Artur Włodarczyk

Cyanobacteria, which use solar energy to convert carbon dioxide into biomass, are potential solar biorefineries for the sustainable production of chemicals and biofuels. However, yields obtained with current strains are still uncompetitive compared to existing heterotrophic production systems. Here we report the discovery and characterization of a new cyanobacterial strain, Synechococcus sp. PCC 11901, with potential for green biotechnology. It is transformable, has a short doubling time of ≈2 hours, grows at high light intensities and a wide range of salinities and accumulates up to 18.3 g dry cell weight/L of biomass – 2-3 times more than previously described for cyanobacteria - when grown in a modified medium containing elevated nitrate, phosphate and iron. As a proof of principle, PCC 11901 engineered to produce free fatty acids did so at unprecedented levels for cyanobacteria, with final yields reaching over 6 mM (1.5 g/L), comparable to those achieved by heterotrophic organisms.

Cyanobacteria, such as Synechococcus sp. PCC 7002 (Syn7002), are promising chassis strains for “green” biotechnological applications as they can be grown in seawater using oxygenic photosynthesis to fix carbon dioxide into biomass. Their other major nutritional requirements for efficient growth are sources of nitrogen (N) and phosphorus (P). As these organisms are more economically cultivated in outdoor open systems, there is a need to develop cost-effective approaches to prevent the growth of contaminating organisms, especially as the use of antibiotic selection markers is neither economically feasible nor ecologically desirable due to the risk of horizontal gene transfer. Here we have introduced a synthetic melamine degradation pathway into Syn7002 and evolved the resulting strain to efficiently use the nitrogen-rich xenobiotic compound melamine as the sole N source. We also show that expression of phosphite dehydrogenase in the absence of its cognate phosphite transporter permits growth of Syn7002 on phosphite and can be used as a selectable marker in Syn7002. We combined these two strategies to generate a strain that can grow on melamine and phosphite as sole N and P sources, respectively. This strain is able to resist deliberate contamination in large excess and should be a useful chassis for metabolic engineering and biotechnological applications using cyanobacteria.

Summary: Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO₂ and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high‐value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non‐limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts.

Microalgae are a diverse group of single-cell photosynthetic organisms that include cyanobacteria and a wide range of eukaryotic algae. A number of microalgae contain high-value compounds such as oils, colorants, and polysaccharides, which are used by the food additive, oil, and cosmetic industries, among others. They offer the potential for rapid growth under photoautotrophic conditions, and they can grow in a wide range of habitats. More recently, the development of genetic tools means that a number of species can be transformed and hence used as cell factories for the production of high-value chemicals or recombinant proteins. In this article, we review exploitation use of microalgae with a special emphasis on genetic engineering approaches to develop cell factories, and the use of synthetic ecology approaches to maximize productivity. We discuss the success stories in these areas, the hurdles that need to be overcome, and the potential for expanding the industry in general.

Histidine triad nucleotide-binding protein 2 (HINT2) is a mitochondrial adenosine phosphoramidase mainly expressed in the pancreas, liver and adrenal gland. HINT2 possibly plays a role in apoptosis, as well as being involved in steroid biosynthesis, hepatic lipid metabolism and regulation of hepatic mitochondria function. The expression level of HINT2 is significantly down-regulated in hepatocellular carcinoma patients. To date, endogenous substrates for this enzyme, as well as the three-dimensional structure of human HINT2, are unknown. In this study, human HINT2 was cloned, overexpressed in Escherichia coli and purified. Crystallization was performed at 278 K using PEG 4000 as the main precipitant; the crystals, which belonged to the tetragonal space group P4₁2₁2 with unit-cell parameters a = b = 76.38, c = 133.25 Å, diffracted to 2.83 Å resolution. Assuming two molecules in the asymmetric unit, the Matthews coefficient and the solvent content were calculated to be 2.63 ų Da^-1 and 53.27%, respectively.

Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch of the histidine triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the structure of the human HINT1-adenosine 5'-monophosphate (AMP) complex at 1.38 Å resolution obtained from a new monoclinic crystal form is reported. The final structure has R _cryst = 0.1207 (R _free = 0.1615) and the model exhibits good stereochemical quality. Detailed analysis of the high-resolution data allowed the details of the protein structure to be updated in comparison to the previously published data.