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Metabolic Engineering of Synechococcus Elongatus PCC Hirokawa et al. Microb Cell Fact (2017) 16:212 DOI 10.1186/s12934-017-0824-4 Microbial Cell Factories RESEARCH Open Access Metabolic engineering of Synechococcus elongatus PCC 7942 for improvement of 1,3‑propanediol and glycerol production based on in silico simulation of metabolic fux distribution Yasutaka Hirokawa1, Shingo Matsuo1, Hiroyuki Hamada1, Fumio Matsuda2 and Taizo Hanai1* Abstract Background: Production directly from carbon dioxide by engineered cyanobacteria is one of the promising tech- nologies for sustainable future. Previously, we have successfully achieved 1,3-propanediol (1,3-PDO) production using Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. The strain into which the synthetic metabolic pathway was introduced produced 3.48 mM (0.265 g/L) 1,3-PDO and 14.3 mM (1.32 g/L) glycerol during 20 days of incubation. In this study, the productivities of 1,3-PDO were improved by gene disruption selected by screening with in silico simulation. Methods: First, a stoichiometric metabolic model was applied to prediction of cellular metabolic fux distribution in a 1,3-PDO-producing strain of S. elongatus PCC 7942. A genome-scale model of S. elongatus PCC 7942 constructed by Knoop was modifed by the addition of a synthetic metabolic pathway for 1,3-PDO production. Next, the metabolic fux distribution predicted by metabolic fux balance analysis (FBA) was used for in silico simulation of gene disrup- tion. As a result of gene disruption simulation, NADPH dehydrogenase 1 (NDH-1) complexes were found by screening to be the most promising candidates for disruption to improve 1,3-PDO production. The efect of disruption of the gene encoding a subunit of the NDH-1 complex was evaluated in the 1,3-PDO-producing strain. Results and Conclusions: During 20 days of incubation, the ndhF1-null 1,3-PDO-producing strain showed the highest titers: 4.44 mM (0.338 g/L) 1,3-PDO and 30.3 mM (2.79 g/L) glycerol. In this study, we successfully improved 1,3-PDO productivity on the basis of in silico simulation of gene disruption. Keywords: Cyanobacteria, Flux balance analysis, Synthetic metabolic pathway, 1,3-Propanediol Background biological production using heterotrophic microorgan- Direct production of chemicals and fuels from car- isms, biological production using cyanobacteria can bon dioxide by cyanobacteria is one of the promising avoid various complicated processes for harvesting and technologies to reduce carbon dioxide emissions and decomposition of biomass. Te introduction of a syn- the dependence on fossil fuels [1, 2]. Compared with thetic metabolic pathway composed of genes from other organisms into cyanobacteria is a key technology for production of various chemicals directly from carbon *Correspondence: [email protected]‑u.ac.jp dioxide. Some of these valuable chemicals include isobu- 1 Laboratory for Bioinformatics, Graduate School of Systems Biosciences, tyraldehyde, isobutanol [3, 4], ethanol [5], 2,3-butanediol Kyushu University, 804 Westwing, 3‑1‑1 Maidashi, Higashi‑ku, Fukuoka 812‑8582, Japan [6, 7], 2-methyl-1-butanol [8], 1-butanol [9, 10], ace- Full list of author information is available at the end of the article tone [11, 12], 3-hydroxybutyrate [13], ethylene [14, 15], © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hirokawa et al. Microb Cell Fact (2017) 16:212 Page 2 of 12 isoprene [16, 17], 1,2-propanediol (1,2-PDO) [18], and gene manipulation, various strategies have been estab- glycerol [19, 20]. In addition, we successfully developed lished for prediction of the efects of a gene knockout, Synechococcus elongatus PCC 7942 to produce 1,3-pro- knockdown, and overexpression [28]. Shastri and Mor- panediol (1,3-PDO) by introduction of a synthetic meta- gan [29] were the frst to construct a stoichiometric met- bolic pathway [21]. 1,3-PDO is a valuable chemical widely abolic model of cyanobacteria composed of the central used for manufacture of polymers, paint, solvents, and metabolism and photosystem of Synechocystis sp. PCC antifreeze agents [22]. In the introduced synthetic meta- 6803. So far, the metabolic models of some cyanobacteria bolic pathway, dihydroxyacetone phosphate (DHAP) in used for bioproduction (Synechocystis sp. PCC 6803, Syn- the Calvin cycle is converted to 1,3-PDO via glycerol echococcus sp. PCC 7002, Synechococcus elongatus PCC (Fig. 1). Te strain into which the synthetic pathway was 7942, Arthrospira platensis, Cyanothece sp. ATCC 51142) introduced was found to produce 3.79 mM (0.29 g/L) 1,3- have been constructed on the basis of an annotated PDO and 12.6 mM (1.16 g/L) glycerol during 14 days of genome sequence [30–32]. Anfelt et al. [9] reported that incubation [21]. Although chemical production directly introduction of the phosphoketolase pathway increases from carbon dioxide is a key advantage of cyanobacteria cellular acetyl-CoA level and butanol productivity in Syn- compared with other heterotrophic organisms, even the echocystis sp. PCC 6803. FBA was used for validation of ethanol production by cyanobacteria showing the highest this genetic modifcation to increase theoretical produc- titer (5.5 g/L) [5] among various chemicals has not been tivity of butanol. Furthermore, some simulations based increased over 10 g/L. Terefore, the improvements of on FBA have predicted various metabolic modifcations productivity are key issues to make production by engi- (knockout and overexpression) to increase theoretical neered cyanobacteria practical and cost efective. production by cyanobacteria, for example, of biofuels In silico simulation is an powerful methodology to (1-butanol, 1-octanol, limonene, ethanol) and succinic improve productivity with low experimental costs. Te acid by Synechocystis sp. PCC 6803 [33–35] and biofuels stoichiometric metabolic model not requiring kinetic (butanol, ethanol) by Synechococcus sp. PCC 7002 [36]. parameters of metabolic reactions is comparatively Nonetheless, there are only a few reports that these pre- applicable to analysis of whole-cell metabolism. Flux dicted strategies for productivity improvement applied balance analysis (FBA) can predict metabolic fux distri- experimentally [35]. bution under constraint conditions [23]. FBA using the In this study, we applied FBA to prediction of meta- genome-scale metabolic model (GSM) has been applied bolic fux distribution in a 1,3-PDO-producing strain of to improvement, for example, of succinic acid [24], thre- S. elongatus PCC 7942 constructed in our previous work onine [25], valine [26], and lycopene [27] production by [21]. Te candidates for the gene knockout to increase Escherichia coli. For screening of efective candidates for 1,3-PDO productivity were estimated by FBA with the H2O NADH Calvin cycle O2 NAD+ Photosynthesis Glycerol-3P DHAP ATP NADPH ATP Glycerol CO 2 NAPD+ Cyclic electron flow NADPH 3-HPA O NADPH TCA 2 H2O ATP + cycle NADP + NAD Respiration 1,3-PDO NADH 1,3-PDO Native metabolism of pathway S. elongatus PCC 7942 Fig. 1 The metabolic pathway of the 1,3-PDO-producing strain Hirokawa et al. Microb Cell Fact (2017) 16:212 Page 3 of 12 minimization of metabolic adjustment (MOMA) algo- T2787–T2726, T2721–2722, and T2713–T2714, respec- rithm [37]. Te positive efect of the gene knockout on tively. Te amplifed upstream and downstream regions 1,3-PDO productivity after screening by in silico simula- except for ndhF1 were digested by KpnI–HindIII and tion was experimentally validated in the 1,3-PDO-pro- HindIII–BamHI, respectively, and ligated into KpnI– ducing strain. BamHI sites of pTA703. Only for ndhF1, the upstream and downstream regions were digested with SalI–Hin- Methods dIII and HindIII–BamHI, respectively, and ligated into Chemicals and reagents SalI–BamHI sites of pTA703. A chloramphenicol resist- All the chemicals were purchased from Wako Pure ance cassette was amplifed from pZA31-luc (Expressys; Chemical Industries, Ltd. (Osaka, Japan) unless speci- Ruelzheim, Germany) by PCR using primers T2128– fed otherwise. Restriction enzymes, phosphatase (New T2129. Te amplifed fragment digested with HindIII England Biolabs; Ipswich, MA, USA), ligase (Rapid DNA was ligated into the HindIII site of each plasmid harbor- Ligation Kit, Roche; Mannheim, Germany), and DNA ing upstream and downstream regions of a target gene, polymerase (KOD Plus Neo DNA polymerase, TOYOBO thus creating plasmids pTA1811 (ndhD1 disruption), Co., Ltd.; Osaka, Japan) were used for cloning. Oligonu- pTA1812 (ndhD2 disruption), pTA1641 (ndhD3 disrup- cleotides were synthesized by Life Technologies Japan, tion), pTA1640 (ndhD4 disruption), and pTA1828 (ndhF1 Ltd. (Tokyo, Japan). disruption). Culture media Strain construction A modifed BG11 medium supplemented with 20 mM Te constructed plasmids (pTA1811, 1812, 1641, 1640, HEPES–NaOH (pH 7.5) for pH stabilization was used and 1828) were used for gene disruption in TA1297 for cultivation of S. elongatus PCC 7942 (Life Technolo- (wild-type strain), TA2984 (1,3-PDO-producing strain), gies Corporation; Carlsbad, CA, USA). Hereafter, this and TA3800 (glycerol-producing strain). TA2984 and medium is referred to as the BG11 medium. Te com- TA3800 were constructed in our previous studies [21, position of the BG11 medium used in this study was the 38]. Te strains used in this study are listed in Additional same as previously reported [21]. Antibiotics (10 μg/mL fle 1: Table S1. kanamycin, 20 µg/mL spectinomycin, and 5 µg/mL chlo- ramphenicol) were added to the BG11 medium when Product analysis appropriate. Culture supernatants were centrifuged (20,000×g, 10 min) and fltered using Minisart RC4 (Sartorius; Production conditions Goettingen, Germany).
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