A promoter engineering-based strategy enhances polyhydroxyalkanoate production in Pseudomonas putida KT2440

Zhang, Yiting; Liu, Honglu; Liu, Yujie; Huo, Kaiyue; Wang, Shufang; Liu, Ruihua; Yang, Chao

doi:10.1016/j.ijbiomac.2021.09.142

“…In addition, several strategies have been tested to engineer PHA biosynthesis. These include metabolic engineering approaches such as the overproduction of the PHA monomer (R)−3HA-CoA through overexpression of PhaG that significantly increased PHA yields, but reduced overall cell dry weights [49], the combination of weakening phenazine and fatty acid beta-oxidation pathways with knockouts of PHA depolymerase PhaZ and overexpression techniques [50], and rational incorporation of strong promoters [51][52][53][54]. Moreover, few studies have examined the effect of differential degrees of incorporation of different monomers.…”

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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“…Genetic manipulation of the transcriptional level of BGCs has been used successfully to improve the production of natural products, and the most direct and convenient approach is to replace the native promoter of a gene cluster of interest with well-characterized inducible or constitutive promoters. ,, Zhang et al screened strong promoters from P. putida KT2440 by RNA-seq analysis and applied them to enhance the biosynthesis of polyhydroxyalkanoate in the strain KT2440 itself . In another study, native promoters were identified by RNA-seq and used to improve the production of actinorhodin and oxytetracycline in Streptomyces.…”

Section: Resultsmentioning

confidence: 99%

Enhancing the Production of Xenocoumacin 1 in Xenorhabdus nematophila CB6 by a Combinatorial Engineering Strategy

Qin

¹

,

Jia

²

,

Zheng

³

et al. 2023

J. Agric. Food Chem.

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Xenocoumacin 1 (Xcn1) is an excellent antimicrobial natural product against Phytophthora capsici. However, the commercial development of Xcn1 is hindered by the low yield, which results in high application costs. In this study, multiple metabolic strategies, including blocking the degradation pathway, promoter engineering, and deletion of competing biosynthetic gene clusters, were employed to improve the production of Xcn1, which was increased from 0.07 to 0.91 g/L. The formation of Xcn1 reached 1.94 g/L in the TB medium with the final strain T3 in a shake flask and further reached 3.52 g/L in a 5 L bioreactor, which is the highest yield ever reported. The engineered strain provides a valuable platform for production of Xcn1, and the possible commercial development of the biofungicide. We anticipate that the metabolic engineering strategies utilized in this study and the constructed constitutive promoter library can be widely applied to other bacteria of the genera Xenorhabdus and Photorhabdus.

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“…Based on the metabolic pathway of P. putida KT2440 to synthesize mcl-PHA, we applied various metabolic engineering strategies with the goal of producing mcl-PHA from lignocellulosic hydrolysate in the artificial microbial consortium. These included: 1) elimination of mcl-PHA solubilization ( Salvachua et al, 2020a ), 2) weakening of the fatty acid β-oxidation pathway to reduce the flux of intermediates to FFAs degradation ( Liu et al, 2011 ), 3) inhibition of competing pathways ( Borrero-de Acuña et al, 2014 ), and 4) enhancement of the conversion of β-oxidation intermediates to synthesize mcl-PHA precursors ( Zhang et al, 2021 ). To eliminate mcl-PHA depolymerization, the phaZ gene encoding PHA depolymerase was knocked out in the wild-type strain P. putida KT2440 as well as the engineered KTΔAB strain in which the fadA and fadB genes had been knocked out previously, resulting in the engineered strains KT2440ΔZ and KTΔABZ, respectively.…”

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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A promoter engineering-based strategy enhances polyhydroxyalkanoate production in Pseudomonas putida KT2440

Cited by 25 publications

References 37 publications

In silico identification of bacterial seaweed-degrading bioplastic producers

In silico identification of bacterial seaweed-degrading bioplastic producers

Enhancing the Production of Xenocoumacin 1 in Xenorhabdus nematophila CB6 by a Combinatorial Engineering Strategy

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

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