The thesis investigated the metabolism of xylose and arabinose in Clostridium acetobutylicum ATCC 824 and Clostridium saccharobutylicum NCP 262. It disrupted genes encoding xylulose kinase (xylB) and arabinose kinase (araK) and found that strains without these functional genes could not utilize or grow on the respective sugars. The thesis also focused on developing genetic manipulation systems for solventogenic clostridia but was unable to achieve a clean deletion of the upp gene in C. acetobutylicum. It created methylation plasmids and demonstrated conjugation of DNA to C. saccharobutylicum using its own methyltransferases

1. Justyna Maria Lesiak, PhD
Thesis title: Investigation of xylose and arabinose metabolism in Clostridium
acetobutylicum ATCC 824 and Clostridium saccharobutylicum NCP 262.
Thesis summary
Solventogenic clostridia are able to use various pentoses to produce energy in the
process called Acetone-Butanol-Ethanol fermentation. Clostridium acetobutylicum ATCC
824 can ferment two common plant-derived pentoses, xylose and arabinose. However, their
utilization is blocked by the presence of glucose, due to the carbon catabolite repression.
Arabinose, compared to xylose, was more efficient as a carbon and energy source and its
fermentation resulted in much higher solvent yields. The acetone-to-butanol ratio of cultures
grown on arabinose was much higher compared to cultures grown on glucose or xylose, and it
was suggested that the carbon flux in C. acetobutylicum grown on arabinose is directed
through the phosphoketolase pathway, which would also explain the differences between the
growth and fermentation profiles on both pentoses. Disruption of the phosphoketolase gene
resulted in an impaired growth of the mutant strain on arabinose during the entire exponential
phase of growth, and most probably it directed the carbon flux through the pentose phosphate
pathway.
In the metabolism of xylose and arabinose in C. acetobutylicum ATCC 824 and
C. saccharobutylicum NCP 262, two important genes, encoding xylulose kinase (xylB) and
arabinose kinase (araK) are involved. In this work intron mutants of the kinase genes were
created in both solventogenic clostridia, and additionally the araK and xylB genes of
C. acetobutylicum were deleted through the allelic exchange (ACE). Clostridium strains
without functional arabinose kinase were not able to utilize arabinose as a sole carbon and
energy source, but it was possible to complement the intron-derived mutation effect by
introducing a functional copy of the araK gene under the promoter of the ferredoxin gene in
C. acetobutylicum. Similarly, strains with disrupted xylulose kinase were not able to grow on
xylose, but introducing a functional copy of the xylB gene under the ferredoxin promoter in
the C. acetobutylicum intron mutant reversed this phenotype. A C. acetobutylicum ΔaraK
ΔxylB strain was incapable of fermenting either xylose or arabinose.

2. Although in batch cultures C. acetobutylicum showed an impaired growth and fermentation
profile on xylose compared to glucose, no such observation was made in the continuous
culture experiment. DNA microarray-based transcriptional analysis of the genes regulated
during the two metabolic states of the cell: acidogenesis and solventogenesis, and during the
transition between both, revealed many similarities in the regulation of important cellular
processes between glucose- (previous work from this group) and xylose-supplemented
continuous cultures. Among genes regulated in a similar manner on xylose and glucose, the
genes involved in solvent production (sol-operon, thlB and aad), cellulosome formation
(celgenes), stress response (groEL/ES, dnaKJ, grpE or hrcA) and the tricarboxylic acid cycle
(citBC) were described. However, the transcriptional pattern of some genes differed between
both cultures. Significant regulation of genes involved in amino acid biosynthesis (aro and trp
genes), redox state (nadABC) and sporulation (spoIII genes, sigFgenes, sigG, sigE or spo0A)
was observed on xylose, but not on glucose.
Solvent production depends not only on productive sugar fermentation, but also on the
effective energy conversion directed by, inter alia, the Rnf complex. This complex is
common among anaerobic Gram-positive species and is used for energy conservation by
converting the transfer of electrons from reduced ferredoxin to NAD into a sodium or proton
gradient. In C. beijerinckii, however, disruption of the rnfC gene, coding for the first gene of
the rnf cluster and a subunit of the complex, resulted in no visible changes in the growth curve
or glucose consumption rates when compared to the wild type strain.
Additionally, this study focused on the need for an accessible clean deletion system for
solventogenic clostridia and the creation of an upp-based system for C. acetobutylicum.
Despite the construction of several variants of deletion vectors and the application of different
conditions to improve the integration rate, no clean deletion of the upp gene was achieved,
although in several attempts the plasmid integrants, representing the first recombination event
in the clean deletion method, were observed.
Furthermore, for the genetic manipulations of C. saccharobutylicum NCP262, a methylation
system and tri-parental conjugation approach were created. The pJL1 and pJL2 methylation
plasmids, based on the native methyltransferase genes from the restriction-modification
systems RM1 and RM2 of the bacterium, were constructed and it was shown that it is possible
to transconjugate DNA to C. saccharobutylicum using in vivo methylation by its own
methyltransferases. Experiments with three clostridial recipients: the wild type strain and

3. mutants in the restriction subunits (hsdR) of the restriction-modification systems RM1 and
RM2, with and without in vivo methylation of donor DNA suggested the RM2 of
C. saccharobutylicum has a higher significance for restriction, as well as for methylation.
Conjugation with an unmethylated plasmid showed higher rates for the hsdR2-disrupted
recipient, while methylation with pJL2 containing the methyltransferase from RM2 was more
effective for conjugation with wild type recipients.