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

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 (PctCp, Pct532) and a modified Pseudomonas sp. MBEL 6–19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19, 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.


Introduction
Physicochemical properties of P(3HB-co-LA) including the molecular weight, thermal 79 properties, and melt viscosity were taken into account as well. The mole fraction of LA monomer 80 has been demonstrated to have an inverse relationship with the molecular weight and the 81 crystallinity of P(3HB-co-LA), and direct relationship with the glass transition temperature (Tg) 82 of the polymer (14). Tg was largely affected by the LA fraction while the melting temperature was 83 only slightly modified, staying at around 160ºC. Copolymer showed a decreased molecular weight 84 (M n ) of 29,000 compared to that of PHA which had a molecular weight of 126,000 (24). The 85 copolymer exhibited more favourable changes in thermal behavior as well as the glass and 86 crystallization transition. Overall the copolymer P(3HB-co-LA) had improved mechanical 87 properties, such as lower viscosity and dynamic moduli (which PLA alone does not possess). A 88 different copolymer, produced by blending MCL PHA with PLA using a melt-mixing method, 89 showed even more improved properties, such as increased toughness, ductility and optical clarity 90 (25). 91 We have recently reported the expression of two codon-optimized engineered genes 92 encoding propionate CoA transferase (pct) and PhaC synthase (phaC) which were integrated into 93 S. meliloti chromosome (26). In an attempt to broaden the range of novel polymer types that could 94 be produced, we have now introduced these engineered genes into a broad-host-range plasmid, 95 and expressed them in the two PHA-producing platforms S. meliloti and P. putida, which exhibit 96 differences in the range and type of PHA that they are able to produce naturally. All strains used in this study are indicated in Table 1. Strains SmUW499 and SmUW501 102 were provided by R. Nordeste, who constructed them as recently described (27) followed by 103 introduction of exoF::Tn5 by transduction from Rm7055. Escherichia coli and Pseudomonas 104 putida were cultured in Luria-Bertani (LB), while Sinorhizobium meliloti was cultured in Tryptone 105 Yeast (TY), supplemented with tetracycline (10 µg ml -1 ) as necessary for plasmid maintenance.

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Rapid screening of polymer production was performed by adding Nile Red (0.5 µg/mL) to YM 107 agar plate, and observing its mucoid phenotype or visualizing by fluorescence (28).   The OD at 600 nm of the culture was also recorded for normalization. Intracellular polymer production was evaluated by gas chromatography following a 137 protocol which has been described in previous studies (15,38). Briefly, cells were harvested from 138 flask culture after a 3-day incubation by centrifuging at 4,000 g for 20 min, then washed twice 139 with distilled water, and finally dried at 100C overnight. The dried cell weight (DCW) was 140 recorded before methanolysis in 2 ml chloroform and 1 ml PHA solution containing 8 g benzoic 141 acid l -1 as an internal standard and 30% sulfuric acid in methanol. The reaction was carried out at 142 96C for 6 h, cooled, and then 1 ml of water was added, the mixture was vortexed, and the solution 143 was allowed to separate into two phases. One µl of the chloroform phase was taken for analysis 144 by GC as previously described (15).

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Construction of plasmid expressing synthetic codon optimized pct532 and phaC1400 genes.
The broad host range vector pTH1227 (39), containing the inducible tac promoter and lacI q 148 along with a downstream gusA gene to use as a reporter, was digested with XhoI and PstI, then  to about half the wild-type levels. In both conditions, LA was not detected (Fig. 4). To 189 investigate whether this was due to issues related to competition between PHB and PLA 190 precursors, we blocked the PHB synthesis pathway by using a phbAB mutant background.

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Feeding the strain with lactic acid (10 g l -1 ) instead of mannitol resulted in PLA homopolymer production of 3.2% DCW (   being produced (Fig. 6a); however, both DCW and yield decreased relative to increased IPTG 214 concentration (data not shown), hence leading to the overall decreasing levels of PHB.

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Interestingly, the basal level of expression in absence of IPTG resulted in the best growth and PHB 216 production based on the highest DCW, yield and PHB percentage.  added into the culture, the lower the GusA activity (Fig. 5b). Therefore, if we only consider 225 reporter gene expression level, induction at the beginning of cultivation is the most optimal; 226 however, whether reporter gene expression is directly proportional to PHB production is a separate 227 issue that we sought to resolve. Once again, the results show that this relationship is inversely 228 proportional (Fig. 6b). Both DCW and yield were significantly decreased when the induction 229 occurred at the beginning of cultivation, resulting in the lowest PHB percentage (S1). Induction at 230 Day 1 or 2 of cultivation did not make a significant difference compared to no induction, 231 suggesting that the basal level is still the ideal for PHB production.

Introduction of engineered synthesized genes into P. putida
233 a. Phenotypic analysis of strain constructs 234 The phenotype of P. putida harboring the pTAM and pTH1227 plasmids when grown on 235 LB supplemented with sodium octanoate was examined. Interestingly, we found that the strains 236 carrying the pTAM plasmid showed a distinguishable phenotype from the strains only carrying 237 the empty plasmid pTH1227 (Fig. 7). Both the wild type and mutant strains containing the pTAM   was abundant carbon source.

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By using two codon-optimized genes placed in tandem on a broad-host range vector and 302 expressed under an inducible promoter in S. meliloti and P. putida backgrounds, we were able to 303 demonstrate the production of LA containing polymers. In S. meliloti, we demonstrated that this 304 plasmid construct was able to complement the phbC mutant strain, but unlike the genome-305 engineered S. meliloti strain which has been shown to be able to produce copolymer P(3HB-co-306 LA) (40), it only produced homopolymer P(3HB). This could be due to insufficient provision of 307 LA precursor. Genetic removal of the ability to produce 3-hydroxybutyryl-CoA substrate for the 308 PHA synthase resulted in production of PLA homopolymer up to 4% when growing in YM 309 supplemented with lactic acid.

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The question of how much synthase enzyme protein level is optimal for polymer 311 production is still unresolved. A strategy of maximizing expression does not necessarily translate 312 into higher levels of metabolic end product (41). For instance, it was reported that E. coli XL1-

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Blue harboring the low-copy-number plasmid pJRDTrcphaCAB Re produced P(3HB) more 314 efficiently than the strain harboring the high copy number pTrcphaCAB Re (42). There is still not 315 a good understanding of the relationship between synthase gene expression and levels of polymer 316 accumulated. In our study, we have provided an example where the relationship between protein 317 expression and target products is inversely proportional. Increasing the levels of IPTG or 318 lengthening the induction time resulted in production of less polymer.

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Upon introduction of pTAM into P. putida, the strain was able to produce a novel 320 quaterpolymer derived from 4 different monomers (LA, 3HB, 3HHx and 3HO). To our knowledge, 321 a copolymer of this type has never been reported. The production of P(LA-co-3HB-co-3HHX) was previously demonstrated in E. coli via a reverse reaction of the β-oxidation pathway (43 only when provided with a PHA polymerase and a modified thioesterase I (44). In addition, the 330 polymer content of P(LA-co-3HB-co-3HHX) produced in E. coli was extremely low (<5% DCW).

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In comparison, we were able to incorporate 4 different monomers in a polymer and increase the 332 polymer content up to 42%. It has been suggested that the engineered PhaC enzyme has a broader 333 substrate than the native P. putida PhaC. The engineered PhaC synthase was originally Type II 334 PhaC1 synthase that accepts and polymerizes MCL-3HA (C6-C14) monomers (14). This enzyme 335 was engineered to broaden the substrate towards SCL-3HAs (specifically, 3HB) and LA.

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Nonetheless, whether it still retains its ability to accept MCL-3HA has not previously been 337 demonstrated.

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The fraction of LA and 3HB monomers in the copolymer increased substantially with the 339 presence of engineered PhaC synthase, irrespective of media. This fraction was increased in mutant 340 PhaC strain to a greater degree than the strain that still contained the wild type phaC genes, likely 341 due to a precursor competition between the polymerase enzymes in the wild-type strain or the 342 higher overall polymerase enzyme activity towards MCL PHAs in the wild-type resulting in the increase of MCL fraction over LA and 3HB fraction. In addition to SCL-3HA and LA monomers,