Applied and Environmental Microbiology, September 2004, p . 5685-5687, Vol . 70, No . 9

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) .

 
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 .

 
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 .

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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