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Metabolic Engineering of Cupriavidus Necator for Heterotrophic and Autotrophic Alka(E)Ne Production Lucie Crepin, Eric Lombard, Stéphane Guillouet

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Metabolic Engineering of Cupriavidus Necator for Heterotrophic and Autotrophic Alka(E)Ne Production Lucie Crepin, Eric Lombard, Stéphane Guillouet Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production Lucie Crepin, Eric Lombard, Stéphane Guillouet To cite this version: Lucie Crepin, Eric Lombard, Stéphane Guillouet. Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production. Metabolic Engineering, Elsevier, 2016, 37, pp.92- 101. 10.1016/j.ymben.2016.05.002. hal-01886395 HAL Id: hal-01886395 https://hal.archives-ouvertes.fr/hal-01886395 Submitted on 14 Apr 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Metabolic Engineering 37 (2016) 92–101 Contents lists available at ScienceDirect Metabolic Engineering journal homepage: www.elsevier.com/locate/ymben Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production Lucie Crépin, Eric Lombard, Stéphane E Guillouet n LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, 31077 Toulouse CEDEX 04, France article info abstract Article history: Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, Received 18 March 2016 microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in Received in revised form non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic 15 April 2016 bacterium Cupriavidus necator has great potential for producing chemicals from CO2: it is known to have Accepted 5 May 2016 one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs Available online 20 May 2016 most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up Keywords: to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding Cupriavidus necator an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a Alkane C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose Alkene we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced Hydrocarbon up to 670 mg/L total hydrocarbons containing 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, Biofuels Metabolic engineering 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the fi Fermentation highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the rst reported alka(e)nes production by a non-native alkane producer from CO2 as the sole carbon source. & 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. 1. Introduction In cyanobacteria alkane biosynthetic pathway is composed by 2 enzymes an acyl-ACP reductase (AAR, EC:1.2.1.80) and an alde- Increasing concerns about limited fossil fuels have stimulated hyde deformylating oxygenase (ADO, EC:4.1.99.5) (Li et al., 2012; interest in microbial production of biofuel from renewable carbon Schirmer et al., 2010; Warui et al., 2011). AAR catalyzes the sources. Biofuels must be both compatible with existing liquid fuel NADPH-dependent reduction of acyl-ACP or acyl-CoA into corre- infrastructure and produced at a competitive level for industrial sponding fatty aldehyde. Because it appears that aar has a higher production. Alkanes and alkenes are the predominant constituents affinity for acyl-ACP, this enzyme was named acyl-ACP reductase of gasoline, diesel and jet fuels (Peralta-Yahya et al., 2012). Alka(e) (Schirmer et al., 2010). ADO transforms Cn fatty aldehyde to a CnÀ1 nes synthesis have been reported in several microorganisms in- alka(e)ne with the same degree of unsaturation as the parent cluding cyanobacteria, bacteria, yeast and fungi (Ladygina et al., substrate and C1-derived formic acid. ADO activity needs oxygen, 2006). So far there have been several reported pathways for en- NADPH and the auxiliary reducing system ferredoxin and ferre- gineering alkane and alkene production derived from the fatty doxin reductase (N. Li et al., 2012; Warui et al., 2011). acid metabolism. Progress in metabolic engineering and identifi- Heterologous expression into Escherichia coli of these two cation of these pathways enabled biofuels production in native genes from Nostoc punctiforme PCC73102 allowed alka(e)ne pro- and non-native biofuel producing microorganisms (Beller et al., duction up to 300 mg/l (primarily C15 alkane and C17 alkene) with 2015; Wang and Lu, 2013). more than 80% of the hydrocarbons found outside of the cells (Schirmer et al., 2010). Alka(e)ne content in Synechocystis sp. PCC6803 mutants overexpressing native alkane biosynthetic genes Abbreviations: C. necator, Cupriavidus necator; S. elongatus, Synechococcus elon- gatus; E. coli, Escherichia coli; AAR, acyl-ACP reductase; ADO, aldehyde deformy- was 1.1% of dry weight cell and alka(e)ne production in photo- lating oxygenase; PHA, polyhydroxyalkanoates; PHB, polyhydroxybutyrate; RBS, bioreactor reached 26 mg/l in 10 days (Wang et al., 2013). In Sy- ribosome-binding site nechococcus sp. PCC 7002, overexpressing a heterologous alkane n Corresponding author. pathway allowed to reach 5% of cell dry weight (CDW) of alka(e)ne E-mail addresses: [email protected] (L. Crépin), [email protected] (E. Lombard), (Reppas and Ridley, 2010). Alka(e)ne production was also obtained [email protected] (S. Guillouet). in Saccharomyces cerevisiae and Cupriavidus necator mutant strains http://dx.doi.org/10.1016/j.ymben.2016.05.002 1096-7176/& 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. L. Crépin et al. / Metabolic Engineering 37 (2016) 92–101 93 expressing a heterologous alkane pathway from Synechococcus (Müller et al., 2013). Engineering C. necator strain for medium elongatus (Bi et al., 2013; Buijs et al., 2015). In S. cerevisae mutant chain fatty acids (MCFA) production was constructed in a mutant strain the biosynthesis was 2.7 mg/gCDW of heptadecane. And in C. unable to produced PHB, deleted for one of several acyl-CoA necator around 6 mg/l of a mixture of pentadecane and heptade- synthase, the first enzyme of β-oxidation and expressing a med- cene was produced. So far the highest reported titer of 580 mg/l of ium-chain-length-specific acyl-ACP thioesterase from Umbellularia alka(e)ne was obtained with an engineered E. coli strain producing californica UcFatB2. This mutant was reported to produced up to short chain fatty acids. In this work the authors overexpressed 60 mg/l MCFA on fructose (Chen et al., 2015). Bi et al. reported the similar pathway with alternative enzymes, an acyl-CoA reductase development of a genetic toolbox for the metabolic engineering of (acr) from Clostridium acetobutylicum and a fatty aldehyde dec- C. necator. The usefulness of this toolbox was further demon- arbonylase (CER1) from Arabidopsis thaliana (Choi and Lee, 2013). strated by performing an optimization of hydrocarbon production Lately, a wide range of short chain alka(e)nes from C3 to C10 was in C. necator resulting in 6 mg/l of alka(e)ne production on fructose successfully produced from engineered E.coli (Sheppard et al., (Bi et al., 2013). All together these works demonstrated that C. 2015). necator is a good candidate for biofuel and chemicals productions Recently, Cupriavidus necator has been identified as a potential from CO2. candidate for biofuel production from carbon dioxide (CO2). This In this work, we designed a synthetic production pathway facultative chemolithoautotrophic bacterium is long known as through codon, gene copy number and gene level optimization in capable of using CO2 and H2 as carbon and energy sources re- order to efficiently redirect the carbon flow from PHB toward alka spectively (Pohlmann et al., 2006; Schuster and Schlegel, 1967). (e)nes production. The resulting strain constructed Re2061-pLC10 Under nutrient limitation in excess of carbon, C. necator redirected was characterized in term of alka(e)nes and fatty aldehydes pro- carbon flux toward polyhydroxyalkanoates (PHA) (mainly poly- duction. Production was optimized by extractive fermentation hydroxybutyrate (PHB)) as carbon storage material up to 80% of its strategy using a surfactant to facilitate alka(e)nes excretion out of cell dry weight (CDW) (Grousseau et al., 2013). Under autotrophic the cells in C. necator. We report here the highest level of alka(e) growth condition on H2/CO2/O2, C. necator was reported to pro- nes production by an engineered C. necator to date. We also de- duce 62 g/l of PHB in 40 h (Tanaka et al., 1995). Because of PHB is a monstrated the first alka(e)nes production by C. necator mutant biobased and biodegradable polymeric material that can be an strain from CO2 as the sole carbon source. interesting substitute to petroleum derived plastics C. necator has been extensively studied for modeling PHB production from CO2 (Islam Mozumder et al., 2015) and industrially exploited in the 2. Material and methods production of biodegradable plastic for more than a decade (Chen, 2009). While, most efforts to engineer C. necator have focused on 2.1. Strain the bioplastics production (Insomphun et al., 2015, 2014) recently, it has been shown that C. necator can be used as a bioproduction For alkane production the parent strain of C. necator used was platform of valuable molecules such as short-chain alcohols and deleted for the PHA biosynthesis: Re2061 (H16 ΔphaCAB)(Lu et al., fatty acids related compounds (Bi et al., 2013; Chen et al., 2015; 2012).
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