Abstract
A high-throughput screening method based on the degree of polymerization (DP) of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC). In this method, PHA production was achieved using recombinant Escherichia coli supplemented with benzyl alcohol as a chain terminal compound. The cultured cells containing benzyl alcohol-capped PHA were decomposed by alkaline treatment, and the peaks of the decomposed monomer and benzyl alcohol were detected using HPLC. The DP of PHA could be determined from the peak ratio of the decomposed monomer to terminal benzyl alcohol. The measured DP was validated by other instrumental analyses using purified PHA samples. Using this system, mutants of PHA synthase from Bacillus cereus YB-4 (PhaRCYB4) were screened, and some enzymes capable of producing PHA with higher DP than the wild-type enzyme were obtained. The PHA yields of two of these enzymes were equivalent to the yield of the wild-type enzyme. Therefore, this screening method is suitable for the selection of beneficial mutants that can produce high molecular weight PHAs.
Highlights
A high-throughput screening method based on the degree of polymerization (DP) of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC)
We demonstrated that benzyl alcohol could be introduced at the PHA carboxyl end following addition to the culture medium of recombinant Escherichia coli expressing PHA synthase from Bacillus cereus YB-4 (PhaRCYB4 ) [16,17]
The molecular weight was determined by gel permeation chromatography (GPC), and the terminal modification yield was calculated from 1 H-nuclear magnetic resonance (NMR) spectra [17]
Summary
Chemicals were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) and TokyoChemical Industry Co., Ltd. (Tokyo, Japan). Poly(3-hydroxybutyrate) (P(3HB)) with an aromatic group at the carboxyl terminal (P(3HB)-93%, number-average molecular weight, Mn = 9600, benzyl alcohol terminal modification yield = 93%) was biosynthesized as follows: recombinant E. coli BW25113 ∆adhE (JW1228-KC), an alcohol dehydrogenase deletion strain, harboring pGEM-phaRCYB4 AB (carrying phaRCYB4 from B. cereus YB-4 and a monomer supplying phaAB from Ralstonia eutropha H16) [18] was cultured at 37 ◦ C for 72 h in Luria. Bertani (LB) medium (10 g/L sodium chloride [NaCl], 10 g/L tryptone, and 5 g/L yeast extract) supplemented with 20 g/L glucose and 1 mL/L benzyl alcohol. The mutant library of PhaRCYB4 from B. cereus YB-4 was constructed as follows: mutations were introduced by Diversify PCR Random Mutagenesis Kit (TaKaRa Bio Inc., Shiga, Japan) using two primers As previously described [20], for efficient gene evolution, polymerase chain reaction (PCR) buffer conditions #1, #3, and #5 were selected to introduce 2.0-, 2.7-, and
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