3.1. Comparative Analysis of A. vinelandii Wild Type and ∆Avin_16040 Mutant Strains Links PHA Biosynthesis Efficiency to the S-Layer
PHA synthases from both
A. vinelandii strains were evaluated in terms of expression activity and gene sequences. The PHA synthase activity was analyzed by measuring the release of CoA from 3HB-CoA using a spectrophotometer. According to
Table 2, the PHA synthase activities of the wild type and mutant strains were determined to be 395 and 485 U/g of protein, respectively. Previous studies also reported that PHA synthase activities of
Chromobacterium sp. USM2 and
C.
necator harboring the PHA synthase gene from
Aeromonas caviae were 2462 and 1600 U/g, respectively [
26,
27]. These synthase activities were much higher than the PHA synthase activities of
A.
vinelandii.
Although the cell dry weight (4.9 g/L) and PHA concentration (2.6 g/L) of the mutant strain was higher than that of the wild type strain (3.2 and 1.1 g/L, respectively) (
Table 3; no precursor), the activities of synthases in both strains were considerably similar. These results were supported by the number and size of PHA granules in both
A. vinelandii strains as seen in the transmission electron micrographs (
Figure S1). The diameter and number of PHA granules for both
A. vinelandii strains were comparable, indicating that the PHA synthase activities from both bacterial strains were also similar. Additionally, the molecular weight and polydispersities of both bacterial strains were also similar (
Table 4).
According to Sim and colleagues, the molecular weight of PHA is affected by the PHA synthase activity [
28]. For more information, both wild type and mutant strains were cultivated under the same conditions including culture medium, food source, cultivation duration, and environment. These results suggested that higher PHA production from the mutant strain compared to the wild type strain was due to the absence of the S-layer. Without the S-layer, the cells could ingest the food source more easily as compared to the wild type strain surrounded by the S-layer.
Since the
A. vinelandii mutant strain could produce a higher PHA concentration compared to that by the
A. vinelandii wild type strain, it was essential to compare the PHA synthases that played a role in PHA generation based on their gene sequences. The ability to generate PHA with a different monomer was checked by expressing the gene encoding the PHA synthase from
A. vinelandii in
C.
necator for PHA biosynthesis. Previous studies showed the evaluation of the PHA synthase’s expression in different hosts, and the most frequently employed hosts were
C.
necator strains [
18,
29]. In this study, the PHA synthase of
A. vinelandii was evaluated by carrying out PHA biosynthesis using
C.
necator as a host strain. Prior to that, the gene sequences of the PHA synthase between the wild type and mutant strains of
A. vinelandii were compared. The only difference between the gene sequences of the two bacterial strains was nucleotide 1166, whereby guanine and adenine were found to be present in the wild type strain and mutant strain, respectively. However, the difference was negligible because the nucleotide sequences for both strains translate to valine (GUG and GUA). Since this study focused on the
A. vinelandii mutant strain due to the higher PHA accumulation, the PHA synthase from the mutant strain was selected for further analyses.
3.2. Identification of Carbon Sources Usable for PHA Production by the PhaC of A. vinelandii in Various Host Strains
Different host strains were used for PHA production in this study.
C. necator PHB¯4 is a PHA negative mutant that is commonly used for the evaluation of synthases for PHA polymerization from various carbon sources. Meanwhile,
C.
necator Re2058 that heterologously expresses a
phaJ was employed for synthase characterization due to its enhanced ability to accumulate PHA compared to
C. necator PHB¯4 (Tan et al., 2020, Int. J. Biol. Mol.) and to investigate the possibility of medium-chain-length (mcl) monomer incorporation such as 3-hydroxyhexanoate (3HHx) from oil as a carbon source [
18].
Around 4 and 2.1 g/L of dry cell and PHA could be produced by both transconjugated strains using 10 g/L of fructose as a carbon source (
Table 5). Similar amounts of PHA (around 2.4 g/L) could be produced by the
A.
vinelandii Δ
Avin_16040 mutant strain using 30 g/L of fructose as a carbon source (
Table S1). This might be due to competition of carbon source for the production of PHA, alginate, and other components in the
A.
vinelandii mutant strain [
30]. For
C.
necator Re2058 transconjungant, when 10 g/L of CPKO was used as a carbon source, up to 8.6 and 7.7 g/L of dry cell and PHA could be obtained while lesser dry cell and PHA were obtained using
C. necator PHB¯4 transconjugant. No other mcl monomer was detected, suggesting that this PHA synthase is only able to polymerize PHA with a short-chain-length (scl) monomer.
Glucose, sucrose, and glycerol did not contribute to biomass increment as
C.
necator is not able to consume these carbon sources for growth. Molasses, a by-product formed during the sugar production process, was utilized, but only a small increase in biomass was observed. The cells could utilize molasses because molasses was composed of a mixture of monosaccharides, disaccharides, or other complex sugar compounds. Monosaccharides such as fructose in the molasses were consumed, but the amount of fructose might not be sufficient for the enhancement of cell growth and PHA production, as molasses contain other sugars as well. The possibility for
A. vinelandii to utilize other inexpensive, renewable carbon sources for PHA biosynthesis should be evaluated in future studies, as this not only enables cost-efficient production of PHA, but also serves as a disposal strategy for waste substrates. Soya waste and malt waste have been used by
Alcaligenes latus as a carbon source to produce P(3HB) [
31], showing that renewable resources as such are able to be converted to PHA.
As fructose contributed to better cell growth and PHA accumulation by the transconjugated strains, different concentrations of fructose were also screened to evaluate the best concentration for PHA accumulation (
Table S1). Around 20 g/L of fructose yielded the highest PHA amount. The higher fructose concentration did not increase the PHA production but slowly decreased the cell biomass, which might be due to substrate inhibition [
32]. After that, a different monomer incorporation was evaluated by adding different precursors during bacterial cultivation. Since the
A.
vinelandii wild type strain and
A.
vinelandii Δ
Avin_16040 used 30 g/L of fructose for bacterial cell cultivation, the ability of monomer incorporation by transformed strains was also screened using 30 g/L of fructose. Although 20 g/L of fructose yielded the highest PHA amount by the transconjugants, in order to standardize the carbon concentration for the
A.
vinelandii wild type strain and
A.
vinelandii Δ
Avin_16040 as well as the transconjugants, 30 g/L of fructose was selected to be used as the carbon source for further analysis.
3.3. PhaC of A. vinelandii Shows Ability for the Utilization of 4-Hydroxybutyrate
Sodium valerate, sodium 4-hydroxybutyrate, 1,4-butanediol, gamma-butyrolactone, and sodium heptanoate could be utilized for the production of PHA consisting of monomers other than 3HB (
Table 3).
A. vinelandii was known to produce PHA consisting of 3HB and 3HV [
11,
33]. Interestingly, PHA containing 4HB was found to be produced from structurally related precursors. This is a new finding as this bacterium is not previously known to synthesize PHA containing 4HB. In both native and heterologous host strains, PhaC of
A.
vinelandii can synthesize PHA containing 4HB, indicating the inherent ability of the synthase to accommodate and utilize the 4HB substrate.
PHA containing approximately 10 mol% of 4HB was produced by the
A.
vinelandii Δ
Avin_16040 mutant strain from 2 g/L of sodium 4HB. PHA that consists of 4HB is known to be degraded by lipase and can be applied in the medical field as a scaffold or other surgical materials. Incorporation of 4HB not only alters the properties of PHA but also increases the value of PHA for application [
34]. The wild type strain of
A.
vinelandii also could utilize the precursors and produce PHA containing similar composition of 4HB, as shown in
Table 3. Previous studies showed that 4HB could be polymerized by several wild type strains including
C.
necator,
Delftia acidovorans,
A.
latus,
Rhodococcus ruber,
Comamonas testosteronii, and
Hydrogenophaga pseudoflava [
35,
36,
37,
38,
39].
The presence of 4HB monomer in the PHA produced by the
A.
vinelandii Δ
Avin_16040 mutant strain was further confirmed by
1H NMR analysis (
Figure S2). Around 9 mol.% of 4HB was measured based on the peaks on the chromatogram, and the results did not deviate much compared to the results obtained from GC analysis (10 mol%). Since there is a lack of publications on the production of PHA with different monomers by
A.
vinelandii, there might be unknown inhibition on the PHA biosynthesis pathway by other components in the cell, such as competition of carbon source for both PHA and alginate productions. The metabolic pathway for 4HB production was usually more direct compared to that for 3HB production since 4HB could be formed directly via a reaction between synthase and a structurally related carbon source or precursor, whereas formation of 3HB requires three main reactions through enzyme catalysis [
40,
41]. Hence, the carbon source used for 3HB production was channeled towards the production of other components, while the precursor was directly used to generate 4HB, thus, increasing the 4HB monomer composition and reducing the 3HB monomer composition.
The substrate specificity of the PHA synthase was further evaluated in
C.
necator transconjugants (
Table 3). Although more dry cells and PHA could be obtained from the transconjugants as compared to the
A.
vinelandii strains, the monomeric composition of produced PHA remains the same. For
A.
vinelandii strains, PHA containing up to 10 mol% of 4HB was obtained by utilizing sodium 4-hydroxybutyrate, while
C. necator transconjugants could only synthesize PHA with a lower 4HB fraction, which was approximately 4–5 mol%. The cell dry weight and PHA content of
C. necator transconjugants grown on 1,4-butanediol is relatively high, and 4HB fraction is approximately 5 mol%. As discussed previously, a higher 4HB molar fraction in the PHA synthesized by
A.
vinelandii strains could be obtained from structurally related precursors such as sodium 4-hydroxybutyrate, as 3HB monomer production is decreased from carbon sources such as fructose, which is partially used for the production of alginate. Meanwhile,
C.
necator transconjugants do not possess pathways for alginate production, and since there is no competition for carbon source used for 3HB production, the 4HB fraction in the resultant PHA is comparatively lower. Of notable interest is that PHA with a higher 3HV molar fraction (6 to 15 mol%) could be synthesized by
A.
vinelandii strains and
C. necator PHB
−4 transconjugants when sodium heptanoate is used as precursor.
Table 6 shows that increasing concentrations of sodium 4-hydroxybutyrate could increase the 4HB monomer content in the PHA produced by the
A.
vinelandii Δ
Avin_16040 mutant strain and
C.
necator transconjugants. However, the cell dry weight exhibited a significant reduction from 4 to 2 g/L when sodium 4-hydroxybutyrate concentration was increased from 2 to 10 g/L. The reduction in cell dry weight was primarily due to the decrease in PHA concentration since the obtained residual cell biomass was similar regardless of sodium 4-hydroxybutyrate concentration. Toxicity of precursor compounds were previously reported to impact cell growth and PHA accumulation negatively [
42]. Although the cell dry weight decreased due to increased precursor concentration, the 4HB molar fraction increased from 10 to 22 mol%. Increments in 4HB molar fraction could change the polymer properties to suit a wider range of applications. Taken together, the data suggest that the PhaC of
A.
vinelandii has a preference for 4HB monomer utilization.