Using synthetic biological parts and microbioreactors to explore the protein expression characteristics of Escherichia coli.

Synthetic biology has developed numerous parts for the precise control of protein expression. However, relatively little is known about the burden these place on a host, or their reliability under varying environmental conditions. To address this, we made use of synthetic transcriptional and translational elements to create a combinatorial library of constructs that modulated expression strength of a green fluorescent protein. Combining this library with a microbioreactor platform, we were able to perform a detailed large-scale assessment of transient expression and growth characteristics of two Escherichia coli strains across several temperatures. This revealed significant differences in the robustness of both strains to differing types of protein expression, and a complex response of transcriptional and translational elements to differing temperatures. This study supports the development of reliable synthetic biological systems capable of working across different hosts and environmental contexts. Plasmids developed during this work have been made publicly available to act as a reference set for future research.

Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

The industrial production of ß-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

Genetic circuit performance under conditions relevant for industrial bioreactors.

Synthetic genetic programs promise to enable novel applications in industrial processes. For such applications, the genetic circuits that compose programs will require fidelity in varying and complex environments. In this work, we report the performance of two synthetic circuits in Escherichia coli under industrially relevant conditions, including the selection of media, strain, and growth rate. We test and compare two transcriptional circuits: an AND and a NOR gate. In E. coli DH10B, the AND gate is inactive in minimal media; activity can be rescued by supplementing the media and transferring the gate into the industrial strain E. coli DS68637 where normal function is observed in minimal media. In contrast, the NOR gate is robust to media composition and functions similarly in both strains. The AND gate is evaluated at three stages of early scale-up: 100 mL shake flask experiments, a 1 mL MTP microreactor, and a 10 L bioreactor. A reference plasmid that constitutively produces a GFP reporter is used to make comparisons of circuit performance across conditions. The AND gate function is quantitatively different at each scale. The output deteriorates late in fermentation after the shift from exponential to constant feed rates, which induces rapid resource depletion and changes in growth rate. In addition, one of the output states of the AND gate failed in the bioreactor, effectively making it only responsive to a single input. Finally, cells carrying the AND gate show considerably less accumulation of biomass. Overall, these results highlight challenges and suggest modified strategies for developing and characterizing genetic circuits that function reliably during fermentation.

Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in deltalig4 or deltaku70 mutants.

Inactivating the non-homologous end-joining (NHEJ) pathway is a well established method to increase gene targeting (GT) efficiencies in filamentous fungi. In this study we have compared the effect of inactivating the NHEJ genes ku70 or lig4 on GT in the industrial penicillin producer Penicillium chrysogenum. Deletion of both genes resulted in strongly increased GT efficiencies at three different loci but not higher than 70%, implying that other, yet uncharacterized, recombination pathways are still active causing a part of the DNA to be integrated via non-homologous recombination. To further increase the GT efficiency we applied the bi-partite approach, in which the DNA fragment for integration was split in two non-functional overlapping parts that via homologous recombination invivo can form a functional selection marker. The combined NHEJ mutant and bi-partite approach further increased GT frequencies up to approximately 90%, which will enable the efficient high throughput engineering of the P. chrysogenum genome. We expect that this combined approach will function with similar high efficiencies in other filamentous fungi.

Functional characterization of the penicillin biosynthetic gene cluster of Penicillium chrysogenum Wisconsin54-1255.

Industrial strain improvement via classical mutagenesis is a black box approach. In an attempt to learn from and understand the mutations introduced, we cloned and characterized the amplified region of industrial penicillin production strains. Upon amplification of this region Penicillium chrysogenum is capable of producing an increased amount of antibiotics, as was previously reported [Barredo, J.L., Diez, B., Alvarez, E., Martín, J.F., 1989a. Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high yielding strains of Penicillium chrysogenum. Curr. Genet. 16, 453-459; Newbert, R.W., Barton, B., Greaves, P., Harper, J., Turner, G., 1997. Analysis of a commercially improved Penicillium chrysogenum strain series, involvement of recombinogenic regions in amplification and deletion of the penicillin gene cluster. J. Ind. Microbiol. 19, 18-27]. Bioinformatic analysis of the central 56.9kb, present as six direct repeats in the strains analyzed in this study, predicted 15 Open Reading Frames (ORFs). Besides the three penicillin biosynthetic genes (pcbAB, pcbC and penDE) only one ORF has an orthologue of known function in the database: the Saccharomyces cerevisiae gene ERG25. Surprisingly, many genes known to encode direct or indirect steps beta-lactam biosynthesis like phenyl acetic acid CoA ligase and transporters are not present. Detailed analyses reveal a detectable transcript for most of the predicted ORFs under the conditions tested. We have studied the role of these in relation to penicillin production and amplification of the biosynthetic gene cluster. In contrast to what was expected, the genes encoding the three penicillin biosynthetic enzymes alone are sufficient to restore full beta-lactam synthesis in a mutant lacking the complete region. Therefore, the role of the other 12 ORFs in this region seems irrelevant for penicillin biosynthesis.

Laboratory-evolved vanadium chloroperoxidase exhibits 100-fold higher halogenating activity at alkaline pH: catalytic effects from first and second coordination sphere mutations.

Directed evolution was performed on vanadium chloroperoxidase from the fungus Curvularia inaequalis to increase its brominating activity at a mildly alkaline pH for industrial and synthetic applications and to further understand its mechanism. After successful expression of the enzyme in Escherichia coli, two rounds of screening and selection, saturation mutagenesis of a "hot spot," and rational recombination, a triple mutant (P395D/L241V/T343A) was obtained that showed a 100-fold increase in activity at pH 8 (k(cat) = 100 s(-1)). The increased K(m) values for Br(-) (3.1 mm) and H(2)O(2) (16 microm) are smaller than those found for vanadium bromoperoxidases that are reasonably active at this pH. In addition the brominating activity at pH 5 was increased by a factor of 6 (k(cat) = 575 s(-1)), and the chlorinating activity at pH 5 was increased by a factor of 2 (k(cat) = 36 s(-1)), yielding the "best" vanadium haloperoxidase known thus far. The mutations are in the first and second coordination sphere of the vanadate cofactor, and the catalytic effects suggest that fine tuning of residues Lys-353 and Phe-397, along with addition of negative charge or removal of positive charge near one of the vanadate oxygens, is very important. Lys-353 and Phe-397 were previously assigned to be essential in peroxide activation and halide binding. Analysis of the catalytic parameters of the mutant vanadium bromoperoxidase from the seaweed Ascophyllum nodosum also adds fuel to the discussion regarding factors governing the halide specificity of vanadium haloperoxidases. This study presents the first example of directed evolution of a vanadium enzyme.