Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2

Wang, Bo; Pugh, Shawn; Nielsen, David R.; Zhang, Weiwen; Meldrum, Deirdre R.

doi:10.1016/j.ymben.2013.01.001

Cited by 150 publications

(73 citation statements)

References 51 publications

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“…We conclude that in Synechocystis, the quantity of produced PHB is limited not by the activity of the PHB synthase but presumably by the supply of its substrate 3-hydroxybutyrate, which is provided by primary metabolism. This result sets a starting point for further metabolic engineering approaches to divert more carbon from primary metabolism toward this pathway for increased synthesis of PHB, 3-hydroxybutyrate, or 1-butanol as a precursor for fine chemicals (45)(46)(47).…”

Section: Resultsmentioning

confidence: 93%

Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803

Hauf

Watzer

Roos

et al. 2015

Appl Environ Microbiol

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Cyanobacteria are photoautotrophic microorganisms which fix atmospheric carbon dioxide via the Calvin-Benson cycle to produce carbon backbones for primary metabolism. Fixed carbon can also be stored as intracellular glycogen, and in some cyanobacterial species like Synechocystis sp. strain PCC 6803, polyhydroxybutyrate (PHB) accumulates when major nutrients like phosphorus or nitrogen are absent. So far only three enzymes which participate in PHB metabolism have been identified in this organism, namely, PhaA, PhaB, and the heterodimeric PHB synthase PhaEC. In this work, we describe the cyanobacterial PHA surface-coating protein (phasin), which we term PhaP, encoded by ssl2501. Translational fusion of Ssl2501 with enhanced green fluorescent protein (eGFP) showed a clear colocalization to PHB granules. A deletion of ssl2501 reduced the number of PHB granules per cell, whereas the mean PHB granule size increased as expected for a typical phasin. Although deletion of ssl2501 had almost no effect on the amount of PHB, the biosynthetic activity of PHB synthase was negatively affected. Secondary-structure prediction and circular dichroism (CD) spectroscopy of PhaP revealed that the protein consists of two ␣-helices, both of them associating with PHB granules. Purified PhaP forms oligomeric structures in solution, and both ␣-helices of PhaP contribute to oligomerization. Together, these results support the idea that Ssl2501 encodes a cyanobacterial phasin, PhaP, which regulates the surface-to-volume ratio of PHB granules. C yanobacteria are photosynthetic microorganisms capable of oxygenic photosynthesis. ATP and reduction equivalents derived from photosynthetic electron flow are utilized to fix carbon dioxide and generate 3-phosphoglycerate (1). This metabolite can be utilized for either gluconeogenesis or glycolysis, providing the necessary carbon skeletons for biosynthesis of amino acids and other metabolites required for cell growth (2), when growth conditions are suitable and nutrients are abundant. In fact, carbon flux is greatly affected by the availability of macronutrients like nitrogen (3-5) and phosphorus, which may limit growth (6). Under nutrient-limiting conditions, cyanobacteria undergo a stress adaptation process termed chlorosis (7). This process leads to the degradation of light-harvesting complexes, causing reduced photosynthetic activity and thereby reduced metabolic activity (8). Furthermore, carbon flux is redirected toward glycogen synthesis upon macronutrient starvation (5). In addition, some cyanobacterial strains like Synechocystis sp. strain PCC 6803 (referred to here as Synechocystis) accumulate polyhydroxybutyrate (PHB) as a carbon and redox storage compound (9). PHB is synthesized in three biosynthetic steps, and all three enzymes catalyzing the reactions are known (10, 11). The first step involves a condensation of two acetyl coenzyme A (acetyl-CoA) groups to acetoacetyl-CoA by PhaA (slr1993). In the second step, PhaB (slr1994) reduces acetoacetyl-CoA to hydroxybutyryl-CoA, utilizing NADPH...

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Section: Resultsmentioning

confidence: 93%

Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803

Hauf

Watzer

Roos

et al. 2015

Appl Environ Microbiol

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“…On the example of the strain Synechocystis PCC 6803 25 the pathway can be drawn as given in Figure 1. Four enzymes are necessary to synthesize polymeric PHB from Acetyl-CoA.…”

Section: Key Enzymes In Phb Biosynthesismentioning

confidence: 99%

“…Wang et al identified phaEC to be the bottleneck in PHB synthesis and replaced the native genes with the more efficient thioesterase II (TesB) deriving from E. coli. 25 …”

Section: Key Enzymes In Phb Biosynthesismentioning

confidence: 99%

Photo-autotrophic Production of Poly(hydroxyalkanoates) in Cyanobacteria

Drosg¹,

Fritz²,

Gattermayr³

et al. 2015

Chem.Biochem.Eng.Q.

100

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In the last two decades, poly(hydroxyalkanoates) (PHA) were solely produced using heterotrophic bacteria in aerobic cultivation. With respect to the great potential (500 Mt yr -1 ) of raw industrial CO 2 streams and even greater potential of flue gases, the focus on photo-autotrophic biotechnological processes is increasing steadily. Primarily, PHA-gene transfer from heterotrophic bacteria into algae and plant cells was attempted, with the intention to combine the known biosynthesis pathway with autotrophic cultivation. The natural occurrence of PHA in cyanobacteria is known at least since 1966. However, cyanobacteria were never considered for commercial production because the PHA amount based on cell mass and based on volumetric productivity is generally very low. Therefore, strain improvements were suggested, either by gene amplification or by suppression of biochemical pathways competing for the cell's acetate pool. In the late 1990s, the success of genetic modification was confirmed experimentally, elevating the cyanobacteria cell's PHA content. With additional optimization, PHB amounts up to 50 % w/w of biomass dry matter or up to about 2.4 g L -1 bioreactor volume could be produced within 11 days. Considering the land use for agriculture and the competition for plant biomass between food, feed, fuel and energy production, the binding of CO 2 in a biotechnological process using photo-autotrophic microorganisms may become a promising option.

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“…Although mutations engineered at such sites could segregate, disruption of coding DNA sequences may decrease the robustness of the organisms, since evolution has likely selected for the maintenance of the entirety of a genome on the basis of some advantage under some conditions. Other investigators have turned to intergenic regions for inserting genes of interest (33). However, transcriptome-sequencing (RNA-seq) analysis of the genome of Synechocystis PCC 6803 has revealed that many regions of DNA previously thought to be noncoding are actually transcribed with unknown or antisense function (34).…”

mentioning

confidence: 99%

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Berla

Pakrasi

2015

Appl Environ Microbiol

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Cyanobacteria are photosynthetic cell factories that use solar energy to convert CO 2 into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous genes in cyanobacteria are often integrated at presumptively "neutral" chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to identify neutral sites from which no transcripts are expressed. We characterized the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the P cpc560 or the P trc1O promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria.C yanobacteria are oxygenic photosynthetic prokaryotes that use solar energy to fix CO 2 , converting it into biomass and valuable products. Since these organisms do not require fixed carbon feedstocks, they have great potential for synthetic biology and metabolic engineering applications (1-4). Several strains of cyanobacteria have been engineered to act as microbial cellular factories for the production of fuels and chemicals, such as isobutanol, 2,3-butanediol, free fatty acids, and D-lactate (5-7). Despite these advances, none of these engineered strains has been able to achieve industrially relevant levels of productivity. A lack of effective and well-characterized tools for synthetic biology in cyanobacteria has limited progress relative to that with other established microbial chassis, such as Escherichia coli or Saccharomyces cerevisiae (1). Synechocystis sp. strain PCC 6803 is a naturally transformable cyanobacterial chassis for synthetic biology. This strain carries several endogenous plasmids in addition to its single chromosome (8-11). Since there are a limited number of self-replicating exogenous plasmids for the expression of heterologous genes in Synechocystis PCC 6803 (12) and other cyanobacteria, these endogenous plasmids may prove to be attractive parts for synthetic biology (13). Additionally, these plasmids increase their copy numbers during the transition from the exponential-growth phase to the stationary phase (14). Thus, we have hypothesized that the expression of heterologous genes from sites in these plasmids could be autoinduced by such a growth transition. This induction could be advantageous in a production system that first yields a dense culture of light-harvesting cyanobacteria and later uses those microbial cell factories to churn out a product of interest.One strat...

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Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2

Cited by 150 publications

References 51 publications

Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803

Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803

Photo-autotrophic Production of Poly(hydroxyalkanoates) in Cyanobacteria

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

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