In vitro synthesis of polyhydroxyalkanoates using thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase from thermotorelant bacteria

Tajima, Kenji; Han, Xuerong; Hashimoto, Yoshiki; Satoh, Yasuharu; Satoh, Toshifumi; Taguchi, Seiichi

doi:10.1016/j.jbiosc.2016.06.001

Cited by 29 publications

(18 citation statements)

References 34 publications

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“…Tajima et al [113] reported PHA in vitro synthesis by the engineered thermostable PHA synthase PhaC1SG(STQK). In this system, however, also non-thermostable enzymes from mesophilic organisms, namely acetyl-CoA synthetase (ACS) and CoA transferase (CT), were used to biosynthesize the monomers.…”

Section: Thermophile Pha Producersmentioning

confidence: 99%

Production of Polyhydroxyalkanoate (PHA) Biopolyesters by Extremophiles?

Koller¹

2017

MOJPS

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The article reviews the current state of knowledge of the production of polyhydroxyalkanoate (PHA) biopolyesters under extreme environmental conditions.Although PHA production by extremophiles is not realized yet at industrial scale, significant PHA accumulation under high salinity or extreme pH-or temperature conditions was reported for diverse representatives of the microbial domains of both Archaea and Bacteria. Several mechanisms were proposed to explain the mechanistic role of PHA and their monomers as microbial cell-and enzyme protective chaperons and the factors boosting PHA biosynthesis under environmental stress conditions. The potential of selected extremophile strains, isolated from extreme environments like glaciers, hot springs, saline brines, or from habitats highly polluted with heavy metals or solvents, for efficient future PHA production on an industrially relevant scale is assessed based on the basic data available in the scientific literature. The article reveals that, beside the needed optimization of other cost-decisive factors like inexpensive raw materials or efficient downstream processing, the application of extremophile production strains can drastically safe energy costs, are easily accessible towards long-term cultivation in chemostat processes, and therefore might pave the way towards cost-efficient PHA production, even combined with safe disposal of industrial waste streams. However, further challenges have still to be overcome in terms of strain improvement and process engineering aspects.

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Section: Thermophile Pha Producersmentioning

confidence: 99%

Production of Polyhydroxyalkanoate (PHA) Biopolyesters by Extremophiles?

Koller¹

2017

MOJPS

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“…Examples of invitro synthesis include the production of PHB from R3HBA by a three-enzyme system that only consumes ATP and the monomer . The yield of this system was recently improved by using a thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase (Tajima et al, 2016). A polymer incorporating lactate and R3HBA residues was produced by Tajima et al (2012) by using an engineered PHA synthase capable of lactate polymerization.…”

Section: Potential Uses Of Polyhydroxyalkanoates and Its Monomersmentioning

confidence: 99%

Beyond Intracellular Accumulation of Polyhydroxyalkanoates: Chiral Hydroxyalkanoic Acids and Polymer Secretion

Yáñez

Conejeros

Vergara-Fernández

et al. 2020

Front. Bioeng. Biotechnol.

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Polyhydroxyalkanoates (PHAs) are ubiquitous prokaryotic storage compounds of carbon and energy, acting as sinks for reducing power during periods of surplus of carbon source relative to other nutrients. With close to 150 different hydroxyalkanoate monomers identified, the structure and properties of these polyesters can be adjusted to serve applications ranging from food packaging to biomedical uses. Despite its versatility and the intensive research in the area over the last three decades, the market share of PHAs is still low. While considerable rich literature has accumulated concerning biochemical, physiological, and genetic aspects of PHAs intracellular accumulation, the costs of substrates and processing costs, including the extraction of the polymer accumulated in intracellular granules, still hampers a more widespread use of this family of polymers. This review presents a comprehensive survey and critical analysis of the process engineering and metabolic engineering strategies reported in literature aimed at the production of chiral (R)-hydroxycarboxylic acids (RHAs), either from the accumulated polymer or by bypassing the accumulation of PHAs using metabolically engineered bacteria, and the strategies developed to recover the accumulated polymer without using conventional downstream separations processes. Each of these topics, that have received less attention compared to PHAs accumulation, could potentially improve the economy of PHAs production and use. (R)-hydroxycarboxylic acids can be used as chiral precursors, thanks to its easily modifiable functional groups, and can be either produced de-novo or be obtained from recycled PHA products. On the other hand, efficient mechanisms of PHAs release from bacterial cells, including controlled cell lysis and PHA excretion, could reduce downstream costs and simplify the polymer recovery process.

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“…CoA is relatively modest and did not result in increased (R)-3HB-CoA content (data not shown), these data suggest that coupled cell-free biotransformation and TX-TL prototyping could be useful in helping to identify metabolite recycling enzymes that might improve PHAs production in vivo. Indeed, in other experimental contexts pct genes from both C. propionicum and Megasphaera elsdenii have been shown to enhance in vivo Acetyl-CoA content (Ken'ichiro Matsumoto et al, 2013aMatsumoto et al, , 2013bSelmer et al, 2002;Tajima et al, 2016).…”

Section: Biotransformation Of Whey Permeate In Phacab-engineered Cellmentioning

confidence: 99%

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

Kelwick

Ricci²,

Chee

et al. 2017

Preprint

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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.

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In vitro synthesis of polyhydroxyalkanoates using thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase from thermotorelant bacteria

Cited by 29 publications

References 34 publications

Production of Polyhydroxyalkanoate (PHA) Biopolyesters by Extremophiles?

Production of Polyhydroxyalkanoate (PHA) Biopolyesters by Extremophiles?

Beyond Intracellular Accumulation of Polyhydroxyalkanoates: Chiral Hydroxyalkanoic Acids and Polymer Secretion

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

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