**7. Biotechnological production**

Previous ventures to produce CGP in high amounts were mainly focused on heterotrophic bacteria, yeasts and plants as production host. These recombinant production hosts heterologously express CGP synthetase genes, mostly from cyanobacteria. In this way, heterotrophic bacteria, which are established in biotechnological industry including *E. coli*, *Corynebacterium glutamicum*, *Cupriavidus necator* (formally known as *Ralstonia eutropha*) and *Pseudomonas putida*, were used for heterologous production of CGP [93].

Strain *E. coli* DH1, containing *cphA* from *Synechocystis* sp. PCC6803, was used for large-scale production of CGP in a culture volume of up to 500 liter, allowing the isolation of CGP in a kilogram scale. During process optimization, the highest observed CGP content was 24% (w/w) per CDM. However, the synthesis of CGP was strongly dependent on the presence of complex components in the medium (terrific broth complex medium). In mineral salt medium, CGP accumulation only occurs in the presence of casamino acids [27]. An engineered version of CphA from *Nostoc ellipsosporum,* transformed in *E. coli*, shows a further increase in CGP production, up to 34.5% (w/w) of CDM. However, this production strain also requires expensive complex growth media to yield such a high amount of CGP [94].

*Cupriavidus necator* and *Pseudomonas putida* are known as model organisms for the industrial scale production of polyhydroxyalkanoates (PHA). Therefore, they have been considered as candidates for large scale CGP production [93, 95]. Metabolic engineering and process optimization studies of *Cupriavidus necator* and *Pseudomonas putida* harboring *cphA* from *Synechocystis* sp. PCC 6803 or *Anabaena* sp. PCC 7120 were performed. In these organisms, the accumulation of CGP is mainly depending on the origin of the *cphA* gene, the accumulation of other storage compounds like PHA as well as the addition of precursor components like arginine to the medium [96]. PHA-deficient mutants of *Cupriavidus necator* and *Pseudomonas putida* accumulate in general more CGP compared to the PHA containing strains [96]. During genetic modification of *cphA* expression in *Cupriavidus necator*, CGP accumulation turned out to be strongly affected by the expression system. A stabilized multi-copy *cphA* expression system, using the KDPG-aldolase gene (*eda*)-dependent addiction system, allows cultivation without antibiotic selection. The multi-copy *cphA* expression results in a CGP yield between 26.9% and 40.0% (w/w) of CDM. The maximum amount of 40.0% (w/w) of CDM was observed in a 30- and 500-l pilot plant. In the absence of the amino acids arginine and aspartic acid in the medium, the CGP amount was still between 26.9% and 27.7% (w/w) of CDM [97].

CGP can also serve as a source for dipeptides and amino acids in food, feed and pharmaceutical industry. The amino acids arginine (semi-essential), aspartate (non-essential) and lysine (essential) derived from CGP have a broad spectrum of nutritional or therapeutic applications. Large-scale production of these amino acids, as mixtures or dipeptides, is established in industry, with various commercial products already available on the market (reviewed by

Potential applications of non-modified CGP have been discussed but remain so far largely unexplored. This can partially be explained by the lack of research being conducted on the material properties of CGP. Recently in 2017, the first study regarding CGP material properties has been published. In this study, Khlystov et al. focused on the structural, thermal, mechanical and solution properties of CGP produced by recombinant *E. coli*, giving new insights in the nature of this polymer as bulk chemical [91]. They describe CGP as an amorphous, glassy polyzwitterion with high thermostability. The dry material is stiff and brittle. According to these properties, CGP could be used to synthesize zwitterionomeric copolymers or as reinforcing fillers [91].

Previous ventures to produce CGP in high amounts were mainly focused on heterotrophic bacteria, yeasts and plants as production host. These recombinant production hosts heterologously express CGP synthetase genes, mostly from cyanobacteria. In this way, heterotrophic bacteria, which are established in biotechnological industry including *E. coli*, *Corynebacterium glutamicum*, *Cupriavidus necator* (formally known as *Ralstonia eutropha*) and *Pseudomonas* 

Strain *E. coli* DH1, containing *cphA* from *Synechocystis* sp. PCC6803, was used for large-scale production of CGP in a culture volume of up to 500 liter, allowing the isolation of CGP in a kilogram scale. During process optimization, the highest observed CGP content was 24% (w/w) per CDM. However, the synthesis of CGP was strongly dependent on the presence of complex components in the medium (terrific broth complex medium). In mineral salt medium, CGP accumulation only occurs in the presence of casamino acids [27]. An engineered version of CphA from *Nostoc ellipsosporum,* transformed in *E. coli*, shows a further increase in CGP production, up to 34.5% (w/w) of CDM. However, this production strain also

requires expensive complex growth media to yield such a high amount of CGP [94].

*Cupriavidus necator* and *Pseudomonas putida* are known as model organisms for the industrial scale production of polyhydroxyalkanoates (PHA). Therefore, they have been considered as candidates for large scale CGP production [93, 95]. Metabolic engineering and process optimization studies of *Cupriavidus necator* and *Pseudomonas putida* harboring *cphA* from *Synechocystis* sp. PCC 6803 or *Anabaena* sp. PCC 7120 were performed. In these organisms, the accumulation of CGP is mainly depending on the origin of the *cphA* gene, the accumulation of other storage compounds like PHA as well as the addition of precursor components like arginine to the medium [96]. PHA-deficient mutants of *Cupriavidus necator* and *Pseudomonas putida* accumulate in general more CGP compared to the PHA containing strains [96]. During genetic modification of *cphA* expression in *Cupriavidus necator*, CGP accumulation turned out to be

Sallam and Steinbuchel [92]).

96 Cyanobacteria

**7. Biotechnological production**

*putida*, were used for heterologous production of CGP [93].

The industrially established host *Saccharomyces cerevisiae* has also been used for CGP production, by expression of *cphA* from *Synechocystis* sp. PCC 6803. *S. cerevisiae* harboring *cphA* accumulated up to 6.9% (w/w) of CDM. Two CGP species were observed in this strain: water-soluble and the typical water-insoluble CGP. Furthermore, the isolated polymer from this transgenic yeast contained 2 mol% lysine, which can be increased up to 10 mol% when cultivation occurs with lysine in the medium [31]. During metabolic engineering studies, several arginine biosynthesis mutants have been analyzed concerning their CGP accumulation abilities. Surprisingly, strains with defects in arginine degradation accumulated only 4% CGP (w/w) of CDM; however, arginine auxotrophic strains were able to accumulate up to 15.3%. Depending on the cultivation conditions, between 30 and 90% of the extracted CGP was soluble at neutral pH. In addition to arginine, aspartate and lysine, further amino acids, such as citrulline and ornithine, have been detected in isolated CGP from different arginine biosynthesis mutants [98]. Furthermore, it was also possible to produce CGP and CGP derivates in *Pseudomonas putida* and the yeast *Pichia pastoris* [99, 100].

CGP and CGP derivates are important sources for β-dipeptides for several applications. A large-scale method was developed to convert CGP into its constituting β-dipeptides by using CphE from *Pseudomonas alcaligenes*. This allows the large-scale production of customized β-dipeptides, depending on the composition of the CGP derivates [92, 101].

Production of CGP has also been attempted in several transgenic plants. Here, ectopic expression of the primer-independent CphA from *Thermosynechococcus elongatus* BP-1 leads to an accumulation of CGP up to 6.8% (w/w) in tobacco leafs and to 7.5% (w/w) of CDM in potato tubers [102, 103]. CGP production and extraction in plants can be coupled with the production of other plant products like starch [103]. The peculiarities and challenges of plant-produced CGP have been reviewed by Nausch et al. [32].

Compared to bacteria that are used so far in biotechnological industry, cyanobacteria are unique as they use sunlight and CO2 as energy and carbon source. Cyanobacteria have been identified as rich source of various biologically active compounds, biofertilizers, bioplastics, energy, food and feed [104]. Obviously, the importance of environmentally friendly production processes increases more and more. Hence, Cyanobacteria are expected to play a major role in future industry. *Synechocystis* sp. PCC 6803 strain BW86 is the first reported bulk chemical producing cyanobacterial strain in the literature. CGP production in *Synechocystis* BW86 does not require organic carbon or CGP precursor substances. Growth limiting conditions like phosphate and potassium starvation can further increase the CGP production up to 47.4 ± 2.3% and 57.3 ± 11.1% per CDM, respectively. The studies of Trautmann et al. showed that strain BW86 can be cultivated in flat plate photobioreactors (Midiplate reactor system [105]). During this optimization study, the optimal light intensity as well as the phosphate concentration was determined to maximize CGP synthesis. Under optimal production conditions, highest amount of CGP was around 40% of CDM with a total yield of 340 mg CGP per liter in 9 days [106].

The main bottleneck of CGP production in Cyanobacteria is the relatively slow growth rate, which is much lower than in biotechnologically established bacteria. Conventional cultivation methods of cyanobacteria reach a biomass of roughly 1 g dry mass per liter [107]. To overcome this limitation, a new cultivation method was developed, using a two-tier vessel with membrane-mediated CO2 supply. By using this cultivation setup, it was possible to enable rapid growth of *Synechocystis* sp. PCC 6803 and *Synechococcus* sp. PCC 7002 up to 30 g CDM per liter [108]. *Synechocystis* sp. PCC 6803 strain BW86 was also used in this high-density cultivation setup. During this study, CGP amounts up to 1 g per liter were reached in 96 h. This is approximately four times higher compared to the maximum CGP yield observed during conventional cultivation after 12 days [106, 109].

**Conflict of interest**

**Author details**

**References**

**22**:15-46

Björn Watzer and Karl Forchhammer\*

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Universität Tübingen, Tübingen, Germany

The authors declare that they have no competing interests.

\*Address all correspondence to: karl.forchhammer@uni-tuebingen.de

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Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Eberhard Karls

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In comparison, the recombinant *E. coli* strain DH1 harboring *cphA* from *Synechocystis* sp. PCC 6803 produces between 6.7 and 8.3 g CDM per liter culture in 16 h. CGP amounts during this fed-batch fermentations were between 21 and 24% of the CDM [27], resulting in a CGP production rate of 87.9 to 124.5 mg/l and hour. Although this exceeds the production rate in *Synechocystis* sp. PCC 6803 strain BW86 by a factor of 10, the recombinant *E. coli* requires terrific broth complex medium, while *Synechocystis* sp. PCC 6803 strain BW86 is cultivated in simple mineral medium and additionally sequesters hazardous greenhouse gas CO2 . Considering these super ordinate factors, production of biopolymers with cyanobacteria may in fact become an alternative to heterotrophic bacteria.
