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

Abstract

The polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcriptiontranslation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the polyhydroxyalkanoates biosynthetic pathways. Here we have developed several Escherichia coli cell-free transcription-translationbased systems for in vitro prototyping of polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcriptiontranslation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising in vivo polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through . CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/225144 doi: bioRxiv preprint first posted online 2 enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3µM (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a Clostridium propionicum propionyl CoA transferase enzyme that can recycle Acetyl-CoA. Together our data demonstrate that cell-free approaches can be used to complement in vivo workflows for identifying additional strategies for optimising polyhydroxyalkanoates production.

Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor

“…For example, environmental conditions can be varied within a wider range to favor product formation [ 4 ]; transportation issues across cell membranes are eliminated [ 5 ]; toxic compounds can be produced at much higher titers than the cell’s tolerance limit [ 6 ]. Because components of the biosynthetic pathways can be readily mix-and-matched in a combinatorial fashion, cell-free biosynthesis has also been used as a high-throughput prototyping tool to inform pathway design in whole-cell biosynthesis [ 7 , 8 ].…”

Cell-Free Biocatalysis for the Production of Platform Chemicals

“…Biologically based bioplastics to replace those derived from petroleum have been a source of interest for many research groups. Kelwick et al (2018) described an E. coli-based cell-free system for the production of polyhydroalkanoate (PHA) plastics from whey permeate in an enzyme cascade with constitutive phaCAB operon products, β-galactosidase and acetyl-CoA provided by glycolysis and aided by a plasmid carrying T 7 RNA polymerase. The authors note that the biodegradable PHA plastics are more expensive to produce than the petroleumsourced products and suggested that the system proposed would be valuable at the microplate level in the search for more efficient producer combinations (see section "Cell-Free Enzyme Pathways").…”

Custom-made transcriptional biosensors for metabolic engineering

“…These systems can also be an appropriate platform for production because of lower noise and toxicity and absence of resource competition between pathway and cell growth. To date, cell-free systems have been applied to implement pathways for violacein [49], 4-BDO [50], polyhydroxyalkanoates bioplastics [51], mevalonate [52], n-butanol [53] and raspberry ketone [54], using either transcriptiontranslation (TX-TL) systems, overexpressed enzymes in the crude extract or purified enzymes. Advantages and possibilities of cell-free systems for metabolic engineering has been reviewed elsewhere [55], and a methods chapter for pathway prototyping in cell-free systems has recently been published [56].…”