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

Traditional plastics made from non-renewable fossil fuels have posed a threat to environment and human health. New materials, which are environment-friendly and easily biodegradable, are being searched to substitute for the traditional plastics. Those materials, such as polyhydroxylalkanoate (PHA), polylactic acid (PLA), polyglycolic acid (PGA), and blends of these polymers, can be produced through either the chemical process or biological process [1,2]. However, the chemical process has some drawbacks such as high temperature, long reaction time, uncontrollable chain length or monomer restriction [3,4]. In addition, chemical synthesis results in harmful left-over chemical residues of metal catalysts for the reaction [5]. Metal catalysts used in ring-opening polymerization stage are heavy metals, such as Cu-based catalyst ({2-[1-(2,6-diethylphenylimino)ethyl]phenoxy}2Cu(II)) [6]. Therefore, bioprocess has been considered as an alternative way to produce novel polymers.

PHAs are able to be produced naturally in bacteria such as Cupriavidus necator H16 (formerly, Ralstonia eutropha) [7] [8], Alcaligenes latus [9], Pseudomonas [10] [11] and the rhizobia [12]. However, PLA and its derivatives have only been produced through genetic engineering [4,5,13-16], by introduction and expression of modified genes for two enzymes, propionate CoA-transferase (e.g. pct532) from Clostridium propionicum and PHA synthase (e.g. phaC1400) from Pseudomonas. These studies, including recent reports of PLA/PHA copolymer production [13,14] have mainly focused on engineering Escherichia coli strains that are common model systems in the metabolic engineering field. E. coli strains have been engineered to produce a broad range of bioproducts such as biopolymers, biofuels, amino acids, and organic acids because molecular tools are intensively studied and widely available. However, E. coli strains are not native polymer producers and hence lack the metabolic pathways for the production of usable polymer precursors. Also, it was shown that E. coli strains suffer from the stress of polymer production, and they produce side products that might decrease the overall yield of target products. For these above reasons, we focused our study on engineering two representative native polymer producers, S. meliloti which produces short-chain-length (SCL) PHA, and P. putida which produces medium-chain-length (MCL) PHA, for efficient novel polymer production systems [12,17]. Both organisms are preferred choices for engineering PHA polymer production because of their broad growth substrate capability, relatively fast growth and fully annotated genomes.

Depending on PHA synthase as well as substrate availability, a variety of different polymer types can be achieved, such as homo-polymer (P(3HB), 3-hydroxybutyrate), co-polymer (P(3HB-co-3HV, 3-hydroxybutyrate-co-3-hydroxyvalerate), ter-polymer (P(LA-co-3HB-co-3HV, lactic- acid-co-3-hydroxybutyrate-co-3-hydroxyvalerate), P (3HB-co-3HV-co-3HHx, 3-hydroxybutyrate-co-3-hydroxyvalerate-co-hydroxyhexanoate), P (LA-co-3HB-co-3HP, lactic acid-co-3-hydroxybutyrate-co-3-hydroxypentanoate) [18-21]. Based on the type, content, monomer composition, and molecular weight distribution, different polymers possess diverse properties allowing a broader spectrum of application. P (3HB) homopolymer exhibits undesirably high melting temperature as well as high crystallinity, resulting in hard and brittle properties [22]. Meanwhile, MCL PHAs (6–14 carbons) are described as having low melting point and high elasticity [23]. Since the melting temperature of MCL PHAs is relatively low, varying from 39ºC to 61ºC, materials made from MCL PHAs are not rigid enough and tend to lose their coherence at rather low temperature [24]. Incorporating a small amount of MCL PHAs into P (3HB) showed some improved properties of the target polymers. Their properties are similar to polyethylene, which is relatively tough and ductile. The crystallinity and melting temperature (Tm) of P (SCL-co-MCL 3HA) are reduced in comparison with the traditional PHA polymers, such as P (3HB) or PHBV copolymers. As a result this type of copolymer is able to be processed efficiently and in a cost-effective way. The complete biodegradation can be achieved in either aerobic or anaerobic environments.

Physicochemical properties of P (3HB-co-LA) including the molecular weight, thermal properties, and melt viscosity were taken into account as well. The mole fraction of LA monomer has been demonstrated to have an inverse relationship with the molecular weight and the crystallinity of P (3HB-co-LA), and direct relationship with the glass transition temperature (Tg) of the polymer [16]. Tg was largely affected by the LA fraction while the melting temperature was only slightly modified, remaining at around 160ºC. Copolymer showed a decreased molecular weight (Mn) of 29,000 compared to that of PHA which had a molecular weight of 126,000 [25]. The copolymer exhibited more favourable thermal behavior as well as glass and crystallization transition. Overall the copolymer P (3HB-co-LA) had improved mechanical properties, such as lower viscosity and dynamic moduli (which PLA alone does not possess). A different copolymer, produced by blending MCL PHA with PLA using a melt-mixing method, showed even more improved properties, such as increased toughness, ductility and optical clarity [26].

We have recently reported the expression of the two codon-optimized engineered genes encoding propionate CoA transferase (pct532) and PHA synthase (phaC1400) which were integrated into S. meliloti chromosome [27]. In an attempt to broaden the range of novel polymer types that could be produced, we have now introduced these engineered genes into a broad-host-range plasmid, and expressed them in the two PHA-producing platforms S. meliloti and P. putida, which exhibit differences in the range and type of PHA that they are able to produce naturally.

Material and methods

Bacterial strains and cell culture

All strains used in this study are indicated in Table 1. Strains SmUW499 and SmUW501 were provided by R. Nordeste (University of Waterloo), who constructed them as recently described [28] followed by introduction of exoF::Tn5 by transduction from Rm7055. Escherichia coli and Pseudomonas putida were cultured in Luria-Bertani (LB), while Sinorhizobium meliloti was cultured in Tryptone Yeast (TY), supplemented with tetracycline (10 μg ml-1) as necessary for plasmid maintenance. Rapid screening of polymer production was performed by adding Nile Red (0.5 μg/mL) to YM agar plate, and observing its mucoid phenotype or visualizing by fluorescence [29]. Media formulations were as previously described [30].

Table 1

Bacterial strains used in this study.

Strains	Genotype	Reference
Escherichia coli
DH5α	supE44 lacU169(w80lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1	Lab collection
DH5α(pRK600)	Helper strain harboring plasmid pRK600	[31]
Sinorhizobium meliloti
Rm1021	Spontaneous Sm resistant isolate of S. meliloti SU47	[32]
Rm7055	Rm1021 exoF::Tn5	[33]
SmUW499	Rm1021 ΔphbC exoF::Tn5	Ricardo Nordeste
SmUW501	Rm1021 ΔphbAB ΔphbC exoF::Tn5	Ricardo Nordeste
SmUW255	SmUW499 harboring pTH1227	This study
SmUW256	SmUW499 harboring pTAM	This study
SmUW257	Rm7055 harboring pTH1227	This study
SmUW558	Rm1021 ΔphbAB ΔphbC exoF::Tn5 harboring pTH1227	This study
SmUW559	Rm1021 ΔphbAB ΔphbC exoF::Tn5 harboring pTAM	This study
Pseudomonas putida
KT2440	Wild type (ATCC 47054)
PPUW1	A spontaneous RifR mutant of KT2440	[17]
PPUW2	ΔphaC1-phaZ-phaC2/ ΩKm in PPUW1(Kmr)	[17]
PPUW18	PPUW2 harboring pTH1227	This study
PPUW19	PPUW2 harboring pTAM	This study
PPUW20	PPUW1 harboring pTH1227	This study
PPUW21	PPUW1 harboring pTAM	This study

For polymer production in S. meliloti, strains were initially inoculated in TY, and then 1% overnight culture was transferred to YM media in flasks on a shaker at 180 rpm, 30°C for 3 days [34,35]. IPTG at 0.4 mM was added into the culture for induction as described.

For polymer production in P. putida, strains were initially inoculated in LB, and then 1% overnight culture was transferred to defined media (0.5X E2) [36] in flasks on a shaker at 180 rpm, 30°C for 3 days [37]. Nile Red (0.5 μg/mL) was added to LB or 0.5X E2 agar plate for rapid screening of polymer production. Sodium octanoate (0.5% w/v) was added in the media when needed.

β-glucuronidase activity assay

β-glucuronidase (GusA) levels were determined as an indication of the transcript level of synthesized genes in S. meliloti strains. The assay was carried out as previously described [37,38] by adding culture into assay buffer at the ratio of 1:4, and incubation at room temperature until the yellow colour developed, at which time the reaction was terminated by the addition of sodium carbonate. Then the absorbance of reaction mixture was measured at 420 nm. The OD at 600 nm of the culture was also recorded for normalization.

GC analysis

Intracellular polymer production was evaluated by gas chromatography following a protocol which has been described in previous studies [4,39]. Briefly, cells were harvested from flask culture after a 3-day incubation by centrifuging at 4,000 g for 20 min, then washed twice with distilled water, and finally dried at 100°C overnight. The dried cell weight (DCW) was recorded before methanolysis in 2 ml chloroform and 1 ml PHA solution containing 8 g benzoic acid l-1 as an internal standard and 30% sulfuric acid in methanol. The reaction was carried out at 96°C for 6 h, cooled, and then 1 ml of water was added, the mixture was vortexed, and the solution was allowed to separate into two phases. One μl of the chloroform phase was taken for analysis by GC as previously described [4].

Results

Construction of plasmid expressing synthetic codon optimized pct532 and phaC1400 genes

The broad host range vector pTH1227 [40], containing the inducible tac promoter and lacI along with a downstream gusA gene to use as a reporter, was digested with XhoI and PstI, then ligated with the XhoI / PstI -cut synthesized DNA containing the previously described codon optimized sequences [27] encoding the Pct532 and PhaC1400 of Jung et al [4] as shown in Fig 1. This resulted in the plasmid construct pTAM. The pTAM plasmid and the empty vector control pTH1227 were separately introduced into S. meliloti and P. putida strains by triparental conjugation. Transcription of the introduced genes was confirmed by assay of gusA-encoded β-glucuronidase activity.

Fig 1

Construction of expression plasmid pTAM.

Two synthesized genes (pct532 and phaC1400) were inserted as a XhoI-PstI fragment into pTH1227 to create pTAM. The synthesized gene sequences have been previously deposited in GenBank and can be accessed via accession Nos. KT382270–KT382273.

Expression of engineered synthesized genes in S. meliloti

a. Phenotypic analysis of strain constructs

Initial confirmation of polymer production in the constructed strain SmUW256 was performed as previously described for the chromosome engineered strain SmUW254 [27]. The strain was streaked out on agar media alongside phbC-positive and phbC-negative controls strains (Fig 2). Strain SmUW256, which is the phbC mutant containing pTAM, exhibited fluorescence similar to the control strain SmUW257, which contains pTH1227 in a non-mutant phbC background. The phbC mutant strain SmUW255, containing pTH1227, which cannot produce P (3HB), was deficient in fluorescence, as expected. Interestingly, in the absence of inducer IPTG, we found that SmUW256 exhibited much more fluorescence than with IPTG. This suggested that the gene expression from the plasmid construct was sufficient without IPTG induction to allow complementation of the PHB synthesis deficiency.

Fig 2

Phenotypic complementation of plasmid-based S. meliloti strains.

a and b demonstrate phenotype difference between plasmid-engineered S. meliloti strains and other S. meliloti strains: SmUW255 (ΔphbC exoF::Tn5 harboring empty plasmid pTH1227), SmUW256 (ΔphbC exoF::Tn5 harboring pTAM (#1, 3)), SmUW257 (exoF::Tn5 harboring pTH1227). a: induced condition, b: non-induced condition.

b. Polymer production

The engineered genes were expressed from pTAM in a S. meliloti phbC mutant background under the control of inducible promoter Ptac by means of the plasmid-encoded lacIq. Surprisingly, in the absence of IPTG supplementation in YM, levels of PHB produced were similar to the wild-type levels, but in the presence of 0.4 mM IPTG, PHB was only accumulated to about half the wild-type levels. In both conditions, LA was not detected (Fig 3). To investigate whether this was due to issues related to competition between PHB and PLA precursors, we blocked the PHB synthesis pathway by using a phbAB mutant background. Feeding the strain with lactic acid (10 g l-1) instead of mannitol resulted in PLA homopolymer production of 3.2% DCW (Table 2), demonstrating that in the absence of competing PHB precursors, the PLA precursors could be incorporated into polymer. Higher concentrations of lactic acid resulted in reduced growth, perhaps due to reduction in medium pH.

Fig 3

Polymer content % DCW of SmUW255 (phbC−), SmUW257 (phbC+) and SmUW256 (engineered phbC).

Black bars represent inducing condition (0.4 mM IPTG was added to cultures); grey bars represent non-inducing condition (no IPTG). Experiment was performed in biological duplicate, and error bars represent the range of the mean.

Table 2

PLA homopolymer production in S. meliloti phbAB phbC mutant background harbouring pTAM.

	Lactic acid concentration
	10 g/l	20 g/l	30 g/l	10 g/l	20 g/l	30 g/l
Strain	PLA homopolymer content (% DCW)			DCW (g/l)
SmUW558 (control)	ND	-	-	0.43 ± 0.03	-	-
SmUW559	3.2 ± 1.8	0.18 ± 0.13	ND	0.35 ± 0.05	0.16 ± 0.05	ND

Strains were cultured in YM with mannitol substituted by lactic acid at the indicated concentrations.

SmUW558: ΔphbAB ΔphbC (pTH1227), SmUW559: ΔphbAB ΔphbC (pTAM).—: Not available. ND: Not detectable. The experiment was performed in duplicate. Results are presented as mean ± standard deviation.

c. Investigation of IPTG concentration and induction time

In light of the observation that more polymer was produced in the absence of IPTG than in the presence of IPTG, we decided to further investigate the PHB production under different induction conditions. We also measured GusA expression from the downstream gusA gene as a proxy for transcription across the engineered genes. A range of IPTG concentrations from 0 to 1 mM were used to induce gene expression (Fig 4). At an IPTG concentration of 0.05 mM, GusA activity in the SmUW256 strain increased markedly compared to that in the absence of IPTG. However, further addition of IPTG did not substantially increase GusA activity in recombinant strain SmUW256. Meanwhile, the control strain SmUW255 which harbors the empty plasmid pTH1227 showed regulated gusA expression which was more proportional to IPTG concentration. Contrary to expectation, increased gene expression did not result in more PHB being produced (Fig 5); however, both DCW and yield decreased relative to increased IPTG concentration (data not shown), hence leading to the overall decreasing levels of PHB. Interestingly, the basal level of expression in absence of IPTG resulted in the best growth and PHB production based on the highest DCW, yield and PHB percentage.

Fig 4

Measurement of β-glucuronidase activity of strain SmUW255 (phbC), SmUW256 (containing pTAM) induced with different concentrations of IPTG (a) and induced with 0.4 mM IPTG at different points in time for 3 day incubation (b).

Fig 5

PHB content produced by strain SmUW256 induced at different concentrations of IPTG (a) and induced at 0.4 mM IPTG at different points in time for 3 day incubation (b). The experiment was performed in duplicate. Results are presented as mean ± standard deviation (SD). Error bars indicate SD.

Next, we investigated the effect of induction timing to see if the cells behave differently when 0.4 mM IPTG was added at a later point in time. We observed that the later the IPTG was added into the culture, the lower the GusA activity (Fig 4B). Therefore, if we only consider reporter gene expression level, induction at the beginning of cultivation is the most optimal; however, whether reporter gene expression is directly proportional to PHB production is a separate issue that we sought to resolve. Once again, the results do not show this relationship (Fig 5B). Both DCW and yield were substantially decreased when the induction occurred at the beginning of cultivation, resulting in the lowest PHB percentage. Induction at Day 1 or 2 of cultivation did not make a difference compared to no induction, suggesting that the basal level is still the ideal for PHB production.

Introduction of engineered synthesized genes into P. putida

The phenotype of P. putida harboring the pTAM and pTH1227 plasmids when grown on LB supplemented with sodium octanoate was examined. Interestingly, we found that the strains carrying the pTAM plasmid showed a distinguishable phenotype from the strains only carrying the empty plasmid pTH1227 (Fig 6). Both the wild type and mutant strains containing the pTAM plasmid showed a milky white colony phenotype, while the strains that carried only the empty plasmid pTH1227 had a yellowish colony color. The milky white colour is likely due to polymer accumulation enabled by pTAM. It appears that the function of the native PHA synthase, and not other enzymes in the P. putida PHA biosynthesis pathway, is affected when the strain is cultured in complex media with excess carbon source because the engineered PHA synthase was able to direct the accumulation of PHA on complex media.

Fig 6

Phenotype of strains on LB supplemented with sodium octanoate.

A milky white color was observed for PPUW19 (phaC mutant harboring pTAM) and PPUW 21 (wild-type strain harboring pTAM). Yellowish color was observed for PPUW18 (phaC mutant harboring pTH1227) and PPUW20 (wild-type strain harboring pTH1227).

We also investigated colony fluorescence on Nile Red containing plates with different growth media (Fig 7). On LB plates without the addition of extra carbon source, no fluorescence was observed for any of strains. Only strains harboring pTAM plasmid showed a strong fluorescence when grown on LB plates with an excess carbon source (eg. sodium octanoate). Meanwhile, the addition of lactic acid did not result in observed fluorescence.

Fig 7

Fluorescence observation of different P. putida strain backgrounds on different PHA and non-PHA accumulating agar plates.

PPUW18 (phaC mutant harboring pTH1227), PPUW19 (phaC mutant harboring pTAM), PPUW20 (wild-type KT2440 harboring pTH1227), PPUW21 (wild-type KT2440 harboring pTAM). a: LB + Nile Red, b: LB + sodium octanoate + Nile Red, c: LB + lactic acid + Nile Red.

b. Polymer production in P. putida strains growing in defined media supplemented with either sodium octanoate or lactic acid as a substrate

Strains were cultured in defined media 0.5X E2 supplemented with either sodium octanoate or lactic acid. The negative control strain PPUW18, which is the P. putida phaC deletion mutant strain carrying the empty plasmid pTH1227, did not produce detectable polymers on either media. Overall, the media containing sodium octanoate as sole carbon source supported both the growth and the polymer production much better than media with lactic acid as sole carbon source (Table 3). The phaC mutant with pTAM plasmid showed the highest DCW and polymer content up to 1.28 g/l and 42%, respectively, using sodium octanoate. The strains carrying the pTAM plasmid were able to incorporate LA monomers to generate a novel quaterpolymer composed of LA, 3HB, 3HHx and 3HO. We found that growth in lactic acid supplied more LA precursors than did sodium octanoate, with the wild-type strain and the mutant strain carrying pTAM accumulating LA of 2.8 and 1.9% mol respectively on lactic acid, while lactic acid was barely detectable on sodium octanoate. Lactic acid is a direct substrate of the modified propionate-CoA transferase, whose function is to add the CoA moiety onto the substrate. Even though lactic acid did not support the cell growth as well as sodium octanoate, it supplied more LA precursor toward novel polymer production. This implies that in P. putida very little lactyl-CoA was produced during growth on sodium octanoate substrate.

Table 3

PHA copolymer production in P. putida harboring pTAM and control plasmid vector.

Strain	Culture Media
	0.5X E2 + Lactic Acid
	LA	3HB	3HHx	3HO	DCW (g/l)	Polymer content (%)	Yield (g/l)
PPUW20	-	3.3	7.3	89.4	1 ± 0.074	5.4 ± 0.178	0.057 ± 0.002
PPUW21	2.8	9.5	12.6	75.1	1 ± 0.163	4.9 ± 0.192	0.04 ± 0.01
PPUW18	-	-	-	-	0.8 ± 0.043	<0.5	-
PPUW19	1.9	22	10.2	65.9	0.7 ± 0.088	3.4 ± 0.477	0.024 ± 0.006
	0.5X E2 + Sodium Octanoate
	LA	3HB	3HHx	3HO	DCW (g/l)	Polymer content (%)	Yield (g/l)
PPUW20	-	0.9	5.7	93.4	0.94 ± 0.003	26.5 ± 1.5	0.25 ± 0.01
PPUW21	0.28	20.5	13.4	65.7	0.91 ± 0.135	35.4 ± 3.3	0.32 ± 0.07
PPUW18	-	-	-	-	0.72 ± 0.025	<0.5	-
PPUW19	0.4	23.9	14.2	61.4	1.28 ± 0.567	42 ± 6.1	0.53 ± 0.27

The experiment was performed in duplicate. Results are presented as mean ± standard deviation.

Discussion

By using two codon-optimized genes placed in tandem on a broad-host range vector and expressed under an inducible promoter in S. meliloti and P. putida backgrounds, we were able to demonstrate the production of LA containing polymers. In S. meliloti, we demonstrated that this plasmid construct was able to complement the phbC mutant strain, but unlike the genome-engineered S. meliloti strain which has been shown to be able to produce copolymer P(3HB-co-LA) [27], it only produced homopolymer P (3HB). This could be due to insufficient provision of LA precursor. Genetic removal of the ability to produce 3-hydroxybutyryl-CoA substrate for the PHA synthase resulted in production of PLA homopolymer up to 4% when growing in YM supplemented with lactic acid.

The question of how much synthase enzyme protein level is optimal for polymer production is still unresolved. A strategy of maximizing expression does not necessarily translate into higher levels of metabolic end product. For instance, it was reported that E. coli XL1-Blue harboring the low-copy-number plasmid pJRDTrcphaCABRe produced P(3HB) more efficiently than the strain harboring the high copy number pTrcphaCABRe [41]. There is still not a good understanding of the relationship between synthase gene expression and levels of polymer accumulated. In our study, we have provided an example where the relationship between protein expression and target products is inversely proportional. Increasing the levels of IPTG or lengthening the induction time resulted in production of less polymer.

Upon introduction of pTAM into P. putida, the strain was able to produce a novel quaterpolymer derived from 4 different monomers (LA, 3HB, 3HHx and 3HO). To our knowledge, 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 [42]. In that study, the E. coli strain was equipped with LA- polymerizing enzyme (LPE) encoding a mutant phaC1 gene from Pseudomonas sp. 61–3, propionyl-CoA transferase (Pct) and (R)-specific enoyl-CoA hydratase (PhaJ4). It is difficult to understand how in that particular study 100% P (3HB) was produced during growth in LB supplemented with glucose since there is no link between the fatty acid synthesis pathway and PHA precursor formation in E. coli. In previous studies, E. coli was able to produce a small amount of MCL PHA from non-related carbon source only when provided with a PHA polymerase and a modified thioesterase I [43]. In addition, the polymer content of P (LA-co-3HB-co-3HHX) produced in E. coli was extremely low (<5% DCW). In comparison, in our study we observed incorporation of 4 different monomers and an increase of the polymer content up to 42%. In addition, the fraction of LA and 3HB monomers was increased in the complemented mutant PhaC strain to a greater degree than the strain that still contained the wild type phaC genes. This is likely due to competition between the polymerase enzymes in the wild-type strain or the higher overall polymerase enzyme activity towards MCL PHAs in the wild-type, resulting in the increase of MCL fraction over LA and 3HB fraction. It has been suggested that the engineered PhaC enzyme has a broader substrate than the native P. putida PhaC. The engineered PHA synthase was originally Type II PhaC1 synthase that accepts and polymerizes MCL-3HA (C6-C14) monomers [16]. This enzyme was engineered to broaden the substrate towards SCL-3HAs (specifically, 3HB) and LA. Nonetheless, whether it still retains its ability to accept MCL-3HA has not previously been demonstrated.

17 Jul 2019

PONE-D-19-15052

Lactic Acid Containing Polymers Produced in Engineered Sinorhizobium meliloti and Pseudomonas putida

PLOS ONE

Dear %Dr% %Charles%,

Thank you for submitting your manuscript to PLOS ONE. After the requirement of three reviewers for the evaluation, two of them present a strong criticism as it currently stands. Therefore, we invite you to carefully study all their considerations and submit a deeply revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Aug 31 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Francisco Martinez-Abarca, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

1. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript describes the production of lactic acid-containing polymers with medium-chain length units in Sinorhizobium meliloti and Pseudomonas putida. Many cases have been reported in various monomeric constituents using some bacterial platforms starting from 2008. In such a case, this is an example of the series study. Most recently Goto et al. reported the similar paper on the polymer with higher molar-fraction of lactic acid (> 4% in this paper) using Escherichia coli. Thy also provides the polymer properties. On the other hands, any new finding cannot be found in this paper. For example, in several points, new synthetic pathway, higher production titter, enriched lactic fraction, high molecular weight, new polymer properties. The reviewer has to consider this as a not-permitted assessment for publication in the present form.

Reviewer #2: General Comments

This manuscript describes the engineering of Sinorhizobium meliloti and Pseudomonas putida to synthesize novel co-polymers containing both polyhydroxyalkanoates (PHAs) and polylactic acid (PLAs). This work is novel and innovative. The manuscript is well written, the experimental approach and methods are appropriate and well described, and the conclusions are supported by the data. This manuscript will make an excellent contribution to the literature. There are only a few minor issues that need to be addressed before the manuscript may be published.

Specific Comments

Page 6, line 102: In the sentence, “…were provided by R. Nordeste, who constructed them as recently described (27)…”, please provide the institutional affiliation of R. Nordeste.

Page 7, line 115: Please provide the vendor/manufacturer and its location for the culture medium “0.5*E2”

Page 7, lines 120 to 121: In the sentence, “β-glucuronidase levels were determined as an indication of the transcript level of synthesized genes in both E. coli and S. meliloti strains.” Firstly, do the authors really mean E. coli here? Should this not be P. putida?

Second, the assay described is protein (gene product) based. The authors cite two references, one which describes the use of the β-glucuronidase (GusA) assay in actinomycetes (reference 36) and the other describes the use of this assay as a measure of promoter activity in Lactic Acid bacteria (reference 37). In the current paper, the authors suggest that the assay is a direct indicator of the level of transcription of the genes of interest, but this is based on the assumption that the level of protein activity detected is proportional to the level of translation, and this in turn is proportional to the level of transcription. Is this actually the case? Can the authors provide some additional clarification about the relationship between transcription, translation, and enzyme activity in S. meliloti and P. putida?

Figure 3: The resolution of the image for Figure 3 is very poor, and the bands in each lane have merge with each other so it is difficult to tell which lane is which. Can the authors replace this gel image with a better one?

Page 11, lines 202 to 209: The authors refer to GusA, which is the gene product β-glucuronidase. For clarity, please revised the sentence on page 7, line 120, as follows: “β-glucuronidase (GusA) levels were determined….

Reviewer #3: The authors present a plasmid containing the genes encoding the propionate CoA transferase of C. propionicum and a modified PHA synthase I of Pseudomonas sp. MBEL 6-19. They introduced this into phaC mutants of S. melioti and P. putida and report the production of PLA homopolymer in S. meloti and of a novel quaterpolymer P(3HB-co-LA-co-3HH-co-3HO) in P. putida in the presence of octanoate. Though the production of a novel quaterpolymer is potentially interesting for applied processes, the manuscript, in its present state, has a number of shortcomings.

p. 9, ll. 147-152: It would be helpful to have some more information on the genetic arrangement of the plasmid, e.g. how does the gusA reporter work, is it a transcriptional or translational fusion? How was the codon optimization achieved? What does the 532 or the 1400 stand for in the gene names? What RBSs are used and where were they located?

p. 9, ll. 161-167 and Fig. 2: Why is there such a big difference in the fluorescence of SmUW257 comparing Fig. 2a to b? It should be comparable, as only IPTG is added in a) but this strain carries the empty plasmid. At the same time in 2b) it is hard to see a difference between the negative control (SmUW255) and the positive control (SmUW257). In my opinion this assay is not suitable to draw quantitative conclusions, only qualitative if any, as fluorescence depends on the amount and density of cells, which is hard to equal in solid phase medium. To get a rough estimation on the amount of polymer produced, the authors could repeat this plate experiment ensuring that the same amount of cells is plated or stain liquid cultures with the same OD (better cell count).

p.10, ll. 174-179 and Fig. 3: In MHO nothing can be concluded form this WB. Apart from the fact, that it is hard to distinguish the different lanes, no band is observed in the positive control, and the rest is mainly smear. How did the authors ensure that the same amount of protein is loaded in each lane, this should be stated in the M&M part or a Coomassie stained gel should be shown. Which lanes show the sample without IPTG, which is mentioned in the manuscript?

p. 10, Polymer production (Fig.4): How many independent replicates where analyzed? What do the error bars represent? Is the % polymer referred to CDW?

p. 11, Tab. 2: Why is the PLA production negatively correlated to the lactic acid concentration in the medium? Is that expected? Please comment on this.

Fig. 5 and 6: Information on how many replicates were analyzed and what the error bar represent is missing. It would be more coherent to have in Fig. 5b and 6b the same order of the samples (from day 1 to 3 or vice versa).

p. 12, ll. 227-228: The authors state that the gusA reporter expression is inversely proportional to PHB production. I don’t see a correlation between the expression and the polymer content. The GusA activity, which serves a s a proxy for gene expression, seems to be either on or off. The authors should perform a correlation and include a correlation coefficient to support this statement

p.12, l. 229: Reference is made to S1, however it is not available.

p.12, l. 230: How was the significance determined?

Fig. 7a is redundant with Fig. 7b. and can be omitted from the manuscript.

p.14, l. 265: Did the authors mean sodium octanoate?

Fig 9a: When were the samples taken? Is this result of only one analysis? Please comment on this.

Fig. 9b: How do the authors explain the difference in CDW in PPU20, PPU21, and PPU19? PPU19 reaches more than twice the CDW of the wt strain in LB medium. Why is this not observed with minimal medium? Is that a common phenotype, or just observed in this specific experiment?

Technical comments:

1. Information on media composition is completely missing (YM, TY, 0.5*E2)

2. A list of abbreviations would be helpful (especially for the polymer abbreviations, HB, LA, HH. HV etc.).

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

11 Dec 2019

Revisions for Lactic Acid Containing Polymers Produced in Engineered Sinorhizobium meliloti and Pseudomonas putida

Comments by Reviewer #1:

The manuscript describes the production of lactic acid-containing polymers with medium-chain length units in Sinorhizobium meliloti and Pseudomonas putida. Many cases have been reported in various monomeric constituents using some bacterial platforms starting from 2008. In such a case, this is an example of the series study. Most recently Goto et al. reported the similar paper on the polymer with higher molar-fraction of lactic acid (> 4% in this paper) using Escherichia coli. Thy also provides the polymer properties. On the other hands, any new finding cannot be found in this paper. For example, in several points, new synthetic pathway, higher production titter, enriched lactic fraction, high molecular weight, new polymer properties. The reviewer has to consider this as a not-permitted assessment for publication in the present form.

Response:

We thank the reviewer for this comment. As stated in the comment, our study is another instance of a continuing effort to seek out new platforms for novel polymer production. There have been many reports using Escherichia coli as a host to produce different types of biopolymer (a given example is the paper of Goto et al. mentioned by the reviewer, and which we have now cited). However, as mentioned in our manuscript, using E. coli as a host has shown some drawbacks, such as lacking native system for polymer precursor production or suffering from the stress of polymer production. Therefore, our study has demonstrated the feasibility to produce these types of polymers in native polymer producers such as Sinorhizobium meliloti and Pseudomonas putida.

Comments by Reviewer #2:

General Comments

This manuscript describes the engineering of Sinorhizobium meliloti and Pseudomonas putida to synthesize novel co-polymers containing both polyhydroxyalkanoates (PHAs) and polylactic acid (PLAs). This work is novel and innovative. The manuscript is well written, the experimental approach and methods are appropriate and well described, and the conclusions are supported by the data. This manuscript will make an excellent contribution to the literature. There are only a few minor issues that need to be addressed before the manuscript may be published.

General response:

We apologize for those minor issues. We have addressed them as described following each comment.

Specific Comments

Page 6, line 102: In the sentence, “…were provided by R. Nordeste, who constructed them as recently described (27)…”, please provide the institutional affiliation of R. Nordeste.

Response:

R. Nordeste, University of Waterloo. This has been added to the manuscript.

Page 7, line 115: Please provide the vendor/manufacturer and its location for the culture medium “0.5*E2”

Response:

We have added the reference for 0.5X E2.

Page 7, lines 120 to 121: In the sentence, “β-glucuronidase levels were determined as an indication of the transcript level of synthesized genes in both E. coli and S. meliloti strains.” Firstly, do the authors really mean E. coli here? Should this not be P. putida?

Second, the assay described is protein (gene product) based. The authors cite two references, one which describes the use of the β-glucuronidase (GusA) assay in actinomycetes (reference 36) and the other describes the use of this assay as a measure of promoter activity in Lactic Acid bacteria (reference 37). In the current paper, the authors suggest that the assay is a direct indicator of the level of transcription of the genes of interest, but this is based on the assumption that the level of protein activity detected is proportional to the level of translation, and this in turn is proportional to the level of transcription. Is this actually the case? Can the authors provide some additional clarification about the relationship between transcription, translation, and enzyme activity in S. meliloti and P. putida?

Response:

We should be more clear about the rationale for this experiment. At first, we aimed to develop the plasmid-based system in our primary short-chain-length PHA producer host S. meliloti. Therefore, we conducted this experiment in S. meliloti only. We added E. coli as a positive control along with our host because the reporter gene was originally obtained from E. coli.

Regarding the second question, we again performed the assay in E. coli and S. meliloti to determine the transcriptional level of the genes of interest. The assay has been applied to quantitatively analyse the activity of the promoter (in terms of gusA expression), hence inferring the transcription of the genes that are constructed under the same promoter with the gusA gene fusions. This assay has been used in different systems such as plants, mosses, algae, fungi and various bacteria (E. coli, lactic acid bacteria, actinomycetes). We obtained similar results in S. meliloti compared to E. coli (data not shown) which strongly suggested the validity of this assay in our host.

Figure 3: The resolution of the image for Figure 3 is very poor, and the bands in each lane have merge with each other so it is difficult to tell which lane is which. Can the authors replace this gel image with a better one?

Response:

We have removed this figure, as we agree that the quality is poor, and it does not provide much useful information.

Page 11, lines 202 to 209: The authors refer to GusA, which is the gene product β-glucuronidase. For clarity, please revised the sentence on page 7, line 120, as follows: “β-glucuronidase (GusA) levels were determined….

Response:

We have edited the sentence at the reviewer’s suggestion.

Comments by Reviewer #3:

The authors present a plasmid containing the genes encoding the propionate CoA transferase of C. propionicum and a modified PHA synthase I of Pseudomonas sp. MBEL 6-19. They introduced this into phaC mutants of S. melioti and P. putida and report the production of PLA homopolymer in S. meloti and of a novel quaterpolymer P(3HB-co-LA-co-3HH-co-3HO) in P. putida in the presence of octanoate. Though the production of a novel quaterpolymer is potentially interesting for applied processes, the manuscript, in its present state, has a number of shortcomings.

General response:

We apologize for these shortcomings. We have addressed them as described following each comment.

p. 9, ll. 147-152: It would be helpful to have some more information on the genetic arrangement of the plasmid, e.g. how does the gusA reporter work, is it a transcriptional or translational fusion? How was the codon optimization achieved? What does the 532 or the 1400 stand for in the gene names? What RBSs are used and where were they located?

Response:

Figure 1 describes the genetic arrangement of the plasmid. It is a transcriptional fusion driven by the tac promoter under the regulation of lacIq on the vector; both genes of interest were introduced into the multiple cloning site (MCS) in front of the gusA gene. We have described how we constructed the plasmid in the text.

We described previously (Tran and Charles 2015) that these genes were codon-optimized for S. meliloti using the codon adaptation tool JCat (Grote et al., 2005). The optimized sequences, with a His tag of six histidine residues at the end of each gene (before the stop codon), and a S. meliloti ribosome binding site (RBS) at the beginning of each gene, were then synthesized on a single fragment, with phaC1400 immediately following pct532. The pct gene which was originally from Clostridium propionicum had an increase of GC content from 40.38% to 53.65% after codon-optimization. Meanwhile, the phaC gene which was originally from P. putida did not show any drastic change of GC content (62.14% compared to initial 58.86%) after codon-optimization since P. putida has GC content similar to S. meliloti. In contrast, the overall number of codons drastically decreased across both genes (for the pct gene, the number of codons decreased from 52 to 37; for the phaC gene, the number of codon decreased from 56 to 39). Both numbers (532 and 1400) are used to be consistent with the gene names used by Jung et al. who originally mutated these genes and then screened them for altered enzyme activities.

Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D. 2005. JCat : a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 33:526–531.

Jung YK, Kim TY, Park SJ, Lee SY. Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers. Biotechnol Bioeng [Internet]. 2010;105(1):161–71.

p. 9, ll. 161-167 and Fig. 2: Why is there such a big difference in the fluorescence of SmUW257 comparing Fig. 2a to b? It should be comparable, as only IPTG is added in a) but this strain carries the empty plasmid. At the same time in 2b) it is hard to see a difference between the negative control (SmUW255) and the positive control (SmUW257). In my opinion this assay is not suitable to draw quantitative conclusions, only qualitative if any, as fluorescence depends on the amount and density of cells, which is hard to equal in solid phase medium. To get a rough estimation on the amount of polymer produced, the authors could repeat this plate experiment ensuring that the same amount of cells is plated or stain liquid cultures with the same OD (better cell count).

Response:

We also agree with the reviewer that this assay should only be used to qualitatively determine polymer production in bacteria. We used this technique as our initial rapid screening for assessment of polymer production in the different strains. For quantitative analysis, we used Gas Chromatography method to determine how much polymer was produced, as well as to identify polymer composition.

p.10, ll. 174-179 and Fig. 3: In MHO nothing can be concluded form this WB. Apart from the fact, that it is hard to distinguish the different lanes, no band is observed in the positive control, and the rest is mainly smear. How did the authors ensure that the same amount of protein is loaded in each lane, this should be stated in the M&M part or a Coomassie stained gel should be shown. Which lanes show the sample without IPTG, which is mentioned in the manuscript?

Response:

We have removed the figure.

p. 10, Polymer production (Fig.4): How many independent replicates where analyzed? What do the error bars represent? Is the % polymer referred to CDW?

Response:

The experiment was done in biological duplicate. Error bars represent the range of the mean. The % polymer content is referred to DCW.

p. 11, Tab. 2: Why is the PLA production negatively correlated to the lactic acid concentration in the medium? Is that expected? Please comment on this.

Response:

Even though lactic acid is theoretically an ideal substrate to produce polymer precursor, it does not seem to be a good substrate to support cell growth. Also, the more lactic acid we added, the more adverse effects on growth were observed, perhaps due to lowering pH in the medium. Evidently, we observed a significant decrease in DCW, as shown in Table 2. As a consequence, the overall PLA production is inversely proportional to the lactic acid concentration.

Fig. 5 and 6: Information on how many replicates were analyzed and what the error bar represent is missing. It would be more coherent to have in Fig. 5b and 6b the same order of the samples (from day 1 to 3 or vice versa).

Response:

The figures have been revised, as recommended. The experiment was done in duplicate. Results are presented as mean ± standard deviation (SD). Error bars indicate SD.

p. 12, ll. 227-228: The authors state that the gusA reporter expression is inversely proportional to PHB production. I don’t see a correlation between the expression and the polymer content. The GusA activity, which serves a s a proxy for gene expression, seems to be either on or off. The authors should perform a correlation and include a correlation coefficient to support this statement

Response:

We thank the reviewer for this comment. What we observed in our experiment that the longer we induced the promoter by adding IPTG at different time points, the lower polymer content detected there appeared to be. Meanwhile GusA activity increased in terms of induction time. More importantly, the strains were still able to produce polymer quite efficiently at base level (without induction). It is merely a suggestion that enzyme activity might not always be directly proportional to polymer production.

p.12, l. 229: Reference is made to S1, however it is not available.

Response:

We have removed this reference to S1

p.12, l. 230: How was the significance determined?

Response:

We have altered the statement to better reflect that we did not determine statistical significance.

Fig. 7a is redundant with Fig. 7b. and can be omitted from the manuscript.

It has been omitted.

p.14, l. 265: Did the authors mean sodium octanoate?

Response:

It is indeed sodium octanoate.

Fig 9a: When were the samples taken? Is this result of only one analysis? Please comment on this.

Response:

The time of sampling is provided in the Methods section. The experiment was done in duplicate.

Fig. 9b: How do the authors explain the difference in CDW in PPU20, PPU21, and PPU19? PPU19 reaches more than twice the CDW of the wt strain in LB medium. Why is this not observed with minimal medium? Is that a common phenotype, or just observed in this specific experiment?

Response:

We have removed these data from the paper.

Technical comments:

1. Information on media composition is completely missing (YM, TY, 0.5*E2)

Response:

We have provided references.

2. A list of abbreviations would be helpful (especially for the polymer abbreviations, HB, LA, HH. HV etc.).

Response:

We have now provided this on first use within the text.

Submitted filename: Response to reviewers Tam PLOS.docx

Click here for additional data file.

20 Dec 2019

PONE-D-19-15052R1

Lactic Acid Containing Polymers Produced in Engineered Sinorhizobium meliloti and Pseudomonas putida

PLOS ONE

Dear %Dr% %Charles%,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Feb 03 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Francisco Martinez-Abarca, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have adequately address the concerns of this reviewer, and is now acceptable for publication.

Reviewer #3: The manuscript in its present state is more a short communication than a research article. In their revised version the authors have deleted all controversial data, and replaced part of figure 9 with table 3. In the first part the authors show that an already published pathway for polymer production works plasmid-based in S. meliloti, however there is no benefit over the wildtype strain neither in terms of polymer content nor in composition. Only when the PHB pathway is blocked, small amounts of PLA are detectable. Furthermore, there is the unexplained paradox of induction of transcription with IPTG and polymer accumulation. It might be due to metabolic burden, simultaneous degradation of the polymer, or a disbalance of the enzymatic machinery. The authors did not analyze this in more detail. Growth curves and rates could give a hint on this. In my opinion, this first part of the manuscript is marginally innovative and does not contribute significantly to the field.

In the second part, the authors show that by implementing this pathway in P. putida a novel co-polymer can be produced up to around 40% of the CDW. This is new and interesting and a good starting point for further investigations.

Some issues should be corrected before acceptance:

1) Material and methods need to be adapted to the shortens version of this article, e.g. no data is shown for P. putida grown in liquid LB medium for polymer production, however this is still part of this section.

2) The number of analyzed replicates should be stated, either in the figure or table legends or in the Material and methods section.

3) p. 11 l. 195/196: how can there be error bars in a table? Delete this sentence.

4) p.15 l. 238 and table 3: see above and numbers are not presented with +/- standard deviation. Why do the authors use a different nomenclature for the P. putida strains in this table? Or are these other strains?

5) p. 17 ll. 314-320: This sentence is hard to understand as it is very long.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

17 Feb 2020

We have addressed Reviewer 3’s concerns as follows.

Some issues should be corrected before acceptance:

1) Material and methods need to be adapted to the shortens version of this article, e.g. no data is shown for P. putida grown in liquid LB medium for polymer production, however this is still part of this section

We have removed the information as appropriate.

2) The number of analyzed replicates should be stated, either in the figure or table legends or in the Material and methods section

This information has been added where it had been missing.

3) p. 11 l. 195/196: how can there be error bars in a table? Delete this sentence

This has been corrected.

4) p.15 l. 238 and table 3: see above and numbers are not presented with +/- standard deviation. Why do the authors use a different nomenclature for the P. putida strains in this table? Or are these other strains

We have added standard deviation values and changed the strain nomenclature to be more consistent.

5) p. 17 ll. 314-320: This sentence is hard to understand as it is very long.

We have revised the sentence structure.

Submitted filename: Response to reviewers Tam PLOS rev.pdf

Click here for additional data file.

19 Feb 2020

Lactic Acid Containing Polymers Produced in Engineered Sinorhizobium meliloti and Pseudomonas putida

PONE-D-19-15052R2

Dear Dr. Charles,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Francisco Martinez-Abarca, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

28 Feb 2020

PONE-D-19-15052R2

Lactic Acid Containing Polymers Produced in Engineered Sinorhizobium meliloti and Pseudomonas putida

Dear Dr. Charles:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Francisco Martinez-Abarca

Academic Editor

PLOS ONE