Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

doi:10.21203/rs.2.23886/v2

Background: Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-D-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis.

Results: This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed ¹³C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways.

Conclusions: Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth-conditions.

Biotechnology and Bioengineering

Rhodobacter sphaeroides

isoprenoid biosynthesis

PHB

MEP

MVA growth-independent production

Isoprenoids (also known as terpenoids) have great industrial value as ingredients of pharmaceuticals, perfumes, food flavourings and most recently biofuels (1–6). They are formed by the condensation of the five-carbon monomers isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). The two naturally existing IPP/DMAPP production pathways are the 2-C-methylerythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway (7). While the former branches in the central metabolism from glyceraldehyde 3-phosphate and pyruvate, the latter uses acetoacetyl-CoA (AA-CoA) as precursor (Fig. 1). These two pathways are, with few exceptions, phylogenetically distinct: the MEP pathway is present in prokaryotes, while the MVA pathway is found in archaea and eukaryotes (8). Plants express both metabolic routes, with the MEP pathway compartmentalized in the chloroplasts and the MVA pathway expressed in the cytosol (9). Since the first implementation of a heterologous MVA pathway in Escherichia coli (10), the number of studies focusing on the synthesis of isoprenoids via microbial cell factories has increased a lot. Efforts for improving bioproduction have been focusing either on engineering of the endogenous MEP pathway (11–13), or by co-expressing the heterologous MVA counterpart (10,14,15). Moreover, to limit the effect of unfavorable regulatory control of the endogenous pathway, substitution by an orthogonal isoprenoid pathway has been reported (16).

In microorganisms, isoprenoids are often membrane-bound molecules - like carotenoids, ubiquinones, chlorophylls and sterols – which are indispensable for growth (17,18). Therefore, during a batch cultivation, their volumetric concentration is expected to increase as consequence of microbial growth inside the reactor. Such type of metabolism for isoprenoid biosynthesis is defined as growth-coupled. For strain improvement purposes, growth-coupled production has been largely employed. In such a scenario, the product of interest becomes a mandatory by-product of growth, and therefore microbial growth becomes the driving force of production (19). This production-growth association has already been exploited for enhancing isoprenoid biosynthesis by laboratory evolution (20).

Nevertheless, growth-coupled production contains an inherent trade-off between substrate use for i) biomass production and maintenance, and ii) product formation (21). Thus, if biomass formation is prevented, in principle more substrate is available for product synthesis.

Microbial metabolism has been engineered to produce non-native isoprenoid molecules as pharmaceuticals, perfumes, food flavourings and biofuels (1–6). These compounds are not required for growth, and often excreted. Coupling of product formation to microbial growth is therefore not a necessity, and growth-uncoupled production would be an advantageous option.

Apart from C, H and O, isoprenoids do not contain other biomass-specific elements like P, S or N. Therefore, under P, S or N limited incubation conditions, glucose can in principle be converted into isoprenoid but not into microbial biomass. Improved isoprenoid/biomass ratios have already been obtained by using nutrient-limited culturing conditions (22–25). Nonetheless, as a highly-regulated primary metabolism, isoprenoid biosynthesis has been largely optimized for growth-coupled production, and decoupling isoprenoid synthesis from microbial growth has been overlooked as a production strategy.

In a recent study, the concept of redesigning biosynthetic networks based on orthogonality principles was introduced (26). This idea entails that a non-native metabolic route that minimizes the interaction with the endogenous biomass-producing pathways can be exploited for bioproduction. Intrinsic independence exists between the two isoprenoid pathways MEP and MVA. Therefore, regulatory control affecting the MEP pathway should not affect the MVA pathway, and vice versa. Moreover, in a recent study, functional replacement of the native MEP with the heterologous MVA pathway was described in the bacterium Rhodobacter sphaeroides (16), a microbial platform organism that is gaining interest for isoprenoid biosynthesis. This organism is able to synthesize intracellular membranes which can accommodate isoprenoids such as as carotenoids and bacteriochlorophylls. Moreover, it is also a natural producer of coenzyme Q₁₀. Apart from these native isoprenoid molecules, heterologous production of lycopene (27) and sesquiterpenes (28) has also been reported in this species.

Growth and isoprenoid synthesis in R. sphaeroides has been studied using defined medium, where the introduction of the heterologous MVA pathway revealed potential for growth-independent isoprenoid biosynthesis (29). Under these conditions, the storage compound poly-β-hydroxybutyrate (PHB) was also accumulated (29). Additionally, a mutual stimulating effect between the MEP and MVA pathways has been observed (30).

In this study, we investigate the behaviour of the two isoprenoid pathways for amorphadiene production in R. sphaeroides during different growth modes, as well as their interaction with the pathway for the carbon- and energy reserve material poly-β-hydroxybutyrate, PHB. By means of ¹³C metabolic flux ratio analysis, we assess the effect of genetic modifications (i.e. for elimination of PHB accumulation) and environmental changes (nitrogen limitation) on isoprenoid pathways capacities. Ultimately, we demonstrate that exclusive use of the orthogonal MVA pathway in combination with elimination of PHB synthesis is a promising strategy for attaining growth-independent production of isoprenoids.

Strains and standard cultivation conditions

The strains and plasmids used in this study are listed in Table 1 and Table 2, respectively. The Rs265_∆phaC1∆phaC2 strain was kindly donated by Isobionics BV. Preculturing of R. sphaeroides was performed in 250-mL Erlenmeyer flasks containing 25 mL of modified Sistrom’s minimal medium (SMM). As previously described (30), the medium contained glucose (3.0 g/L) as carbon source, and NH₄Cl (1.0 g/L) as nitrogen source. Moreover, the SMM contained (per liter): 3.48 g KH₂PO₄, 0.5 g/L NH₄Cl, 0.1 g glutamic acid, 0.04 g L-aspartic acid, 0.5 g NaCl, 0.02 g nitrilotriacetic acid, 0.3 g MgSO₄·7H₂O, 0.0334 g CaCl₂·2H₂O, 0.002 g FeSO₄·7H₂O, and 0.0002 g (NH₄)₆Mo₇O₂₄. Trace elements were added (0.01% v/v) from a stock solution containing: 17.65 g/L disodium EDTA, 109.5 g/L ZnSO₄·7H₂O, 50 g/L FeSO₄·7H₂O, 15.4 g/L MnSO₄·7H₂O, 3.92 g/L CuSO₄·5H₂O, 2.48 g/L Co(NO₃)₂·6H₂O, and 0.0114 g/L H₃BO₃. Vitamins were added (0.01% v/v) from a stock containing: 10 g/L nicotinic acid, 5 g/L thiamine HCl, and 0.1 g/L biotin.

Generation of double-KO strain via CRISPR-Cas9 counter-selection

The primers used in this study are listed in Additional file 1: Table S1. Deletion of phaB was performed as previously described (31), using the pBBR_Cas9_∆phaB_HR plasmid. Such plasmid was transferred from E. coli S17 cells to Rs265-MVA_∆dxr via diparental conjugation, resulting in the double mutant strain Rs265-MVA_∆dxr∆phaB. By employing the pBBR_Cas9_∆phaB_HR plasmid, phaB could be removed by homologous-recombination, and Cas9-based counter-selection of cells with intact genomic copies of phaB.

Diparental conjugation of R. sphaeroides

Diparental conjugation for transferring amorphadiene producing plasmids was performed as previously described (30) using RÄ medium. Such medium contained, per liter: 3 g malic acid, 0.2 g MgSO₄·7H₂O, 1.2 g (NH₄)₂SO₄, 0.07 g CaCl₂·2H₂O, 1.5 mL of microelements stock solution, 2 mL of vitamin stock solution and 5 mL of phosphate buffer. In case of RÄ agar medium, 15 g/L agar was added. The microelements solution contained: 0.5 g/L Fe(II)-Citrate, 0.02 g/L MnCl₂·4H₂O, 0.005 g/L ZnCl₂, 0.0025 g/L KBr, 0.0025 g/L KI, 0.0023 g/L CuSO₄·5H₂O, 0.041 g/L Na₂MoO, 0.005 g/L CoCl₂·6H₂O, 0.0005 g/L SnCl₂·2H₂O, 0.0006 g/L BaCl₂·2H₂O, 0.031 g/L AlCl, 0.41 g/L H₃BO₃, 0.02 g/L EDTA. The vitamin solution contained: 0.2 g/L nicotinic acid, 0.4 g/L thiamine HCl, 0.008 g/L biotin, 0.2 g/L nicotinamide. The phosphate buffer contained 0.6 g/L KH₂PO₄ and 0.9 g/L K₂HPO₄.

Cultivation in nitrogen excess and nitrogen limited conditions

After overnight preculturing on SMM, R. sphaeroides cultures were transferred to fresh SMM with a starting OD₆₀₀ of 0.1, and incubated at 30 ˚C with 250 rpm. Cultivations were performed for biological triplicates in 250-mL Erlenmeyer flasks, each filled with 45-mL of SMM medium and 5-mL of filter-sterilized dodecane. SMM composition differed between nitrogen excess condition and nitrogen limited conditions only in the initial NH₄Cl concentration: 1.0 g/L and 0.25 g/L, respectively. Initial glucose concentration remained 3.0 g/L in both cases. Amorphadiene titers were measured after glucose depletion. This occurred after 24 h (for nitrogen excess condition) or after 48 h (for nitrogen limited condition). At the same time, the content of the flasks was harvested by centrifugation and further processed for analytical measurements.

¹³C-metabolic flux ratio analysis of isoprenoid biosynthesis

Isoprenoid flux ratios analyses were performed as previously described (30) in 10-mL Erlenmeyer flasks containing 1.8-mL of labeled SMM medium and 0.2-mL of filter-sterilized dodecane. [1-¹³C]- and [4-¹³C]-glucose tracers had an initial concentration of 3.0 g/L. For nitrogen excess and limited conditions, the initial NH₄Cl concentration used was the same as for the cultivations in 250-mL flasks: 1.0 and 0.25 g/L, respectively. Samples for GC-MS measurement were taken at glucose depletion. MEP and MVA pathways capacities were obtained by multiplying the flux ratios determined via GC-MS with the amorphadiene titers measured via GC-FID for the 250-mL cultivations.

Cultivation under resting cell conditions

For resting cell cultivations, exponentially growing cells on SMM with nitrogen excess conditions were incubated until mid-exponential phase. Then, cells were pelleted by centrifugation at 4255 g for 10 min at room temperature. Subsequently, pellets were washed twice with sterile physiologic solution (NaCl 9 g/L) and centrifuged at 4255 g for 5 min. Washed pellets were inoculated with a starting OD₆₀₀ of 1.0 on ‘nitrogen free SMM’ containing 5.0 g/L glucose and 0.0 g/L NH₄Cl. Initial biomass concentration was determined at the moment of the inoculum with a sample of 5-mL for TOC-L analysis. Then, incubation proceeded at 30 ˚C with 250 rpm. Samples for OD₆₀₀, pH and amorphadiene measurements were taken at a regular interval until the pH dropped below 5.5. Determination of productivity and yields were performed for samples taken within the first 24 h.

Analytics

Cell density was monitored by measuring the optical density at 600nm (OD₆₀₀). Amorphadiene concentration was measured via GC-FID as previously described (29). Glucose, PHB and organic acid concentrations were determined via (U)HPLC as previously described (29). For pyruvate and crotonic acid (resulting from PHB hydrolysis) identification, DAD detector was used, while for glucose, 2-oxoglutarate and 3-hydroxybutyrate determination, the RID detector was used. Determination of biomass concentration via TOC-L was performed by measuring the nitrogen content of the pellet, which was then used to calculate the active biomass concentration using the elemental composition of R. sphaeroides of CH_1.99O_0.5N_0.19(29). Identification of unknown compounds in the spent medium was obtained via ¹H-nuclear magnetic resonance spectroscopy (¹H-NMR) measurements performed in D₂O on a Bruker Avance III 400 MHz NMR spectrometer.

Prevention of PHB formation and its effect on amorphadiene biosynthesis

Culturing R. sphaeroides under nitrogen-limited conditions could theoretically result in growth-independent isoprenoid synthesis via the native MEP and the heterologous MVA pathways. However, upon consumption of the limited available nitrogen, R. sphaeroides stores excess carbon intracellularly as PHB, a nitrogen-free carbon and energy storage compound (29). Aiming to increase isoprenoid production, we reasoned that deletion of one of the PHB synthesis genes would block PHB production under nitrogen-limited conditions and could therefore increase the flux through the MVA pathway. Deletion of the phaC1 and phaC2 genes, that code for the PHB polymerase, is the established approach for eliminating PHB biosynthesis in R. sphaeroides (32–35). Nonetheless, this does not prevent activity of the NADPH-dependent acetoacetyl-CoA reductase PhaB, which could result in the undesired accumulation of 3-hydroxybutyryl-CoA or excretion of 3-hydroxybutyrate (Fig. 1). In a recent study, we demonstrated that deletion of the phaB gene prevents PHB biosynthesis (31). Here, we confirmed that the deletion of either the phaB gene (Rs265_∆phaB strain) or the combined deletion of the phaC1 and phaC2 genes (Rs265_∆phaC1∆phaC2 strain) prevents PHB formation both under nitrogen excess and nitrogen-limited conditions (Fig. 2A). As observed before (29), the wild type (Rs265) strain produced substantial amounts of PHB, especially under nitrogen-limiting conditions.

The pBBR-ads plasmid harbouring the heterologous amorphadiene synthase gene was transferred to the various R. sphaeroides strains by conjugation. The resulting strains were cultured under both nitrogen excess and nitrogen-limited conditions. At glucose depletion, we determined (Table 3): amorphadiene titers (C_P), yield of amorphadiene on glucose (Y_P/S), active biomass concentration (C_X) and the amorphadiene on biomass ration (Y_P/X). We observed that, for the Rs265_∆phaB:pBBR-ads and Rs265_∆phaC1∆phaC2:pBBR-ads strains that use only the endogenous MEP pathway (MEP-only strains), elimination of PHB synthesis does not result in higher amorphadiene/biomass ratios compared to the Rs265 wt strain (Fig. 2B, Table 3). In fact, these ratios remained unaffected when moving from nitrogen excess to nitrogen-limited conditions (Table 3) at a value of 2.9 ± 0.2 mg ∙ g of biomass^-1. Interestingly, the Rs265_∆phaC1∆phaC2:pBBR-ads strain showed an even lower amorphadiene/biomass ratio compared to the Rs265:pBBR-ads and the Rs265_∆phaB:pBBR-ads strains (Table 3). In summary, when only the endogenous MEP pathway was active, isoprenoid biosynthesis appeared to be strictly growth-coupled. Moreover, the MEP flux was insensitive or negatively affected by the impaired PHB synthesis.

We subsequently transformed the available Rs265, Rs265_∆phaC1∆phaC2 and Rs265_∆phaB strains with the orthogonal MVA pathway, cloned in the pBBR-MVA-ads plasmid. The amorphadiene/biomass ratio increased 10- to 20-fold for all the strains tested (Fig. 2C, Table 3). The highest increase was observed for the ∆phaB strain (Rs265_∆phaB:pBBR-MVA-ads), reaching a ratio of 63.7 ± 4.0 mg ∙ g of biomass^-1(Fig. 2C, Table 3). This value was significantly higher than the ratio reached by the strain with a functional PHB synthesis (Rs265:pBBR-MVA-ads), which was 35.9 ± 3.6 mg ∙ g of biomass^-1(Fig. 2C, Table 3).

Increase of the MVA pathway flux, as consequence of the phaB deletion, was confirmed also for the Rs265-MVA_∆dxr strain, for which the MEP pathway is inactivated via the deletion of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) gene, after genomic integration of the MVA pathway (Fig. 1). This strain relies exclusively on the non-native isoprenoid route (MVA-only) (16). Also here, a substantial increase in the amorphadiene/biomass ratio was observed during both nitrogen excess and nitrogen-limited conditions (Fig. 2D, Table 3). The highest value observed was for the Rs265-MVA_∆dxr∆phaB:pBBR-MVA-ads strain, with 36.9 ± 1.6 mg ∙ g of biomass^-1during nitrogen limitation.

In summary, although both the ∆phaC1∆phaC2 and ∆phaB knockouts were equally effective in reducing PHB synthesis, the ∆phaB knockout strain produced more amorphadiene, both volumetrically and per biomass unit (Table 3).

Organic acids secretion as consequence of PHB deletion

We reasoned that comparing the secretion profiles between ∆phaB and ∆phaC1∆phaC2 could provide additional insights on the beneficial effect of ∆phaB on isoprenoid synthesis. Therefore, we quantified by HLPC analysis the organic acids in the spent medium of Rs265, Rs265_∆phaC1∆phaC2 and Rs265_∆phaB harbouring either pBBR-ads or pBBR-MVA-ads plasmids (Fig. 2E-H). For both ∆phaB and ∆phaC1∆phaC2 strains, 2-oxoglutarate (80 to 340 mg ∙ g of biomass^-1, Fig. 2F) and pyruvate (150 to 500 mg ∙ g of biomass^-1, Fig. 2G) were the main by-products. Both compounds require coenzyme A (CoA) for proceeding further in the metabolism via oxidative decarboxylation (Fig. 1). Excretion of these compounds suggests that free CoA is limiting when PHB biosynthesis is prevented.

The ∆phaC1∆phaC2 strains secreted an additional unknown compound, which was identified as 3-hydroxybutyrate (3HB) by NMR (Additional file 1: Fig. S1). The spent medium for the Rs265_∆phaC1∆phaC2:pBBR-ads strain showed a value of 0.002 ± 0.001 mg 3HB ∙ g of biomass^-1 In contrast, a value of 0.004 ± 0.001 mg 3HB ∙ g of biomass^-1 was observed for the Rs265_∆phaC1∆phaC2:pBBR-MVA-ads strain, which expressed the heterologous MVA pathway (Fig. 2H). Therefore, under nitrogen limitation and upon expression of the MVA pathway, the amount of 3HB secreted increased significantly compared to when only the MEP pathway was active.

¹³C metabolic flux ratio analysis of isoprenoid biosynthesis under different growth conditions

The Rs265_∆phaB:pBBR-MVA-ads strain, that overexpresses the MVA pathway and still has an active MEP pathway, showed the highest amorphadiene/biomass ratio under nitrogen-limited conditions (Fig. 2C, Table 3). Additionally, we observed that, independent from the cultivation conditions and the presence of an active PHB synthesis pathway, the dual-pathway (co-expressing MEP and MVA pathways) strains largely outperformed the single-pathway strains (Fig. 2B-D, Table 3). We therefore decided to further investigate the separate and combined contribution of the isoprenoid pathways to the amorphadiene production by ¹³C flux ratio analysis.

MEP and MVA pathways are known to exert a reciprocal stimulation (30,36). To better understand their mode of interaction under the conditions tested, we determined their contribution via ¹³C metabolic flux ratio analysis of the Rs265:pBBR-MVA-ads and Rs265_∆phaB:pBBR-MVA-ads strains. We therefore compared the resulting amorphadiene/biomass ratios for each pathway with the ones determined for i) the Rs265:pBBR-ads and Rs265_∆phaB:pBBR-ads (MEP-only) strains, and ii) the Rs265-MVA_∆dxr:pBBR-MVA-ads and Rs265-MVA_∆dxr∆phaB:pBBR-MVA-ads (MVA-only) strains. Previously, this ¹³C-method provided important insights on the flux ratios upon co-expression of the MEP and MVA pathways (30). Under nitrogen excess condition, we observed that the dual-pathway strains with active MEP and MVA pathways showed a higher amorphadiene/biomass ratio for each isoprenoid route compared to when these were active individually (Fig. 3A). We therefore confirmed that, during nitrogen excess conditions, co-expression of the two isoprenoid pathways resulted in enhancement of their capacities in the Rs265 and Rs265_∆phaB strains harbouring the pBBR-MVA-ads plasmid (Fig. 3A, Table 4). Moreover, the capacity of the MVA pathway in the Rs265_∆phaB and Rs265-MVA_∆dxr∆phaB strains was even further enhanced by the phaB deletion, as made obvious by comparison to strains that still contain the phaB gene (Fig. 3A, Table 4). In contrast, the flux through the native MEP pathway remained unaffected by the phaB deletion. Thus, under nitrogen excess conditions, deletion of the phaB gene results in an increase of the isoprenoid flux exclusively via the MVA pathway (Table 4).

We further studied the isoprenoid flux ratio under nitrogen-limited conditions (Fig. 3B). The Rs265:pBBR-ads and Rs265_∆phaB:pBBR-ads strains, which rely exclusively on the MEP pathway for isoprenoid production did not show any increase in the amorphadiene/biomass ratio when compared to nitrogen excess conditions (Fig. 3B). In contrast, the amorphadiene/biomass ratio for the strains that express only the MVA pathway was increased 3-fold when compared to this value under nitrogen excess conditions (Rs265-MVA_∆dxr and Rs265-MVA_∆dxr∆phaB strains harbouring pBBR-MVA-ads plasmid, Fig. 3). Interestingly, for the strains that express both pathways (Rs265_:pBBR-MVA-ads and Rs265_∆phaB:pBBR-MVA-ads strains, Fig. 3B), there was only a minor increase of the MVA pathway capacity in the Rs265_∆phaB strain when compared to nitrogen excess conditions (Table 4). On the other hand, the MEP pathway capacity increased by 80% for the Rs265 strain (from 9.4 ± 0.3 to 16.9 ± 0.4 mg amorphadiene ∙ g of biomass^-1) and by 300% for the Rs265_∆phaB strain (from 9.2 ± 1.3 to 28.7 ± 1.3 mg amorphadiene ∙ g of biomass^-1). Thus, for the dual-pathway strain, the increase of the amorphadiene/biomass ratio under nitrogen limitation conditions is attributed to the endogenous MEP pathway (Table 4).

Amorphadiene biosynthesis during resting cells conditions

Under nitrogen-limited conditions a short exponential growth phase occurred, and therefore a short growth-associated amorphadiene production phase could not be avoided. This resulted in non-linear growth and production kinetics, making it difficult to assess yields (mg amorphadiene ∙ glucose^-1) and productivities (mg amorphadiene ∙ L^-1 ∙ h^-1). In order to focus exclusively on growth-uncoupled production, and to obtain linear kinetics, we decided to assess amorphadiene production during resting cell conditions in nitrogen-free medium. This cultivation setup simulates the production phase of a two-stage fermentation setup where growth and production are separated.

Since deletion of phaB and expression of the MVA pathway increased production during nitrogen limitation, we reasoned to assess the amorphadiene production levels in the presence of also an active MEP pathway. Therefore, we further cultivated the strains Rs265, Rs265_∆phaB, Rs265-MVA_∆dxr and Rs265-MVA_∆dxr∆phaB under resting cells condition. All these strains contained the pBBR-MVA-ads plasmid (Fig. 4A).

A linear increase was observed in the OD₆₀₀ of the strains with a functional PHB biosynthetic pathway (Rs265 and Rs265-MVA_∆dxr, Fig. 4B). This trend is known to be associated with the accumulation of this storage compound (29), and it is associated with cell expansion rather than cell division. Accordingly, the corresponding ∆phaB strains (Rs265_∆phaB and Rs265-MVA_∆dxr∆phaB) did not show any increase in OD₆₀₀. Glucose consumption (Fig. 4C, Additional file 1: Fig. S2), pH and amorphadiene concentrations (Fig. 4D, E) were followed over time. A decrease in the pH of the Rs265_∆phaB and Rs265-MVA_∆dxr∆phaB strains was observed (Fig. 4D), which can be explained by the secretion of organic acids upon prevention of PHB accumulation, mainly of pyruvate and 2-oxoglutarate (Fig. 4F).

Amorphadiene samples were collected over time from all the cultures (Fig. 4E), and yields and productivities were calculated for the first 24 h (Fig. 4G-I). The corresponding values for the Rs265 strain were the lowest among the tested strains. Deletion of phaB (Rs265_∆phaB) resulted in a 2-fold increase of the amorphadiene/biomass ratio (Fig. 4G, Additional File 1: Table S2), the volumetric productivity (Fig. 4H) and the yield on glucose (Fig. 4I) compared to the Rs265 strain. Also, inactivation of the endogenous MEP pathway in the Rs265-MVA_∆dxr strain resulted in an increase of those values compared to Rs265 strain (Fig. 4H, I). Hence, inactivation of either the PHB production pathway or the endogenous MEP pathway stimulates growth-independent production. Combined inactivation of the MEP and PHB production pathways (Rs265-MVA_∆dxr∆phaB strain) allowed this strain to reach the highest amorphadiene/biomass ratio, volumetric amorphadiene productivity (Fig. 4H) and yield on glucose (Fig. 4I). All these values were 2,5-fold higher in the Rs265-MVA_∆dxr∆phaB strain, compared to the Rs265 control strain. Hence, deletion of the endogenous MEP and PHB biosynthetic pathways resulted in the best metabolic setup for exploiting non-growing conditions for amorphadiene production.

Strain optimization for improved bioproduction often relies on strategies that couple production to microbial growth (19,37). Nevertheless, an emerging approach for metabolic engineering strategies is the one of dissociating production and growth (26). Following this view, in this work we engineered the isoprenoid and the PHB biosynthetic pathways in R. sphaeroides. Therefore, we could demonstrate that isoprenoid biosynthesis, a typically growth-coupled type of metabolism in microorganisms, can be uncoupled from biomass production by means of rational metabolic engineering.

A previous work indicated that isoprenoid synthesis is strictly growth-coupled via the endogenous MEP pathway (29). In the same study, the authors reported the first TRY parameters for this species, on a defined medium. They demonstrated that cultivation conditions can affect TRY values. By co-expressing both isoprenoid pathways, the yield of amorphadiene on glucose was highest under micro-aerobic conditions, reaching a value of about 0.005 mol ∙ mol glucose^-1(29). This value is still considerably low compared to the maximum theoretical yield that could be obtained from glucose (0.285 mol ∙ mol glucose^-1). Moreover, the maximal amorphadiene productivity was attained during exponential growth and reached 2 mg ∙ L^-1 ∙ h^-1. We applied nitrogen-limited conditions to a strain relying only on this isoprenoid pathway, but this did not result in increased amorphadiene/biomass ratio (Fig. 2B). We reasoned that targeting the storage compound (PHB) synthesis could increase the flux via the MEP pathway during this condition. We attempted to enhance the isoprenoid flux via this pathway because it presents the highest theoretical yield on glucose. Nevertheless, inactivation of the PHB synthetic pathway did not result in any improvement (Fig. 2B), thereby indicating that the endogenous MEP pathway is inhibited during non-growing conditions.

Differently from the MEP pathway, the MVA pathway is non-native in R. sphaeroides. Therefore, albeit its lower theoretical yield, this heterologous pathway is expected to be less affected by endogenous regulation. Introduction of a heterologous MVA pathway was described to allow isoprenoid synthesis also during non-growing conditions (29). Here, we confirmed that nitrogen limited conditions increased the amorphadiene/biomass ratio in a strain relying exclusively on the MVA pathway (Fig. 2D). Moreover, inactivation of PHB synthesis by targeting phaB increased amorphadiene production when the MVA pathway was present (Fig. 2C, D). Because the current production of isoprenoids in R. sphaeroides is far from the maximum theoretical yield, the beneficial effect of MVA pathway expression on the overall isoprenoid flux compensated its lower theoretical yield. In order to calculate titers, rates and yields (TRY) of growth-independent amorphadiene synthesis, we performed cultivation under resting cells condition (Fig. 4). The experimental data confirmed that exclusive isoprenoid flux via the MVA pathway combined with inactivation of PHB synthesis result in maximal TRY values (Fig. 4E, G-I).

¹³C metabolic flux ratio analysis was performed to understand the interaction between the two isoprenoid pathways during nitrogen limitation. The analysis indicated that in the dual-pathway strain the endogenous MEP pathway capacity is substantially enhanced during nitrogen limitation (Fig. 3B). Therefore, presence of the MVA pathway helps in deregulating the endogenous MEP pathway during nitrogen limitation.

Despite the increase in flux, only a small part of the carbon that originally went to PHB production could be redirected to amorphadiene. Organic acids - especially pyruvate and 2-oxoglutarate – were excreted instead. The accumulation of these two organic acids indicates that their downstream reactions, catalysed by the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes respectively, were inhibited by the inability to produce PHB. These reactions require input of free CoA (Fig. 1), the co-enzyme released by the conversion of AA-CoA into PHB. We therefore speculate that knocking out either phaB or phaC1 and phaC2 decreased the availability of free CoA, that the capacity of the mevalonate pathway was insufficient to remedy this, and that this low CoA availability resulted in the accumulation of both pyruvate and 2-oxoglutarate. Possibly, secretion of 3HB after deletion of phaC1 and phaC2 allowed to release free CoA from the intermediate 3HB-CoA.

PHB synthesis results in (38): i) carbon storage, ii) regeneration of NADP⁺ and iii) regeneration of free CoA for cellular homeostasis. Knocking out the ability to produce PHB should therefore increase the availability of precursors (AA-CoA) and NADPH for the MVA pathway. The relatively small increase in flux through the MVA pathway indicated that this pathway benefited from the increased amounts of AA-CoA and NADPH, but its capacity is limiting isoprenoid production. One of the rate-determining enzymes of the heterologous pathway is HMG-CoA reductase, already described as crucial enzyme for enhancing isoprenoid flux (15,39). We speculate that improving the catalytic efficiency of this enzyme might allow the MVA pathway to benefit from the higher availability of NADPH and AA-CoA. Such an improvement could allow to increase carbon flux towards isoprenoids, while reducing by-products secretion. Therefore, rational engineering strategies can build upon the findings of this work for further improving growth-independent isoprenoid biosynthesis in R. sphaeroides.

In this work, we assessed the contribution of the MEP and MVA pathway to amorphadiene biosynthesis under different culturing conditions. We confirmed that application of the heterologous MVA pathway holds potential for growth-independent production. In a dual-pathway strain, enhancement of the endogenous MEP pathway capacity was confirmed during nitrogen-limited conditions via ¹³C metabolic flux ratio analysis.

Nevertheless, isoprenoid synthesis during resting cells condition was limited by the presence of an active endogenous MEP pathway. On the other hand, exclusive isoprenoid flux via the MVA pathway increased amorphadiene synthesis during this condition. Additionally, prevention of PHB synthesis via phaB resulted in the highest TRY values for growth-independent amorphadiene production.

Ultimately, this work proposed a metabolic engineering design for increasing growth-independent isoprenoid biosynthesis in R. sphaeroides, while providing novel insights about the interaction occurring between the two isoprenoid pathways.

Availability of data and material

The dataset supporting the conclusions of this article are included within the article and in the additional file.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Competing interests

Not applicable

Funding

This project was financially supported by The Netherlands Ministry of Economic Affairs, via a public–private NWO‑Green Foundation for sustainable production and supply chains in agriculture and horticulture (870.15.130,2015/05279/ALW). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, grant agreement No. 834279.

Authors' contributions

EO, IM, SWMK and RAW designed the work. EO, IM, WP JB and MD conducted, analyzed, and interpreted the experiments. EO and IM drafted and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Isobionics BV for providing the Rhodobacter sphaeroides Rs265 strain used in this study. Any request for the strain and its derivatives should be directed to Isobionics BV. Moreover, we thank Rosyida Arifah for technical assistance in generating the Rs265-MVA_∆dxr∆phaB strain, and Frank E. Fluitman for providing the Rs265_∆phaC1∆phaC2 strain.

PHB: poly-β-hydroxybutyrate;

MEP: 2-methyl-D-erythritol 4-phosphate;

MVA: mevalonate;

CRISPR-Cas: Clustered Regularly Inter‑ spaced Short Palindromic Repeats‑CRISPR associated proteins;

PYR: pyruvate;

CoA: coenzyme-A;

NADPH: dihydronicotinamide-adenine dinucleotide phosphate;

Ac-CoA: acetyl-CoA;

AA-CoA: acetoacetyl-CoA;

HMG-CoA: hydroxymethylglutaryl-CoA;

3-HB-CoA: 3-hydroxybutyryl-CoA;

3HB: 3-hydroxybutyrate;

2-OXO: 2-oxoglutarate;

phaB: acetoacetylreductase (gene);

dxr: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (gene);

ads: amorphadiene synthase (gene);

Ajikumar PK, Tyo K, Carlsen S, Mucha O, Phon TH, Stephanopoulos G. Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol Pharm. 2008;5(2):167–90.
Schempp FM, Drummond L, Buchhaupt M, Schrader J. Microbial cell factories for the production of terpenoid flavor and fragrance compounds. J Agric Food Chem. 2018;66(10):2247–58.
Park SY, Yang D, Ha SH, Lee SY. Metabolic engineering of microorganisms for the production of natural compounds. Vol. 2, Advanced Biosystems. 2018. p. 1–41.
Kirby J, Keasling JD. Biosynthesis of plant isoprenoids: perspectives for microbial engineering. Annu Rev Plant Biol. 2009;60(1):335–55.
Mewalal R, Rai DK, Kainer D, Chen F, Kulheim C, Peter GF, et al. Plant-derived terpenes: a feedstock for specialty biofuels. Trends Biotechnol. 2016;35(3):227–40.
Niu FX, Lu Q, Bu YF, Liu JZ. Metabolic engineering for the microbial production of isoprenoids: carotenoids and isoprenoid-based biofuels. Synth Syst Biotechnol. 2017;2(3):167–75.
Kuzuyama T, Seto H. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis. Proc Japan Acad Ser B. 2012;88(3):41–52.
Lombard J, Moreira D. Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of life. Mol Biol Evol. 2011;28(1):87–99.
Vranová E, Coman D, Gruissem W. Network analysis of the MVA and MEP pathways for isoprenoid synthesis. Annu Rev Plant Biol. 2013 Apr 29;64(1):665–700.
Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol. 2003;21(7):796–802.
Yuan LZ, Rouvière PE, LaRossa RA, Suh W. Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in Escherichia coli. Metab Eng. 2006;8(1):79–90.
Ajikumar PK, Xiao WH, Tyo KEJ, Wang Y, Simeon F, Leonard E, et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science (80- ). 2010 Oct 1;330(6000):70–4.
Li Q, Fan F, Gao X, Yang C, Bi C, Tang J, et al. Balanced activation of IspG and IspH to eliminate MEP intermediate accumulation and improve isoprenoids production in Escherichia coli. Metab Eng. 2017;44(July 2016):13–21.
Anthony JR, Anthony LC, Nowroozi F, Kwon G, Newman JD, Keasling JD. Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4, 11-diene. Metab Eng. 2009;11(1):13–9.
Pitera DJ, Paddon CJ, Newman JD, Keasling JD. Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab Eng. 2007;9(2):193–207.
Orsi E, Beekwlider J, van Gelder D, van Houwelingen A, Eggink G, Kengen SWM, et al. Functional replacement of isoprenoid biosynthetic pathways in Rhodobacter sphaeroides. Microb Biotechnol. 2020;13(4):1082–93.
Kannenberg EL, Poralla K. Hopanoid biosynthesis and function in bacteria. Naturwissenschaften. 1999;86(4):168–76.
Volkman JK. Sterols in microorganisms. Appl Microbiol Biotechnol. 2003;60(5):495–506.
Von Kamp A, Klamt S. Growth-coupled overproduction is feasible for almost all metabolites in five major production organisms. Nat Commun. 2017;8(May):1–10.
Reyes LH, Gomez JM, Kao KC. Improving carotenoids production in yeast via adaptive laboratory evolution. Metab Eng. 2014;21:26–33.
Klamt S, Mahadevan R, Hädicke O. When do two-stage processes outperform one-stage processes? Biotechnol J. 2018;13(2).
Willrodt C, Hoschek A, Bühler B, Schmid A, Julsing MK. Decoupling production from growth by magnesium sulfate limitation boosts de novo limonene production. Biotechnol Bioeng. 2016;113(6):1305–14.
Tsuruta H, Paddon CJ, Eng D, Lenihan JR, Horning T, Anthony LC, et al. High-level production of amorpha-4, 11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli. PLoS One. 2009;4(2):e4489.
Li S, Jendresen CB, Nielsen AT. Increasing production yield of tyrosine and mevalonate through inhibition of biomass formation. Process Biochem. 2016;51(12):1992–2000.
Zhang L, Liu L, Wang KF, Xu L, Zhou L, Wang W, et al. Phosphate limitation increases coenzyme Q10 production in industrial Rhodobacter sphaeroides HY01. Synth Syst Biotechnol. 2019;4(4):212–9.
Pandit AV, Srinivasan S, Mahadevan R. Redesigning metabolism based on orthogonality principles. Nat Commun. 2017;8(May):1–11.
Su A, Chi S, Li Y, Tan S, Qiang S, Chen Z, et al. Metabolic redesign of Rhodobacter sphaeroides for lycopene production. J Agric Food Chem. 2018;66:5879–85.
Beekwilder J, van Houwelingen A, Cankar K, van Dijk ADJ, de Jong RM, Stoopen G, et al. Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene. Plant Biotechnol J. 2014;12(2):174–82.
Orsi E, Folch PL, Monje-López VT, Fernhout BM, Turcato A, Kengen SWM, et al. Characterization of heterotrophic growth and sesquiterpene production by Rhodobacter sphaeroides on a defined medium. J Ind Microbiol Biotechnol. 2019;46(8):1179–90.
Orsi E, Beekwilder J, Peek S, Eggink G, Kengen SWM, Weusthuis RA. Metabolic flux ratio analysis by parallel 13C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides. Metab Eng. 2020;57:228–38.
Mougiakos I, Orsi E, Ghiffari MR, De Maria A, Post W, Adiego-Perez B, et al. Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering. Microb Cell Fact. 2019;18(204):1–13.
Kim M, Baek J, Lee JK. Comparison of H₂ accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase deficient mutant. Int J Hydrogen Energy. 2006;31:121–7.
Ryu M-H, Hull NC, Gomelsky M. Metabolic engineering of Rhodobacter sphaeroides for improved hydrogen production. Int J Hydrogen Energy. 2014 Apr;39(12):6384–90.
Shimizu T, Teramoto H, Inui M. Introduction of glyoxylate bypass increases hydrogen gas yield from acetate and L-glutamate in Rhodobacter sphaeroides. Appl Environ Microbiol. 2019;85(2):1–17.
Lee IH, Park J, Kho D, Kim MS, Lee J. Reductive effect of H₂ uptake and poly-β-hydroxybutyrate formation on nitrogenase-mediated H₂ accumulation of Rhodobacter sphaeroides according to light intensity. Appl Microbiol Biotechnol. 2002;60(1–2):147–53.
Yang C, Gao X, Jiang Y, Sun B, Gao F, Yang S. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli. Metab Eng. 2016 Sep;37:79–91.
Mans R, Daran JMG, Pronk JT. Under pressure: evolutionary engineering of yeast strains for improved performance in fuels and chemicals production. Curr Opin Biotechnol. 2018;50:47–56.
Leaf TA, Srienc F. Metabolic modeling of polyhydroxybutyrate biosynthesis. Biotechnol Bioeng. 1998;57(5):557–70.
Sasaki Y, Eng T, Herbert RA, Trinh J, Chen Y, Rodriguez A, et al. Engineering Corynebacterium glutamicum to produce the biogasoline isopentenol from plant biomass hydrolysates. Biotechnol Biofuels. 2019;12(1):41.

Table 1. Strains used in this study.

Strain	Description	Source of reference
E. coli
S17-1	Host strain for conjugation, thi pro recA hsdR [RP4‑2Tc::Mu‑Km::Tn7] Tp^r Sm^r	Laboratory stock
R. sphaeroides
Rs265	wild-type	Derivative of ATCC35035, Isobionics BV
Rs265_∆phaC1∆phaC2	Rs265 with the PHB biosynthetic pathway knocked-out via phaC1 and phaC2 deletion	Isobionics BV
Rs265_∆phaB	Rs265 with the PHB biosynthetic pathway knocked-out via phaB deletion	(31)
Rs265-MVA_∆dxr	Rs265 with chromosomally integrated the MVA pathway operon and knocked-out the endogenous MEP pahtway via dxr deletion	(16)
Rs265-MVA_∆dxr∆phaB	Rs265 with chromosomally integrated the MVA pathway operon and knocked-out the endogenous MEP (via dxr deletion) and PHB (via phaB deletion) biosynthetic pathways	This study

Table 2. Plasmids used in this study.

Plasmid	Description	Source of reference
pBBR-ads	pBBR1MCS-2 + crtE promoter and ads (amorphadiene synthase)	(29)
pBBR-MVA-ads	pBBR1MCS-2 + crtE promoter controlling MVA enzymes and ads	(28)
pBBR_ Cas9_ΔphaB_HR	pBBR1MCS-2 + codon harmonized cas9 sequence, sgRNA targeting phaB and 1kb homologous-recombination flanks for recombination with phaB	(31)

Table 3. Titers and yields obtained from shaking flasks experiments of strains Rs265 and Rs265-MVA. Data was obtained from end-point measurements under nitrogen excess and nitrogen limited conditions.

Plasmid	Condition	Genomic background	Amorphadiene titers, *C_P* (mg ∙ L^-1)		Amorphadiene yield on glucose, *Y_P/S* (mg ∙ g^-1)		Final Biomass concentration, *C_X* (g ∙ L^-1)		*Amorphadiene on Biomass, Y_P/X* (mg ∙ g^-1)**
Plasmid	Condition	Genomic background	value	st. dev.	value	st. dev.	value	st. dev.	value	st. dev.
pBBR-ads	Nitrogen excess	wt	4.4	0.3	1.41	0.15	1.533	0.062	2.9	0.2
		ΔphaC1 ΔphaC2	3.3	0.1	N.D.	N.D.	1.491	0.000	2.2	0.1
		ΔphaB	4.7	0.2	1.49	0.07	1.724	0.110	2.7	0.1
	Nitrogen limited	wt	2.4	0.2	0.73	0.05	0.805	0.122	2.9	0.2
		ΔphaC1 ΔphaC2	1.4	0.2	N.D.	N.D.	0.693	0.034	2.0	0.3
		ΔphaB	2.2	0.2	0.69	0.04	0.693	0.112	3.2	0.2
pBBR-MVA-ads	Nitrogen excess	wt	41.8	1.8	13.64	0.56	1.289	0.019	32.4	1.4
		ΔphaC1 ΔphaC2	39.3	4.4	N.D.	N.D.	1.060	0.856	37.1	4.2
		ΔphaB	56.4	2.1	18.25	0.68	1.466	0.036	38.5	1.5
	Nitrogen limited	wt	23.6	2.3	7.65	0.76	0.656	0.115	35.9	3.6
		ΔphaC1 ΔphaC2	24.1	2.1	N.D.	N.D.	0.539	0.260	44.8	3.9
		ΔphaB	34.4	2.2	11.38	0.73	0.539	0.022	63.7	4.0
pBBR-MVA-ads	Nitrogen excess	MVAint_Δdxr	15.6	3.9	3.41	0.23	2.229	0.144	7.0	1.7
	Nitrogen excess	MVAint_ΔdxrΔphaB	28.0	1.0	9.67	0.86	2.187	0.080	12.8	0.5
	Nitrogen limited	MVAint_Δdxr	13.5	1.9	3.80	0.82	0.597	0.142	22.6	3.1
	Nitrogen limited	MVAint_ΔdxrΔphaB	19.5	0.8	5.65	0.29	0.528	0.161	36.9	1.6

N.D.: not determined.

Table 4. Isoprenoid flux ratio calculated for the Rs265 and Rs265_∆phaB strains harboring the pBBR-MVA-ads plasmid after growth on different initial nitrogen concentrations.

	Isoprenoid flux ratios				Pathway capacities (mg ∙ g biomass^-1)
	Nitrogen excess		Nitrogen limitation		Nitrogen excess		Nitrogen limitation
Genomic background	MEP	MVA	MEP	MVA	MEP	MVA	MEP	MVA
Rs265	0.29 ± 0.01	0.71 ± 0.01	0.46 ± 0.01	0.54 ± 0.1	9.4 ± 0.3	23.0 ± 0.3	16.9 ± 0.4	19.9 ± 0.5
Rs265_∆phaB	0.24 ± 0.03	0.76 ± 0.03	0.45 ± 0.02	0.55 ± 0.03	9.2 ± 1.3	29.3 ± 1.3	28.7 ± 1.3	35.0 ± 1.9

Orsietal.2020RevAdditionalfile1.docx

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Status:

Journal Publication

Version 2

Abstract

Figures

Background

Materials And Methods

Strains and standard cultivation conditions

Generation of double-KO strain via CRISPR-Cas9 counter-selection

Diparental conjugation of R. sphaeroides

Cultivation in nitrogen excess and nitrogen limited conditions

¹³C-metabolic flux ratio analysis of isoprenoid biosynthesis

Cultivation under resting cell conditions

Analytics

Results

Prevention of PHB formation and its effect on amorphadiene biosynthesis

Organic acids secretion as consequence of PHB deletion

¹³C metabolic flux ratio analysis of isoprenoid biosynthesis under different growth conditions

Amorphadiene biosynthesis during resting cells conditions

Discussion

Conclusions

Declarations

Availability of data and material

Consent for publication

Ethics approval and consent to participate

Competing interests

Funding

Authors' contributions

Acknowledgements

Abbreviations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 2

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Status:

Journal Publication

Version 2

Abstract

Figures

Background

Materials And Methods

Strains and standard cultivation conditions

Generation of double-KO strain via CRISPR-Cas9 counter-selection

Diparental conjugation of R. sphaeroides

Cultivation in nitrogen excess and nitrogen limited conditions

13C-metabolic flux ratio analysis of isoprenoid biosynthesis

Cultivation under resting cell conditions

Analytics

Results

Prevention of PHB formation and its effect on amorphadiene biosynthesis

Organic acids secretion as consequence of PHB deletion

13C metabolic flux ratio analysis of isoprenoid biosynthesis under different growth conditions

Amorphadiene biosynthesis during resting cells conditions

Discussion

Conclusions

Declarations

Availability of data and material

Consent for publication

Ethics approval and consent to participate

Competing interests

Funding

Authors' contributions

Acknowledgements

Abbreviations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 2

¹³C-metabolic flux ratio analysis of isoprenoid biosynthesis

¹³C metabolic flux ratio analysis of isoprenoid biosynthesis under different growth conditions