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

Nils Hanik,Shuzo Arai,Camila Utsunomia,Ken'Ichiro Matsumoto,Manfred Zinn

doi:10.3389/fbioe.2019.00301

Nils Hanik, Shuzo Arai + Show 3 more

Open Access

https://doi.org/10.3389/fbioe.2019.00301

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Abstract

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 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 ( = 0.25). In addition, PHA isolated from cultivations with PhVA resulted in the number average molecular weight 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.

Highlights

Tailor-made production of polyhydroxyalkanoates (PHA) bearing unconventional functional groups in the side chain is of high interest since their presence enables the control of the mechanical and physical properties already during biosynthesis (Hany et al, 2005; Tortajada et al, 2013)
We investigated in a two-stage chemostat the accumulation of medium-chain-length PHA (mcl-PHA) in P. putida KTQQ20 from unusual substrates as carbon sources in a co-feed experiment
While a change in propagation rate would lead to different amounts of polymer per gram of biomass, a higher chain transfer rate of the growing polymer chains for cultures accumulating on phenylvaleric acid (PhVA) would explain this observation

Summary

Introduction

Tailor-made production of polyhydroxyalkanoates (PHA) bearing unconventional functional groups in the side chain is of high interest since their presence enables the control of the mechanical and physical properties already during biosynthesis (Hany et al, 2005; Tortajada et al, 2013). These integrated groups enable a further fine-tuning of the PHA using mild, Unusual Co-substrates in Polyhydroxyalkanoate Biosynthesis chemical conditions (Levine et al, 2016). Growth substrates that went through β-oxidation build up a pool of different 3-hydroxyalkanoates that can serve for the synthesis of PHA. They differ in their number of C2 units removed during the β-oxidation process and result typically in copolymeric PHAs (Huisman et al, 1989; Ouyang et al, 2007)

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