Abstract
The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimize several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimizing PHAs biosynthetic pathways. To this end, we developed several Escherichia coli cell-free systems for in vitro prototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customized our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimize in vivo PHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by approximately 50%. In cell-free transcription–translation prototyping reactions, gas chromatography–mass spectrometry quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the Native ΔPhaC_C319A (1.18 ± 0.39 µM), C104 ΔPhaC_C319A (4.62 ± 1.31 µM) and C101 ΔPhaC_C319A (2.65 ± 1.27 µM) phaCAB operons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the Native phaCAB operon in both in vitro and in vivo assays. Coupled cell-free biotransformation/transcription–translation reactions produced greater yields of 3HB (32.87 ± 6.58 µM), and these reactions were also used to characterize a Clostridium propionicum Acetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complement in vivo workflows for identifying additional strategies for optimizing PHAs production.
Highlights
The mass production of oil-derived plastics has resulted in widespread and potentially irreversible global ecological damage (Eriksen et al, 2014; Suaria et al, 2016)
Summary and Conclusions Despite the environmental consequences that are associated with the mass production of oil-derived plastics, global demand is likely to continue to increase unless viable economic alternatives are developed (Eriksen et al, 2014; Geyer et al, 2017)
The polyhydroxyalkanoates are a family of biodegradable biopolymers, that could potentially be used as sustainable alternatives to replace several widely used oilderived plastics
Summary
The mass production of oil-derived plastics has resulted in widespread and potentially irreversible global ecological damage (Eriksen et al, 2014; Suaria et al, 2016). Despite these environmental consequences, oil-derived plastics are still in very highdemand, due to their versatility and low-cost of production, that even with improvements in recycling processes, de novo plastic production has not been reduced (Geyer et al, 2017). 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 Alternative, enzymatic approaches for the production of PHAs have been investigated (Han et al, 2011; Opgenorth et al, 2016; Thomson et al, 2010)
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