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Polyhydroxybutyrate production by recombinant Escherichia coli based on genes related to synthesis pathway of PHB from Massilia sp. UMI-21

Abstract

Background

Polyhydroxybutyrate (PHB) is currently the most common polymer produced by natural bacteria and alternative to conventional petrochemical-based plastics due to its similar material properties and biodegradability. Massilia sp. UMI-21, a newly found bacterium, could produce PHB from starch, maltotriose, or maltose, etc. and could serve as a candidate for seaweed-degrading bioplastic producers. However, the genes involved in PHB metabolism in Massilia sp. UMI-21 are still unclear.

Results

In the present study, we assembled and annotated the genome of Massilia sp. UMI-21, identified genes related to the metabolism of PHB, and successfully constructed recombinant Escherichia coli harboring PHB-related genes (phaA2, phaB1 and phaC1) of Massilia sp. UMI-21, which showed up to 139.41% more product. Also, the vgb gene (encoding Vitreoscilla hemoglobin) was introduced into the genetically engineered E. coli and gained up to 117.42% more cell dry weight, 213.30% more PHB-like production and 44.09% more product content. Fermentation products extracted from recombinant E. coli harboring pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb were identified as PHB by Fourier Transform Infrared and Proton nuclear magnetic resonance spectroscopy analysis. Furthermore, the decomposition temperature at 10% weight loss of PHB extracted from Massilia sp. UMI-21, recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb was 276.5, 278.7 and 286.3 °C, respectively, showing good thermal stability.

Conclusions

Herein, we presented the whole genome information of PHB-producing Massilia sp. UMI-21 and constructed novel recombinant strains using key genes in PHB synthesis of strain UMI-21 and the vgb gene. This genetically engineered E. coli strain can serve as an effective novel candidate in E. coli cell factory for PHB production by the rapid cell growth and high PHB production.

Background

The heavy application of petrochemical-based plastics causes severe environmental and health issues. Polyhydroxyalkanoates (PHAs) serve as important alternative sources because of their excellent physical properties, such as similar to synthetic plastics, biodegradability and thermoplasticity [1, 2]. That kind of polymer are accumulated by numerous bacteria as carbon and energy storage substances and can be commercially produced by microbial fermentation [3]. However, the high-cost of PHA production becomes restrictions on the large-scale production and application of PHAs [4]. Therefore, exploring new and better PHA producers and genetic modification of fermentation bacteria have become important focus.

PHA-producing microbes are isolated from diverse habitats such as soil, water, waste streams and even extreme environments and about 92 bacterial genera were found can produce PHAs [5,6,7]. Ralstonia eutropha H16 is considered the model organism for PHA metabolism [8]. Also, species in Pseudomonas, Azotobacter, Alcaligenes, etc. are widely studied for PHA production [7, 9]. For the past few years, members in a recent descripted genus, Massilia [10], were reported to show a capacity of PHA accumulation [11,12,13,14]. Those Massilia isolates produce polyhydroxybutyrate (PHB, the most common polymer of PHAs) and have a product content from trace (lower than 1%) to about 45% of cell dry weight (Additional file 1: Table S1). Some strains from the genus Massilia which could grow and produce PHAs using seaweed-derived carbohydrates are considered highly promising candidates for seaweed-degrading bioplastic producers [15]. Of them, Han et al. isolated one PHA-producing microorganism from green algae Ulva and identified as Massilia sp. UMI-21, which could produce PHB from starch, maltotriose or maltose as a sole carbon source. Only polysaccharides instead of monosaccharides can be utilized by strain UMI-21, which makes it very special [14]. However, although its PHA synthase gene (phaC) was cloned, the genes involved in PHA metabolism in Massilia sp. UMI-21 are still unclear thus hindering the application.

Producing PHAs by its natural producers can be difficult since the long time and non-uniform condition to grow, accumulate and extract PHA polymers from their cells by fermentation [16]. As a widely used bacterial cell factory, Escherichia coli performs excellently in generating higher yields of the biopolymer because of its fast growth and easy cell lysing [17, 18]. Therefore, many PHA synthetic genes have been cloned into E. coli and more PHA yield and lower cost were successfully gained by this way [19, 20]. During the fermentation improving processes of genetically engineered E. coli, the application of vgb gene, encoding Vitreoscilla hemoglobin (VHb), plays an important role [21, 22]. VHb can efficiently bind and transport oxygen to the respiratory chain by interacting with terminal oxidase, especially under oxygen-limited conditions, showing powerful oxygen transport capacity and thus excellent performance in cell growth enhancement and PHA accumulation [23].

Here, we assembled and annotated the genome of Massilia sp. UMI-21 using long read sequencing, and identified genes related to the metabolism of PHB based on the genome. Then, PHB-related genes (phaA2, phaB1 and phaC1) of strain UMI-21 were cloned and introduced into E. coli to produce PHB. Also, the vgb gene was employed in genetically engineered E. coli for more PHB production. The fermentation products extracted from recombinant E. coli were finally identified by Fourier Transform Infrared and Proton nuclear magnetic resonance spectroscopy analysis and their thermal properties were also analyzed by thermogravimetric analysis.

Results

Genome sequencing and identification of PHB-related genes in Massilia sp. UMI-21

The genome of the PHB-producing bacterium Massilia sp. UMI-21 was sequenced using PacBio RS II and Illumina 4000 platform, and 2871 Mb PacBio subreads and 1153 Mb Illumina data were generated respectively, resulting into a 541 × and 217 × genomic depth. The estimated genome size was 5.30 Mb with a GC content of 67.06% (Table 1; Fig. 1A). A total of 4672 genes were predicted with a total length of 4.70 Mb, accounting for 88.61% of the whole genome.

Table 1 General feature statistics of the Massilia sp. UMI-21 genome
Fig. 1
figure 1

Genes related to the metabolism of PHB in Massilia sp. UMI-21 genome. (A) Circos plot of Massilia sp. UMI-21 genome assembly. From outside to inside: genome size, forward strand gene, reverse strand gene, forward strand ncRNA, reverse strand ncRNA, repeat units, GC content and GC-Skew. (B) The organization of PHB relevant genes in the genome of Massilia sp. UMI-21

To identified genes relevant to PHB metabolism in the genome of Massilia sp. UMI-21, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), NCBI Non-redundant (Nr), SwissProt, Gene Ontology (GO), TrEMBL, and EggNOG databases were used in gene function annotation. As a result, a total of 14 genes encoded for enzymes involved in PHB metabolic pathways were annotated, including 2 β-ketothiolase genes (phaA), 3 acetoacetyl-CoA reductase genes (phaB), 3 PHA synthase genes (phaC), 3 PHA depolymerase genes (phaZ), 1 PHA synthesis repressor/regulator gene (phaR), and 2 phasin genes (phaP, Table 2). To investigate the phylogenetic relationship of three important enzymes, PhaA, PhaB and PhaC, the protein sequences of the predicted 2 phaA, 3 phaB and 3 phaC genes in Massilia sp. UMI-21 and their homologs in other strains were analyzed. The phylogenetic tree suggested that PHA synthases in Massilia sp. UMI-21 might be Class I PhaC (Additional file 1: Fig. S1). For gene distribution, in bacterial genomes, PHA-related genes usually distribute and function in gene clusters [24]. However, those genes were scattered throughout the genome of Massilia sp. UMI-21. Only phaA2 and phaB1 constituted a phaAB operon, and phaC1 and phaR formed a phaCR operon (Fig. 1B).

Table 2 Putative PHB-related genes in the genome of Massilia sp. UMI-21

Construction of genetically engineered Escherichia coli for PHB production

Previous study has shown that the polymer produced by Massilia sp. UMI-21 was PHB [14]. The synthesis of PHB polymer from acetyl-CoA is sequentially catalyzed by three kinds of enzymes including β-ketothiolase, acetoacetyl-CoA reductase and PHA synthase, which are encoded by phaA, phaB, and phaC, respectively. We aligned the predicted 2 phaA, 3 phaB and 3 phaC genes from the genome of Massilia sp. UMI-21 to their homologous genes of the model PHA-producing strain Ralstonia eutropha H16 [25, 26], and found that genes UMI-21GL000317 (phaA2, percent identity at 75.8% in DNA and 74.9% in protein), UMI-21GL000318 (phaB1, percent identity at 67.7% in DNA and 60.7% in protein) and UMI-21GL002338 (phaC1, percent identity at 65.6% in DNA and 60.3% in protein) showed the highest sequence identity (Additional file 1: Fig. S2S4). Also, in consideration of PHA-related genes usually cluster into operons and function in PHA synthesis [24], those three genes phaA2, phaB1 and phaC1 from phaAB and phaCR operon in Massilia sp. UMI-21 genome were cloned for construction of recombinant E. coli strains for PHB production, with the vector pETDuet1 as the plasmid backbone (Fig. 2A). First, gene fragments phaA2phaB1 was amplified from the genome of Massilia sp. UMI-21, and inserted into the pETDuet1, producing pETDuet1-phaA2phaB1. Gene phaC1 was then cloned into pETDuet1 and pETDuet1-phaA2phaB1, producing pETDuet1-phaC1 and pETDuet1-phaA2phaB1-phaC1, respectively. It has been accepted that, Vitreoscilla hemoglobin encoded by vgb gene has a great help to enhance cell growth and product synthesis [21], and vgb gene was widely used in improving PHB accumulation [22]. Therefore, we synthesized the vgb gene fragment, based on the coding sequence of vgb gene in Vitreoscilla sp. C1 (NCBI accession: L21670), into recombinant plasmid pETDuet1-phaA2phaB1-phaC1, producing pETDuet1-phaA2phaB1-phaC1-vgb (Fig. 2B). Finally, genetically engineered E. coli DH5α harboring each plasmid of pETDuet1-phaA2phaB1, pETDuet1-phaC1, pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb were constructed, where the latter two were for the PHB production with the former two as controls. Results of qRT-PCR of phaA2, phaB1 and phaC1 from the two PHB-producing strains, E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb, validated that all three genes successfully expressed in both recombinant strains (Additional file 1: Fig. S5).

Fig. 2
figure 2

Construction process of recombinant plasmids. (A) Map of pETDuet1 as the plasmid backbone. (B) Schematic of four recombinant plasmids

PHB accumulation by E. coli expressing PHB-related genes of Massilia sp. UMI-21

Fermentation products were extracted after 72 h shake flask experiments in Mineral Salt (MS) medium. The carbon source of recombinant E. coli DH5α harboring each plasmid was glucose. Since Massilia sp. UMI-21 could not grow when glucose was used as a carbon source [14], we employed starch and sucrose instead. For E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1phaC1-vgb, Luria-Bertani (LB) medium supplemented with glucose was also used to investigate the influence of different medium (Fig. 3).

Fig. 3
figure 3

Cell growth and PHB accumulation of Massilia sp. UMI-21 and recombinant E. coli after 72 h fermentation. (A) Cell dry weight (CDW) of Massilia sp. UMI-21 and four recombinant E. coli. (B) and (C) PHB titer and PHB content (wt%) of Massilia sp. UMI-21, E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb

The cell dry weight (CDW) of Massilia sp.UMI-21 with starch and sucrose as carbon sources had comparable results of 0.439 g/L and 0.445 g/L, respectively. Similarly, genetically engineered E. coli DH5α-pETDuet1-phaA2phaB1 and DH5α-pETDuet1-phaC1 could also grow on the glucose contained MS medium with a 0.487 g/L and 0.421 g/L CDW. In the MS medium, recombinant E. coli DH5α harboring pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb had the highest CDW with 0.596 g/L and 0.668 g/L, increasing by 34.84% and 51.13% than strain UMI-21, respectively. When cultured in LB medium, those two recombinant strains showed higher CDW, up to 0.801 g/L and 0.961 g/L with approximately 81.22% and 117.42% increase (Fig. 3A).

As expected, no PHA was extracted from E. coli DH5α-pETDuet1-phaA2phaB1 and DH5α-pETDuet1-phaC1 since lack of complete set of genes for PHB synthesis. The PHA titer and content (wt%) of recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb in MS medium was 0.157 g/L (26.34%) and 0.195 g/L (29.19%) respectively, which were higher than Massilia sp. UMI-21, 0.105 g/L (23.91%) and 0.098 g/L (22.02%) with starch and sucrose as carbon sources. That means about 54.68% and 92.12% increase in product titer, and 14.70% and 27.11% increase in product content. In LB medium, the performance of the two PHB-producing genetically engineered E. coli were even better, with 0.243 g/L and 0.318 g/L product titer and 30.33% and 33.09% PHB content. Compared with Massilia sp. UMI-21, PHB production increased 139.41% and 213.30%, and PHB content increased 32.07% and 44.09% (Fig. 3B, C).

Characterization of fermentation products

In order to identify structure and determine properties of fermentation products by genetically engineered E. coli, Fourier Transform Infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1H-NMR), and thermogravimetric analysis (TGA) were conducted. Fermentation products in MS medium from recombinant E. coli DH5α, harboring plasmid pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb, and Massilia sp. UMI-21 with glucose and sucrose as the carbon source were used in this study. Consistent with the characteristic absorption bands of PHB products produced by Massilia sp. UMI-21, the FT-IR results of E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb products showed absorption bands at 3420 cm− 1 (-OH), 1719 cm− 1 (C = O), and 2850–2965 cm− 1 (C-H), indicating the fermentation products of recombinant E. coli was PHB (Fig. 4A). Also, the results of 1H-NMR analysis suggested that the polymer produced by recombinant E. coli was PHB, same with the fermentation products from strain UMI-21 (Fig. 4B).

Fig. 4
figure 4

Characterization of fermentation products. (A) Results of FT-IR analysis. (B) Results of 1H-NMR analysis. Plots from top to bottom show results of Massilia sp. UMI-21, E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb, respectively

Thermal stability of PHAs is important for their melt processing. The results of TGA showed that the initial decomposition temperature (T10%, temperature at 10% weight loss) of PHB extracted from recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb was 278.7 and 286.3 °C, respectively, which was in approximately the same T10% of PHB produced by Massilia sp. UMI-21 with 276.5 °C (Fig. 5).

Fig. 5
figure 5

TGA of fermentation products from Massilia sp. UMI-21 and recombinant E. coli harboring pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb

Discussion

Researches focusing on screening and construction of excellent, new and genetically engineered PHA-producing strains provide a theoretical basis for large-scale popularization and application of PHAs. In this study, we assembled and annotated the genome of Massilia sp. UMI-21, a PHB-producing bacterium isolated from previous study [14], and successfully constructed recombinant E. coli harboring PHB-related genes (phaA2, phaB1 and phaC1) of strain UMI-21, which showed more PHB accumulation than Massilia sp. UMI-21. Also, the vgb gene was introduced into the genetically engineered E. coli and the PHB production was further increased.

We identified 14 genes encoded for enzymes involved in PHB metabolic pathways in the genome of Massilia sp. UMI-21, including 2 phaA, 3 phaB, 3 phaC, 3 phaZ, 1 phaR, and 2 phaP (Table 2). With those clues, we can reasonably conclude that in Massilia sp. UMI-21, two Acetyl Coenzyme A molecules condense into one acetoacetyl-CoA molecule with the help of β-ketothiolase (PhaA), then acetoacetyl-CoA reductase (PhaB) reduces Acetoacetyl-CoA to 3-hydroxybutyryl-CoA (3HB-CoA), and finally 3HB-CoA is polymerized into PHB by PHA synthase PhaC [25, 26]. Since the discovery of phaCAB operon in model PHA-producing R. eutropha H16, genes relevant to PHA metabolism were usually found clustered together in bacterial genome [26,27,28,29]. Interestingly, in the genome of Massilia sp. UMI-21, PHA-related genes were disjointed except for phaA2B1 and phaC1R (Fig. 1B). That scattered distribution pattern was also found in the genome of other PHA-producing bacteria, such as Neptunomonas concharum JCM17730, which could accumulate PHB using fructose as the carbon source [30, 31]. However, further investigations are still needed to provide insights into whether and how gene distribution affects PHA synthesis.

Fig. 6
figure 6

Putative PHB metabolism pathway in E. coli DH5α-pETDuet1-phaA2phaB1-phaC1-vgb. (+) in green denotes activation; (-) in red denotes repression; Glucose-6P: glucose-6-phosphate; Glk: glucokinase; PTS: phosphotransferase system; Cyt: cytochrome; TCA: tricarboxylic acid cycle; VHb: Vitreoscilla hemoglobin

Heterologous expression of phaA2, phaB1 and phaC1 in E. coli resulted in PHB accumulation, showing that those three genes are crucial in the PHB metabolic pathway in Massilia sp. UMI-21. The putative PHB metabolism pathway in recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1-vgb was shown in Fig. 6, where VHb encoded by vgb gene enhances aerobic respiration and consequently increases the consumption of NADH and generation of NAD+, which contributes to PHB synthesis under the cooperation of PhaA2, PhaB1 and PhaC1. E. coli as a widely used engineered bacterium has more advantages than other bacteria in PHB production. For example, mature and versatile E. coli cultivation techniques enable mass production [32], PHA production in E. coli can be carried out using a wide range of carbon sources making it possible to reduce the cost of raw materials [33], and since the absence of depolymerase in the cell of E. coli, PHA polymers do not degrade during fermentation resulting in more product accumulation [34]. In this work, we obtained 54.68–139.41% more PHB accumulation than in Massilia sp. UMI-21 in cultivation of recombinant E. coli harboring pETDuet1-phaA2phaB1-phaC1 in MS or LB medium. Compared with similar work from de Almeida et al. (2010), in which they produced PHB using recombinant E. coli carrying phaBAC of Azotobacter sp. FA8 and obtained 20.4% PHB content in MYA medium with glucose, our work got higher production in both MS and LB medium with the same carbon source [35], showing PHB-related genes in Massilia sp. UMI-21 has the potential for applications. In addition, the further improvement, namely the introduction of vgb gene, resulted into 92.12% (in MS medium) and 213.30% (in LB medium) increase of PHB production (Fig. 3). Similar important roles of vgb gene for improving microbial fermentation processes have been widely observed [22]. For example, Horng et al. (2010) overexpressed the artificial phaCAB-vgb operon in E. coli and got 23%, 57% and 93% more CDW, PHB content and PHB concentration, respectively [36]. Products extracted from E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb was identified as PHB by FT-IR and 1H-NMR analysis (Fig. 4), which was identical to that from Massilia sp. UMI-21. Also, the thermal properties of PHB from our recombinant E. coli and strain UMI-21 were resembled with an approximately 280 °C initial decomposition temperature (Fig. 5), similar with the standard PHB (T10% ~270–290 °C) and about 20–65 °C higher than PHB extract from other organism, such as Burkholderia sacchari (T10% ~ 263 °C), Bacillus sp. CYR1 (T10% ~ 230 °C) and Micrococcus luteus (T10% ~ 224 °C), showing better thermal stability [9, 37, 38].

Conclusions

In this work, we presented genome assembly of PHB-producing Massilia sp. UMI-21 and further constructed recombinant E. coli based on genes related to synthesis pathway of PHB mined from the genome for PHB production. The results that PHB successfully and more accumulated in the recombinant E. coli harboring phaA, phaB and phaC showed these three might be key genes for PHB synthesis in strain UMI-21. Also, we introduced the vgb gene into recombinant E. coli and gained more PHB production and the fermentation products showed good properties. PHB synthesis by recombinant E. coli using monosaccharides is more efficient than that by Massilia sp. UMI-21 using starch, which could reduce the cost of PHB production. Thus, the genetically engineered E. coli DH5α-pETDuet1-phaA2phaB1-phaC1-vgb constructed in the present study can serve as an effective new strain for PHB synthesis by its rapid cell growth and high PHB production. Although further examination is needed to realize large scale PHB production, during the improving process such as fermentation optimization, this novel recombinant strain could contribute to the industrial low-cost PHA production. Moreover, the association study of other PHA-related genes will provide molecular basis for future synthetic biology research in novel PHAs.

Methods

Genome sequencing, assembly and annotation

Bacterial cells of Massilia sp. UMI-21 from overnight cultures were used for de novo genome sequencing with PacBio RS II and Illumina HiSeq 4000 platform at the Beijing Genomics Institute (BGI, Shenzhen, China). Genomic DNA was extracted using the FastPure Bacteria DNA Isolation Mini Kit (Vazyme, Nanjing, China). Draft genome was assembled using the Celera Assembler against a high-quality corrected circular consensus sequence subreads set [39]. To improve the accuracy of the genome sequences, GATK and SOAP tool packages (SOAP2, SOAPsnp, SOAPindel) were used to make single-base corrections [40, 41]. To trace the presence of any plasmid, the filtered Illumina reads were mapped using SOAP to the bacterial plasmid database (http://www.ebi.ac.uk/genomes/plasmid.html, last accessed July 8, 2016). Finally, the genome assembly of Massilia sp. UMI-21 was submitted to GenBank under the BioProject PRJNA669114.

For gene prediction, Glimmer3 with a Hidden Markov Model was employed [42]. Non-coding RNA, including tRNA, rRNA and sRNA was recognized with tRNAscan-SE, RNAmmer, and Rfam database [43,44,45]. The tandem repeats annotation was obtained using the Tandem Repeat Finder (http://tandem.bu.edu/trf/trf.html), and the minisatellite DNA and microsatellite DNA selected based on the number and length of repeat units. For gene function annotation, the best hit abstracted using BLAST alignment tool based on seven databases (Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), NCBI Non-redundant (Nr), SwissProt, Gene Ontology (GO), TrEMBL, and EggNOG database) was used [46,47,48,49,50,51,52]. Sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and pairwise sequence alignment (https://www.ebi.ac.uk/Tools/psa/) tools available at EMBL-EBI for multiple and pairwise sequence alignment, respectively. Phylogenetic trees were displayed by the online tool of iTOL v5 (https://itol.embl.de).

Construction of genetically engineered Escherichia coli

Molecular cloning and manipulation of plasmids were done with E. coli DH5α. For construction of recombinant plasmid, the vector pETDuet1 was used as a backbone. Gene fragments were amplified using KOD-Plus-Neo DNA polymerase (TOYOBO, Osaka, Japan) and primers listed in Additional file 1: Table S2, which were synthesized by Jilin KuMei company (Changchun, China). Restriction enzymes used for vector linearization were purchased from the New England Biolabs (Beijing) LTD (Additional file 1: Table S2). All seamless cloning between gene fragments and linear vectors was using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). First, PHB-related genes phaA2B1 was amplified from the genome of Massilia sp. UMI-21 and seamless cloned into pETDuet1 vector, which resulted in pETDuet1-phaA2phaB1. Second, gene phaC1 was inserted into pETDuet1 and pETDuet1-phaA2phaB1, producing pETDuet1-phaC1 and pETDuet1-phaA2phaB1-phaC1. The same method was used in the construction of recombinant plasmid pETDuet1-phaA2phaB1-phaC1-vgb. Based on the coding sequence of vgb gene from Vitreoscilla sp. C1 in NCBI (accession L21670), the vgb gene fragment was synthesized and connected to the pUC57 vector by Jilin KuMei company (Changchun, China). Then, the vgb gene fragment was amplified using the pUC57-vgb plasmid as template and seamless cloned into pETDuet1-phaA2phaB1-phaC1 (Additional file 1: Table S2). Finally, pETDuet1-phaA2phaB1, pETDuet1-phaC1, pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb was transformed into E. coli DH5α using the heat shock method, respectively.

Cultivation of genetically engineered E. coli and extraction of products

PHB accumulation were carried out in Massilia sp. UMI-21 and four genetically engineered E. coli DH5α harboring each plasmid. For strain UMI-21, cells were activated in R2A liquid medium (Hopebio, Qingdao, China) at 30 °C, 150 rpm for 24 h followed by a 24 h pre-culture with a 5% (v/v) inoculation in the same condition. A total of 5% (v/v) pre-culture was then inoculated into nitrogen-limiting Mineral Salt (MS) medium with 1% (w/v) cold water-soluble starch or sucrose and incubated at 30 °C, 150 rpm for 72 h (all reagents without explicitly stated were purchased from Beijing Chemical Plant and of analytical grade) [14]. For recombinant strains, cell activation and pre-culture were carried out in Luria-Bertani (LB) liquid medium at 37 °C, 180 rpm for 12 and 24 h, respectively. Then, 5% (v/v) pre-culture was inoculated into nitrogen-limiting MS with 1% (w/v) glucose as a carbon source and incubated at 37 °C, 180 rpm for 72 h. LB medium supplemented with 1% (w/v) glucose were also used for PHB accumulation in E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1phaC1-vgb.

After 72 h shake flask fermentation, the culture was harvested by centrifugation at 4 °C, 10,000 rpm for 10 min and washed twice with deionized water. The bacteria were rapid frozen at -80 °C and finally lyophilized to constant weight by FD-1B-80 vacuum freeze dryer (Biocool, Beijing, China). The cell dry weight (CDW) was used to assess the growth of strains.

PHB was extracted from the bacterial cell by the chloroform/methanol method. The lyophilized bacteria were ground into powder, mixed with chloroform and ultrasonic crushed for 10 min followed by shaking at 30 °C, 110 rpm for 48 h. After suction filtration and rotatory evaporation, prechilled methanol (five times the volume) was mixed in and placed at 4 °C overnight for precipitation. PHB was gained after a second suction filtration and drying. PHB production was determined by the dry weight of extracted PHB. The PHB content was defined as the percent ratio of PHB production to CDW.

Fourier Transform Infrared spectroscopy (FT-IR) analysis

A total of 2 mg dry products extracted from Massilia sp. UMI-21, E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1phaC1-vgb were mixed with potassium bromide (KBr) in 1:50 ratio to form samples and qualitative characterized by FT-IR analysis, which was carried out in IRAffinity-1 S instrument (SHIMADZU, Japan) for 15 scans and a resolution of 1 cm− 1.

Proton nuclear magnetic resonance (1H-NMR) spectroscopy

The 1H-NMR spectroscopy was also used to identify the structure of fermentation products. Thoroughly dissolved in deuterated chloroform (CDCl3), 5 mg polymers were used to obtain the 1H-NMR spectra using the AVANCE NEO (400 MHz) spectrometer (Bruker, Germany) at 90 °C with a 4 ms, 3,000 Hz spectral width and a 4 s repetition rate.

Thermogravimetric analysis (TGA)

TGA was performed to determine the thermal stability in terms of weight loss of extracted polymer as a function of temperature. A total of 10 mg samples were analyzed by STA449C thermal analyzer (Netzsch, Germany) using a scanning rate of 10 °C/min with temperature increasing from 50 to 350 °C under nitrogen.

Data Availability

Genome data of Massilia sp. UMI-21 were submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under the BioProject PRJNA669114.

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Acknowledgements

We thank the Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences for multiple technical help.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31971252), the Development and Reform Commission of Jilin Province (No. 2020C028-3), the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (No. TSBICIP-CXRC-006), and the Key Research and Development Project of Jilin Province (No. 20230204090YY).

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Contributions

XH conceived and designed the experiments. NJ, MW and LS constructed the recombinant strains. MW, DY and SZ performed the PHB extraction and property determination. NJ, YL and HL analyzed the data. NJ wrote the manuscript, which was modified by XH and approved by all authors.

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Correspondence to Xuerong Han.

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Additional file 1

: Table S1. PHB-producing strains in the genus Massilia. Table S2. Primers used in recombinant plasmid construction. Fig. S1. Phylogenetic tree of PHA-related proteins from Massilia sp. UMI-21 and related taxa. (A) PhaA. (B) PhaB. (C) PhaC. The GenBank accession numbers were shown in the parentheses. Green denotes Class III and IV PhaC; orange denotes Class I and II PhaC. Fig. S2. Results of phaA gene and amino acid (AA) sequence alignment between Ralstonia eutropha H16 and Massilia sp. UMI-21. (A) Alignment of DNA sequences. (B) Alignment of AA sequences. Fig. S3. Results of phaB gene and amino acid (AA) sequence alignment between Ralstonia eutropha H16 and Massilia sp. UMI-21. (A) Alignment of DNA sequences. (B) Alignment of AA sequences. Fig. S4. Results of phaC gene and amino acid (AA) sequence alignment between Ralstonia eutropha H16 and Massilia sp. UMI-21. (A) Alignment of DNA sequences. (B) Alignment of AA sequences. Fig. S5. Relative expression of phaA2 (A), phaB1 (B) and phaC1 (C) genes in wild E. coli DH5α strain and the recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb strains by qRT-PCR. 16S rRNA serves as the internal control

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Jiang, N., Wang, M., Song, L. et al. Polyhydroxybutyrate production by recombinant Escherichia coli based on genes related to synthesis pathway of PHB from Massilia sp. UMI-21. Microb Cell Fact 22, 129 (2023). https://doi.org/10.1186/s12934-023-02142-x

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Keywords

  • Genome of Massilia sp. UMI-21
  • PHB metabolism-related genes
  • Genetically engineered bacteria
  • Vgb gene
  • PHB synthesis