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Construction of a novel d -lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2017 www.elsevier.com/locate/jbiosc
Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942 Yasutaka Hirokawa, Ryota Goto, Yoshitaka Umetani, and Taizo Hanai* Laboratory for Bioinformatics, Graduate School of Systems Biosciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Received 27 January 2017; accepted 23 February 2017 Available online xxx
Using engineered cyanobacteria to produce various chemicals from carbon dioxide is a promising technology for a sustainable future. Lactate is a valuable commodity that can be used for the biodegradable plastic, polylactic acid. Typically, lactate production using engineered cyanobacteria was via the conversion of pyruvate in glycolysis by lactate dehydrogenase. In cyanobacteria, the metabolic flux in the Calvin cycle is higher than that in glycolysis under photoautotrophic conditions. The construction of a novel lactate producing pathway that uses metabolites from the Calvin cycle could potentially increase lactate productivity in cyanobacteria. In order to develop such a novel lactate production pathway, we engineered a cyanobacterium Synechococcus elongatus PCC 7942 strain that produced lactate directly from carbon dioxide using dihydroxyacetone phosphate (DHAP) via methylglyoxal. We confirmed that wild-type strain of S. elongatus PCC 7942 could produce lactate using exogenous methylglyoxal. A methylglyoxal synthase gene, mgsA, from Escherichia coli was introduced into Synechococcus elongates PCC 7942 for conversion of DHAP to methylglyoxal. This engineered strain produced lactate directly from carbon dioxide. Genes encoding intrinsic putative glyoxalase I, II (Synpcc7942_0638, 1403) and the lactate/HD symporter from E. coli (lldP) were additionally introduced to enhance the production. For higher lactate production, it was important to maintain elevated extracellular pH due to the characteristics of lactate exporting system. In this study, the highest lactate titer of 13.7 mM (1.23 g/l) was achieved during a 24day incubation with the engineered S. elongatus PCC 7942 strain possessing the novel lactate producing pathway. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Carbon dioxide; Cyanobacteria; D-Lactate; Photosynthesis; Glyoxalase; Dihydroxyacetone phosphate]
The world’s energy supply is highly dependent on fossil resources despite the excessive consumption of such fuel, leading to numerous environmental issues. It is crucial to reduce our dependency on fossil resources for both energy generation and chemical production for a sustainable future. One option to solve such problems is the use of biomass for the biological production of fuels and chemicals (1,2). This approach has become feasible by advances in genetic engineering that allow modification of microorganisms such as Escherichia coli and yeast to either enhance production of natural chemicals or to even generate entirely foreign substances (3,4). One of the most important limiting factors affecting the use of these organisms is the need for an abundant carbon source to feed the manufacturing process. So far edible biomass (for example, corn and sugarcane) (5) and inedible lignocellulosic biomass (for example, wood wastes and energy crops) (6) were used for bioproduction. However, commonly used heterotrophic microorganisms are unable to directly assimilate these biomass, so it must first be enzymatically or chemically saccharified to simple sugars (7,8). An additional issue is that growing the required biomass can take a considerable amount of time, again limiting potential.
* Corresponding author. Tel.: þ81 92 642 6751; fax: þ81 92 642 6744. E-mail address: [email protected] (T. Hanai).
In contrast to heterotrophic microorganisms, cyanobacteria are photoautotrophic and feed by fixing carbon dioxide to organic compounds using solar energy. Similar to other organisms, genetic engineering approaches have also been developed in cyanobacteria for the production of chemicals, although in this case, products are manufactured directly from carbon dioxide. Some of these valuable products include isobutyraldehyde, isobutanol (9,10), ethanol (11), 2,3-butanediol (12,13), 2-methyl-1-butanol (14), 1-butanol (15,16), acetone (17,18), 3-hydroxybutyrate (19), ethylene (20,21), isoprene (22,23), 1,2-propanediol (1,2-PDO) (24), and glycerol (25,26). Our prior work has also demonstrated biological production of isopropanol (27) and 1,3-PDO (28) using S. elongatus PCC 7942. However, the currently achievable chemical titers by engineered cyanobacteria are significantly lower than those from heterotrophic microorganisms (29,30). For example, even the most efficient ethanol production with engineered cyanobacteria has not been able to exceed 10 g/l (11). Therefore, while direct production of chemicals from carbon dioxide is an attractive and promising technology for a sustainable future, increased production titers are necessary if it is to become practical and cost effective. Lactate is one of the major chemicals produced by engineered microorganisms. It is widely used and can be found in a range of food additives, cosmetics, and various pharmaceuticals (31,32). Demand as a substrate for biodegradable plastics (in the form of poly-lactic acid; PLA) is expected to increase as the world moves
1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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towards a sustainable future (33,34). Structurally, the asymmetric carbon in the lactate molecule yields two optical isomers, L-lactate and D-lactate. Importantly, the physical properties of PLA are affected by optical purity so it is vital to produce optically pure lactate for PLA synthesis (35). Lactate can be produced chemically by hydrolysis of lactonitrile, a byproduct of acrylonitrile production. However, lactate produced by this method is a mixture of the two optical isomers, affecting the quality of any resultant PLA (36). When produced biologically, lactate is generated from conversion of pyruvate by lactate dehydrogenase (LDH) (37). The specific optical character of the lactate isomer produced strictly depends on enzymatic characteristics, allowing optically pure lactate to be manufactured. The lactic acid bacteria, Lactobacillus sp. and Lactococcus sp. can naturally produce lactate (38), but these strains have complex auxotrophic requirements and the purity of produced lactate depends on incubation conditions. To overcome these drawbacks, LDH has been introduced into more user friendly microorganisms such as E. coli and yeast. This has led to more effective biological production of optically pure lactate (39). Lactate can be produced directly from carbon dioxide using genetically engineered cyanobacteria. As with E. coli and yeast, the introduction of LDH enables cyanobacteria to produce lactate from cellular pyruvate. The highest lactate titer of 20.5 mM (1.84 g/l) has been achieved using an engineered Synechocystis sp. PCC 6803 strain possessing LDH (40). The highest production rate (2.2 mM/ day) was obtained using another cyanobacterium strain, Synechococcus sp. PCC 7002 (41). This production rate was achieved by combining LDH introduction with additional genetic engineering using CRISPRi to increase central carbon flux. Another approach to increase lactate production may be to utilize a metabolic pathway that naturally possesses a higher carbon flux. One example is the Calvin cycle of photoautotrophically grown cyanobacteria, which typically shows a greater flux than glycolysis (42,43). The reason of greater flux in Calvin cycle is that many parts of fixed carbon by photosynthesis were utilized to reproduce ribulose-1,5bisphosphate for next carbon fixation. Chemical production using metabolites in the Calvin cycle of cyanobacteria would therefore be expected to lead to higher titers of chemical production. Previously, production of 1,2-PDO, sucrose, glycerol, and 1,3-PDO have all been achieved using metabolites (DHAP or fructose-6-phosphate) in the Calvin cycle (19,24,25,28,44). The titers of these chemicals were indeed higher than the other chemicals produced using metabolites from other pathways. In this study, we constructed a novel lactate producing pathway that uses dihydroxyacetone phosphate (DHAP), a metabolite from the Calvin cycle, rather than pyruvate (Fig. 1). The pathway composed of methylglyoxal synthase, lactate/Hþ symporter, and glyoxalase I, II was constructed in S. elongatus PCC 7942. In the constructed pathway, any reducing power like as NADH is not
FIG. 1. Overview of the novel lactate producing pathway constructed in this study. Introduced genes are highlighted in italics. Shaded background indicates exogenous gene (mgsA and lldP) from E. coli. White background indicates intrinsic gene (gloA and gloB). 3PG, 3-phosphoglycerate; DHAP, dihydroxyacetone phosphate; MG, methylglyoxal; s-LG, s-lactoylglutathione; GSH, glutathione.
J. BIOSCI. BIOENG., necessary for lactate production and DHAP in Calvin cycle showing greater flux (42,43) is used as substrate. Previously, we constructed 1,3-PDO and glycerol producing pathway from DHAP in this strain (28). We fully investigated the effectiveness of the novel pathway in an engineered cyanobacterium S. elongatus PCC 7942 strain. To the best of our knowledge, it is the first time to construct a lactate producing pathway using DHAP for lactate production in cyanobacteria and even any bacteria.
MATERIALS AND METHODS Chemicals and reagents All chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless otherwise specified. Restriction enzymes, phosphatase (New England Biolabs; Ipswich, MA, USA), ligase (Rapid DNA Ligation Kit, Roche, Mannheim, Germany), and DNA polymerase (KOD Plus Neo DNA polymerase, Toyobo Co., Ltd., Osaka, Japan) were used for cloning. Oligonucleotides were synthesized by Life Technologies Japan, Ltd. (Tokyo, Japan). Culture media Modified BG11 medium supplemented with 20 mM HEPESNaOH (pH 7.5) for pH stabilization was used for cultivation of S. elongatus PCC 7942 (Life Technologies Corporation, Carlsbad, CA, USA). Hereafter, this medium was referred to as BG11-HEPES medium. The composition of BG11-HEPES medium used in this study was the same as previously reported (27). In some experiments, HEPES-NaOH (pH 7.5) buffer was eliminated and substituted for 20 mM CHESNaOH (pH 9.0) or CAPS-NaOH (pH 10.0). Hereafter, these buffer replaced mediums were referred to as BG11-CHES and BG11-CAPS medium. To prepare the plate medium, 1.5% (w/v) of Bacto Agar (Difco Laboratories, Franklin Lakes, NJ, USA) was added. Antibiotics (10 mg/ml kanamycin and 20 mg/ml spectinomycin) were added to BG11 mediums as appropriate. Growth and production conditions All cyanobacterial cultures were grown under fluorescent light (100 mmol photon m2 s1) at 30 C in a growth chamber (MLR-325H-PJ; Panasonic Corporation, Osaka, Japan). Photon flux density was measured with an IKS-27 (Koito Manufacturing, Tokyo, Japan). Cell density (OD730) was measured using an Infinite 200 PRO (Tecan, Männedorf, Switzerland). For preculture, cells were inoculated into 20 mL BG11-HEPES medium in a 50-mL flask, and incubated under fluorescent light on a rotor shaking at 150 rpm (NR-30 shaker; Taitec Corporation, Saitama, Japan). Pre-cultured cells at OD730 of 1.0e2.0 were inoculated into 20 mL BG11 mediums containing 1 mM of IPTG at an initial OD730 of 0.025. Cultures were grown under same conditions as pre-culture. Strain construction The bacterial strains used in this study are listed in Table S1. Genes were integrated into the S. elongatus PCC 7942 genome by homologous recombination. For gene integration into the neutral site (NS) I (45) and NS II (46), the previously constructed and newly generated plasmids were used. The integrated genes in the engineered strains were confirmed by sequence analysis. Plasmids construction Plasmids and primers used in this study are listed in Tables S1 and S2. The gene encoding methylglyoxal synthase (mgsA) was amplified from E. coli BW25113 genome using primers T1514-T1590. The amplified sequence was digested by Acc65I-BglII. The digested PCR products were ligated into Acc65I-BamHI site of pTA424 to create pTA1501 (PLlacO1::mgsA, NS II-targeting plasmid). mgsA amplified from E. coli BW25113 genome using primers T1514-T1515 was digested by KpnI-NheI. The gene encoding lactate/Hþ symporter (lldP) amplified from E. coli BW25113 genome with primers T2576-T2577 was digested by NheIBamHI. Digested PCR products were ligated into the KpnI-BamHI site of pTA424 to create pTA1567 (PLlacO1::mgsA-lldP, NS II-targeting plasmid). A NS I targeting plasmid (pTA1562) was newly constructed based on commercial pZ vectors purchased from EXPESSYS. The antibiotic resistant cassette of pZE22-MCS was switched from kanamycin resistance to spectinomycin resistance to create pTA1471. The upper and lower regions of NS I were amplified from S. elongatus PCC 7942 genome using primers T2489-T2490, and T2491-T2492, respectively. The amplified fragments were digested by AvrII-SpeI. The digested NS I upper and NS I lower fragments were ligated into the SpeI and AvrII sites of pTA1471 respectively to create pTA1476. lacI regulated by a PlacIq promoter was amplified using primers T2662-T2664 and digested by AvrII-SpeI. The digested fragment was ligated into the AvrII site of pTA1476 to create pTA1562. The genes encoding putative glyoxalase [gloA (Synpcc7942_0638) and gloB (Synpcc7942_1403)] were amplified from the S. elongatus PCC 7942 genome using primers T2783-T2784 and T2785-T2786, respectively. The amplified gloA and gloB sequences were digested by KpnI-NheI and NheI-BamHI, respectively. The digested sequences were ligated into KpnI-BamHI site of pTA1562 to create pTA1697 (PLlacO1::gloAB, PlacIq::lacI, NS I-targeting plasmid). Product analysis The supernatant was obtained by centrifugation (20,000 g, 10 min, 4 C) and filtered using Minisart RC4 (Sartorius, Goetingen, Germany). The filtered sample was subjected to quantitative analysis. Lactate was analyzed using a high-performance liquid chromatograph (LC-20AD, Shimadzu, Kyoto, Japan) equipped with an autosampler and electric conductivity detector
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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(CDD-10A, Shimadzu). The detailed conditions of analysis were described previously (47). A D-/L-Lactic Acid (D-/L-Lactate) (Rapid) Assay Kit (K-DLATE, Megazyme, Ireland) was used to determine the optical purity of the produced lactate. Measurement of intracellular metabolites Intracellular metabolites of TA1297 (wild-type strain) and TA3739 (lactate producing strain) were measured using a LC-QqQ-MS system (high-performance liquid chromatography: Agilent 1290 Infinity, MS: Agilent 6460 with Jet Stream Technology) (Agilent Technologies, Waldbronn, Germany) controlled by MassHunter Workstation Data Acquisition software (Agilent Technologies). Culture at 7 days was used to measure intracellular metabolites. Sample preparation and LC-QqQ-MS analysis were performed using conditions described previously (48).
RESULTS AND DISCUSSION Lactate producing pathway from DHAP in the Calvin cycle Methylglyoxal is a toxic metabolite that can cause cell death at high concentrations (49). Because cellular production of methylglyoxal can occur both enzymatically and non-enzymatically in organisms, several detoxification systems have evolved. The glutathione dependent glyoxalase IeII system is one of the most conserved methylglyoxal detoxification systems (50). It is composed of two enzymes, glyoxalase I (EC 4.4.1.5) and glyoxalase II (EC 3.1.2.6), that convert methylglyoxal to D-lactate. Enzymatically, methylglyoxal is produced from DHAP by the action of methylglyoxal synthase (EC 4.2.3.3). Previously, DHAP has been used in engineered cyanobacteria as a substrate for both 1,2-PDO (24) and glycerol production (25,26). We have also constructed a 1,3-PDO producing pathway using DHAP in S. elongatus PCC 7942 (28). DHAP is one of the metabolites in the Calvin cycle that exhibits a particularly high metabolic flux in cyanobacteria under photoautotrophic conditions (42,43). A higher production rate could be achieved using DHAP in engineered cyanobacteria rather than using metabolites from other pathways. To test this hypothesis, we designed a novel production pathway that will generate lactate from DHAP via methylglyoxal rather than pyruvate (Fig. 1). Evaluation of native glyoxalase system in S. elongatus PCC 7942 In Synechocystis sp. PCC 6803, Slr0381 (gloA) and Sll1019 (gloB) were identified as genes encoding glyoxalase I and glyoxalase II, respectively (51). And the kinetic parameters of these gene products were characterized, suggesting that this strain possesses glyoxalase activity converting methylglyoxal to D-lactate. Similar genes, Synpcc7942_0638 (gloA) and Synpcc7942_1403 (gloB), have also been found in S. elongatus PCC 7942, encoding for glyoxalase I and glyoxalase II, respectively. Between S. elongatus PCC 7942 and Synechocystis sp. PCC 6803, the identities of amino acid sequence of glyoxalase I and glyoxalase II are 72.1% and 61.6%, respectively (refer to Cyanobase; http://genome.microbedb. jp/cyanobase). However, glyoxalase activity has not been confirmed in S. elongatus PCC 7942. We therefore examined the natural glyoxalase activity of S. elongatus PCC 7942 in the presence of exogenous methylglyoxal (Fig. 2).
FIG. 2. Lactate production by TA1297 (wild-type strain) in the presence of exogenous methylglyoxal. (A) Cell density (OD730), (B) lactate concentration. Open, shaded, and black circles represent the data with 0, 1, and 5 mM methylglyoxal, respectively. Presented data are means S.D. from three individual experiments.
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Methylglyoxal (1 or 5 mM) was added to TA1297 (wild-type S. elongatus PCC 7942) during the late exponential growth phase. After addition of methylglyoxal, lactate was detectable in the culture. No lactate was detected in the control culture without methylglyoxal (Fig. 2B). These are the results indicating that S. elongatus PCC 7942 possesses an ability to convert methylglyoxal to lactate. Lactate concentration increased for 6 h after the addition of methylglyoxal and then plateaued until the end of the experiment at 24 h. Lactate concentrations peaked at 0.65 mM after the addition of 1 mM methylglyoxal and at 1.28 mM after the addition of 5 mM methylglyoxal. Deletion of ldh (Synpcc7942_1347), effectively limiting the conversion of pyruvate to lactate, did not affect lactate production from exogenous methylglyoxal (data not shown). These results strongly suggest the presence of glyoxalase activity in S. elongatus PCC 7942. In glyoxalase system, one molecule of lactate is produced from one molecule of methylglyoxal (50). The lower conversion yield (about 60%) in 1 mM methylglyoxal addition would be caused of methylglyoxal property being instability and high reactivity to protein, lipid, and nucleic acid (49). Similar growth rates were observed in cultures without methylglyoxal and with 1 mM methylglyoxal, however addition of 5 mM methylglyoxal resulted in a serious growth defect (Fig. 2A). It is possible that 5 mM methylglyoxal exceeds the natural detoxification capacity of S. elongatus PCC 7942 and this was a reason why the conversion yield in 5 mM methylglyoxal was lower than that in 1 mM methylglyoxal. Prior work with a Synechococcus sp. PCC 7002 strain demonstrated that cell growth gradually decreased at methylglyoxal concentrations above 1.5 mM and concentrations above 3 mM completely inhibit cell growth (52). The methylglyoxal tolerance of S. elongatus PCC 7942 appears consistent with that observed in Synechococcus sp. PCC 7002. Lactate production from DHAP via methylglyoxal In order to generate lactate from cellular DHAP, genes encoding methylglyoxal synthase (mgsA), the lactate/Hþ symporter (lldP), and glyoxalase I, II (gloAB) were integrated into the genome of S. elongatus PCC 7942 by homologous recombination. The specific mgsA and lldP sequences used were obtained from E. coli. These genes have been previously validated for 1,2-PDO and lactate production in engineered S. elongatus PCC 7942 (24,53). Due to the newly identified existence of intrinsic glyoxalase activity (Fig. 2), we cloned putative glyoxalase genes from S. elongatus PCC 7942. In total, three strains were constructed for testing lactate production: TA3335 (a strain with mgsA only), TA3504 (a strain with mgsA and lldP), and TA3739 (a strain with mgsA, lldP, and gloAB) (Table S1, Fig. S1). The engineered strains were incubated under photoautotrophic conditions and tested for lactate production (Fig. 3). Compared to wild-type S. elongatus PCC 7942 (TA1297), a growth defect was observed in the TA3335, TA3504, and TA3739 strains (Fig. 3A). Approximately equivalent concentrations of lactate were produced by each of the three engineered strains (Fig. 3C). During the twoweek incubation, lactate concentration reached a peak of 2.85 mM, 2.18 mM, and 2.79 mM in cultures of TA3335, TA3504, and TA3739, respectively. No lactate production was detected in the culture of wild-type TA1297. This result indicates that, through the addition of methylglyoxal synthase, we have successfully introduced a lactate producing pathway that uses DHAP in the Calvin cycle. However, other genes that were added (lldP, gloAB) to improve productivity had no effect under these conditions. This may be because the direction of lactate transport by the lactate/Hþ symporter LldP is dependent on differences between the extracellular and intracellular concentration of Hþ. A relationship between transport direction and extracellular pH has previously been described for this transporter (44,54). Specifically, to facilitate export of lactate from the cell to the media, an extracellular pH that
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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FIG. 3. Lactate production directly from carbon dioxide in BG11-HEPES (pH 7.5). (A) Cell density (OD730), (B) pH, (C) lactate concentration. Circles, squares, triangles, and diamonds represent the data of TA1297 (wild-type strain), TA3335 (mgsA), TA3504 (mgsA, lldP), and TA3739 (mgsA, lldP, gloAB), respectively. Presented data are means S.D. from three individual experiments.
is higher than intracellular pH is necessary. The HEPES-NaOH buffer (pH 7.5) is basically supplemented in medium for incubation of cyanobacteria to keep experimental conditions stable in our previous works. In lactate production, the extracellular pH kept nearly neutral, however, would affect lactate transport (Fig. 3B). Optimization of culture condition for effective lactate production To determine if culture pH influences lactate export, we examined the effect of different media conditions on our engineered strains. In BG11 medium without HEPES-NaOH buffer (BG11-buf()), a growth defect observed in all lactate producing strains (TA3335, TA3504, and TA3739) was decreased compared with that in BG11-HEPES (Figs. 3A and 4A). The pH values of the control culture media (BG11-HEPES) were consistently pH 7.5 (Fig. 3B), whereas cultures without HEPES-NaOH buffer (BG11-buf()) observed a dramatic increase to pH 11.0 during the
J. BIOSCI. BIOENG.,
FIG. 4. Lactate production directly from carbon dioxide in medium without HEPESNaOH buffer (BG11-buf()). (A) Cell density (OD730), (B) pH, (C) lactate concentration. Squares, triangles, and diamonds represent the data of TA3335 (mgsA), TA3504 (mgsA, lldP), and TA3739 (mgsA, lldP, gloAB), respectively. Presented data are means S.D. from three individual experiments.
first 5 days and then gradually decreased to pH 7.0 during the 25 day incubation (Fig. 4B). pH increase observed in BG11-buf() would be caused of the reduction of nitrate to basic ammonia for amino acid synthesis by cyanobacteria metabolism (55). Under these pH conditions in BG11-buf() media, lactate production was improved and concentrations reached 8.97 mM, 10.7 mM, and 11.8 mM in cultures of TA3335, TA3504, and TA3739, respectively (Fig. 4C). The correlation of growth and lactate titers strongly suggest that the observed growth defect is likely caused by accumulation of metabolites involved in lactate producing pathway in the cell, while the acceleration of lactate export was due to higher extracellular pH. Unexpectedly, elimination of HEPES-NaOH buffer increased lactate production in a strain without additional lldP (TA3335) (Fig. 4C). These results
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
VOL. xx, 2017 suggested that the lactate export system exist in S. elongatus PCC 7942 and this system is similar to E. coli lldP. The existence of organic acid transporter that depends on extracellular pH in S. elongatus PCC 7942 has recently been discussed in a report examining succinate production (56). In summary, the lactate titers of three strains were increased by elimination of HEPESNaOH (pH 7.5) with or without lldP introduction. The effect of lldP introduction was indicated by the difference between TA3335 without lldP introduction (8.97 mM) and TA3504 with lldP introduction (10.7 mM) (Fig. 4C). Among all three strains, the highest titer of lactate (11.8 mM) was obtained from TA3739. This strain has both mgsA and lldP introduced and an overexpressed gloAB. Overexpression of the intrinsic putative glyoxalases IeII would stimulate conversion of methylglyoxal to lactate. Therefore, this result indicates that the cloned gloAB gene from S. elongatus PCC 7942 does in fact encode a glyoxalase. The BG11-buf() data demonstrates the importance of pH for lactate production, with increased pH offering the optimal conditions for lactate production and export. We therefore examined the effects of a constantly high pH applied to the TA3739 strain (Fig. 5). To generate a consistently high pH, CHES-NaOH (pH 9.0) and CAPS-NaOH (pH 10.0) buffers were used instead of HEPESNaOH (pH 7.5) buffer. TA3739 showed improved growth in both BG11-CHES and BG11-CAPS compared to standard BG11-HEPES (Figs. 3A and 5A). There was also no indication of toxicity to TA1297 (Fig. S2A). The pH of BG11-CHES and BG11-CAPS showed a moderate decrease during the 24 days of incubation (Fig. 5B). However, compared to BG11-buf(), the pH change was drastically suppressed. The moderate decrease of pH detected during growth of the TA3739 strain was not observed in TA1297 (Fig. S2B). It is likely that the lactate produced during the incubation causes a decrease in pH, as the difference of buffering conditions (without buffer, or with CHES or CAPS) did not affect production rate but did affect the final concentration of lactate (Fig. 5C). The final lactate concentrations produced by TA3739 in BG11-buf(), BG11-CHES, and BG11-CAPS, reached 11.3 mM, 10.8 mM, and 13.7 mM, respectively. Culture media from strain TA3739 grown in BG11CAPS was used to determine lactate optical purity. No L-lactate was detected in the media, demonstrating that highly optically pure D-lactate can be produced by our genetically engineered TA3739 strain. In BG11-buf(), a decrease in pH was observed at 20 days, with a correlating decrease in lactate production. A similar decrease in pH was also observed in BG11-CAPS, but the pH values in BG11-CAPS after 20 days were still higher than in BG11-buf() and BG11-CHES (Fig. 5B). Thus, the importance of maintaining a high extracellular pH is demonstrated by the experiment in different buffering conditions. Intracellular metabolite analysis of lactate producing strain Culture of wild-type TA1297 grown in BG11-HEPES and cultures of engineered TA3739 grown in BG11-HEPES and BG11-CAPS were harvested at 7 days to measure intracellular metabolites using LC-QqQ-MS. The concentrations of metabolites in TA3739 were normalized to those in TA1297 (Fig. 6). Compared to wild-type TA1297, TA3739 cultures showed an increase in lactate concentration and a corresponding decrease in DHAP concentration. This result is consistent with the introduction of the novel lactate producing pathway. Almost all other metabolites, except for ADP-glucose (ADP-Glc) and 3-phosphoglycerate (3PG), were decreased by introduction of the lactate producing pathway and/or buffer changing from HEPES-NaOH (pH 7.5) to CAPS-NaOH (pH 10.0) in TA3739. In comparison of cultures in BG11HEPES and BG11-CAPS of TA3739, there was a decrease in metabolites involved in both the Calvin cycle and glycolysis. This correlated with the increased lactate production seen after buffer changing. These results indicate that the metabolic fluxes of both
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FIG. 5. Lactate production by TA3739 (a strain with mgsA, lldP, gloAB) in BG11 medium with buffers with various pH. (A) Cell density (OD730), (B) pH, (C) lactate concentration. Open and closed symbols represent the data obtained in medium without and with buffer, respectively. Closed circles, squares, and triangles represent the medium with HEPES-NaOH (pH 7.5), CHES-NaOH (pH 9.0), and CAPS-NaOH (pH 10.0) buffer, respectively. Presented data are means S.D. from three individual experiments.
the Calvin cycle and glycolysis are being utilized for lactate production. Generally, chemical production by engineered microorganisms results in a growth defect due to product toxicity, competition of metabolic flux for growth, and redox imbalance. However, our results with the TA3739 strain demonstrate an increase in lactate production combined with an improvement in growth, after buffer changing (Fig. 5). Increased lactate production in TA3739 may be the result of accelerated lactate export due to higher extracellular pH. TA3739 showed increased growth in BG11CAPS compared to BG11-HEPES despite a decrease in the abundance of many important metabolites, including pyruvate and acetyl-CoA. As no obvious differences were detected in intracellular lactate concentration in cultures of TA3739, the growth defect is likely due to accumulation of other intermediate metabolites in the
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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FIG. 6. Measurement of intracellular metabolites in TA1297 (wild-type strain) and TA3739 (a strain with mgsA, lldP, gloAB). Bar charts representing the abundance of metabolites from wild-type TA1297 in BG11-HEPES (left), engineered TA3739 in BG11-HEPES (center), and in BG11-CAPS (right). All concentrations of metabolites in TA3739 were normalized to TA1297. Presented data are means S.D. from three individual experiments. ADP-Glc, ADP-glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F16P, ructose-1,6bisphosphate; S7P, sedoheptulose-7-phosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; R5P, ribose-5-phosphate; 3PG, 3-phosphoglycerate; RuBP, ribulose-1,5-bisphosphate; Ribu5P, ribulose-5-phosphate.
lactate producing pathway, such as methylglyoxal or s-lactoylglutathione. Because the intracellular methylglyoxal and s-lactoylglutathione were not determined in this study, no confirmed result indicating the accumulation of these metabolites was observed. But methylglyoxal is known as toxic metabolite caused of its high reactivity to cellular macromolecules (49). And its toxicity to cyanobacteria was observed in S. elongatus PCC 7942 (Fig. 2) and in Synechococcus sp. PCC 7002 (52). Because the glyoxalase system is glutathione dependent, the enhancement of glutathione content might improve cell growth and lactate productivity. Buffer changing from HEPES-NaOH (pH 7.5) to CAPS-NaOH (pH 10.0) recovered the growth defect present in TA3739, with growth profiles almost identical to wild-type TA1297 grown in BG11-HEPES over 7 days. After 7 days, the growth profiles of the cultures diverged. The cell density of TA1297 linearly increased until approximately 14 days (Figs. 3 and S2), but TA3739 plateaued after 7 days. This result suggests that lactate production competes with cell growth over longer time scales. The newly constructed lactate producing pathway enabled S. elongatus PCC 7942 to produce high titers of lactate (13.7 mM, 1.23 g/l at 24 days), comparable to previous studies using engineered cyanobacteria (Table S3). However, our strain produces lactate from DHAP in the Calvin cycle via methylglyoxal (Fig. 1) rather than conversion from pyruvate. Glyoxalase that converts methylglyoxal to lactate is broadly conserved in most organisms as methylglyoxal detoxification system (50). To our knowledge, this is the first report to demonstrate that this detoxification pathway can be utilized for lactate production, in not only cyanobacteria but also other microorganisms. Lactate production is not dependent on
simple sugars, a key advantage that cyanobacteria production has over other heterotrophs. Production titers higher than 1 g/l, using carbon dioxide fixation, have only been achieved for ethanol (11), sucrose (44), 2,3-butanediol (13), isoprene (23) glycerol (25,26,57), 1,3-PDO (57), and lactate (Table S3). Increasing production titers is therefore an issue that must be solved for efficient chemical production using engineered cyanobacteria. Our data indicate that a combination of lactate producing pathways utilizing both DHAP and pyruvate may increase overall lactate productivity. Our measurements of intracellular metabolites however, indicate that lactate export is likely to be a major bottleneck for further production (Fig. 6). To resolve this, the lactate/Hþ symporter from E. coli was introduced into our engineered cyanobacteria to facilitate lactate export. Unfortunately, the effect of adding this transporter was unexpectedly small (Fig. 4C). Increased titers of lactate under higher pH conditions in a strain without the E. coli transporter suggest the existence of an equivalent intrinsic transporter in S. elongatus PCC 7942. The highest lactate production rate achieved so far using cyanobacteria is 2.2 mM/day. This study used an engineered Synechococcus sp. PCC 7002 strain (41). This rate is about three-fold higher than that of our TA3739 strain over a period of 19 days (day 5 to day 24) (Fig. 5). The high production rates observed in Synechococcus sp. PCC 7002 suggest that this strain possesses a highly effective lactate export system. One way to maximize lactate productivity in TA3739 may be the introduction of the more efficient export system from Synechococcus sp. PCC 7002. Improvements in the lactate exporting system, in combination with our novel pathway could be an effective way to increase lactate titer. In studies specifically examining lactate production from
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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glycolysis in cyanobacteria, metabolic flux to pyruvate was enhanced by knockdown of phosphoenolpyruvate carboxylase and overexpression of pyruvate kinase (62), and conditional downregulation of the glnA gene by CRISPRi (41). Similarly, methods to increase the metabolic flux into DHAP may improve the productivity of the alternative lactate pathway. Our results provide strong evidence for the existence of glyoxalase activity in S. elongatus PCC 7942 that converts methylglyoxal to D-lactate. We have constructed a novel lactate producing pathway that utilizes DHAP in the Calvin cycle and uses intrinsic glyoxalase activity in S. elongatus PCC 7942. This pathway was created by the introduction of a methylglyoxal synthase from E. coli. To improve lactate production further, a lactate/Hþ symporter from E. coli was introduced and an intrinsic putative glyoxalase was overexpressed. The highest lactate titer of 13.7 mM (1.23 g/l) was achieved under high extracellular pH conditions by the engineered TA3739 strain in which methylglyoxal synthase, lactate/Hþ symporter, and glyoxalase were introduced. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2017.02.016.
ACKNOWLEDGMENTS This study was financially supported by the Core Research of Evolutional Science and Technology program (CREST) from the Japan Science and Technology Agency (JST).
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Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942 Yasutaka Hirokawa, Ryota Goto, Yoshitaka Umetani, and Taizo Hanai* Laboratory for Bioinformatics, Graduate School of Systems Biosciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Received 27 January 2017; accepted 23 February 2017 Available online xxx
Using engineered cyanobacteria to produce various chemicals from carbon dioxide is a promising technology for a sustainable future. Lactate is a valuable commodity that can be used for the biodegradable plastic, polylactic acid. Typically, lactate production using engineered cyanobacteria was via the conversion of pyruvate in glycolysis by lactate dehydrogenase. In cyanobacteria, the metabolic flux in the Calvin cycle is higher than that in glycolysis under photoautotrophic conditions. The construction of a novel lactate producing pathway that uses metabolites from the Calvin cycle could potentially increase lactate productivity in cyanobacteria. In order to develop such a novel lactate production pathway, we engineered a cyanobacterium Synechococcus elongatus PCC 7942 strain that produced lactate directly from carbon dioxide using dihydroxyacetone phosphate (DHAP) via methylglyoxal. We confirmed that wild-type strain of S. elongatus PCC 7942 could produce lactate using exogenous methylglyoxal. A methylglyoxal synthase gene, mgsA, from Escherichia coli was introduced into Synechococcus elongates PCC 7942 for conversion of DHAP to methylglyoxal. This engineered strain produced lactate directly from carbon dioxide. Genes encoding intrinsic putative glyoxalase I, II (Synpcc7942_0638, 1403) and the lactate/HD symporter from E. coli (lldP) were additionally introduced to enhance the production. For higher lactate production, it was important to maintain elevated extracellular pH due to the characteristics of lactate exporting system. In this study, the highest lactate titer of 13.7 mM (1.23 g/l) was achieved during a 24day incubation with the engineered S. elongatus PCC 7942 strain possessing the novel lactate producing pathway. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Carbon dioxide; Cyanobacteria; D-Lactate; Photosynthesis; Glyoxalase; Dihydroxyacetone phosphate]
The world’s energy supply is highly dependent on fossil resources despite the excessive consumption of such fuel, leading to numerous environmental issues. It is crucial to reduce our dependency on fossil resources for both energy generation and chemical production for a sustainable future. One option to solve such problems is the use of biomass for the biological production of fuels and chemicals (1,2). This approach has become feasible by advances in genetic engineering that allow modification of microorganisms such as Escherichia coli and yeast to either enhance production of natural chemicals or to even generate entirely foreign substances (3,4). One of the most important limiting factors affecting the use of these organisms is the need for an abundant carbon source to feed the manufacturing process. So far edible biomass (for example, corn and sugarcane) (5) and inedible lignocellulosic biomass (for example, wood wastes and energy crops) (6) were used for bioproduction. However, commonly used heterotrophic microorganisms are unable to directly assimilate these biomass, so it must first be enzymatically or chemically saccharified to simple sugars (7,8). An additional issue is that growing the required biomass can take a considerable amount of time, again limiting potential.
* Corresponding author. Tel.: þ81 92 642 6751; fax: þ81 92 642 6744. E-mail address: [email protected] (T. Hanai).
In contrast to heterotrophic microorganisms, cyanobacteria are photoautotrophic and feed by fixing carbon dioxide to organic compounds using solar energy. Similar to other organisms, genetic engineering approaches have also been developed in cyanobacteria for the production of chemicals, although in this case, products are manufactured directly from carbon dioxide. Some of these valuable products include isobutyraldehyde, isobutanol (9,10), ethanol (11), 2,3-butanediol (12,13), 2-methyl-1-butanol (14), 1-butanol (15,16), acetone (17,18), 3-hydroxybutyrate (19), ethylene (20,21), isoprene (22,23), 1,2-propanediol (1,2-PDO) (24), and glycerol (25,26). Our prior work has also demonstrated biological production of isopropanol (27) and 1,3-PDO (28) using S. elongatus PCC 7942. However, the currently achievable chemical titers by engineered cyanobacteria are significantly lower than those from heterotrophic microorganisms (29,30). For example, even the most efficient ethanol production with engineered cyanobacteria has not been able to exceed 10 g/l (11). Therefore, while direct production of chemicals from carbon dioxide is an attractive and promising technology for a sustainable future, increased production titers are necessary if it is to become practical and cost effective. Lactate is one of the major chemicals produced by engineered microorganisms. It is widely used and can be found in a range of food additives, cosmetics, and various pharmaceuticals (31,32). Demand as a substrate for biodegradable plastics (in the form of poly-lactic acid; PLA) is expected to increase as the world moves
1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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towards a sustainable future (33,34). Structurally, the asymmetric carbon in the lactate molecule yields two optical isomers, L-lactate and D-lactate. Importantly, the physical properties of PLA are affected by optical purity so it is vital to produce optically pure lactate for PLA synthesis (35). Lactate can be produced chemically by hydrolysis of lactonitrile, a byproduct of acrylonitrile production. However, lactate produced by this method is a mixture of the two optical isomers, affecting the quality of any resultant PLA (36). When produced biologically, lactate is generated from conversion of pyruvate by lactate dehydrogenase (LDH) (37). The specific optical character of the lactate isomer produced strictly depends on enzymatic characteristics, allowing optically pure lactate to be manufactured. The lactic acid bacteria, Lactobacillus sp. and Lactococcus sp. can naturally produce lactate (38), but these strains have complex auxotrophic requirements and the purity of produced lactate depends on incubation conditions. To overcome these drawbacks, LDH has been introduced into more user friendly microorganisms such as E. coli and yeast. This has led to more effective biological production of optically pure lactate (39). Lactate can be produced directly from carbon dioxide using genetically engineered cyanobacteria. As with E. coli and yeast, the introduction of LDH enables cyanobacteria to produce lactate from cellular pyruvate. The highest lactate titer of 20.5 mM (1.84 g/l) has been achieved using an engineered Synechocystis sp. PCC 6803 strain possessing LDH (40). The highest production rate (2.2 mM/ day) was obtained using another cyanobacterium strain, Synechococcus sp. PCC 7002 (41). This production rate was achieved by combining LDH introduction with additional genetic engineering using CRISPRi to increase central carbon flux. Another approach to increase lactate production may be to utilize a metabolic pathway that naturally possesses a higher carbon flux. One example is the Calvin cycle of photoautotrophically grown cyanobacteria, which typically shows a greater flux than glycolysis (42,43). The reason of greater flux in Calvin cycle is that many parts of fixed carbon by photosynthesis were utilized to reproduce ribulose-1,5bisphosphate for next carbon fixation. Chemical production using metabolites in the Calvin cycle of cyanobacteria would therefore be expected to lead to higher titers of chemical production. Previously, production of 1,2-PDO, sucrose, glycerol, and 1,3-PDO have all been achieved using metabolites (DHAP or fructose-6-phosphate) in the Calvin cycle (19,24,25,28,44). The titers of these chemicals were indeed higher than the other chemicals produced using metabolites from other pathways. In this study, we constructed a novel lactate producing pathway that uses dihydroxyacetone phosphate (DHAP), a metabolite from the Calvin cycle, rather than pyruvate (Fig. 1). The pathway composed of methylglyoxal synthase, lactate/Hþ symporter, and glyoxalase I, II was constructed in S. elongatus PCC 7942. In the constructed pathway, any reducing power like as NADH is not
FIG. 1. Overview of the novel lactate producing pathway constructed in this study. Introduced genes are highlighted in italics. Shaded background indicates exogenous gene (mgsA and lldP) from E. coli. White background indicates intrinsic gene (gloA and gloB). 3PG, 3-phosphoglycerate; DHAP, dihydroxyacetone phosphate; MG, methylglyoxal; s-LG, s-lactoylglutathione; GSH, glutathione.
J. BIOSCI. BIOENG., necessary for lactate production and DHAP in Calvin cycle showing greater flux (42,43) is used as substrate. Previously, we constructed 1,3-PDO and glycerol producing pathway from DHAP in this strain (28). We fully investigated the effectiveness of the novel pathway in an engineered cyanobacterium S. elongatus PCC 7942 strain. To the best of our knowledge, it is the first time to construct a lactate producing pathway using DHAP for lactate production in cyanobacteria and even any bacteria.
MATERIALS AND METHODS Chemicals and reagents All chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless otherwise specified. Restriction enzymes, phosphatase (New England Biolabs; Ipswich, MA, USA), ligase (Rapid DNA Ligation Kit, Roche, Mannheim, Germany), and DNA polymerase (KOD Plus Neo DNA polymerase, Toyobo Co., Ltd., Osaka, Japan) were used for cloning. Oligonucleotides were synthesized by Life Technologies Japan, Ltd. (Tokyo, Japan). Culture media Modified BG11 medium supplemented with 20 mM HEPESNaOH (pH 7.5) for pH stabilization was used for cultivation of S. elongatus PCC 7942 (Life Technologies Corporation, Carlsbad, CA, USA). Hereafter, this medium was referred to as BG11-HEPES medium. The composition of BG11-HEPES medium used in this study was the same as previously reported (27). In some experiments, HEPES-NaOH (pH 7.5) buffer was eliminated and substituted for 20 mM CHESNaOH (pH 9.0) or CAPS-NaOH (pH 10.0). Hereafter, these buffer replaced mediums were referred to as BG11-CHES and BG11-CAPS medium. To prepare the plate medium, 1.5% (w/v) of Bacto Agar (Difco Laboratories, Franklin Lakes, NJ, USA) was added. Antibiotics (10 mg/ml kanamycin and 20 mg/ml spectinomycin) were added to BG11 mediums as appropriate. Growth and production conditions All cyanobacterial cultures were grown under fluorescent light (100 mmol photon m2 s1) at 30 C in a growth chamber (MLR-325H-PJ; Panasonic Corporation, Osaka, Japan). Photon flux density was measured with an IKS-27 (Koito Manufacturing, Tokyo, Japan). Cell density (OD730) was measured using an Infinite 200 PRO (Tecan, Männedorf, Switzerland). For preculture, cells were inoculated into 20 mL BG11-HEPES medium in a 50-mL flask, and incubated under fluorescent light on a rotor shaking at 150 rpm (NR-30 shaker; Taitec Corporation, Saitama, Japan). Pre-cultured cells at OD730 of 1.0e2.0 were inoculated into 20 mL BG11 mediums containing 1 mM of IPTG at an initial OD730 of 0.025. Cultures were grown under same conditions as pre-culture. Strain construction The bacterial strains used in this study are listed in Table S1. Genes were integrated into the S. elongatus PCC 7942 genome by homologous recombination. For gene integration into the neutral site (NS) I (45) and NS II (46), the previously constructed and newly generated plasmids were used. The integrated genes in the engineered strains were confirmed by sequence analysis. Plasmids construction Plasmids and primers used in this study are listed in Tables S1 and S2. The gene encoding methylglyoxal synthase (mgsA) was amplified from E. coli BW25113 genome using primers T1514-T1590. The amplified sequence was digested by Acc65I-BglII. The digested PCR products were ligated into Acc65I-BamHI site of pTA424 to create pTA1501 (PLlacO1::mgsA, NS II-targeting plasmid). mgsA amplified from E. coli BW25113 genome using primers T1514-T1515 was digested by KpnI-NheI. The gene encoding lactate/Hþ symporter (lldP) amplified from E. coli BW25113 genome with primers T2576-T2577 was digested by NheIBamHI. Digested PCR products were ligated into the KpnI-BamHI site of pTA424 to create pTA1567 (PLlacO1::mgsA-lldP, NS II-targeting plasmid). A NS I targeting plasmid (pTA1562) was newly constructed based on commercial pZ vectors purchased from EXPESSYS. The antibiotic resistant cassette of pZE22-MCS was switched from kanamycin resistance to spectinomycin resistance to create pTA1471. The upper and lower regions of NS I were amplified from S. elongatus PCC 7942 genome using primers T2489-T2490, and T2491-T2492, respectively. The amplified fragments were digested by AvrII-SpeI. The digested NS I upper and NS I lower fragments were ligated into the SpeI and AvrII sites of pTA1471 respectively to create pTA1476. lacI regulated by a PlacIq promoter was amplified using primers T2662-T2664 and digested by AvrII-SpeI. The digested fragment was ligated into the AvrII site of pTA1476 to create pTA1562. The genes encoding putative glyoxalase [gloA (Synpcc7942_0638) and gloB (Synpcc7942_1403)] were amplified from the S. elongatus PCC 7942 genome using primers T2783-T2784 and T2785-T2786, respectively. The amplified gloA and gloB sequences were digested by KpnI-NheI and NheI-BamHI, respectively. The digested sequences were ligated into KpnI-BamHI site of pTA1562 to create pTA1697 (PLlacO1::gloAB, PlacIq::lacI, NS I-targeting plasmid). Product analysis The supernatant was obtained by centrifugation (20,000 g, 10 min, 4 C) and filtered using Minisart RC4 (Sartorius, Goetingen, Germany). The filtered sample was subjected to quantitative analysis. Lactate was analyzed using a high-performance liquid chromatograph (LC-20AD, Shimadzu, Kyoto, Japan) equipped with an autosampler and electric conductivity detector
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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(CDD-10A, Shimadzu). The detailed conditions of analysis were described previously (47). A D-/L-Lactic Acid (D-/L-Lactate) (Rapid) Assay Kit (K-DLATE, Megazyme, Ireland) was used to determine the optical purity of the produced lactate. Measurement of intracellular metabolites Intracellular metabolites of TA1297 (wild-type strain) and TA3739 (lactate producing strain) were measured using a LC-QqQ-MS system (high-performance liquid chromatography: Agilent 1290 Infinity, MS: Agilent 6460 with Jet Stream Technology) (Agilent Technologies, Waldbronn, Germany) controlled by MassHunter Workstation Data Acquisition software (Agilent Technologies). Culture at 7 days was used to measure intracellular metabolites. Sample preparation and LC-QqQ-MS analysis were performed using conditions described previously (48).
RESULTS AND DISCUSSION Lactate producing pathway from DHAP in the Calvin cycle Methylglyoxal is a toxic metabolite that can cause cell death at high concentrations (49). Because cellular production of methylglyoxal can occur both enzymatically and non-enzymatically in organisms, several detoxification systems have evolved. The glutathione dependent glyoxalase IeII system is one of the most conserved methylglyoxal detoxification systems (50). It is composed of two enzymes, glyoxalase I (EC 4.4.1.5) and glyoxalase II (EC 3.1.2.6), that convert methylglyoxal to D-lactate. Enzymatically, methylglyoxal is produced from DHAP by the action of methylglyoxal synthase (EC 4.2.3.3). Previously, DHAP has been used in engineered cyanobacteria as a substrate for both 1,2-PDO (24) and glycerol production (25,26). We have also constructed a 1,3-PDO producing pathway using DHAP in S. elongatus PCC 7942 (28). DHAP is one of the metabolites in the Calvin cycle that exhibits a particularly high metabolic flux in cyanobacteria under photoautotrophic conditions (42,43). A higher production rate could be achieved using DHAP in engineered cyanobacteria rather than using metabolites from other pathways. To test this hypothesis, we designed a novel production pathway that will generate lactate from DHAP via methylglyoxal rather than pyruvate (Fig. 1). Evaluation of native glyoxalase system in S. elongatus PCC 7942 In Synechocystis sp. PCC 6803, Slr0381 (gloA) and Sll1019 (gloB) were identified as genes encoding glyoxalase I and glyoxalase II, respectively (51). And the kinetic parameters of these gene products were characterized, suggesting that this strain possesses glyoxalase activity converting methylglyoxal to D-lactate. Similar genes, Synpcc7942_0638 (gloA) and Synpcc7942_1403 (gloB), have also been found in S. elongatus PCC 7942, encoding for glyoxalase I and glyoxalase II, respectively. Between S. elongatus PCC 7942 and Synechocystis sp. PCC 6803, the identities of amino acid sequence of glyoxalase I and glyoxalase II are 72.1% and 61.6%, respectively (refer to Cyanobase; http://genome.microbedb. jp/cyanobase). However, glyoxalase activity has not been confirmed in S. elongatus PCC 7942. We therefore examined the natural glyoxalase activity of S. elongatus PCC 7942 in the presence of exogenous methylglyoxal (Fig. 2).
FIG. 2. Lactate production by TA1297 (wild-type strain) in the presence of exogenous methylglyoxal. (A) Cell density (OD730), (B) lactate concentration. Open, shaded, and black circles represent the data with 0, 1, and 5 mM methylglyoxal, respectively. Presented data are means S.D. from three individual experiments.
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Methylglyoxal (1 or 5 mM) was added to TA1297 (wild-type S. elongatus PCC 7942) during the late exponential growth phase. After addition of methylglyoxal, lactate was detectable in the culture. No lactate was detected in the control culture without methylglyoxal (Fig. 2B). These are the results indicating that S. elongatus PCC 7942 possesses an ability to convert methylglyoxal to lactate. Lactate concentration increased for 6 h after the addition of methylglyoxal and then plateaued until the end of the experiment at 24 h. Lactate concentrations peaked at 0.65 mM after the addition of 1 mM methylglyoxal and at 1.28 mM after the addition of 5 mM methylglyoxal. Deletion of ldh (Synpcc7942_1347), effectively limiting the conversion of pyruvate to lactate, did not affect lactate production from exogenous methylglyoxal (data not shown). These results strongly suggest the presence of glyoxalase activity in S. elongatus PCC 7942. In glyoxalase system, one molecule of lactate is produced from one molecule of methylglyoxal (50). The lower conversion yield (about 60%) in 1 mM methylglyoxal addition would be caused of methylglyoxal property being instability and high reactivity to protein, lipid, and nucleic acid (49). Similar growth rates were observed in cultures without methylglyoxal and with 1 mM methylglyoxal, however addition of 5 mM methylglyoxal resulted in a serious growth defect (Fig. 2A). It is possible that 5 mM methylglyoxal exceeds the natural detoxification capacity of S. elongatus PCC 7942 and this was a reason why the conversion yield in 5 mM methylglyoxal was lower than that in 1 mM methylglyoxal. Prior work with a Synechococcus sp. PCC 7002 strain demonstrated that cell growth gradually decreased at methylglyoxal concentrations above 1.5 mM and concentrations above 3 mM completely inhibit cell growth (52). The methylglyoxal tolerance of S. elongatus PCC 7942 appears consistent with that observed in Synechococcus sp. PCC 7002. Lactate production from DHAP via methylglyoxal In order to generate lactate from cellular DHAP, genes encoding methylglyoxal synthase (mgsA), the lactate/Hþ symporter (lldP), and glyoxalase I, II (gloAB) were integrated into the genome of S. elongatus PCC 7942 by homologous recombination. The specific mgsA and lldP sequences used were obtained from E. coli. These genes have been previously validated for 1,2-PDO and lactate production in engineered S. elongatus PCC 7942 (24,53). Due to the newly identified existence of intrinsic glyoxalase activity (Fig. 2), we cloned putative glyoxalase genes from S. elongatus PCC 7942. In total, three strains were constructed for testing lactate production: TA3335 (a strain with mgsA only), TA3504 (a strain with mgsA and lldP), and TA3739 (a strain with mgsA, lldP, and gloAB) (Table S1, Fig. S1). The engineered strains were incubated under photoautotrophic conditions and tested for lactate production (Fig. 3). Compared to wild-type S. elongatus PCC 7942 (TA1297), a growth defect was observed in the TA3335, TA3504, and TA3739 strains (Fig. 3A). Approximately equivalent concentrations of lactate were produced by each of the three engineered strains (Fig. 3C). During the twoweek incubation, lactate concentration reached a peak of 2.85 mM, 2.18 mM, and 2.79 mM in cultures of TA3335, TA3504, and TA3739, respectively. No lactate production was detected in the culture of wild-type TA1297. This result indicates that, through the addition of methylglyoxal synthase, we have successfully introduced a lactate producing pathway that uses DHAP in the Calvin cycle. However, other genes that were added (lldP, gloAB) to improve productivity had no effect under these conditions. This may be because the direction of lactate transport by the lactate/Hþ symporter LldP is dependent on differences between the extracellular and intracellular concentration of Hþ. A relationship between transport direction and extracellular pH has previously been described for this transporter (44,54). Specifically, to facilitate export of lactate from the cell to the media, an extracellular pH that
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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FIG. 3. Lactate production directly from carbon dioxide in BG11-HEPES (pH 7.5). (A) Cell density (OD730), (B) pH, (C) lactate concentration. Circles, squares, triangles, and diamonds represent the data of TA1297 (wild-type strain), TA3335 (mgsA), TA3504 (mgsA, lldP), and TA3739 (mgsA, lldP, gloAB), respectively. Presented data are means S.D. from three individual experiments.
is higher than intracellular pH is necessary. The HEPES-NaOH buffer (pH 7.5) is basically supplemented in medium for incubation of cyanobacteria to keep experimental conditions stable in our previous works. In lactate production, the extracellular pH kept nearly neutral, however, would affect lactate transport (Fig. 3B). Optimization of culture condition for effective lactate production To determine if culture pH influences lactate export, we examined the effect of different media conditions on our engineered strains. In BG11 medium without HEPES-NaOH buffer (BG11-buf()), a growth defect observed in all lactate producing strains (TA3335, TA3504, and TA3739) was decreased compared with that in BG11-HEPES (Figs. 3A and 4A). The pH values of the control culture media (BG11-HEPES) were consistently pH 7.5 (Fig. 3B), whereas cultures without HEPES-NaOH buffer (BG11-buf()) observed a dramatic increase to pH 11.0 during the
J. BIOSCI. BIOENG.,
FIG. 4. Lactate production directly from carbon dioxide in medium without HEPESNaOH buffer (BG11-buf()). (A) Cell density (OD730), (B) pH, (C) lactate concentration. Squares, triangles, and diamonds represent the data of TA3335 (mgsA), TA3504 (mgsA, lldP), and TA3739 (mgsA, lldP, gloAB), respectively. Presented data are means S.D. from three individual experiments.
first 5 days and then gradually decreased to pH 7.0 during the 25 day incubation (Fig. 4B). pH increase observed in BG11-buf() would be caused of the reduction of nitrate to basic ammonia for amino acid synthesis by cyanobacteria metabolism (55). Under these pH conditions in BG11-buf() media, lactate production was improved and concentrations reached 8.97 mM, 10.7 mM, and 11.8 mM in cultures of TA3335, TA3504, and TA3739, respectively (Fig. 4C). The correlation of growth and lactate titers strongly suggest that the observed growth defect is likely caused by accumulation of metabolites involved in lactate producing pathway in the cell, while the acceleration of lactate export was due to higher extracellular pH. Unexpectedly, elimination of HEPES-NaOH buffer increased lactate production in a strain without additional lldP (TA3335) (Fig. 4C). These results
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
VOL. xx, 2017 suggested that the lactate export system exist in S. elongatus PCC 7942 and this system is similar to E. coli lldP. The existence of organic acid transporter that depends on extracellular pH in S. elongatus PCC 7942 has recently been discussed in a report examining succinate production (56). In summary, the lactate titers of three strains were increased by elimination of HEPESNaOH (pH 7.5) with or without lldP introduction. The effect of lldP introduction was indicated by the difference between TA3335 without lldP introduction (8.97 mM) and TA3504 with lldP introduction (10.7 mM) (Fig. 4C). Among all three strains, the highest titer of lactate (11.8 mM) was obtained from TA3739. This strain has both mgsA and lldP introduced and an overexpressed gloAB. Overexpression of the intrinsic putative glyoxalases IeII would stimulate conversion of methylglyoxal to lactate. Therefore, this result indicates that the cloned gloAB gene from S. elongatus PCC 7942 does in fact encode a glyoxalase. The BG11-buf() data demonstrates the importance of pH for lactate production, with increased pH offering the optimal conditions for lactate production and export. We therefore examined the effects of a constantly high pH applied to the TA3739 strain (Fig. 5). To generate a consistently high pH, CHES-NaOH (pH 9.0) and CAPS-NaOH (pH 10.0) buffers were used instead of HEPESNaOH (pH 7.5) buffer. TA3739 showed improved growth in both BG11-CHES and BG11-CAPS compared to standard BG11-HEPES (Figs. 3A and 5A). There was also no indication of toxicity to TA1297 (Fig. S2A). The pH of BG11-CHES and BG11-CAPS showed a moderate decrease during the 24 days of incubation (Fig. 5B). However, compared to BG11-buf(), the pH change was drastically suppressed. The moderate decrease of pH detected during growth of the TA3739 strain was not observed in TA1297 (Fig. S2B). It is likely that the lactate produced during the incubation causes a decrease in pH, as the difference of buffering conditions (without buffer, or with CHES or CAPS) did not affect production rate but did affect the final concentration of lactate (Fig. 5C). The final lactate concentrations produced by TA3739 in BG11-buf(), BG11-CHES, and BG11-CAPS, reached 11.3 mM, 10.8 mM, and 13.7 mM, respectively. Culture media from strain TA3739 grown in BG11CAPS was used to determine lactate optical purity. No L-lactate was detected in the media, demonstrating that highly optically pure D-lactate can be produced by our genetically engineered TA3739 strain. In BG11-buf(), a decrease in pH was observed at 20 days, with a correlating decrease in lactate production. A similar decrease in pH was also observed in BG11-CAPS, but the pH values in BG11-CAPS after 20 days were still higher than in BG11-buf() and BG11-CHES (Fig. 5B). Thus, the importance of maintaining a high extracellular pH is demonstrated by the experiment in different buffering conditions. Intracellular metabolite analysis of lactate producing strain Culture of wild-type TA1297 grown in BG11-HEPES and cultures of engineered TA3739 grown in BG11-HEPES and BG11-CAPS were harvested at 7 days to measure intracellular metabolites using LC-QqQ-MS. The concentrations of metabolites in TA3739 were normalized to those in TA1297 (Fig. 6). Compared to wild-type TA1297, TA3739 cultures showed an increase in lactate concentration and a corresponding decrease in DHAP concentration. This result is consistent with the introduction of the novel lactate producing pathway. Almost all other metabolites, except for ADP-glucose (ADP-Glc) and 3-phosphoglycerate (3PG), were decreased by introduction of the lactate producing pathway and/or buffer changing from HEPES-NaOH (pH 7.5) to CAPS-NaOH (pH 10.0) in TA3739. In comparison of cultures in BG11HEPES and BG11-CAPS of TA3739, there was a decrease in metabolites involved in both the Calvin cycle and glycolysis. This correlated with the increased lactate production seen after buffer changing. These results indicate that the metabolic fluxes of both
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FIG. 5. Lactate production by TA3739 (a strain with mgsA, lldP, gloAB) in BG11 medium with buffers with various pH. (A) Cell density (OD730), (B) pH, (C) lactate concentration. Open and closed symbols represent the data obtained in medium without and with buffer, respectively. Closed circles, squares, and triangles represent the medium with HEPES-NaOH (pH 7.5), CHES-NaOH (pH 9.0), and CAPS-NaOH (pH 10.0) buffer, respectively. Presented data are means S.D. from three individual experiments.
the Calvin cycle and glycolysis are being utilized for lactate production. Generally, chemical production by engineered microorganisms results in a growth defect due to product toxicity, competition of metabolic flux for growth, and redox imbalance. However, our results with the TA3739 strain demonstrate an increase in lactate production combined with an improvement in growth, after buffer changing (Fig. 5). Increased lactate production in TA3739 may be the result of accelerated lactate export due to higher extracellular pH. TA3739 showed increased growth in BG11CAPS compared to BG11-HEPES despite a decrease in the abundance of many important metabolites, including pyruvate and acetyl-CoA. As no obvious differences were detected in intracellular lactate concentration in cultures of TA3739, the growth defect is likely due to accumulation of other intermediate metabolites in the
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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FIG. 6. Measurement of intracellular metabolites in TA1297 (wild-type strain) and TA3739 (a strain with mgsA, lldP, gloAB). Bar charts representing the abundance of metabolites from wild-type TA1297 in BG11-HEPES (left), engineered TA3739 in BG11-HEPES (center), and in BG11-CAPS (right). All concentrations of metabolites in TA3739 were normalized to TA1297. Presented data are means S.D. from three individual experiments. ADP-Glc, ADP-glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F16P, ructose-1,6bisphosphate; S7P, sedoheptulose-7-phosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; R5P, ribose-5-phosphate; 3PG, 3-phosphoglycerate; RuBP, ribulose-1,5-bisphosphate; Ribu5P, ribulose-5-phosphate.
lactate producing pathway, such as methylglyoxal or s-lactoylglutathione. Because the intracellular methylglyoxal and s-lactoylglutathione were not determined in this study, no confirmed result indicating the accumulation of these metabolites was observed. But methylglyoxal is known as toxic metabolite caused of its high reactivity to cellular macromolecules (49). And its toxicity to cyanobacteria was observed in S. elongatus PCC 7942 (Fig. 2) and in Synechococcus sp. PCC 7002 (52). Because the glyoxalase system is glutathione dependent, the enhancement of glutathione content might improve cell growth and lactate productivity. Buffer changing from HEPES-NaOH (pH 7.5) to CAPS-NaOH (pH 10.0) recovered the growth defect present in TA3739, with growth profiles almost identical to wild-type TA1297 grown in BG11-HEPES over 7 days. After 7 days, the growth profiles of the cultures diverged. The cell density of TA1297 linearly increased until approximately 14 days (Figs. 3 and S2), but TA3739 plateaued after 7 days. This result suggests that lactate production competes with cell growth over longer time scales. The newly constructed lactate producing pathway enabled S. elongatus PCC 7942 to produce high titers of lactate (13.7 mM, 1.23 g/l at 24 days), comparable to previous studies using engineered cyanobacteria (Table S3). However, our strain produces lactate from DHAP in the Calvin cycle via methylglyoxal (Fig. 1) rather than conversion from pyruvate. Glyoxalase that converts methylglyoxal to lactate is broadly conserved in most organisms as methylglyoxal detoxification system (50). To our knowledge, this is the first report to demonstrate that this detoxification pathway can be utilized for lactate production, in not only cyanobacteria but also other microorganisms. Lactate production is not dependent on
simple sugars, a key advantage that cyanobacteria production has over other heterotrophs. Production titers higher than 1 g/l, using carbon dioxide fixation, have only been achieved for ethanol (11), sucrose (44), 2,3-butanediol (13), isoprene (23) glycerol (25,26,57), 1,3-PDO (57), and lactate (Table S3). Increasing production titers is therefore an issue that must be solved for efficient chemical production using engineered cyanobacteria. Our data indicate that a combination of lactate producing pathways utilizing both DHAP and pyruvate may increase overall lactate productivity. Our measurements of intracellular metabolites however, indicate that lactate export is likely to be a major bottleneck for further production (Fig. 6). To resolve this, the lactate/Hþ symporter from E. coli was introduced into our engineered cyanobacteria to facilitate lactate export. Unfortunately, the effect of adding this transporter was unexpectedly small (Fig. 4C). Increased titers of lactate under higher pH conditions in a strain without the E. coli transporter suggest the existence of an equivalent intrinsic transporter in S. elongatus PCC 7942. The highest lactate production rate achieved so far using cyanobacteria is 2.2 mM/day. This study used an engineered Synechococcus sp. PCC 7002 strain (41). This rate is about three-fold higher than that of our TA3739 strain over a period of 19 days (day 5 to day 24) (Fig. 5). The high production rates observed in Synechococcus sp. PCC 7002 suggest that this strain possesses a highly effective lactate export system. One way to maximize lactate productivity in TA3739 may be the introduction of the more efficient export system from Synechococcus sp. PCC 7002. Improvements in the lactate exporting system, in combination with our novel pathway could be an effective way to increase lactate titer. In studies specifically examining lactate production from
Please cite this article in press as: Hirokawa, Y., et al., Construction of a novel D-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.02.016
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glycolysis in cyanobacteria, metabolic flux to pyruvate was enhanced by knockdown of phosphoenolpyruvate carboxylase and overexpression of pyruvate kinase (62), and conditional downregulation of the glnA gene by CRISPRi (41). Similarly, methods to increase the metabolic flux into DHAP may improve the productivity of the alternative lactate pathway. Our results provide strong evidence for the existence of glyoxalase activity in S. elongatus PCC 7942 that converts methylglyoxal to D-lactate. We have constructed a novel lactate producing pathway that utilizes DHAP in the Calvin cycle and uses intrinsic glyoxalase activity in S. elongatus PCC 7942. This pathway was created by the introduction of a methylglyoxal synthase from E. coli. To improve lactate production further, a lactate/Hþ symporter from E. coli was introduced and an intrinsic putative glyoxalase was overexpressed. The highest lactate titer of 13.7 mM (1.23 g/l) was achieved under high extracellular pH conditions by the engineered TA3739 strain in which methylglyoxal synthase, lactate/Hþ symporter, and glyoxalase were introduced. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2017.02.016.
ACKNOWLEDGMENTS This study was financially supported by the Core Research of Evolutional Science and Technology program (CREST) from the Japan Science and Technology Agency (JST).
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