Background

The increasing concerns regarding global warming and the depletion of fossil fuel resources have prompted researchers to explore sustainable methods for chemical production from renewable carbon resources. Microbial fermentation has emerged as a promising approach, aided by the recent advancements in synthetic biology-guided metabolic engineering tools [1]. Polyphenolic compounds, including resveratrol, bisdemethoxycurcumin, and naringenin, have been extensively studied due to their diverse biological activities [2]. Resveratrol (trans-3,5,4′-transhydroxy-stilbene), a naturally occurring stilbene, has been widely used in various applications, including as a flavoring, fragrance, medicinal, and nutritional supplement [3, 4]. Commercially, these bioactive compounds are either extracted from plants such as grapes, berries, peanuts, and other vine plants in trace amounts or synthesized chemically [3, 5]. Owing to their health benefits and the increasing demand, the microbial production of plant-derived natural products has mainly been achieved by microbial metabolic engineering. Considering the number of benefits such as low production cost, high product purity, and sustainability, microbial fermentation process of resveratrol production provides a promising alternative to plant extraction or chemical synthesis [3, 6].

Resveratrol is synthesized in plants via the phenylpropanoid pathway, beginning with the synthesis of phenylpropanoids (such as coumaric and ferulic acids) from the aromatic amino acids phenylalanine and tyrosine [2, 7]. Phenylpropanoids are converted into various polyphenolic compounds via type III polyketide synthases (PKS). PKS include stilbene synthase for the synthesis of stilbenes such as resveratrol and chalcone synthase for the synthesis of flavonoids [7]. Recently, significant progress has been made in the de novo synthesis of polyphenolic compounds using engineered microorganisms, such as Escherichia coli and yeast [3].

Cupriavidus necator H16 (formerly Ralstonia eutropha H16), a well-studied H2-oxidizing lithoautotrophic bacterium, is capable of fixing CO2 via the Calvin–Benson–Bassham (CBB) cycle and storing large amounts of fixed carbon in the form of poly(3-hydroxybutyrate) (PHB) [1, 8]. Since this microbial platform is genetically tractable and has versatile metabolic capabilities, it has been engineered to produce a variety of valuable compounds from CO2 demonstrating its potential as an industrial workhorse [1, 9,10,11,12,13]. Moreover, C. necator H16 exhibits a faster autotrophic growth rate (doubling time = 4.2 h) than the photoautotrophic cyanobacteria (doubling time = 7–12 h) [14,15,16] and has been employed as a central biocatalyst in the microbial electrosynthetic systems that can use solar energy to convert CO2 into value added chemicals [13].

While C. necator H16 is considered a suitable candidate for autotrophic and electro-autotrophic chemical production, it has never been engineered for the production of polyphenolic compounds, such as resveratrol. We report here for the first time on lithoautotrophic production of resveratrol by engineered C. necator strains. The best-performing strains were initially screened under heterotrophic conditions, and then evaluated under lithoautotrophic conditions. An artificial phenylpropanoid pathway consisting of tyrosine ammonia lyase (TAL), 4-coumarate-coA ligase (4CL), and stilbene synthase (STS) was introduced into C. necator H16 with supplementation of L-tyrosine. Various metabolic strategies, including the overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes, were implemented to enhance resveratrol production. Finally, we produced 1.9 mg/L of resveratrol using CO2 as the sole carbon source via lithoautotrophic production of engineered C. necator strains.

Methods

Strains and plasmids

All strains and plasmids used in this study are listed in Table 1. Cupriavidus necator H16 (KCTC 22,469; Korean Collection for Type Cultures, Daejeon, South Korea) and PHB−4 (DSM 541, KACC 11,970; Korean Agricultural Culture Collection, Wanju, South Korea) were used as host strains for resveratrol production.

While ACC gene was amplified from the genomic DNA of Corynebacterium glutamicum ATCC 13,032, STS, TAL and 4CL genes were codon-optimized and synthesized by IDT KOREA (Additional file: Table S2). All genes were expressed in the pBBR1-MCS2 vector under the control of the arabinose-inducible araBAD promoter. The primers used to construct the recombinant plasmids are listed in Additional file: Table S1. Plasmid preparation was performed using Mini Exprep plasmid SV (Geneall, South Korea). A QIAquick gel extraction kit (Qiagen, Germany) was used for the gel purification of DNA fragments. The restriction enzyme-based cloning method, Gibson Assembly (New England Biolabs, Massachusetts, USA), or Hifi DNA Assembly (New England Biolabs, Massachusetts, USA) was used to assemble the recombinant plasmids. To transform the constructed plasmids into C. necator, bacterial conjugation using Escherichia coli S17-1 donor strain harboring the desired plasmid was performed [17].

Table 1 Strains and plasmids used in this work
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Heterotrophic and lithoautotrophic cultures

Cupriavidus necator H16 was routinely grown in Luria-Bertani (LB) broth at 30 ℃ and 200 rpm. For heterotrophic resveratrol fermentation, C. necator H16 strains were aerobically grown in 100 mL flasks containing 40 mL minimal media (MM) with 10 g/L fructose, 1.5 g/L KH2PO4, 6.74 g/L Na2HPO4•7H2O, 1.0 g/L (NH4)2SO4, 80 mg/L MgSO4•7H2O, 0.56 mg/L NiSO4•7H2O, 0.4 mg/L ferric citrate, 1 mg/L CaSO4•2H2O, and 0.5 g/L NaHCO3 supplemented with 5 mM tyrosine. The heterologous gene expression in the recombinant strains was induced at 6 h by the addition of 0.2% (w/v) L-arabinose unless otherwise indicated. For lithoautotrophic fermentation, the preculture was cultivated in MM with 10 g/L fructose at 30 ℃ and 200 rpm for 24 h. Cultured cells were collected by centrifugation at 4,200 rpm for 10 min, washed with MM without fructose and then inoculated into MM supplemented with 5 mM tyrosine in serum bottles. The strains were incubated at 30 ℃ and 200 rpm with the initial OD600 of 1 and 5, and filled with a 150 kPa of mixture gas (H2:O2:CO2 = 70:20:10 or 78:2:10; Airkorea Corporation, South Korea). The gas mixture was pressurized into the headspace of the 157 mL-serum bottles containing 20 mL MM every 24 h. The expression of biosynthetic genes was induced by adding 0.2% (w/v) L-arabinose after 24 h of autotrophic culture. During the cultivation of recombinant strains harboring resveratrol synthetic plasmids, kanamycin was also supplemented at a final concentration of 200 µg/mL.

Genetic manipulation

For the preparation of the △phaCAB strain based on the sacB-knockout system, pJQ200mp18Km plasmids were constructed by amplifying homologies to the 500 bp regions upstream and downstream of the phaCAB operon and transferred into C. necator H16 via conjugation as described previously with slight modifications [15, 17]. After culture in low salt-LB broth (2.5 g/L NaCl) supplemented with 15% (w/v) sucrose and gentamicin, the deletion strains were screened by PCR with diagnostic primers (Additional file 1: Table S1).

Analytical procedures

For the analysis of resveratrol and p-coumaric acid, 2 ml of the supernatant was centrifuged at 4,200 rpm for 10 min and extracted with an equal volume of ethyl acetate by vortexing. After extraction, the ethyl acetate layer was transferred to a glass tube and evaporated. The remaining residue was then dissolved in methanol. The samples filtered through a 0.2 μm syringe filter were analyzed by high-performance liquid chromatography (HPLC; Agilent technology 1100 Infinity, CA, USA) equipped with a ZORBAX SB-C18 column (4.6 × 150 mm, 3.5 μm, Agilent technology, CA, USA) maintained at 30 °C using a mobile phase composed of 40% acetonitrile and 60% water at a flow rate of 0.6 ml/min. The PHB content was quantified as previously described by Kim et al. (2022) [8].

RNA sequencing analysis

For verification of p-coumaric acid utilization by C. necator H16, sequencing experiments were performed by E-biogen, Inc. (Seoul, South Korea). RNA-seq samples were prepared after 20 h of aerobic fermentation in a minimal medium containing 10 g/L fructose with or without 1 g/L p-coumaric acid. RNA-seq analysis was performed as described previously by Kim et al. (2022) [8].

Results and discussion

Construction of a lithoautotrophic platform for production of resveratrol from CO2

A lithoautotrophic microbial platform was designed to produce resveratrol from CO2 via a phenylpropanoid biosynthetic pathway (Fig. 1). As the starting point, we expressed tyrosine ammonia lyase (TAL), which converts L-tyrosine to p-coumaric acid. In previous studies, microbial production of resveratrol was achieved with the supplementation of the precursor p-coumaric acid or aromatic amino acids such as L-tyrosine [2, 18]. The aromatic amino acid tyrosine is formed via the shikimate pathway in plants and microorganisms [19]. In this study, 5 mM tyrosine was used as the starting precursor for resveratrol synthesis. Consequently, p-coumaroyl-CoA is formed from the conversion of p-coumaric acid by expressing 4-coumarate: coenzyme ligase (4CL). Stilbene synthase (STS) sequentially directs the condensation of one molecule of p-coumaroyl-CoA with three malonyl-CoA molecules to produce resveratrol [18] (Fig. 1).

Fig. 1
figure 1

Synthetic metabolic pathways for de novo biosynthesis of resveratrol from CO2 and tyrosine in engineered C. necator H16. An artificial phenylpropanoid biosynthetic pathway engineered for resveratrol biosynthesis are shown in the colored box. The abbreviations are as follows: TAL, tyrosine ammonia lyase; 4CL, 4-coumarate-CoA ligase; STS, stilbene synthase; ACC, acetyl-CoA carboxylase; 3-PGA, 3-phosphoglycerate; G3P, glyceraldehyde-3-phosphate; RuBP, ribulose biphosphate. The dashed lines indicates omitted reaction steps

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To achieve resveratrol production from CO2, we constructed three recombinant plasmids carrying an STS gene from Vitis vinifera (VvSTS), a TAL gene from Flavobacterium johnsoniae (FjTAL), a 4CL gene from Arabidopsis thaliana (At4CL) and a ACC gene from Corynebacterium glutamicum (CgACC) under the control of arabinose-inducible araBAD promoter (Fig. 2A). To enhance the carbon flux of p-coumaroyl-CoA towards the resveratrol biosynthetic pathway, the recombinant plasmid containing a second copy of VvSTS was also constructed. Since the distance from the promoter affects the gene expression level [20], another VvSTS gene was placed in the last order to ensure the strong expression of FjTAL, At4CL and CgACC. The recombinant strains tested in this study are shown in Fig. 2B. First, the cell growth of C. necator H16 in the presence of resveratrol ranging from 0 to 50 mg/L was tested to evaluate the potential problems with the toxicity of resveratrol to cells (Additional file 1: Fig. S1). Notably, the presence of 50 mg/L resveratrol caused severe retardation of C. necator H16 cell growth.

Fig. 2
figure 2

Construction of recombinant C. necator strains. (A) Genetic map of a resveratrol biosynthetic plasmid containing a synthetic ribosome-binding site (RBS) and a nucleotide linker sequence inserted between each gene [20]. (B) List of engineered strains with different combinations of heterologous genes. The sources of heterologous genes are indicated: VvSTS, Vitis vinifera STS; FjTAL, Flavobacterium johnsoniae; At4CL, Arabidopsis thaliana 4CL; CgACC, Corynebacterium glutamicum ACC

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Evaluation of heterotrophic resveratrol production by engineered strains

The engineered strains were evaluated for their resveratrol production capabilities, using fructose as the carbon source (Fig. 3). When the CR-1 strain carrying FjTAL, At4CL, and VvSTS genes was cultured in a minimal medium supplemented with 5 mM tyrosine, resveratrol synthesis was not detected (Fig. 3B). Since the biosynthesis of resveratrol requires three moles of malonyl-CoA, limited malonyl-CoA availability may be the decisive bottleneck in the native metabolism of C. necator H16. Most microorganisms generate malonyl-CoA solely from irreversible acetyl-CoA carboxylation catalyzed by acetyl-CoA carboxylases (ACCs), which is a key precursor of de novo fatty acid synthesis (FAS) [21]. Since the malonyl-CoA pool is highly controlled and consumed by FAS [21, 22], FAS is regarded as an undesired metabolic pathway in terms of resveratrol biosynthesis [22]. Therefore, we used two strategies to increase intracellular malonyl-CoA availability: inhibition of fatty acid synthesis and overexpression of acetyl-CoA carboxylase (ACC) (Fig. 3A).

To inhibit the synthesis of fatty acids that incorporate malonyl-CoA, 0.05 mM of the FAS inhibitor cerulenin was supplemented into the medium (Fig. 3B). The CR-1 strain supplemented with cerulenin produced 1.5 mg/L of resveratrol. This indicates that the limited availability of malonyl-CoA is a major bottleneck in resveratrol synthesis in C. necator H16. Cerulenin supplementation has been shown to enhance polyphenolic compound production in E. coli and C. glutamicum in prior studies [7, 23, 24]. However, it is expensive and can inhibit cell growth due to irreversible and non-selective FAS inhibition [22, 25,26,27]. To address this, gene downregulation strategies using antisense RNA or CRISPR interference have been used in previous studies for malonyl-CoA-derived compounds [28, 29].

Instead of reducing undesired malonyl-CoA consumption by adding cerulenin, we constructed a CR-2 strain overexpressing ACC derived from C. glutamicum to increase the intracellular malonyl-CoA pool. Since ACC of C. glutamicum comprises only two subunits (accBC and dtsR1) instead of a four-subunit protein complex of ACC derived from E. coli and others for catalytic activity [30], this enzyme was overexpressed in C. necator H16. The expression of heterologous ACC genes in C. necator H16 resulted in successful resveratrol synthesis, producing 3.5 mg/L (Fig. 3D).

To further channel more carbon flux from acetyl-CoA towards malonyl-CoA, we disrupted the PHB synthesis pathway. Using the sacB-based gene knock-out plasmid, the PHB non-producing strain ΔphaCAB was obtained with the deletion of the entire phaCAB operon encoding the essential enzymes for PHB synthesis (Table 1). We also employed a representative PHB-negative mutant, PHB−4 (DSM 541), which has a single nonsense mutation in the PHA synthase gene phaC [31]. Although PHB accumulation was not detected, both the PHB-negative PHB−4 and △CAB strains showed retarded cell growth in media containing fructose (10 g/L) as the carbon source (Additional file 1: Fig. S2). Since acetyl-CoA is not consumed in the PHB synthetic pathway, mutant cells might have altered cellular metabolism with the accumulation of metabolites, such as acetyl-CoA and pyruvate [31]. When C. necator H16, △CAB, and PHB−4 strains carried the pBAD-STS-TAL-4CL-ACC plasmid, the resulting strains CR-2, CR-3, and CR-4 produced 3.5, 4.1, and 3.5 mg/L of resveratrol, respectively (Fig. 3D). Although the cell growth rate of CR-3 was lower than that of CR-2, its resveratrol titer was obtained as 4.1 mg/L, 17% higher than that of CR-2. Disruption of the competing pathway by deleting the entire phaCAB operon effectively enhanced resveratrol synthesis. However, resveratrol production by the PHB−4 strain (CR-4) did not show any positive result and its growth rate was lower than that of CR-3. Therefore, the CR-3 strain with the highest resveratrol synthesis capability was selected for further engineering. Since cell growth and PHB synthesis in C. necator H16 depend on the nitrogen concentration with excess carbon available, the effect of nitrogen supplementation on resveratrol production was investigated. Also, the resveratrol production performances of the different strains with or without the PHB synthetic pathway were compared. While nitrogen limitation generally boosts PHB accumulation [32], the resveratrol synthesis was negatively affected under the nitrogen limitation condition (0.5 (NH4)2SO4-g/L). Although the CR-2 strain showed the enhanced cell growth at 24 h under nitrogen limitation (Fig. 3C), it led to the decreased resveratrol production. For the PHB-negative CR-3 and CR-4 strains, nitrogen limitation led to decreased cell growth, resulting in reduced resveratrol production (Fig. 3C, D). When the competing pathway was disrupted to avoid the depletion of acetyl-CoA, which is the precursor to PHB, in CR-3 and CR-4 strains, the redirected carbon flow towards acetyl-CoA under nitrogen limitation could not increase resveratrol synthesis. While the sufficient nitrogen supply is required for enhancing cell growth and resveratrol production, the > 2-fold enhancement of 1,3-butanediol production by engineered C. necator H16 was observed under nutrient-limiting conditions [15]. Despite decreased cell growth, the availability of 3-hydroxybutyryl-CoA precursor boosted by nitrogen limitation enabled the enhanced 1,3-butanediol production [15].

Fig. 3
figure 3

Resveratrol production using engineered strains under heterotrophic conditions. (A) Strategies for increased malonyl-CoA availability and resveratrol synthesis. The strategies used in this study including the overexpression of ACC, addition of cerulenin and disruption of PHB synthetic pathway are stated as red colors. The dashed lines indicates omitted reaction steps. The abbreviations are as follows: ACC, acetyl-CoA carboxylase; PEP, phosphoenolpyruvate; DHAP, 3-deoxy-D-arabinoheptulosanate-7-phosphate; FAS, fatty acid synthesis. (B) Resveratrol production using the CR-1 strain with or without cerulenin supplementation. (C) Cell growth and (D) resveratrol production using the CR-2, CR-3, and CR-4 strains. Cells were grown in either nitrogen-rich MM medium (solid lines, dark purple) containing 10 g/L fructose and 1 g/L (NH4)2SO4 or nitrogen-limiting MM (dashed lines, light purple) containing 10 g/L fructose and 0.5 g/L (NH4)2SO4. The MM medium was supplemented with 5 mM tyrosine for resveratrol synthesis. The resveratrol titer was measured at the end of 96 h-fermentation. Error bars represent the standard deviation from three biological replicates. Student’s two-tailed t-test was performed to determine the significance of differences (*, p < 0.05)

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Additional STS copy increases the flux of p-coumaroyl CoA towards resveratrol biosynthesis

While malonyl-CoA availability was regulated by overexpression of ACC and disruption of the PHB synthetic pathway, yielding 4.1 mg/L of resveratrol, one molecule of p-coumaroyl-CoA via coumaric acid conversion by the action of 4CL was still required for resveratrol synthesis. Although high levels of p-coumaric acid are toxic to cells, the possibility of coumaric acid as a carbon source of C. necator H16 at low concentrations has been suggested in previous studies [33]. The catabolic mechanisms of lignin-derived aromatic compounds, including coumaric, ferulic, and cinnamic acids, have not been completely elucidated in C. necator H16. It has been suggested that p-coumarate is converted to 4-hydroxybenzoate and then fed into the TCA cycle [33]. As shown in Fig. 4A, the growth of C. necator H16 was observed in the presence of 3–5 mM p-coumaric acid as the sole carbon source, although a high concentration of p-coumaric acid (5 mM) led to a prolonged lag phase. Although the genes responsible for the conversion of p-coumaric acid to 4-hydroxybenzoate were not completely identified, cell growth on 4-hydroxybenzoate and the upregulation of the genes involved in the reactions starting from 4-hydroxybenzoate to succinyl-CoA were observed when the cells were cultured with supplementation of p-coumaric acid (Additional file 1: Fig. S3). This indicates that tyrosine supplemented in the medium as the precursor for resveratrol synthesis might be converted to primary metabolites in the engineered strains. As the activation of coumarate catabolic pathways in C. necator H16 causes the loss of p-coumaroyl CoA, an important precursor for resveratrol synthesis, we aimed to redirect the carbon flux of p-coumaroyl-CoA towards the resveratrol biosynthetic pathway by overexpressing two copies of VvSTS (Fig. 4B). The addition of a second copy of VvSTS in the CR-2 and CR-3 strains, resulting in CR-5 and CR-6 strains, increased resveratrol production from 3.5 to 4.1 mg/L to 6.4 and 6.9 mg/L, respectively (Fig. 5B). The final OD600 values at 96 h of CR-5 and CR-6 strains were not significantly different from those of CR-2 and CR-3 strains, respectively (Fig. 5A). The > 70% increase in resveratrol synthesis in the CR-5 and CR-6 strains demonstrated that increasing STS activity is important to make use of the available p-coumaroyl-CoA. With further efforts to understand the metabolism of aromatic compounds in C. necator H16, the identification, and deletion of key genes involved in the conversion of p-coumaroyl-CoA to 4-hydroxybenzoate could be an alternative strategy for increasing p-coumaroyl-CoA availability for resveratrol production. Incha et al. (2020) identified genes involved in the coumarate catabolic pathways of Pseudomonas putida KT2440 that could potentially impact p-coumaroyl-CoA-derived products. In engineered P. putida, deletion of the gene ech (enoyl-CoA hydrolase-lyase) in coumarate catabolism increased type III polyketide bisdemethoxycurcumin [34].

Microbial production of resveratrol has been reported in various microorganisms (Table 2). In particular, significant efforts in metabolic engineering toward high-level resveratrol production have been achieved in E. coli, C. glutamicum, Saccharomyces cerevisiae, and Yarrowia lipolytica [3]. While the first production of resveratrol with the titer of 1.45 µg/L was achieved in engineered yeast [35], a high level of resveratrol production reaching approximately 800 mg/L was found in engineered S. cerevisiae by optimized fed-batch fermentation [36]. With advancements in synthetic biology tools, there have been numerous efforts to regulate metabolic pathways and optimize resveratrol production; E. coli and C. glutamicum have yielded 300 and 110 mg/L of resveratrol titer, respectively [7, 18, 25, 36, 37]. Other bacterial hosts, such as Streptomyces venezuelae and Aspergillus niger have also been used to produce resveratrol by introducing a heterologous phenylpropanoid biosynthetic pathway [38, 39]. It was reported that engineered S. venezuela produced resveratrol for the first time, with a yield of just 0.4 mg/L [39]. In this study, we achieved 6.8 mg/L of resveratrol from fructose in engineered C. necator H16, which is the first time that a polyphenolic compound has been synthesized in this host. Taken together, the CR-6 strain constructed in this study represents a promising starting point for further engineering towards a more efficient resveratrol production.

Fig. 4
figure 4

Coumarate catabolic pathways in C. necator H16 causes the loss of p-coumaroyl CoA, an important precursor for resveratrol synthesis. (A) Cell growth of C. necator H16 strain on various aromatic compounds as the sole carbon source. PCA: p-coumaric acid, 4-HBA: 4-hydroxybenzoic acid. The initial optical density at 600 nm (OD600) was 0.2 at 600 nm and the initial p-coumaric acid and 4-hydroxybenzoic acid concentrations were 3 and 5 mM, respectively. (B) Metabolic engineering of C. necator H16 for re-directing malonyl-CoA flux towards resveratrol synthesis by addition of VvSTS

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Fig. 5
figure 5

Effects of increasing the copy number of VvSTS and disrupting the PHB pathway on resveratrol production under heterotrophic conditions. (A) Cell growth curve and (B) resveratrol production in the CR-2, CR-3, CR-5, and CR-6 strains. The initial OD600 was 5 and the initial fructose concentration was 10 g/L. The MM medium was supplemented with 5 mM tyrosine for resveratrol synthesis. The resveratrol titer was measured at the end of 96 h-fermentation

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Table 2 Heterotrophic and autotrophic microbial production of resveratrol in various engineered strains
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Lithoautotrophic production of resveratrol from CO2

The final goal of this study was to produce resveratrol from CO2. For autotrophic production of resveratrol, the recombinant strains CR-2, CR-3, CR-5, and CR-6 were cultivated in serum bottles supplemented with CO2, H2, and O2 (Fig. 6). Although the cell growth rates of the CR-2 and CR-3 strains were significantly higher than those of the CR-5 and CR-6 strains with initial cell densities (OD600) of 1 (Fig. 6A), resveratrol synthesis was not observed in the CR-2 and CR-3 strains (Fig. 6B). Approximately 1.2 mg/L of resveratrol was produced from CO2 and tyrosine by the CR-5 and CR-6 strains carrying a second copy of VvSTS. However, differences in autotrophic performance between the CR-5 and CR-6 strains were not observed (Fig. 6B). Since the high cell density culture offers an efficient way to enhance the microbial fermentation productivities [45], the autotrophic cultures with the higher initial cell densities were performed. When the initial cell density was increased from 1 to 5, both the CR-5 and CR-6 strains produced slightly more resveratrol, yielding 1.4 and 1.5 mg/L, respectively (Fig. 6C and D). In addition, autotrophic fermentation under oxygen stress (H2:O2:CO2 = 88:2:10) was performed to promote resveratrol synthesis. Although C. necator H16 is typically cultured in a gas composition of H2:O2:CO2 = 70:20:10 for autotrophic growth, some previous works have reported that oxygen stress (< 3% O2) induced enhanced PHB production while restricting cell growth [46]. When oxygen stress (H2:O2:CO2 = 88:2:10) was induced in the CR-5 and CR-6 strains, resveratrol production increased from 1.4 to 1.5 mg/L to 1.8–1.9 mg/L.

Although C. necator H16 has been used as a host for the potential commercial production of polyhydroxyalkanoates (PHAs), it has been engineered to produce a variety of chemicals, such as isopropanol, acetoin, humulene, methyl ketone, (R)-1,3-butanediol, and alkene, under both heterotrophic and autotrophic conditions (Table 3) [1, 9, 10, 12, 15]. More recently, the CO2 conversion into sugars and value-added compounds by engineered C. necator strains using H2 as an energy source produced 470 mg/L trehalose, 250 mg/L glucose, and 1.7 mg/L lycopene [11, 13, 47]. This work is the first step towards the production of plant type III PKS-derived compounds in C. necator, but the synergistic combination of metabolic and fermentation optimization strategies for increasing productivity should be addressed in the future. With further studies on improving efficiencies of carbon fixation and resveratrol synthesis, a better understanding of carbon flux of CO2 in resveratrol-producing C. necator strains should allow us to enhance the viability of lithoautotrophic biochemical production. Due to the inhibitory effect of resveratrol on the cell growth of C. necator H16 (Additional file: Fig. S1), further metabolic engineering strategies for enhancing microbial tolerance towards resveratrol as well as the application of in situ product removal strategies are required to avoid the product toxicity limitations. Since the engineered C. necator strains still require the supplementation of exogenous tyrosine as a precursor, the metabolic engineering efforts on de novo tyrosine biosynthetic pathway is also needed to produce resveratrol solely from CO2.

Fig. 6
figure 6

Resveratrol production using engineered strains from CO2 and tyrosine. Lithoautotrophic fermentation profiles of the engineered strains for resveratrol production under different gas compositions of (A, B) H2:O2:CO2 = 70:20:10 vs. (C, D) H2:O2:CO2 = 88:2:10 (O2-limited) with different initial cell densities. The MM medium was supplemented with 5 mM tyrosine for resveratrol synthesis and the expression of resveratrol-biosynthetic genes was induced by adding 0.2% (w/v) L-arabinose after 24 h of autotrophic culture. The resveratrol titer was measured at the end of 144 h-fermentation

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Table 3 Valorization of CO2 into valuable chemicals by engineered C. necator H16
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Conclusions

In this study, a microbial lithoautotrophic platform was developed by introducing an artificial resveratrol biosynthetic pathway into C. necator H16. Implementation of phenylpropanoid pathways consisting of tyrosine ammonia lyase (TAL), 4-coumarate-coA ligase (4CL), and stilbene synthase (STS) with supplementation of L-tyrosine in combination with disrupting PHB biosynthesis and enhancing carbon flux by increasing copies of STS enabled the strain to produce 1.9 mg/L of resveratrol from CO2 and tyrosine. Further intensive efforts on the metabolic engineering of phenylpropanoid and tyrosine-biosynthetic pathways will be necessary to realize the industrial production of resveratrol from CO2. The recombinant C. necator strain developed in this study is considered a promising starting strain for further engineering to produce polyphenolic compounds. With the continued engineering of C. necator H16, this strain will become a more attractive lithoautotrophic platform, providing a sustainable route for the valorization of CO2 to high-value natural products.