Conventional oil-derived polyolefin plastics exhibit useful characteristics that have extensive commercial applications. However, the accumulation of plastics in the environment and the non-renewable source of polyolefin plastics have stimulated interest in sustainable sources of plastic production. 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 [1,2]. Furthermore, P(3HB) has the added advantages of biodegradability, biocompatibility and, based upon several life cycle analyses, P(3HB) production is more environmentally sustainable than polyolefin plastic production [1,3,4]. 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 [5].

Genetic engineering approaches—in which the P(3HB)-producing operon, phaCAB, is cloned into Escherichia coli—have pioneered the industrial production of P(3HB) [6]. More recently, synthetic biology approaches involving the rational engineering of the phaCAB operon [7], and metabolic engineering strategies [8] have continued to increase the efficiency of P(3HB) production. In this paper we report on the forward-design and experimental validation of a rationally engineered phaCAB operon with a hybrid promoter design. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB).

The phaCAB operon from Ralstonia eutropha H16, is the most extensively studied P(3HB) synthesis operon [2,9]. It consists of three enzymes, which through a multi-stage enzymatic process generate P(3HB) inside the cell from the central metabolite acetyl-CoA [1]. The process is shown in Fig. 1A and is briefly summarised here. Firstly, PhaA (3-ketothiolase) combines two molecules of acetyl-CoA to form acetoacetyl-CoA. Next, PhaB (acetoacetyl-CoA reductase) reduces acetoacetyl-CoA to form (R)-3-hydroxybutyl-CoA, which is then polymerised by PhaC (PHA synthase) to form poly-3-hydroxybutyrate P(3HB). The phaCAB operon from R. eutropha H16 was originally cloned into E. coli in the late 1980s [2]. More recently the Tokyo Tech 2012 iGEM team (http://2012.igem.org/Team:Tokyo_Tech) cloned, characterised and standardized the native phaCAB operon (BBa_K934001) into a biobrick-compatible format for the synthetic biology community.

In order to increase P(3HB) production, this study used a forward-design approach that is based upon the engineering principles of synthetic biology [10]. Several phaCAB operons were rationally engineered (Fig. 1B). The constitutive phaCAB operon (BBa_K1149052) was designed such that the native promoter and RBS were replaced with the constitutive promoter J32104 from the Anderson collection [11] and the RBS B0034. The hybrid phaCAB operon design (BBa_K1149051) was constructed in parallel to the constitutive operon and was noted for its dual promoter and RBS combinations (Fig. 1B). Design considerations were based upon modeling simulations of the PhaCAB pathway in engineered E. coli. The model was comprised of the glycolysis pathway, the tricarboxylic acid (TCA) cycle and the phaCAB synthetic pathway (S1 Supporting Information; S2 Supporting Information). These pathways were coupled in order to reflect the metabolic flux [12] of several metabolites and species between them and to provide a qualitative indication of their influence on P(3HB) production.

A sensitivity analysis of species within the synthetic pathway revealed that increasing expression of phaB would be critical for directing the reaction flow towards the end product—an increase in P(3HB) production (S1 Supporting Information; S2 Supporting Information). In order to increase expression of phaB, further simulations predicted that of the several simulated phaCAB operon designs, an operon that incorporates the constitutive promoter, J23104, would result in the greatest increase in P(3HB) production (Fig. 2).

To experimentally validate our forward-design approach, E. coli MG1655 carrying either the empty vector (BBa_K608002), native phaCAB (BBa_K934001), constitutive phaCAB (BBa_K1149052) or hybrid phaCAB (BBa_K1149051) operons were cultured with Lysogeny broth (LB) media, supplemented with 3% glucose (w/v) for either 24 or 48 hours. P(3HB) was purified from each of the engineered populations using sodium hypochlorite and were weighed for comparison (Fig. 3A). Average P(3HB) production at 24 hours was around two-fold higher in constitutive phaCAB-engineered E. coli (0.49 g/L S.D. ± 0.06) compared to native phaCAB (0.22 g/L S.D. ± 0.18), while average P(3HB) production was six-fold higher in hybrid phaCAB-engineered E. coli (1.47 g/L S.D. ± 0.48). At 48 hours, P(3HB) production was around three-fold higher in constitutive phaCAB-engineered E. coli (1.05 g/L S.D. ± 0.26) and hybrid phaCAB-engineered E. coli (0.94 g/L S.D. ± 0.14), compared to native phaCAB (0.30 g/L S.D. ± 0.14) (Fig. 3A). In hybrid phaCAB-engineered E. coli we observed a decrease in P(3HB) content, expressed as a percentage of the cell dry weight (CDW) from 50% at 24 hours to 32% at 48 hours (Fig. 3B). Similar observations have been previously reported, and is understood as a consequence of competition for cellular metabolites between cell growth and P(3HB) production [13–15]. The empty vector-engineered E. coli did not produce detectable levels of P(3HB).

Qualitative analysis of Nile Red stained E. coli also confirmed that the constitutive and hybrid phaCAB operon designs increased P(3HB) production (S3 Supporting Information). It is likely that the dual promoter and RBS design (Fig. 1B) of the hybrid system results in a higher level of mRNA transcript production and/or ribosome recruitment and thus an increase in the expression of the PhaCAB enzymes. qRT-PCR analysis, confirmed that both the hybrid and constitutive phaCAB operon designs result in higher levels of phaCAB transcripts than that of the native phaCAB operon (S6 Supporting Information).

Interestingly, as shown in Fig. 3A, P(3HB) production from the hybrid operon at 24 h was greater than the combined P(3HB) production of the native and constitutive operons. Together, these data suggest that the hybrid promoter performs a multiplicative, rather than an additive combination of the native and constitutive promoter designs. Unlocking the design rules of the hybrid promoter may have applicability that extends beyond the phaCAB operon. For instance, Li et al. 2012, suggest that engineered promoter clusters may be a useful metabolic engineering approach for increasing metabolite production. Additionally, model-guided optimisation of the PhaCAB pathway led to the generation of the constitutive operon design, which in combination with the hybrid promoter, represent an emerging family of rationally engineered P(3HB)-producing operons.

The broader project aim was to exploit non-recyclable waste as a low-cost carbon source that could be utilized by engineered E. coli tasked with the de novo synthesis of P(3HB). Industrial processing of non-recyclable mixed waste into solid and liquefied fractions of mixed cellulosic (~80%) and plastic (~20%) streams is currently possible [16]. However, these processes could be made more efficient through the use of natural and genetically engineered organisms that can both degrade and use these waste streams as a low-cost carbon source for the production of bioplastics. Beneficially, this approach may also result in the diversion of non-recyclable waste away from environmentally damaging activities such as landfill or incineration [3]. To this end, phaCAB-engineered E. coli were cultured in waste-media, which is analogous to industrially processed liquefied cellulosic waste; and from these cultures, P(3HB) was purified according to a modified sodium hypochlorite protocol, where waste-media cultures were filtered prior to centrifugation to remove residual waste material. Purified P(3HB) from each culture was depolymerized with recombinant phaZ1 into monomeric 3-hydroxybutyrate (3HB), which could then be detected as a yellow colour change when these samples were analysed with the β-Hydroxybutyrate colorimetric assay kit (S4 Supporting Information). PhaZ1-treated P(3HB) from E. coli carrying either the empty vector or native phaCAB operon resulted in a negligible color change, suggesting that little or no P(3HB) was produced in those cultures. Whilst, PhaZ1-treated P(3HB) from E. coli carrying either the constitutive phaCAB or hybrid phaCAB operon, depolymerized into 3HB, as indicated by a detectable, yellow color change. Additionally, flow cytometry analysis of Nile Red stained, [17,18] phaCAB-engineered E. coli from waste-media cultures, confirmed an increase in P(3HB) content (Fig. 4).

In conclusion, our study used a forward-design approach to rationally engineer novel phaCAB operon designs that were experimentally validated to increase the production of P(3HB) in genetically engineered E. coli. Additionally, we have described the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB). With further development, these strategies, in combination with additional synthetic biology approaches [7,8], will further increase the efficiency and commercial viability of P(3HB) production.