77NISR Research GRANT
2005 Young Investigator Research Grant

Molecular Mechanism of Strategy for Minimization of theIntracellular Formaldehyde Level in Microorganisms and Its
Application
Hiroya YURIMOTO
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University

Research aims
Formaldehyde, which is a cytotoxic compound, is a central metabolic intermediate in the metabolic pathway for assimilation and dissimilation of one-carbon (C1) compounds, such as methane, methanol, and formaldehyde, inmethylotrophs. To achieve efficient industrial use of C1compounds by microorganisms, it is important to elucidate
the molecular basis of how methylotrophs cope with thetoxicity of formaldehyde. Recently, several biochemical andgenetic approaches have established the molecular mechanisms by which the intracellular level of formaldehyde isregulated during microbial growth on C1 compounds1,2). In
this study, I described the physiological significance of theribulose monophosphate (RuMP) pathway, which arewidely distributed not only in methylotrophic bacteria but
also in non-methylotrophic bacteria and archaea, in a hyperthermophilic archaeon and the molecular mechanism ofgene regulation of this pathway in Bacillus subtilis. I also
described the characterization of a transcriptional factor involved in C1 compounds-induced gene expression inmethylotrophic yeast.
Results and Discussion
1) Physiological role of the ribulose monophosphate pathway enzymes in archaea
The RuMP pathway, involving 3-hexulose-6-phosphatesynthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is
now recognized as a widespread prokaryotic pathway forformaldehyde assimilation and detoxification. Interestingly,
HPS and PHI orthologs are also found in archaeal strains,and recent biochemical and genome analyses have proposedthat the reverse reaction of formaldehyde fixation, i.e. ribulose 5-phosphate (Ru5P) synthesis from fructose 6-phosphate, may possibly function in the biosynthesis of Ru5P inseveral archaeal strains whose pentose phosphate pathways
are incomplete. In this study, we have taken a genetic approach to address this proposal using the hyperthermophilicarchaeon Thermococcus kodakaraensis KOD1 in whichgene disruption system has been developed and HPS andPHI are encoded in a single open reading frame (TK0475).
The recombinant HPS-PHI fused enzyme exhibited theexpected HPS and PHI activities in both directions(formaldehyde-fixing and Ru5P-synthesizing). The
TK0475-deletion mutant did not exhibit any growth in min-imal medium, while growth of the mutant strain could berecovered by the addition of nucleosides to the medium.
This auxotrophic phenotype together with the catalytic
properties of the HPS-PHI-fused enzyme reveal that HPS
and PHI are essential for the biosynthesis of Ru5P, the precursor of nucleotides, showing that the RuMP pathway is
the sole pathway for Ru5P biosynthesis which substitutes
for the conventional pentose phosphate pathway3).
2) Analysis of the novel DNA-binding protein that acts as apositive regulator of the formaldehyde-inducible RuMP
operon in Bacillus subtilis
In B. subtilis, hxlA and hxlB, coding for HPS and PHI,
respectively, make up the hxlAB operon and expression ofthis operon is induced by the presence of formaldehyde.
Upstream of the hxlAB operon, the open reading frame ofhxlR is oriented in the opposite direction relative to hxlAB.
The HxlR protein belongs to the DUF24 protein family ofunknown function which contains DNA-binding helix-turnhelix motif. The hxlR gene was essential for the formaldehyde-induced expression of HPS and PHI because the hxlR
disruptant strain did not exhibit HPS or PHI activity after
exposure to formaldehyde.
Purified recombinant HxlR showed specific binding to
the upstream region of the hxlAB operon. Using gel-retardation and DNase I-foot printing assays, two 25-bp binding
regions for HxlR within the upstream DNA were identified.
Surface plasmon resonance analyses suggested that twoHxlR dimers sequentially bound to these regions. Finally,
we showed that each of the two binding regions for HxlR
was necessary for formaldehyde-induced expression of thehxlAB operon4). Thus, I have shown that HxlR is a novel
DNA-binding protein that activates the formaldehyde-induced expression of hxlAB. HxlR is now designated as atranscriptional activator of the “helix-turn-helix, HxlR type”(http://www.ebi.ac.uk/interpro/IEntry?ac=IPR002577).
3) Trm1 is a transcriptional activator involved in regulationof methanol-inducible gene expression in yeast
Methylotrophic yeasts have been widely used as a hostfor the heterologous gene expression system because thepromoters of the genes encoding methanol-metabolizing
enzymes are highly inducible by C1 compounds5). However, the molecular mechanism of C1 compounds-inducible
gene expression is still unclear. We have screened for themutants that transcriptional activity of C1 compounds-in-

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ducible promoters was reduced using Candida boidinii as ahost strain and acid phosphatase from Saccharomyces cerevisiae as a reporter. As a result, the TRM1 gene was foundand its properties were analyzed.
Trm1 belongs to Zinc finger protein family of a Zn(II)2
Cys6 type restricted to eucaryotic microbe. The TRM1 genedisrupted strain was not able to grow on methanol, butshowed normal growth on glucose or other non-fermentative carbon sources. In TRM1 gene disrupted strain, thetranscriptional activity of several methanol-inducible promoters was examined. As a result, the drastic reduction oftranscriptional activity was observed compared with wildtype strain when methanol or formaldehyde is used for induction substrate. In addition, Trm1 was localized in nucleus. From these results, it was suggested that Trm1 was amain transcriptional factor of C1 compounds-inducible
gene expression.
Conclusion
Molecular basis of formaldehyde metabolism in microorganisms was investigated in this study. First, it was clearlyindicated that the reverse reaction of formaldehyde fixation
catalyzed by HPS and PHI play an indispensable role inpentose phosphate biosynthesis in archaea. The fact that theforward and reverse reactions have different physiologicalroles in bacteria and archaea is interesting from the view-point of evolution of metabolic pathways. Second, as for themolecular mechanism of regulation of gene expression in
response to C1 compounds, I have obtained and characterized two DNA-binding proteins which activate C1-compunds-inducible gene expression from B. subtilis and C.
boidinii, respectively. Further analyses are necessary to understand the molecular mechanism such as recognition ofC1 substrates and signal transduction.
References
1) Kato, N., Yurimoto, H., and Thauer, R. K., The physiological role of
the ribulose monophosphate pathway in bacteria and archaea. Biosci.
Biotechnol. Biochem., 70: 10–21 (2006).
2) Yurimoto, H., Kato, N., and Sakai, Y., Assimilation, dissimilation,and detoxification of formaldehyde, a central metabolic intermediateof methylotrophic metabolism. Chem. Rec., 5: 367–375 (2005).
3) Orita, I., Sato, T., Yurimoto, H., Kato, N., Atomi, H., Imanaka, T.,
and Sakai, Y., The ribulose monophosphate pathway substitutes forthe missing pentose phosphate pathway in the archaeon Thermococcus kodakaraensis. J. Bacteriol., 188: 4698–4704 (2006).
4) Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N., andSakai, Y., HxlR, a member of the DUF24 protein family, is a DNAbinding protein that acts as a positive regulator of the formaldehydeinducible hxlAB operon in Bacillus subtilis. Mol. Microbiol., 57:511–519 (2005).
5) Yurimoto, H., Komeda, T., Lim, C. R., Nakagawa, T., Kondo, K.,
Kato, N., and Sakai, Y., Regulation and evaluation of five methanolinducible promoters in the methylotrophic yeast Candida boidinii.
Biochim. Biophys. Acta, 1493: 56–63 (2000).