A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli

Abstract

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB).

Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

“…The constitutive C104 phaCAB operon construct (C104, BBa_K1149052; pRK61) was transformed into E. coli MG1655 to create strain, S_RK003. The Native (pRK51) and C104 (pRK61) phaCAB constructs are also available in the iGEM Registry of Standard Biological Parts (partsregistry.org) and were also reported in our previous study ( 13 ). The constitutive C101 phaCAB operon construct (C101—pRK14) is derived from C104, such that PCR (primer pairs RK024 CH101F and RK025 CH101R) was used to change the Anderson constitutive promoter to J23101.…”

“…Nile red plate assays for qualitative detection of PHAs were carried out as previously described ( 13 ). Briefly, E. coli MG1655 transformed with either a negative control plasmid (EV104 or EV101) or a phaCAB operon (Native, C104, C101 or Δ PhaC_C319A inactive PhaC operon versions) were grown in 5 ml 2×YT+P media (supplemented with 34 μg/ml Chloramphenicol) overnight at 37°C with shaking (220 rpm).…”

“…PHAs share several material characteristics with some of the most widely used oil-derived plastics, but beneficially, PHAs are also biodegradable ( 16 , 17 ). In order to develop PHAs as viable alternatives to oil-derived plastics, great efforts have been undertaken to design more efficient microbial PHAs production processes through the rational engineering of biosynthetic pathways ( 13 , 18–20 ), metabolite recycling processes (e.g. Acetyl-CoA) ( 21 , 22 ) and the use of industrially-sourced, low-cost feedstocks (e.g.…”

Biological Materials: The Next Frontier for Cell-Free Synthetic Biology

“…Unfortunately, the commercial impact of PHA-based bioplastics has been historically prohibited by their higher production costs than oil-derived plastics (Chen et al, 2020). However, more efficient PHAs production processes have been devised through the rational design of phaCAB biosynthetic pathways (Hiroe et al, 2012;Kelwick et al, 2015b;Li et al, 2016;Tao et al, 2017;Zhang X. et al, 2019), key metabolite recycling processes (e.g., Acetyl-CoA) (Matsumoto et al, 2013;Beckers et al, 2016), alternative microbial production hosts (e.g., Halomonas sp., Tan et al, 2011) and the use of industrially sourced, low-cost feedstocks (e.g., whey permeate) (Wong and Lee, 1998;Ahn et al, 2000;Kim, 2000;Nikel et al, 2006;Cui et al, 2016;Nielsen et al, 2017). Interestingly, several of these microbial PHAs production strategies are also compatible with cell-free synthetic biology reactions.…”

Auxotrophic Selection Strategy for Improved Production of Coenzyme B12 in Escherichia coli

“…The resultant strains (EDMB1-5 cob ) were cultivated, and the expression of metH was measured in the same manner (Figure 4). Although there was a gap between predicted strength and measured metH expression levels (Iverson et al, 2016;Kelwick et al, 2015;Noh et al, 2017), varied metH expression levels (up to 7.84fold) were successfully identified in these engineered strains as intended. Moreover, these strains showed higher metH expression up to 28.6-fold than the EDMB cob strain at an early stage of cultivation and appeared to maintain a relatively constant level during cultivation.…”