a RubisCO chaperonin.

4. A genetically modified cyanobacterial host cell which produces C2H5OH and O2 comprising:
(a) a first genetic modification which comprises transforming the cyanobacterial host cell with a nucleic acid encoding a protein with RubisCO activity; and
(b) a second genetic modification which comprises transforming the cyanobacterial host cell with an alcohol dehydrogenase gene and a pyruvate decarboxylase gene.

5. A genetically modified cyanobacterial host cell which produces ethanol and oxygen comprising:
(a) at least one first genetic modification which results in an increase in the level of a first metabolic intermediate in the genetically modified cyanobacterial host cell compared to the level of said first metabolic intermediate in the corresponding cyanobacterial host cell lacking the first genetic modification, wherein said first genetic modification comprises transforming the cyanobacterial host cell with a nucleic acid encoding a protein with RubisCO activity; and
(b) at least a second genetic modification which comprises transforming the cyanobacterial host cell with a nucleic acid encoding a pyruvate decarboxylase enzyme for the formation of ethanol.

6. The genetically modified cyanobacterial host cell of claim 5 wherein the first metabolic intermediate is pyruvate.

7. The genetically modified cyanobacterial host cell of claim 1, wherein:
(a) the nucleic acid encoding the protein with RubisCO activity encodes a small subunit and a large subunit of a RubisCO;
(b) the nucleic acid encoding the protein with RubisCO activity encodes a small subunit, a large subunit and a chaperonin of a RubisCO; or
(c) the cyanobacterial host cell has been transformed with a conjugative, self-replicating pVZ plasmid containing a RubisCO (rbcLXS) operon and a Z. mobilis pyruvate decarboxylase gene.

8. The genetically modified cyanobacterial host cell of claim 1, wherein the protein with RubisCO activity is from a cyanobacterial cell.

9. The genetically modified cyanobacterial host cell of claim 8, wherein the nucleic acid encoding the protein with RubisCO activity has at least 95% sequence identity to the Synechocystis rbcL-rbcX-rbcS operon of SEQ ID NO: 65.

10. The genetically modified cyanobacterial host cell of claim 9, wherein the rbcL gene encodes a RubisCO large subunit having an amino acid sequence at least 95% identical to SEQ ID NO: 66; the rbcS gene encodes a RubisCO small subunit having an amino acid sequence at least 95% identical to SEQ ID NO: 68; and the rbcX gene encodes a RubisCO chaperonin having an amino acid sequence at least 95% identical to SEQ ID NO: 67.

11. A method for the production of ethanol comprising:
(a) providing the genetically modified cyanobacterial host cell of claim 1;
(b) growing the genetically modified cyanobacterial host cell of (a) under conditions whereby ethanol is produced; and
(c) isolating the ethanol produced in step (b).

12. A method for the production of ethanol comprising:
(a) providing the genetically modified cyanobacterial host cell of claim 4;
(b) growing the genetically modified cyanobacterial host cell of (a) under conditions whereby ethanol is produced; and
(c) isolating the ethanol produced in step (b).