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Influence of Unusual Co-substrates on the Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates Produced in Multistage Chemostat.

ABSTRACT: A two-stage chemostat cultivation was used to investigate the biosynthesis of functionalized medium-chain-length polyhydroxyalkanoate (mcl-PHA) in the ?-oxidation weakened strain of Pseudomonas putida KTQQ20. Chemostats were linked in sequence and allowed separation of biomass production in the first stage from the PHA synthesis in the second stage. Four parallel reactors in the second stage provided identical growth conditions and ensured that the only variable was the ratio of decanoic acid (C10) to an unusual PHA monomer precursor, such as 10-undecenoic acid (C11:1) or phenylvaleric acid (PhVA). Obtained PHA content was in the range of 10 to 25 wt%. When different ratios of C10 and C11:1 were fed to P. putida, the produced PHA had a slightly higher molar ratio in favor of C11:1-based 3-hydroxy-10-undecenoate. However, in case of PhVA a significantly lower incorporation of 3-hydroxy-5-phenylvalerate over 3-hydroxydecanoate took place when compared to the ratio of their precursors in the feed medium. A result that is explained by a less efficient uptake of PhVA compared to C10 and a 24% lower yield of polymer from the aromatic fatty acid ( y P H A - M P h V A = 0.25). In addition, PHA isolated from cultivations with PhVA resulted in the number average molecular weight M n ¯ two times lower than the PHA produced from C10 alone. Detection of products from PhVA metabolism in the culture supernatant showed that uptaken PhVA was not entirely converted into PHA, thus explaining the difference in the yield polymer from substrate. It was concluded that PhVA or its related metabolites increased the chain transfer rate during PHA biosynthesis in P. putida KTQQ20, resulting in a reduction of the polymer molecular weight.

SUBMITTER: Hanik N

PROVIDER: S-EPMC6848797 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Influence of Unusual Co-substrates on the Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates Produced in Multistage Chemostat.

Hanik Nils N Utsunomia Camila C Arai Shuzo S Matsumoto Ken'ichiro K Zinn Manfred M

Frontiers in bioengineering and biotechnology 20191105

A two-stage chemostat cultivation was used to investigate the biosynthesis of functionalized medium-chain-length polyhydroxyalkanoate (mcl-PHA) in the β-oxidation weakened strain of <i>Pseudomonas putida</i> KTQQ20. Chemostats were linked in sequence and allowed separation of biomass production in the first stage from the PHA synthesis in the second stage. Four parallel reactors in the second stage provided identical growth conditions and ensured that the only variable was the ratio of decanoic ...[more]

PMID: 31750296

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Project description:Polyhydroxyalkanoate (PHA) can be produced by microorganisms from renewable resources and is regarded as a promising bioplastic to replace petroleum-based plastics. Pseudomonas mendocina NK-01 is a medium-chain-length PHA (mcl-PHA)-producing strain and its whole-genome sequence is currently available. The yield of mcl-PHA in P. mendocina NK-01 is expected to be improved by applying a promoter engineering strategy. However, a limited number of well-characterized promoters has greatly restricted the application of promoter engineering for increasing the yield of mcl-PHA in P. mendocina NK-01. In this work, 10 endogenous promoters from P. mendocina NK-01 were identified based on RNA-seq and promoter prediction results. Subsequently, 10 putative promoters were characterized for their strength through the expression of a reporter gene gfp. As a result, five strong promoters designated as P4, P6, P9, P16 and P25 were identified based on transcriptional level and GFP fluorescence intensity measurements. To evaluate whether the screened promoters can be used to enhance transcription of PHA synthase gene (phaC), the three promoters P4, P6 and P16 were separately integrated into upstream of the phaC operon in the genome of P. mendocina NK-01, resulting in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1. As expected, the transcriptional levels of phaC1 and phaC2 in the recombinant strains were increased as shown by real-time quantitative RT-PCR. The phaZ gene encoding PHA depolymerase was further deleted to construct the recombinant strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1 and NKU-∆phaZ-16C1. The results from shake-flask fermentation indicated that the mcl-PHA titer of recombinant strain NKU-∆phaZ-16C1 was increased from 17 to 23 wt% compared with strain NKU-∆phaZ. This work provides a feasible method to discover strong promoters in P. mendocina NK-01 and highlights the potential of the screened endogenous strong promoters for metabolic engineering of P. mendocina NK-01 to increase the yield of mcl-PHA.

Project description:BackgroundMedium-chain-length polyhydroxyalkanoates (mcl-PHAs) containing various chain length monomers from C6 to C14 have more applications besides sustainable and environmental-friendly biomaterials owing to their superior physical and mechanical properties. We engineered a reversed fatty acid β-oxidation pathway in Escherichia coli that can synthesize mcl-PHA directly from glucose and achieved high yield. However, there were only even-chain monomers in the biosynthetic polymers. The need for mcl-PHA harboring both even- and odd-chain monomers with better and wider utility impels us to develop the biosynthetic routes for the production of the novel and unnatural mcl-PHA through rewiring the basic metabolism.ResultsIn the present study, a propionate assimilation and metabolic route was integrated into the reversed fatty acid β-oxidation in order to produce mcl-PHA consisting of both even- and odd-numbered monomers. The content of odd-numbered monomers in mcl-PHA was improved with the increased propionate addition. After further deletion of pyruvate oxidase (PoxB) and pyruvate formate-lyase (PflB), the metabolically engineered chassis E. coli LZ08 harboring pQQ05 and pZQ06 (overexpression of prpP and prpE genes from Ralstonia eutropha H16) innovatively accumulated 6.23 wt% mcl-PHA containing odd-chain monomers ranging from 7 to 13 carbon atoms about 20.03 mol%.ConclusionsThis is the first successful report on production of mcl-PHA harboring both even- and odd-chain monomers (C6-C14) synthesized from glucose and propionate in recombinant E. coli. This present study achieved the highest yield of de novo production of mcl-PHA containing odd-numbered monomers in E. coli at shake-flask fermentation level. Continued engineering of host strains and pathway enzymes will ultimately lead to more economical production of odd-chain monomers based on market demand. The synthetic pathway can provide a promising platform for production of other value-added chemicals and biomaterials that use acetyl-CoA and propionyl-CoA as versatile precursors and can be extended to other microorganisms as intelligent cell factories.

Project description:BACKGROUND:Our study aimed to search for novel bacteria capable of producing polyhydroxyalkanoates (PHAs) using crude glycerol residue obtained from biodiesel production in which used cooking oils were the substrates. RESULTS:Newly isolated bacteria from soils in Thailand were screened for the efficient production of PHAs from crude glycerol. The bacterial strains were cultivated on glucose, refined glycerol, crude glycerol, or various cooking oils (canola oil, palm oil, soybean oil, sunflower oil, corn oil, grape seed oil, olive oil, rice bran oil, camellia seed oil) for growth and PHA production. The effects of the total organic carbon (TOC) concentration and the mole ratio of carbon to nitrogen were investigated in batch cultivation. (1)H NMR, two dimensional-(1)H-correlation spectroscopy (2D-(1)H-COSY) and (13)C NMR analyses confirmed four bacterial strains were capable of producing medium-chain-length PHAs (mcl-PHAs), consisting of 3-hydroxyoctanoate (3HO) and 3-hydroxy-5-cis-dodecanoate (3H5DD), from crude glycerol. On the basis of phenotypic features and genotypic investigations, the bacterial strains were assigned as: ASC1, Acinetobacter genus (94.9% similarity); ASC2, Pseudomonas genus (99.2% similarity); ASC3, Enterobacter genus (99.2% similarity); ASC4, Bacillus genus (98.4% similarity). The highest amount of mcl-PHAs, 17.5 ± 0.8 g/L (content 61.8 ± 3.3% wt), with 3HO (14.7 ± 2.2 mol %), 3H5DD (85.3 ± 2.2 mol%), and a total biomass of 32.3 ± 0.3 g/L, was obtained from Pseudomonas sp. ASC2 in batch cultivation after 36 h. The mcl-PHAs recovered had a number-average molecular weight (M N) of 3.6 × 10(4) Da. Homopolymeric 3H5DD was obtained when the cultivation time was prolonged to 96 h. CONCLUSIONS:Novel PHA-producing strains were isolated and identified. These bacterial strains are able to produce mcl-PHAs from crude glycerol. The mcl-PHAs produced contained a high percentage of 3H5DD, which suggests their future application as softeners mixed with other biomaterials. The unsaturated side chain of 3H5DD monomers containing double bounds offers additional potential for improving the properties of the mcl-PHAs or extending their applications to the food industry.

Dataset Information

Influence of Unusual Co-substrates on the Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates Produced in Multistage Chemostat.

Publications

Influence of Unusual Co-substrates on the Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates Produced in Multistage Chemostat.

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