Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design.

The development of Pseudomonas strains for industrial production of fuels and chemicals will require the integration of heterologous genes and pathways into the chromosome. Finding the most appropriate integration site to maximize strain performance is an essential part of the strain design process. We characterized seven chromosomal loci in Pseudomonas putida KT2440 for integration of a fluorescent protein expression construct. Insertion in five of the loci did not affect growth rate, but fluorescence varied by up to 27-fold. Three sites displaying a diversity of phenotypes with the fluorescent reporter were also chosen for the integration of a gene encoding a muconate importer. Depending on the integration locus, expression of the importer varied by approximately 3-fold and produced significant phenotypic differences. This work demonstrates the impact of the integration location on host viability, gene expression, and overall strain performance.

Biotechnology of cyanobacterial isoprene production.

Heterologous cyanobacterial production of isoprene (C5H8) presents an opportunity to develop renewable resources for fuel and industrial chemicals. Isoprene can be generated photosynthetically in these microorganisms from dimethylallyl-diphosphate (DMAPP) by the recombinant enzyme isoprene synthase (ISPS), as a transgenic product of the isoprenoid biosynthetic pathway. The present work sought to combine recent enhancements in the cellular level of reactant (DMAPP) and enzyme (ISPS), as a means in the further development of this technology. This objective was approached upon the heterologous overexpression of fni, an isopentenyl isomerase from Streptococcus pneumoniae, which increased the amount of the DMAPP reactant at the expense of its isomer, isopentenyl-diphosphate (IPP), in the cells. In addition, the cellular concentration of ISPS was substantially enhanced upon expression of the ISPS gene, as a fusion construct with the highly expressed in cyanobacteria cpcB gene, encoding the abundant ß-subunit of phycocyanin. Synergy between these two modifications, i.e., enhancement in DMAPP substrate availability and enhancement in the concentration of the ISPS enzyme, improved the isoprene-to-biomass production ratio in cyanobacteria from 0.2:1 mg g-1 (w:w), attained with the ISPS transgene alone, up to 12.3:1 mg g-1 (w:w), measured when the combined two modifications were applied to the same cell. This is the highest verifiable yield of heterologous photosynthetic isoprene production reported so far. Findings in this work constitute a step forward in the development of the cyanobacterial biotechnology for isoprene production.

Engineering isoprene synthesis in cyanobacteria.

The renewable production of isoprene (Isp) hydrocarbons, to serve as fuel and synthetic chemistry feedstock, has attracted interest in the field recently. Isp (C5 H8 ) is naturally produced from sunlight, CO2 and H2 O photosynthetically in terrestrial plant chloroplasts via the terpenoid biosynthetic pathway and emitted in the atmosphere as a response to heat stress. Efforts to institute a high capacity continuous and renewable process have included heterologous expression of the Isp synthesis pathway in photosynthetic microorganisms. This review examines the premise and promise emanating from this relatively new research effort. Also examined are the metabolic engineering approaches applied in the quest of renewable Isp hydrocarbons production, the progress achieved so far, and barriers encountered along the way.

Engineering Isoprene Synthase Expression and Activity in Cyanobacteria.

Efforts to heterologously produce quantities of isoprene hydrocarbons (C5H8) renewably from CO2 and H2O through the photosynthesis of cyanobacteria face barriers, including low levels of recombinant enzyme accumulation compounded by their slow innate catalytic activity. The present work sought to alleviate the "expression level" barrier upon placing the isoprene synthase (IspS) enzyme in different fusion configurations with the cpcB protein, the highly expressed ß-subunit of phycocyanin. Different cpcB*IspS fusion constructs were made, distinguished by the absence or presence of linker amino acids between the two proteins. Composition of linker amino acids was variable with lengths of 7, 10, 16, and 65 amino acids designed to test for optimal activity of the IspS through spatial positioning between the cpcB and IspS. Results showed that fusion constructs with the highly expressed cpcB gene, as the leader sequence, improved transgene expression in the range of 61 to 275-fold over what was measured with the unfused IspS control. However, the specific activity of the IspS enzyme was attenuated in all fusion transformants, possibly because of allosteric effects exerted by the leader cpcB fusion protein. This inhibition varied depending on the nature of the linker amino acids between the cpcB and IspS proteins. In terms of isoprene production, the results further showed a trade-off between specific activity and transgenic enzyme accumulation. For example, the cpcB*L7*IspS strain showed only about 10% the isoprene synthase specific-activity of the unfused cpcB-IspS control, but it accumulated 254-fold more IspS enzyme. The latter more than countered the slower specific activity and made the cpcB*L7*IspS transformant the best isoprene producing strain in this work. Isoprene to biomass yield ratios improved from 0.2 mg g-1 in the unfused cpcB-IspS control to 5.4 mg g-1 in the cpcB*L7*IspS strain, a 27-fold improvement.

Role of isopentenyl-diphosphate isomerase in heterologous cyanobacterial (Synechocystis) isoprene production.

Heterologous production of isoprene (C5H8) hydrocarbons in cyanobacteria, emanating from sunlight, CO2, and water, is now attracting increasing attention. The concept entails application of an isoprene synthase transgene from terrestrial plants, heterologously expressed in cyanobacteria, aiming to reprogram carbon flux in the terpenoid biosynthetic pathway toward formation and spontaneous release of this volatile chemical from the cell and liquid culture. However, flux manipulations and carbon-partitioning reactions between isoprene (the product) and native terpenoid biosynthesis for cellular needs are not yet optimized for isoprene yield. The primary reactant for isoprene biosynthesis is dimethylallyl diphosphate (DMAPP), whereas both DMAPP and its isopentenyl diphosphate (IPP) isomer are needed for cellular terpenoid biosynthesis. The present work addressed the function of an isopentenyl diphosphate (IPP) isomerase in cyanobacteria and its role in carbon partitioning between IPP and DMAPP, both of which serve, in variable ratios, as reactants for the synthesis of different cellular terpenoids. The work was approached upon the heterologous expression in Synechocystis of the "isopentenyl diphosphate isomerase" gene (FNI) from Streptococcus pneumoniae, using isoprene production as a "reporter process" for substrate partitioning between DMAPP and IPP. It is shown that transgenic expression of the FNI gene in Synechocystis resulted in a 250 % increase in the "reporter isoprene" rate and yield, suggesting that the FNI isomerase shifted the endogenous DMAPP-IPP steady-state pool size toward DMAPP, thereby enhancing rates and yield of isoprene production. The work provides insight into the significance and functional role of the IPP isomerase in these photosynthetic microorganisms.