# Patent

## Title
Reagents and methods for cyanobacterial production of bioplastics and biomaterials

## Abstract

The present invention provides reagents and methods for biomaterial production from cyanobacteria.

## Background
CROSS REFERENCE
This application is a continuation of U.S. application Ser. No. 13/911,814 filed Jun. 6, 2013, which is a divisional application of Ser. No. 12/664,433 filed Mar. 24, 2010, now U.S. Pat. No. 8,465,965 issued Jun. 18, 2013, which claims priority under 35 USC 371 of PCT application serial number PCT/US2008/068623 filed Jun. 27, 2008, which claims priority to U.S. Patent Application Ser. No. 60/937,400 filed Jun. 27, 2007, incorporated by reference herein in its entirety.


BACKGROUND OF THE INVENTION
In the coming decades, renewables are expected to gradually replace petrochemical-based industrial products, including polymers. Production of plastics from biopolymers offers the potential to replace non-renewable materials derived from petroleum with renewable resources, resulting in reliable (domestic) supplies, jobs in rural communities, sustainable production, lower greenhouse gas production, and competitive prices.
In response to an increased awareness of global environmental problems, PHA (Polyhydroxyalkanoates (PHA)) is gaining serious attention as a potential substitute for non-biodegradable polymers. The current rise of the oil and natural gas prices is reflected in the plastics market, and is making renewable bioplastics more competitive. However, prices of raw materials for the production of bioplastics based on bacterial fermentation are also increasing.
According to some reports, the cost of production of bioplastics by bacterial fermentation, especially when energy and materials consumed for the production of fertilizers, pesticides, transport, and process energy are factored in, is higher than that of photosynthetically produced plastics, for which no raw material and fossil-fuel energy is required and that take up CO2 from the environment. Thus, biopolymers produced from autotrophic cyanobacteria that generate their own fixed-carbon sources are likely to have an ever-increasing advantage over the production of biopolymers by fermentation.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides methods for producing biomaterials, comprising:
(a) culturing cyanobacterial host cells that are deficient in Slr1125 expression;
(b) harvesting the cyanobacterial host cells; and
(c) preparing biomaterials from the harvested cyanobacterial host cells.
The methods of the invention can be used, for example, to produce quantities of biomaterials not previously possible using known cyanobacterial host cells. In one embodiment the cyanobacterial host cells have been genetically engineered to reduce or eliminate Slr1125 expression. In another embodiment, the biomaterials comprise biomaterials selected from the group consisting of polyhydroxyalkanoates (PHA) and cyanophycin. In a further embodiment, the cyanobacterial host cells are selected from the group consisting of Synechocystis, Arthrospira maxima, Synechococcus, Trichodesmium; and Crocosphaera. In another embodiment, the cyanobacterial host cells are Synechocystis sp. PCC 6803 cells. In various further embodiments, the cyanobacterial host cells have been recombinantly engineered to delete the slr1125 gene; the cyanobacterial host cells have been genetically engineered to overexpress NAD synthetase and/or NAD+ kinase; the cyanobacterial host cells are deficient in cyanophycin production; the cyanobacterial host are deficient in expression of Slr1993; the cyanobacterial host cells are deficient in cyanophycinase expression, and the cyanobacterial host cells are recombinantly engineered to reduce or eliminate expression of one or more of Slr1994, Slr1829, and Slr1830.
In a further aspect, the present invention comprises isolated recombinant nucleic acids, comprising:
(a) a first nucleic acid comprising an inducible cyanobacterial promoter; and
(b) a second nucleic acid operably linked to the first nucleic acid, wherein the second nucleic acid encodes an inhibitory nucleic acid complementary to a target nucleic acid sequence that encodes an amino acid sequence of a polypeptides selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. The isolated nucleic acids of the invention can be used, for example, to down-regulate expression of Slr1125 in cyanobacterial cells, which is useful, for example, in producing biomaterials according to the methods of the present invention. In one embodiment, the inhibitory nucleic acid comprises and antisense nucleic acid. In another embodiment, the target nucleic acid sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. In a further embodiment, the inducible cyanobacterial promoter comprises SEQ ID NO:9. In a further embodiment, the isolated nucleic acid further comprises a third nucleic acid that encodes an NAD synthetase protein selected from the group consisting of SEQ ID NO:10, 11, and 12. In another embodiment, the third nucleic acid consists of a NAD synthetase coding sequence according to SEQ ID NO:13, 14, or 15. In another embodiment, the isolated nucleic acids of the invention are provided in a vector for replication and expression.
In another aspect, the present invention provides recombinant host cells comprising isolated nucleic acids or expression vectors according to the invention. Such host cells can be used, for example, to produce large amounts of the isolated nucleic acids of the invention, of to carry out the methods of the invention. In one embodiment, the host cell is a cyanobacterial host cell; in another embodiment, the recombinant host cell is a bacterial host cell. In a further embodiment, the recombinant cyanobacterial host cell is selected from the group consisting of Synechocystis, Arthrospira maxima, Synechococcus, Trichodesmium; and Crocosphaera. In another embodiment, the recombinant cyanobacterial host cell is a Synechocystis PCC 6803 cell. In a further embodiment, the recombinant nucleic acid is chromosomally integrated into the cyanobacterial genome. In various further embodiments, the cyanobacterial host cells have been recombinantly engineered to delete the slr1125 gene; the cyanobacterial host cells have been genetically engineered to overexpress NAD synthetase and/or NAD+ kinase; the cyanobacterial host cells are deficient in cyanophycin production; the cyanobacterial host are deficient in expression of Slr1993; the cyanobacterial host cells are deficient in cyanophycinase expression, and the cyanobacterial host cells are recombinantly engineered to reduce or eliminate expression of one or more of Slr1994, Slr1829, and Slr1830.
In a further aspect, the present invention provides recombinant cyanobacterial host cell, comprising:
(a) a deficiency in Slr1125 expression; and
(b) one or more of the following recombinantly generated phenotypes:

    
    
        (i) a deficiency in cyanophycin production;
        (ii) a deficiency in poly-β-hydroxyalkanoate (PHA) production;
        (iii) overexpression of NAD synthetase;
        (iv) overexpression of NAD+ kinase;
        (v) deficiency in cyanophycin synthetase expression;
        (vi) deficiency in Slr1993 expression;
        (vii) deficiency in cyanophycinase expression;
        (viii) deficiency in PHB production;
        (ix) deficiency in Slr 1994 expression;
        (x) deficiency in Slr 1829 expression; and
        (xi) deficiency in Slr 1830 expression.
    
    


Such host cells can be used, for example, to carry out the methods of the invention. In one embodiment, the recombinant cyanobacterial host cell has been genetically engineered to reduce or eliminate Slr1125 expression. In another embodiment, the cyanobacteria is selected from the group consisting of Synechocystis, Arthrospira maxima, Synechococcus, Trichodesmium; and Crocosphaera. In a further embodiment, the recombinant cyanobacterial host cell is a Synechocystis sp. PCC 6803 cell.
These aspects and embodiments of the invention are described in more detail below, each of which can be combined except where the context of the specification clearly indicates otherwise.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a flow chart for PHB biosynthesis in Synechocystis. 
FIG. 2 is a schematic diagram of an exemplary recombinant nucleic acid construct that can be used, for example, to create a stable transfected cyanobacterial strain for PHA production.
FIG. 3 is a schematic diagram of a recombinant nucleic acid that is effective for creating a stable cyanophycin-producing cyanobacterial strain.
FIG. 4 is a schematic diagram of a recombinant nucleic acid suitable for production of 3-hydroxybutyrate.



DETAILED DESCRIPTION OF THE INVENTION
Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.)
In one aspect, the present invention provides recombinant nucleic acids, comprising:
(a) a first nucleic acid comprising an inducible cyanobacterial promoter; and
(b) a second nucleic acid operably linked to the first nucleic acid, wherein the second nucleic acid encodes an inhibitory nucleic acid complementary to a target nucleic acid sequence that encodes an amino acid sequence of one or more polypeptides selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
The recombinant nucleic acids of this first aspect of the invention can be used, for example, as constructs for producing recombinant cyanobacteria to control Slr1125 (or orthologue thereof) production. The Slr1125 protein is a carotenoid glycosyl transferase involved in the biosynthesis of myxoxanthophyll and the recombinant nucleic acids of the invention can be used, for example, to produce bioplastics (such as polyhydroxyalkanoates (“PHA”), which include, for example, PHBs as discussed below) and cyanophycin in larger quantity in cyanobacteria than previously possible, based at least in part on the induction of cell conversion into granule formation and biopolymer production by Slr1125 down-regulation, due to interruption of the carotenoid biosynthesis/degradation pathway.
SEQ ID NO:2 is the amino acid sequence of Synechocystis sp. PCC 6803 Slr1125. The filamentous cyanobacterium Trichodesmium erythraeum IMS 101 has an Slr1125 orthologue (SEQ ID NO:8), with 55% identity at the amino acid level, while the Crocosphaera watsonii WH 8501 (previously known as Synechocystis sp. WH 8501) Slr1125 apparently has been split up into two open reading frames, recited in SEQ ID NO:4 and SEQ ID NO:6.
Thus, the recombinant nucleic acids incorporating a second nucleic acid encoding an inhibitory nucleic acid complementary to a nucleic acid sequence encoding one or more of the Slr1125 orthologues can also be used to produce bioplastics in larger quantity in cyanobacteria than previously possible.
As used herein, “recombinant nucleic acids” are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences. Such recombinant nucleic acid sequences may comprise additional sequences useful for promoting expression of the inhibitory nucleic acid, or any other useful signals.
The term “operably linked” refers to the association of the first and second nucleic acids in a single recombinant nucleic acid so that the expression of the second nucleic acid is activated by the first nucleic acid. Thus, the first nucleic acid is operably linked to the second nucleic acid when it is capable of affecting the expression of the second nucleic acid.
As used herein, the term “expression” refers to the transcription of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
The term “inhibitory nucleic acid” means any type of nucleic acid that could inhibit expression (transcription or translation), or accumulation of the expression product (RNA or protein), of its target nucleic acid sequence. Such inhibitory nucleic acids include, but are not limited to, antisense nucleic acids, small interfering nucleic acids, ribozymes, and aptamers that bind the target nucleic acid.
“Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes. “Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell.
“Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020).
The term “complementary” is used to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. The inhibitory nucleic acid is complementary to the target nucleic acid over a region large enough to inhibit expression or accumulation of the target nucleic acid expression product. In one embodiment, the inhibitory nucleic acid is complementary to at least 20 contiguous nucleotides of the target nucleic acid; in various further embodiments, the inhibitory nucleic acid is complementary to at least 30, 50, 100, 250, or 500 contiguous nucleotides of the target nucleic acid, or is complementary to the entire nucleic acid sequence of the target nucleic acid.
“Promoter” refers to a DNA sequence capable of controlling the expression of the second nucleic acid. In general, the second nucleic acid is located 3′ to the inducible promoter, although any arrangement that permits an operable linkage of the first and second nucleic acids can be used. “Inducible” means that the promoter does not constitutively activate expression of the second nucleic acid, but allows for regulated expression. The inducible promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
The term “Cyanobacterial promoter” means that the promoter is capable of directing expression of the second nucleic acid in a cyanobacteria (for example, Synechocystis, Trichodesmium, and Crocosphaera), and is not limited to promoters derived from cyanobacteria.
In one embodiment of this first aspect, the target nucleic acid sequence comprises or consists of a nucleic acid selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, which are the coding DNA for SEQ ID NOS: 2, 4, 6, and 8, respectively.
As will be understood by those of skill in the art, the inhibitory nucleic acid can be used in different cyanobacterial species, so long as it has sufficient identity with the target nucleic acid sequence. In a preferred embodiment, the strain from which the inhibitory nucleic acid is derived is used. In prokaryotes, the gene is translated during its transcription, so the RNA is usually connected from one side with the transcription enzymes and the other end has the ribosome attached to it for translation. Therefore, in further embodiments, a full length antisense-RNA molecule is used so it can bind to whatever exposed segment of the RNA molecule during the transcription-translation process, in addition to full binding to the free RNA molecules. These embodiments also ensure the maximum specificity of inhibition.
The first nucleic acid comprises or consists of an inducible cyanobacterial promoter. As used herein, “inducible” means that the expression of the inhibitory nucleic acid from the promoter can be regulated and thus increased or decreased as desired by application of an appropriate stimulus to cells in which the promoter is functional, Any inducible promoter capable of replicating in cyanobacteria can be used, while those inducible in cyanobacteria selected from the group consisting of Synechocystis, Arthrospira maxima Trichodesmium, and Crocosphaera are preferred. In one embodiment, the inducible cyanobacterial promoter comprises a plastocyanin promoter (inducible by copper), according to SEQ ID NO:9, or a functional equivalent thereof. In other embodiments, the inducible cyanobacterial promoter comprises or consists of an inducible promoter as disclosed in US20040157331 or US20020164706.
The recombinant nucleic acids of this aspect of the invention can comprise further functional components as desired for a given application. For example, the constructs can comprise one or more further nucleic acids that encode expression products of interest; such further nucleic acids can be operably linked to the inducible promoter, or can be operably linked to one or more further promoters present in the recombinant nucleic acid. In one such embodiment, the recombinant nucleic acids further comprise a third nucleic acid that encodes an NAD+ synthetase protein (Slr1691) that comprises or consists of the amino acid sequence of SEQ ID NO:10 (Synechocystis), 11 (Crocosphaera Watsonii WH 8501), or 12 Trichodesmium Erythraeum IMS101). In a further embodiment of this first aspect of the invention, the third nucleic acid comprises or consists of a NAD synthetase coding sequence according to SEQ ID NO:13 (Synechocystis), 14 (Crocosphaera Watsonii WH 8501), or 15 (Trichodesmium Erythraeum IMS101).
In another embodiment of this first aspect of the invention, the recombinant nucleic acids further comprise a fourth nucleic acid that encodes Sll1415, the putative NAD+-kinase, which comprises or consist of the amino acid sequence of SEQ ID NO:16 (Synechocystis sp. PCC 6803), 18 Crocosphaera watsonii WH 8501), or 20 (Trichodesmium erythraeum IMS101), together with slr1691. In a further embodiment, the fourth nucleic acid comprises or consists of a NAD+-kinase coding sequence according to SEQ ID NO:17 (Synechocystis sp. PCC 6803)), 19 Crocosphaera watsonii WH 8501)), or 21 (Trichodesmium erythraeum IMS101).
PHA biosynthesis requires NADPH as a cofactor; therefore increasing NAD biosynthesis could enhance PHA production. Thus, the present invention provides cyanobacterial recombinants in which a copy of the NAD synthetase and/or the NAD+-kinase, can be introduced in front of the inducible promoter (such as the copper-controlled promoter), to over express NAD synthetase and/or the NAD+-kinase in coordination with the induction of PHA biosynthesis, thereby increase the availability of the cofactor required for PHA biosynthesis.
The nucleic acids of the first aspect of the invention can further comprise “recombination sequences” for promoting double homologous recombination in a cyanobacterial genome. As used herein, “Double Homologous recombination” means the exchange of DNA fragments between two DNA molecules at two sites of identical nucleotide sequences. Thus, the “recombination sequences” comprise two nucleic acids that flank the regions desired to be recombined into the genome, wherein the recombination sequences are identical to sequences in the cyanobacterial genome that are targeted for insertion of the recombinant nucleic acids of the invention. See, for example, Mes and Stal, Gene. 2005 Feb. 14; 346:163-71; and Mes and Doeleman, J Bacteriol. 2006 October; 188(20):7176-85. In one embodiment, the region of the cyanobacterial genome target for insertion can be a non-protein coding region; in other embodiments, the targeted region of the cyanobacterial genome can be a protein-encoding gene to be disrupted, thus providing the ability to generate mutant cyanobacteria that cannot express the disrupted protein-encoding gene, and which also express the recombinant nucleic acids of the invention. Further examples of these embodiments are provided below.
The recombinant sequences of any of the embodiments of this first aspect can further comprise sequences to promote replication in an organism of choice. Such sequences are well known in the art. For example, commercially available vectors (plasmid or viral) can be used with such replication capabilities, and the recombinant nucleic acids of the invention can be cloned into the vector. Such replication competent vectors are useful, for example, to produce large quantities of the recombinant nucleic acids of the invention. The organism of choice can be any organism in which replication of the recombinant nucleic acids would be useful, including but not limited to E. coli. 
The recombinant nucleic acids of the first aspect of the invention, and vectors comprising the recombinant nucleic acids of the first aspect of the invention, may further comprise nucleic acid sequences encoding a selectable marker to, for example, facilitate selection of host cells expressing the vector. The recombinant nucleic acids and vectors may contain other promoter sequences and other encoded nucleic acids or polypeptides, as discussed in more detail below, as well as relevant control signals (ie, leader, transcriptional and translational stop signals), and polylinkers for introducing specific restriction sites facilitating ligation in specific regions of the recombinant nucleic acids.
These embodiments of the first aspect of the invention can be combined except where the context of the specification clearly indicates otherwise.
In a second aspect, the present invention provides recombinant host cells that (a) possess chromosomally integrated recombinant nucleic acids of the first aspect of the invention; and/or (b) are transfected with replication competent vectors comprising the recombinant nucleic acids of the first aspect of the invention. Such recombinant host cells can be either prokaryotic or eukaryotic, with prokaryotic host cells preferred. For example, the recombinant host cells transfected with recombinant expression vectors constructed to permit expression in, for example, E. coli, can be used for production of large quantities of the recombinant expression vectors and the recombinant nucleic acids of the invention. Double homologous recombination is discussed above. Recombinant cyanobacterial host cells including, but not limited to, cyanobacteria selected from the group consisting of Chlorococcales (including Synechocystis and Synechococcus, with Synechocystis sp. PCC 6803 and Synechococcus MA19 being preferred), Trichodesmium; and Crocosphaera, and specific strains disclosed in the Examples below, can be used, for example, to produce large quantities of bioplastics, as discussed in more detail below. Transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.). Double homologous recombination for producing recombinant cyanobacterial host cells is discussed above; see also Koksharova and Wolk, Appl Microbiol Biotechnol. 2002 February; 58(2):123-37; and Golden, Methods Enzymol. 1988; 167:714-27.
Recombinant cyanobacteria of the second aspect of the invention produce large amounts of PHA and cyanophycin. In some cases, it may be desirable to produce large amounts of only one of these products to, for example, facilitate isolation of the product of interest. This can be accomplished by further modifying the recombinant cyanobacteria of the second aspect of the invention to produce only one of these two major products. Thus, in various further embodiments of any of the above embodiments, the recombinant cyanobacterial cells of the second aspect of the invention may further be deficient in one of:

    
    
        (a) Cyanophycin expression; or
        (b) poly-β-hydroxyalkanoate (PHA) expression.
    
    


Thus, in one set of further embodiments of the second aspect of the invention, a recombinant cyanobacterial host cell according to the invention is further deficient in expression of cyanophycin. In an alternative set of further embodiments of the second aspect of the invention, any of the recombinant host cells are further deficient in PHA expression. As used herein, “PHA” (also referred to herein as a “bioplastic”, which is a polymer of biological origin) includes any PHA in the cyanobacteria being manipulated, including but not limited to 3-hydroxybutyryl-CoA, and poly(3-hydroxybutyrate) (“PHB”).
Any mechanism for creating the recited deficiency can be used, including but not limited to gene knockouts using double homologous recombination and the construction of recombinant nucleic acids with a promoter operably linked to an inhibitory nucleic acid that is complementary to the expression product of a gene involved in cyanophycin expression (including but not limited to cyanophycin synthetase) or PHA expression (including but not limited to PHA synthetase); exemplary genes are discussed below. If inhibitory nucleic acids are used, the operably linked promoter can be a constitutive or inducible promoter; in either case the recombinant nucleic acid can be linked in a single construct with the recombinant nucleic acids of the first aspect of the invention, or can be constructed as a recombinant nucleic acid separate from the recombinant nucleic acids of the first aspect of the invention.
In one embodiment, recombinant host cells of the second aspect of the invention are deficient in cyanophycin expression; this strain is particularly useful for PHA production. In another embodiment, the cyanophycin expression deficiency results from deletion of the cyanophycin synthetase gene from Synechocystis (SEQ ID NO:22) in the cyanobacteria. Examples of these embodiments are provided below.
Alternatively, the recombinant host cell of the second aspect of the invention may further comprise an expression vector comprising a nucleic acid construct comprising a promoter sequence operatively linked to a nucleic acid encoding an inhibitory nucleic acid complementary to a target nucleic acid sequence that encodes an amino acid sequence of Synechocystis cyanophycin synthetase (SEQ ID NO:23) (Slr2002). In a further embodiment, the inhibitory nucleic acid is an antisense transcript that comprises at least 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:22 (slr2002). In various further embodiments, the antisense transcript comprises at least a contiguous 30, 50, 100, 250, 500, or the entire nucleic acid sequence of SEQ ID NO:22. In a preferred embodiment, the nucleic acid encoding the antisense transcript is operably linked to the cyanobacterial inducible promoter.
In an alternative embodiment, the recombinant cyanobacterial cells of the second aspect of the invention are deficient in PHA expression, wherein the PHA expression deficiency results from deletion of the slr1993 gene (SEQ ID NO:24; from Synechocystis). This embodiment is particularly useful for cyanophycin production. An example of this embodiment is provided below. Alternatively, the recombinant host cell may further comprise an expression vector comprising a nucleic acid construct comprising a promoter sequence operatively linked to a nucleic acid encoding an inhibitory nucleic acid complementary to a target nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:25 (Slr1993). In a further embodiment, the inhibitory nucleic acid is an antisense transcript that comprises at least 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:24. In various further embodiments, the antisense transcript comprises at least a contiguous 30, 50, 100, 250, 500, or the entire nucleic acid sequence of SEQ ID NO:24. In a preferred embodiment, the nucleic acid encoding the antisense transcript is operably linked to a cyanobacterial inducible promoter, such as the plastocyanin promoter discussed above.
Host cells combining the slr1125 mutant and the PHA deficient mutants can be used, for example, for production/isolation of cyanophycin, since PHA will not be abundantly expressed. In a further embodiment, these host cells may further include a second inhibitory nucleic acid whose expression is under control of the inducible promoter, wherein the second inhibitory nucleic acid is complementary to a target nucleic acid that encodes cyanophycinase (Slr2001; see SEQ ID NO:26 from Synechocystis), which degrades cyanophycin granules. In this embodiment, it is preferred that the second inhibitory nucleic acid targeting cyanophycinase and the inhibitory nucleic acid targeting slr1125 are both under control of the same inducible promoter. The second inhibitory nucleic acid may be an antisense transcript that comprises at least 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:27. In various further embodiments, the antisense transcript comprises at least a contiguous 30, 50, 100, 250, 500, or the entire nucleic acid sequence of SEQ ID NO:27. In an alternative embodiment, the cyanophycinase deficiency results from deletion of the slr2001 gene (SEQ ID NO:27), or orthologue thereof.
PHB Pathway in Synechocystis. 
Polyhydroxybutyrates (PHB), are intracellular reserve materials produced by a large number of bacteria including cyanobacteria. In Synechocystis, three enzymes are involved in the conversion of acetyl-CoA to PHB: beta ketothiolase (Slr1993), acetoacetyl-CoA reductase (Slr1994) and PHB polymerase (Slr1829 and Slr1830; these two ORFs encode two polypeptides which form the PHB heterodimer; absence of one or both is sufficient to completely eliminate PHB polymerase activity). Two acetyl-CoA groups are condensed by beta-ketothiolase to form acetoacetyl-CoA. The acetoacetyl-CoA is then reduced by an NADP-specific reductase to form D(−)-beta-hydroxybutyryl-CoA, the substrate for PHB polymerase.
In further embodiments, the recombinant cyanobacteria of the second aspect of the invention can be rendered deficient for expression of other PHA (such as PHB) biosynthesis pathway gene(s), resulting in a desired PHA pathway end product. These embodiments can preferably be combined with embodiments in which cyanophycin expression is inhibited, thus resulting in recombinant cyanobacteria that produce a desired PHA. A flow chart for PHB biosynthesis in Synechocystis is provided in FIG. 1.
Thus, in various further embodiments, the recombinant cyanobacteria of the second aspect of the invention are further rendered deficient in expression of one or more of:
Slr1994 (SEQ ID NO:28);
Slr1829 (SEQ ID NO:29); and
Slr1830 (SEQ ID NO:30). (Taroncher-Oldenburg et al., Appl Environ Microbiol. 2000 October; 66(10):4440-8; Hein et al., Arch Microbiol. 1998 September; 170(3):162-70)
Those cells rendered deficient in slr1994 expression can be used, for example, to produce acetoacetyl-CoA, which is useful, in one example, for feed stock for other bacteria to produce other desired chemicals; those rendered deficient in one or both of slr1829 and slr1830 can be used, for example, to produce poly(3-hydroxybutyryl-CoA), which can be used to produce 3-hydroxybutyryl-CoA and or 3-hydroxybutyryl monomer which is valuable as feed stock for other bacteria or for direct use as a biofuel.
In one embodiment, the expression deficiency results from deletion of the relevant gene (SEQ ID NO:31, 32, and/or 33) in the cyanobacteria. Examples of this embodiment are provided below. Alternatively, the recombinant cyanobacteria of the second aspect of the invention may further comprise an expression vector comprising a nucleic acid construct comprising a promoter sequence operatively linked to a nucleic acid encoding an inhibitory nucleic acid complementary to a target nucleic acid sequence that encodes an amino acid sequence of one or more of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30. In a further embodiment, the inhibitory nucleic acid is an antisense transcript that comprises at least 20 contiguous nucleotides of the nucleic acid sequence of one or more of SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In various further embodiments, the antisense transcript comprises at least a contiguous 30, 50, 100, 250, 500, or the entire nucleic acid sequence of SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In a preferred embodiment, the nucleic acid encoding the antisense transcript is operably linked to a cyanobacterial inducible promoter, such as the plastocyanin promoter discussed above.
In an alternative embodiment, the recombinant cyanobacterial cells of the second aspect of the invention are deficient in PHA expression, wherein the PHA expression deficiency results from deletion of the slr1993 gene (SEQ ID NO:24), or orthologue thereof. An example of this embodiment is provided below. Alternatively, the recombinant host cell may further comprise an expression vector comprising a nucleic acid construct comprising a promoter sequence operatively linked to a nucleic acid encoding an inhibitory nucleic acid complementary to a target nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:25. In a further embodiment, the inhibitory nucleic acid is an antisense transcript that comprises at least 20 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:24. In various further embodiments, the antisense transcript comprises at least a contiguous 30, 50, 100, 250, 500, or the entire nucleic acid sequence of SEQ ID NO:24. In a preferred embodiment, the nucleic acid encoding the antisense transcript is operably linked to a cyanobacterial inducible promoter, such as the plastocyanin promoter discussed above.
The following are specific examples of recombinant nucleic acid constructs that fall within the scope of the present invention, together with specific uses for such constructs.
FIG. 2 provides a schematic diagram of an exemplary construct that can be used, for example, to create a stable transfected cyanobacterial strain for PHA production. The construct can be cloned into any standard vector for purposes of propagation in, for example, E. coli. Any antibiotic resistance marker suitable for use with the host cells of the invention can be used, including but not limited to chloramphenicol.
Each box represents a segment of DNA. The “N-terminal” and “C-terminal” of slr2002 is an abbreviation for the upstream and downstream sites for double homologous recombination with the genomic slr2002 gene. Slr2002 is the cyanophycin synthetase gene discussed above (SEQ ID NO:22). As will be understood by those of skill in the art, the use of 600 bp of the 5′ sequence of slr2002 (SEQ ID NO:34) or 3′ sequence of slr2002 (SEQ ID NO:35) is exemplary. In various embodiments, 200 or more nucleotides can be used, with a larger number of nucleotides preferred.
Inserting the nucleic acid construct comprising a nucleic acid encoding an antisense-slr1125 operably linked to the copper-controlled petE promoter inside the slr2002 homologous recombination sequences and transfecting a cyanobacterium with the construct via double homologous recombination results in recombinant cyanobacteria that produce large amounts of PHA in the presence of copper, but are unable to synthesize cyanophycin granules, thus facilitating exclusive production and purification of PHA from the host cells without interference from cyanophycin.
FIG. 3 provides a schematic of a recombinant nucleic acid according to the invention that is effective for creating a stable cyanophycin-producing strain for pure cyanophycin production. In this case, the recombination sequences are derived from slr1993 (PHA-specific beta-ketothiolase gene) (SEQ ID NO:24), so a recombinant cyanobacterium transfected with this construct via double homologous recombination is deleted for this gene and does not synthesize PHA. The construct further comprises nucleic acids encoding antisense-slr1125 and also antisense slr2001, the gene for cyanophycinase, which degrades cyanophycin granules (see above), each operably linked to the copper-controlled petE promoter.
Thus, exposure of the cells to copper will result in antisense expression from the construct, which will down regulate cyanophycinase, while antisense-slr1125 will induce the cyanophycin granule formation in the absence of PHB biosynthesis and cyanophycin degradation.
FIG. 4 shows a schematic diagram of a recombinant nucleic acid of the invention suitable for production of 3-hydroxybutyrate. In this case, the recombination sequences are derived from slr1829 (poly(3-hydroxyalkanoate synthase gene) (SEQ ID NO:32)), so a recombinant cyanobacterium transfected with this construct via double homologous recombination is deleted for this gene and does not synthesize PHB.
The construct further comprises nucleic acids encoding antisense-slr1125 and also antisense slr2002, the gene for cyanophycin synthetase (SEQ ID NO: 22), each operably linked to the copper-controlled petE promoter. Thus, exposure of the cells to copper will result in antisense expression from the construct, which will down regulate cyanophycin expression, while antisense-slr1125 will induce the accumulation of 3-hydroxybutyryl acid as a monomer without polymerization due to the absence of a functional slr1829 gene.
These embodiments of the second aspect of the invention can be combined as desired, as well as combined with various embodiments of the first aspect of the invention except where the context of the specification clearly indicates otherwise.
In a third aspect, the present invention provides methods for producing biomaterials, comprising:
(a) culturing cyanobacterial host cells that are deficient in Slr1125 expression;
(b) harvesting the cyanobacterial host cells; and
(c) preparing biomaterials from the harvested cyanobacterial host cells.
The methods of the invention can be used, for example, to produce quantities of biomaterials not previously possible using known cyanobacterial host cells. The cyanobacterial host cells may be naturally deficient in Slr1125 expression, or may be genetically engineered to reduce or eliminate Slr1125 expression. In one embodiment, such engineering comprises deleting the slr1125 gene. In another embodiment, such engineering comprises use of host cells as disclosed above, in which slr1125 is under control of an inducible promoter, such that expression of Slr1125 can be controlled as desired. For example, antisense technology can be achieved using the native copper-controlled plastocyanin promoter with slr1125 in the antisense direction behind it, as discussed above, permitting down-regulating the expression of Slr1125 by adding μM amounts of copper to the medium. This protocol provides a direct and cheap way of controlling slr1125 gene expression.
The inventors have discovered that cyanobacteria deficient in Slr1125 expression are capable of producing large amounts of granules and releasing their granules content, in which a mixture of PHA and cyanophycin are the predominant products, facilitating production of much large amounts of these products than was possible in the art.
All of the embodiments of the recombinant nucleic acids and host cells of the first and second aspects of the invention are equally applicable for use in this third aspect of the invention. As disclosed above, the various embodiments were all based on combinations with the inducible slr1125 inhibitor construct; in this third aspect, all of the various embodiments above are equally compatible, alone or in combination, for use with the slr1125 deletion mutant.
Thus, in one exemplary embodiment, the biomaterials produced comprise biomaterials selected from the group consisting of polyhydroxyalkanoates (PHA) and cyanophycin. In another embodiment, the cyanobacterial host cells are selected from the group consisting of Synechocystis, Arthrospira maxima, Synechococcus, Trichodesmium, and Crocosphaera. In a further embodiment, the cyanobacterial host cells are Synechocystis PCC 6803 cells. In another embodiment, the cyanobacterial host cells have been genetically engineered to overexpress NAD synthetase and/or NAD+ kinase, wherein the culturing comprises culturing the recombinant cyanobacterial host cells under conditions suitable to overexpress NAD synthetase and/or NAD+ kinase.
In a further embodiment, the method comprises preparation of PHA, wherein the cyanobacterial host cells are deficient in cyanophycin production, for example, by recombinantly engineering the cyanobacterial host cells to reduce or eliminate expression of cyanophycin synthetase in the cyanobacterial host cells. In another embodiment, the method comprises preparation of cyanophycin, wherein the cyanobacterial host cells are deficient in PHA production, for example, by recombinantly engineering the cyanobacterial host cells to reduce or eliminate expression of Slr1993 in the cyanobacterial host cells. In another embodiment, the cyanobacterial host cells are deficient in cyanophycinase expression.
In another embodiment, the method comprises preparation of PHA, wherein the cyanobacterial host cells are deficient in production of polyhydroxybutyrates (PHB), for example, by recombinantly engineering the cyanobacterial host cells to reduce or eliminate expression of one or more of Slr1994, Slr1829, and Slr1830. In one embodiment, the cyanobacterial host cells have been genetically engineered to reduce or eliminate expression of Slr1994, and wherein the method comprises production of acetoacetyl-CoA. In another embodiment, the cyanobacterial host cells have been genetically engineered to reduce or eliminate expression of one or both of Slr1829 and Slr1830, and wherein the method comprises production of poly(3-hydroxybutyryl-CoA).
Preparation of nucleic acid constructs and recombinant cyanobacterial host cells according to these various embodiments are described in detail above.
The culture conditions used can be any that are suitable for production of the biomaterials of interest. Exemplary culture conditions for slr1125 deletion mutants are provided in the examples below. A major advantage in using cyanobacteria for bioplastics production is that solar energy provides the energy input. In one example, cyanobacteria can be grown at between 25° C. and 34° C. (for example, Synechocystis sp. PCC 6803 grows between 25° C.-34° C. with optimum temperature (30° C.)), with shaking in a media such as buffered BG-11 medium (40) in the presence of appropriate light conditions, such as between 50 to 200 μmol of photons m−2 s−1, where 50 is low light and 200 is high light. As will be understood by those of skill in the art, large scale production cells can be adapted to different light regimes according to location and the bioreactor specifications, for example, up to 600 μmol of photons m−2 s−1 which is approximately equivalent to a bright sunny day by taking into account the self shading effect of the cells. Light conditions can vary as appropriate for a given purpose, and can be continuous or periodic; for large scale and outdoor cultivation, light/dark cycling is preferred to minimize the cost and avoid extra cost from artificial lighting. Under such conditions, large scale cyanobacterial growth can result in high density cultures.
In one embodiment, granulation is induced during cell growth by substituting ammonia for nitrates as the nitrogen source in the growth medium. Cyanobacteria do not fix nitrogen and thus a nitrogen source is needed in the growth medium; using ammonia as the nitrogen source eliminates the need for cyanobacterial conversion of nitrates to ammonia, limits consumption of NADPH reducing power, and permitting increased NADPH reserves in the cells for granulation and biomaterials biosynthesis during the induction phase. In one embodiment, the amount of ammonia is approximately equimolar to the amount of nitrates in the standard growth medium; in another embodiment, the amount of ammonia ranges from 0.75 g/L to 1.5 g/L; in another embodiment, it ranges from 0.75 g/L to 1.25 g/L; in another embodiment, it ranges from 0.75 g/L to 1.0 g/L. In a further embodiment of any of these granulation embodiments, cells can be grown to maximal density, and granulation induced to maximize production of biomaterials of interest, such as PHB, cyanophycin and 3-hydroxybutyrate. In a further embodiment, the cyanobacterial cells have been recombinantly engineered to inducibly down-regulate expression of the slr1125 gene, such as by inclusion of a copper-inducible promoter, as disclosed above. In a further embodiment, NAD synthetase and/or NAD kinase can be overexpressed in conjunction with sh-1125 down-regulation or deletion. In one non-limiting example, Synechocystis sp. PCC 6803 has a doubling time of 8-10 hours. Therefore, biomass of 4-5 OD730/Liter can be divided into two halves: one can be further grown to allow a continuous supply of cell-biomass, while the other half can be used to stimulate granule production as discussed above.
In a further embodiment, granulation can be induced by including an inhibitor of lycopene cyclase in the growth medium; examples of such inhibitors include, but are not limited to, nicotinic acid (5-50 uM), chlorophenoxytriethylamine (COPTA), 2-(4-chlorophenylthio)-triethylamine (CAPT), 2-(3,4-dichlorophen-oxy)-triethylamine (DCPTA), 2-(3,5-dimethylphenoxy)-triethylamine (DMPTA), 2-(4-methyl-phenoxy)-triethylamine (MPTA), aminotriazole, azasqualene, dodecyltrimethylammonium, N,N-dimethyldodecylamine, imidazole, piperonyl butoxide, piperidine, triethylamine, and pyridine. The use of such inhibitors enhances granule formation and reduces granulation time after the cell culture reaches its maximum density. In one embodiment, the culture media includes nitrates as a nitrogen source; in another embodiment, ammonia is provided as a nitrogen source. Any of the slr1125 constructs can be used with the deletion mutant being preferred for embodiments employing carotenoid biosynthesis inhibitors (e.g. desaturases and cyclases).
Carotenoid is a group of C40 hydrocarbons that is synthesized from polymerization of Isopentenyl pyrophosphate (IPP) with its isomer, dimethylallyl pyrophosphate (DMPP), both are C5 hydrocarbon molecules, through a sequential steps until it form phytoene (C40 molecules). This molecule is the first committed carotenoid molecule synthesized in the carotenoid biosynthesis pathway of the cyanobacterium Synechocystis sp. PCC 6803. Introduction of four double bounds to the phytoene molecule produces a desaturated C40 lycopene by the action of two carotene desaturases enzymes (phytoene desaturase and zeta-carotene desaturase). Lycopene is further cyclized by lycopene cyclase to produce monocyclic (Gamma-carotenel) or dicyclic carotenes (Beta-carotene). In the case of myxoxanthophyll additional enzymes are required to further modify monocyclic carotenoid molecules to produce the glycosylated molecule (sll0254, slr1293 and slr1125), the product of these genes are major enzymes required for the final formation of myxoxanthophyll carotenoid glycoside. The final major carotenoids are further processed to smaller carotenoid products (e.g. retinal group). The inhibitors listed below inhibit one or more of the carotenoid biosynthesis/degradation enzymes and block the biosynthesis of myxoxanthophyll. Therefore, using one or more of these inhibitors with combinations of the host cells of the invention provides additional control to produce and increase biomaterials (such as PHB and cyanophycin) and reduces the granulation time needed for full conversion of cell to granules. In one embodiment, one or more of the inhibitor are used for large scale production of PHB and cyanophycin from cyanobacteria to further improve both quantity and the quality of the final product and minimize the cost. Table 1 provides preferred concentration ranges in culture media for the inhibitors.








TABLE 1






Preferred 


Inhibitors
concentration range







1-COPTA, chlorophenoxytriethylamine,
 5 ug/L-25 ugl/L


2-CAPT (2-(4-chlorophenylthio)-triethylamine);
 5 ug/L-25 ugl/L


3-DCPTA, 2-(3,4-dichlorophen-oxy)-triethylamine
25 ug/L-50 ugl/L


4-DMPTA, 2-(3,5-dimethylphenoxy)-triethylamine
25 ug/L-50 ugl/L


5-MPTA, 2-(4-methyl-phenoxy)-triethylamine
15 ug/L-50 ugl/L


6-Aminotriazole
0.5 mg/L-1.5 mgl/L


7-Azasqualene
 5 ug/L-15 ugl/L


8-Azasqualene
 50 ug/L-250 ugl/L


9-Dodecyltrimethylammonium
 2 ug/L-25 ugl/L


10-N,N-Dimethyldodecylamine
 25 ug/L-250 ugl/L


11-imidazole
250 ug/L-500 ugl/L


12-piperonyl butoxide
 5 ug/L-50 ugl/L


13-piperidine
 50 ug/L-100 ugl/L


14-triethylamine
 50 ug/L-150 ugl/L


15-pyridine
100 ug/L-150 ugl/L









The dramatic change in availability of electrons induced by the various conditions disclosed above, accumulation of NADPH, and change in light due to self shading effects greatly promote increased granule formation in the cyanobacterial host cells of the invention.
Similar culture conditions can be used for recombinant cyanobacteria that carry an inducible promoter linked to an inhibitory nucleic acid whose expression down-regulates expression of the open reading frame of slr1125 (or orthologues thereof), except that appropriate conditions for induction are used when appropriate. The relevant conditions under which to reduce slr1125 expression will be dependent on the inducible promoter used, as well as other factors, including but not limited to the specific cyanobacteria used, cyanobacterial concentration, media, pH, temperature, light exposure, etc. However, those of skill in the art can determine the specific conditions to be used, in light of the teachings herein. In one exemplary embodiment, the inducible promoter comprises the petE promoter (SEQ ID NO:9), and expression of the inhibitory nucleic acid is induced by the addition of μM amounts of copper to the media. (See, for example, (Briggs, et al., 1990)) While those of skill in the art can determine an optimal concentration of copper, ranges between 2 and 10 μM have been used under laboratory conditions. In one embodiment, an antisense slr1125 construct is downstream, and under the control, of the petE promoter. Thus, adding μM amounts of copper to the medium will result in down-regulation of slr1125 expression, granule production, and the ability to harvest the cells and prepare PHA and cyanophycin from the cells.
Harvesting of the cyanobacterial cells can be accomplished by any technique known to those of skill in the art, including but not limited to centrifugation and filtration.
Similarly, methods for preparing biomaterials from the harvested recombinant cyanobacteria can be carried out by any means known in the art, such as those described in the examples below. In one non-limiting embodiment, PHA polyesters can be recovered and purified in a procedure consisting of acidic non-PHA cell mass dissolution, pH adjustment (pH 10), and final decolorization in a bleaching solution. The major product produced by the recombinant cyanobacteria of the invention is a mixture of PHA and cyanophycin. As noted above, in various embodiments, separate cyanobacterial strains (cyanohphycin-deficient and PHA synthetase-deficient) are produced to discriminate between the biosynthesis of these two polymers.
Cyanophycin, a copolymer of L-aspartic acid and L-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. In a further embodiment, the isolated biomaterial comprises cyanophycin, which is then partially hydrolyzed using any suitable method, including but not limited to boiling at high pH, to produce polyaspartate, which is a biodegradable substitute for chemically synthesized polycarboxylate. The latter is an anionic polyelectrolyte, which can be used as a highly effective pigment dispersing agent for use in waterborne industrial, protective coatings, gloss dispersion paints as well as printing inks. It is also used in pigment concentrates used for tinting paints, leather finishes, textiles, plastics, inks, etc. It is used in conjunction with a wide variety of binders such as physically drying acrylic dispersions, air-drying alkyd emulsions, polyester-melamine, 2-pack epoxies, acrylates etc. Therefore, this embodiment of the invention provides a very cost-effective way for cyanophycin (polyaspartate) production to replace toxic-polycarboxylates, and which can also be used as energy and water savers (ie: forming a thin film on water surface of lakes and pools to prevent water evaporation).
Thus, in various embodiments noted above, the recombinant cyanobacteria for use in the third aspect of the invention may further be deficient in cyanophycin expression or PHA expression, as disclosed above. Separation and purification of PHA polymers from non-PHA cell mass presents a technical challenge due to the solid phase of both PHA granules and non-PHA cell mass. The purity, yield, and molecular size are three major factors in PHA recovery. PHA polyesters (such as PHB) can be recovered and purified in a procedure consisting of acidic non-PHA cell mass dissolution, pH adjustment (pH 10), and final decolorization in a bleaching solution. Thus, using cells that are deficient in cyanophycin expression facilitates separation and purification of PHA produced by the recombinant cyanobacteria of the invention. Similarly, using cells that are deficient in PHA synthetase expression facilitates separation and purification of the cyanophycin produced by the recombinant cyanobacteria of the invention. PHA biosynthesis requires NADPH as a cofactor; therefore increasing NAD biosynthesis could enhance PHA production and reduce the time for full conversion to granules. Thus, as discussed above, the present invention provides cyanobacterial recombinants in which a copy of the NAD(+) synthetase can be introduced in front of the copper-controlled promoter to over express in coordination with the induction of PHA biosynthesis, thereby increasing the availability of the cofactor required for PHA biosynthesis.
In one non-limiting example, Synechocystis sp. PCC 6803 has a doubling time of 8-10 hours. Therefore, biomass of 4 OD730/Liter can be divided into two halves: one can be further grown to allow a continuous supply of cell-biomass, while the other half can be used to granule production phase. The continuous supply of the biomass of 4-5 OD730/Liter of cells can be achieved daily using sun light. For example: A plastic bag of 20 cm×100 cm×250 cm (width×length×height) provides a 500 L which yield approximately 1.5 kg/bag/day biomass that converted to 0.75-1.0 kg/bag/day PHA (50%) wt/wt dry biomass. The methods of the invention achieve very high and pure yield that is approximately 80-90% wt/wt dry biomass that provides 1.2-1.35 kg/bag/day PHA, which represents an unprecedented yield for these biomaterials from biologically photoautotrophic organisms to date.
In a fourth aspect, the present invention provides recombinant cyanobacterial host cells, comprising:
(a) a deficiency in Slr1125 expression; and
(b) one or more of the following recombinantly generated phenotypes:

    
    
        (i) a deficiency in cyanophycin production;
        (ii) a deficiency in poly-β-hydroxyalkanoate (PHA) production;
        (iii) overexpression of NAD synthetase;
        (iv) overexpression of NAD+ kinase;
        (v) deficiency in cyanophycin synthetase expression;
        (vi) deficiency in Slr1993 expression;
        (vii) deficiency in cyanophycinase expression;
        (viii) deficiency in PHB production;
        (ix) deficiency in Slr 1994 expression;
        (x) deficiency in Slr 1829 expression; and
        (xi) deficiency in Slr 1830 expression.
    
    


The host cells of this aspect of the invention can be used, for example, to prepare large amounts of biomaterials according to the methods of the invention disclosed above.
Embodiments for generating recombinant cyanobacteria) host cells with any of the recited expression deficiencies or overexpression are disclosed above and are equally applicable for use in this fourth aspect of the invention. In this aspect, the recombinant cyanobacteria) host cell has a deficiency in Slr1125 expression and at least one further altered phenotype from the recited list, which increases the capacity of the recombinant to produce biomaterials, such as PHAs and cyanophycin. In one embodiment, the deficiency in Slr1125 expression may be based on a naturally occurring deficiency. In another embodiment, the host cell is engineered to cause the deficiency, such as by deletion of the slr1125 gene. In another embodiment, such engineering comprises use of host cells as disclosed above, in which slr1125 is under control of an inducible promoter, such that expression of Slr1125 can be controlled as desired, and as disclosed in detail above.
The at least one further recombinant alteration in expression in the cyanobacterial host comprises one or more of the recited alterations, each of which is disclosed in detail above. In various further embodiments, the cyanobacteria is selected from the group consisting of Synechocystis, Arthrospira maxima, Synechococcus, Trichodesmium; and Crocosphaera; in a further embodiment, the recombinant cyanobacteria) host cell is a Synechocystis PCC 6803 cell.
EXAMPLES
Cloning of Slr1125 and Construction of the pΔslr1125S Plasmid—
The Synechocystis sp. PCC 6803 slr1125 gene and its flanking regions were cloned by polymerase chain reaction (PCR) based on the available Synechocystis genomic sequence (CyanoBase; web site: kazusa.or.jp/cyano/cyano.html) (Kaneko et al.). The forward primer was 5′ CTAGAAACGGGAATTCAAGCGGAAT 3′ (SEQ ID NO: 39) with an engineered EcoR I site (underlined) and corresponding to base number 85721-85745 in CyanoBase; the reverse primer was 5′ GTTTAATAGCATGCTTTGCCAGC 3′ (SEQ ID NO: 40) with an engineered Sph I restriction site (underlined) and a sequence corresponding to CyanoBase bases 87845-87867 (base changes to introduce restriction sites have been bolded). The PCR-amplified sequence corresponds to slr1125 with approximately 430-450 bp flanking sequence on both sides of the ORF. A PCR product of the expected size (2.147 kb) was purified, restricted with EcoR I and Sph I (using the introduced restriction sites in the primers) and cloned into pUC 19 creating pslr1125 construct. The slr1125 gene was deleted by restriction at internal Sty I sites near the beginning and end of the slr1125 open reading frame and replacing the Sty I fragment (1.2 kb) by a 1.5 kb streptomycin resistance cassette. This creates the pΔslr1125S construct, which was used for transformation of Synechocystis sp. PCC 6803, carried out according to Vermaas et al. 1987. Transformants were propagated on BG-11/agar plates supplemented with 5 mM glucose and increasing concentrations of up to 300 ug/ml of streptomycin dissolved in sterile water. The segregation state of the transformants was monitored by PCR of transformant DNA using primers recognizing sequences upstream and downstream of the slr1125-coding region. Synechocystis sp. PCC 6803 genomic DNA used for PCR analysis of mutants was prepared as described in He et al. The sequence of the cloned slr1125 ORF with flanking regions up and down stream was verified (SEQ ID NO:41).
Growth Conditions.
Synechocystis sp. strain PCC 6803 was cultivated on a rotary shaker at 30° C. in BG-11 medium (40), buffered with 5 mM N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid-NaOH (pH 8.2). For growth on plates, 1.5% (wt/vol) Difco agar and 0.3% (wt/vol) sodium thiosulfate were added. Flux densities of 40, and 100 μmol of photons m−2 s−1 from cool-white fluorescent tubes were used for growth in continuous light in liquid medium. The normal BG11 media described by Rippka et al. 1979 contains nitrate as a source of nitrogen for the growth of Synechocystis strains; this medium composition provides cell propagation to its maximum limit (3-5 OD730).
Granulation Conditions.
To start the granule induction phase, the cells were diluted to 0.75 OD730 with BG11 medium containing an equimolar amount of ammonia (1 g/L) as a nitrogen source instead of nitrate; this strategy saves approximately 40% of the reducing power of NADPH, which promotes increased granule biosynthesis. Cells were cultures for an additional 48 hours under the same culture conditions discussed above. Under these culture conditions, little further cell growth occurred, as the growth conditions favored large scale granule production.
Extraction of PHB
One of the benefits of the current invention is that once granulation is induced, PHB granules float on the media surface and can be collected directly from the surface in the large scale. A one liter culture of 0.75 OD730 cells was collected by centrifugation to collect everything in the media and precisely analyze the amount of PHB. The collected granules and cell materials were suspended in methanol (4° C., overnight) for the removal of pigments. The pellet obtained after centrifugation was dried at 60° C. and PHB was extracted in hot chloroform followed by precipitation with cold diethyl ether. The precipitate was centrifuged at 11 000 g for 20 min, washed with acetone and dissolved again in hot chloroform.
Spectrophotometric Measurement of PHB.
The spectrophotometric assay was performed as per Law and Slepecky (1961). The sample containing the polymer in chloroform was transferred to a clean test tube. The chloroform was evaporated and 10 ml of concentrated H2SO4 was added. The solution was heated in a water bath for 20 min. After cooling and thorough mixing the absorbance of the solution was measured at 235 nm against H2SO4 blank. To further confirm the presence of PHB, absorption spectra (200-1000 nm) of the sample as well as the standard (dl-β-hydroxybutyric acid, Sigma Chemical Co., USA) were compared by Spectrophotometer following acid digestion. These spectra were further compared with the spectrum of crotonic acid which is the byproduct of acid hydrolysis of PHB and the compound that absorb at 235 nm in case of standard PHB or the PHB from the cell. A total of 200 mg was collected from a 0.75 OD730 culture flask of the slr1125 deletion strain. The absorption at 235 nm of the extracted granules was compared to standard curve generated by β-hydroxybutyric acid, Sigma Chemical Co., USA. 105 mg PHB was measured out of 200 mg cell materials which approximately give 52% PHB/Dry weight of material collected.
REFERENCES


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    Danner R L, Joiner K A, Rubin M, Patterson W H, Johnson N, Ayers K M and Parrillo J E. 1989. Purification, toxicity, and antiendotoxin activity of polymyxin B nonapeptide. Antimicrob Agents Chemother. 1989 September; 33(9): 1428-1434
    Duhring U, Axmann I M, Hess W R, Wilde A. 2006. An internal antisense RNA regulates expression of the photosynthesis gene isiA. PNAS, USA. 103:7054-7058.
    He, Q., Brune, D., Nieman, R., and Vermaas, W. (1998) Eur J. Biochem. 253, 161-172.
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## Claims

1. A recombinant cyanobacterial host cell, comprising:
(a) an insertion or deletion in an endogenous slr1125 gene encoding the protein of SEQ ID NO: 2 or SEQ ID NO: 4, such that the host cell is deficient in expression of the endogenous Slr1125 protein of SEQ ID NO: 2 or SEQ ID NO: 4; and
(b) an insertion or deletion in a gene encoding an endogenous cyanophycin synthetase gene such that the hos cell is deficient in production of endogenous cyanophycin.

2. The recombinant cyanobacterial host cell of claim 1, wherein the cyanobacterial host cell is genetically engineered to delete the endogenous slr1125 gene.

3. The recombinant cyanobacterial host cell of claim 1, further comprising a disruption by insertion or deletion of one or more endogenous genes encoding enzymes selected from the group consisting of poly-β-hydroxyalkanoate synthetase, beta ketothiolase, acetoacetyl-CoA reductase, and poly(3-hydroxybutyrate) polymerase.

4. The recombinant cyanobacterial host cell of claim 1, wherein the cyanobacterial host cell is selected from the group consisting of Synechocystis, Arthrospira maxima, Synechococcus, Trichodesmium; and Crocosphaera.

5. The recombinant cyanobacterial host cell of claim 1, wherein the cyanobacterial host cell is a Synechocystis PCC 6803 cell.

6. A method for producing a biomaterial, comprising:
(a) culturing the cyanobacterial host cell of claim 1;
(b) harvesting the cyanobacterial host cell; and
(c) preparing a biomaterial from the harvested cyanobacterial host cell.

7. The method of claim 6, wherein the cyanobacterial host cell has been genetically engineered to delete the endogenous slr1125 gene.

8. The method of claim 6, wherein the biomaterial is polyhydroxyalkanoate (PHA).

9. The method of claim 6, wherein the cyanobacterial host cell is selected from the group consisting of Synechocystis, Arthrospira maxima, Synechococcus, Trichodesmium, and Crocosphaera.

10. The method of claim 6, wherein the cyanobacterial host cell is Synechocystis PCC 6803.