Modification of acetoacetyl-CoA reduction step in Ralstonia eutropha for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from structurally unrelated compounds

Background Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is a bacterial polyester with high biodegradability, even in marine environments. Ralstonia eutropha has been engineered for the biosynthesis of P(3HB-co-3HHx) from vegetable oils, but its production from structurally unrelated carbon sources remains unsatisfactory. Results Ralstonia eutropha strains capable of synthesizing P(3HB-co-3HHx) from not only fructose but also glucose and glycerol were constructed by integrating previously established engineering strategies. Further modifications were made at the acetoacetyl-CoA reduction step determining flux distribution responsible for the copolymer composition. When the major acetoacetyl-CoA reductase (PhaB1) was replaced by a low-activity paralog (PhaB2) or enzymes for reverse β-oxidation, copolyesters with high 3HHx composition were efficiently synthesized from glucose, possibly due to enhanced formation of butyryl-CoA from acetoacetyl-CoA via (S)-3HB-CoA. P(3HB-co-3HHx) composed of 7.0 mol% and 12.1 mol% 3HHx fractions, adequate for practical applications, were produced at cellular contents of 71.4 wt% and 75.3 wt%, respectively. The replacement by low-affinity mutants of PhaB1 had little impact on the PHA biosynthesis on glucose, but slightly affected those on fructose, suggesting altered metabolic regulation depending on the sugar-transport machinery. PhaB1 mostly acted in the conversion of acetoacetyl-CoA when the cells were grown on glycerol, as copolyester biosynthesis was severely impaired by the lack of phaB1. Conclusions The present results indicate the importance of flux distribution at the acetoacetyl-CoA node in R. eutropha for the biosynthesis of the PHA copolyesters with regulated composition from structurally unrelated compounds.


Supporting Information
Table S4

Construction of expression plasmids for N-His6-tagged PhaBs
The coding regions of phaB1, phaB2, and phaB3 were amplified by PCR using primer sets shown in Supplementary Table S4 and R. eutropha gDNA, and the amplified fragments were individually cloned into pUC118. The phaB1 fragment was excised by digestion with NdeI and BamHI, and then inserted into pCold II (Takara Bio) at the corresponding sites to obtain pColdII-phaB1. pET15b-phaB3 was constructed by insertion of an NdeI-BamHI-restricted fragment of phaB3 into pET15b (Novagen), because the gene expression using a pCold II-based vector was not observed by unknown reason. The phaB2 fragment was excised by KpnI and inserted into pCold II at the corresponding site. The extra region between the His6-tag sequence and phaB2 was removed by inverse PCR and successive self-ligation to obtain pColdII-phaB2.

Preparation of N-His6-tagged recombinant proteins
E. coli BL21(DE3) transformed with pColdII-phaB1 or pColdII-phaB2 was cultivated in LB medium at 37˚C on a reciprocal shaker (115 strokes/min). When the cell growth reached to OD600 of 0.4, the culture broth was cooled at 15˚C for 30 min and IPTG was added at the final concentration of 0.5 mM for induction of gene expression. The cultivation was continued for further 24 h at 15˚C. In the case of expression of phaB3, E. coli BL21(DE3) harboring pET15b-phaB3 was cultivated in LB medium at 37˚C on a reciprocal shaker and the gene expression was induced by addition of 0.5 mM IPTG when OD600 reached to 0.5, and the cells were cultivated for further 16 h at 37˚C.
The cells were harvested, washed and resuspended within 20 mM sodium phosphate buffer (pH7.4) containing 0.5 M NaCl and 30 mM imidazole, and then disrupted by sonication. The soluble fraction prepared by centrifugation and filtration (pore size 0.20 µm) was subjected to Niaffinity chromatography using HisTrap FF crude 1 mL (GE Healthcare Life Sciences). The Nterminal His6-tagged recombinant proteins were eluted by linear gradient of imidazole from 30 mM to 500 mM in sodium phosphate buffer (pH7.4) containing 0.5 M NaCl. The protein fraction was desalted using HiTrap Desalting (GE Healthcare Life Sciences) with 50 mM Tris-HCl (pH7.5), and then used for enzyme assay.

Site-directed mutagenesis of PhaB1
NADPH-acetoacetyl-CoA reductase (PhaB) is belonging to a short-chain dehydrogenase/ reductase family along with NADPH-3-oxoacyl-ACP reductase (FabG). It is known that the former shows strict specificity to acetoacetyl-CoA, while the latter can accept 3-oxoacyl-ACPs with medium-chain-length as substrates. We had attempted protein engineering of PhaB1 based on comparison of the crystal structures of PhaB1 and FabG, aiming to obtain the enzyme capable of catalyzing (R)-specific reduction of the C6 substrate, 3-oxohexanoyl-CoA.
The sequence alignment of three PhaB paralogs from R. eutropha (PhaB1, PhaB2, and PhaB3) [1] and FabGs from E. coli and Pseudomonas sp. 61-3 [2] identified residues that were conserved in the three PhaBs but not in FabGs, and comparison of the three-dimensional structures of FabG from E. coli complexed with NADP + (1Q7B) [3] and PhaB1 complexed with NADP + (not containing the substrate, obtained by personal communication from Hokkaido Univ., before deposition of that complexed with NADP + and acetoacetyl-CoA (3VZS) [4]). This suggested that a cavity near from the probable substrate binding pocket was filled by the side chains of asparagine 142 and tyrosine 185 in PhaB1 ( Supplementary Fig. S2) possibly forming a hydrogen bond. We therefore replaced these residues by valine and phenylalanine correspondingly conserved in FabGs, respectively.
The site directed mutagenesis was carried out by QuickChange protocol using primer sets N142V_Fw/N142V_Re and Y185F_Fw/Y185F_Re for N142V and Y185F mutations, respectively. The NdeI-BamHI restricted fragments of the mutagenized genes were inserted into pCold II at the corresponding sites. The N-terminal His6-tagged PhaB1s with N142V, Y185F, or N142V/Y185F mutations (designated as PhaB1NV, PhaB1YF, and PhaB1NVYF, respectively) were produced by recombinant strains of E. coli BL21(DE3) harboring each the expression plasmid, and then purified by the same procedure for PhaB1 as described in the main text. NADPHdependent reduction activities towards acetoacetyl-CoA and 3-oxohexanoyl-CoA were determined as described previously [5].
Unfortunately, expansion of the substrate specificity was not achieved by the site-directed mutagenesis, since the ratios of activity to the C6 and C4 substrates of the three mutants were 0.020-0.023 (C6/C4) that was only slightly higher than 0.013 of the parent wild-type enzyme. Further characterization of the mutants clarified that catalytic efficiencies for NADPH-dependent reduction of acetoacetyl-CoA were markedly reduced by the mutations (