Table 2.

Economical and technological bottlenecks and proposed measures

Bottleneck	Proposed measure(s)
Investments, including costs for fermentation and downstream processing equipment	The calculation provided here suggests that these may be acceptable
Costs for the production of cyanophycin, cyanophycin-derived products and for downstream processing of biomass	Construction of a sufficiently productive microbial strain to convert or simply utilize constituents of plant waste streams like Protamylasse™ and to incorporate these compounds, presumably amino acids, into the cyanophycin polymer chain during cyanophycin biosynthesis
Phenotypic instability of E. coli production strains used until now, DH1 and DH5α, containing plasmid pMa/c5-914::cphA 6803	Construction of stable strains with integrated copies of the cyanophycinsynthesis genes
Low biomass yields of the E. coli strains used	Since not all components present in the current source of Protamylasse™ may have the proper concentration for current laboratory strain(s), an optimization may require the addition of substrates other than Protamylasse™, for example other plant waste streams. Sufficient provision of amino acids like arginine should be ensured during the production phase
Optimization of microbial biomass formation	By using yeasts as alternative production organisms biomass yields could be increased to 100 g/l CDM for S. cerevisiae [factor 20×] or 150 g/l CDM for Pichia pastoris [factor 30×] if in Protamylasse™ the same yields can be obtained as in dedicated growth media
Sub-optimal fermentation processes	Fermentation technology and feeding regimes have to be developed for optimum amino acid utilization or biosynthesis from Protamylasse™ or other plant waste streams
Generation of valuable side stream particle fraction of Protamylasse™	Alternative use of the side stream particle fraction of Protamylasse™, e.g. by using cyanophycin producing filamentous fungi
Co-production with, e.g., ethanol	When using S. cerevisiae as the production organisms and (semi-) anaerobic fermentation both cyanophycin and ethanol could possibly be produced during the same run
Costs for cyanophycin extraction	Development of alternative cheap cyanophycin extraction methods using, e.g., hydro-cyclone equipment for the non-soluble fraction
Cost-efficient production of cyanophycin in plants	The transfer of the bacterial cyanophycin synthetase gene (cphA) into eukaryotic hosts, mostly plants and its effective expression in suitable organs or cell compartments is a major step (see below)
Efficacy of downstream processing	Downstream processing has to be adapted and optimized for cyanophycin or cyanophycin derivatives containing biomass, which will be either bacterial cells or eukaryotic (mostly plant) cells or tissues
Lack of insight in possible modifications of cyanophycin, their impact on cyanophycin properties and market potential	The diverse possibilities to modify the cyanophycin molecule chemically or enzymatically has to be exhaustingly explored to identify all potential key applications for cyanophycin-derived products and to find the most suitable products with regard to market potential and the possibility of their commercialization
Lack of knowledge concerning properties of known cyanophycin synthetases and their genetic engineering	The possibility to modify the active sites of the cyanophycin synthetases in order to change its substrate specificity and to allow the production of cyanophycin derivatives has to be determined
Insufficient insight in all possible applications for cyanophycin as a polymer or as a starting material for chemical syntheses	The exploitation of cyanophycins and cyanophycin-derived molecules as substitutes for well established industrial products or as renewable raw materials has to be determined precisely