Engineering microorganisms for improving polyhydroxyalkanoate biosynthesis

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ScienceDirect Engineering microorganisms for improving polyhydroxyalkanoate biosynthesis Guo-Qiang Chen1,2,3,4 and Xiao-Ran Jiang1,2,3 Biosynthesis of polyhydroxyalkanoates (PHA) has been studied since the 1920s. The biosynthesis pathways have been well understood and various attempts have been made to improve the PHA biosynthesis efficiency. Recent progresses have been focused on systematic improvements on PHA biosynthesis including changing growth pattern for rapid proliferation, engineering to enlarge cell sizes for more PHA accumulation space, reprogramming the PHA synthesis pathways using optimized RBS and promoter, redirecting metabolic flux to PHA synthesis using CRISPR/Cas9 tools, and very importantly, the employment of non-traditional host such as halophiles for reduced complexity on PHA production. All of the efforts should lead to ultrahigh PHA accumulation, controllable PHA compositions and molecular weights, open and continuous PHA production with gravity separation processes, resulting in competitive PHA production cost.

Addresses 1 MOE Lab of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China 2 Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China 3 Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China 4 Manchester Institute of Biotechnology, Centre for Synthetic Biology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK Corresponding author: Chen, Guo-Qiang ([email protected]. cn)

Current Opinion in Biotechnology 2018, 53:20–25 This review comes from a themed issue on Chemical biotechnology Edited by Patrick Cirino and Mattheos Koffas

https://doi.org/10.1016/j.copbio.2017.10.008 0958-1669/ã 2017 Elsevier Ltd. All rights reserved.

Introduction Microbial synthesis of polyhydroxyalkanoates (PHA) has been studied as biodegradable materials for many years with a hope to at least partially replace the non-degradable petroleum-based plastics [1–3]. Only limited successes have been achieved. Many efforts have been made to improve the biosynthesis efficiency [4,5,6–8]. Current Opinion in Biotechnology 2018, 53:20–25

Challenges are still to be met so that PHA can be synthesized more efficient [7,9,10,11,12–14]. PHA biosynthesis pathways and their related enzymes have been extensively studied [15–17], especially related to the multiple pathways leading to the formations of various PHA monomers (Figure 1). A lot of studies have been conducted to improve metabolic flux to PHA synthesis [4,5,6,7], such as essential element limitation such as nitrogen, phosphorus, sulfur or iron [18], oxygen limitation [19–21], the weakening of beta-oxidation cycle [22,23,24], over-expression of NADH (or NADPH) synthesis enzymes [25,26] and new pathway construction for non-3-hydroxybutyrate (non-3HB) monomer synthesis such as 4-hydroxybutyrate (4HB) or 3-hydroxyvalerate (3HV) from glucose alone [27–31], as well as the deletion or repression on pathways competing for PHA monomers (Figure 1) [24,26,32,33]. On the other hand, the DNA reprogramming technology has been developed very fast. Now the PHA synthesis can also be improved by reprogramming RBS to achieve optimized levels in the PHA synthesis operon [34]. The CRISPR/Cas9 technology, especially the CRISPRi, has been used successfully to manipulate the PHA synthesis-related genes [13,35] for controlling the PHA structures and molecular weights. On the other hand, it is becoming very important to control cell growth rate and cell size for improving PHA synthesis. As growth rate will determine the PHA synthesis yield in terms of g/L/h, cell sizes will decide the amount of PHA accumulated intracellularly [36,37]. Last but not least, the PHA synthesis operon host organisms, namely, the chassis, is one of the most critical factors for PHA biosynthesis. It is suggested that robust microorganisms, especially bacteria that are resistant to contamination, are more useful for the above engineering to add new properties for effective PHA production and applications [38,39]. The coming Next Generation Industrial Biotechnology (NGIB), which will be discussed in other places, will rely on contamination resistant microorganisms for the open and continuous production of bulk chemicals, materials and fuels. This paper review progresses made in the past few years to improve PHA biosynthesis efficiency with an aim to economic production of PHA (Table 1).

Improving metabolic flux to PHA synthesis Since PHA biosynthesis is competing with many other metabolites and intermediates, it is important to remove www.sciencedirect.com

Polyhydroxyalkanoates (PHA) biosynthesis Chen and Jiang 21

Figure 1

Glucose

Glycerol

Pyruvate 3-Hydroxypropionaldehyde

Malonyl-CoA 3-Hydroxypropionate

3-Hydroxypropionyl-CoA

Ga

Succinate

Acetyl-CoA Citrate

bD

4-Hydroxybutyryl-CoA

TCA Succinate Semialdehyde

Succinyl-CoA

Acetoacetyl-CoA NADPH UdhA NADP

(R)-2-methylmalonyl-CoA

Succinate PrpC

Propionyl-CoA

(R)-3-Hydroxybutyryl-CoA

4-Hydroxybutyryl

MCC 2-Methylcitrate Malate

3-Ketovaleryl-CoA

(R)-3-Hydroxyacyl-CoA (S)-3-Hydroxyacyl-CoA

(R)-3-Hydroxyvaleryl-CoA

FadB Enoyl-CoA

β-Oxidation Ketoacyl-CoA

fadBA

dCas9

PVgb udhA

Genome

ori

pPvgbudhA-Pcl857 -gRNA

FadA Acyl-CoA

gRNA

Cm

pr p

C

R

bD ga

Pcl857 Fatty acid Current Opinion in Biotechnology

Engineering metabolic pathways to channel more carbon source to PHA synthesis. The cost of PHA production was reduced by engineering the extremophiles for consuming low-cost substrates (such as glucose or glycerol) to produce various PHA monomers (highlighted by different colored boxes). At the same time, the weakening of beta-oxidation cycle, the deletion or repression (CRISPR/Cas9 tool) on pathways competing for PHA monomers and the over-expression of NADH (or NADPH) synthesis enzymes help to improve metabolic flux to PHA synthesis. Abbreviations: UdhA, soluble pyridine nucleotide transhydrogenase; GabD, succinate semialdehyde dehydrogenase; PrpC, 2-methylcitrate synthase; FadA, thiolase; FadB, hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase.

Table 1 Various possibilities to improve PHA biosynthesis. Approach Substrate limitation such as N, P, S, Fe, and so on Anaerobic PHA synthesis Over-expression on PHA synthesis operon Chromosomal over-expression of PHA synthesis genes Enhanced NADH or NADPH supply for PHA synthesis Shutdown competing pathways: deleting b-oxidation Morphology engineering to increase cell sizes Controllable morphology engineering

Advantages

References

Limitation affects cell growth

[18]

High substrate to PHA conversion and saving energy Most convenient Convenient and stable

Slow growth and low biomass

[19–21]

Plasmid instability Low gene copy

[27–31] [25,38]

Convenient

Upset other metabolic balances

[25,26]

Improving fatty acids to PHA conversion

Fatty acids are only PHA precursors but not cell growth substrates Cell number can be low

[24,33]

Difficulty to induce morphology changes at high cell density Promoter difficult to induce at high cell density Difficulty to conduct molecular engineering Large Cas9 a burden for cell growth

[47]

Increase PHA content Increase PHA content and biomass

Optimization of RBS or/and promoter of PHA operon Engineering extremophiles

Convenient

CRISPR/Cas9 or/and CRISPRi to program PHA synthesis

Convenient for multiple gene deletions or repressions

www.sciencedirect.com

Disadvantages

Convenient

Reduce PHA process complexity

[37,45]

[34] [43,58] [35]

Current Opinion in Biotechnology 2018, 53:20–25

22 Chemical biotechnology

or weaken competing pathways so that more resources are channeled to the PHA synthesis pathways. For example, if glucose is used as a sole carbon source, TCA cycle is the main competing pathway [40,41]. Yet TCA is essential, it can only be weakened but not be deleted. Weakening TCA resulted in a slight increase in PHB synthesis [42]. For biosynthesis of copolymer PHBV consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), propionate is added to form 3HV monomers. However, expensive propionate is also consumed by MCC cycle. The deletion on gene prpC encoding enzyme bridging propionylCoA to MCC cycle significantly improves conversion of propionate to 3HV [25,43]. If fatty acids are to be used as precursors for PHA synthesis, beta-oxidation consuming fatty acids should be weakened or deleted so that fatty acids are completely channeled to PHA synthesis pathways [22,44].

Engineering the cell sizes for enhanced PHA synthesis Since PHA are inclusion bodies stored in tiny intracellular spaces, the amount and the size of PHA granules are limited by the small intracellular space [36] regardless of how much flux is directed to PHA biosynthesis. Therefore, a lot of efforts have been made to enlarge the sizes of the PHA producing bacteria [37,45]. It was found that E. coli elongates itself to become fiber shapes when the fission ring protein FtsZ is inhibited [46], longer cell shapes liberate more space for E. coli to store more PHA granules. Similarly, E. coli accumulated more PHA granules when the cell diameter is increased by 2–5 folds resulted from the weakening of the cell skeleton protein MreB [37]. Furthermore, E. coli cells lose their regular bar shapes to show diverse morphologies including various sizes of gourds, bars, coccus, spindles, multi-angles and ellipsoids once their FtsZ and MreB expressions are simultaneously weakened by CRISPRi [45], this leads to even more PHA storages compared with single repression of FtsZ or MreB. The results achieved in E. coli have been successfully transferred to a more industrially interesting Halomonas campaniensis, which demonstrated similar improvements on PHA accumulation [47]. On the other hand, FtsZ and MreB are essential proteins for cell growth, Jiang et al. [47] were able to grow the H. campaniensis properly with a normal size in the presence of MreB, and then enlarged itself to become very large spheres upon removal of MreB encoded on a mobile plasmid. Large cell sizes not only increase PHA accumulation but also enhance substrate (glucose) to PHA conversion. In addition, gravity separation of PHA containing cells and fermentation broth is possible, allowing a low-cost downstream separation process to occur. Current Opinion in Biotechnology 2018, 53:20–25

CRISPR/Cas9 tool for engineering PHA biosynthesis CRISPRi derived from CRISPR/Cas9 has been successfully used to direct metabolic flux to PHA biosynthesis [35]. Competing pathways for 4-hydroxybutyrate (4HB) were simultaneously repressed using CRISPRi, allowing the flux generated from glucose as a substrate to flow to 4HB. Intensities of CRISPRi repression decide ratios of 4HB to 3HB. While CRISPRi repression intensity can be designed by the binding location of sgRNA, this permits the regulation of compositions of copolymers of 3HB and 4HB [35]. In another study, Tao et al. [42] were able to use CRISPRi to repress competition pathway consuming propionate for forming 3-hydroxyvalerate (3HV), the propionate was then mostly converted to 3HV. By weakening acetyl-CoA consumption by the TCA cycle, more acetyl-CoA could be directed to PHB synthesis, improving the substrate to PHB conversion efficiency [42]. Very interestingly, PHA synthase (PhaC) activity was found to influence PHA molecular weights [48]. By designing CRISPRi binding sites using various sgRNA, the PhaC activity can be regulated. Thus, molecular weights of PHB could be well controlled ranging from 2 to 6 millions Dalton [13]. CRISPRi has been proven very useful for controlling metabolic flux in multiple pathways [13,35,42]. Combined with the CRISPR/Cas9 which is suitable for gene deletion in most non-model organisms, PHA biosynthesis can be conveniently manipulated.

Engineering gene expression for improving PHA biosynthesis Reprogramming the PHA synthesis operons (RBS optimization), new powerful promoter for enhancing PHA synthesis. PHB synthesis is a multistep enzymatic reaction involving over-expression of three genes phbC, phbA, and phbB [49,50]. Pathway optimization by adjusting expression levels of the three genes leads to improved PHB accumulation [51]. A semi-rational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon was reported [34]. Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains accumulating 0% to 92% PHB contents in cell dry weight (CDW) were found. PHB with various weight-average molecular weights (Mw) of 2.7– 6.8  106 was also efficiently produced in relatively high contents, suggesting that the semi-rational approach combining library design, construction, and proper screening is efficient to optimize PHB and other multiple enzyme pathways [34]. Genetic parts are sometimes host strain sensitive. Novel T7-like RNA polymerase-promoter pairs were obtained by mining many phage genomes [52], followed by in vivo characterization in non-model strains Halomonas www.sciencedirect.com

Polyhydroxyalkanoates (PHA) biosynthesis Chen and Jiang 23

bluephagenesis (short as H. bluephagenesis), Pseudomonas entomophila and model organism E. coli. Three expression systems, namely, MmP1, VP4, and K1F, were developed displaying high efficiency for the above model and nonmodel organisms, implying suitability of broad-host range. Three recombinant H. bluephagenesis strains were then constructed based upon these expression systems to express the cell-elongation cassette (minCD genes) and PHB operon under IPTG induction, resulting in a 100fold increase in cell lengths and 92% PHB accumulation of cell dry weight, respectively. These T7-like expression systems would be very useful for metabolic engineering in other non-model organisms [52]. H. bluephagenesis lacks a constitutive promoter. Porin, a highly expressed pore protein in H. bluephagenesis, was first identified with a core promoter region, including -10 and -35 elements. By randomizing the sequence between the -35 and -10 elements, a constitutive promoter library was obtained with 310-fold variation in transcriptional activity. It was employed to successfully regulate PHB synthesis in H. bluephagenesis [53]. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a chromosomal expression method was developed for H. bluephagenesis on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was successful for stable and inducible or constitutive expression of PHB or PHBV synthesis pathway chromosomally [25,38].

Extremophiles for PHA synthesis Extremophilic bacteria including acidophiles, alkaliphiles, psychrophiles, thermophiles, xerophiles and halophiles as well as methanotrophs, gaseous substrates or cellulose utilizers, are able to grow under some unique conditions prevented contaminations by normal microorganisms [54–57,58]. They can be engineered to become production strains for the next generation industrial biotechnology (NGIB). However, most of these NGIB bacteria cannot bio-synthesize PHA, or can only produce simple PHA such as PHB. A PHA synthesis pathway must be constructed into the above chassis. The above strategies including redirection of metabolic flux to PHA synthesis, enlarging cell sizes, acceleration of cell growth, CRISPR/Cas9 reprogramming PHA biosynthesis and engineering other biological mechanisms can be adopted. If succeeded, the reprogrammed organisms should be able to grow under open and continuous conditions for economic PHA production. The most successful example of engineering extremophiles for PHA biosynthesis is the halophilic Halomonas spp. able to grow contamination free under open www.sciencedirect.com

conditions for up to 65 days [25,43,58]. Most halophilic bacteria are both alkaliphilic and halophilic, they provide double barriers to prevent microbial contaminations [38,59]. So far, H. bluephagenesis has been successfully developed as a platform for NGIB to produce PHA in a pilot scale of 1000 L fermentor vessel [60]. H. bluephagenesis and H. campaniensis can grow in seawater under open unsterile and continuous conditions without contamination for weeks to months [59,61]. Engineering technology including conjugation procedure, plasmids, chromosome engineering, CRISPR/Cas9 and strong inducible promoter has been developed for Halomonas spp. [42,43,52,62], allowing multiple product productions and controllable morphology engineering [47].

Future prospects Since PHA are bulk materials, the cost is an issue for large-scale application. To reduce production cost, NGIB based on extremophiles should be employed to save energy, water and reduce process complexity [54– 56,58]. Artificial intelligence for PHA production should be installed for maintaining the process consistency and thus PHA quality consistency. Since substrates, especially carbon source constitutes the largest part of PHA production cost, a high substrate to PHA conversion efficiency is required. To achieve this goal, the extremophiles should be redesigned to channel more carbon flux to PHA synthesis without sacrificing the cell growth rate. In situ design of PHA synthesis bacteria should become possible due to the rapid development of synthetic biology. The designed PHA producer should be able to conduct ultrahigh PHA accumulation inducible under oxygen limitation. Controllable morphology changes should be achievable after cells grow to high density [47]. In summary, future PHA synthesis should be based on extremophiles redesigned to accumulate ultrahigh content of PHA in large cell sizes inducible under oxygen limitation. Its associated NGIB should be able to produce the PHA with a price competitive to petrochemical plastics.

Conflict of interest The authors declare no financial or commercial conflict of interest.

Acknowledgements This research was financially supported by a grant from Ministry of Sciences and Technology (Grant No. 2016YFB0302504), and grants from National Natural Science Foundation of China (Grant No. 31430003). Tsinghua President Fund also supported this project (Grant No. 2015THZ10).

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Polyhydroxyalkanoates (PHA) biosynthesis Chen and Jiang 25

34. Li T, Ye J, Shen R, Zong Y, Zhao X, Lou C, Chen GQ: Semirational  approach for ultrahigh poly(3-hydroxybutyrate) accumulation in Escherichia coli by combining one-step library construction and high-throughput screening. ACS Synth Biol 2016, 5:13081317. This paper reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha. Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Applying this approach, strains accumulating 0%–92% PHB contents in cell dry weight (CDW) were achieved. 35. Lv L, Ren Y-L, Chen J-C, Wu Q, Chen G-Q: Application of  CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: controllable P (3HB-co-4HB) biosynthesis. Metab Eng 2015, 29:160-168. This paper shows that CRISPRi can also be used for fine-tuning prokaryotic gene expression while simultaneously regulating multiple essential gene expression with less labor and time consumption. CRISPRi was used to control polyhydroxyalkanoate (PHA) biosynthesis pathway flux and to adjust PHA composition in this study. 36. Jiang XR, Chen GQ: Morphology engineering of bacteria for bio-production. Biotechnol Adv 2016, 34:435-440. 37. Jiang XR, Wang H, Shen R, Chen GQ: Engineering the bacterial shapes for enhanced inclusion bodies accumulation. Metab Eng 2015, 29:227-237. 38. Yin J, Chen J-C, Wu Q, Chen G-Q: Halophiles, coming stars for industrial biotechnology. Biotechnol Adv 2014, 33:1433-1442. 39. Li T, Chen XB, Chen J.C., Wu Q, Chen GQ: Open and continuous fermentation: products, conditions and bioprocess economy. Biotechnol J 2014, 9:1503-1511. 40. Lu¨tke-Eversloh T, Steinbu¨chel A: Biochemical and molecular characterization of a succinate semialdehyde dehydrogenase involved in the catabolism of 4-hydroxybutyric acid in Ralstonia eutropha. FEMS Microbiol Lett 1999, 181:63-71. 41. Heinrich D, Raberg M, Steinbu¨chel A: Synthesis of poly (3 hydroxybutyrate-co-3-hydroxyvalerate) from unrelated carbon sources in engineered Rhodospirillum rubrum. FEMS Microbiol Lett 2015, 362:fnv038. Authors constructed a recombinant strain Rhodospirillum rubrum S1 by introducing the gene encoding the membrane-bound transhydrogenase PntAB from E. coli MG1655 and the phaB1 gene coding for an NADHdependent acetoacetyl-CoA reductase from Ralstonia eutropha H16, which accumulated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [Poly(3HB-co-3HV)] with a 3HV fraction of up to 13 mol% from fructose. Moreover, the engineered R. rubrum strain was also able to synthesize this industrially relevant copolymer from CO2 and CO of artificial synthesis gas (syngas) with a 3HV content of 56 mol%. 42. Tao W, Lv L, Chen G-Q: Engineering Halomonas species TD01 for enhanced polyhydroxyalkanoates synthesis via CRISPRi. Microb Cell Fact 2017, 16:48. 43. Tan D, Wu Q, Chen JC, Chen GQ: Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates. Metab Eng 2014, 26C:34-47. 44. Wang Y, Chung A, Chen GQ: Synthesis of medium-chain-length polyhydroxyalkanoate homopolymers, random copolymers, and block copolymers by an engineered strain of Pseudomonas entomophila. Adv Healthc Mater 2017, 6 http:// dx.doi.org/10.1002/adhm.201601017. 45. Elhadi D, Lv L, Jiang XR, Wu H, Chen GQ: CRISPRi engineering E.  coli for morphology diversification. Metab Eng 2016, 38:358369. Clustered regularly interspaced short palindromic repeats interference, abbreviated as CRISPRi, was for the first time used to regulate expression intensities of ftsZ or/and mreB in E. coli. Combined repressions on expressions of ftsZ and mreB generated long and larger E. coli with diverse morphologies including various sizes of gourds, bars, coccus, spindles, multi-angles and ellipsoids. 46. Wang Y, Wu H, Jiang X, Chen GQ: Engineering Escherichia coli for enhanced production of poly(3-hydroxybutyrate-co-4hydroxybutyrate) in larger cellular space. Metab Eng 2014, 25:183-193. www.sciencedirect.com

47. Jiang XR, Yao ZH, Chen GQ: Controlling cell volume for efficient  PHB production by Halomonas. Metab Eng 2017, 44:30-37. This paper developed a temperature-responsible plasmid expression system for compensated expression of relevant gene(s) in mreB or ftsZ disrupted recombinants H. campaniensis LS21, allowing mreB or ftsZ disrupted recombinants to grow normally at 30  C in a bioreactor for 12 h so that a certain cell density can be reached, followed by 36 h cell size expansions or cell shape elongations at elevated 37  C at which the mreB and ftsZ encoded plasmid pTKmf failed to replicate in the recombinants and thus lost themselves. 48. Tsuge T: Fundamental factors determining the molecular weight of polyhydroxyalkanoate during biosynthesis. Polym J 2016, 48:1051-1057. 49. Leong YK, Show PL, Ooi CW, Ling TC, Lan JCW: Current trends in polyhydroxyalkanoates (PHAs) biosynthesis: insights from the recombinant Escherichia coli. J Biotechnol 2014, 180:52-65. 50. Pen˜a C, Castillo T, Garcı´a A, Milla´n M, Segura D:  Biotechnological strategies to improve production of microbial poly-(3-hydroxybutyrate): a review of recent research work. Microb Biotechnol 2014, 7:278-293. This paper summarized the recent trends in the bacterial production of PHB using novel fermentation strategies combined with the use of genetic engineering to improve productivity and molecular weight of PHB that could be applied for its commercial production. 51. Slater SC, Voige W, Dennis D: Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-betahydroxybutyrate biosynthetic pathway. J Bacteriol 1988, 170:4431-4436. 52. Zhao H, Zhang HM, Chen X, Li T, Wu Q, Ouyang Q, Chen GQ: Novel T7-like expression systems used for Halomonas. Metab Eng 2017, 39:128-140. 53. Li T, Li T, Ji W, Wang Q, Zhang H, Chen GQ, Lou C, Ouyang Q: Engineering of core promoter regions enables the construction of constitutive and inducible promoters in Halomonas sp. Biotechnol J 2016, 11:219-227. 54. Dumorne K, Co´rdova DC, Astorga-Elo´ M, Renganathan P: Extremozymes: a potential source for industrial applications. J Microbiol Biotechnol 2017, 27:649-659. 55. Sharma A, Kawarabayasi Y, Satyanarayana T: Acidophilic bacteria and archaea: acid stable biocatalysts and their potential applications. Extremophiles 2012, 16:1-19. 56. Wernick DG, Pontrelli SP, Pollock AW, Liao JC: Sustainable biorefining in wastewater by engineered extreme alkaliphile Bacillus marmarensis. Scient Rep 2016:6. 57. Jiang H, Chen Y, Jiang P, Zhang C, Smith TJ, Murrell JC, Xing XH: Methanotrophs: multifunctional bacteria with promising applications in environmental bioengineering. Biochem Eng J 2010, 49:277-288. 58. Waditee-Sirisattha R, Kageyama H, Takabe T: Halophilic  microorganism resources and their applications in industrial and environmental biotechnology. AIMS Microbiol 2016, 2:4254. In this paper, the authors discussed on recent efforts on discovery and utilization of halophiles for biotechnological interest and future perspective in biotechnology. 59. Yue H, Ling C, Yang T, Chen X, Chen Y, Deng H, Wu Q, Chen J, Chen GQ: A seawater-based open and continuous process for polyhydroxyalkanoates production by recombinant Halomonas campaniensis LS21 grown in mixed substrates. Biotechnol Biofuels 2014, 7:108-120. 60. Chen XB, Yin J, Ye J, Zhang H, Che X, Ma Y, Li M, Wu LP, Chen GQ: Engineering Halomonas bluephagenesis TD01 for non-sterile production of poly (3-hydroxybutyrate-co-4hydroxybutyrate). Bioresour Technol 2017, 244:534-541. 61. Tan D, Xue YS, Aibaidula G, Chen GQ: Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Bioresour Technol 2011, 102:8130-8136. 62. Yin J, Fu XZ, Wu Q, Chen JC, Chen GQ: Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp. Appl Microbiol Biotechnol 2014, 98:8987-8997. Current Opinion in Biotechnology 2018, 53:20–25