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Noda Institute for Scientific Research GRANT2014 Young Investigator Research GrantOutline of Research Result

Enhancement of a platform chemical (catechol) productionfrom cellulosic materialsTomonori SonokiDepartment of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University

Research aims

tract, and it was used to evaluate the initial characteristicsof the enhanced Pdc.

In this study, we focused on improving the production ofcatechol (CL), which is a platform chemical for the synthesis of pesticides, pharmaceuticals, antioxidants etc. It hasbeen reported that CL can be produced by shunting the carbon flow to protocatechuate from 3-dehydroshkimate(DHS) in the shikimate biosynthetic pathway (Fig. 1).1) Theproduced protocatechuate is then decarboxylated to catechol. However, the decarboxylation step has been reportedto be a bottleneck in CL production, and to the best of myknowledge, no study till date has been able to resolve thisissue. The aim of this study is to find this solution, and develop an efficient strategy for catechol production from renewable cellulosic feedstocks.

Construction and evaluation of a catechol-producingstrain with the enhanced Pdc activityA pheA-deficient mutant, E. coli strain JD23488 (obtained from NBRP-E. coli), was transformed with pTS066to express aroFFBR and qutC. The recombinant, JD23488/pTS066 was further transformed with either pTS036 (to express aroY) or pTS052 (to express aroY and kpdB).
JD23488/pTS066+pTS036 and JD23488/pTS066+pTS052were cultured in M9 medium containing 1 mM IPTG and10 μM phenylalanine in addition to appropriate antibiotics,and evaluate the effect of the enhanced Pdc activity on CLproduction. Glucose was used as the sole carbon source.

MethodsResults

Identification of an enhancer-like protein for protocatechuate decarboxylation and characterization of theenhanced PdcThe deletion clones of the region encoding KpdBCD, a4-hydroxybenzoate decarboxylase complex, were prepared,and an Escherichia coli XL-1 Blue recombinant harboringa plasmid to express AroY was transformed with each deletion clone. The resulting transformants were used to evaluate in vivo PCA decarboxylation activity. The cells expressing both AroY and KpdB were ground using an ice-coldpestle and mortar with alumina. Phosphate buffer (100 mM,pH 7.5) was used to extract enzymes from the cells. Aftercentrifugation, the supernatant was collected as a crude ex-Identification of an enhancer-like protein for protocatechuate decarboxylationOther decarboxylases of aromatic compounds such asvanillate demethylase (VdcBCD) and a 4-hydroxybenzoatedecarboxylase (BsdBCD) are hetero-oligomeric enzymesconsisting of three subunits. Pdc has been reported to be ahomo-oligomeric enzyme, and its activity was low and unstable.1–3) Methods for enhancing the activity to increasethe productivity of CL and other valuable chemicals are required; however, no efficient techniques have been reportedtill date.
KpdC, a subunit of the 4-hydroxynenzoate decarboxylasefrom Klebsiella pneumoniae NBRC14940 (KpdBCD),showed 24% identity with AroY. We hypothesized thatAroY could make a hetero-oligomeric protein complex withKpdB and KpdD, thereby increasing Pdc activity. Deletionclones of the KpdBCD-encoding region were prepared andtheir effects of their expression on Pdc activity were evaluated. Pdc activity increased when both KpdB and KpdDwere expressed with AroY, and it was also enhanced withthe coexpression of only KpdB and AroY (Fig. 2).

Fig. 1. CL production by carbon flow shunting in the shikimate pathway.

Initial characterization of the enhanced protocatechuatedecarboxylaseThe effects of pH and temperature on Pdc activity underthe condition that AroY was expressed either with or without KpdB are shown in Fig. 3 (a and b). Regardless of the34

Fig. 2.

Fig. 3.

Effects of the expression of tne subunits of KpdBCD on PCA decarboxylation.

Effects of KpdB expression on Pdc activity (a, pH; b, Temperature) and CL production (c).

expression of KpdB, the optimal pH and temperature forPdc activity were 5.5 and 35°C, respectively, although Pdcactivity increased approximately 20-fold than that withoutthe KpdB expression. AroY and KpdB were separatelyeluted at different ion concentrations via DEAE-anion chromatography. These results suggested that AroY and KpdBwould not form a hetero-oligomeric protein complex likeVdcBCD and BsdBCD, and the activity may be enhancedby a different and novel mechanism.
Effect of the enhanced Pdc activity on catechol productionThe expression of the enhanced Pdc (AroY+KpdB)
solved the temporary accumulation of PCA, as observedwhen the ordinary Pdc (AroY) was used to decarboxylatePCA (Fig. 3c). The enhanced Pdc-expressing strain produced catechol with 2-fold higher yield than the ordinarystrain.

ConclusionIn this study, a novel factor, which enables efficient production of CL by enhancing Pdc activity, was identified. CLis converted to muconic acid via ortho cleavage reaction,and muconic acid also acts as a platform chemical foradipic acid and terephalic acid, which are in great demandin the polymer industries.4) Our results will also contributeto efficient muconate production.
References1)

Draths, K.M., Frost, J.W. Biocatalytic synthesis of catechol fromglucose. US 5272073, 1993.
2) He, Z.Q., Wiegel, J. Purification and characterization of an oxygensensitive, reversible 3,4-dihydroxybenzoate decarboxylase fromClostridium hydroxybenzoicum. J. Bacteriol. 178, 3539–3543, 1996.
3) Yoshida, T., Inami, Y., Matsui, T., Nagasawa, T. Regioselective carboxylationof catechol by 3,4-dihydroxybenzoate decarboxylase ofEnterobacter cloacae P. Biotechnol. Lett. 32, 701–705, 2010.
4) Anon. Myriant Corporation. http://www.myriant.com/products/productpipeline.cfm.