Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2017 www.elsevier.com/locate/jbiosc

Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance Yukinori Tanaka,1, z Ken Kasahara,2, z Yutaka Hirose,2 Yu Morimoto,1 Masumi Izawa,1 and Kozo Ochi1, * Department of Life Sciences, Hiroshima Institute of Technology, Saeki-ku, Hiroshima 731-5193, Japan1 and Chitose Laboratory Corp., Biotechnology Research Center, Nogawa, Miyamae-ku, Kawasaki 216-0001, Japan2 Received 8 March 2017; accepted 6 May 2017 Available online xxx

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5 g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C. saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Keywords: Butanol; Ribosome engineering; rpsL mutation; rpoB mutation; Tolerance; Clostridium saccharoperbutylacetonicum]

Drug resistance mutation technology, often called ribosome engineering (1,2), has been widely utilized for microbial strain improvement, especially in the overproduction of antibiotics and the activation of silent genes involved in bacterial secondary metabolite production (3e5). Ribosome engineering is characterized by simplicity, consisting of the isolation of spontaneously developed drug-resistant mutants. Therefore, this method does not require the induction of mutagenesis or any genomic information and provides a rational approach of enhancing bacterial capabilities for industrial applications. To date, however, few reports have focused on utilization of ribosome engineering in the production of primary metabolites (3). The notion of ribosome engineering was derived from results obtained with Streptomyces lividans. Although S. lividans normally does not produce antibiotics, it possesses dormant antibiotic biosynthesis genes (6). The introduction of ribosome engineering into Streptomyces strains isolated from soil samples was found to activate the dormant abilities of these bacteria to produce antibiotics (7). Furthermore, the bacterial alarmone ppGpp (guanosine 50 -diphosphate 30 -diphosphate), produced on ribosomes in response to nutrient starvation, was found to bind to RNA polymerase, eventually initiating the production of antibiotics (8e10). These observations led to the development of a ribosome

* Corresponding author. Tel.: þ81 82 921 6923; fax: þ81 82 921 6961. E-mail address: [email protected] (K. Ochi). z The first two authors contributed equally to this work.

engineering technology targeting S12, RNA polymerase, and other ribosomal proteins and translation factors, thus activating or enhancing the production of secondary metabolites. Ribosome engineering technology was found applicable to strain improvement and silent gene activation, resulting in the identification of novel secondary metabolites (7,11e13), as well as to the enhancement of enzyme production and tolerance to toxic chemicals (14,15). Streptomycin-resistant (SmR) rpsL-mutant ribosomes, which carry an amino acid substitution in the ribosomal S12 protein that confers high level resistance to streptomycin, are more stable than wild-type ribosomes, indicating that increased stability may enhance protein synthesis during the late growth phase of bacteria (15e17). Increased expression of the translation factor ribosome recycling factor also contributed to the enhanced synthesis of the rpsL K88E mutant protein during the transition and stationary growth phase (18). That is, both the greater stability of the 70S ribosomes and the elevated levels of ribosome recycling factor resulting from the rpsL K88E mutation were responsible for enhanced protein synthesis during the late growth phase, with the latter being responsible for antibiotic overproduction and silent gene activation. In contrast, the activation of silent genes by rifampicin resistance (RifR) rpoB mutations in Streptomyces has been attributed, at least in part, to the increased affinity of mutant RNA polymerase for silent gene promoters (7). A recent study showed the broad applicability of the RifR rpoB mutation method to the expression of cryptic secondary metabolite-biosynthetic gene clusters (19).

1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2017.05.003

Please cite this article in press as: Tanaka, Y., et al., Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.003

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Butanol can be efficiently produced by the genus Clostridium through acetone-butanol-ethanol fermentation. Commercial butanol fermentation utilizes the classical solventogenic strains of clostridial strains, including Clostridium acetobutylicum, C. beijerinckii, C. saccharobutylicum, and C. saccharoperbutylacetonicum (20). Several challenges to the industrial production of butanol must still be overcome, such as overall cost competitiveness and development of higher performance strains with greater butanol tolerance (21,22). Butanol is highly toxic to all microorganisms, including clostridia, and at high concentrations can lead to reduced substrate consumption and decreased overall cellular metabolism. Overcoming butanol toxicity has become a major challenge in the economic production of butanol (23e26). Details of metabolic pathways and biochemistry of clostridial species are well documented in review papers (24,27,28). Employing random chemical mutagenesis and butanol exposure, butanol-tolerant strains of C. acetobutylicum were selected and exhibited enhanced butanol production. Genome shuffling and selection in the presence of high butanol concentrations was also effective (29,30). Several studies have demonstrated that butanol production can be improved by manipulating genes other than those directly involved in acetonebutanol-ethanol fermentation pathways (reviewed by Moon et al. (20) and Zheng et al. (31)). However, clostridial strains are, in general, rather difficult to genetically engineer. Strain development through metabolic engineering has also been explored for enhanced production of butanol (32e34). After 100 years of investigations, level of butanol production reached 25.7 g/L by batch fermentation (35e39) (reviewed by Moon et al. (20)). Although several studies have assessed the ability of ribosome engineering technology to enhance ethanol and butanol production, this study assessed the applicability of ribosome engineering technology (and sequential introduction of mutations conferring butanol tolerance) to improve butanol production as a representative example because of its industrial importance.

MATERIALS AND METHODS Bacterial strains and culture conditions The wild-type strain C. saccharoperbutylacetonicum ATCC 27021 (N1-4) was used for acetone-butanol

fermentation. C. saccharoperbutylacetonicum was grown in tryptone-yeast extractacetate (TYA) medium (40) at 30  C for 1 day under anaerobic conditions, using an anaeropack system (Mitsubishi Gas Chemical Co., Tokyo, Japan). TYA medium was composed of (per liter); 20 g glucose, 2 g yeast extract, 6 g tryptone, 3 g ammonium acetate, 0.3 g MgSO4$7H2O, 0.5 g KH2PO4, and 10 mg FeSO4$7H2O. Subsequently, 10% (v/v) volume of culture was inoculated into fresh TYA medium, followed by incubation for 4 days under anaerobic conditions. After 1 day cultivation, glucose was added to a final concentration of 6% (w/v) to enhance acetone-butanol fermentation. Butanol, acetone and ethanol produced in the medium were measured as described below. Determination of minimum inhibitory concentrations To determine minimum inhibitory concentrations (MICs) of various drugs,10 ml of full grown culture of C. saccharoperbutylacetonicum was spotted onto TYA and inoculation plates, respectively, containing various concentrations of drug. Plates containing C. saccharoperbutylacetonicum were incubated at 30  C for 3 days. The minimum drug concentration able to fully inhibit growth was defined as the MIC. Resistance to butanol and acetone was determined by inoculating 1% (v/v) of full grown culture into TYA liquid medium containing various concentrations of butanol or acetone. After incubation for 3 days at 30  C, the presence or absence of growth was determined. Mutagenesis and screening procedure The disparity mutagenesis cocktail (Chitose Laboratory Corp., Kawasaki, Japan), which generates point mutations at higher efficiencies on dispersive chromosome areas than general mutagens (41e43), was used to introduce mutations into C. saccharoperbutylacetonicum. The disparity mutagenesis method allows accumulation of the replication errors during the overnight growth in liquid culture. Strains were cultivated to full growth in 5 ml of culture medium containing the mutagen cocktail. C. saccharoperbutylacetonicum mutants resistant to butanol, streptomycin, rifampicin or fusidic acid were selected using TYA plates containing each chemical, which were incubated at 30  C for 3e7 days. The antibiotic concentrations used for mutant isolation are described in Table 1. Mutations in the rpoB and rpsL genes were determined by DNA sequencing using the primers listed in Table S1. Measurement of butanol, ethanol, and acetone Culture broth of C. saccharoperbutylacetonicum was centrifuged at 15,000 g for 1 min, and the supernatant was analyzed directly by gas chromatographyemass spectrometry (GC/MS). The analytical conditions were: device, Shimadzu GC-2010; column, GL Science Inert Cap WAX (15 m, inner diameter 0.25 mm); oven temperature, increased from 40  C to 240  C at a rate of 10  C/min; injector temperature, 220  C carrier gas, helium; flow rate, 2.29 ml/min; detection, m/z between 29 and 1090 using a Shimadzu GCMSQP2010; ion source temperature, 200  C; interface temperature, 220  C. Transcriptional analysis by real-time qPCR Total RNAs were extracted and purified from cells grown for the indicated times, using Isogen reagent (Nippon Gene) according to the manufacturer’s protocol. Real-time qPCR was performed as described (44). Following the removal of contaminating DNA by incubation of 2 mg total RNA with 2 U of DNase I (Invitrogen, Carlsbad, CA, USA) for 15 min at 25  C, RNAs were reverse transcribed using a High Capacity RNA-to-cDNA Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. After terminating the reaction by incubation for 5 min at 95  C, samples were analyzed using a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) and

TABLE 1. Characteristics of mutants of Clostridium saccharoperbutylacetonicum. Strain

Butanol or antibiotic concentration used for selection

Resistance to: (mg/ml)a

Resistance to butanolb (g/L)

Streptomycin (50) Rifampicin (1) Fusidic acid (0.5)

17.8

e

e

21.1

NDd

ND

Position of mutation in rpsL or rpoB

Amino acid substitution

27021 (wild-type)

ec

KO-1255 (ButR) (selected by 8 steps of disparity mutagenesis from 27021)

Butanol in plate (23.5e35.6 g/L)

KO-1256 (ButR SmR) (selected for streptomycin resistance from KO1255)

Streptomycin (100 mg/ml)

Streptomycin (>1000)

21.9

129G/A (rpsL)

Lys43/Asn

KO-1257 (ButR RifR) (selected for rifampicin resistance from KO1255)

Rifampicin (5 mg/ml)

Rifampicin (200)

20.3

1514G/A (rpoB)

Arg505/Lys

KO-1258 (ButR FusR) (selected for fusidic acid resistance from KO1255)

Fusidic acid (5 mg/ml)

Fusidic acid (20)

20.3

ND

ND

a b c d

After incubation for 3 days on TYA solid medium. After incubation for 3 days in TYA liquid medium. Not applicable. Not detected.

Please cite this article in press as: Tanaka, Y., et al., Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.003

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Thunder Bird Syber qPCR Mix (Toyobo, Osaka, Japan). Each transcription assay was normalized relative to the corresponding transcriptional level of 16S rRNA gene. Primers used for real-time qPCR are listed in Table S1. Assay of butanol dehydrogenase and butyraldehyde dehydrogenase Butanol dehydrogenase and butyraldehyde dehydrogenase were assayed as described by Welch et al. (45) and Dürre et al. (46), respectively, with slight modifications as described bellow. For cell lysis, frozen cells (1 g) were suspended in potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 0.1 mM ZnSO4. The cell suspension was lysed by sonication (treated for 30e40 s) and treated with TurboNuclease (250 units/ ml) (Accelagen, San Diego, CA, USA) for 10 min, and the extract was centrifuged for 1 h at 15,000 g to remove cell debris and unlysed cells. Assay of butanol dehydrogenase was performed at 30  C in 50 mM 2-(N-morpholino) ethanesulfonic acid solution (pH 6.0) containing 0.4 mM NADH. Butyraldehyde suspended in methanol was added at a final concentration of 50 mM. The assay volume was 1 ml, and the reaction was initiated by adding enzyme. Methanol without butyraldehyde was used as a control. Changes in absorption at 340 nm were measured. One unit (U) of enzyme activity was defined as the amount of enzyme that oxidized 1 mmol of NADH per minute. Assay of butyraldehyde dehydrogenase was performed at 30  C in 50 mM 2-(Nmorpholino) ethanesulfonic solution (pH 6.0) containing 0.4 mM NADH, 1 mM dithiothreitol, and 70 mM semicarbazide. Butyryl-CoA suspended in water was added at a final concentration of 0.2 mM. The assay volume was 0.5 ml, and the reaction was initiated by adding enzyme. Water without butyryl-CoA was used as a control. Changes in absorption at 340 nm were measured. One unit (U) of enzyme activity was defined as the amount of enzyme that oxidized 1 mmol of NADH per minute. Statistic treatment of data The data from experiments of butanol production and of transcriptional analysis were treated statistically, showing with p-values (n ¼ 3). p-values lower than 0.05 and 0.01 are shown by asterisk and double asterisk, respectively, on each figure. p-values higher than 0.05 are without symbols.

RESULTS Development of C. saccharoperbutylacetonicum mutants with increased butanol tolerance Resistance of an organism to butanol is crucial for microbial butanol overproduction because butanol, unlike ethanol, is highly cytotoxic, resulting in cell death (26,47). We, therefore, first attempted to increase the resistance of C. saccharoperbutylacetonicum to butanol by sequential mutagenesis. The disparity mutagenesis method has been shown to induce point mutations at high efficiencies on dispersive chromosome areas (41e43). C. saccharoperbutylacetonicum wildtype strain 27021 was subjected to disparity mutagenesis, with mutant strains selected for increased butanol tolerance (ButR) on TYA plates containing 2.35% butanol. The strain with the highest level of butanol tolerance was subsequently subjected to a repeat disparity mutagenesis step, followed by selection of mutants with increased butanol tolerance. Following eight steps of disparity mutagenesis and strain selection, strain KO-1255 (ButR), which

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was able to grow in the presence of 3.56% butanol, was obtained (Fig. 1). Strikingly, strain KO-1255 displayed not only increased butanol tolerance but markedly enhanced butanol production, a 48% higher (p ¼ 0.001) butanol titer than the wild-type (Fig. 2B), although its growth rate and final biomass were somewhat lower (Fig. 2A). Butanol yields (g butanol/g glucose) were 13.2% and 18.9% in the wild-type strain 27021 and KO-1255 (ButR), respectively. These results prompted us to further enhance butanol titer using ribosome engineering. Effects of introducing the SmR, RifR or FusR mutation on butanol production Ribosome engineering technology, which can activate bacterial secondary metabolism, has focused on certain mutations in the rpsL and rpoB genes, which encode the ribosomal protein S12 and RNA polymerase b-subunit, respectively (1,3,5). As these rpsL and rpoB mutants can be readily detected among streptomycin-resistant (SmR) and rifampicin-resistant (RifR) mutants, respectively, we isolated, starting from KO-1255 (ButR), mutants resistant to streptomycin or rifampicin (see Materials and methods). Mutants (FusR) resistant to fusidic acid (an inhibitor of peptidyl-tRNA translocation by binding to elongation factor EF-G) were also isolated since a certain mutation conferring resistance to fusidic acid results in overproduction of antibiotic actinorhodin in Streptomyces coelicolor (13). Assessment of these drug-resistant mutants (15e20 isolates resistant to each drug) for butanol production revealed that several mutants produced butanol in higher titer than the parent strain KO-1255. These strains were designated KO-1256 (SmR), KO-1257 (RifR) and KO-1258 (FusR) as the representative mutants (Table 1). Of these strains, KO-1256 produced the highest titer of butanol, 16.5 g/L (Fig. 2B), 6.3% higher (p ¼ 0.01) than that of the parent strain KO1255, despite the growth of KO-1256 being markedly impaired (Fig. 2A). In contrast, KO-1257 and KO-1258 produced slightly higher (p > 0.05) titers of butanol than KO-1255. Butanol yield (g butanol/g glucose) of KO-1256 (ButR SmR) was 19.8%. Importantly, KO-1255 (ButR) and KO-1256 (ButR SmR) both displayed a significantly higher (p < 0.01) per cell ability to produce butanol (i.e., butanol/OD600), suggesting that the ButR and SmR mutations markedly activated butanol biosynthesis (Fig. 2C). Characterization of mutants with increased butanol production As expected from previous studies (3,5), KO-1256 with streptomycin resistance had a mutation (Lys43/Asn) in the rpsL gene encoding ribosomal protein S12, whereas KO-1257 with rifampicin resistance had a mutation (Arg505/Lys) in the rpoB gene encoding RNA polymerase b-subunit (Table 1). Resistance levels to butanol were less pronounced in liquid culture than on

FIG. 1. Diagram of strain improvement by disparity mutagenesis. C. saccharoperbutylacetonicum wild-type strain 27021 and its progenies were subjected, in each step, to increasing concentrations (23.5e35.6 g/L) of butanol after disparity mutagenesis, eventually yielding the butanol-tolerant (ButR) strain KO-1255.

Please cite this article in press as: Tanaka, Y., et al., Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.003

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J. BIOSCI. BIOENG., agar plates, although KO-1255, KO-1256 and KO-1257 all showed greater tolerance to butanol (Table 1). In addition, KO-1255 (ButR) and KO-1256 (ButR SmR) showed increased tolerance to acetone (Table S2) and produced greater amounts of acetone and ethanol, consuming greater amounts of glucose (Fig. S1). Notably, KO-1255 (ButR) and KO-1256 (ButR SmR) both displayed increased sensitivity to vancomycin, but not to erythromycin, fusidic acid or penicillin (Table S2). Although the wild-type strain 27021 produced spores (2% spores/viable cell titer) when cultured for 4 days in TYA medium containing a reduced amount (0.4%) of glucose, KO-1255 and KO-1256 showed less ability to form spores (1/10 and 1/100 that of wild-type, respectively), as determined microscopically. The importance of butanol tolerance (ButR) for butanol overproduction was confirmed by cultivating the wild-type (27021) and ButR mutant (KO-1255) strains in the presence of added butanol. When strains were grown, as same as the legend to Fig. 2, in TYA medium containing 1.2% (v/v) butanol, which was added at the inoculation time, the wild-type strain produced butanol in low titer (1.7 g/L), whereas the ButR strain KO-1255 produced 5.7-fold higher (p < 0.01) titer (9.7 g/L) of butanol, accentuating the importance of butanol tolerance in enhancing the butanol production. Transcriptional analysis of genes involved in butanol fermentation The fermentation metabolism of clostridia can be divided into two phases. Exponentially growing cells mainly generate the acids butyrate and acetate (acidogenic phase), whereas stationary cells take up these acids, converting them to butanol and acetone, respectively. Many genes of C. acetobutylicum are involved in this process (Fig. 3), with the genes encoding hbd, crt, bcd, ctfA, and ctfB clustered on the chromosome of C. acetobutylicum, forming the bcs operon. The bifunctional aldehyde/alcohol dehydrogenase encoded by adhE is essential for the initiation of solvent formation (reviewed by Moon et al. (20) and Lütke-Eversloh and Bahl (21)). We therefore utilized realtime qPCR to assess the transcription of the adc, adhE1, bcd1, bdhA, buk1, crt, ctfA, hbd1, pfl1, ptb1, and thlA1 genes of wild-type and mutant strains (Fig. 3, Table 2) (48). Several genes involved in amino acid synthesis (aspB, glnA), nucleotide synthesis (purC, pyrB), ribosomal protein (rplD, rpsL) and glycolysis (glcK1, pgi) (Table 2) were analyzed as a reference. Strains were grown in TYA liquid medium to late-exponential phase (24 h for wild-type, 48 h for KO-1255, and 72 h for KO-1256), and total RNA was prepared and real-time qRCR performed. As summarized in Fig. 4A, the expression of genes involved in acetone-butanol fermentation was markedly enhanced in the mutants KO-1255 (ButR) and KO1256 (ButR SmR), except for hbd1, which is involved in the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. Enhancement of expression was most pronounced for bcd1, bdhA, buk1, crt, ptb1 and thlA1 with all increasing 7e10-fold, in good agreement with a higher yield of butanol (Fig. 2C). Except for glcK1, the other genes involved in primary metabolism were also markedly activated at the transcriptional level in mutant cells (Fig. 4B). Although the expression analysis was conducted only at one growth point (i.e., late-exponential phase), the results suggest that metabolism was generally activated in the mutants, especially in KO-1256 (ButR SmR).

FIG. 2. Growth and butanol production by C. saccharoperbutylacetonicum wild-type and mutant strains. Strains were grown in TYA medium at 30  C for 4 days as described in Materials and methods. (A) Growth of wild-type (27021) and mutant (KO-1255 and KO-1256) strains. (B) Butanol production by the wild-type and mutant strains. (C) Butanol production as a function of cell mass (OD600). Doube asterisk indicates the pvalue lower than 0.01.

Activity of enzymes involved in butanol synthesis As the conversion of butyryl-CoA to butyraldehyde and butanol is the key route in butanol synthesis (Fig. 3) (49), we measured the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB) involved in these steps. Strains were grown in TYA liquid medium to late-exponential phase (24 h for 27021, 48 h for KO-1255, and 72 h for KO-1256), and enzyme activity was determined as described in Materials and methods. Strikingly, mutants KO-1255 (ButR) and KO-1256 (ButR SmR)

Please cite this article in press as: Tanaka, Y., et al., Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.003

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FIG. 3. Outline of acetone-butanol fermentation pathway in C. saccharoperbutylacetonicum. The gene products are listed in Table 2. The figure was drawn based on Schiel-Bengelsdorf et al. (22).

TABLE 2. Genes of C. saccharoperbutylacetonicum analyzed. Gene

a

Gene product

Function

adc

Putative acetoacetate decarboxylase adhE1 Butyraldehyde dehydrogenase aspB bcd1

Aspartate aminotransferase Acyl-CoA dehydrogenase, shortchain specific bdhA NADH-dependent butanol dehydrogenase buk1 Butyrate kinase crt

3-Hydroxybutyryl-CoA dehydratase ctfA Butyrate-acetoacetate CoAtransferase subunit A glcK1 Glucokinase glnA1 Glutamine synthetase hbd1 3-Hydroxybutyryl-CoA dehydrogenase pfl1 Formate acetyltransferase

pgi pyrB ptb1 purC

Glucose-6-phosphate isomerase Aspartate carbamoyltransferase Phosphate butyryltransferase

Phosphoribosylaminoimidazolesuccinocarboxamide synthase rplD 50S ribosomal protein L4 rpsL 30S ribosomal protein S12 thlA1 Acetyl-CoA acetyltransferase a

Acetone-butanol-ethanol fermentation pathway Acetone-butanol-ethanol fermentation pathway Biosynthesis of amino acids Acetone-butanol-ethanol fermentation pathway Acetone-butanol-ethanol fermentation pathway Acetone-butanol-ethanol fermentation pathway Acetone-butanol-ethanol fermentation pathway Acetone-butanol-ethanol fermentation pathway Glycolysis/Gluconeogenesis Biosynthesis of amino acids Acetone-butanol-ethanol fermentation pathway Acetone-butanol-ethanol fermentation pathway Glycolysis/Gluconeogenesis Pyrimidine biosynthesis Acetone-butanol-ethanol fermentation pathway Purine biosynthesis Ribosomal protein Ribosomal protein Acetone-butanol-ethanol fermentation pathway

Gene names are from Del Cerro et al. (48).

showed, in this order, markedly higher (p < 0.01) enzyme activity than the wild-type strain (Fig. 5), in agreement with the activation of the relevant genes, adhE and bdhAB, at the transcriptional level (Fig. 4A).

DISCUSSION Ribosome engineering is characterized by its feasibility, allowing the identification of antibiotic-overproducing mutants among drug-resistant isolates at a high frequency of 10e30% (3,5). Combinations of various ribosome engineering can further enhance bacterial productivity, as demonstrated, for example, by

introducing eight ribosome engineering into S. coelicolor or triple ribosome engineering into Bacillus subtilis (13,50). The mutant C8 resistant to eight drugs produced huge amounts (1.63 g/L) of the antibiotic actinorhodin, 180-fold higher than that produced by the wild type 1147 strain. More recent study demonstrated that acquisition of certain erythromycin resistance (EryR) mutation or lincomycin resistance (LinR) mutation, resulting in formation of the hybrid gene linR, renders cells more active in secondary metabolism of S. coelicolor (51,52). Combined with the results of previous studies on ethanol and butanol production (29,53,54), the present study demonstrated that ribosome engineering is effective in activating not only secondary metabolism but primary metabolic activity, such as the production of butanol, especially when assessed on a per cell activity basis (i.e., yield). Although ribosome engineering often results in somewhat reduced growth rate, enhancement of growth rate and final biomass of the mutants by fermentation technology or gene engineering may further enhance the production of butanol. The marked increases not only in gene transcription but in the activity of enzymes involved in butanol fermentation by the mutants indicate that ribosome engineering can enhance primary metabolite production. In Nonomuraea terrinata strain S58, a strain with a duplicated (rpoB(S) þ rpoB(R)) rpoB showed much greater growth, sporulation and antibiotic production than the strain S114 with a single (rpoB(R)), especially under stressful conditions, suggesting the physiological significance of rpoB duplication (55). Construction of merodiploids harboring both mutant-type and wild-type rpoB (or rpsL) genes may therefore resolve this issue because the presence of the wild-type gene would overcome the reduced growth rate and/or fitness cost caused by the rpoB or rpsL mutation (3). In the butanol fermentation, cell tolerance to butanol was readily increased using the disparity mutagenesis method, perhaps due to its ability to generate mutations at high efficiencies in the dispersive chromosome area (Fig. 1). It is an important fact that acquisition of butanol tolerance gave rise to marked increase in butanol production (Fig. 2). This concomitant increase may signify the close genetic and physiological interrelationship between butanol tolerance and butanol production, as has been pointed out previously (20,23,26). Stationary events in gram-positive bacteria, including B. subtilis and clostridia, are regulated by Spo0A, a response regulator (22,56e58). A spo0A knockout strain of C. acetobutylicum neither sporulates nor produces solvents, while overexpression of spo0A results in increased tolerance and prolonged metabolism (59). Asporogenous solvent-producing strains

Please cite this article in press as: Tanaka, Y., et al., Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.003

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FIG. 4. Transcription analysis of C. saccharoperbutylacetonicum mutant strains. Strains were grown in TYA liquid medium to late-exponential phase (24 h for wild-type, 48 h for KO1255, and 72 h for KO-1256). Total RNA was prepared and real-time qPCR performed as described in Materials and methods. (A) Transcriptional analysis of genes involved in acetone-butanol fermentation. (B) Transcriptional analysis of genes involved in primary metabolism (glycolysis, ribosomal proteins, and biosynthesis of amino acids and nucleic acids). The gene products are listed in Table 2. The error bars indicate the standard deviations of the means of three samples. Asterisk and double asterisk indicate the p-values lower than 0.05 and 0.01, respectively.

are, however, of great importance for butanol fermentation, as spores are metabolically inactive. These findings are supported by the reduced, but not completely abolished, sporulation of KO-1255 (ButR) and KO-1256 (ButR SmR). Moreover, KO-1255 and KO-1256 showed increased sensitivity to vancomycin. As vancomycin is a glycopeptide antibiotic that inhibits peptidoglycan synthesis in cell wall formation (60), KO-1255 and KO-1256 may have altered cell wall structures. This alteration may, in turn, contribute to their enhanced tolerance to butanol. In this regard, butanol toxicity and tolerance are reportedly known to be associated with effects on the cell membrane’s biophysical properties (61). Certain streptomycin-resistant (SmR) rpsL-mutant ribosomes (e.g., K43N, K88E), carrying an amino acid substitution in the ribosomal protein S12 that confers a high level of resistance to streptomycin, are characterized by greater stability than wild-type ribosomes, indicating that increased stability could enhance protein synthesis during the late growth phase (17,18,62). Strikingly, mutants of C. saccharoperbutylacetonicum, especially KO-1256 (ButR SmR), showed marked enhancement of the expression of genes

involved in representative primary metabolism at the level of transcription (Fig. 4B). These results may indicate that metabolism in SmR rpsL mutant cells was fully activated during the late growth phase (including late-exponential growth phase), eventually resulting in butanol overproduction. In this regard, it is reported recently that certain antimicrobial drug resistance (e.g., SmR, RifR) effects broad changes in metabolomics and physiological phenotype in addition to secondary metabolism (63,64). Apart from the solvent production, C. saccharoperbuty lacetonicum produces detectable amounts of organic acids in addition to solvents, mainly acetate and butyrate (65) (Fig. 3). Although we did not measure these organic acids in the present study, it is notable that the expression of buk1 and ptb1, the genes devoted to butyrate synthesis (Fig. 3), was also enhanced markedly at the transcriptional level in the ButR and ButR SmR mutants (Fig. 4A), denoting the widespread metabolic effects of ribosome engineering. Of the SmR rpsL mutations conferring high level resistance to streptomycin, three mutations, K43N (Lys43/Asn), K88E (Lys88/Glu), and K88R (Lys88/Arg), were most often found to be

Please cite this article in press as: Tanaka, Y., et al., Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.003

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Promotion of Basic and Applied Research for Innovations on Biooriented Industry) and MEXT-Supported Program for Strategic Research Foundation at Private Universities, 2014 to 2016 (grant S1413002). The authors thank Yasuko Tanaka and Izumi Yamada for providing experimental assistance. Y.T. conducted mutation work, transcriptional analysis and enzyme assays. K.K. and Y.H. constructed cultivation conditions for butanol production and assayed butanol produced in the medium. Y.M. and M.I. supported the study in various experiments. K.O. designed the study and wrote the article. References

FIG. 5. Activity of butanol dehydrogenase and butyraldehyde dehydrogenase in wildtype (27021) and mutant (KO-1255 and KO-1256) C. saccharoperbutylacetonicum strains. Strains were grown to late-exponential phase (24 h for 27021, 48 h for KO1255, and 72 h for KO-1256) in TYA liquid medium. Enzyme activity was determined as described in Materials and methods. Double asterisk indicates the p-value lower than 0.01.

associated with antibiotic overproduction in actinomycetes (3). Similarly, the K43N mutation was associated with increases in ethanol production by Klebsiella variicola (54) and butanol production by C. saccharoperbutylacetonicum (this study). The rpsL mutations (e.g., K43N) may therefore be widely effective in eubacteria at enhancing primary metabolite production. In contrast, of the RifR rpoB mutations conferring high level resistance to rifampicin, two, i.e., H437Y (His437/Tyr) and H437R (His437/Arg), were most often associated with antibiotic overproduction (3). The rpoB R505K (Arg505/Lys) mutation, which slightly enhanced butanol production, corresponds to mutations at position Arg485 and Arg440 in B. subtilis and S. coelicolor, respectively. Certain mutations at this position (e.g., R440C, R440H, R485H) have been reported to enhance antibiotic production by these organisms (reviewed by Ochi (3)). Taken together, these findings emphasize the importance and efficacy of specific SmR and RifR mutations for the overproduction of microbial metabolites, although establishment of casual relationship between the identified mutations and the observed overproduction of butanol, for example by gene replacement experiments, is required. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2017.05.003. ACKNOWLEDGMENTS This work was supported by grants to K. O. from the National Agriculture and Food Research Organization (Program for

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Please cite this article in press as: Tanaka, Y., et al., Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.05.003