Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5562-5
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Development of a deletion mutant of Pseudomonas denitrificans that does not degrade 3-hydroxypropionic acid Shengfang Zhou & Somasundar Ashok & Yeounjoo Ko & Dong-Myung Kim & Sunghoon Park
Received: 15 October 2013 / Revised: 18 January 2014 / Accepted: 20 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Pseudomonas denitrificans is a gram-negative bacterium that can produce vitamin B12 under aerobic conditions. Recently, recombinant strains of P. denitrificans overexpressing a vitamin B12-dependent glycerol dehydratase (DhaB) were developed to produce 3-hydroxypropionic acid (3-HP) from glycerol. The recombinant P. denitrificans could produce 3-HP successfully under aerobic conditions without an exogenous supply of vitamin B12, but the 3-HP produced disappeared during extended cultivation due to the 3-HP degradation activity in this strain. This study developed mutant strains of P. denitrificans that do not degrade 3-HP. The following eight candidate enzymes, which might be responsible for 3-HP degradation, were selected, cloned, and studied for their activity in Escherichia coli: four (putative) 3-hydroxyisobutyrate dehydrogenases (3HIBDH), a putative 3-HP dehydrogenase (3HPDH), an alcohol dehydrogenase (ADH), and two choline dehydrogenases (CHDH). Among them, 3HIBDHI, 3HIBDHIV, and 3HPDH exhibited 3-HP degrading activity when expressed heterologously in E. coli. When 3hpdh alone or along with 3hibdhIV were disrupted from P. denitrificans, the mutant P. denitrificans exhibited greatly reduced 3-HP degradation activity that could not grow on 3-HP as the sole carbon and energy source. When the double mutant P. denitrificans Δ3hpdhΔ3hibdhIV was transformed with DhaB, an improved 3-HP yield (0.78 mol/mol) compared to that of the wild-type counterpart (0.45 mol/mol) was obtained from a 24-h flask culture. This study indicates that 3hpdh and 3hibdhIV (to a S. Zhou : S. Ashok : Y. Ko : S. Park (*) School of Chemical and Biomolecular Engineering, Pusan National University, San 30, Jangeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea e-mail:
[email protected] D.32 % amino acid similarity were identified and called 3HIBDHI, 3HIBDHII, 3HIBDHIII, and 3 H I B D H I V. S i m i l a r l y, u s i n g t h e p u t a t i v e 3 hydroxypropionate dehydrogenase (DddA) from Halomonas sp. HTNKK1 as a query (Todd et al. 2010), four enzymes were identified and designated as 3HPDH, ADH, CHDHI, and CHDHII. The latter four enzymes showed >36 % amino acid similarity to DddA with 3HPDH showing the highest (60 %).
Analytical methods Quantitative real-time PCR The cell concentration was determined in a 10-mm-pathlength cuvette using a double-beam spectrophotometer (Lambda 20, PerkinElmer, Norwalk, CT). The concentrations of glycerol, glucose, 3-HP, and other metabolites were determined by high-performance liquid chromatography (HPLC) using a slight modification of the method described elsewhere (Raj et al. 2008). Briefly, the supernatant obtained by centrifugation of the culture samples at 10,000×g for 10 min were filtered through a Tuffryn membrane (Acrodisc, Pall Life Sciences, Port Washington, NY) and eluted through a 300mm×7.8-mm Aminex HPX-87H (Bio-Rad, USA) column at 65 °C using 2.5 mmol/L H2SO4 as the mobile phase.
Results Identification of enzymes related with 3-HP degradation in P. denitrificans The genome sequence of P. denitrificans was searched by NCBI BLAST using two dehydrogenases as queries
Figure 1 shows the mRNA expression levels of the eight candidate enzymes determined by quantitative real-time PCR [reverse transcription-polymerase chain reaction (RTPCR)]. The analysis was carried out for the cells cultivated in either the absence (3-HP non-exposed) or presence of 3-HP at 25 mmol/L (3-HP exposed). The housekeeping gene, rpoD, encoding sigma factor 70, was used as a reference. The mRNA expression levels of the four 3hydroxyisobutyrate dehydrogenases have been reported previously and are shown here for comparison (Zhou et al. 2013b). All four 3-hydroxyisobutyrate dehydrogenases (3HIBDHs), particularly 3hibdhI, showed weaker expression than rpoD, whereas 3hpdh, adh, and chdhI exhibited stronger expression. The expression of many of the genes was enhanced (3hibdhII, 11.5-fold; 3hibdhI, 7.5fold; 3hpdh, 4.5-fold; adh, 2-fold; chdhI, 2.1-fold) when the cells were exposed to 3-HP during cultivation. The mRNA level of 3hpdh was the highest among the genes examined, particularly when P. denitrificans was cultured in the presence of 3-HP.
Table 2 Enzymes potentially involved in 3-hydroxypropionic acid degradation in P. denitrificans ORF no. (Annotated function)
Query enzyme name P. fluorescens Pf5/ Halomonas sp. HTNK1
3-Hydroxyisobutyrate dehydrogenase
3HIBDH
3-Hydroxyisobutyrate dehydrogenase Putative 3-hydroxyisobutyrate dehydrogenase 3-Hydroxyisobutyrate dehydrogenase 3-Hydroxyisobutyrate dehydrogenase Putative 3-hydroxypropionate dehydrogenase Putative GMC-type oxidoreductase Alcohol dehydrogenase Choline dehydrogenase Choline dehydrogenase/alkyl sulfatase
Enzyme name P. denitrificans
3HIBDHI 3HIBDHII 3HIBDHIII 3HIBDHIV DddA 3HPDH ADH CHDHI CHDHII
Size (AA)
Identity (%)
Accession no.
298
100
AAY90588
294 299 292 291 579 554 528 560 562
50 76 32 34 100 60 43 39 36
YP_007656737 YP_007659067 YP_007658212 YP_007658098 ACV84069 YP_007659112 YP_007657852 YP_007660285 YP_007657871
The potential enzymes were identified by performing a blast search with the known 3-hydroxyisobutyrate dehydrogenase from P. fluorescens and putative 3-hydroxypropionate dehydrogenase from Halomonas sp. HTNKK1 as queries. The sequences were deposited in GenBank and the accession no. is listed in the table
Appl Microbiol Biotechnol
Fig. 1 Relative mRNA levels for the potential 3-hydroxypropinatedegrading genes in wild type P. denitrificans ATCC 13867. P. denitrificans cells were cultivated in M9 medium supplemented with 3-HP at 25 mmol/ L (gray bar) or without 3-HP (black bar) and harvested during the exponential growth period. The standard deviation of the measurements of the mRNA levels was
