Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

Podsumowanie

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

Streszczenie

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Wprowadzenie

Reductive whole-cell biotransformation using microorganisms has become a widespread method for the chemo-enzymatic synthesis of commercially and therapeutically important biomolecules^1-3. It presents several advantages over the use of isolated enzymes, especially the elimination of cost-intensive downstream purification processes and the demonstration of an extended lifetime^4-7. For biocatalytic pathways where cofactors are required for product formation, whole-cell systems have the potential to provide in situ cofactor regeneration via the addition of inexpensive electron-donating co-substrates^5,8,9. However, this capacity is diminished for reactions that require a stoichiometric concentration of rare or expensive co-substrates^10-13. Together with poor reusability of whole cells, this impedes the establishment of a scalable and continuous production system. Strategic modifications of whole-cell systems for these cofactor-dependent biotransformations are required to overcome the aforementioned limitations. Specifically, the combination of whole-cell biocatalysts that work cooperatively have been shown to significantly enhance the productivity and stability of the harbored enzymes¹⁴. These factors, which are often critical for enabling large-scale production of commercially viable products, can be optimized further by co-immobilizing biocatalytic microbes¹⁵. We have recently developed a simple and reusable whole-cell biocatalytic system that allows both cofactor regeneration and biocatalyst immobilization for the L-xylulose production¹⁶. In this study, this system was utilized as an example to illustrate the experimental procedures of applying these two strategies for improved biotransformation production yield and biocatalyst reusability.

L-xylulose belongs to a class of biologically useful molecules named rare sugars. Rare sugars are unique monosaccharides or sugar derivatives that occur very rarely in nature, but play crucial roles as recognition elements in bioactive molecules^17,18. They have a variety of applications ranging from sweeteners, functional foods to potential therapeutics¹⁹. L-xylulose can be used as a potential inhibitor of multiple α-glucosidases, and may also be used as an indicator of hepatitis or liver cirrhosis^17,20. High efficiency conversion of xylitol to L-xylulose in whole-cell systems has been reported previously in Pantoea ananatis^21,22, Alcaligenes sp. 701B²³, Bacillus pallidus Y25^24,25 and Escherichia coli²⁶. In E. coli, however, it was achieved only using low (<67 mM) xylitol concentrations²⁶ due to potential inhibitory effects of an initial xylitol concentration higher than 100 mM on xylitol-4-dehydrogenase activity^21,26. The thermodynamic equilibrium between xylulose and xylitol has been shown to strongly favor the formation of xylitol^25,27. Additionally, xylulose yield is limited by the amount of expensive cofactors that have to be supplied in the absence of an in situ cofactor regeneration system. Together, these factors limit the potential for scaling into sustainable systems for L-xylulose biosynthesis.

To overcome these limitations and improve the L-xylulose biotransformation yield, the strategy of cofactor regeneration was employed first by establishing a coupled whole-cell biocatalytic system. Specifically, L-Arabinitol 4-dehydrogenase (EC 1.1.1.12) from Hypocrea jecorina (HjLAD), an enzyme in the L-arabinose catabolic pathway of fungi, was selected to catalyze the conversion of L-arabinitol into L-xylulose^28,29. Like many biosynthetic enzymes, a major limitation of HjLAD is that it requires a stoichiometric amount of the expensive nicotinamide adenine dinucleotide cofactor (NAD⁺, the oxidized form of NADH) to carry out this conversion. NADH oxidase found in Streptococcus pyogenes (SpNox) has been shown to display high cofactor-regeneration activity^30,31. Taking advantage of this attribute of SpNox, E. coli cells expressing HjLAD for the production of L-xylulose were coupled with E. coli cells expressing SpNox for the regeneration of NAD⁺ to boost the L-xylulose production depicted by the coupled reaction shown in Figure 1A. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, making this system much more efficient than those reported in literature.

The strategy of whole-cell immobilization was employed next to further enhance the reusability of the coupled biocatalytic system. Commonly used methods for whole-cell immobilization include adsorption/covalent linking to solid matrices, cross-linking/entrapment and encapsulation in polymeric networks³². Among these approaches, the most suitable method for cell immobilization is encapsulation in calcium alginate beads. Their mild gelation properties, inert aqueous matrix and high porosity help preserve the physiological properties and functionality of the encapsulated biologicals³³. Therefore, the coupled biocatalyst system containing both E. coli cells harboring HjLAD or SpNox was immobilized in calcium alginate beads to enable multiple cycles of L-xylulose production (Figure 2).The immobilized biocatalyst system demonstrated good operational stability, maintaining 65% of the conversion yield of the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost its catalytic activity.

Protokół

1. Whole-cell Biocatalysts Preparation  

NOTE: The recombinant E. coli cells harboring pET28a-SpNox³¹ or pET28a-HjLAD²⁸ are hereafter referred to as E. coli_SpNox and E. coli_HjLAD, respectively.

1. Inoculate a single colony of E. coli_HjLAD in 3 ml of Luria-Bertani (LB) medium supplemented with kanamycin (50 µg/ml) and incubate in an incubator shaker O/N at 37 ^oC, 250 rpm.
2. Dilute the culture by 1:100 in 200 ml of fresh LB containing 50 µg/ml kanamycin and incubate at 37 ^oC, 250 rpm until the OD₆₀₀ reaches ~0.6.
3. Induce HjLAD protein expression by adding 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to the culture medium and incubate at 16 ^oC, 180 rpm for 16 hr.
   1. Alternatively, perform induction at 25 ^oC, 200 rpm for 6 hr if the L-xylulose biosynthesis (Step 2 below) is performed the same day.
4. Harvest the induced E. coli_HjLAD cells by centrifugation at 3,200 x g for 20 min at 4 ^oC. Discard the supernatant and proceed to Step 2 to process the cell pellet.
5. In parallel, perform steps 1.1 - 1.4 for E. coli_SpNox.

2. Biosynthesis of L-xylulose by Coupling E. coli_HjLAD and E. coli_SpNox for Cofactor Regeneration

1. Resuspend the cell pellets of E. coli_HjLAD and E. coli_SpNox separately in 50 mM Tris-HCl buffer (pH 8.0) at a cell density of 5.0 g dry cell weight (gDCW)/L.
   Note: A correlation between gDCW and the optical density measured at 600 nm (OD₆₀₀) can be established to facilitate the experiment. The formula used in this protocol is 1 gDCW/L=0.722*OD₆₀₀ - 0.0965, which can vary among different spectrometers.
2. Mix 600 µl of 5.0 gDCW/L E. coli_HjLAD, 600 µl of 5.0 gDCW/L E. coli_SpNox, 100 µl of 20 mM NAD⁺, and 150 µl of 2 M L-arabinitol in a 14 ml round-bottom tube and bring the reaction volume to 2 ml by adding 550 µl of 50 mM Tris-HCl (pH 8.0).
   Note: The ratio of the two whole-cell biocatalysts amount can be optimized to improve the biosynthesis of the product. For the described system, a ratio of E. coli_SpNox:E. coli_HjLAD = 1:1 was found to be optimal for L-xylulose biosynthesis (Figure 1B).
3. Incubate the reaction mixture at 30 ^oC, 200 rpm for 8 hr.
4. Collect the supernatant after centrifugation at 4 ^oC, 3,200 x g for 10 min and proceed to quantify the L-xylulose production as described in Step 3 below.

3. Colorimetric Assay for L-xylulose Quantification

1. Aspirate 100 µl of the reaction supernatant collected from step 2.4 into a 1.5 ml tube.
2. Add 50 µl of 1.5% cysteine, 900 µl of 70% sulfuric acid, and 50 µl of 0.1 % carbazole dissolved in ethanol and mix gently by inverting the tube 3 times.
3. Incubate the reaction mixture at 37 ^oC, 200 rpm for 20 min.
4. Measure the optical absorbance of the reaction mixture at 560 nm (A₅₆₀) using a spectrophotometer.
   1. Dilute the reaction mixture if the A₅₆₀ reading is above 1.

4. Immobilization of Recombinant Whole-cell Catalysts in Calcium Alginate Beads

1. Dissolve 4 g sodium alginate in 100 ml of distilled water. Prepare alginate solution by adding sodium alginate to water to avoid the formation of clumps. Heat the mixture if needed.
2. Add 600 µl of 5.0 gDCW/L E. coli_HjLAD and 600 µl of 5.0 gDCW/L E. coli_SpNox in 1.2 ml 4% alginate prepared in step 4.1 and mix the cells and alginate by gentle pipetting to avoid bubble formation.
3. Aspirate the alginate/cell suspension into a syringe using a needle and add the mixture drop-wise into a 0.3 M calcium chloride (CaCl₂) solution in a 100 ml beaker with continuous stirring.
   Note: The volume of CaCl₂ solution used for alginate bead formation should be enough for the alginate droplets to be completely submerged. Additionally, the distance between the syringe needle and the surface of the calcium chloride solution must be maintained within an optimal range to ensure formation of uniformly spherical beads. The optimum distance range can be determined experimentally and depends on the inner diameter of the needle. As an estimate, a distance of ~15 ± 5 cm was found to be optimal for a 0.6 cm (inner diameter) syringe needle.
4. Leave the beads in the CaCl₂ solution for 2 - 3 hr at RT without stirring to allow crosslinking and gel beads formation.
5. Decant the CaCl₂ solution without disturbing the beads by carefully pouring the CaCl₂ solution into a 50 ml conical tube, and transfer the remaining beads in CaCl₂ solution into another 50 ml conical tube.
6. Wash the beads with 10 ml of 50 mM Tris-HCl (pH 8.0) buffer three times to remove excessive CaCl₂ and un-encapsulated cells.
   NOTE: At any given step, do not centrifuge the beads as doing so will rupture them. To separate the beads from solution, allow the suspension to stand undisturbed for 3 - 5 min. The beads will settle at the bottom and the wash buffer can be decanted into another container.
   1. Do not discard the used wash buffer. Pool the used Tris-HCl wash buffer (30 ml) with the used CaCl₂ solution collected from step 4.5.
7. Pellet the un-immobilized E. coli cells by centrifugation of the pooled CaCl₂ and Tris-HCl solution collected from step 4.6 at 3,200 x g for 20 min. To determine the immobilization efficiency, calculate the density of the pelleted un-encapsulated cells in gDCW/L as described in step 2.1.
8. Transfer the washed beads from step 4.6 into a tube. Follow steps 2.2 - 3.4 to evaluate the L-xylulose biosynthesis using all of the washed beads in place of cell pellets.

5. Stability Assay of Immobilized Biocatalysts for L-xylulose Production

1. Collect the beads from step 4.8 and wash twice with 10 ml of 50 mM Tris-HCl (pH 8.0) buffer without centrifugation (as described in Step 4.6).
2. Use all of the washed beads to perform the reaction as described in steps 2.2 - 3.4.
3. Repeat steps 5.1 - 5.2 for desired number of production cycles and measure the amount of L-xylulose produced in the reaction supernatant in each cycle.

Wyniki

To enable cofactor regeneration, L-xylulose synthesis was carried out in a coupled whole-cell biocatalytic system containing E. coli_HjLAD and E. coli_SpNox cells. Following the optimization of various parameters, the reusability of this system was improved by immobilizing it in calcium alginate beads (Figure 2).

L-xylulose Production with Cofactor ...

Dyskusje

Recent technological advancements have enabled a surge in the commercialization of recombinant biotherapeutics, resulting in a gradual rise in their market value in the biotechnology industry. One such advancement is the advent of metabolic engineering in recombinant microorganisms, which has shown a great promise in establishing scalable industrial systems³⁸. As with most processes, the successful commercialization of recombinant biomolecules produced by genetically engineered microbes is highly dependent on ...

Materiały

Name	Company	Catalog Number	Comments
LB broth	Sigma Aldrich	L3022-6X1KG
Kanamycin	Fisher	BP906-5
Isopropyl β-D-thiogalactopyranoside (IPTG)	Sigma Aldrich	I6758-10G
Tris base	Fisher	BP1521
B-Nicotinamide adenine dinucleotide hydrate	Sigma Aldrich	N7004-1G
L-Arabinitol	Sigma Aldrich	A3506-10G
L-Cysteine	Sigma Aldrich	168149
Sulfuric acid	Sigma Aldrich	320501-500ML
Carbazole	Sigma Aldrich	C5132
Ethanol	Fisher	BP2818-4
Sodium alginate	Sigma Aldrich	W201502
Calcium chloride dihydrate	Sigma Aldrich	223506-500G
Excella E24 shaker incubator	New Brunswick Scientific
Cary 60 UV-Vis Spectrophotometer	Agilent Technologies
Centrifuge 5810R	Eppendrof
Beakers	Fisher
Syringe	Fisher
Needle	Fisher
Pioneer Analytical and Precision Weighing Balance	Ohaus