Methanol-based γ-aminobutyric acid (GABA) production by genetically engineered Bacillus methanolicus strains

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Methanol-based ␥-aminobutyric acid (GABA) production by genetically engineered Bacillus methanolicus strains Marta Irla a , Ingemar Nærdal b , Trygve Brautaset c , Volker F. Wendisch a,∗ a b c

Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany SINTEF Materials and Chemistry, Department of Biotechnology and Nanomedicine, Trondheim, Norway NTNU, Norwegian University of Science and Technology, Department of Biotechnology, Trondheim, Norway

a r t i c l e

i n f o

Article history: Received 21 June 2016 Received in revised form 14 November 2016 Accepted 27 November 2016 Available online xxx Keywords: Bacillus methanolicus Thermophile Methanol ␥-Aminobutyric acid Fed-batch fermentation

a b s t r a c t The use of methanol as a carbon source for biotechnological processes has recently attracted great interest due to relatively low price, high abundance, high purity of methanol, and the fact that it is a nonfood raw material. In this study, methanol-based production of ␥-aminobutyric acid (GABA), which is a component of drugs and functional foods and is used as monomer for production of the biodegradable plastic polyamide 4, was established using recombinant Bacillus methanolicus strains. This was achieved by heterologous overexpression of glutamate decarboxylase genes from Sulfobacillus thermosulfidooxidans (gadSt ) or Escherichia coli (gadB) in methylotrophic B. methanolicus MGA3. Strains expressing either gadSt or gadB accumulated between 0.03 and 0.4 g/L of GABA in shake flask experiments. Initially, controlled methanol fed-batch fermentations yielded low GABA concentration (0.1 g/L). However, employing a two-phase production strategy with an initial high-cell-density fermentation phase for growth and lglutamate accumulation followed by pH reduction from 6.5 to 4.6 after 27 h for enzymatic conversion of glutamate to GABA led to 90-fold increased GABA accumulation to a final titer of 9 g/L. To the best of our knowledge, this study represents the first demonstration of methanol-based production of GABA. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Gamma (␥)-aminobutyric acid (GABA) is a four-carbon nonprotein amino acid, distributed in a variety of organisms, which belong to all kingdoms of life. In bacteria, decarboxylation of lglutamate to GABA is a part of the acid stress response and possibly participates in sporulation processes (Aronson et al., 1975; Foerster and Foerster, 1973; Lin et al., 1996). In plants, GABA is involved in maintaining the C:N balance, regulation of cytosolic pH, storage of nitrogen, cell signaling and protection against various stress agents (Bouche and Fromm, 2004; Shelp et al., 1999). In mammals, GABA serves as a major inhibitory neurotransmitter and is involved in control of growth and maturation of neurons. A disrupted GABA-glutamate balance can lead to diverse disorders such as epilepsy, seizures, motoric disorders, schizophrenia, anxiety and stress (Goddard, 2016; Obata, 2013; Wong et al., 2003). Due to its tranquilizing, pain-killing and diuretic properties GABA is an interesting candidate for becoming a food additive or a drug (Bowery and Smart, 2006; Hayakawa et al., 2004). GABA is a precursor of 2-pyrrolidone, a monomer of polyamide 4 (PA4) also known as

∗ Corresponding author at: Universitätsstr. 25, 33615 Bielefeld, Germany. E-mail address: [email protected] (V.F. Wendisch).

nylon 4 which is a bio-based polymer with remarkable mechanical and thermal properties resulting from its high melting point (Kawasaki et al., 2005). PA4 is degraded in soil and activated sludge by diverse microorganisms including Pseudomonas sp., Fusarium solani, F. oxysporum, and Clonostachys rosea (Hashimoto et al., 2004, 2002, 1994; Kawasaki et al., 2005; Yamano et al., 2008). GABA is naturally produced by different lactic acid bacteria (LAB) strains such as Lactobacillus lactis, L. brevis or L. plantarum (Franciosi et al., 2015; Nomura et al., 1998; Tajabadi et al., 2015b). Its biosynthesis by natural isolates depends on different factors: carbon and nitrogen sources, pyridoxal phosphate (cofactor) availability, Tween-80 supplementation, initial l-glutamate concentration, temperature, pH and incubation time (Cho et al., 2007; Komatsuzaki et al., 2005; Li et al., 2010a,b; Tajabadi et al., 2015b; Villegas et al., 2016). The efficiency of natural GABA producers can be improved by overexpression of additional glutamate decarboxylase (gad) gene (Kook et al., 2010; Park et al., 2005, 2013; Plokhov et al., 2000; Tajabadi et al., 2015a; Le Vo et al., 2014, 2013a), supported by the overexpression of gadC coding for the glutamate:GABA antiporter (Le Vo et al., 2013b) and/or deletion of the GABA degradation genes (Le Vo and Kim Hong, 2012). However, neither LAB nor Escherichia coli naturally synthesize high concentrations of the GABA precursor l-glutamate; for this reason the fermentations carried out by these bacteria have to be sup-

http://dx.doi.org/10.1016/j.indcrop.2016.11.050 0926-6690/© 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: Irla, M., et al., Methanol-based ␥-aminobutyric acid (GABA) production by genetically engineered Bacillus methanolicus strains. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.050

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plemented with this compound. To tackle this issue, E. coli was engineered for overproduction of l-glutamate directly from glucose (Pham et al., 2016, 2015). To our knowledge, the highest GABA titer achieved so far by engineered E. coli strain is 1.3 g/L from 10 g/L glucose (Pham et al., 2015). Corynebacterium glutamicum is an industrial workhorse for the production of amino acids, in particular of l-glutamate (Kimura, 2003) and has been engineered for glucose-based production of GABA (Heider and Wendisch, 2015). Heterologous expression of the glutamate decarboxylase gene gadB2, the GABA:glutamate antiporter gene gadC, and the regulator gene gadR from L. brevis in C. glutamicum led to accumulation of about 2 g/L of GABA within 72 h (Shi and Li, 2011). The titer was increased to 7 g/L (after 84 h) when the activity range of glutamate decarboxylase was broadened towards a near-neutral pH by mutagenesis (Shi et al., 2014) or to 19 g/L (after 84 h cultivation) by co-expression of two glutamate decarboxylase genes gadB1 and gadB2 from L. brevis (Shi et al., 2013). Overexpression of E. coli-derived glutamate decarboxylase gene gadB in C. glutamicum yielded 8 g/L of GABA after 96 h (Takahashi et al., 2012). Changing the genetic background of C. glutamicum by deleting pknG coding protein kinase G increased GABA production to 30 g/L during 120 h fermentation (Okai et al., 2014). As consequence of engineering of glutamate decarboxylase from E. coli towards activity at neutral pH the GABA titers of 39 g/L were achieved in this host (Choi et al., 2015). Finally, a C. glutamicum strain was constructed that produced 71 g/L of GABA during 70 h of fermentation under a two-stage pH control strategy. The latter strain lacked genes argB, proB and dapA encoding enzymes involved in the formation of the by-products l-arginine, l-proline and l-lysine, and it had two copies of the gad gene from L. plantarum inserted into its genome (Zhang et al., 2014). Besides glutamate decarboxylase-based production of GABA, a new metabolic route to GABA via putrescine has been described in C. glutamicum and the highest volumetric productivity reported so far for fermentative production of GABA from glucose in shake flasks was achieved (Jorge et al., 2016a,b). Methanol has recently attracted interest as a potential feedstock for biotechnological processes due to its numerous advantages over conventional carbon sources such as availability, chemical purity and lack of competition with food industry (Linton and Niekus, 1987; Müller et al., 2015; Ochsner et al., 2015; Schrader et al., 2009). To date, methanol is mostly produced from syngas; however, a lot of progress has been made in last years in development of alternative, renewable routes for methanol synthesis for example from crude glycerol which is a by-product in production of biodiesel from plant-derived triglycerides (Haider et al., 2015). For this reason methanol is expected to emerge as a base of methanol bio-economy in the near future (Olah, 2013). In the present study, we have established methanol-based production of GABA by recombinant Bacillus methanolicus. A wild-type strain of this Gram-positive, endospore-forming, thermophilic and methylotrophic bacterium is known to secrete up to 60 g/L of the GABA precursor l-glutamate during high-cell-density fed-batch methanol fermentation (Heggeset et al., 2012; Schendel et al., 2000) and 0.8 g/L of l-glutamate in shake flaks cultivations (Krog et al., 2013). The overexpression of heterologous glutamate decarboxylase genes led to conversion of l-glutamate to GABA in the two phase production process.

All primers (Metabion or BASF SE) used in this research are listed in the Supplementary Table S1. 2.2. Molecular cloning The E. coli competent cells were prepared according to the calcium chloride protocol as described in Chan et al. (2013). All standard molecular cloning procedures were carried out as described in Sambrook and Russell (2001) or according to manuals provided by producers. Chromosomal DNA of E. coli and Corynebacterium terpenotabidum was isolated as described in Eikmanns et al. (1994). The genomic DNA (gDNA) of S. thermosulfidooxidans, Bacillus megaterium, and L. brevis was obtained from German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen; DSMZ). PCR product purifica® tion and gel extraction were performed with the NucleoSpin Gel and PCR Clean-up kit (Machery-Nagel). GeneJET Plasmid Miniprep Kit from (Thermo Fisher Scientific) was used for plasmids isolation. Restriction enzymes were produced by Thermo Fisher Scientific. The DNA amplification was carried out with ALLinTM HiFi DNA Polymerase (highQu). Antarctic Phosphatase from New England Biolabs was used for the dephosphorylation of plasmid DNA. The DNA fragments were joined either with Rapid DNA Ligation Kit (Roche) or by the means the isothermal DNA assembly (Gibson et al., 2009). The plasmids pTH1mp-gadBm and pTH1mp-gadCt were constructed by amplifying the respective gad gene from gDNA with primers as described in Table S1. The PCR product and pTH1mp plasmid were digested with PciI and KpnI, and both fragments were ligated. The plasmids pTH1mp-gadB, pTH1mp-gadBTM , pTH1mpgadSt , pTH1mp-gadB1Lb , pTH1mp-gadB2Lb , pTH1mp-gadB3Lb were constructed by amplifying respective gad gene from gDNA with primers as described in Table S1, and joining the resulting PCR product with PciI and BamHI digested pTH1mp by means of the isothermal DNA assembly method. For the plasmids pTH1mp-gadB1Ao , pTH1mp-gadB3Ao the synthetic genes were codon optimized for B. methanolicus by GeneArt (Thermo Fisher Scientific). The sequences of the codon optimized genes are available in the Supplementary material. The colony PCR was done with Taq polymerase (New England Biolabs). All cloned DNA fragments were confirmed by sequencing. The competent cells of B. methanolicus were prepared and the electroporation was performed as described before (Jakobsen et al., 2006). 2.3. Media and conditions for shake flask cultivations E. coli strains were cultivated at 37 ◦ C in Lysogeny Broth (LB) or on LB agar plates supplemented with antibiotics when necessary. For transformations B. methanolicus strains were cultured at 50 ◦ C in SOBsuc (SOB medium supplemented with 0.25 M sucrose). For GABA production screening experiments, recombinant B. methanolicus strains were grown in MVcM minimal medium with 200 mM methanol supplemented with 5 ␮g/ml chloramphenicol and 20 ␮M pyridoxal 5 -phosphate (PLP) and no yeast extract (Jakobsen et al., 2009). The medium optimized for l-glutamate and GABA production was MVcM medium in which the final magnesium concentration was reduced from 1 mM to 0.04 mM, 200 ␮M PLP and no yeast extract were added. 2.4. Determination of amino acid concentration

2. Materials and methods 2.1. Strains, plasmids, and primers Bacterial strains and plasmids used in this study are listed in the Supplementary Table S1. The E. coli strain DH5␣ was used as general cloning host and MG1655 was source for cloning of the gadB gene.

For the analysis of amino acids concentrations, 1 ml of the culture sample was taken from the bacterial cultures and centrifuged for 10 min at 13,000g. Extracellular amino acids were quantified by means of high-pressure liquid chromatography (1200 series; Agilent Technologies Deutschland GmbH). The samples underwent pre-column derivatization with ortho-phthaldialdehyde (OPA),

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and were separated on a system consisting of a pre-column (LiChrospher 100 RP18 EC-5 ␮ (40 × 4 mm), CS-Chromatographie Service GmbH) and a main column (LiChrospher 100 RP18 EC-5 ␮ (125 × 4 mm), CS-Chromatographie). The detection was performed with a fluorescence detector, with excitation at 230 nm and emission at 450 nm (FLD G1321A, 1200 series, Agilent Technologies). Derivatization and quantification was carried out as published before (Jones and Gilligan, 1983; Schrumpf et al., 1991) with some modifications. 2.5. High-cell-density fed-batch methanol fermentation Fed-batch fermentation was performed at 50 ◦ C (unless stated differently) in UMN1 medium using Applikon 3 l fermenters with an initial volume of 0.75 l medium essentially as previously described (Jakobsen et al., 2009). Chloramphenicol (5 ␮g/ml) was added to the initial batch growth medium, the pH was maintained at 6.5 by automatic addition of 12.5 % (w/v) NH3 solution (unless stated differently), and the dissolved oxygen level was maintained at 30 % saturation by increasing the agitation speed and using enriched air (up to 60 % O2 ). The methanol concentration in the fermenter was monitored by online analysis of the headspace gas with a mass spectrometer (Balzers Omnistar GSD 300 02). The headspace gas was transferred from the fermenters to the mass spectrometer in insulated heated (60 ◦ C) stainless steel tubing. The methanol concentration in the medium was maintained at a set point of 150 mM by automatic addition of methanol feed solution containing methanol, trace metals and antifoam 204 (Sigma), as previously described (Brautaset et al., 2010). Bacterial growth was monitored by measuring OD600 . Dry cell weight was calculated using a conversion factor of one OD600 unit corresponding to 0.24 g dry cell weight per liter (Jakobsen et al., 2009; Nærdal et al., 2015). Due to an increase in the culture volume throughout the fermentation, the biomass, GABA and l-glutamate concentrations were corrected for the increase in volume and subsequent dilution. Volume correction factors from 1.4 to 1.6 were used and the actual concentrations measured in the bioreactors were therefore accordingly lower as described previously (Jakobsen et al., 2009). Samples for determination of GABA and l-glutamate concentrations were collected from early exponential phase and throughout the cultivation. 3. Results and discussion 3.1. B. methanolicus traits favorable for GABA biosynthesis An efficient GABA producing bacterium should be an efficient l-glutamate producer, display high tolerance to the product, lack a product degradation pathway and enable cofactor regeneration. To test the influence of GABA on growth of B. methanolicus we performed a series of shake flask growth experiments in methanol minimal medium with increasing concentrations of GABA added to the medium before the beginning of the experiment. As shown in Fig. 1, B. methanolicus grew in the presence of GABA in the growth medium, however, growth arrested completely at a concentration of 16.5 g/L and the growth rate was reduced to 50 % at 7.2 g/L. In some bacteria, GABA is degraded by ␥-aminobutyric acid transaminase (encoded by gabT), which together with succinate semialdehyde dehydrogenase (encoded by gabD) and glutamate decarboxylase form a GABA shunt converting l-glutamate to the Krebs cycle intermediate succinate. The biological function of the GABA shunt in bacteria is not fully clear, but in B. megaterium it is involved in spore formation (Foerster, 1971) and in B. subtilis it is required for utilization of GABA as nitrogen source (Ferson et al., 1996). The gabT and gabD genes commonly form a gabTD operon in bacteria (Belitsky and Sonenshein, 2002; Metzner et al.,

Fig. 1. Influence of GABA supplementation on growth of B. methanolicus MGA3. GABA was added to the growth medium at the beginning of the experiment and the growth was measured until reaching of the end of exponential phase. The means of triplicate shake flask cultures with standard deviations are shown.

2004; Zhu et al., 2010). We have not found gabT in the genome of B. methanolicus, however, gabD is present. This indicates that B. methanolicus does not possess the necessary genetic repertoire for GABA degradation. This finding was experimentally confirmed in a series of shake flask experiments where GABA was tested as substitute of methanol (carbon source), ammonium sulphate (nitrogen source) or both. GABA could neither be metabolized as carbon nor as nitrogen source (data not shown). A BLAST search revealed that B. methanolicus lacks a glutamate decarboxylase gene in its genome (Altschul et al., 1990; Heggeset et al., 2012; Irla et al., 2014). Accordingly, we did not observe GABA formation by B. methanolicus MGA3 control strains. Glutamate decarboxylase requires PLP as a cofactor for its activity (Strausbaucht and Fischer, 1970), which means that this compound needs to be effectively regenerated to support efficient glutamate decarboxylation. To be certain that supplementation with expensive PLP will not be limiting factor for GABA biosynthesis we searched the genome of B. methanolicus for pdxK gene coding for pyridoxine kinase (EC 2.7.1.35), which catalyzes phosphorylation of pyridoxal, pyridoxine and pyridoxamine to their corresponding 5 -phosphates (Newman et al., 2006), and in fact we found that pdxK is present in its genome. Summarized, a natural high l-glutamate production, a pyridoxine kinase, and lack of a GABA degradation pathway should make B. methanolicus MGA3 a suitable host for production of GABA from methanol. 3.2. Screening heterologous glutamate decarboxylases for activity in B. methanolicus at 50 ◦ C Despite the obvious advantages of B. methanolicus for GABA biosynthesis, we have anticipated some difficulties in finding the optimal glutamate decarboxylase (Gad) source for two particular reasons. First, due to the fact that B. methanolicus is a thermophile, the Gad should be active during cultivation at 50 ◦ C. Secondly B. methanolicus does not grow at a pH lower than 5.5 (Irla et al., 2015). However, the best studied glutamate decaroxylases originate from mesophilic bacteria and most of them are catalytically active at low pH because they are part of the bacterial acid stress response system (Lin et al., 1996).

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Table 1 Properties of characterized Gad used to establish GABA production by B. methanolicus. Strain of B. methanolicus

MGA3(pTH1mp-gadB) MGA3(pTH1mp-gadSt ) MGA3(TH1mp-gadBm ) MGA3(pTH1mp-gadCt ) MGA3(pTH1mp-gadB1Lb ) MGA3(pTH1mp-gadB2Lb ) MGA3(pTH1mp-gadB3Lb ) MGA3(pTH1mp-gadB1Ao ) MGA3(pTH1mp-gadB3Ao )

Overexpressed gad (donor organism)

gadB (E. coli)# gadSt (S. thermosulfidooxidans) gadBm (B. megaterium) gadCt (Corynebacterium terpenotabidum) gadB1 (L. brevis)* gadB2 (L. brevis)† gadB3 (L. brevis) gadB1 (Aspergillus oryzae)§ gadB3 (Aspergillus oryzae)

GABA produced by recombinant strain [g/L]‡

Characterization of Gad in literature

Sp. act. [U/mg]

Optimal pH

Optimal T [◦ C]

4.6

37 65–230 Not characterized

KM [mM] 2–20

Not characterized Not characterized 4.0–5.2 4.2–5.0 5.5

37–50 6.59–33.3 30 6.0 Not characterized 60 48 Not characterized

0.03 ± 0.00 0.03 ± 0.00 Not detected Not detected

1.75–10.26 1.4–9.3 13.3

Not detected Not detected Not detected Not detected Not detected

#

Ho et al. (2013), Jun et al. (2014), Pennacchietti et al. (2009), Tramonti et al. (1998). Fan et al. (2012), Huang et al. (2007b), Lee and Jeon (2014), Seo et al. (2013). † Hiraga et al. (2008), Ueno et al. (1997). § Tsuchiya et al. (2003). ‡ B. methanolicus MGA transformed with a pTH1mp derivative for methanol-inducible expression of the respective gad genes was used for GABA production in shake flasks containing MVcM medium with 200 mM methanol and 20 ␮M PLP. *

For this reason we have compared GABA production in the B. methanolicus strains overexpressing nine different gad genes from six different organisms; only five of these enzymes encoded by genes have been characterized before (Table 1). The other donor organisms included B. megaterium whereof Gad is active during spore germination (Foerster and Foerster, 1973), Corynebacterium terpenotabidum (Rückert et al., 2014; Takeuchi et al., 1999) and S. thermosulfidooxidans, a thermo- and acidophilic, sulphuroxidising bacterium (Golovacheva and Karava˘ıko, 1987; Travisany et al., 2012). The respective gad genes were cloned into the vector pTH1mp under transcriptional control of the methanol dehydrogenase promoter, the corresponding plasmids were transformed to B. methanolicus MGA3 and the resulting strains tested for GABA production in shake flasks. The results of these screening experiments indicated that two of the tested strains accumulated GABA; one expressing gadB from E. coli and one expressing gad from S. thermosulfidooxidans (Table 1). These two strains were therefore chosen for further experiments and optimization.

3.3. Influence of growth temperature on recombinant strains expressing GadB and GadSt on GABA production in shake flasks According to Jun et al. (2014), the wild-type GadB from E. coli retains over 90 % of its activity after 10 min incubation at 50 ◦ C. However, since no data was available about its folding and stability during prolonged incubation at elevated temperatures, we decided to test two alternative approaches for GABA biosynthesis in B. methanolicus MGA3 expressing gadB from E. coli. To test whether the temperature influences GABA accumulation by strain MGA3(pTH1mp-gadB), we developed a two-phase production protocol, where the cells were grown at 50 ◦ C for 10–12 h until the maximal OD600 and l-glutamate concentration were reached. Then, the temperature was shifted to 37 ◦ C, the optimal temperature for E. coli GadB activity. As shown in Fig. 2, the temperature shift led to full conversion of l-glutamate to GABA after 24 h in contrast to incubation at 50 ◦ C, where only about 50 % of the l-glutamate was converted. These initial results indicated that thermal stability of GadB may, indeed, be limiting for GABA synthesis and encouraged us to test a thermostable version of this enzyme. Jun et al. (2014) described that introduction of three mutations (Gln5Asp/Val6Ile/Thr7Glu) into the amino acid sequence of E. coliderived GadB led to increased thermostability in comparison to

Fig. 2. Production of GABA (green) and l-glutamate (grey) by recombinant B. methanolicus MGA3 strains cultivated in shake flasks at different temperatures. Part A: growth and production at 50 ◦ C; part B: temperature shift to 37 ◦ C after 12 h of initial growth at 50 ◦ C. The means of triplicates with standard deviations are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

the wild-type GadB. As shown in Fig. 2, the introduction of the above-mentioned mutations into the GadB sequence (GadBTM ) had no beneficial effect on GABA accumulation. In fact, the GABA titer after 24 h of incubation at 50 ◦ C was lower in comparison to the titer obtained with the strain synthesizing wild-type GadB protein. Possibly, the increased thermostability of the enzyme was achieved at the cost of its activity at the near to neutral pH values of 7.2 used as initial medium pH in our experiments. Because the temperature shift led to full conversion of lglutamate in shake flask cultivations of the MGA3(pTH1mp-gadB) strain, we next used a modified minimal medium containing a

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Fig. 3. Production of GABA and l-glutamate by B. methanolicus strains MGA3(pTH1mp) (A), MGA3(pTH1mp-gadB) (B) and MGA3(pTH1mp-gadSt ) (C) cultivated in shake flasks. After 12 h of cultivation at 50 ◦ C the temperature for strain MGA3(pTH1mp-gadB) was shifted to 37 ◦ C in (B). Green squares, GABA concentration; grey circles, l-glutamate concentration; closed triangles, OD600 . The means of triplicates with standard deviations are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

decreased magnesium concentration which previously has been shown to stimulate l-glutamate production in B. methanolicus MGA3 (Schendel et al., 2000; Krog et al., 2013). l-Glutamate accumulation by strain MGA3(pTH1mp) in the modified medium was 8-fold higher (0.63 ± 0.05 g/L; Fig. 3) as compared to the base medium (0.08 ± 0.00 g/L; Fig. 2). The gadB expressing strain was able to convert almost all l-glutamate to GABA over the course of 72 h in the experiment, when the temperature was shifted from 50 ◦ C to 37 ◦ C. A final GABA titer of 0.41 ± 0.00 g/L was obtained using the modified medium (Fig. 3). The Gad from S. thermosulfidooxidans (GadSt ) has not been characterized biochemically so far. We have decided to use S. thermosulfidooxidans as a donor organism because it is a thermophilic bacilli and we expected S. thermosulfidooxidans-derived GadSt to be active at 50 ◦ C (Golovacheva and Karava˘ıko, 1987; Travisany et al., 2012). In fact, as shown in Fig. 3, MGA3(pTH1mp-gadSt ) produced 0.35 ± 0.00 g/L of GABA within 72 h of flask cultivation at 50 ◦ C. However, the conversion of l-glutamate to GABA was not complete. Contrary to flask cultivations of MGA3(pTH1mp-gadB), the temperature was maintained at 50 ◦ C during the whole cultivation time of MGA3(pTH1mp-gadSt ), which is beneficial because of potentially decreased cooling costs during up-scaled fermentation. The flask experiment was carried out with the initial pH of the medium at the level of 7.2 which is not maintained during the cultivation and decreases to final level of 5.5. Most bacterial glutamate decarboxylases are active at low pH and for this reason the growth conditions might have deterred the Gad activity. Due to the fact that it is difficult to control the pH during flask cultivations, we decided to test pH influence on GABA production during controlled fed-batch fermentations.

GABA was produced. In shake flasks, GABA accumulation lagged behind l-glutamate accumulation (Fig. 3). This observation may be explained by GABA formation involving re-uptake of l-glutamate from the growth medium and/or by cell lysis leading to release of GadB to the medium followed by conversion of l-glutamate to GABA. Since GadB undergoes a conformational change at neutral pH which decreases its activity (Capitani et al., 2003), pH 6.5 conventionally used during fermentations of B. methanolicus might lead to deterioration of GadB activity. For this reason, a pH shift from 6.5 to 4.6 was performed after 26 h but still no GABA formation was observed (Table 2). To investigate if the high cultivation temperature may be the reason, we applied a temperature shift from 50 ◦ C to 37 ◦ C after 20 h of cultivation, but GABA was not formed. GadB from E. coli has been successfully applied for GABA biosynthesis before, including the use of the purified enzyme in biocatalytic conversion of l-glutamate to GABA (Dinh et al., 2013; Huang et al., 2007a; Kang et al., 2013; Lee and Jeon, 2014; Lee et al., 2013) and the use of recombinant strains producing GadB heterologously (Takahashi et al., 2012). However, our experiments show that E. coli-derived GadB was not suitable for methanol-based GABA production at 50 ◦ C in fed-batch fermentations of B. methanolicus although the immediate precursor l-glutamate accumulated and, thus, was available (24–38 g/L; Table 2). Decarboxylation of l-glutamate to GABA by GadB did not occur, possibly due to effects such as a too high pH, too high temperature or the dilution of the enzyme’s cofactor PLP that was provided only by the yeast extract initially added to the fermentation broth (Rubin et al., 1947).

3.4. Fed-batch methanol fermentation of B. methanolicus MGA3(pTH1mp-gadB)

Based on the gad gene from the thermophilic S. thermosulfidooxidans, we performed fed-batch methanol fermentations of the GABA producing strain MGA3(pTH1mp-gadSt ) at a temperature of 50 ◦ C. Three different conditions were chosen: at a constant pH of 6.5 (control), at a reduced constant pH of 6.0 and as a two-phase process with a pH shift from 6.5 to 4.6 after 27 h (Table 2). The comparison of the obtained results clearly indicated an influence of medium pH on the Gad activity. In the control fermentation at pH 6.5, only 0.1 g/L GABA accumulated. Similarly, at the reduced pH of 6.0 only 0.3 g/L GABA were produced. Interestingly, when the pH was shifted from 6.5 to 4.6 after 27 h, GABA accumulated to 9 g/L (Table 2 and Fig. 4). The conversion of l-glutamate to GABA appeared to be quick

We carried out fed-batch methanol fermentations with strain MGA3(pTH1mp-gadB) under three different conditions: constant pH and temperature, with a temperature shift from 50 ◦ C to 37 ◦ C after 20 h of fermentation, or with a pH shift from pH 6.5–4.6 after 26 h of fermentation. As compared to constant pH and temperature conditions, both a pH shift and a temperature shift decreased biomass formation and l-glutamate accumulation (Table 2). However, while about 25 g/L l-glutamate still accumulated under pH or temperature shift conditions, unexpectedly no

3.5. GABA production by MGA3(pTH1mp-gadSt ) under optimized fed-batch methanol cultivation

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Table 2 Fed-batch methanol fermentation data for strains MGA3(pHP13), MGA3(pTH1mp-gadB) and MGA3(pTH1mp-gadSt ). Strain

Conditions of fermentation

CDW [g/L]

Glua [g/L]

GABAa [g/L]

MGA3(pHP13) MGA3(pTH1mp-gadB) MGA3(pTH1mp-gadB) MGA3(pTH1mp-gadB) MGA3(pTH1mp-gadSt ) MGA3(pTH1mp-gadSt ) MGA3(pTH1mp-gadSt )

Shift to pH 4.6 Control conditions Shift to 37 ◦ C Shift to pH 4.6 Control conditions Constant pH 6.0 Shift to pH 4.6

48.6 45.5 31.0 34.2 31.6 41.0 47.5

33.9 37.9 25.5 24.3 28.8 31.7 12.9

0.0 0.0 0.0 0.0 0.1 0.3 9.0

Production data from early or late stationary growth phase are presented. The fermentations were performed at control conditions (50 ◦ C and pH 6.5) or with modifications. For MGA3(pHP13) pH was shifted after 26 h from pH 6.5 to pH 4.6 (Shift to pH 4.6); for MGA3(pTH1mp-gadB) control conditions were used, temperature was shifted from 50 ◦ C to 37 ◦ C after 20 h of cultivation (Shift to 37 ◦ C) or pH shifted after 26 h from pH 6.5 to pH 4.6 (Shift to pH 4.6); for MGA3(pTH1mp-gadSt ) control conditions (50 ◦ C and pH 6.5), with constant pH of 6.0 (constant pH 6.0) or with pH shift after 27 h from pH 6.5 to pH 4.6 (Shift to pH 4.6). CDW, cell dry weight; Glu, l-glutamate, GABA, ␥-aminobutyric acid. a GABA and amino acid concentrations and CDW are volume corrected (see “Materials and Methods”).

activity at 50 ◦ C and at pH 4.6, full conversion of l-glutamate was not observed for B. methanolicus MGA3(pTH1mp-gadSt ). It remains to be studied what factor(s) limit GABA production from methanol under these conditions. Possibly, GadSt is subject to degradation by proteases released from B. methanolicus rendering it inactive. The gadSt expression level may be increased or regulated by using alternative expression systems (Irla et al., 2016). Alternatively, the cofactor PLP might be degraded more rapidly or its regeneration is less efficient under conditions of high temperature coupled with low pH. 4. Conclusions

Fig. 4. Two-phase methanol-based GABA production process using B. methanolicus MGA3(pTH1mp-gadSt ). The fed-batch methanol fermentation was performed at 50 ◦ C and after 27 h the initial pH of 6.5 was shifted to pH 4.6. CO2 , CO2 evolution rate as a function of time [mmol/L/h] (cyan line); pH (purple line); CDW, cell dry weight [g/L] (filled triangles) calculated from OD600 measurements by correlation to selected measurements of dry weights of cells; Glu, l-glutamate concentration in the medium [g/L] (grey circles); GABA, GABA concentration in the medium [g/L] (green squares). Values for CO2 , CDW, Glu, and GABA are corrected for volume changes during the cultivation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

as 9 g/L of GABA was obtained within 4 h after the pH shift. As expected, the l-glutamate concentration was reduced from 22.5 g/L to 12.9 g/L during the same time. From Fig. 4 it is clear that pH 4.6 was not compatible with B. methanolicus metabolism as the culture respiration dropped to near zero. We therefore assume that the conversion of l-glutamate to GABA is an enzymatic process occurring after the pH shift. Despite the fact that GadSt has not yet been characterized biochemically, its activity at acidic pH and at 50 ◦ C was not surprising. First of all, bacterial glutamate decarboxylases are known to be active at low pH because they are often part of acid stress response systems. In fact, GadSt shares 50 % identical amino acids with GadB of E. coli and 47 % with GadB1 of L. brevis. More importantly, S. thermosulfidooxidans is an acidophilic bacterium with a growth optimum between pH 2.0 and 2.5 (Golovacheva and Karava˘ıko, 1987; Travisany et al., 2012), which confirms the need of an active acid stress response system. This mixotrophic, acidophilic, moderately thermophilic bacterium has been isolated from copper mining environments in the north of Chile and has been shown to enhance bioleaching of the copper sulfide chalcopyrite at 50 ◦ C and pH 1.6 (Bobadilla-Fazzini et al., 2014). However, despite the

A number of recombinant B. methanolicus strains expressing heterologous glutamate decarboxylase genes were constructed and tested for production of GABA from methanol. A one-pot, two-phase production process with recombinant thermophilic B. methanolicus strains for methanol-based GABA production was developed and operated at 50 ◦ C involving a pH shift from nearneutral to acid pH. The well-studied glutamate decarboxylases from mesophilic enteric bacteria proved inefficient, but performance at 50 ◦ C and a pH shift to 4.6 proved successful for B. methanolicus expressing gadSt from S. thermosulfidooxidans. The enzyme has not yet been studied biochemically, but its performance in the methanol-based GABA production process fits well to the extreme lifestyle of S. thermosulfidooxidans, since it is used for copper bioleaching at 50 ◦ C and pH 1.6. To the best of our knowledge, this study describes methanol-based GABA production for the first time which can potentially be a basis for development of environmentally-friendly process of production of biodegradable plastics derived from GABA. Funding sources This work was supported in part by the EU7 FWP project PROMYSE and by ERASysAPP project MetApp. Acknowledgments M. Irla acknowledges support from the CLIB Graduate Cluster Industrial Biotechnology at Bielefeld University, Germany, which is financed in part by a grant from the Federal Ministry of Innovation, Science and Research (MIWF) of the federal state North Rhine-Westphalia, Germany. We thank Dr. Oskar Zelder, Dr. Robert Thummer and Dr. Trond E. Ellingsen for discussions, Per Odin Hansen for technical assistance during fed-batch methanol fermentations, Hans Fredrik Kvitvang and Marianne Kjos for performing amino acid analysis of fermentation samples.

Please cite this article in press as: Irla, M., et al., Methanol-based ␥-aminobutyric acid (GABA) production by genetically engineered Bacillus methanolicus strains. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.050

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2016.11. 050.

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Please cite this article in press as: Irla, M., et al., Methanol-based ␥-aminobutyric acid (GABA) production by genetically engineered Bacillus methanolicus strains. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.050