Butanol is an important commodity chemical and due to its properties considered to represent an ideal advanced biofuel. In recent years, in addition to petrochemical production of butanol, fermentation as a means of biobutanol production has been revived. This so called ABE fermentation is not a novel process, and has already been employed commercially for most of the first half of the 20th century. While most of the principles are the same, the rise of genetic tools and metabolic engineering allowed optimization of this process. Through synthetic biology, industrial workhorses as E. coli and yeast have been successfully modified for production of n-butanol, and through a non-fermentative pathway also for production of iso-butanol, a process that is currently commercialized. While all these approaches rely on use of sugar or starch, alternative processes have been developed for sustainable production of biobutanol from CO2 and light with cyanobacteria or by fermenting syngas using acetogens.

Cyanobacteria require large quantities of iron to maintain their photosynthetic machinery; however, in most environments iron is present in the form of insoluble iron oxides. Whether cyanobacteria can utilize these sources of iron, and the potential molecular mechanisms involved remains to be defined. There is increasing evidence that pili can facilitate electron donation to extracellular electron acceptors, like iron oxides in non-photosynthetic bacteria. In these organisms, the donation of electrons to iron oxides is thought to be crucial for maintaining respiration in the absence of oxygen. Our study investigates if PilA1 (major pilin protein) may also provide a mechanism to convert insoluble ferric iron into soluble ferrous iron. Growth experiments supported by spectroscopic data of a strain deficient in pilA1 indicate that the presence of the pilA1 gene enhances the ability to grow on iron oxides. These observations suggest a novel function of PilA1 in cyanobacterial iron acquisition.

Sequence and ligation-independent cloning (Nat Methods 4:251-256, 2007) is a powerful tool for the construction of multi-fragment complex plasmids in a simple and efficient manner. Plasmids consisting of 6-7 DNA fragments can be assembled in a single day, with additional 2 days for screening and extraction. SLIC requires PCR products with overlapping regions of 30-40 bp at the 5' and 3' ends, T4 DNA polymerase, and an optional RecA protein for construction.

The cyanobacterium Synechocystis sp. PCC 6803 is an attractive target for engineering novel metabolic pathways for the synthesis of useful compounds directly from CO2 as it is a naturally transformable, oxygenic photoautotroph and the genome has been sequenced. The compound n-butanol is a potential bio-fuel for direct replacement of petroleum, with little to no adjustment of the current infrastructure, as n-butanol has similar fuel characteristics to petroleum. To accomplish the aim, an integrative expression system was developed consisting of two plasmids, which integrated at the phaAB and phaEC loci. Integration at the phaEC site eliminated a competing pathway, polyhydroxybutyrate (PHB) biosynthesis. The two plasmids were used to introduce foreign genes into the genome under the control of promoters PphaA and PphaE. The expression system was validated and analysed using a luciferase reporter enzyme. The promoters were found to express under circadian rhythm, expression was increased in the dark and repressed in the light. In addition, under phosphate limitation the luciferase expression was increased three-fold and became constitutively expressed. Expression was strongly up-regulated in the stationary compared to the logarithmic growth phase. The genes for n-butanol biosynthesis pathway from Clostridium beijerinckii were introduced into Synechocystis sp. PCC 6803 under control of the PphaA and PphaE promoters; however, no butanol could be detected above background levels. Enzyme assays of the cell lysate showed that two of the enzymes activities could not be detected, indicating either the enzymes were inactive or being expressed below limits of detection. Replacement of the C. beijerinckii Bcd-complex with crotonyl-CoA reductase (Ccr) from Streptomyces collonius, along with codon optimisation of the remaining C. beijerinckii genes, generated strain SynRH-10, which synthesised 36 μg/L of culture in eight days and had a peak output of 14 μg/day/L of culture. The aldehyde dehydrogenase enzyme was identified as a potential bottleneck and will require replacement to improve n-butanol output. The program Precog was developed to identify additional novel pathways from MetaCyc reaction and compound data. Several novel pathways were identified, in doing so it also identified Ccr as an additional putative bottleneck in the pathway. An engineering strategy and new pathway was proposed, based on the results of this research and from other studies, which could generate a strain capable of synthesising milligram quantities per litre of culture. iii

This study investigated the use of promoters belonging to the phaAB and phaEC operons of the polyhydroxybutyrate (PHB) biosynthetic pathway to drive foreign expression of the luxAB operon, which encodes the reporter enzyme luciferase from Vibrio harveyi. Luciferase expression was successfully achieved under conditions previously reported for PHB production. Expression is controlled by circadian rhythm under growth conditions in BG-11. Under phosphate-limiting conditions luciferase exhibited a three-fold increase in expression levels and was constitutively expressed with circadian rhythm abolished. The PHB promoters for the phaAB and phaEC operons present a potentially useful set of promoters for introducing new metabolic pathways under the control of a circadian rhythm or phosphate availability.

Background Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (Alr Bax ), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native Alr Bax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation. Results B. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1 , is able to complement a D-alanine auxotrophic strain of E. coli . Purified Dal1, which we term Alr Bax , is shown to be a dimer in solution by dynamic light scattering and has a V max for racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified Alr Bax is reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, Alr Bax makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure. Conclusion The chloride ion in Alr Bax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two Alr Bax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.