Streamlined production, purification, and comparison of recombinant extracellular polyhydroxybutyrate depolymerases

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp., Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, strategies were developed to circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15 °C, and rPhaZs from Cupriavidus sp. and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs, and to perform preliminary evaluation for applications that require PHB degradation.


Introduction
The study of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) produced by a variety of microorganisms [1][2][3] remains an important and evolving research area. Their enzymatic activity results in the degradation of PHB, a natural biodegradable polymer with the potential to replace some currently widely used petroleum-based plastics [4] that increasingly accumulate in the environment [5].

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Recombinant protein production is a powerful tool that allows the production of higher levels of proteins in expression systems such as E. coli. Optimized recombinant technologies facilitate purification, the study of proteins in isolation, the conception of a platform to modify and improve them, and the development of new applications. In the case of PhaZs, such applications include biosensors -such as time-temperature indicators [6] and pathogen detection platforms [7] -and recycling of biodegradable polymers [8].
In this study, we established a platform for the rapid expression and purification of extracellular rPhaZs. This was demonstrated with five extracellular PhaZs displaying different properties and of various bacterial origins. Predicted solubility and disulfide bonds (necessary for maintaining proper conformation and activity in many proteins [25]) of the rPhaZs produced were important criteria in selecting the E. coli system, specifically the plasmid vector and expression strains. A single platform with simple ! 3 -Pre-Printstrategies was successfully employed for expression, purification, and preliminary comparison of degradation performance under different conditions.

Bacterial strains and growth conditions
The bacterial strains used for isolation of the PhaZs, cloning and expression, as well as their growth medium and conditions can be found in Table 1. Cell growth was monitored by measuring optical density of the cultures at 600 nm (OD600) using a UV-Vis spectrophotometer (Biochrom, Ultrospec 50). Plating was performed on 1.5% w/v agar supplemented with the medium of interest and plates were incubated in a temperature-controlled incubator (Isotemp 500 Series, Fisher Scientific). ! 4 Table 1 Bacterial strains, conditions, and primers. Growth conditions and information of (a) PhaZ-producing strains and (b) cloning and expression strains; (c) rPhaZs primers.

Induction screening and His-tag verification
Starter cultures from single colonies of E. coli Rosetta-gami B(DE3) or T7 Express lysY/I q E. coli containing the constructs were grown in 5 ml LB with corresponding antibiotics (Table 1b)   Assay (microassay procedure, Bio-Rad) using bovine serum albumin as standard.

Expression and purification of PhaZs
Amicon Ultra 0.5-mL filters (Millipore) were used for PhaZCsp, PhaZMal, and PhaZRsp, which required further purification.

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Buffer exchange was done prior to assays in which imidazole and glycerol caused interference with Amicon Ultra 0.5-mL filters or dialysis (Slide-A-Lyzer MINI Dialysis Devices, 20K MWCO, Thermo Scientific).

PHB plates rPhaZs activity comparison
Rapid
Sequencing of mature phaZ inserts revealed the sequences of PhaZMal and PhaZPst were the same as in the Genbank registers, while some changes were found for PhaZCte, PhaZCsp, and PhaZRsp, that resulted in 3, 1, and 7 amino acid changes, likely due to variations in the taxonomic strains [45] since their deposition.  Removing the native signal peptide from the PhaZ sequence is a key step in diminishing the formation of inclusion bodies and avoiding completely insoluble PhaZs when using pET-22b(+) or plasmids that add signal sequences. As an example, Figure   !  Relative expression levels in the SF of each PhaZ can be observed in Figure 1 (v).

Conditions for induction were established with the expression strain E. coli
These were qualitatively classified as very high for PhaZCte, high for PhaZPst, low for PhaZMal and PhaZRsp, and very low for PhaZCsp (Table 2). Purification, which was especially challenging for PhaZMal, could be further improved using a combination of strategies, including doing the equilibration, sample application, and wash steps with solutions containing 50 mM imidazole -this improved purity of PhaZCte, PhaZMal, and PhaZRsp, at the expense of recovery -adding an elution step with 150 mM imidazole instead of 500 mM for PhaZMal, and using size exclusion columns. ! 13 (v) SDS-PAGE of rPhaZs after imidazole elution (E: consecutive elution; * indicates a 10-fold reduction factor in the loading volume of the elution fraction). Equilibration, sample application, and wash contained 20 mM imidazole, while the elution buffer contained 500 mM imidazole, except for PhaZMal, for which 150 mM were used for E1 and E2, 100 mM for E3 and E4, 200 mM for E5 and E6, and 500 mM for E7. Arrows point to the location of rPhaZs.

Comparison of rPhaZ activity
While PHB plates have been mostly used to screen for PHB degrading bacteria [1], activity from expressed PhaZs has also been estimated by the diameter of clear ! 14 zones on glass slides covered by PHB-agar mix [9,13] (this test is limited to short-term incubations due to agar drying, but is advantageous for preliminary assessment and when only small volumes of sample are available).
In this study, a rapid method using PHB plates was used to compare PhaZ activity at various pH and temperatures and provided semi-quantitative assessments of activity based on the diameter of degradation halos formed (Figure 2). At 37 °C, degradation was observed on the first day of incubation for all rPhaZs tested, while longer incubation periods were required at 15 °C. PhaZMal showed the highest activity at 15 °C (halo observed after 1 day) compared to the other rPhaZs (halos observed after 6 days), which could be explained by its marine origin [41]. All enzymes were rendered inactive at pH 4.3 (no halos discernable) but PhaZCsp and PhaZRsp retained significant activity at pH 5.4. This is consistent with their broad pH working ranges (PhaZCsp is stable when stored at pH 5.0-8.0 [50] with optimum activity at pH 7.5 [51], and the optimum pH range of PhaZRsp is 5.0-6.0 [52]).
These results could be confirmed and semi-quantified by comparing the rate of change of the degradation halos under the different conditions tested (Figure 2 (ii) )).
For example, similar degradation rates were observed for all rPhaZs at 37 °C and pH 7.0, but PhaZMal had a noticeably greater rate at 15 °C and pH 7.0 (leading to ≈ 33% more degradation after 28 h). Such methods represent powerful tools for screening

Concluding remarks
This study presents a streamlined platform for the rapid production of rPhaZs.
Five active PHB-degrading extracellular PhaZs (PhaZCte, PhaZPst, PhaZCsp PhaZRsp, and PhaZMal), originating from bacteria from diverse environments, were successfully produced in the SF of Rosetta-gami B(DE3) E. coli. An important aspect of the method requires the removal of the native signal peptide sequence of PhaZ to avoid production ! 16 -Pre-Printof insoluble proteins and inactive enzymes. Expression levels and purity varied for each enzyme -PhaZCte and PhaZPst saw highest expression -but they could all be recovered and retained activity. In addition, degradation activity could easily be assessed by determining the diameter of degradation halos in PHB plates. This assay can be done in parallel for the initial screening of PhaZs and conditions for diverse applications. Both the rPhaZ production platform and the modified PHB plates assay are versatile and reliable, and could be employed with other PhaZs reported in the literature or novel ones to be discovered or synthesized.