Biosynthesis and Characterization of Medium-Chain-Length Polyhydroxyalkanoate with an Enriched 3-Hydroxydodecanoate Monomer from a <i>Pseudomonas chlororaphis</i> Cell Factory

Li, Huiling; Deng, Ru-Xiang; Wang, Wei; Liu, Kaiquan; Hu, Hongbo; Huang, Xianqing; Zhang, Xuehong

doi:10.1021/acs.jafc.1c00500

“…Experimentation with multiple PHA-producing strains has shown this to be an effective approach at producing PHA with different properties and yields [39,47,48]. In addition, several strategies have been tested to engineer PHA biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, few studies have examined the effect of differential degrees of incorporation of different monomers. Enriched ratios of 3-hydroxydodecanoate in PHA produced by metabolically engineered Pseudomonas chloroaphis HT66 exhibit enhanced thermophysical and mechanical properties [50].…”

Section: Discussionmentioning

confidence: 99%

In silico identification of bacterial seaweed-degrading bioplastic producers

Leadbeater

¹

,

Bruce

²

,

Tonon

³

2022

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There is an urgent need to replace petroleum-based plastic with bio-based and biodegradable alternatives. Polyhydroxyalkanoates (PHAs) are attractive prospective replacements that exhibit desirable mechanical properties and are recyclable and biodegradable in terrestrial and marine environments. However, the production costs today still limit the economic sustainability of the PHA industry. Seaweed cultivation represents an opportunity for carbon capture, while also supplying a sustainable photosynthetic feedstock for PHA production. We mined existing gene and protein databases to identify bacteria able to grow and produce PHAs using seaweed-derived carbohydrates as substrates. There were no significant relationships between the genes involved in the deconstruction of algae polysaccharides and PHA production, with poor to negative correlations and diffused clustering suggesting evolutionary compartmentalism. We identified 2 987 bacterial candidates spanning 40 taxonomic families predominantly within Alphaproteobacteria, Gammaproteobacteria and Burkholderiales with enriched seaweed-degrading capacity that also harbour PHA synthesis potential. These included highly promising candidates with specialist and generalist specificities, including Alteromonas , Aquisphaera , Azotobacter , Bacillus , Caulobacter , Cellvibrionaceae , Duganella , Janthinobacterium , Massilia , Oxalobacteraceae , Parvularcula , Pirellulaceae , Pseudomonas , Rhizobacter , Rhodanobacter , Simiduia , Sphingobium , Sphingomonadaceae , Sphingomonas , Stieleria , Vibrio and Xanthomonas . In this enriched subset, the family-level densities of genes targeting green macroalgae polysaccharides were considerably higher (n=231.6±68.5) than enzymes targeting brown (n=65.34±13.12) and red (n=30.5±10.72) polysaccharides. Within these organisms, an abundance of FabG genes was observed, suggesting that the fatty acid de novo synthesis pathway supplies (R)−3-hydroxyacyl-CoA or 3-hydroxybutyryl-CoA from core metabolic processes and is the predominant mechanism of PHA production in these organisms. Our results facilitate extending seaweed biomass valorization in the context of consolidated biorefining for the production of bioplastics.

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“…To further increase the conversion efficiency of acetic acid and FFAs in these knockout strains, thus contributing to the availability of mcl-PHA precursors, the acs gene encoding acetyl coenzyme A synthase and the phaJ gene encoding enoyl coenzyme A hydratase were overexpressed to produce the strains KTΔABZF (p2-a-J) and KTΔABZFJ (p2-a-J). To enhance the polymerization of the PHA precursor 3-hydroxyacyl-CoA to mcl-PHA, the phaC1 and phaC2 genes encoding PHA polymerase ( Li et al, 2021 ) were expressed using the T3 promoter or Tac promoter, to produce strains KTΔABZF (p2 T3 -C1C2) and KTΔABZF (p2 Tac -C1C2), respectively. Furthermore, the acs and phaJ genes were overexpressed using the T3 promoter, while the phaC1 and phaC2 genes were overexpressed using the Tac promoter, resulting in the strain KTΔABZF (p2-a-J-C1C2).…”

Section: Resultsmentioning

confidence: 99%

Reconstruction and optimization of a Pseudomonas putida-Escherichia coli microbial consortium for mcl-PHA production from lignocellulosic biomass

Qin

¹

,

Zhu

²

,

Ai

³

et al. 2022

Front. Bioeng. Biotechnol.

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The demand for non-petroleum-based, especially biodegradable plastics has been on the rise in the last decades. Medium-chain-length polyhydroxyalkanoate (mcl-PHA) is a biopolymer composed of 6–14 carbon atoms produced from renewable feedstocks and has become the focus of research. In recent years, researchers aimed to overcome the disadvantages of single strains, and artificial microbial consortia have been developed into efficient platforms. In this work, we reconstructed the previously developed microbial consortium composed of engineered Pseudomonas putida KT∆ABZF (p2-a-J) and Escherichia coli ∆4D (ACP-SCLAC). The maximum titer of mcl-PHA reached 3.98 g/L using 10 g/L glucose, 5 g/L octanoic acid as substrates by the engineered P. putida KT∆ABZF (p2-a-J). On the other hand, the maximum synthesis capacity of the engineered E. coli ∆4D (ACP-SCLAC) was enhanced to 3.38 g/L acetic acid and 0.67 g/L free fatty acids (FFAs) using 10 g/L xylose as substrate. Based on the concept of “nutrient supply-detoxification,” the engineered E. coli ∆4D (ACP-SCLAC) provided nutrient for the engineered P. putida KT∆ABZF (p2-a-J) and it acted to detoxify the substrates. Through this functional division and rational design of the metabolic pathways, the engineered P. putida-E. coli microbial consortium could produce 1.30 g/L of mcl-PHA from 10 g/L glucose and xylose. Finally, the consortium produced 1.02 g/L of mcl-PHA using lignocellulosic hydrolysate containing 10.50 g/L glucose and 10.21 g/L xylose as the substrate. The consortium developed in this study has good potential for mcl-PHA production and provides a valuable reference for the production of high-value biological products using inexpensive carbon sources.

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“…Biorefinery and biomanufacturing processes for producing value-added biofuel, biobased materials, and chemicals are widely investigated with the development of metabolic engineering. Genetic engineering strategies for the high production of phenazines mainly include byproduct pathway elimination, , transcription factor engineering, , reinforcement of rate-limiting enzymes, ,, and efflux pump engineering .…”

Section: Introductionmentioning

confidence: 99%

“…One interesting phenomenon is that most Pseudomonas strains only produce PCA, whereas most Streptomyces strains have been reported as PDC derivatives producers, 22,23 which inspires us to search potential BGCs or strains for PDC production in the Streptomyces genus. Biorefinery and biomanufacturing processes for producing value-added biofuel, 24 biobased materials, 25 and chemicals 26 are widely investigated with the development of metabolic engineering. Genetic engineering strategies for the high production of phenazines mainly include byproduct pathway elimination, 27,28 transcription factor engineering, 28,29 reinforcement of rate-limiting enzymes, 27,28,30 and efflux pump engineering.…”

Section: ■ Introductionmentioning

confidence: 99%