Respiration-deficient mutants of Zymomonas mobilis show improved growth and ethanol fermentation under aerobic and high temperature conditions

Journal of Bioscience and Bioengineering VOL. 111 No. 4, 414 – 419, 2011 www.elsevier.com/locate/jbiosc

Respiration-deficient mutants of Zymomonas mobilis show improved growth and ethanol fermentation under aerobic and high temperature conditions Takeshi Hayashi,1,⁎ Yoshifumi Furuta,2 and Kensuke Furukawa1 Department of Food and Fermentation Science, Faculty of Food and Nutrition, Beppu University, Kitaishigaki 82, Beppu, Oita 874-8501, Japan 1 and Research Laboratory, Sanwa Shurui Co. Ltd., Usa, Oita 879-0495, Japan 2 Received 22 September 2010; accepted 7 December 2010 Available online 13 January 2011

Respiration-deficient mutant (RDM) strains of Zymomonas mobilis were isolated from antibiotic-resistant mutants. These RDM strains showed various degrees of respiratory deficiency. All RDM strains exhibited much higher ethanol fermentation capacity than the wild-type strain under aerobic conditions. The strains also gained thermotolerance and exhibited greater ethanol production at high temperature (39°C), under both non-aerobic and aerobic conditions, compared with the wild-type strain. Microarray and subsequent quantitative PCR analyses suggest that enhanced gene expression involved in the metabolism of glucose to ethanol resulted in the high ethanol production of RDM strains under aerobic growth conditions. Reduction of intracellular oxidative stress may also result in improved ethanol fermentation by RDM strains at high temperatures. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Zymomonas mobilis; Aerobic fermentation; Ethanol fermentation; Respiration-deficient mutant; Thermotolerant mutant]

Zymomonas mobilis is a gram-negative facultatively anaerobic bacterium. This organism can ferment certain sugars to ethanol via the Entner–Doudoroff (ED), glyceraldehyde-3-phosphate-to-pyruvate (GP), and pyruvate-to-ethanol (PE) pathways (1,2). Z. mobilis possesses an incomplete TCA cycle, lacking certain genes for 2-oxoglutarate dehydrogenase complex and malate dehydrogenase (3–5), but features strong ED–GP pathways (6). This organism also exhibits a high uptake rate of sugars, fermenting these to ethanol at specific rates (1). In addition, the production of ethanol approaches theoretical maximum yields (97%), while only 90–93% can be achieved for Saccharomyces cerevisiae (3). These are advantages for generating higher ethanol production. Thus, Z. mobilis is a promising alternative microorganism to S. cerevisiae as an ethanol fuel producer. The complete components of the respiratory chain remain to be clarified in Z. mobilis. Recently, the ndh gene (ZMO1113) was found, which is the only component of the functional respiratory NAD(P)H dehydrogenase in Z. mobilis (7). Furthermore, the bd-type ubiquinol oxidase genes (ZMO1571 and ZMO1572) produced the sole terminal

Abbreviations: DO, dissolved oxygen; ED pathway, Entner–Doudoroff pathway; GP pathway, glyceraldehyde-3-phosphate-to-pyruvate pathway; HSP, heat shock protein; OD660, optical density at 660 nm; PCR, polymerase chain reaction; PE pathway, pyruvate-to-ethanol pathway; pO2, dissolved oxygen percent saturation; qPCR, quantitative PCR; RDM, respiration-deficient mutant. ⁎ Corresponding author. Tel.: + 81 977 66 9630; fax: + 81 977 66 9631. E-mail address: [email protected] (T. Hayashi).

oxidase (8). The availability of the Z. mobilis genome sequence (5) allows us to search the candidate gene sequences for respiratory chain components. Twenty-eight candidate genes were identified in the genome, although genes of cytochrome c oxidase (electron transport complex IV) could not be ascertained (5,8). The structure and function of the respiratory chain are important because these may provide clues to improving ethanol production. Respiration-deficient mutant (RDM) strains are best characterized in yeasts (9). These mutants exhibited a higher rate of fermentation and higher yield of ethanol (10–12). Such RDM yeast strains are known as “petite mutants” (9). The petite mutants spontaneously appear in ordinary yeast cultures, although some chemical agents, such as acriflavine, increase the frequency of mutation. Unlike in yeast, the method for obtaining RDM strains has not been established for Zymomonas, except for the ndh::cmr strain, which was generated artificially (7). Fermentation at high temperatures reduces cooling costs and prevents contamination by mesophilic microorganisms. Thus, thermotolerant Z. mobilis could be beneficial for ethanol fermentation at high temperatures. Sootsuwan et al. successfully isolated three strains of thermotolerant Z. mobilis in Thailand (13). One such strain, TISTR 405, exhibits high growth activity and ethanol fermentation at 39°C. Here, we report the RDM strains of Z. mobilis for the first time, and show high ethanol fermentation even under aerobic and high temperature conditions. Also discussed here are the results of microarray analyses of the RDM strain in comparison with the wildtype (wt) strain.

1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.12.009

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Strains and culture media Z. mobilis ZM6 (ATCC 29191) was used as the wt strain, from which various RDM strains were obtained (Table 1). Cellular growth and ethanol production were examined using medium containing 0.5% (w/v) yeast extract (Wako Pure Chemical Industries, Osaka) and 2% (w/v) glucose (Sigma-Aldrich, St. Louis, MO, USA); for solid medium, 1.5% (w/v) agar (Kishida Chemical, Osaka, Japan) was used. Antibiotics, such as streptomycin sulfate (Sm), gentamicin sulfate (Gm), and rifampicin (Rif), were purchased from Wako Pure Chemical Industries, and kanamycin monosulfate (Km) was purchased from Sigma-Aldrich. Isolation of RDM strains The antibiotic-resistant mutants were isolated from the wt strain of Z. mobilis ZM6. The wt strain of a stationary phase culture was inoculated into the liquid media at 1/60 volume, or 100 μL was spread onto the solid media containing various antibiotics. The minimum inhibitory concentrations (MIC) of the wt strain in liquid media and solid media were as follows: Rif, 10 μg/mL and 5 μg/mL; Sm, 300 μg/mL and 150 μg/mL; Gm, 15 μg/mL and 10 μg/mL; Km, 30 μg/mL and 5 μg/mL, respectively. The isolated resistant strains were repeatedly and routinely grown on both liquid and solid media with enhanced concentrations of the respective antibiotics to obtain mutants with higher resistance. Furthermore, double antibiotic-resistant mutants such as Sm/Km, Sm/Rif, Sm/Gm, Rif/Km, and Rif/Gm were also isolated on both liquid and solid media. The various mutant strains were cultured overnight in the liquid media without antibiotics, and then 100 μL of each culture was spread onto three types of solid media containing ethanol and glucose [4% (v/v) ethanol/16% (w/v) glucose, 5%/16%, and 5%/14%, respectively]. The solid media were sealed and incubated at room temperature until colonies appeared. Aerobic and non-aerobic cultivation of RDM strains Non-aerobic cultivations (3 mL) were performed without shaking in test tubes (16.5 φ mm × 165 mm) at 30°C and 39°C (high temperature). Aerobic cultivations were carried out in the same manner, but these were shaken at 220 rpm. The results obtained here are expressed as the means of at least three experiments. Aerobic batch cultures were carried out with a 2-L working volume in a 5-L jar fermenter (Takasugi, Tokyo, Japan) under 300 rpm stirring at 30°C; dissolved oxygen (DO) was continuously monitored. Overnight seed cultures were inoculated for use in all growth experiments. Cell growth was monitored by optical density at 660 nm (OD660). Analytical methods pO2 (dissolved oxygen percent saturation) was measured by a Galvanic-type oxygen electrode (ABLE & Biott, Tokyo, Japan). Overnight cultures (100 mL) were centrifuged and washed in 10 mM phosphate buffer (pH 6.9). Next, the cells were resuspended in 1 L of the same buffer (OD660 0.1–0.2), and then transferred to the 5-L jar fermenter (Takasugi). The oxygen consumption of the cells was measured using the oxygen electrode after the addition of 2% (w/v) glucose at 30°C. Cell concentration was determined as OD660, and dry cell mass was calculated by reference to a calibration curve. The results are expressed as the means of four replicates. Ethanol concentration was determined by gas chromatography (GC-2014, Shimadzu, Kyoto, Japan). The column was a coiled column (2 m by 3 mm internal diameter) packed with a Porapak Q (Waters, Milford, MA, USA), and used at 155°C. The ethanol concentration was calculated by reference to a calibration curve. Preparation of total RNA Total RNA for the DNA microarray experiments was extracted from the shaking cultures of wt and RDM-4 strains grown at 30°C, a nonaerobic culture of wt strain grown at 30°C, and a non-aerobic culture of RDM-4 strain grown at 38°C. Because of the low yield of total RNA extracted at 39°C, the hightemperature condition was adopted as 38°C. Cells were grown until the late logarithmic phase and were immediately treated with RNAprotect bacterial reagent (Qiagen, Valencia, CA, USA) for the stabilization of RNA. Total RNA was extracted with an RNeasy midi kit (Qiagen) and concentrated with an RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Extracted RNA was quantified spectrophotometrically by OD260. DNA microarray experiments The gene expression experiments using DNA microarray were performed by NimbleGen Systems, Inc. (Madison, WI, USA). Briefly, the whole-genome array of Z. mobilis was designed from the open reading frame (ORF) of Z. mobilis Zm4 strain. The arrays were composed of 60-mer oligonucleotide probes with two replicates and 18 unique probes per gene (i.e., a total of 36 probes per gene). The extracted RNA was tested for purity and integrity, and then Cy3 labeling, microarray hybridization, data acquisition, and normalization were conducted by NimbleGen. Student's t test for the expression data of each gene, followed by the Bonferroni correction for multiple testing (total of 1998 ORFs on arrays), were used to evaluate genes showing differential expression patterns (p b 0.05).

TABLE 1. Strains used in this study and the antibiotic treatments used in the isolation of these strains. Strains Zymomonas mobilis ZM6 (wt) RDM-1 RDM-4 RDM-7 RDM-8 RDM-9 LM; liquid media, SM; solid media.

Treatments for mutant isolation – Ethanol (SM 9 w/v%) Rif (LM 60 μg/mL) Sm (LM 600 μg/mL), Gm (SM 40 μg/mL) Rif (LM 60 μg/mL), Gm (SM 10 μg/mL) Sm (LM 600 μg/mL), Km (SM 10 μg/mL)

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Microarray data accession number Microarray data analyzed in this study have been deposited in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm. nih.gov/geo/) and were assigned series record GSE22355. Quantitative polymerase chain reaction (qPCR) Primers were designed by using Primer-3 software (http://frodo.wi.mit.edu/primer3/), and the nucleotide sequences were shown in Supplementary material Table S1. RNA samples stored at − 80°C (the same samples used for the DNA microarray experiments) were used for qPCR. Reverse transcriptase-polymerase chain reactions (RT-PCR) were conducted using the One-step qPCR kit (Toyobo, Osaka, Japan) with the following composition. The total reaction volume was 20 μL: 10 μL of RNA-direct SYBR Green Realtime PCR Master Mix, 0.2 μM of each primer, 50 mM Mn(OAc)2, and 20 pmol of total RNA as template. RT-PCR, including the reverse transcription (RT) and the polymerase chain reaction (PCR) steps, was carried out using a DNA polymerase (rTth DNA polymerase). The mixtures were first denatured at 95°C for 30 s, and then RT was carried out at 61°C for 20 min. The PCR cycle was performed in 55 cycles at 95°C for 5 s, 55°C for 10 s, and 74°C for 15 s. Reactions were performed in a Light-Cycler PCR instrument (Roche Diagnostics, Mannheim, Germany). Intracellular hydrogen peroxide assay Cells grown until the late logarithmic phase were harvested and lysed by sonication. The intracellular hydrogen peroxide level was determined by ferrous ion oxidation in the presence of a ferric ion indicator, xylenol orange (14). Fifty µL of crude cell extract was added to 950 μL of FOX reagent [100 μM xylenol orange (Sigma), 250 μM ammonium ferrous sulfate, 100 mM sorbitol, and 25 mM sulfuric acid]. The mixture was incubated at room temperature for 30 min and then centrifuged to remove any flocculated material before measuring the OD560. The hydrogen peroxide concentration was represented by nmol per mg protein.

RESULTS Isolation of mutant strains with high ethanol fermentation capacity under aerobic conditions Mutant strains possessing a high fermentation capacity under aerobic conditions were unexpectedly obtained during screening of high ethanol and glucose tolerant mutants from various antibiotics-resistant mutants (15). Spontaneous antibiotic-resistant mutants against Sm, Rif, Gm, or Km were first isolated from solid or liquid media. Double mutants, resistant to two kinds of antibiotics, were also isolated. These mutants were then inoculated onto the solid media containing glucose (14–16%) and ethanol (4–5%), and incubated at room temperature. A few colonies appeared on the media after 3 days to 3 weeks at frequencies of 2– 30 × 10− 8. The single high antibiotic-resistant strains and double antibiotic-resistant strains tended to show higher colony formation on the solid media containing glucose and ethanol. We expected that these mutants would acquire tolerance against ethanol and glucose; however, this did not turn out to be the case. These strains did not show any significant tolerance to ethanol and glucose compared with the wt strain. However, more interestingly, these mutants exhibited a much higher ethanol fermentation capacity under aerobic conditions than did the wt strain. On the other hand, a mutant strain possessing similar characteristic to those strains could also be isolated directly from the wt strain on the solid media containing only 9% (w/v) ethanol, although the isolation frequency was very low. Growth and ethanol production of mutant strains under aerobic and non-aerobic conditions The representative isolated mutants (Table 1) were further investigated in more detail for their growth and ethanol production under aerobic (shaking) and non-aerobic (nonshaking) conditions. Under non-aerobic conditions, the mutant strains exhibited similar growth and ethanol production compared to the wt strain (Figs. 1A and B). Thus, there were no remarkable differences between the wt and mutant strains in biomass yield and ethanol production under non-aerobic culture (Table 2, YX/S and YP/S, respectively). Theoretical ethanol yields of mutant strains reached 94.5–97.4%. On the other hand, the wt strain exhibited strikingly reduced growth and ethanol production under aerobic conditions (Figs. 1C and D). In contrast, the mutant strains reached maximal growth (OD660 N 1.1) under aerobic conditions (Fig. 1C). All mutant strains produced greater biomass under aerobic conditions than nonaerobic conditions (Table 2, YX/S). Furthermore, the ethanol production of these mutants was almost the same as that under the non-aerobic

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FIG. 1. Growth and ethanol fermentation of wt (open box), RDM-1 (open lozenge), RDM-4 (closed box,) RDM-7 (closed circle), RDM-8 (open circle), and RDM-9 (closed triangle) strains at 30°C under non-aerobic and aerobic conditions. Cell growth without shaking (A) and with shaking at 220 rpm (C). Ethanol production without shaking (B) and with shaking at 220 rpm (D).

condition (Fig. 1D and Table 2, YP/S). Among these mutant strains, RDM-4 and RDM-8 exhibited the highest growth and ethanol production under aerobic conditions. These strains produced a 1.7fold increase in biomass and 3-fold increase in ethanol compared to the wt strain under aerobic growth conditions (Table 2, YX/S and YP/S, respectively). Oxygen uptake of mutant strains To evaluate respiration in the mutant strains, pO2 was continuously monitored under aerobic culture conditions in a jar fermentor attached to a DO electrode. All mutant strains exhibited lower oxygen consumption than did the wt strain (Fig. 2). Among the mutant strains, RDM-4 and RDM-8 showed extremely low oxygen consumption. The specific rates of respiration, expressed as international units (μmol O2 min− 1) per mg dry weight,

are shown in Table 3. The oxygen consumption value of the wt strain (0.131 ± 0.006) was nearly equivalent to that of the previous study (0.103 ± 0.020) (7). On the other hand, RDM-4 and RDM-8 showed

TABLE 2. Cell mass and ethanol production of wt and RDM strains cultivated under non-aerobic and aerobic conditions. YX/S [g dry weight (mol glucose)− 1] Non-aerobic wt RDM-1 RDM-4 RDM-7 RDM-8 RDM-9

6.494 5.278 6.829 5.224 6.767 5.138

(± 0.352) (± 0.339) (± 0.455) (± 0.326) (± 0.349) (± 0.332)

Aerobic 5.427 7.837 8.977 7.324 9.097 6.909

(± 0.159) (± 0.497) (± 0.644) (± 0.673) (± 0.519) (± 0179)

YP/S [g ethanol (g glucose)− 1] Non-aerobic 0.483 0.483 0.498 0.483 0.494 0.490

YP/S: g ethanol synthesized per g glucose consumed. YX/S: g dry weight per mole glucose consumed. Data are means ± SEM.

(± 0.005) (± 0.011) (± 0.017) (± 0.015) (± 0.024) (± 0.007)

Aerobic 0.153 0.387 0.468 0.438 0.453 0.440

(± 0.001) (± 0.007) (± 0.012) (± 0.031) (± 0.010) (± 0.019) FIG. 2. Oxygen consumption of wt and RDM strains. The Z. mobilis strains were grown in a jar fermentor with aeration and pO2 was continuously monitored. The experiments were performed three times for each strain, with less than 10% difference in measurement.

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TABLE 3. Oxygen consumption of wt and RDM strains. QO2 (glucose) [U (mg dry weight)− 1] wt RDM-1 RDM-4 RDM-7 RDM-8 RDM-9

0.131 0.082 0.027 0.074 0.041 0.063

(±0.006) (±0.005) (±0.005) (±0.007) (±0.002) (±0.002)

QO2: mmol oxygen per minute per g dry weight.

drastically reduced oxygen consumption, as low as 20–30% of the wt strain. Oxygen consumption in RDM-7 and RDM-9 was reduced by 50%, whereas RDM-1 showed less than a 40% reduction. These results indicate that the mutant strains all exhibit respiratory deficiency to differing extents. Here, we designated these mutant strains as RDM. Growth and ethanol production of the RDM strains under aerobic and non-aerobic conditions at high temperature The RDM strains were further investigated using additional stressors such as heat, acid, base, and organic solvents (acetaldehyde and toluene) (data not shown). Among these stressors, all RDM strains showed increased thermotolerance. Thus, growth and ethanol fermentation capacity were examined under aerobic and non-aerobic conditions at higher temperatures. The wt strain grew at up to 39°C. However, the growth and ethanol production of the wt strain were much lower at 39°C than

those at 30°C (Fig. 3). Meanwhile, all RDM strains exhibited high growth and ethanol production, even at 39°C, under both non-aerobic and aerobic conditions (Fig. 3). Among the RDM strains, RDM-4 exhibited the greatest growth and ethanol production. The ethanol production of RDM-4 reached more than 8-fold of that of the wt strain at 36 h (Figs. 3B and D). The ethanol production of RDM-4, under aerobic conditions at 39°C, achieved 80% of that of the wt strain grown under non-aerobic conditions at 30°C. The value observed for RDM-4 could be higher if increased ethanol evaporation, as a result of the higher temperature (39°C) incubation with shaking, was taken into consideration. It was reported that acetaldehyde accumulation inhibits the growth and ethanol production in aerobically grown Z. mobilis (7,16,17). However, acetaldehyde was not detected in either the wt or RDM strain cultures at high temperature (39°C) (data not shown). DNA microarray analyses of high fermentation capacity under aerobic conditions and thermotolerance of RDM-4 To investigate how the RDM strains exhibit higher ethanol fermentation under aerobic conditions, DNA microarray analysis was performed. For this purpose RDM-4 was used; this mutant showed the most deficient respiration capacity, grew well at 39°C and produced ethanol well under both non-aerobic and aerobic conditions, even at 39°C. Gene expression with RNA from the cells grown at 30°C under aerobic growth conditions was compared between wt and RDM-4 strains (Supplementary material Table S2). Fifteen genes were differentially transcribed, N1.5-fold (p b 0.05), in which eight genes were up-

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FIG. 3. Growth and ethanol fermentation of wt (open box), RDM-1 (open lozenge), RDM-4 (closed box), RDM-7 (closed circle), RDM-8 (open circle), and RDM-9 (closed triangle) strains at high temperature (39°C) under non-aerobic and aerobic conditions. Cell growth without shaking (A) and with shaking at 220 rpm (C). Ethanol production without shaking (B) and with shaking at 220 rpm (D).

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regulated and seven were down-regulated in RDM-4. Among the 15 genes, three cysteine biosynthetic-related genes (metE, cysK, and cysD) and six hypothetical genes were included. Z. mobilis ferments glucose to ethanol via the ED pathway using five genes, the GP pathway using five genes, and the PE pathway using three genes (1,2). The expression of these genes from the cells grown at 30°C under aerobic growth conditions was compared between the wt and RDM-4 strains. All 13 genes examined showed 16–53% higher expression in RDM-4 than in the wt strain (Table 4). qPCR was performed for these 13 genes to confirm the microarray results. The results confirmed that the expression of all 13 genes was 15–99% higher in RDM-4 than in the wt strain under aerobic growth conditions (Table 4). To examine the mechanism of the thermotolerance of RDM strains, the gene expression of RDM-4 from the cells grown at high temperature (38°C) under non-aerobic condition was compared with that of the wt strain from the cells grown at optimum temperature (30°C) under nonaerobic condition. A total of 103 genes showed significant changes of more than 3-fold (p b 0.05); among these, 43 genes were up-regulated and 60 genes were down-regulated in RDM-4 (Supplementary material Table S3). Those up-regulated were mainly hypothetical protein genes, whereas those down-regulated were almost exclusively translationrelated genes, such as 50S and 30S ribosomal protein genes. In the Z. mobilis ZM4 genome, eight heat shock proteins (HSPs) and their related genes (ZMO0016, ZMO0336, ZMO0410, ZMO0660, ZMO0661, ZMO0989, ZMO1928, and ZMO1929) are present. These HSPs were not significantly induced by heat stress except for ZMO1928 (groES, 1.93-fold, p = 9.0E−06). Microorganisms possess certain genes to protect against oxidative stress, which include antioxidant genes encoding superoxide dismutase, catalase, thioredoxin, and glutaredoxin (18). In addition, DNA repair genes are also induced by oxidative stress (19). However, nine antioxidant genes (ZMO0918, ZMO1060, ZMO1211, ZMO1573, ZMO0070, ZMO0573, ZMO1142, ZMO1136, and ZMO0367) and 17 DNA repair genes in Z. mobilis (ZMO0199, ZMO0354, ZMO0362, ZMO0589, ZMO0663, ZMO0672, ZMO0673, ZMO0812, ZMO1114, ZMO1166, ZMO1187, ZMO1231, ZMO1582, ZMO1648, ZMO1677, ZMO1956, and ZMO1907) showed no significant alterations in response to heat stress, except for ZMO0672 (uvrC, 1.79-fold, p=1.8E−04) and ZMO0673 (recO, 2.06-fold, p=0.046). Intracellular hydrogen peroxide level of RDM strains under high temperature condition The several studies of S. cerevisiae suggested that heat stress causes oxidative stress which is major stressor under high temperature condition (18–20). To estimate the thermotolerance mechanism of RDM strains, intracellular hydrogen peroxide levels of RDM strains were measured under high temperature condition (Fig. 4). RDM-4 and RDM-8 showed significantly reduced levels of intracellular hydrogen peroxide. The levels reached to as low as 10–15% of that in the wt strain. The levels in RDM-7 and RDM-9 were 60–70%, whereas RDM-1 showed more than 90%

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activities of that in the wt strain. These results indicate that RDM strains, particular in RDM-4 and RDM-8 suffer much lower oxidative stress than wt strain under high temperature condition. DISCUSSION In this study the RDM strains were serendipitously isolated from antibiotic-resistant strains. The RDM strains were grown on solid media containing glucose and ethanol, in which the wt strain cannot form colonies. We expected from this result that RDM strains might acquire tolerance against high concentrations of ethanol and glucose. However, the RDM strains show no significant tolerance against these stressors (data not shown). The RDM of Escherichia coli and Lactococcus lactis exhibited a remarkable reduction in intracellular oxygen incorporation, resulting in the prevention of oxidative stress (21,22). The oxygen uptake experiments allowed us to postulate that the RDM strains are exposed to lower oxidative stress than the wt strain, due to a reduction in intracellular oxygen concentrations (Fig. 2 and Table 3). The RDM of yeast (petite mutants) have been well documented (9). The petite mutants are useful as ethanol producers, because the loss of carbon source to respiratory chain can be reduced (10–12). However the petite mutant is less efficient in the utilization of carbon sources than normal yeast; therefore, they grow slower than the wt strain (9). Unlike petite yeast mutants, the RDM strains of Z. mobilis grew well under both non-aerobic and aerobic culture conditions. More interestingly, the RDM strains produce similar levels of ethanol under aerobic conditions, as does the wt strain under non-aerobic conditions. Another interesting feature is that the respiratory activities are different among the RDM strains. These RDM strains exhibit various extents of growth and ethanol production, suggesting that the mutations in the respiratory chain are different from each other. Among the RDM strains, RDM-4 showed the lowest respiratory activities (Fig. 2 and Table 3) and highest ethanol production under both non-aerobic

TABLE 4. Microarray and qPCR analyses of ED, GP, and PE pathway genes.

ED pathway

GP pathway

PE pathway

a

ORF

GenBank accession no.

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Proteins

qPCR change (n-fold) a

Microarray change (n-fold)

ZMO0369 ZMO0367 ZMO1478 ZMO0368 ZMO0997 ZMO0177 ZMO0178 ZMO1240 ZMO1608 ZMO0152 ZMO1360 ZMO1236 ZMO1596

YP_162104.1 YP_162102.1 YP_163213.1 YP_162103.1 YP_162732.1 YP_161912.1 YP_161913.1 YP_162975.1 YP_163343.1 YP_161887.1 YP_163095.1 YP_162971.1 YP_163331.1

glk zwf pgl edd eda gap pgk gpm eno pyk pdc adhA adhB

Glucokinase Glucose-6-phosphate 1-dehydrogenase 6-Phosphogluconolactonase Phosphogluconate dehydratase 2-Dehydro-3-deoxy-phosphogluconate aldolase Glyceraldehyde 3-phosphate dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase Enolase Pyruvate kinase Pyruvate decarboxylase Alcohol dehydrogenase Alcohol dehydrogenase II

1.99 1.62 1.39 1.80 1.44 1.19 1.48 1.32 1.53 1.93 1.94 1.15 1.18

1.26 1.16 1.24 1.16 1.20 1.17 1.33 1.22 1.53 1.05 1.50 1.22 1.16

qPCR data represent the mean of three independent experiments.

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and aerobic conditions (Figs. 1, 3, and Table 2). Contrary to RDM-4 and RDM-8, RDM-1 showed the highest respiratory activity among RDM strains and lowest ethanol production. Thus, it is important to note that the degree of respiratory deficiency reflects the growth and ethanol fermentation, particularly at high temperatures (Fig. 3). Recently, three strains of thermotolerant Z. mobilis, TISTR 548, TISTR 550, and TISTR 551, were isolated in Thailand (13). These strains grew well at 39°C, but not at 40°C. These TISTR strains are similar to RDM-4 and RDM-8 in thermotolerance and ethanol production. However, the growth and ethanol production of TISTR strains under aerobic conditions have not been reported. We have attempted to investigate the expression of the respiratory genes of RDM-4 strain by microarray analysis. Twenty-eight possible components of the respiratory chain (8) were validated for this purpose. However, the results showed that all of these genes were fully expressed under the aerobic condition. Thus, no significant differences were found in the expression of respiratory genes between wt and RDM-4 strains, suggesting that the respiratory deficiency of RDM-4 is not due to the gene expression of at least these 28 respiratory genes examined. More study is needed to elucidate the respiratory deficiency in RDM strains, but possible explanations could be the reduced uptake of oxygen through the modified cell wall and/or mutations in certain respiratory proteins. Also considered is the poor post-transcription for certain respiratory protein(s) in the RDM strains. Kalnenieks et al. demonstrated that reduced accumulation of acetaldehyde improved aerobic growth and ethanol fermentation (7,16). In our study, no acetaldehyde was detected in aerobic cultures of the mutant strains, whereas the wt strain accumulated approximately 0.3% (data not shown). DNA microarray and the following qPCR analyses revealed that the expression of ED, GP, and PE pathway genes was higher in the RDM-4 strain than in the wt strain under aerobic growth conditions. The higher expression of 13 genes involved in the metabolism of glucose to ethanol may allow the RDM strains to improve ethanol production under aerobic growth conditions. From these results, we considered that the reduction of intracellular oxidative stress and reduced acetaldehyde accumulation in the RDM strains produces enhanced growth and higher energy flow from glucose to ethanol under aerobic growth conditions. Additionally, thermotolerance in the RDM strains is also likely to be the result of oxidative stress. Our efforts are currently focused on characterizing the genes and proteins responsible for the generation of the RDM strains in Z. mobilis. ACKNOWLEDGMENTS We thank Yukine Eto, Yukari Ono, and Ayana Sato for their valuable help in cultivation experiments. This work was supported in part by Japan Science and Technology Agency (JST) in Research for Promoting Technological Seeds. APPENDIX A. SUPPLEMENTARY DATA Supplementary materials related to this article can be found online at doi:10.1016/j.jbiosc.2010.12.009.

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