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Applied and Environmental Microbiology, September 2004, p. 5685-5687, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5685-5687.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Increasing the Carbon Flux toward Synthesis of Short-Chain-Length—Medium-Chain-Length Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae through Modification of the ß-Oxidation Cycle

Valeria Cora de Oliveira, Isamu Maeda, Syndie Delessert, and Yves Poirier^*

Département de Biologie Moléculaire Végétale, Université de Lausanne, Lausanne, Switzerland

Received 20 April 2004/ Accepted 19 May 2004

ABSTRACT

Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in Saccharomyces cerevisiae from intermediates of the ß-oxidation cycle by expressing the polyhydroxyalkanoate synthases from Aeromonas caviae and Ralstonia eutropha in the peroxisomes. The quantity of polymer produced was increased by using a mutant of the ß-oxidation-associated multifunctional enzyme with low dehydrogenase activity toward R-3-hydroxybutyryl coenzyme A.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxy acids naturally synthesized as intracellular inclusions by a wide variety of bacteria (11, 14, 15). These polymers have attracted considerable attention because of their properties as biodegradable plastics and elastomers. PHAs can be subdivided into three main groups: namely, short-chain-length PHAs (SCL PHAs) containing mainly 3-hydroxy acids ranging from 3 to 5 carbons, medium-chain-length PHAs (MCL PHAs) containing 3-hydroxy acids ranging from 6 to 16 carbons, and the hybrid SCL-MCL PHAs containing 3-hydroxy acids from 4 to 12 carbons.

One of the main limitations for the use of PHAs as biodegradable plastics used in high-volume, low-value commodity products is their relatively high production cost through bacterial fermentation relative to petroleum-derived plastics such as polypropylene. Synthesis of PHAs has been demonstrated in several genetically engineered plants as well as in recombinant yeast (4, 8-10, 13). Synthesis of PHAs in crop plants has been seen as a promising alternative approach for their production on a large scale and at a low cost (7, 11). However, much remains to be learned on how to improve the yield and monomer composition of PHA produced in eukaryotic hosts, such as plants or yeast.

Synthesis of the elastomer MCL PHA from the polymerization of the R-3-hydroxyacyl coenzyme A (CoA) intermediates of the ß-oxidation cycle has recently been demonstrated in Saccharomyces cerevisiae and Pichia pastoris expressing the Pseudomonas aeruginosa PHA synthase in the peroxisomes (9, 10). In this work, we explored the synthesis in S. cerevisiae of SCL-MCL PHA containing 3-hydroxyacids of 4 and 6 carbons, since this PHA combines the properties of flexibility and toughness similar to those of polypropylene and desired for bulk commodity plastics (1). We have targeted two distinct PHA synthases to yeast peroxisomes to access the R-3-hydroxyacyl-CoA intermediates of the ß-oxidation cycle and have examined the effects of the expression of variants of the ß-oxidation multifunctional enzyme on the quantity and monomer composition of PHA synthesized.

The PHAC synthases from Ralstonia eutropha (PHAC_Re) and Aeromonas caviae (PHAC_Ac), two bacteria known to produce SCL or SCL-MCL PHA, were modified at the N termini by using oligonucleotides to add the first 16 amino acids derived from the S. cerevisiae peroxisomal 3-ketothiolase protein (PTO1, or FOX3), which harbors a peroxisomal targeting sequence (PTS2) (3) (Fig. 1). Previous experiments have shown that these amino-terminal 16 amino acids are necessary and sufficient to target cytoplasmic proteins to the peroxisome (3). The chimeric genes were cloned into the yeast centromeric vector p416GPD, putting the genes under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter, resulting in the constructs p415GPD::PTS2-PHAC_Re and p415GPD::PTS2-PHAC_Ac (6). Mutants with mutations in the 3-hydroxyacyl-CoA dehydrogenase A and B domains of the S. cerevisae multifunctional enzyme (MFE-2) encoded by the FOX2 gene have been previously described by Qin et al. (12). Briefly, the MFE-2(a) mutant retains a broad activity towards short (C4)-, medium (C10)-, and long (C16)-chain R-3-hydroxyacyl-CoAs, while the MFE-2(b) mutant shows highest activity with medium- and long-chain R-3-hydroxyacyl-CoAs and does not accept the short-chain R-3-hydroxybutyryl-CoA (12). The plasmid pYE352::ScMFE-2 containing the intact multifunctional gene from S. cerevisiae, as well as the plasmids pYE352::ScMFE-2(a) and pYE352::ScMFE-2(b) containing the mutated variants of the MFE-2 gene, have been previously described (12). All MFE-2 gene constructs were expressed in the vector pYE352, placing the genes under the control of the catalase A (CTA1) promoter and terminator (12). Plasmids harboring the various PHA synthases and MFE-2 constructs were transformed by the lithium acetate procedure (2) into the S. cerevisiae mutant fox20 (YKR009C::kanMX4) in the BY4742 background (MAT his31 leu20 lys20 ura30) obtained from EUROSCARF (http://www.uni-frankfurt.de/fb15/mikro/euroscarf/index.html). For the synthesis of PHA, cells were grown in selective medium containing 0.1% (vol/vol) oleic acid, and the polymer was analyzed as previously described (9).

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FIG. 1. DNA constructs used to express the PHA synthase of R. eutropha or A. caviae in S. cerevisae. The 16 amino acids of the S. cerevisiae 3-ketothioase (FOX3) harboring the peroxisome targeting sequence (PTS2) are indicated in capital letters in the first open box. The cloning strategy used to make an in-frame fusion between PTS2 and either PHACAc or PHACRe synthase (top and bottom lines of the second open box, respectively) created a histine amino acid, indicated in italics. The promoter of the glyceraldehyde-3-phosphate dehydrogenase promoter (GPD-Pr) and terminator of the cytochrome c oxidase (CYC1-Tr) are indicated by shaded boxes. B, BamHI; H, HindIII. The figure was not drawn to scale.

Expression of the PTS2-modified PHAC_Re or PHAC_Ac in the fox20 deletion mutant resulted in no PHA accumulation (data not shown), consistent with the requirement of a functional ß-oxidation cycle for SCL-MCL PHA synthesis. Thus, fox20 cells expressing PHAC_Re or PHAC_Ac were retransformed with the plasmid pYE352::ScMFE-2, pYE352::ScMFE-2(a), or pYE352::ScMFE-2(b). The quantity and monomer composition of PHA synthesized in the various strains grown in media containing 0.1% oleic acid as the main carbon source are shown in Table 1.

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TABLE 1. Synthesis of SCL-MCL PHA in S. cerevisiae through coexpression of R. eutropha or A. caviae PHA synthase and variants of S. cerevisiae MFE-2

Both the PHA quantity and monomer composition remained relatively unchanged in the strains fox20/PHAC_Re/ScMFE-2 and fox20/PHAC_Re/ScMFE-2(a). In contrast, strain fox20/PHAC_Re/ScMFE-2(b) accumulated considerably more PHA then the two other strains, increasing from 1.5 x 10^–4 g of PHA per g of cell (dry weight) for fox20/PHAC_Re/ScMFE-2 and fox20/PHAC_Re/ScMFE-2(a) to 1.1 x 10^–3 g of PHA/g of cell (dry weight) for strain fox20/PHAC_Re/ScMFE-2(b). No significant differences were observed in the PHA monomer composition between cells expressing the wild type and the variant MFE-2. In parallel to the data obtained with the PHAC_Re, the quantity and monomer composition of the PHA produced in the fox20/PHAC_Ac/ScMFE-2 and fox20/PHAC_Ac/ScMFE-2(a) remained unchanged, while there was a 3.8-fold increase in the quantity of PHA in fox20/PHAC_Ac/ScMFE-2(b), from 1.3 x 10^–4 g of PHA/g of cell (dry weight) to 5.0 x 10^–4 g of PHA/g of cell (dry weight). Furthermore, a small but significant decrease in the quantity of the 3-hydroxyhexanoic acid monomer is observed, going from 14 mol% in the fox20/PHAC_Ac/ScMFE-2 and fox20/PHAC_Ac/ScMFE-2(a) strains to 8 mol% for fox20/PHAC_Ac/ScMFE-2(b).

It has been previously shown for MCL PHA synthesized in recombinant S. cerevisiae expressing the PHA synthase from P. aeruginosa (PHAC_Pa) in the peroxisomes that coexpression of the MFE-2(b) variant resulted in an approximate twofold increase in the proportion of the 5- and 6-carbon monomers but had no impact on the quantity of PHA synthesized (5). The effect of the expression of the MFE-2(b) on PHA quantity and monomer composition can be explained by the potential impact of the expression of MFE-2(b) on the R-3-hydroxyacyl-CoA pools. In vitro measurements of the k_cat value of the MFE-2(b) revealed an undetectable dehydrogenase activity toward R-3-hydroxybutyryl-CoA, while k_cat values toward R-3-hydroxydecanoyl-CoA and R-3-hydroxyhexadecanoyl-CoA were minimally changed compared to those of the wild type (12). Thus, expression of MFE-2(b) would result in a shift in the relative abundance of the various R-3-hydroxyacyl-CoAs, with the short-chain R-3-hydroxyacyl-CoAs being relatively more abundant compared to the medium- or long-chain R-3-hydroxyacyl-CoAs, and thus more available for their incorporation into PHA. In the case of coexpression of PHAC_Pa with MFE-2(b), although the k_cat values for R-3-hydroxyvaleryl-CoA and R-3-hydroxyhexanoyl-CoA were not measured, the fact that the proportion of the 5- and 6-carbon monomers increased indicates that the affinity of the MFE-2(b) for these substrates is also probably reduced, and thus the availability of these intermediates for PHA synthesis increased. However, since the 5- and 6-carbon monomers represent only a small fraction of the monomers present in MCL PHA, the impact on the total amount of PHA is very limited. In contrast, in the case of the synthesis of SCL-MCL PHA from the coexpression of PHAC_Re or PHAC_Ac with MFE-2(b), the increased availability of short-chain R-3-hydroxyacyl-CoAs has a significant impact on PHA quantity, since monomers between 4 and 6 carbons form the entirety of the SCL-MCL PHA synthesized in these cells. The fact that a small but significant decrease in the proportion of the R-3-hydroxyhexanoic acid monomer is observed in cells expressing the PHAC_Ac and MFE-2(b) indicates that the variant enzyme may retain some activity toward R-3-hydroxyhexanoyl-CoA, thus decreasing the relative abundance of R-3-hydroxyhexanoyl-CoA compared to R-3-hydroxybutyryl-CoA.

In conclusion, this work demonstrates that improvement in the quantity of SCL-MCL PHA synthesized in S. cerevisiae peroxisomes from ß-oxidation intermediates is possible through the use of a mutant MFE-2 enzyme having reduced dehydrogenase activity for short-chain R-3-hydroxyacyl-CoAs.

ACKNOWLEDGMENTS

V.C.D.O. was a recipient of a bursary from the Commission Fédérale des Bourses pour Étudiants Étrangers, and I.M. was a recipient of a fellowship of the Fonds National Suisse de la Recherche Scientifique (83JS-067392). This research was also partially funded by the Université de Lausanne, by the Canton de Vaud, and by a grant from the Fonds National Suisse de la Recherche Scientifique (3100-061731).

We thank Y. Doi (RIKEN, Japan) for providing the PHA synthase from A. caviae, as well as Silvia Marchesini and Simon Goepfert (Université de Lausanne) for help with the gas chromatography-mass spectrometry.

 
* Corresponding author. Mailing address: Département de Biologie Moléculaire Végétale, Université de Lausanne, CH-1015 Lausanne, Switzerland. Phone: 41 21 692 4222. Fax: 41 21 692 4195. E-mail: yves.poirier{at}ie-bpv.unil.ch.

FOOTNOTES

Present address: Instituto de Ciências Biomédicas II, Universidade de So Paulo, São Paulo, Brazil.

Present address: Laboratory of Applied Microbiology, Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya 321-8505, Japan.

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Applied and Environmental Microbiology, September 2004, p. 5685-5687, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5685-5687.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Zhang, B., Carlson, R., Srienc, F. (2006). Engineering the Monomer Composition of Polyhydroxyalkanoates Synthesized in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 72: 536-543 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Oliveira, V. C. Articles by Poirier, Y. Search for Related Content PubMed PubMed Citation Articles by de Oliveira, V. C. Articles by Poirier, Y. Agricola Articles by de Oliveira, V. C. Articles by Poirier, Y.