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

ABSTRACT Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in Saccharomyces cerevisiae from intermediates of the β-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 β-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)(9)(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 ␤-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 ␤-oxidation cycle and have examined the effects of the expression of variants of the ␤-oxidation multifunctional enzyme on the quantity and monomer composition of PHA synthesized.
Expression of the PTS2-modified PHAC Re or PHAC Ac in the fox2⌬0 deletion mutant resulted in no PHA accumulation (data not shown), consistent with the requirement of a functional ␤-oxidation cycle for SCL-MCL PHA synthesis. Thus, fox2⌬0 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.
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-3hydroxydecanoyl-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-3hydroxyacyl-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 ␤-oxidation intermediates is possible through the use of a mutant MFE-2 enzyme having reduced dehydrogenase activity for short-chain R-3-hydroxyacyl-CoAs.