Biosynthesis of novel lactate-based polymers containing medium-chain-length 3-hydroxyalkanoates by recombinant Escherichia coli strains from glucose

Goto, Saki; Hokamura, Ayaka; Shiratsuchi, Hideki; Taguchi, Seiichi; Matsumoto, Ken’ichiro; Abe, Hideki; Tanaka, Kenji; Matsusaki, Hiromi

doi:10.1016/j.jbiosc.2019.01.009

Cited by 11 publications

(14 citation statements)

References 35 publications

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“…PhaC1 Ps STQK incorporates lactate (LA, 2-hydroxypropionate) [ 23 ], glycolate (GL, 2-hydroxyacetate) [ 26 ], 2-hydroxybutyrate (2HB) [ 27 , 28 ], and amino acid-derived 2HAs [ 29 ]. A random copolymer of LA and medium-chain-length (MCL) 3HA-CoAs (C 6–12 ) was also synthesized using the same enzyme [ 30 ]. Strict enantio-specificity of PHA synthase has enabled the synthesis of highly isotactic polymers from inexpensive racemic precursors, which is an advantage over ROP.…”

Section: Introductionmentioning

confidence: 99%

Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaCAR

et al. 2022

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Background Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized by PHA synthases. Naturally occurring PHA copolymers possess a random monomer sequence. The development of PhaCAR, a unique sequence-regulating PHA synthase, has enabled the spontaneous biosynthesis of PHA block copolymers. PhaCAR synthesizes both a block copolymer poly(2-hydroxybutyrate)-b-poly(3-hydroxybutyrate) [P(2HB)-b-P(3HB)], and a random copolymer, poly(3HB-co-3-hydroxyhexanoate), indicating that the combination of monomers determines the monomer sequence. Therefore, in this study, we explored the substrate scope of PhaCAR and the monomer sequences of the resulting copolymers to identify the determinants of the monomer sequence. PhaCAR is a class I PHA synthase that is thought to incorporate long-main-chain hydroxyalkanoates (LMC HAs, > C3 in the main [backbone] chain). Thus, the LMC monomers, 4-hydroxy-2-methylbutyrate (4H2MB), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HHx), as well as 2HB, 3HB, and 3-hydroxypropionate (3HP) were tested. Results Recombinant Escherichia coli harboring PhaCAR, CoA transferase and CoA ligase genes was used for PHA production. The medium contained the monomer precursors, 2HB, 3HB, 3HP, 4H2MB, 5HV, and 6HHx, either individually or in combination. As a result, homopolymers were obtained only for 3HB and 3HP. Moreover, 3HB and 3HP were randomly copolymerized by PhaCAR. 3HB-based binary copolymers P(3HB-co-LMC HA)s containing up to 2.9 mol% 4H2MB, 4.8 mol% 5HV, or 1.8 mol% 6HHx were produced. Differential scanning calorimetry analysis of the copolymers indicated that P(3HB-co-LMC HA)s had a random sequence. In contrast, combining 3HP and 2HB induced the synthesis of P(3HP)-b-P(2HB). Similarly, P(2HB) segment-containing block copolymers P(3HB-co-LMC HA)-b-P(2HB)s were synthesized. Binary copolymers of LMC HAs and 2HB were not obtained, indicating that the 3HB or 3HP unit is essential to the polymer synthesis. Conclusion PhaCAR possesses a wide substrate scope towards 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates. 3HB or 3HP units are essential for polymer synthesis using PhaCAR. The presence of a 2HB monomer is key to synthesizing block copolymers, such as P(3HP)-b-P(2HB) and P(3HB-co-LMC HA)-b-P(2HB)s. The copolymers that did not contain 2HB units had a random sequence. This study’s results provide insights into the mechanism of sequence regulation by PhaCAR and pave the way for designing PHA block copolymers.

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Section: Introductionmentioning

confidence: 99%

Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaCAR

et al. 2022

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“…PHAs are able to be produced naturally in bacteria such as Cupriavidus necator H16 (formerly, Ralstonia eutropha) [7] [8], Alcaligenes latus [9], Pseudomonas [10] [11] and the rhizobia [12]. However, PLA and its derivatives have only been produced through genetic engineering [4,5,[13][14][15][16], by introduction and expression of modified genes for two enzymes, propionate CoA-transferase (e.g. pct532) from Clostridium propionicum and PHA synthase (e.g.…”

Section: Introductionmentioning

confidence: 99%

“…phaC1400) from Pseudomonas. These studies, including recent reports of PLA/PHA copolymer production [13,14] have mainly focused on engineering Escherichia coli strains that are common model systems in the metabolic engineering field. E. coli strains have been engineered to produce a broad range of bioproducts such as biopolymers, biofuels, amino acids, and organic acids because molecular tools are intensively studied and widely available.…”

Section: Introductionmentioning

confidence: 99%

Lactic acid containing polymers produced in engineered Sinorhizobium meliloti and Pseudomonas putida

Tran

Charles

2020

PLoS ONE

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This study demonstrates that novel polymer production can be achieved by introducing pTAM, a broad-host-range plasmid expressing codon-optimized genes encoding Clostridium propionicum propionate CoA transferase (Pct Cp , Pct532) and a modified Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 Ps6-19 , PhaC1400), into phaC mutant strains of the native polymer producers Sinorhizobium meliloti and Pseudomonas putida. Both phenotypic analysis and gas chromatography analysis indicated the synthesis and accumulation of biopolymers in S. meliloti and P. putida strains. Expression in S. meliloti resulted in the production of PLA homopolymer up to 3.2% dried cell weight (DCW). The quaterpolymer P (3HB-co-LA-co-3HHx-co-3HO) was produced by expression in P. putida. The P. putida phaC mutant strain produced this type of polymer the most efficiently with polymer content of 42% DCW when cultured in defined media with the addition of sodium octanoate. This is the first report, to our knowledge, of the production of a range of different biopolymers using the same plasmid-based system in different backgrounds. In addition, it is the first time that the novel polymer (P(3HB-co-LA-co-3HHx-co-3HO)), has been reported being produced in bacteria.

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“…Optically pure lactic acid is a feedstock for the production of poly-lactic acid (PLA), a biobased and biodegradable plastic [1], and of lactate-based polyesters containing 3-hydroxyalkanoates [2]. Bacterial fermentation from sustainable lignocellulosic biomass is a promising alternative source to fossil fuel-derived chemicals.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome profile of carbon catabolite repression in an efficient l-(+)-lactic acid-producing bacterium Enterococcus mundtii QU25 grown in media with combinations of cellobiose, xylose, and glucose

et al. 2020

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Enterococcus mundtii QU25, a non-dairy lactic acid bacterium of the phylum Firmicutes, is capable of simultaneously fermenting cellobiose and xylose, and is described as a promising strain for the industrial production of optically pure l-lactic acid (≥ 99.9%) via homo-fermentation of lignocellulosic hydrolysates. Generally, Firmicutes bacteria show preferential consumption of sugar (usually glucose), termed carbon catabolite repression (CCR), while hampering the catabolism of other sugars. In our previous study, QU25 exhibited apparent CCR in a glucose-xylose mixture phenotypically, and transcriptional repression of the xylose operon encoding initial xylose metabolism genes, likely occurred in a CcpA-dependent manner. QU25 did not exhibit CCR phenotypically in a cellobiose-xylose mixture. The aim of the current study is to elucidate the transcriptional change associated with the simultaneous utilization of cellobiose and xylose. To this end, we performed RNA-seq analysis in the exponential growth phase of E. mundtii QU25 cells grown in glucose, cellobiose, and/or xylose as either sole or co-carbon sources. Our transcriptomic data showed that the xylose operon was weakly repressed in cells grown in a cellobiose-xylose mixture compared with that in cells grown in a glucose-xylose mixture. Furthermore, the gene expression of talC, the sole gene encoding transaldolase, is expected to be repressed by CcpA-mediated CCR. QU25 metabolized xylose without using transaldolase, which is necessary for homolactic fermentation from pentoses using the pentose-phosphate pathway. Hence, the metabolism of xylose in the presence of cellobiose by QU25 may have been due to 1) sufficient amounts of proteins encoded by the xylose operon genes for xylose metabolism despite of the slight repression of the operon, and 2) bypassing of the pentose-phosphate pathway without the TalC activity. Accordingly, we have determined the targets of genetic modification in QU25 to metabolize cellobiose, xylose and glucose simultaneously for application of the lactic fermentation from lignocellulosic hydrolysates.

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Biosynthesis of novel lactate-based polymers containing medium-chain-length 3-hydroxyalkanoates by recombinant Escherichia coli strains from glucose

Cited by 11 publications

References 35 publications

Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaCAR

Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaCAR

Lactic acid containing polymers produced in engineered Sinorhizobium meliloti and Pseudomonas putida

Transcriptome profile of carbon catabolite repression in an efficient l-(+)-lactic acid-producing bacterium Enterococcus mundtii QU25 grown in media with combinations of cellobiose, xylose, and glucose

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