Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction

Alsiyabi, Adil; Brown, Brandi; Immethun, Cheryl M.; Long, Dianna; Wilkins, Mark R.; Saha, Rajib

doi:10.1016/j.ymben.2021.08.008

Cited by 21 publications

(38 citation statements)

References 54 publications

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“…TEM was conducted to visualize granules inside the wild type and phaP1 strains grown on p- coumarate in both aerobic and anaerobic conditions as described previously ( Alsiyabi et al, 2021 ). Briefly, the samples were fixed in a 2% glutaraldehyde and 1.5% paraformaldehyde in 100 mM sodium cacodylate buffer and submitted to the Nebraska Center for Biotechnology Morrison Microscopy Core Research Facility at the University of Nebraska-Lincoln.…”

Section: Methodsmentioning

confidence: 99%

“…The trade-off plot between PHB and biomass production for the phaP1 strain under aerobic conditions was generated by simulating (via flux balance analysis) aerobic growth on p -coumarate ( Orth et al, 2010 ) using R. palustris' genome-scale metabolic model iRpa940 ( Alsiyabi et al, 2019 , 2021 ). The maximal PHB and biomass generation rates were first determined by maximizing their respective reactions in the model.…”

Section: Methodsmentioning

confidence: 99%

“…Next, biomass production was maximized while PHB production rate was set to increasing values between zero and maximal production. The maximum rate of carbon fixation was assumed to be equal to the rate in anaerobic growth ( Alsiyabi et al, 2021 ). Furthermore, arbitrary uptake rates were set for p- coumarate and oxygen since the actual uptake rates are not known.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, R. palustris can break down numerous aromatic compounds derived from lignin while also potentially accomplishing complete degradation of these compounds to intermediates in the citric acid cycle that are important building blocks for bioplastic development. Our recent works show that R. palustris can utilize the major LBPs p- coumarate and coniferyl alcohol to produce bioplastics ( Alsiyabi et al, 2021 ; Brown et al, 2020 ). We also revealed that R. palustris can create a copolymer of PHB called poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), which is a copolymer of the 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomers that has more ideal thermomechanical properties compared to PHB alone ( Li et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

“…R. palustris’ ability to produce PHBV from lignocellulosic biomass renders it an ideal candidate for engineering toward industrial PHA production from lignin. However, our recent efforts also revealed that granule formation may be a limiting factor for maximum PHBV production due to one large PHBV granule formed inside the cells ( Alsiyabi et al, 2021 ). Thus, heterologous expression of phap1 was implemented in R. palustris with the aim of overproducing PHBV from lignocellulosic biomass by fostering smaller and more abundant granules to utilize more of the intracellular space.…”

Section: Introductionmentioning

confidence: 99%

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Heterologous phasin expression in Rhodopseudomonas palustris CGA009 for bioplastic production from lignocellulosic biomass

Brown

Immethun

Alsiyabi

et al. 2022

Metabolic Engineering Communications

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Rhodopseudomonas palustris CGA009 is a metabolically robust microbe that can utilize lignin breakdown products to produce polyhydroxyalkanoates (PHAs), biopolymers with the potential to replace conventional plastics. Our recent efforts suggest PHA granule formation is a limiting factor for maximum production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by R. palustris. The Phap1 phasin ( phaP1 ) from the PHB-producing model bacterium Cupriavidus necator H16 was expressed in R. palustris with the aim of overproducing PHBV from the lignin breakdown product p- coumarate by fostering smaller and more abundant granules. Expression of phaP1 yielded PHBV production from R. palustris aerobically (0.7 g/L), which does not occur in the wild-type strain, and led to a significantly higher PHBV titer than wild-type anaerobic production (0.41 g/L). The 3HV fractions were also significantly increased under both anaerobic and aerobic conditions, which boosts thermomechanical properties and potential for application. Thus, heterologous phasin expression in R. palustris provides flexibility for industrial processing and could foster compositional changes in copolymers with better thermomechanical properties compared to PHB alone.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Heterologous phasin expression in Rhodopseudomonas palustris CGA009 for bioplastic production from lignocellulosic biomass

Brown

Immethun

Alsiyabi

et al. 2022

Metabolic Engineering Communications

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Eliminating genes for a two‐component system increases PHB productivity in Cupriavidus basilensis 4G11 under PHB suppressing, nonstress conditions

Sander,

Abel,

Friedline

et al. 2023

Biotech & Bioengineering

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Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly‐hydroxybutyrate (PHB) making them attractive candidates for bioplastic production. The native synthesis of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB‐TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two‐component system, which when omitted from the genome enable increased PHB productivity in balanced, nonstress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain upon deleting each gene individually from the genome. The increased PHB phenotype disappears, however, in nitrogen‐free unbalanced growth conditions suggesting the phenotype is specific to fast‐growing, replete, nonstress growth. Bioproduction modeling suggests this phenotype could be due to a decreased reliance on metabolic stress induced by nitrogen limitation to initiate PHB production in the mutant strains. Due to uncertainty in the two‐component system's input signal and regulon, the mechanism by which these genes impart this phenotype remains unclear. Such strains may allow for the use of single‐stage, continuous bioreactor systems, which are far simpler than many PHB bioproduction schemes used previously, given a similar product yield to batch systems in such a configuration. Bioproductivity modeling suggests that omitting this regulation in the cells may increase PHB productivity up to 24% relative to the wildtype organism when using single‐stage continuous systems. This work expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes.

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Phosphate-inducible poly-hydroxy butyrate production dynamics in CO2 supplemented upscaled cultivation of engineered Phaeodactylum tricornutum

et al. 2022

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Diatoms such as Phaeodactylum tricornutum are emerging as sustainable alternatives to traditional eukaryotic microbial cell factories. In order to facilitate a viable process for production of heterologous metabolites, a rational genetic design specifically tailored to metabolic requirements as well as optimised culture conditions are required. In this study we investigated the effect of constitutive and inducible expression of the heterologous poly-3-hydroxybutyrate (PHB) pathway in P. tricornutum using non-integrative episomes in 3 different configurations. Constitutive expression led to downregulation of at least one individual gene out of three (phaA, phaB and phaC) and was outperformed by inducible expression. To further asses and optimise the dynamics of PHB accumulation driven by the inducible alkaline phosphatase 1 promoter, we upscaled the production to lab-scale bioreactors and tested the effect of supplemented CO2 on biomass and PHB accumulation. While ambient CO2 cultivation resulted in a maximum PHB yield of 2.3% cell dry weight (CDW) on day 11, under elevated CO2 concentrations PHB yield peaked at 1.7% CDW on day 8, coincident with PHB titres at 27.9 mg L−1 that were approximately threefold higher than ambient CO2. With other more valuable bio-products in mind, these results highlight the importance of the genetic design as well as substrate availability to supply additional reduction equivalents to boost biomass accumulation and relieve potential enzymatic bottlenecks for improved product accumulation.

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Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction

Cited by 21 publications

References 54 publications

Heterologous phasin expression in Rhodopseudomonas palustris CGA009 for bioplastic production from lignocellulosic biomass

Heterologous phasin expression in Rhodopseudomonas palustris CGA009 for bioplastic production from lignocellulosic biomass

Eliminating genes for a two‐component system increases PHB productivity in Cupriavidus basilensis 4G11 under PHB suppressing, nonstress conditions

Phosphate-inducible poly-hydroxy butyrate production dynamics in CO2 supplemented upscaled cultivation of engineered Phaeodactylum tricornutum

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