Skip to main content
Download PDF

Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase

Microbial Cell Factories volume 23, Article number: 227 (2024) Cite this article

Abstract

Background

The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer–Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L−1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.

Results

Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer–Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW−1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW− 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.

Conclusions

Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

Highlights

  • Heterologous co-expression of an invertase gene improved the capacity of E. coli to utilize sucrose as an electron donor for whole-cell biotransformations.

  • The invertase improved the efficiency of the Baeyer–Villiger oxidation of cyclohexanone catalyzed by a Baeyer–Villiger Monooxygenase both in the laboratory strain, E. coli BL21 (DE3) and in E. coli W ΔcscR, an E. coli strain bearing a deregulated sucrose operon.

  • Periplasmic production of invertase for both hosts was prominent at a sucrose concentration of 10 mM.

  • E. coli strains with invertase exported to the periplasm showed full conversion of 5 mM cyclohexanone within less than three hours using photosynthetically generated sucrose from Synechocystis S02.

Introduction

Oxidoreductases comprise a large number of enzymes catalysing oxidation and reduction reactions. Due to their high efficiency and selectivity, these enzymes find wide application in biocatalysis for pharmaceutical, fine chemical, polymer synthesis, hydrocarbon oxyfunctionalization, pollutant degradation and biosensor construction [1]. During the catalytic reaction, external compounds such as nicotinamide cofactors [NAD(P)H], ferredoxin, or adenosine triphosphate (ATP) are required in stoichiometric amounts [2]. Due to the high cost of these cofactors, their recycling by enzyme cascade reactions or through cellular metabolism is critical for economic viability [3,4,5]. Various chemical, electrochemical, microbial, and enzymatic methods have been developed for cofactor regeneration [2, 6]. Indeed, several metabolic engineering strategies were employed to increase NAPDH generation to improve productivity and titers in E. coli [7]. Three pathways play a major role in NADPH generation in E. coli namely the oxidative part of the pentose phosphate pathway, the NAD(P)+-dependent isocitrate dehydrogenase of the tricarboxylic acid cycle, and the transhydrogenases system [8, 9]. In whole-cell biotransformation reactions, the metabolism of microorganisms (usually heterotrophic) is used for the regeneration of redox cofactors at the expense of glucose or other carbon-rich compounds [10]. However, only a portion of the electron pairs are utilized for cofactor regeneration when glucose dehydrogenase is used [11]. In addition, the heterotrophic metabolism will necessarily dedicate a considerable part of the electrons to respiration and biomass accumulation, which results in a poor atom economy of cofactor-regeneration. Hence, cofactor regeneration using glucose results in poor atom economy [12, 13].

Interestingly, sucrose can be produced by cyanobacteria via photosynthesis which could alleviate this problem. Cyanobacteria are prokaryotes capable of utilizing CO2, water and basic minerals for biomass accumulation through the process of photosynthesis powered by light energy. More than 60 cyanobacterial strains accumulate sucrose as an osmoprotective compound under high salt concentrations [14, 15]. The mechanism of sucrose accumulation was exploited for the engineering of cyanobacterial strains that secrete sucrose in the range of several gram per liter. This was achieved through the expression of the sucrose regulon and by deleting competing catabolic pathways [16,17,18]. The feasibility of coupling microbial production strains to photosynthetic sucrose synthesis was already demonstrated in the production of L-threonine, gluconic acid and sorbitol by E. coli, Zymomonas mobilis and Synechocystis sp. PCC 6803, respectively [19, 20].

Cyanobacterial sucrose production presents itself as a promising option to supply electrons for the production of large-scale whole-cell biotransformations, where the low atom economy associated with the use of sacrificial electron donors is a severe problem. In this context, an industrially-relevant and important organic reaction is the Baeyer–Villiger (BV) oxidation of cyclic ketones producing lactones catalysed by oxidants such as peracids and hydrogen peroxide [21]. Due to the toxicity, instability, and lack of enantioselectivity of these catalysts, enzymatic BV oxidation has been increasingly in focus as an alternative in the last decades. Baeyer Villiger Monooxygenases (BVMOs) are flavin-dependent enzymes catalysing the oxidative transformations of ketones to their corresponding lactones and the oxidation of sulphides [22]. In the majority of BVMOs, classified as type I, molecular oxygen reacts with reduced flavin adenine nucleotide (FAD) in the active site of the enzyme; FAD is in turn regenerated by NADPH resulting in the product and water as the byproduct [23]. A kilogram-scale production of a caprolactone derivative, trimethyl-ε-caprolactone from 3,3,5-trimethyl cyclohexanone was reported by expressing a cyclohexanone monooxygenase (CHMO) from T. municipal in E. coli [24]. A higher amount of glucose relative to the final product was utilized to regenerate the cofactor contributing to higher process costs. Similarly, glycerol was also used in a kilogram-scale synthesis of a lactone relying on another CHMO [25]. Their NADPH dependency and the high relevance of the reactions they catalyse make BVMOs interesting model enzymes for the investigation of sucrose-mediated cofactor regeneration systems [26].

While cyanobacterial sucrose supply would be a suitable alternative instead of glucose for cofactor-recycling, not all industrial E. coli strains can metabolize sucrose. One particular strain that retained a csc (sucrose catabolism) regulon is E. coli W, which allows growth up to 2% sucrose (20 g L−1) [27]. By deleting the cscR (sucrose operon repressor) gene, E. coli W effectively utilized sucrose below 6 mM [28]. The efficient uptake of sucrose in E. coli W via active transport was made possible by a cscB permease, embedded in the cell membrane whereas the outer membrane is generally thought to be permeable to sucrose in E. coli strains and other gram-negative bacteria [29, 30]. However, solely the expression of cscA gene confers the capacity to grow on sucrose to E. coli strains which normally do not metabolize sucrose readily [31,32,33]. Recently, a proof of concept for whole-cell biotransformation using an E. coli W ΔcscR strain harboring a BVMO utilizing photosynthetically-produced sucrose from Synechocystis S02 was demonstrated [26]. Entrapment of Synechocystis S02 in alginate hydrogel beads resulted in ~ 9 mM (3 g L−1) of cumulative sucrose yield after 17 days in a semi-continuous system, which was then utilized in whole-cell biotransformation by E. coli W ΔcscR overexpressing an invertase gene with complete conversions and an average transformation rate of 0.9 mM h−1.

While the utilization of sucrose for whole-cell redox biotransformations can be improved by the overexpression of the invertase gene, several questions remain. In particular, it is unclear whether the permease encoded in the sucrose regulon is essential, or whether simple invertase expression can boost sucrose utilization. The latter would make it easier to use laboratory strains for sucrose-driven biotransformations. The use of highly optimized laboratory strains would be particularly beneficial for difficult-to-express proteins. Herein, we report an analysis of key factors for efficient sucrose utilization of E. coli W ΔcscR strain co-expressing invertase and a BVMO with a fast reaction rate originating from Burkholderia xenovorans (BVMOXeno) for the whole-cell biotransformation of cyclohexanone 1a to the polymer precursor, ε-caprolactone 1b (Fig. 1) [34]. Furthermore, we report that the expression of the invertase gene is a simple strategy to induce sucrose metabolism in a widely-used laboratory strain such as E. coli BL21 (DE3) lacking the csc regulon. Finally, the applicability of both systems was demonstrated in the utilization of photosynthetically-derived sucrose produced by the cyanobacterium Synechocystis S02 for sustainable production of platform chemicals.

Fig. 1
figure 1

Schematic representation of sucrose utilization for cofactor regeneration in whole cell oxyfunctionalization when cscA hydrolyses sucrose in the periplasm. A synthetic operon was constructed containing a BVMO and a cscA fused with a pelB signal sequence. Both genes were controlled by a T5 promoter and cloned in a pQE-30 plasmid. The gene was heterologously expressed in a) E. coli W mutant with a cscR deletion (ΔcscR) and b) a standard E. coli strain BL21 (DE3). E. coli W ΔcscR carries the csc regulon encoded in the genome with a deletion of the cscR repressor to enable low sucrose utilization (< 6 mM) whereby E. coli BL21 (DE3) entirely lacks the csc regulon. The design of the synthetic operon can be found in Figure S1. [Abbreviations: CCM—central carbon metabolism; cscR—repressor, cscA—invertase, cscK—fructokinase, cscB—sucrose permease; pelB—signal sequence for the periplasmic export]. Image created using BioRender

Full size image

Materials and methods

Chemicals

All chemicals were purchased from Sigma-Aldrich (Steinheim, Germany) or Carl Roth (Karlsruhe, Germany) unless otherwise indicated.

Strain and plasmid construction

A synthetic operon containing invertase (cscA, accession: AF084030.1) fused with a pelB leader sequence and a BVMO gene from Burkholderia xenovorans (BVMOXeno, UniProt: Q13I90) was designed and cloned into a pQE-30 vector using Gibson assembly. Both genes were controlled by a T5 bacteriophage promoter (Figure S1). As E. coli W does not contain a gene encoding the T7 polymerase, a T5 promoter was utilized for gene expression. This construct was used as a template for the other two cloning strategies employed in this work, i.e. (i) the construct entirely lacking cscA and (ii) the one carrying the cscA without the pelB signal sequence for the cytosolic gene expression. The vector was linearized using PCR and overhangs were introduced to the respected inserts via primers. E. coli TOP10 strain was used for plasmid propagation and selected mutants were screened using colony PCR with a DreamTaq Green PCR Master Mix (Thermo Scientific: K1081). Both E. coli W ΔcscR and E. coli BL21 (DE3) were transformed with (a) pQE-30 vector encoding BVMOXeno::pelB_cscA cassette for the periplasmic export of cscA, (b) control vector expressing solely BVMOXeno and (c) expressing both BVMO and cscA into the cytosol (BVMOXeno::cscA). Table 1 shows the strains and constructs utilized in this study.

Table 1 Bacterial strains and plasmids utilized in this study
Full size table

Bacterial strains and growth conditions on LB medium

The bacterial strains and plasmids used in this study are listed in Table S1. The E. coli W mutant with a cscR gene deletion (hereafter E. coli W ΔcscR) was provided by Professor Claudia Vickers from the University of Queensland, Australia. Seed cultures were prepared either from glycerol stocks or fresh plates and incubated overnight at 37 °C in liquid Lysogeny broth (LB) medium. Ampicillin (100 µg mL−1) was added to ensure selection pressure. The main culture was grown at 37 °C at 160 rpm until an optical density of 0.6–0.8 was reached.

Growth of E. coli strains on different sugar sources

To test the ability of E. coli W ΔcscR and E. coli BL21 (DE3) to grow on sucrose and glucose, seed cultures (4 mL) were prepared in a minimal medium supplemented with 11 mM glucose. Minimal medium was prepared as follows: 100 mL of 10X M9 salts, 1 mL trace element solution, 0.05 mg mL−1 of thiamine, 0.05 mg mL−1 biotin both sterile filtered, MgSO4 × 7H2O cfinal = 0.0081 mg mL−1 filled up to 1 L with sterile ddH2O. The main cultures were inoculated from the seed cultures to a final volume of 30 mL and OD600 = 0.1 using 100 mL baffled flasks. All components were autoclaved separately to avoid the precipitation of heavy salts. Glucose or sucrose (1 M prepared in ddH2O) was sterile-filtered before adding to the medium. The cells were left to grow at 37 °C, 120 rpm for 2 days. Control inoculum without sugar addition was also prepared in parallel.

Enzyme production and whole-cell biotransformation of 1a

Recombinant gene expression was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the cells reached an OD600 = 0.6–0.8 and further grown at 30 °C using the same shaking conditions. After 4–5 h of expression, the cells were centrifuged at 4 °C, 5000g for 30 min and washed with approximately 30 mL of Tris–HCl buffer (20 mM, pH 8.0). Once harvested, the cell suspension was centrifuged and finally resuspended in M9 salts, which were also used as a medium for whole-cell biotransformation. The cultures were adjusted to OD600 = 5 and pH 7.5 using NaOH in 300 mL baffled flasks (working volume = 30 mL). Sucrose (1 mM) or glucose (2 mM) was added for cofactor regeneration. The substrate 1a (5 mM, 1 M stock solution in EtOH) was added to the cells to initiate the reaction. Control reactions were performed without sugar addition. Reactions were performed in triplicates at 25 °C and 160 rpm. Aliquots (100 µL) of the reaction mixture were taken periodically, quenched in liquid nitrogen and stored at − 20 °C before GC-FID analysis.

Whole-cell biotransformations using sucrose produced by immobilized Synechocystis S02

Photosynthetically-produced sucrose from the cyanobacterium, Synechocystis S02 was utilized to sustain the biotransformation. The cyanobacterial cells were immobilized in alginate beads as described previously and sucrose was produced for 7 days [26]. Cells were then removed from the mixture and the sucrose-enriched BG11 medium was utilized for biotransformation. E. coli seed cultures were prepared overnight in LB medium supplemented with 100 µg mL−1 ampicillin. Main cultures were inoculated in TB medium and grown until an OD600 = 3 at 37 °C. After induction with 1 mM IPTG, the culture was left overnight at 25 °C for protein expression. Cells were harvested at OD600 = 8 and washed once with phosphate buffer and then resuspended in the sucrose-enriched BG11 medium containing 400 mM NaCl. The substrate 1a (5 mM) and IPTG (1 mM) were added and the biotransformation was conducted at 30 °C, 1% CO2 and 100 rpm as previously described. Sampling was done at time points 0, 1, 2, 3 and 24 h after substrate addition and samples were analyzed on GC-FID.

Analytics of target compounds using GC-FID and cell dry weight determination

The concentration of substrates and products was determined using Gas Chromatography with a Flame Ionization Detector (GC-FID, GC-2010 Plus Shimadzu, Japan) equipped with a ZB-5 column. Samples were extracted using 1:3 (v/v) dichloromethane containing 2 mM n-decanol as an internal standard. Samples were centrifuged at maximum speed for 10 min at 4 °C and the organic phase was dried using a spatula-tip of anhydrous MgSO4. A concentration range from 0 to 20 mM was prepared and extracted similarly to determine the concentrations for 1a and 1b. Table S2 shows the GC-FID conditions used in this study and Figure S2 the respective calibration curves with corresponding equations. For reactions utilizing photosynthetically-produced sucrose, compounds were analysed as previously reported [26].

For cell dry weight correlations, cells were grown as described above and the enzyme was produced for up to 5 h. Afterwards, they were washed three times with sterile double distilled water (ddH2O) and resuspended in 30 mL LB medium to mimic biotransformation conditions. The optical density was measured and the cells were centrifuged (4 °C, 5000 × g) for 20 min. The supernatant was discarded and cells were placed in a 60 °C incubator and weighed until no mass difference was observed. An OD600 = 1 correlates to 0.3 gDCW L−1 of cells.

Determination of specific enzyme activity

In whole-cell biotransformations, the specific enzyme activity is given as U gDCW−1, analogous to U mg−1 when using a purified enzyme. Thus, it was determined based on the product formation rate (mM h−1), which is a slope of the curve (time vs. product concentration) and reflects the initial rate, i.e. ≤ 10% conversion. Since U is given in µmol min− 1 the conversion of the mM h− 1 was done and divided by 1.5 gDCW L−1, which is the cell dry weight corresponding to the OD600 = 5 used to perform the whole-cell biotransformation.

Results and discussion

Growth of E. coli strains on sucrose and glucose

To ascertain the metabolism of the two E. coli strains using the sugars tested, their growth was observed for two days in a minimal medium. In particular, we compared the sucrose utilization by E. coli W ΔcscR and E. coli BL21 (DE3) as a representative for frequently applied E. coli laboratory strains. Using native E. coli W, a minimum sucrose concentration of 6 mM was determined wherein catabolism takes place without activating the cscR gene [27, 28]. Hence, we used this concentration to monitor the growth of E. coli W ΔcscR and E. coli BL21 (DE3). As shown in Fig. 2, glucose was the preferred substrate for both E. coli strains. Unsurprisingly, E. coli BL21 (DE3) was not able to utilize sucrose for growth due to the lack of a csc regulon confirmed by a BLAST search (Figure S3) [32, 33].

Fig. 2
figure 2

(1) Growth of (A) E. coli BL21 (DE3) and (B) E. coli W ΔcscR in the presence of glucose (11 mM), sucrose (6 mM) or no sugar addition in the M9 minimal medium. Reaction conditions: 30 mL volume in 100 mL baffled flasks, 37 °C, 120 rpm. (2) Whole-cell biotransformation of 1a using BVMOXeno constructs lacking the invertase in (C) E. coli BL21 (DE3) and (D) E. coli W ΔcscR. Reaction conditions: 30 mL, M9 salts as medium, 1.5 gDCW L−1, 5 mM 1a, 25 °C, 160 rpm, N = 3. All values represent data generated from three biological replicates. Error bars represent standard deviations. P-values were calculated using Welch’s t test (*P < 0.05)

Full size image

Consequently, the biotransformation using sucrose in E. coli BL21 (DE3) (Fig. 2C) did not show any appreciable product formation comparable to negative controls. On the other hand, E. coli W ΔcscR was able to grow using sucrose albeit with a slower growth rate compared to glucose. We also observed product formation with the aforementioned E. coli host using sucrose to regenerate cofactors (Fig. 2D).

Functional co-expression of invertase and BVMO in E. coli W ΔcscR

In order to analyse the utilization of sucrose in the specialized laboratory strain, we investigated the expression of the BVMOXeno gene and its co-expression with the invertase originating from E. coli W. Furthermore, we also compared the allocation of the invertase in the cytosol (cscA) or in the periplasm (pelb_cscA) (Table 1). Eventhough Lee and co-workers reported invertase activity [38] in the extracellular medium after intracellular expression, indicating its leakage across the cell membrane during cultivation [19], it was difficult to anticipate which system would be the most efficient for biotransformations. Successful production of BVMOXeno was achieved for all investigated constructs. A band at 62 kDa corresponding to BVMOXeno was observed in SDS-PAGE (Figure S4) [34]. However, the band for the invertase (cscA) at 54 kDa could not be detected, which was in agreement with previous findings from Lee et al. [19].

The strains were then investigated in the whole-cell oxidation of 1a as shown in Fig. 1. Biotransformations were performed in the presence of either glucose (2 mM) or sucrose (1 mM) and compared with control reactions without any sugar addition. With sucrose, E. coli BL21 (DE3) BVMOXeno (the strain without invertase) did not show any product formation (Fig. 2C). In comparison, the strain had a specific activity of 23 U gDCW−1 in the presence of glucose, which shows that BVMOXeno was produced in soluble and active form. These results underline that E. coli laboratory strains without further modification do not have the capacity to utilize sucrose as also shown in Fig. 2A. We then investigated E. coli W ΔcscR BVMOXeno, E. coli W ΔcscR BVMOXeno::cscA and E. coli W ΔcscR BVMOXeno::pelB_cscA to analyse whether cscA overexpression has an additional effect to the deregulated sucrose operon. All the constructs successfully produced the BVMO enzyme as shown in Figure S4.

E. coli W ΔcscR BVMOXeno showed good activity in whole-cell biotransformations driven by sucrose, indicating that the lack of the sucrose operon in BL21 (DE3) was indeed the most likely explanation for its poor sucrose utilization. The genome-encoded sucrose permease cscB and cscA invertase in E. coli W ΔcscR BVMOXeno appear to be responsible for the efficient utilization of sucrose as an electron source for whole-cell biotransformations [28].

Regarding the co-expression of the invertase gene in the E. coli W ΔcscR strain, both E. coli W ΔcscR BVMOXeno::cscA and E. coli W ΔcscR BVMOXeno::pelB_cscA showed comparable activity rates with glucose and with sucrose. The overall highest specific activity (44 U gDCW−1) was observed when glucose was added to the reaction mediated by E. coli W ΔcscR BVMOXeno::cscA (Fig. 3A). This is not surprising since glucose can be easily imported by the phosphotransferase system (PTS) and is the main substrate for glycolytic pathways, including the reduction of NADP+ in the oxidative part of the pentose phosphate pathway, which regenerates 2 molecules of NADPH per glucose molecule [36]. Without the addition of either glucose or sucrose, only negligible product formation was observed among all constructs indicating its role in cofactor regeneration.

Fig. 3
figure 3

A Whole-cell biotransformation of 1a mediated by different E. coli W ΔcscR constructs harboring BVMOXeno in the presence of various sugars and (B) Effect of different sucrose concentrations in the specific activity. Reaction conditions: 30 mL, M9 salts as medium, 1.5 gDCW L−1, 5 mM 1a, 25 °C, 160 rpm, N ≥ 3. Cofactor regeneration was ensured by adding 2 mM glucose or 1 mM sucrose. All bars represent data generated from reactions stemming from biological replicates with individual values depicted. Error bars represent standard deviations. P values were calculated using Welch’s t test (*P < 0.05)

Full size image

In the utilization of sucrose, the total activity of E. coli W ΔcscR BVMOXeno::cscA of 37 U gDCW−1 was the highest of all strains investigated in this study. The higher activity of E. coli W ΔcscR BVMOXeno::cscA and E. coli W ΔcscR BVMOXeno::pelB_cscA indicated that overexpression of the invertase gene is also beneficial for an E. coli strain with deregulated sucrose operon. The specific activity did not change much with different sucrose concentrations, indicating that quite low sucrose concentrations (0.1 mM) can be utilized efficiently. With higher concentrations, there was a slight increase in activity, reaching a maximum of 31 U gDCW−1 in the presence of 5 mM sucrose. We hypothesize that with the increasing sucrose concentration, more NADP+ is regenerated, thus more NADPH available for the biocatalytic reaction. Consequently, the cofactor is not a limiting factor compared to when little is available within the cells. Compared to wild-type E. coli W that can only utilize at least 6 mM of sucrose [27, 28], the constructed E. coli W ΔcscR BVMOXeno::pelB_cscA could utilize 50-fold lower sucrose concentrations (0.1 mM) delivering at least 15 U gDCW−1 of specific activity in the oxidation of 1a.

In the presence of sucrose, E. coli W ΔcscR BVMOXeno::cscA strain expressing the invertase gene in the cytosol outperformed all other constructs regardless of sugar sources (37 U gDCW−1). On the other hand, periplasmic production of invertase showed 22 U gDCW− 1. Nevertheless, both expression systems for invertase demonstrated the feasibility of utilizing low sucrose concentrations (1 mM), sustaining cofactor regeneration in an ongoing biocatalytic reaction. We hypothesized that the periplasmic expression of cscA would result in higher specific activities compared to the cytosolic expression because once generated in the periplasmic space, glucose, and fructose as monosaccharides can easily pass the inner membrane via the PTS system [37]. Furthermore, it would ensure the sufficient import of sugars in case cscB is downregulated at the investigated concentrations. The initial results at low sucrose concentrations (1 mM) did not confirm this but showed rather comparable activities between constructs with periplasmic or cytosolic gene expression. One explanation might be that when overexpressed cscA tends to leak out of the cells in a non-specific manner, as reported in other studies [26, 33]. Leakage of intracellularly produced cscA leads to sucrose hydrolysis in the extracellular medium. Its monomers glucose and fructose are then easily taken up by the cells.

Transfer of the sucrose-utilizing ability to E. coli BL21 (DE3) by expression of cscA

The transferability of the system was tested in a widely-used laboratory strain, E. coli BL21 (DE3). Similarly, as in E. coli W ΔcscR, various constructs of the genes were expressed in E. coli BL21 (DE3) (Table 1). Figure 4 shows the specific activities using E. coli BL21 (DE3) in the whole-cell biotransformation of 1a in the presence of either sucrose (1 mM) or glucose (2 mM). E. coli BL21 BVMOXeno::pelB_cscA showed a specific activity of 17 U gDCW−1 with sucrose, which is very similar to the specific activities obtained with E. coli W ΔcscR (22 U gDCW−1). Moreover, this is comparable with rates obtained in E. coli BL21 (DE3) with the same enzyme albeit having a different promoter and carbon source [25].

Fig. 4
figure 4

Whole-cell biotransformation of 1a mediated by different E. coli BL21 (DE3) constructs harboring BVMOXeno in the presence of sugars (2 mM glucose or 1 mM sucrose). Reaction conditions: 30 mL, M9 salts as medium, 1.5 gDCW L−1, 5 mM 1a, 25 °C, 160 rpm, N ≥ 3. All bars represent data generated from reactions stemming from biological replicates with individual values depicted. Error bars represent standard deviations. P values were calculated using Welch’s t test (*P < 0.05)

Full size image

Similar to E. coli W ΔcscR, overexpression of the cscA gene as a fusion protein with the pelB leader sequence for periplasmic export would allow hydrolysis of sucrose in the periplasm and thus transport via the phosphotransferase system. Heterologous production of cscA was sufficient for sucrose uptake even without cscB as previously demonstrated [18, 27, 33]. Sucrose can be thus used efficiently for biotransformations, without the need for production of a sucrose permease, which is not present in E. coli BL21 (DE3) constructs. As it is known that invertase is leaking to some extent out of the cells [19], this amount of invertase appears to be sufficient for the extracellular (or periplasmic) hydrolysis of sucrose. At a sucrose concentration of 1 mM, periplasmic or cytosolic production of invertase did not lead to significant differences in the specific activity (Fig. 4). In fact, there were no significant differences when the invertase gene was expressed either in the periplasm or in the cytosol for E. coli BL21 (DE3), indicating that, in principle, both strategies are equally applicable for biotransformation applications in laboratory strains of E. coli BL21 (DE3). We postulated that the outer membrane of E. coli cells is permeable for sucrose and its insertion in the cell could be limited by the inner membrane. However, cscA again showed to have the ability to confer sucrose uptake without significant difference whether produced in the periplasm or in the cytosol. Thus, sucrose can be used as an alternative electron source for BVMO oxidation of 1a in the laboratory strain E. coli BL21 (DE3), simply by co-expressing the heterologous cscA gene.

Photosynthetically-derived sucrose fuels oxidation reaction using recombinant E. coli

Finally, the feasibility of utilizing photosynthetically-derived sucrose to regenerate cofactors during whole-cell biotransformation of 1a in the constructed E. coli strains was demonstrated. Sucrose (ca. 9 mM) was produced over 7 days by Synechocystis S02 immobilized in alginate beads. Afterwards, E. coli strains using different plasmid constructs (Table 1) were inoculated in the sucrose-enriched medium and 1a was added to initialize the reaction. Figure 5 shows the progression of the biotransformation mediated by E. coli W ΔcscR and E. coli BL21 (DE3) with a periplasmic and cytoplasmic transport of invertase.

Fig. 5
figure 5

Progress of whole-cell biotransformation of 1a mediated by BVMOXeno::pelB_cscA produced in (a) E. coli W ΔcscR and (b) E. coli BL21 (DE3) or by BVMOXeno::cscA produced in (c) E. coli W ΔcscR and (d) E. coli BL21 (DE3) supplemented by photosynthetically-derived sucrose from Synechocystis S02. Reaction conditions: sucrose-enriched BG11 medium containing 400 mM NaCl, 1% CO2, 30 °C, 100 rpm, initial concentration of 5 mM 1a, N = 3. All bars represent data generated from reactions stemming from biological replicates with individual values depicted. Error bars represent standard deviations

Full size image

Both E. coli strains with a periplasmic production of invertase (Fig. 5a and b) showed efficient utilization of sucrose with a complete conversion of 1a after 3 h. In contrast, biotransformations performed using strains with cytosolic production of invertase, which requires sucrose import across the inner cell membrane, showed lower product formation and the reaction did not proceed to completion (Fig. 5c and d). Negative controls without the invertase in E. coli BL21 (DE3) (Figure S5) did not show any product formation. E. coli W ΔcscR BVMOXeno::pelB_cscA was deemed to be the best-performing strain resulting in a volumetric productivity of 1.7 mmol L−1 h−1. Interestingly, also E. coli BL21 (DE3) BVMOXeno::pelB_cscA achieved complete conversion within a short time, showing that the introduction of the sucrose-utilizing capacity to this laboratory strain was straightforward.

Using photosynthetically-produced sucrose, constructs expressing the invertase in the periplasm performed better as compared with cytosolic expression (Fig. 5). This was observed both for E. coli W ΔcscR and BL21 (DE) strains. Interestingly, in the experiments using pure sugars, intracellular and periplasmic production had worked equally well (Figs. 3 and 4). A possible explanation is that at the six-fold higher sucrose concentrations used in the system with photosynthetically-derived sucrose, hydrolysis of the sucrose in the periplasm is more efficient than leaking of invertase. Moreover, the leaking of invertase might differ under different conditions of biotransformation, making the periplasmic invertase production the more reliable option. Also, cell permeabilization with the effect of cell activity is not expected, this is more likely when using lyophilized cells. Resting cells, on the contrary, still require the pentose phosphate pathway and perform glycolysis.

In summary, a stronger effect of the periplasmic invertase production was observed when higher concentrations were applied using higher photosynthetically-derived sucrose when determining the initial specific activity of BVMO oxidation reaction using respective strains.

Notably, Fig. 5 shows no particular difference between the E. coli W ΔcscR and BL21 (DE3) strains while carrying the same BVMOXeno::cscA cassette, whereas the same variants resulted in different activities in the initial study (Figs. 3 and 4). We hypothesize that when small amounts of sucrose such as 1 mM are used the genomically encoded sucrose permease cscB and invertase cscA in the E. coli W ΔcscR add to more effective import and hydrolysis, respectively. Thus, enabling the biocatalytic reaction to take place at a higher rate. As a result, more NADPH is available to the enzyme, as opposed to BL21 (DE3), which solely relies on the recombinant invertase. However, when sucrose is abundant the hydrolysis is faster, and NADP+ reduction is taking place at a higher rate than the BVMOXeno oxidation. Thus, NADPH is not the rate-limiting factor and the effect of the genomically encoded enzymes is less prominent reflecting no particular changes in the specific enzyme activity.

Additionally, results in Fig. 5 represent the quantification of a total substrate and product each hour and not the enzyme specific activity, which is determined within the ca. first 15 min (≤ 10% conversion) of the reaction. Consequently, the experiments involving pure sugars focus on reaction rates and directly correlate them to different sugar types and the metabolism of the respective host. On the other hand, when a given amount of photosynthetically-derived sucrose was used instead, the focus was on how long it takes for the enzyme to drive the oxidation of 1a to completion while using the thus far fastest BVMO variant. Additionally, if different E. coli variants have a different effect within this time frame, which was a benchmark based on previous studies [26, 34].

The volumetric productivity of the system using photosynthetically-derived sucrose is still lower than that of the recombinant cyanobacteria (up to 3.7 mmol L−1 h−1), but the high efficiency of E. coli whole-cell catalysts in Baeyer–Villiger oxidation reactions of up to 1.7 mmol L−1 h−1 shows the outstanding potential of this host [34]. We believe that our present work will contribute to a coupling of this system to sustainable photosynthetic sucrose production.

Periplasmic production of invertase is advantageous when high sucrose concentration is used

In the initial experiments, we tested for sucrose utilization at 1 mM concentration because of the assumption that high concentrations are ideal conditions. Also, sugars are costly and hence can be a bottleneck in a biotechnological process. Given that, if a batch of photosynthetically-produced sucrose results in low yields, in the case of effective E. coli strains, they will subsequently perform the biocatalysis at high rates. Furthermore, E. coli ferments sugars when highly abundant and the Crabtree effect is a possible unfavorable outcome [39].

However, we hypothesized that the higher specific enzyme activities with photosynthetically-derived sucrose attributed to the higher sucrose availability, which was hence confirmed by the results shown in Fig. 6B referring to whole-cell biotransformation of 1a and the addition of 10 mM pure sucrose. These indicate that periplasmic invertase production was overall the most efficient for the oxidation of 1a when using 10 mM sucrose. E. coli W ΔcscR and BL21 (DE3) resulted in 26 U gCDW−1 and 17 U gCDW−1, respectively. Same as with the photosynthetically-derived sucrose, E. coli BL21 (DE3) BVMOXeno::cscA was the worst performing under these conditions, and E. coli W ΔcscR BVMOXeno::pelB_cscA the best one. In addition, Fig. 6A shows the growth of both E. coli W ΔcscR and BL21 (DE3) strains in the presence of 10 mM sucrose. Results were comparable to those in Fig. 2A and B.

Fig. 6
figure 6

A Growth of E. coli W ΔcscR and E. coli BL21 (DE3) in the presence 10 mM sucrose in M9 minimal medium. Reaction conditions: 30 mL volume in 100 mL baffled flasks, 37 °C, 120 rpm; and B Specific enzyme activities obtained in whole-cell biotransformation of 1a mediated by BVMOXeno::cscA and BVMOXeno::pelB_cscA produced in E. coli W ΔcscR and E. coli BL21 (DE3) when supplemented with 10 mM sucrose. Reaction conditions: 30 mL, M9 salts as medium, 1.5 gDCW L−1, 5 mM 1a, 25 °C, 160 rpm, N = 3. All values represent data generated from three biological replicates. Error bars represent standard deviations. P-values were calculated using Welch’s t test (*P < 0.05)

Full size image

These final results indicate that indeed if higher concentrations of sucrose in solution are applied to drive the oxidation of 1a, the cofactor regeneration is faster if the recombinant invertase is produced in the periplasmic space thus overcoming a possible bottleneck of NADPH limitation.

Conclusion

The co-expression of invertase and BVMO in two E. coli chassis showed efficient utilization of sucrose to fuel a whole-cell monooxygenase reaction. Coupling sucrose-utilizing strains with sucrose-secreting cyanobacteria poses the challenge that many of the frequently used and well-characterized E. coli laboratory strains do not have the capacity to utilize sucrose. Specific E. coli strains that have retained permeases and invertases for sucrose utilization are available but are less practicable for research since they lack the above-mentioned features (such as gene deletions to facilitate disulphide bond formation). Our results show that on the one hand, the introduction of the capacity of sucrose utilization into laboratory strains allows relying on the large experience with these well-characterized systems and harnessing the diversity of features available for these strains (as a ‘plug-and-play system’) whose introduction into E. coli W ΔcscR would be tedious. This is a great advantage for research purposes. On the other hand, invertase overexpression also improved the performance of E. coli W ΔcscR, showing that a combination of a deregulated sucrose operon with additional periplasmic invertase expression leads to the highest efficiency in the coupling of whole-cell biotransformations in E. coli with photosynthetically-derived sucrose, where alginate immobilized cyanobacteria can easily be removed – which makes this system highly attractive for sustainable, sucrose-driven production processes.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its Additional file.

Abbreviations

1a :

Cyclohexanone

1b :

ε-Caprolactone

ATP:

Adenosine triphosphate

BVMO:

Baeyer Villiger Monooxygenase

BVMOXeno :

BVMO from Burkholderia xenovorans

BVMOParvi :

BVMO from Parvibaculum lavamentivorans

CHMO:

Cyclohexanone monooxygenase

cscR :

Sucrose repressor gene

cscA :

Invertase gene

cscB :

Sucrose permease gene

cscK :

Fructokinase gene

E. coli :

Escherichia coli

E. coli  W ΔcscR :

E. coli W with a deleted sucrose repressor

FAD:

Flavin adenine dinucleotide

GC-FID:

Gas Chromatography equipped with a Flame Ionization Detector

IPTG:

Isopropyl-β-D-thiogalactopyranoside

NAD(P)H:

Nicotinamide adenine dinucleotide (phosphate)

OD600 :

Optical density at 600 nm

pelB:

Signal sequence for the periplasmic export

Synechocystis  sp.:

Synechocystis sp. PCC 6803

References

  1. May SW. Applications of oxidoreductases. Curr Opin Biotechnol. 1999;10(4):370–5.

    Article  CAS  PubMed  Google Scholar 

  2. Liu W, Wang P. Cofactor regeneration for sustainable enzymatic biosynthesis. Biotechnol Adv. 2007;25(4):369–84.

    Article  CAS  PubMed  Google Scholar 

  3. Chenault HK, Simon ES, Whitesides GM. Cofactor regeneration for enzyme-catalysed synthesis. Biotechnol Genet Eng Rev. 1988;6(1):221–70.

    Article  CAS  PubMed  Google Scholar 

  4. Grimm HC, Erdem E, Kourist R. Chapter 8 biocatalytic applications of autotrophic organisms. In: Kourist R, Schmidt S, editors. The autotrophic biorefinery. Berlin: De Gruyter; 2021. p. 207–46.

    Chapter  Google Scholar 

  5. Zhao H, van der Donk WA. Regeneration of cofactors for use in biocatalysis. Curr Opin Biotechnol. 2003;14(6):583–9.

    Article  CAS  PubMed  Google Scholar 

  6. Hollmann F, Schmid A. Electrochemical regeneration of oxidoreductases for cell-free biocatalytic redox reactions. Biocatal Biotransform. 2004;22(2):63–88.

    Article  CAS  Google Scholar 

  7. Engineering of NADPH regenerators in Escherichia coli for enhanced biotransformation | applied microbiology and biotechnology. 7 Mar 2024

  8. Bian M, Zhao J, Xu W, Han X, Chen X, Wang P, et al. Enzymatic characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the marine bacterium umbonibacter marinipuiceus. Int J Mol Sci. 2023;24(14):11428.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Graf SS, Hong S, Müller P, Gennis R, von Ballmoos C. Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli. Sci Rep. 2021;11(1):21234.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Walton AZ, Stewart JD. An efficient enzymatic Baeyer–Villiger oxidation by engineered Escherichia coli cells under non-growing conditions. Biotechnol Prog. 2002;18(2):262–8.

    Article  CAS  PubMed  Google Scholar 

  11. Feyza Özgen F, Runda ME, Burek BO, Wied P, Bloh JZ, Kourist R, et al. Artificial light-harvesting complexes enable rieske oxygenase catalyzed hydroxylations in non-photosynthetic cells. Angew Chem Int Ed. 2020;59(10):3982–7.

    Article  Google Scholar 

  12. Grimm HC, Kourist R. 3 Cyanobacteria as catalysts for light-driven biotransformations. In: Rögner M, editor. Photosynthesis. Berlin: De Gruyter; 2021. p. 57–92.

    Chapter  Google Scholar 

  13. Runda ME, Schmidt S. Light-driven bioprocesses. Phys Sci Rev. 2023. https://doi.org/10.1515/psr-2022-0109.

    Article  Google Scholar 

  14. Du W, Liang F, Duan Y, Tan X, Lu X. Exploring the photosynthetic production capacity of sucrose by cyanobacteria. Metab Eng. 2013;1(19):17–25.

    Article  Google Scholar 

  15. Hagemann M. Molecular biology of cyanobacterial salt acclimation. FEMS Microbiol Rev. 2011;35(1):87–123.

    Article  CAS  PubMed  Google Scholar 

  16. Ducat DC, Avelar-Rivas JA, Way JC, Silver PA. Rerouting carbon flux to enhance photosynthetic productivity. Appl Environ Microbiol. 2012;78(8):2660–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. The potential of Synechococcus elongatus UTEX 2973 for sugar feedstock production | applied microbiology and biotechnology. 7 Mar 2024

  18. Thiel K, Patrikainen P, Nagy C, Fitzpatrick D, Pope N, Aro EM, et al. Redirecting photosynthetic electron flux in the cyanobacterium Synechocystis sp PCC 6803 by the deletion of flavodiiron protein Flv3. Microb Cell Fact. 2019;18(1):189.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Lee JW, Choi S, Park JH, Vickers CE, Nielsen LK, Lee SY. Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for L-threonine production. Appl Microbiol Biotechnol. 2010;88(4):905–13.

    Article  CAS  PubMed  Google Scholar 

  20. Ro HS, Kim HS. Continuous production of gluconic acid and sorbitol from sucrose using invertase and an oxidoreductase of Zymomonas mobilis. Enzyme Microb Technol. 1991;13(11):920–4.

    Article  CAS  Google Scholar 

  21. Assil-Companioni L, Büchsenschütz HC, Solymosi D, Dyczmons-Nowaczyk NG, Bauer KKF, Wallner S, et al. Engineering of NADPH supply boosts photosynthesis-driven biotransformations. ACS Catal. 2020;10(20):11864–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sakoleva T, Austin HP, Tzima C, Dörr M, Bornscheuer UT. Discovery and characterization of a Baeyer-Villiger monooxygenase using sequence similarity network analysis. ChemBioChem. 2023;24(10): e202200746.

    Article  CAS  PubMed  Google Scholar 

  23. Hoschek A, Bühler B, Schmid A. Overcoming the gas–liquid mass transfer of oxygen by coupling photosynthetic water oxidation with biocatalytic oxyfunctionalization. Angew Chem Int Ed. 2017;56(47):15146–9.

    Article  CAS  Google Scholar 

  24. Solé J, Brummund J, Caminal G, Álvaro G, Schürmann M, Guillén M. Enzymatic synthesis of trimethyl-ε-caprolactone: process intensification and demonstration on a 100 L scale. Org Process Res Dev. 2019;23(11):2336–44.

    Article  Google Scholar 

  25. Hilker I, Wohlgemuth R, Alphand V, Furstoss R. Microbial transformations 59: first kilogram scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity using a resin-based in situ SFPR strategy. Biotechnol Bioeng. 2005;92(6):702–10.

    Article  CAS  PubMed  Google Scholar 

  26. Tóth GS, Siitonen V, Nikkanen L, Sovic L, Kallio P, Kourist R, et al. Photosynthetically produced sucrose by immobilized Synechocystis sp. PCC 6803 drives biotransformation in E. coli. Biotechnol Biofuel Bioprod. 2022;15(1):146.

    Article  Google Scholar 

  27. Arifin Y, Sabri S, Sugiarto H, Krömer JO, Vickers CE, Nielsen LK. Deletion of cscR in Escherichia coli W improves growth and poly-3-hydroxybutyrate (PHB) production from sucrose in fed batch culture. J Biotechnol. 2011;156(4):275–8.

    Article  CAS  PubMed  Google Scholar 

  28. Sabri S, Nielsen LK, Vickers CE. Molecular control of sucrose utilization in Escherichia coli W, an efficient sucrose-utilizing strain. Appl Environ Microbiol. 2013;79(2):478–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pilizota T, Shaevitz JW. Fast, multiphase volume adaptation to hyperosmotic shock by Escherichia coli. PLoS ONE. 2012;7(4): e35205.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Decad GM, Nikaido H. Outer membrane of gram-negative bacteria. XII. Molecular-sieving function of cell wall. J Bacteriol. 1976;128(1):325–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sahin-Tóth M, Lengyel Z, Tsunekawa H. Cloning, sequencing, and expression of cscA invertase from Escherichia coli B-62. Can J Microbiol. 1999;45(5):418–22.

    Article  PubMed  Google Scholar 

  32. Tsunekawa H, Azuma S, Okabe M, Okamoto R, Aiba S. Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry. Appl Environ Microbiol. 1992;58(6):2081–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Scholle RR, Coyne VE, Maharaj R, Robb FT, Woods DR. Expression and regulation of a Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli. J Bacteriol. 1987;169(6):2685–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Erdem E, Malihan-Yap L, Assil-Companioni L, Grimm H, Barone GD, Serveau-Avesque C, et al. Photobiocatalytic oxyfunctionalization with high reaction rate using a Baeyer–Villiger monooxygenase from Burkholderia xenovorans in metabolically engineered cyanobacteria. ACS Catal. 2022;12(1):66–72.

    Article  CAS  PubMed  Google Scholar 

  35. Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986;189(1):113–30.

    Article  CAS  PubMed  Google Scholar 

  36. Kruger NJ, von Schaewen A. The oxidative pentose phosphate pathway: structure and organisation. Curr Opin Plant Biol. 2003;6(3):236–46.

    Article  CAS  PubMed  Google Scholar 

  37. Transporters of glucose and other carbohydrates in bacteria | Pflügers Archiv—European journal of physiology. 7 Mar 2024

  38. Park J-B, Bühler B, Habicher T, Hauer B, Panke S, Witholt B, Schmid A. The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation. Biotechnol Bioeng. 2006;95(3):501–12.

    Article  CAS  PubMed  Google Scholar 

  39. Gschaedler A, Robas N, Boudrant J, Branlant C. Effects of pulse addition of carbon sources on continuous cultivation of Escherichia coli containing a recombinant E. Coli gapA Gene Biotechnol Bioengin. 1999;63(6):712–20.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank to Dr. Claudia E. Vickers and Dr. Bingyin Peng from University of Queensland, Australia for kindly providing us with E. coli W ΔcscR strain. Additionally, L.S. would like to thank to Dr. Hanna Grimm, Elske van der Pol, Dr. Marianna Karava, Javier G.-Navarro, Jasmin Zuson and Dr. L. A.-Companioni for great support in GC-FID analytics, critical thinking and daily input, as well as introducing the GraphPad Prism 8.4.0.

Funding

Open access funding provided by Graz University of Technology. This project has received funding from the FET Open grant agreement 899576 (FuturoLEAF) and by the Novo Nordisk Foundation project “PhotoCat” (project no. NNF20OC0064371 to YA). The funding bodies had no role in the design of the study, analysis of the data, or writing of the manuscript.

Author information

Authors and Affiliations

  1. Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14, 8010, Graz, Austria

    Lucija Sovic, Lenny Malihan-Yap & Robert Kourist

  2. Molecular Plant Biology, Department of Life Technologies, University of Turku, 20014, Turku, Finland

    Gábor Szilveszter Tóth, Vilja Siitonen & Yagut Allahverdiyeva

  3. Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France

    Véronique Alphand

  4. ACIB GmbH, 8010, Graz, Austria

    Robert Kourist

Authors
  1. Lucija Sovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lenny Malihan-Yap
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gábor Szilveszter Tóth
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vilja Siitonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Véronique Alphand
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yagut Allahverdiyeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert Kourist
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

LS executed the experiments regarding cloning and generating mutants, tested the growth and the whole-cell biotransformations with different sugars, and analyzed the data. LMY gave feedback on generated data, conceptualized, wrote, reviewed and edited the manuscript. GST performed the experiments using photosynthetically-derived sucrose and reviewed the manuscript. VS established the analytics with GST at their affiliation. VA developed the BVMOXeno gene and reviewed the manuscript. YA reviewed the manuscript. RK was responsible for funding acquisition, supervising the study and writing the manuscript. All authors reviewed the manuscript.

Corresponding author

Correspondence to Robert Kourist.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sovic, L., Malihan-Yap, L., Tóth, G.S. et al. Sucrose as an electron source for cofactor regeneration in recombinant Escherichia coli expressing invertase and a Baeyer Villiger monooxygenase. Microb Cell Fact 23, 227 (2024). https://doi.org/10.1186/s12934-024-02474-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12934-024-02474-2

Keywords

Microbial Cell Factories

ISSN: 1475-2859

Contact us