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# Alka(e)ne synthesis in Cupriavidus necator boosted by the expression of endogenous and heterologous ferredoxin-ferredoxin reductase systems

Lucie Crepin, Manon Barthe, Florence Leray, Stéphane Guillouet

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Lucie Crepin, Manon Barthe, Florence Leray, Stéphane Guillouet. Alka(e)ne synthesis in Cupriavidus necator boosted by the expression of endogenous and heterologous ferredoxin-ferredoxin reductase systems. Biotechnology and Bioengineering, 2018, 115 (10), pp.2576-2584. 10.1002/bit.26805 . hal-02154335

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Alka(e)ne synthesis in *Cupriavidus necator* boosted by the expression of endogenous and heterologous ferredoxin-ferredoxin reductase systems.

Lucie Crépin, Manon Barthe, Florence Leray and Stéphane E Guillouet*

LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 avenue de Rangueil, 31077

Toulouse CEDEX 04, France

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* Corresponding author: stephane.guillouet@insa-toulouse.fr

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**Abstract**

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In order to boost the aldehyde deformylating oxygenase (ADO) activity in a *Cupriavidus necator* strain expressing a synthetic alkane pathway, the expression of two ferredoxin-ferredoxin reductase systems was tested. The genes of a native fd/FNR-like system were identified in *C. necator* and expressed in a previously engineered alka(e)ne producing strain.

The improved production of alka(e)nes in this Re2061-pMAB1 strain confirmed the activity of the native Fd/FNR system in *C. necator*. Concomitantly the expression of the heterologous system from *S. elongatus* was performed identically leading to a second strain, Re2061-pMAB2. In bioreactor, the aldehyde production was strongly reduced compared to the original alka(e)ne producer leading to alka(e)nes production up to 0.37 and 1.48 g/l (22 and 82 mg/gDCW), respectively. The alka(e)ne production yield of Re2061-pMAB2 accounted for 15% of the theoretical yield. We report here the highest level and yield of alka(e)nes production by an engineered bacterium to date.

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**Keywords**

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*Cupriavidus necator*, alkane, alkene, hydrocarbon, biofuels, metabolic engineering, fermentation, ferredoxin, ferredoxin reductase.

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**Abbreviations**

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*Cupriavidus necator* (*C. necator*), *Synechococcus elongatus* (*S. elongatus*), *Escherichia coli* (*E. coli*), acyl-ACP reductase (AAR), aldehyde deformylating oxygenase (ADO), polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB), ribosome-binding site (RBS), ferredoxin (Fd), ferredoxin reductase (FNR), Cell Dry Weight (CDW)

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# 1 Introduction

The facultative chemolithoautotrophic bacterium, *Cupriavidus necator*, also known as

*Ralstonia eutropha*, has regained a lot of interest in recent years particularly due to the

increasing concerns about limited fossil fuels stimulating research in microbial production of

biofuel from renewable carbon sources as CO₂. *C. necator* is known for its abilities (i) to grow

fast under autotrophic conditions in presence of CO₂, H₂ and O₂ and with minimal nutrient

supplement (ii) to naturally and efficiently convert CO₂ into polyhydroxyalkanoates (PHAs),

and (iii) to be easily genetically modified. Specific growth rates in the range of 0.3-0.4 h⁻¹

have been reported for *C. necator* demonstrating its ability to grow efficiently on gas mixture

CO₂/H₂/O₂ (Tanaka et al., 1995). Recently the range of metabolites that can be produced in

*C. necator* has enlarged thanks to the development of the genetic engineering tools.

Successful productions were reported for isopropanol (Grousseau et al., 2014; Marc et al.,

2017), isobutanol (Lu et al., 2012), methyl ketones (Müller et al., 2013), free fatty acids (Chen

et al., 2015), alka(e)nes (Bi et al., 2013; Crépin et al., 2016), fatty aldehydes (Crépin et al.,

2016) and terpenes (Krieg et al., 2018) under heterotrophic conditions. The production of

some of those chemicals was reported under autotrophic conditions from CO₂/H₂/O₂ mixture :

250 mg/l isopropanol (Marc et al., 2017), 180 mg/l of methyl ketones (Müller et al., 2013), 4.4

mg/l alka(e)nes (Crépin et al., 2016) and 6 mg/l α-humulene (Krieg et al., 2018). Up to 216

mg/l isopropanol was also produced from CO₂ in a hybrid microbial-water-splitting catalyst

system as well (Torella et al., 2015) by the strain Re2133-pEG12 (Grousseau et al., 2014).

Furthermore, biotransformations of the CO₂ were demonstrated in electromicrobial systems,

yielding up to 100 mg/l isobutanol by Li et al., (2012) and up to 10 mg/l humulene by Krieg et

al.(2018). Therefore, *C. necator* appears as a potentially strong candidate for the

development of future bioprocesses for the bio-based chemical synthesis using CO₂ as a

sustainable carbon source.

The production of alkane and alkene, the predominant compounds of the gasoline, diesel

and jet fuels, from renewable resources has stimulated interest since the discovery of alkane
synthetic pathways in microorganisms (Peralta-Yahya et al., 2012). Various microorganisms including cyanobacteria, bacteria, yeast and fungi were reported as being natural alkane producers (Ladygina et al., 2006). Consequently, several pathways were reported for engineering alkane and alkene production derived from the fatty acid metabolism. Recently, we successfully expressed an engineered alka(e)ne pathway in C. necator based on a cyanobacterial native pathway (Crépin et al., 2016). In cyanobacteria, 2 enzymes, an acyl-ACP reductase (AAR, EC:1.2.1.80) and an aldehyde deformylating oxygenase (ADO, EC:4.1.99.5) constitute the alkane biosynthetic pathway (Figure 1) (N. Li et al., 2012; Schirmer et al., 2010; Warui et al., 2011). The AAR catalyzes the NADPH-dependent reduction of acyl-ACP or acyl-CoA into the corresponding fatty aldehyde with a higher affinity for acyl-ACP (Schirmer et al., 2010). ADO catalyzes the conversion of a C~n~ fatty aldehyde to a C~n-1~ alka(e)ne with the same degree of unsaturation as the initial substrate releasing a formic acid. The ADO activity requires oxygen, NADPH and an auxiliary reducing system ferredoxin and ferredoxin reductase (Li et al., 2012; Warui et al., 2011; Zhang et al., 2013). An auxiliary reducing system (biological or chemical) is needed for ADO activity indeed as the ADO forms a peroxo intermediate as part of its reaction mechanism as demonstrated by numerous in vitro studies (Das et al., 2011; Li et al., 2012; Schirmer et al., 2010). In total, the reaction requires an input of four electrons for every alkane that is formed. Biologically, ferredoxin-ferredoxin reductase systems (Fd/FNR) serve as the cognate electron supply chain in cyanobacteria (Zhang et al., 2013).

In our previous work (Crépin et al., 2016), the alkane synthesis pathway from *Synechococcus elongatus* was heterologously expressed (2 genes *aar* and *ado*) in a C. *necator* mutant strain deficient in the PHB synthesis pathway. Under nitrogen limiting heterotrophic conditions, a total production of 345 mg/l of hydrocarbons was obtained pointing out the presence of an endogenous ferredoxin and ferredoxin reductase-like activity enable to support the S. *elongatus* ADO activity. The addition of an organic phase (decane) led to an increase in the concentration of hydrocarbons (up to 670 mg/l) containing 435 mg/l
1 of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of
2 heptadecane, and 236 mg/l of hexadecanal. The decane effect was explained by the higher
3 affinity of the hydrocarbons towards the organic phase, pulling then the flux of the molecules
4 out of the cells. (Crépin et al., 2016). The bi-phasic fermentation strategy strongly enhanced
5 total hydrocarbon production, however, a higher fatty aldehyde production rate was mainly
6 observed without an increase in the production rate of the alkanes suggesting a limitation by
7 the activity of the ADO. This limitation by the ADO activity could be either due to an
8 insufficient expression of the ADO or to an insufficient activity of the ferredoxin and
9 ferredoxin reductase endogenous system (Crépin et al., 2016). Some studies demonstrated
10 lately the importance of the electron transfer system in alkane production as well in E. coli
11 (Cao et al., 2016; Kallio et al., 2014).

12 The aim of this study was then (i) to screen for the responsible ferredoxin and ferredoxin
13 reductase-like activity in C. necator, (ii) to overexpress it in our engineered alka(e)ne
14 producer strain and (iii) to compare with a strain expressing a heterologous ferredoxin and
15 ferredoxin reductase system from S. elongatus. All strains were evaluated in a bioreactor
16 under fed-batch mode in the presence of decane.

## Material and methods

### Strain

19 In this study, the parent strain of C. necator used was Re2061 deficient for the PHA
20 biosynthesis (H16 ΔphaCAB) (Lu et al., 2012). The genes encoding for PHA synthase
21 (phaC1, H16_A1437), β ketothiolase A (phaA, H16_A1438), acetoacetyl-CoA reductases
22 (phaB1, H16_A1439) were deleted in the wild type strain C. necator H16 (ATCC17699, Gen')
23 (Table 1). The strain was stored at -80°C in liquid TBS medium (dextrose-free tryptic soy
24 broth, Becton Dickinson, France) with 20 % glycerol (v/v).

25 Plasmid and strain constructions
1 DNA sequence amplifications were achieved using Phusion High-Fidelity PCR Master Mix
2 with GC Buffer (New England Biolabs, Evry, France). All the primers were obtained from
3 Eurogentec, (Angers, France) and are listed in table 1. Restriction enzymes and DNA
4 modification enzymes were purchased from (New England Biolabs, Evry, France). Plasmid
5 purification was performed by using QIAprep Spin Miniprep Kit (QIAGEN, Courtaboeuf,
6 France). PCR products and digested plasmids were purified from agarose gel using
7 QIAquick Gel Extraction Kit (QIAGEN, Courtaboeuf, France).

8 For the construction of pMAB1, the *fd 2Fe-2S* gene (H16_A1164) was amplified from *C. necator* H16 genomic DNA using forward primer (1) and reverse primer (2). A second DNA amplification was performed from this PCR product in order to add homologous ends (for
10 Gibson assembly). This second amplification was carried out using forward primer (3) and
11 reverse primer (4). The *ferredoxin-NADP reductase* gene (H16_B0102) was amplified from
12 *C. necator* H16 genomic DNA using forward primer (5) and reverse primer (6). A synthetic
13 ribosome binding site (RBS) sequence was encoded in the forward primers (1) and (5) and
14 thus incorporated upstream of each gene. The RBS sequence (5'- AAAGGAGGACAACC -3')
15 was already used in a previous work (Crépin et al., 2016). The pLC10 plasmid (Crépin et al.,
16 2016) used as backbone was digested by *HindIII* and treated with Antarctic phosphatase.
17 The plasmid pMAB1 was constructed by one-step isothermal DNA assembly (Gibson et al.,
18 2009). The one-step isothermal assembly products were transformed into *E. coli* Top 10
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21 For the construction of pMAB2 the genes coding for *FNR* and *Fd* (SynPcc7942_1499 and
22 SynPcc7942_0979) from *Synechococcus elongatus* were codon-optimized for *C. necator*
23 H16 and synthesized by GenScript (Piscataway, NJ, USA). These synthetic genes were
24 received in the pUC57 plasmid with a synthetic RBS sequence upstream of each gene and
25 flanked by *HindIII* restriction sites. Both, pLC10 and pUC57-inserts, were digested by *HindIII*
26 and after purification, plasmid assembly was performed by ligation.
For both constructs, transformants were selected on selective plates containing 50 mg/l of kanamycin. The plasmids pMAB1 and pMAB2 were checked by sequencing (GATC, Konstanz, Germany) with forward (7) and reverse (8) primers. Plasmids were then transformed into C. necator Re2061 strain by electroporation using a standard protocol described previously (Crépin et al., 2016). The transformants were selected on TBS plates with 10 mg/l gentamycin and 200 mg/l kanamycin.

## Media

The rich medium consisted of 2.75% (w/v) dextrose-free tryptic soy broth (TSB, Becton Dickinson, France).

The buffered minimal medium used for preculture in flask was previously described by Lu et al. (2012) and Crépin et al. (2016). Fructose (20 g/l) was used as the only carbon source. $NH_4Cl$ (0.5 g/l) was used as the nitrogen source to reach a biomass concentration of about 1 g/l.

The minimal medium used in the fed-batch cultures was previously described by (Aragao et al., 1996) and consisted of 0.19 g/l of nitrilotriacetic acid, 0.06 g/l of ferrous ammonium citrate, 0.5 g/l of $MgSO_4.7H_2O$, 0.01 g/l of $CaCl_2.2H_2O$, and 1 ml of trace element solution. The trace element solution composition was 0.3 g/l of $H_3BO_3$, 0.2 g/l of $COCl_2.6H_2O$, 0.1 g/l of $ZnSO_4.7H_2O$, 0.03 g/l of $MnCl_2.4H_2O$, 0.03 g/l of $Na_2MoO_4.2H_2O$, 0.02 g/l of $NiCl_2.6H_2O$, 0.01 g/l of $CuSO_4.5H_2O$. $(NH_4)_2SO_4$ (5.0 g/l) was used as nitrogen source to reach a biomass concentration of about 7.5 g/l. After autoclaving the above medium, 40 ml of a sterile phosphate solution containing 224 g/l of $Na_2HPO_4.12H_2O$ and 37.5 g/l of $KH_2PO_4$ was aseptically added to the bioreactor. The carbon substrate was added by a sterile fructose solution of 600 g/l to reach 45 g/l just before inoculation.

## Fed-batch culture conditions and bioreactor system
The Fed-Batch cultures were performed in a 6 l (total volume) bioreactor containing an initial medium volume of 3 l. The bioreactor was equipped with pH, dissolved oxygen (DO), temperature and anti-foam controllers. The on-line monitoring and control systems of the reactor were handled by the software BioPAT® MFCS/win. Both CO₂ and O₂ in the inlet and outlet gasses were analyzed using 1313 fermentation monitor INNOVA Airtech Instrument. The DO level in the reactor was controlled above 20 % of air saturation by varying stirring speed and/or inlet air flow rate. Temperature was maintained at 30°C. The pH was maintained at 7.0 by addition of a 16.6 % (v/v) ammonia solution during growth phase and then by addition of 4 N KOH solution or 50 % (v/v) orthophosphoric acid during the nitrogen limiting phase. Nitrogen limitation was controlled by an exponential feeding of a 16.6 % (v/v) NH₃ solution calculated to monitor the growth at a constant specific growth rate of 0.01 h⁻¹. The fructose concentration was maintained in excess at 20 g/l thanks to an exponential feeding of fructose solution (600 g/l).

## 14 Analytical procedures

The biomass concentration was quantified thermo-gravimetrically. The culture medium was sampled and filtrated through a 0.22 µm pore-size polyamide membrane (Sartorius AG, Göttingen, Germany). The membrane was then dried to a constant weight at 60°C under partial vacuum (200 mmHg, i.e. approximately 26.7 kPa). The total biomass growth was also followed by optical density measurement at 600 nm (OD600nm) using a visible spectrophotometer (Hach Lange) with a 2 mm absorption glass cell (HELLMA).

The culture samples were centrifuged at 12000 g for 4 min. The supernatants were then filtrated (0.2 µm Minisart RC4, Sartorius) for substrate and organic acid determination. The residual substrate and organic acid concentrations were quantified by high-performance liquid chromatography (HPLC). The HPLC instrument (e2695, Waters) was equipped with an ion-exchange column (Aminex HPX-87H, 300×7.8 mm, Bio-Rad) protected with a guard column (Cation H+ cartridge, 30×4.6 mm, Bio-Rad) and coupled to a RI detector and an UV
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detector (λ=210 nm). The column was eluted with 2.5 mM H₂SO₄ as a mobile phase at 50°C

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at a flow rate of 0.5 ml/min.

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Alkane, alkene and aldehyde quantification by GC-FID

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The culture broth was centrifuged at 12000 g during 4 min in order to separate the phases.

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The organic phase was then sampled: 50 µl of the decane phase was added to 50 µl of 20

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mg/l tetradecane in hexane (used as internal standard) into a fresh vial and analyzed by Gas

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Chromatography

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Hydrocarbons analysis was performed on a column CP-sil 5 CB (30 m 0.53 mm ID 0.70 µm

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df, Agilent) with GC instrument (6890, Agilent) coupled to a flame ionization detector (FID).

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For GC separation, the following method was used: after 1 µl splitless injection (inlet

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temperature at 250°C) into the column, the oven was held at 50°C for 1 min. The

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temperature was ramped up to 180°C at 15°C/min, up to 185°C at 2.5°C/min, up to 190°C at

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0.25°C/min, up to 305°C at 15°C/min and then held at 305°C for an additional 2 min. The

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flow rate of the carrier nitrogen gas was 1.0 ml/min.

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Statistical analyses

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The concentrations of metabolites and biomass were given as the mean value of 2 to 3

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independent analyses.

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The error on the specific growth rate (determined as being the slope of ln[Biomass]=f(t)) was

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calculated as the standard deviation of the slope.

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For the yield determination, the state variables were plotted pairwise in a scatter plot within

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the considered period of the culture (growth phase or production phase). A linear regression

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was applied to determine the yield (as being the slope) and the error (as being the standard

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deviation of the slope).

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For Carbon balances and yield calculation, a CHON composition for the biomass of

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CH₁.₇七O₀.₄₄N₀.₂₅ (MW 25.3 g/Cmole) was used (Aragao *et al.*, 1996).
## 1 Results and discussion

## 2 Engineering the *C. necator* strain Re2061-pMAB1 and Re2061-pMAB2

In order to boost the ADO activity, the endogenous ferredoxin and ferredoxin reductase genes of *C. necator* needed to be identified and tested for overexpression in the Re2061-pLC10 strain. In *C. necator* H16 genome, the gene H16_B0102 has been annotated as a ferredoxin-NADP reductase gene (Kanehisa et al., 2010) and the fd 2Fe-2S gene (H16_A1164) as a 2Fe-2S activity (Stothard et al., 2005). From the plasmid pLC10 containing *aar* and *ado* genes, we introduced the *fd* (H16_A1164) and *fnr* (H16_B0102) genes from *C. necator* leading to the pMAB1 plasmid (Figure 2). In parallel, the cognate ADO supporting electron transfer system from *S. elongatus* PCC7942, which involves *fnr* (*SynPcc7942_0978*, EC: 1.18.1.2), and *fd* (*SynPcc7942_1499*) (Zhang et al., 2013), was tested. Therefore, a second plasmid pMAB2 (Figure 2) was constructed by introducing the *SynPcc7942_1499* and *SynPcc7942_0979* genes upon codon optimization for *C. necator* into the plasmid pLC10. The plasmids, pMAB1 and pMAB2, were introduced into the Re2061 strain leading to the Re2061-pMAB1 and Re2061-pMAB2 strains, respectively.

## 17 Alka(e)ne production by Re2061-pMAB1 and Re2061-pMAB2 C. necator H16 strains

We previously showed that uncoupling growth and production via nitrogen limitation and adding an organic phase (decane) improved significantly the hydrocarbon production with the engineered *C. necator* H16 Re2061-pLC10 (Crépin et al., 2016). Therefore, the new engineered strains Re2061-pMAB1 and Re2061-pMAB2 were identically evaluated via two-step fed-batch cultivation. During the first step, all nutrients except the nitrogen source were provided in excess allowing maximal growth in order to reach nitrogen depletion at a biomass concentration of about 7.5 gCDW/l. Then, after total N-depletion, the reactor was fed with an ammonia solution to support a residual growth rate of 0.01 h⁻¹ under N limitation. The alkane operon expression was induced by adding 0.1 % (w/w) of arabinose and at the same time 10
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$(v/v)$ decane was added in the reactor. The kinetics of production of biomass and alka(e)nes are presented in Figure 3. HPLC analyses showed that the arabinose was not consumed. Carbon balances were closed at 84% and 86% for the strains Re2061-pMAB1 and Re2061-pMAB2, respectively.

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During the growth phase, Re2061-pMAB1 and Re2061-pMAB2 strains grew both at a maximal growth rate of 0.21 ± 0.01h⁻¹. The biomass production yields on fructose were 0.47±0.03 and 0.50±0.01 Cmole/Cmole, respectively. During the alkane production phase, the N limitation imposed the specific growth rate at 0.0096±0.0012 h⁻¹ and 0.0093±0.0018 h⁻¹ for Re2061-pMAB1 and Re2061-pMAB2, respectively. Both strains showed similar behavior concerning organic acid production. Just after nitrogen depletion, pyruvic acid was first excreted (up to 0.7 g/l), then re-consumed during the first hours of N-limitation phase and reproduced after 60 hours to little amounts up to 0.3 g/l and 0.6 g/l for the strains Re2061-pMAB1 and Re2061-pMAB2, respectively. While no acetic acid and no succinic acid were detected, citric acid excretion was detected at the onset of the N-depletion and continuously produced to reach final titers of 6.1 and 6.4 g/l. After more than 80 h of nitrogen limitation, the final biomass concentrations were 16.8 g/l and 18.0 g/l for the strains Re2061-pMAB1 and Re2061-pMAB2, respectively (Table 2 and Supplementary figure 1).

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Nitrogen limited fed-batch culture of Re2061-pMAB1 strain resulted in a production of 0.49 g/l of total hydrocarbons, consisting of 0.225 g/l of pentadecane, 0.123 g/l of heptadecene, 0.026 g/l of heptadecane, together with 0.113 g/l of hexadecanal. The over-expression of the native *C. necator* H16 putative Fd/FNR led to significant increase in alka(e)ne production by factors of 1.7, 3 and 2 for pentadecane, heptadecane and heptadecene, respectively, compared to our previous strain Re2061-pLC10 under the same cultivation conditions (Crépin et al., 2016). However, the production of fatty aldehyde (hexadecanal) was almost the same

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Comparatively, the N-limited fed-batch culture of Re2061-pMAB2 strain led to a higher production of 1.55 g/l of total hydrocarbons, containing 1.48 g/l of alka(e)nes consisting of 1.1
1 g/l of pentadecane, 0.32 g/l of heptadecene, and 0.06 g/l of heptadecane. Re2061-pMAB2
2 strain produced only 0.068 g/l of hexadecanal. No fatty alcohol was detected in the
3 fermentation broth. The highest performance reported so far was performed in an engineered
4 E. coli strain with 2.5 g/l alka(e)nes concomitantly with 5.7 g/l fatty alcohols from glucose
5 (Fatma et al., 2018). The specific production for alka(e)nes in our best producing strain
6 Re2061-pMAB2 was 82 mg/gCDW. Comparatively, the specific productions of alka(e)nes
7 reported in other engineered microorganisms were 51 mg/gCDW in E. coli (Fatma et al.,
8 2018) and far more lower in yeasts such as S. cerevisiae (22 µg/gCDW) (Buijs et al., 2015)
9 or Y. lipolytica (45 µg/gCDW) (unpublished data). The level of excreted organic acids in our
10 strain can be a good indicator for the potential carbon amount still available for further
11 improvement into targeted products. In our case, this potential carbon amount of 0.44
12 Cmole/CmoleCDW could lead to a significant additional amount of alka(e)nes with further
13 strain engineering.
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15 Our data confirmed that the endogenous Fd/FNR of C. necator H16 was able to provide an
16 electron supply chain for the in vivo activity of the S. elongatus ADO, however the Fd/FNR of
17 S. elongatus performed better. Recently, (Cao et al., 2016) improved alkane production in
18 E.coli from 60 to 100 mg/l by expressing the heterologous Se Fd/FNR system concomitantly
19 with Se aar and ado genes, whereas the overexpression of the endogenous E. coli Fd/FNR
20 had no detectable effect. The authors suggested that ADO was selective for homologous
21 cyanobacterial other than heterologous and chemical ones as observed earlier by Zhang et
22 al. (2013). We demonstrated here that the Se ADO was in vivo functional with the surrogate
23 Fd/FNR system from C. necator, even if it is not fully adequate.
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Kinetics of specific hydrocarbon production rates
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25 In order to analyze the hydrocarbon production at the cell level, the specific production rates
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hexadecanal). The data showed that the specific production rates for alka(e)nes were improved in Re2061-pMAB1 with a maximum at 2 µmole/gCDW.h (Figure 4) compared to around 1 µmole/gCDW.h in our previous strain (Crépin et al., 2016). The expression of the heterologous Se Fd/FNR system in Re2061-pMAB2 showed an even stronger improvement in the alka(e)ne production. The specific production rates were increased for the alka(e)nes with a maximum at 8.5 µmole/gCDW.h, a 8-fold increase compared to Re2061-pLC10.

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In both strains, the specific production rates of fatty aldehydes (hexadecanal) were very low compared to the one obtained in Re2061-pLC10 (up to 0.9 µmole/g.h). Their maximal values were 0.6 and 0.35 µmole/g.h in Re2061-pMAB1 and -pMAB2, respectively, demonstrating that the expression of a ferredoxin-ferredoxin-NADP+ reductase system strongly reduced the C-flow into fatty aldehydes.

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Carbon distribution between organic acids, alka(e)nes and aldehydes

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The carbon distribution into the products (organic acids, hydrocarbons) at the end of the fed-batch of the strains over-expressing a ferredoxin/ferredoxin reductase system (Re2061-pMAB1 and -pMAB2) was shown in Figure 5 and compared with the reference strain bearing the empty plasmid and our previous alkane producer strain Re2061-pLC10 (Crépin et al., 2016). The best improvement was obtained by expressing the heterologous cognate Se Fd/FNR. Its expression strongly increased the C-flow directed towards the alka(e)ne synthetic pathway at the expense of aldehyde and towards organic acids. As showed in Figure 5, the expression of a Fd/FNR system pull out the C-flow into those end-products from 0.11 Cmole/CmoleCDW in the Re2061-pLC10 to 0.56 and 0.72 Cmole/CmoleDCW with the surrogate and cognate Fd/FNR, respectively. Thus, the additional expression of a ferredoxin/ferredoxin reductase system reduced the part of carbon leading into fatty aldehyde (hexadecanal) as end-product from 37% in Re2061-pLC10 to 23% in Re2061-pMAB1 and only 3.5% in Re2061-pMAB2, expressing the cognate Fd/FNR. However, the figure 5 also showed that expressing ferredoxin/ferredoxin reductase systems led to a substantial
redirection of the C-flow into the targeted hydrocarbon and the organic acid metabolism. We
previously observed that expressing a heterologous biosynthetic pathway in C. necator H16
was accompanied not only by the production of the targeted product but also by an increased
C-flow towards organic acids. It held true for the alka(e)ne production (Crépin et al., 2016)
and isopropanol production (Grousseau et al., 2014). However, both producing strains
excreted pyruvate transiently upon N-limitation whereas only the alka(e)ne producing strains
excreted citrate and to a lesser extent pyruvate during the production phase. This fact could
be explained by the different background of the reference strains chosen for isopropanol and
alka(e)ne production. The reference strains were Re2133 deleted in phaB1B2B3 and phaC
genes for isopropanol production and Re2061 deleted in phaA, phaB1 and phaC1 for
alka(e)ne production. Under N limitation and depending on the saturation of the engineered
pathway, the C-flow originally redirected to PHB synthesis in the wild type would lead to
accumulation of acetoacetyl-coA and acetocetate in the isopropanol producing strains
whereas to accumulation of acetyl-coA in the alka(e)ne producing strain. As citrate synthase
and pyruvate carboxylase are known to be activated by high concentration of acetyl-coA in
bacteria, the accumulation of acetyl-coA in the alka(e)ne producing strains could lead to the
excretion of TCA cycle organic acids. This phenomenon could be accentuated by the
overexpression of the ferredoxin. The ferredoxin overexpressing strains, Re 2061-pMAB1
and 2, excreted higher amounts of citric acid, 0.35 and 0.36 gCitrate/gCDW respectively,
compared to the non-overexpressing Re 2061-pLC10 strain, 0.028 gCitrate/gCDW. C.
necator H16 possesses 3 genes annotated for a pyruvate:flavodoxin oxidoreductase
(H16_A3547, porG) and 2-oxoacid:ferredoxin reductases (H16_B1980, H16_A1255). Those
three enzymes catalyse the oxidative decarboxylation of oxo-acids such as pyruvate, oxo-
butyrate, oxo-glutarate, indole pyruvate into coenzyme A intermediates in presence of
flavodoxin or ferredoxin. Based on their specificity for the redox partner (ferredoxin,
flavodoxin) and for the substrate, pyruvate could then be decarboxylated into acetyl-coA.
1

The level of excreted organic acids in our strain could be a good indicator of the quantity of potentially convertible carbon into the targeted alkanes. In our case, this potential carbon amount of 0.44 Cmole/CmoleCDW could lead to a significant additional amount of alka(e)nes with further strain engineering.

2

3

4

5

In terms of hydrocarbon production yields on fructose (Table 3), the expression of an additional Fd/FNR system led to improvement in yields. Re2061-pMAB1 strain had an alka(e)ne production yield from fructose of 15.7 mCmole per Cmole fructose whereas it reached 45 mCmole/Cmole fructose in Re2061-pMAB2 harboring the Se Fd/FNR system.

6

7

8

9

This highest value accounted for 15% of the theoretical alka(e)ne production yield on fructose and represented the highest alka(e)ne production yield from sugar reported so far.

10

11

## Conclusions

12

The overexpression of a ferredoxin/ferredoxin NADP-reductase system (native or heterologous from *Synechococcus elongatus*) in an engineered alkane producing *C. necator* strain improved significantly the alka(e)ne production. The combination of the metabolic engineering and fermentation strategies led to the highest specific production of alka(e)ne reported so far (86 mg/g<sub>CDW</sub>). The alka(e)ne production yield accounted for 15% of the theoretical yield, indicating that an additional engineering work is necessary for approaching the theoretical yield.

13

14

15

16

17

18

19

## Acknowledgements

20

We thank A. J. Sinskey for providing us the Re2061 mutant strain. We also thank the A. J. Sinskey laboratory (MIT) for general technical advice. We are grateful to E. Grousseau and J. Marc for technical advice and helpful discussions. This work has benefited of the financial support from the French Research Ministry managed by the Agence Nationale de la Recherche under the program "Investissements d'Avenir "bearing the ANR-11-BTBR-0003 reference.

21

22

23

24

25
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1

2 **Figure legends**

3 **Figure 1:** Metabolic engineering strategy for alka(e)ne production in C. necator strain.

4
Abbreviations: PHB: poly hydroxyl butyrate, ACC: acetoacetyl-coA carboxylase, FAS: fatty
acid synthase, AAR: acyl-ACP reductase, ADO: aldehyde deformylating oxygenase, Fd :
ferredoxin (oxidized or reduced form), FNR: ferredoxin reductase.

7
8
transformed into strain Re2061 (H16 ΔphaCAB). Abbreviations: ado (aldehyde
decarbonylase oxygenase), aar (acyl-ACP reductase), fdx (ferredoxin), FNR (ferredoxin-
NADP reductase), PBAD (Inducible Arabinose Promoter), RBS (Ribosome Binding Site)

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12
13
14
**Figure 3:** Kinetics of production of biomass (♦), pentadecane (□), heptadecene (Δ) and hexadecanal (○) during nitrogen limited fed-batch of C. necator strains Re2061-pMAB1 and Re2061-pMAB2 with addition of 10 % (v/v) of decane after induction. Induction time is indicated by the arrow. Dark bars indicate standard deviations

15
16
17
18
**Figure 4:** Evolution of the specific production rates of total alka(e)nes and fatty aldehydes (hexadecanal) in µmole of product per g of cell dry weight per hour during nitrogen limited fed-batch of C. necator H16 strains Re2061-pMAB1 (dotted line) and Re2061-pMAB2 (full line) in the presence of 10 % (v/v) of decane.

19
20
21
22
23
24
**Figure 5:** Final carbon repartition (distribution) into extracellular metabolites (organic acids, alka(e)nes and aldehydes) in Cmole per Cmole of cell dry weight for strains Re2061 bearing the empty plasmid pBBAD, the alkane operon plasmid pLC10 (Crepin et al., 2016), the alkane operon plasmid pMAB1 containing the native ferredoxin and ferredoxin reductase of C. necator H16 and the alkane operon plasmid pMAB2 containing the ferredoxin and ferredoxin reductase of S. elongatus.
1 **Supplementary figure 1:** Kinetics of the cumulated amounts of fructose consumed (♦), of

2 pyruvic acid (□) and citric acid (Δ) during nitrogen limited fed-batch of C. necator strain

3 Re2061-pMAB1 and Re2061-pMAB2 with addition of 10 % (v/v) of decane after induction.

4 Induction time is indicated by the arrow.

5
<table><caption>1 Table 1: Strains, plasmids and primers used in this work.</caption><thead><tr><th>Strains</th><th>Relevant characteristics</th><th>References</th></tr></thead><tbody><tr><td>C. necator H16</td><td>Wild type, gentamicin resistant (Gen<sup>r</sup>)</td><td>ATCC17699</td></tr><tr><td>C. necator Re2061</td><td>H16 ΔphaC1AB1 (Gen<sup>r</sup>)</td><td>(Lu et al., 2012)</td></tr><tr><th>Plasmids</th><th>Relevant characteristics</th><th>References</th></tr><tr><td>pBBR1MCS-2</td><td>Broad-Host-Range cloning vector (Kan<sup>r</sup>), P<sub>Lac</sub></td><td>(Kovach et al., 1995)</td></tr><tr><td>pBBad</td><td>pBBR1MCS-2 derivative with L-Arabinose inducible system P<sub>Bad</sub></td><td>(Fukui et al., 2009)</td></tr><tr><td>pLC10</td><td>pBBad with the RBS-ado*-RBS-aar* sequence inserted into the multiple cloning site (MCS) (Kan<sup>r</sup>), P<sub>Bad</sub></td><td>(Crépin et al., 2016)</td></tr><tr><td>pMAB1</td><td>pBBad with the RBS-ado*-RBS-aar*-RBS-fd-RBS-fnr sequence inserted into the multiple cloning site (MCS) (Kan<sup>r</sup>), P<sub>Bad</sub></td><td>This work</td></tr><tr><td>pMAB2</td><td>pBBad with the RBS-ado*-RBS-aar*-RBS-fd*-RBS-fnr* sequence inserted into the multiple cloning site (MCS) (Kan<sup>r</sup>), P<sub>Bad</sub></td><td>This work</td></tr><tr><th>N# primer</th><th>Sequence (5'→3')</th><th></th></tr><tr><td>Primer 1</td><td>GTCGACAAAGGAGGACAACCATGCCACAGATCGTTG</td><td></td></tr><tr><td>Primer 2</td><td>GTTGTCCTCCTTTTCAGTGGCCTTCCTTG</td><td></td></tr><tr><td>Primer 3</td><td>GCCATCTGAAAGCTTGTCGACAAAGGAGGAC</td><td></td></tr><tr><td>Primer 4</td><td>CCGGGATGTGCATGGTTGTCCTCCTTTTCAG</td><td></td></tr><tr><td>Primer 5</td><td>GGAGGACAACCATGCACATCCCGGAAATC</td><td></td></tr><tr><td>Primer 6</td><td>CAAAACAGCCAAGCTTTCAGATGGCCAGCGC</td><td></td></tr><tr><td>Primer 7</td><td>CTCTCTACTGTTTCTCCATAC</td><td></td></tr><tr><td>Primer 8</td><td>GGCTGAAAATCTTCTCTCATC</td><td></td></tr></tbody></table>

2 phaA (H16_A1438), phaB1 (H16_A1439) and phaC1 (H16_A1437) from C. necator, coding for a β-

3 ketothiolase A, an acetoacetyl-CoA reductase and a PHA synthase, respectively

4 ado* and aar* are the codon-optimized versions of the ado (Synpcc7942_1593) and aar

5 (Synpcc7942_1594) heterologous genes of Syneccococcus elongatus, respectively coding for an

6 aldehyde deformylating oxygenase and an acyl-ACP reductase

7 fd and FNR are the Cupriavidus necator genes coding for a ferredoxin and a ferredoxin-NADP

8 reductase

9 fd* and FNR* are the codon-optimized versions of the Synechococcus elongatus genes

10 SynPcc7942_0979 and SynPcc7942_1499 coding for a ferredoxin and a ferredoxin-NADP reductase,

11 respectively.

12 RBS : RBS and nucleotide linker sequence: AAAGGAGGACAACC (Lu et al., 2012)

13
**Table 2:** Final titers of alka(e)nes, aldehydes, organic acids and biomass and specific growth rates. Results are shown for the strains Re2061-pMAB1 and Re2061-pMAB2 grown under nitrogen limiting heterotrophic conditions in the presence of 10 % decane.

<table><thead><tr><th></th><th></th><th colspan="2">pMAB1</th><th colspan="2">pMAB2</th></tr></thead><tbody><tr><td>pentadecane (C15)</td><td>g.L<sup>-1</sup></td><td>0.225</td><td>±0.010</td><td>1.094</td><td>±0.030</td></tr><tr><td>heptadecane (C17)</td><td>g.L<sup>-1</sup></td><td>0.026</td><td>±0.001</td><td>0.060</td><td>±0.003</td></tr><tr><td>heptadecene (C17)</td><td>g.L<sup>-1</sup></td><td>0.123</td><td>±0.006</td><td>0.323</td><td>±0.013</td></tr><tr><td>hexadecanal (C16)</td><td>g.L<sup>-1</sup></td><td>0.113</td><td>±0.005</td><td>0.068</td><td>±0.003</td></tr><tr><td>total alka(e)nes</td><td>g.L<sup>-1</sup></td><td><strong>0.37</strong></td><td>±0.018</td><td><strong>1.48</strong></td><td>±0.044</td></tr><tr><td>total hydrocarbons</td><td>g.L<sup>-1</sup></td><td><strong>0.49</strong></td><td>±0.024</td><td><strong>1.55</strong></td><td>±0.046</td></tr><tr><td>citric acid</td><td>g.L<sup>-1</sup></td><td>6.1</td><td>±0.18</td><td>6.4</td><td>±0.16</td></tr><tr><td>pyruvic acid</td><td>g.L<sup>-1</sup></td><td>0.3</td><td>±0.009</td><td>0.6</td><td>±0.002</td></tr><tr><td>CDW</td><td>g.L<sup>-1</sup></td><td>16.8</td><td>±0.08</td><td>18.0</td><td>±0.09</td></tr><tr><td>μmax</td><td>h<sup>-1</sup></td><td>0.21</td><td>±0.01</td><td>0.21</td><td>±0.01</td></tr><tr><td colspan="6"><strong>Specific production</strong></td></tr><tr><td>total alka(e)nes</td><td>mg.g<sup>-1</sup></td><td><strong>22.0</strong></td><td>±0.09</td><td><strong>82.2</strong></td><td>±2.0</td></tr><tr><td>total hydrocarbons</td><td>mg.g<sup>-1</sup></td><td><strong>29.2</strong></td><td>±1.2</td><td><strong>86.1</strong></td><td>±2.1</td></tr><tr><td colspan="6"><strong>global specific rate</strong></td></tr><tr><td>total alka(e)nes</td><td>mg.g<sup>-1</sup>.h<sup>-1</sup></td><td><strong>0.36</strong></td><td>±0.01</td><td><strong>1.32</strong></td><td>±0.03</td></tr><tr><td>total hydrocarbons</td><td>mg.g<sup>-1</sup>.h<sup>-1</sup></td><td><strong>0.47</strong></td><td>±0.02</td><td><strong>1.39</strong></td><td>±0.03</td></tr></tbody></table>

4

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6

7

<table><caption>**Table 3:** Yields in mCmole of hydrocarbons per Cmole of fructose consumed.</caption><thead><tr><th></th><th>Re2061-pLC10</th><th>Re2061-pMAB1</th><th>Re2061-pMAB2</th></tr></thead><tbody><tr><td>C15ane</td><td>3.2±0.2 (0.94%)</td><td>9.6±1.2 (2.8%)</td><td>33±3 (9.7%)</td></tr><tr><td>C17ane</td><td>0.24±0.02 (0.08%)</td><td>0.8±0.1 (0.2%)</td><td>2±0.2 (0.7%)</td></tr><tr><td>C17ene</td><td>2.0±0.12 (0.7%)</td><td>5.3±0.8 (1.7%)</td><td>10±1 (3.4%)</td></tr><tr><td>C16al</td><td>3.3±0.2 (0.9%)</td><td>5±1 (1.3%)</td><td>5±1 (1.3%)</td></tr><tr><td>Total alka(e)nes</td><td>5.4±0.5 (1.7%)</td><td>15.7 (5%)</td><td>45 (14%)</td></tr></tbody></table>

8

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10

In brackets: % of the theoretical yield. Theoretical yields were calculated by metabolic flux balancing at 0.34 ; 0.30 ; 0.30 and 0.37 Cmole/Cmole for C15ane, C17ane, C17ene and C16al, respectively.

11

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13

14
1 **Figure 1:** Metabolic engineering strategy for alka(e)ne production in C. necator strain.

2 Abbreviations: PHB: poly hydroxyl butyrate, ACC: acetoacetyl-coA carboxylase, FAS: fatty

3 acid synthase, AAR: acyl-ACP reductase, ADO: aldehyde deformylating oxygenase, Fd :

4 ferredoxin (oxidized or reduced form), FNR: ferredoxin reductase.

<figure><img src="image_3.png" /></figure>

5

6
1 **Figure 2:** Schematic of the synthetic alka(e)ne pathway operon incorporated in plasmid and

2 transformed into strain Re2061 (H16 ΔphaCAB). Abbreviations: ado (aldehyde

3 decarbonylase oxygenase), aar (acyl-ACP reductase), fdx (ferredoxin), FNR (ferredoxin-

4 NADP reductase), PBAD (Inducible Arabinose Promoter), RBS (Ribosome Binding Site)

<figure><img src="image_4.png" /></figure>

5

6
**Figure 3:** Kinetics of production of biomass (♦), pentadecane (□), heptadecene (Δ) and hexadecanal (○) during nitrogen limited fed-batch of *C. necator* strains Re2061-pMAB1 and Re2061-pMAB2 with addition of 10 % (v/v) of decane after induction. Induction time is indicated by the arrow. Dark bars indicate standard deviations

<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
**Figure 4:** Evolution of the specific production rates of total alka(e)nes and fatty aldehydes (hexadecanal) in µmole of product per g of cell dry weight per hour during nitrogen limited fed-batch of C. necator H16 strains Re2061-pMAB1 (dotted line) and Re2061-pMAB2 (full line) in the presence of 10 % (v/v) of decane.

<figure><img src="image_6.png" /></figure>
**Figure 5:** Final carbon repartition (distribution) into extracellular metabolites (organic acids, alka(e)nes and aldehydes) in Cmole per Cmole of cell dry weight for strains Re2061 bearing the empty plasmid pBBAD, the alkane operon plasmid pLC10 (Crepin et al., 2016), the alkane operon plasmid pMAB1 containing the native ferredoxin and ferredoxin reductase of C. necator H16 and the alkane operon plasmid pMAB2 containing the ferredoxin and ferredoxin reductase of S. elongatus.

<figure><img src="image_7.png" /></figure>

7

8
1 **Supplementary figure 1:** Kinetics of the cumulated amounts of fructose consumed (♦), of

2 pyruvic acid (□) and citric acid (Δ) during nitrogen limited fed-batch of C. necator strain

3 Re2061-pMAB1 and Re2061-pMAB2 with addition of 10 % (v/v) of decane after induction.

4 Induction time is indicated by the arrow.

<figure><img src="image_8.png" /><figcaption>Figure S1</figcaption></figure>

5

6

7

8

9

10