Co‐Expression of ORF_Cma with PHB Depolymerase (PhaZ_Cma) in Escherichia coli Induces Efficient Whole‐Cell Biodegradation of Polyesters

Abstract

Whole-cell degradation of polyesters not only avoids the tedious process of enzyme separation, but also allows the degraded product to be reused as a carbon source. In this study, Escherichia coli BL21(DE3) harboring phaZ , a gene encoding poly(3-hydroxybutyrate) (PHB) depolymerase from Caldimonas manganoxidans, is constructed. The extra-cellular fraction of E. coli/pPHAZ exhibits a fast PHB degradation rate where it only took 35 h to completely degrade PHB films, while C. manganoxidans takes 81 h to do the same. The co-expression of ORF (a putative periplasmic substrate binding protein that is within the same operon of phaZ ) further improves the PHB degradation. While 28 h is needed for E. coli/pPHAZ to cause an 80% weight loss in PHB films, E. coli/pORFPHAZ needs only 21 h. Furthermore, it is able to degrade at-least four different polyesters, PHB, poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(butylene succinate-co-adipate) (PBSA). Testing of the time course of 3-hydroxybutyrate concentration and the turbidity of the degradation solutions over time shows that PhaZ has both exo- and endo-enzymatic activity. The whole-cell E. coli/pORFPHAZ can be used for recycling various polyesters while ORF can potentially be a universal element for enhancing the secretion of recombinant protein.

Bioderived and degradable polymers for transient electronics

“… 42,55,57,58 The catalytic domain of the enzyme hydrolyzes the polyester chains in PHB to yield a non‐toxic compound known as 3‐hydroxybutyric acid 27,42 . Recently, it was discovered that certain bacteria carry a specific gene ( phaZCma ) encoding for a PHB depolymerase that degrades PHB significantly faster; E.coli /pPHAZ requires 35 h to completely degrade PHB films, while C. manganoxidans requires 81 h to achieve the same 59 . Similar to factors affecting chemical degradation, PHB degradation is influenced by the molecular weight of the polymer as it affects solubility 27 .…”

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

“…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 (Anbukarasu et al, 2017) and pathogen detection platforms (Elias et al, 2018)-and recycling of biodegradable polymers (Lee et al, 2018).…”

“…Examples of expression of rPhaZs in E. coli include PhaZ2-PhaZ3 (Briese, Schmidt, & Jendrossek, 1994) and PhaZ7 (although for this specific PhaZ better expression was achieved with in Bacillus subtilis WB800) (Braaz, Handrick, & Jendrossek, 2003) from Paucimonas lemoignei and PhaZ from Caldimonas manganoxidans (Takeda et al, 2000;Lee et al, 2018). In some cases, purification of rPhaZs has also been performed: several PhaZs from P. lemoignei (PhaZ1-PhaZ5 (Jendrossek, Frisse, et al, 1995;Jendrossek, Müller, & Schlegel, 1993), PhaZ7 and related mutants (Jendrossek, Hermawan, Subedi, & Papageorgiou, 2013)), Pseudomonas stuzeri (Ohura, Kasuya, & Doi, 1999), Alcaligenes faecalis AE122 (Kita et al, 1997), Marinobacter sp.…”

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

“…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].…”

“…Examples of expression of rPhaZs in E. coli include PhaZ2-PhaZ3 [9] and PhaZ7 (although for this specific PhaZ better expression was achieved with in Bacillus subtilis WB800) [10] from Pseudomonas lemoignei, and PhaZ from Caldimonas manganoxidans [8,11]. In some cases, purification of rPhaZs has also been performed:…”