Halophiles, Coming Stars for Industrial Biotechnology☆

Biotechnology Advances 33 (2015) 1433–1442

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Biotechnology Advances

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Research review paper Halophiles, coming stars for industrial biotechnology☆

Jin Yin, Jin-Chun Chen, Qiong Wu, Guo-Qiang Chen ⁎

MOE Key Lab of Bioinformatics, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China article info abstract

Available online 27 October 2014 Industrial biotechnology aims to produce chemicals, materials and biofuels to ease the challenges of shortage on petroleum. However, due to the disadvantages of bioprocesses including energy consuming sterilization, Keywords: high fresh water consumption, discontinuous fermentation to avoid microbial contamination, highly expensive Halophiles stainless steel fermentation facilities and competing substrates for human consumption, industrial biotechnology Polyhydroxyalkanoates is less competitive compared with chemical processes. Recently, halophiles have shown promises to PHB overcome these shortcomings. Due to their unique halophilic properties, some halophiles are able to grow in Ectoines high pH and high NaCl containing medium under higher temperature, allowing fermentation processes to run Bio-surfactants Halomonas contamination free under unsterile conditions and continuous way. At the same time, genetic manipulation Industrial Biotechnology methods have been developed for halophiles. So far, halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants. Increasing effects have been made to develop halophiles into a low cost platform for bioprocessing with advantages of low energy, less fresh water consumption, low ﬁxed capital investment, and continuous production. © 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction...... 1433 2. Biologyofhalophilicmicroorganisms...... 1434 3. Applicationofhalophilesforbiotechnologicalindustries...... 1434 3.1. PHAproductionbyhalophilicmicroorganisms...... 1434 3.2. Productionofectoinesbyhalophilicmicroorganisms...... 1435 3.3. Halophilichydrolasesforbiotechnologicalindustries...... 1437 3.4. Biosurfactants and bioemulsiﬁersfromhalophiles...... 1437 3.5. Otherchemicalsproducedbyhalophiles...... 1438 4. Advancesinmolecularmanipulationforhalophilicmicroorganisms...... 1438 4.1. Developmentofgenetictoolsforhalophilicmicroorganisms...... 1438 4.2. Systemsandsyntheticbiologyforhalophiles...... 1439 5. Futureprospects...... 1439 Acknowledgements...... 1439 References...... 1440

1. Introduction 2010). However, bio-based products such as biochemicals, bioplastics and biofuels are still too expensive due to their high production cost Industrial biotechnology has been developed substantially in the compared with chemical counterparts (Philp et al., 2013). The high pro- past years with an aim to partially replace petroleum based chemical duction cost of bioprocessing is mainly associated with the following is- industry (Chen et al., 2010; Gartland et al., 2013; Otero and Nielsen, sues. First, the price of raw materials (substrates), e.g. glucose from hydrolysis of starch, has increased very fast (Tanadchangsaeng and Yu, 2012). Second, bioprocessing requires large amount of fresh water, ☆ This paper is for“Biotechnology Advances” special issue dedicated to two recent making water shortage even worse (Chen, 2012). Third, most of the fer- conferences: (1) 18th Biochemical and Molecular Engineering Conference (BME XVIII) mentation processes (bioprocessing) are run discontinuously to avoid and (2) 4th International Conference on Bioreﬁnery towards Bioenergy 2013 (ICBB 2013). ﬁ ⁎ Corresponding author. Tel.: +86 10 62783844; fax: +86 10 62794217. microbial contamination, which results in low production ef ciency. E-mail address: [email protected] (G.-Q. Chen). Fourth, the energy intensive sterilization of the fermentors and piping

http://dx.doi.org/10.1016/j.biotechadv.2014.10.008 0734-9750/© 2014 Elsevier Inc. All rights reserved. 1434 J. Yin et al. / Biotechnology Advances 33 (2015) 1433–1442 systems as well as the medium is a very expensive process (Wang et al., culture. Tan et al. (2011) cultivated a Halomonas strain termed TD01 2014). Fifth, also the investment to purchase stainless steel bioprocess for 14 days in an open unsterile process for polyhydroxybutyrate facilities is heavy. Lastly, Procedures to maintain contamination free (PHB) production. More recently, a halophilic bacterium termed and batch processes make the bioprocessing very complicated, leading Halomonas campaniensis LS21 was isolated and continuously cultured to increasing cost for biochemical production. for 65 days under unsterile conditions without contamination (Yue To make industrial biotechnology as competitive as the chemical et al., 2014). industry, it is urgent to develop low cost bioprocessing technology. In Halophilic microorganisms, especially Halomonas spp., have such technology, low energy and fresh water consumptions as well as been the candidates for production of diverse products used in various contamination free continuous fermentation processes are required in industrial fields (Table 1). Many halophiles are found to be able to addition to low cost substrates. accumulate polyhydroxyalkanoates (PHA), a family of biodegradable Halophilic microorganisms have recently been re-discovered to plastics (Quillaguamán et al., 2010). Ectoine and hydroxyectoine from possess advantages for the above mentioned desirable properties halophiles are commercially used as protective agents for mammalian (Wang et al., 2014). Due to their unique halophilic properties, many cells and for skins (Lippert and Galinski, 1992; Pastor et al., 2010). Halomonas spp. can grow in high pH and high NaCl containing medium There are also growing interest in the alkaline enzymes produced at a larger temperature range, making contamination free fermentation by halophilic microorganisms (Setati, 2010). Moreover, many halophilic processes under unsterile conditions and continuous way possible. microorganisms are producers of bio-surfactants (BS) and bio- Recently, genetic manipulation methods have been developed for emulsifiers(BE)(Satpute et al., 2010). Halomonas by the authors’ lab (Fu et al., 2014), allowing diversification of products from Halomonas spp. 3.1. PHA production by halophilic microorganisms This review summaries recent developments of Halomonas spp. as well as other halophiles and efforts made using the species for Polyhydroxyalkanoates (PHA) are a family of biodegradable and bioprocessing with advantages of low energy, less fresh water con- biocompatible polyesters accumulated by many microorganisms, PHA sumption, low fixed capital investment, and continuous production. are developed into an industrial value chain ranging from bioplastics, biofuels, fine chemicals to medicine (Chen, 2009; Chen and Patel, 2. Biology of halophilic microorganisms 2012). PHA have diverse material properties due to over 150 monomer variations (Chen and Wu, 2005; Steinbüchel and Valentin, 1995). Halophiles (salt-loving) are referred to those microorganisms that Among these PHA, poly(3-hydroxybutyrate) (PHB) and poly(3- require salt (NaCl) for growth, and they can be found in all three hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are two of the most domains of life – Archaea, Bacteria and Eukarya (Quillaguamán et al., well studied polymers and have been produced in large scale (Chen, 2010). Halophiles can be found in hypersaline environments which 2009; Steinbüchel and Füchtenbush, 1998). PHB is rigid and brittle are widely distributed in various geographical areas on earth, such as while PHBV is more flexible with wider application potentials as medi- saline lakes, salt pans or salt marshes (Setati, 2010). According to the cal materials, films products, disposable items and packaging materials salt concentration for optimal growth, halophiles can be roughly divid- (Chen, 2009; Philip et al., 2007). ed into two groups, moderate and extreme halophiles (Oren, 2008; After PHA accumulation by halophiles first observed in 1972 Ventosa et al., 1998). A moderate halophile grows at salt concentration (Kirk and Ginzburg, 1972), more and more halophiles have been of 3-15% (w/v) and can tolerate 0-25% (w/v) (Ventosa et al., 1998). A exploited and found to synthesize PHA (Fernandez-Castillo et al., large number of phylogenetic subgroups contain many types of halo- 1986; Han et al., 2007; Koller et al., 2007a; Legault et al., 2006). philic bacteria, most of which belongs to the family Halomonadaceae Among those PHA producing halophiles, the archaeon Haloferax (class Gamma proteobacteria) (Oren, 2002). mediterranei produced 46 wt% PHA (Lillo and Rodriguez-Valera, 1990; To thrive in the hypersaline environment, halophiles have two main Rodriguez-Valera and Lillo, 1992). Further study showed that the PHA adaptation mechanisms to prevent NaCl from diffusing into the synthesized by H. mediterranei was a copolymer of PHBV when carbohy- cells. The first mechanism is accumulation of inorganic ions (mainly drates such as glucose, extruded starch or hydrolyzed whey was used as KCl) for balancing osmotic pressure. This mechanism is mainly utilized substrate (Chen et al., 2006; Don et al., 2006; Koller et al., 2007b). by aerobic and extremely halophilic archaea and some anaerobic Several halophilic strains were also reported to synthesize PHBV from halophilic bacteria (Oren, 1999, 2002). In contrast, most halophilic non-fatty acid carbohydrates as carbon source (Van-Thuoc et al., bacteria and eukarya accumulate water soluble organic compounds of 2012). Han et al. (2013) described four pathways in H. mediterranei low molecular weight, which are referred to as compatible solutes or leading to propionyl-CoA, the precursor for 3-hydroxyvalerate (3HV) osmolytes, to maintain low intracellular salt concentration (Oren, in PHBV, including citramalate/2-oxobutyrate pathway, aspartate/2- 2008; Quillaguamán et al., 2010; Roberts, 2005). Compatible solutes oxobutyrate pathway, the methylmalonyl-CoA pathway and 3- can act as stabilizers for biological structures and allow the cells to hydroxypropionate pathway. adapt not only to salts but also to heat, desiccation, cold or even freezing The halobacterium Halomonas boliviensis, a representative of the conditions (Delgado-García et al., 2012), allowing the halophile to grow family Halomonadaceae, tolerates salt concentration of 0-25% w/v. It at around pH 10 and over 50 °C (Hozzein et al., 2013). Many halophilic grows from 0-45 °C under pH 6-11 (Quillaguamán et al., 2004). The bacteria accumulate ectoine or hydroxyectoine as the predominant strain utilizes carbon sources including glucose, xylose, or sucrose, and compatible solutes. Other intracellular compatible solutes include can produce PHB with high molecular weight of 1,100 kDa under its amino acids, glycine betaine and other osmotic solutes accumulated in optimized condition (Quillaguamán et al., 2005, 2006, 2007, 2008). small amounts (Louis and Galinski, 1997; Vargas et al., 2008; Ventosa Recently, two more Halomonas spp. were isolated and showed a et al., 1998). great potential for low cost PHA production. Halomonas sp. TD01 grows optimally at salt concentration of 5-6% (w/v) at pH of 9.0 (Tan 3. Application of halophiles for biotechnological industries et al., 2011). The strain grew rapidly to over 80 g/L cell dry weight (CDW) in a lab fermentor and accumulated over 80% PHB on glucose Since Halomonas spp. favor a condition of high salt concentration salt medium (Tan et al., 2011). A two-stage open and continuous under which non-halophiles can not grow, they can be used for devel- fermentation process was tested, in which the cultures from the first opment of open unsterile and continuous fermentation process. fermentor was pumped continuously to the second fermentor contain- Johnson et al. (2009) achieved a record of 3-year fermentation process ing nitrogen-deficient glucose salt medium. Halomonas TD01 accumu- without contamination by feeding acetate to the mixed bacterial lated PHB up to 70 wt% CDW with a glucose to PHB conversion ratio J. Yin et al. / Biotechnology Advances 33 (2015) 1433–1442 1435

Table 1 Current and potential industrial applications of halophilic microorganisms.

Product Application Representative producer Reference

Polyhydroxyalkanoates (PHA) Biomaterials Polyhydroxybutyrate (PHB) Plastics Halomonas boliviensis Quillaguamán et al. (2004) Halomonas sp. TD01 Tan et al. (2011) Poly(hydroxybutyrate-co- hydroxyvalerate) (PHBV) Plastics, medical materials Haloferax mediterranei Don et al. (2006) Ectoines Protectants for proteins and cells Ectoine Cell membrane protection, Halomonas elongata Sauer and Galinski (1998) antiageing skin protection Halomonas salina Zhang et al. (2009) Hydroxyectoine Protection of proteins against misfolding, Marinococcus M52 Frings et al. (1995) degradation and freezing

Halophilic hydrolases Amylases Food industry Halomonas sp. Coronado et al. (2000) Halobacillus sp. Amoozegar et al. (2003) Streptomyces sp. Chakraborty et al. (2009) Proteases Additives in pharmaceuticals, laundry Bacillus sp. Shivanand and Jayaraman (2009) detergents, and so on Halobacillus sp. Karbalaei-Heidari et al. (2009) Chromohalobacter sp. Vidyasagar et al. (2009) Xylanases and cellulases Biobleaching, hydrolysis of cellulose Streptomonospora sp. Ren et al. (2013) Halomonas sp. Shivanand et al. (2013) Biosurfactants and bioemulsiﬁers Solubilisation of hydrophobic substrates Halomonas spp. Gutiérrez et al. (2007) Natrialba sp. strain E21 Kebbouche-Gana et al. (2013) Bioemulsiﬁer protein PhaR Halomonas sp. (recombinant) Fu et al. (2014)

Other products β-Carotene Food additive Dunaliella spp. Ben-Amotz and Avron (1981) Glycerol Cosmetic industries Dunaliella spp. Ben-Amotz and Avron (1981) Bacteriorhodopsin Light-driven proton pump protein Halobacterium halobium Oesterhelt and Stoeckenius (1973)

of above 50%, indicating a nitrogen limitation is beneficial for PHB Although the commercial demand for ectoines kept increasing, a production by Halomonas TD01. large-scale production of ectoines was only achieved in mid-1990s H. campaniensis LS21 was found able to grow in artificial seawater (Pastor et al., 2010). A technology of osmotic downshock termed “bac- and kitchen-waste-like mixed substrates consisting of cellulose, pro- terial milking” was successfully developed for extracting intracellular teins, fats, fatty acids and starch (Yue et al., 2014). When cultured in a solutes from several microorganisms (Fischel and Oren, 1993; Reed kitchen-waste-simulating medium, wild type H. campaniensis produced et al., 1986; Tsapis and Kepes, 1977). only 26 wt% PHB, while the recombinant H. campaniensis,withthe A moderate halophile Halomonas elongata was applied as a cell facto- genes of PHB synthesis pathway over-expressed, produced 70 wt% ry for ectoine production (Sauer and Galinski, 1998). H. elongata has a PHB. Remarkably, the H. campaniensis grew contamination free over a broad salt tolerance of ~0.1 to ~4 M NaCl. It rapidly releases ectoine period of 65 days under unsterile and continuous conditions at 40 g/L under hypo-osmotic shock, and synthesis of ectoine was restored NaCl, 37 °C and pH 10. The strain can be further genetically manipulated when hyper-osmotic condition was applied again (Cánovas et al., for production of other chemicals from waste and mixed substrates 1997; Pastor et al., 2010; Vreeland et al., 1980). Sauer and Galinski (Wang et al., 2014; Yue et al., 2014). (1998) reported a procedure of culturing H. elongata first in a medium The application of halophiles for PHA production can reduce the containing 2.57 M NaCl to a high cell density and then treating the costs in fermentation and recovery processes due to the following cells with osmotic downshocks. Cells were retained via filtration while reasons (Fig. 1): first, a high salt concentration prevents contamination the medium was replaced with distilled water. When ectoine was by non-halophiles, hence reducing the costs, energy and process excreted and the intracellular osmolarity was recovered, the ectoine complexity for sterilization (Quillaguamán et al., 2010); second, seawa- containing medium was withdrawn and replaced back to salty medium. ter can be used as salt containing medium, thus reducing the consump- Thus, a new cycle for ectoine synthesis could restart. A final ectoine yield tion of fresh water (Tan et al., 2011; Yue et al., 2014); third, the 155mg(gCDW)-1 (per gram cell dry weight) was obtained. efficiency of a long-lasting and continuous process is much higher Zhang et al. (2009) achieved to produce 220 mg (g CDW)-1 extracel- than that of a batch one (Wang et al., 2014); fourth, low cost substrates lular ectoine using Halomonas salina. They found that H. salina was able including kitchen waste mixed substrates and/or cellulose can be used to excrete ectoine to the medium even when the extracellular osmolar- for some halophiles to reduce substrate cost (Yue et al., 2014); fifth, ity was constant. The optimum salt concentration for synthesis and for some halophiles, cost for PHA recovery can be decreased by cell excretion of ectoine in H. salina was 0.5 M, lower than 2.57 M reported lysis via hypo-osmotic shock treatment (Quillaguamán et al., 2010). for H. elongata.SinceH. salina excretes ectoine in a constant salt concen- Koller et al. (2007a) demonstrated that the cost of PHBV production tration, ectoine should be able to produce without using the “bacterial by H. mediterranei was 30% lower than that by recombinant Escherichia milking” technique. coli. All the above results reveal that halophiles are a low cost platform Continuous bioprocessing was also applied to ectoine production. for industrial biotechnology concerning PHA production. Fallet et al. (2010) developed a two-bioreactor system and optimized the productivity of ectoine in Chromohalobacter salexigens.Thefirst bio- 3.2. Production of ectoines by halophilic microorganisms reactor was used for cell growth and intracellular accumulation of ectoine, while the second one for osmotic downshock to excrete Halophiles accumulate compatible solutes within the cells to ectoine. The culture broth of the first bioreactor was pumped into the maintain osmotic balance under the hyperosmotic condition. Ectoines second one continuously. After optimization, the intracellular ectoine are the most widespread compatible solutes, and are commercially content reached up to 540 mg (g CDW)-1. available as protectants for proteins, DNA and mammalian cells (Kolp Hydroxyectoine is another compatible solute that has attracted com- et al., 2006; Lippert and Galinski, 1992; Pastor et al., 2010). mercial interest due to its better protection capacity than ectoine 1436 J. Yin et al. / Biotechnology Advances 33 (2015) 1433–1442

Fig. 1. Applications of halophiles as low cost production hosts for chemicals, materials and biofuels. Seawater can be used as salt containing medium, thus reducing the consumption of fresh water. Low cost substrates including kitchen waste mixed substrates and/or cellulose can be used to reduce substrate cost. Unsterile and continuous processing can increase the process efﬁciency and reduce the costs, energy and process complexity. Since high pressure sterilization is not needed, low cost materials such as plastics, ceramics and carbon steel can be used to make fermentors and piping systems to avoid corrosion of stainless steel systems by high salt concentration. Pollution is reduced when the water is recycled during the fermentation process.

(Pastor et al., 2010). The Gram-positive halophilic eubacterium hydroxyectoine under high-salinity and high-temperature, ectoine Marinococcus M52 can convert ectoine to hydroxyectoine during the and hydroxyectoine can be separated subsequently (Sauer and stationary phase (Frings et al., 1995). Schiraldi et al. (2006) improved Galinski, 1998; Vargas et al., 2008). the hydroxyectoine yield to 3.6 g/L. However, Marinococcus M52 Many halophiles accumulate not only compatible solutes but also showed resistance to osmotic downshock, thus its hydroxyectoine PHA under certain conditions (Pastor et al., 2010; Quillaguamán et al., remained intracellular unless organic solvents such as methanol or eth- 2010). Methylarcula marina and Methylarcula terricola synthesize anol was used for extraction (Frings et al., 1995). 18 wt% PHB and compatible solutes including ectoine and glutamate A method termed thermal permeabilization was described by in NaCl concentration of 6-10% (w/v) (Doronia et al., 2000). Halomonas Schiraldi et al. (2006) to obtain intracellular hydroxyectoine. Yet both campaniensis accumulates PHB and ectoine in 5.8% (w/v) NaCl solvent extraction and permeabilization do not recycle the cells, (Strazzullo et al., 2008). Co-production of around 40 wt% PHB and 10% resulting in lower productivity (Pastor et al., 2010). Gram-negative ectoine of the cell dry mass was achieved by H. elongata and several strains including H. elongata and C. salexigens produce ectoine and Halomonas spp. (Mothes et al., 2008). Synthesis of PHA and ectoine J. Yin et al. / Biotechnology Advances 33 (2015) 1433–1442 1437 shares the same precursor acetyl-CoA but is triggered by different et al., 2009). These enzymes remain active in the presence of NaCl and factors including nutrient deficiency and salt stress, respectively are tolerant to a pH of 5-10 and temperature of 40-75 °C (Setati, (Guzmán et al., 2009; Mothes et al., 2008). Guzmán et al. (2009) de- 2010). However, the Chromohalobacter protease can retain 100% signed a two-step fed-batch system for improvement on co-production stability even in the absence of NaCl (Vidyasagar et al., 2009). Such of the two products by H. boliviensis.Thefirst step was aimed for proteases can be used in food production under both saline and saline cell growth under its optimal condition. In the second step, a higher free conditions, i.e. fish and meat-based food or soy sauce production NaCl concentration promoted ectoine synthesis while nitrogen sources (Setyorini et al., 2006). Most of halophilic proteases are also alkali toler- limitation led to PHB accumulation. 13.2% ectoine content and 68.5 wt% ant or alkaliphilic that are desirable for laundry industry (Setati, 2010). PHB were produced. Alkaliphilic and thermotolerant xylanases have the potential to be In most cases, wild-type strains were used for ectoine production. used for biobleaching of paper and pulp (Mamo et al., 2009; Setati, Non-halophilic microorganisms such as E. coli were genetically 2010). These enzymes were isolated from Glaciecola mesophila, engineered for heterologous expression of ectoine synthsis genes Chromohalobacter sp., Nesterenkonia sp., and Bacillus pumilus GESF-1 (Louis and Galinski, 1997; Rajan et al., 2008), no higher yield on ectoines (Govender et al., 2009; Guo et al., 2009; Menon et al., 2010; Prakash was obtained compared with that by the wild type (Bestvater et al., et al., 2009). They showed stability at pH 6-11 above 60 °C. Ren et al. 2008). (2013) purified a unique Ag+ and SDS resistant xylanase from Yet engineering halophiles has been proven favorable for increasing Streptomonospora sp. YIM 90494. Recently, Liu et al. (2014) reported a the productivity. Prabhu et al. (2004) used H. elongata harboring halophilic xylanase with low temperature activity from marine the ectoine hydroxylase gene from Streptomyces chrysomallus for Zunongwangia profunda. improvement on hydroxyectoine synthesis. Another study described Cellulases are useful for textile processing and for 2nd generation improvement of ectoine production by H. elongata mutant with ectoine bioethanol production (Aygan and Arikan, 2008; Wang et al., 2009). hydroxylase gene (ectD) deleted (Tanimura et al., 2013). Rodríguez- Halophilic cellulases obtained from Bacillus spp., Salinivibrio spp., Moya et al. (2013) constructed a genetically engineered C. salexigens Halomonas spp., and from metagenomic library of some soil microbial to overproduce hydroxyectoine in a temperature-independent manner. consortia were reported to be thermostable, halostable and alkalostable This temperature effect achieved mixed results. that are all favorable for textile, laundry and food industries (Aygan and Arikan, 2008; Shivanand et al., 2013; Voget et al., 2006; Wang et al., 3.3. Halophilic hydrolases for biotechnological industries 2009). Other halophilic hydrolases produced by halophiles include Halophiles usually thrive in hypersaline environments in which pullulanases, lipases, chitinases, pectinases, inulinases and Dnases plants and animals are present at any given time (Setati, 2010). Conse- (Essghaeir et al., 2010; Hatori et al., 2006; Makhdoumi Kakhki et al., quently, the halophilic microorganisms grown in such environments 2011; Ozcan et al., 2009; Rohban et al., 2009). These enzymes are also are expected able to utilize many different nutrient resources (Setati, commonly polyextremophilic which are halophilic, thermostable and 2010). Many halophiles can secrete extracellular hydrolytic enzymes, alkaliphilic, all these properties are desirable for various processing such as amylases, lipases, proteases, xylanases and cellulases (Govender industries. et al., 2009; Rohban et al., 2009; Sánchez-Porro et al., 2003; Yue et al., 2014). These enzymes are termed halophilic hydrolases, capable of 3.4. Biosurfactants and bioemulsifiers from halophiles catalyzing hydrolytic reactions under high salt concentrations (Delgado-García et al., 2012; Setati, 2010). Moreover, many halophilic Biosurfactants (BS) and bioemulsifiers (BE) are amphiphilic enzymes are also thermostable and adaptable to a wide range of pH compounds from bio-sources (Satputeetal.,2010). Since BS/BE contain (Enache and Kamekura, 2010; Ozcan et al., 2009; Rohban et al., 2009; both a hydrophobic and a hydrophilic moiety, allowing homogeneous Sánchez-Porro et al., 2003). Due to these unique properties, halophilic mixing of hydrophobic and hydrophilic substrates (Desai and Banat, hydrolases are attractive for biotechnological applications under ad- 1997). BS/BE are investigated as potential replacements for chemically verse conditions (Delgado-García et al., 2012; Setati, 2010). synthetic surfactants (Rodrigues et al., 2006). They are expected to Common halophilic amylases are cyclomaltodextrinases (EC play an increasingly important role in emulsification, foaming, 3.2.1.54) (Delgado-García et al., 2012; Setati, 2010), used to hydrolyze detergency, wetting, and dispersion (Dastgheib et al., 2008). starch (Gupta et al., 2003). Halobacteria including Micrococcus halobius, Microorganisms that produce BS/BE can be found inhabiting in Halomonas meridiana, Halobacillus spp., Halothermothrix orenii, different environments including sea, fresh water, soil, sludge, and Streptomyces sp., and Chromohalobacter sp. were reported to produce hypersalinity environment (Satpute et al., 2010). For low cost BS/BE halophilic amylases (Amoozegar et al., 2003; Chakraborty et al., 2009; production, bioresources from industrial wastes and by-products can Coronado et al., 2000; Mitjs and Patel, 2002; Onishi and Sonoda, 1979; be used as the substrates, reducing pollutions at the same time Prakash et al., 2009; Tan et al., 2008). Chromohalobacter amylase re- (Tambekar and Gadakh, 2013). Many industrial wastes contain complex mains stable both in the presence and absence of salt (Prakash et al., organic and inorganic mixture with various salinity, halophilic microor- 2009). A marine Streptomyces amylase is active even in the presence ganisms are thus suitable as BS/BE producers to use these mixed of commercial detergents and thus can be used as additives in laundry substrates (Kebbouche-Gana et al., 2013). detergents (Chakraborty et al., 2009). Amylase from Haloarcula sp. S-1 Marine environment holds rich resource for BS/BE producing micro- is tolerant to organic solvents (Fukishima et al., 2005). Higher stability organisms. Satpute et al. (2010) reviewed various bacteria isolated from of halophilic amylases under adverse conditions is desirable for indus- oil/petroleum contaminated marine sites and looked for BS/BE trial applications, especially for treatment of waste water containing producers. These bacteria include Acinetobacter, Arthrobacter, Pseudo- high salts and starch residues (Margesin and Schinner, 2001). monas, Halomonas, Myroides, and Corynebacterium sp. (Satpute et al., Proteases are widely applied for laundry additives, pharmaceuticals, 2010). Several classes of surfactant molecules were produced by these waste management and food processing (Amoozegar et al., 2007; marine microbes and showed novel structural and functional proper- Karbalaei-Heidari et al., 2009). Halophilic proteases have been isolated ties. For instance, Gutiérrez et al. (2007) reported glycoprotein based from Bacillus spp., Pseudoaltermonas sp., Salinivobrio sp., Halobacillus bioemulsifier from Halomonas sp. TG39 and TG67. These BE were highly spp., Filobacillus sp., Chromohalobacter sp., Nesterenkonia sp., and stable at neutral or acidic pH at high temperature. Halomonas spp. were Virgibacillus sp. (Amoozegar et al., 2007; Bakhtiar et al., 2005; Hiraga also reported to produce glycolipid surfactants (Pepi et al., 2005) et al, 2005; Karbalaei-Heidari et al., 2009; Moreno et al., 2009; and exopolysaccharide (EPS) with various useful properties including Sánchez-Porro et al., 2003; Shivanand and Jayaraman, 2009; Vidyasagar thickening, coagulating, adhesion, stabilizing and gelling (Gutierrez 1438 J. Yin et al. / Biotechnology Advances 33 (2015) 1433–1442 et al., 2009; Satpute et al., 2010). Halophiles from hypersaline environ- even though their copy number are usually low (Argandoña et al., ments other than marine sites can also produce BS/BE with interesting 2012). In haloarchaea Haloferax spp. and Halobacterium spp., shuttle properties. For instance, the BS from halophilic Bacillus sp. BS3 showed vectors pWL102 and pUBP2 have also been proven to be applicable potential pharmacological importance by suppressing the replication for genetic manipulation (Cline and Doolittle, 1992; Han et al., 2007; of shrimp white spot syndrome virus (Donio et al., 2013). Lam and Doolittle, 1992). Kebbouche-Gana et al. (2013) reported using immobilized cells of Both native and heterologous promoters were confirmed to be Natrialba sp. strain E21 for BS production in continuous fermentation. useful in halophiles for purposes of recombinant protein expression.

Their process eliminated cell recovery and recycling and thus offered The native mob gene promoter Pmob from the plasmid pHE1, as well as an economical way for BS/BE production. Recently, Halomonas sp. Pbla (β-lactamase of E. coli) and Ppdc (pyruvate decarboxylase of TD01 was genetically engineered for heterologous expression of a BE Zymomonas mobilis), were successfully used for foreign gene expression protein PhaR (Ma et al., 2013), which showed thermostability after (Afendra et al., 2004; Douka et al., 2001). In Halomonas spp. the outer heat treatment at 115 °C for 20 min (Fu et al., 2014). These studies membrane protein porin is usually expressed at very high level demonstrated that halophilic microorganisms can be used as a platform (Tokunaga et al., 2004), and several groups reported using the promoter for low cost production of endogenous/heterogenous BS/BE. region of this porin gene as a strong promoter (Fu et al., 2014; Nagayoshi et al., 2006, 2009; Tokunaga et al., 2010b). 3.5. Other chemicals produced by halophiles Genetic transfer in halophiles have been successfully achieved by several different methods. Conjugation was widely used for genetic Halophilic microorganisms have been studied for production of transfer, from E. coli to Chromohalobacter and Halomonas (Fu et al., chemicals other than those mentioned above. For instance, Halomonas 2014; Osman et al., 2010; Tan et al., 2014; Vargas et al., 1997; Yue spp. were used to produce antibacterial and cytotoxic compounds et al., 2014), as well as between moderate halophiles (Vargas et al., aminophenoxazinones (Bitzer et al., 2006), a fructose homopolymer 1997). There are few reports on DNA transformation in halophiles. levan (Küçükaşik et al., 2011). Abdelkafi et al. (2006) reported conver- Yanase et al. (2007) used electroporation to transfer plasmids to sion of ferulic acid to vanillic acid by H. elongata. Naziri et al. (2014) iso- Zymobacter palmae. Köcher et al. (2011) used protoplast fusion for lated an extremely halophilic archaeon of Halorubrum sp. able to transformation in Gram-positive moderate halophile Halobacillus produce carotenoids. Kivisto et al. (2013) studied unsterile halophilus. Transduction was also reported by Seaman and Day (2007) bioprocessing for hydrogen and 1,3-propanediol production from raw using Myoviridae ΦgspB and ΦgspC, the first virus found to infect glycerol by Halanaerobium saccharolyticum. The halophilic microalga Halomonas. Dunaliella spp. have been successfully exploited for industrial scale pro- Mutagenesis technique is widely used in characterization of gene duction of β-carotene and glycerol (Ben-Amotz and Avron, 1981, 1990; function and in metabolic engineering. In halophilic bacteria, random Raja et al., 2007; Ventosa and Nieto, 1995). Bacteriorhodopsin is a mutagenesis was achieved by chemical mutagens such as hydroxyl- unique light-driven proton pump protein found in the archaea amine, methylmetane sulfonate and nitrosoguanidine (Cánovas et al., Halobacterium spp., and bacteriorhodopsin-containing purple 1997; Llamas et al., 1999; Ryu et al., 2004). Transposon mutagenesis membranes have been commercially produced (Oesterhelt and was also feasible in Halomonas spp. (Kunte and Galinski, 1995; Llamas Stoeckenius, 1973; Oren, 2010; Ventosa and Nieto, 1995). These et al., 2000). In many cases, site-directed mutagenesis is more favored studies have made halophilic microorganisms an option for a variety than random mutagenesis. Till now, homologous (double cross-overs) of applications in biotechnology industries. recombination is the basis of those reported site-directed mutagenesis techniques (Fu et al., 2014; Grammann and Kunte, 2002; Kraegeloh 4. Advances in molecular manipulation for halophilic microorganisms et al., 2005). DNA regions upstream and downstream of the target gene (to be deleted or replaced by other sequence) are joined by over- Halophiles have promising industrial applications, they are also lap PCR and then cloned into the suicide vector. The suicide vector is candidate hosts for expression of soluble recombinant proteins, as the normally unable to replicate in halophilic bacteria and single cross- accumulation of compatible solutes enhance the solubility and proper over is stimulated by antibiotic as the selective marker. Double cross- folding of proteins (Tokunaga et al., 2010a). Efforts have been made in over happens when stimulated by conditionally lethal gene or helper developing genetic tools including vectors, promoters and mutagenesis plasmid. After double cross-over homologous recombination, both dele- approaches for halophilic bacteria to express foreign genes (Argandoña tion mutants and wild type strains are produced, thus selection and ver- et al., 2012). ification by PCR and sequencing are needed (Argandoña et al., 2012; Fu et al., 2014). In haloarchaea such as Haloferax spp. and Halobacterium 4.1. Development of genetic tools for halophilic microorganisms spp., similar gene knockout systems have been developed by using orotidine-5’-phosphate decarboxylase gene knockout mutants as the Molecular engineering is important for microbial property improve- hosts. Wild type haloarchaea are usually sensitive to 5-fluoroorotic ments (Kim et al., 2012a). For halophilic microorganisms, especially acid (5-FOA) while the mutants are resistant to 5-FOA. The suicide vec- the family Halomonadaceae (including Halomonas and other genera), tor carries the orotidine-5’-phosphate decarboxylase gene, which acts genetic manipulation has become possible (Argandoña et al., 2012). as a positive selection for the single cross-over recombination and coun- Isolation of DNA (plasmid and genome) can be achieved by using ter selection marker for double cross-over in the medium containing standard or modified methods, although the minipreps procedure uracil and 5-FOA (Bitan-Banin et al., 2003; Liu et al., 2011; Peck et al., for E. coli is not suitable (Argandoña et al., 2012; Morelle, 1989). RNA 2000). can also be isolated by standard methods for further investigation Many efforts have been made to develop genetic tools for halophilic (Calderón et al., 2004; Fu et al., 2014). microorganisms. However, improvements are still needed in the Many native plasmids from Gram-negative moderately halophilic following areas: first, a better method to prepare competent halophile bacteria have been isolated for uses as cloning vectors (Argandoña cells (Tokunaga et al., 2010a); second, current available vectors are et al., 2012). Vargas et al. (1999) reported isolation and characteriza- too large in size for effective transformation (Yanase et al., 2007); tion of pHE1 from Chromohalobacter salexigens. This plasmid was third, a tightly controllable expression system is required (Tokunaga found mobilizable from E. coli to Halomonas elongata, and was con- et al., 2010a); fourth, the efficiency of the conjugation process is good structed as the first shuttle vector pHS15 for Gram-negative moderate- enough for vector transformation but poor for constructing a gene ly halophiles (Afendra et al., 2004; Vargas et al., 1995). Broad host library. Currently available genetic tools suffer above disadvantages range plasmids are another choice as vectors for halophilic bacteria (Table 2), they need to be improved. J. Yin et al. / Biotechnology Advances 33 (2015) 1433–1442 1439

Table 2 Current genetic tools for halophiles and current challenges.

Genetic tool Application Challenges

Plasmids Native plasmids Shuttle vectors, expression vectors Too large in size, low transformation efﬁciency Broad host range plasmids Cloning and expression vectors Unstable in halophile bacteria

Promoters Native promoters, heterologous promoters Recombinant gene expression No inducible promoter available yet

Genetic manipulations Conjugation Most commonly used Efﬁciency not high enough for library construction Electroporation Rarely used Most halophiles can not be turned into competent cells Protoplast fusion Used for Gram-positive bacteria Complex procedure Transduction Rarely used No available phage

Mutagenesis Chemical mutagens Random mutagenesis Few mutagens are applicable for halophiles Transposon Random mutagenesis Unable to target on interesting gene(s) Homologous recombination Site-directed mutagenesis Two-step selection needed, time and labor consuming

4.2. Systems and synthetic biology for halophiles biosurfactants/bioemulsiﬁers and several other chemicals. Halophiles are strong candidates for unsterile and continuous bioprocessing Due to the fast advancements in systems and synthetic biology as using seawater and mixed substrates as substrates (Yue et al., 2014). well as genome sequencing technology, more and more halophilic Although the high salt concentration in the medium will corrode the microorganisms have been sequenced. For instance, 16 species from stainless steel fermentors and piping systems (Quillaguamán et al., the family of Halomonas have their genomes sequenced (Table 3). 2010), low cost materials such as plastics, carbon steel, and ceramics Among them, genome sequences of 7 species are published in 2013, can be used to make fermentors and piping systems to avoid corrosion nearly half of the total number available. Other halophiles, including since high pressure sterilization is not needed. Recent advances in Halobacterium sp., Chromohalobacter sp., as well as the halophilic archaea molecular biology methods have made metabolic engineering in halo- Haloferax volcanii and H. mediterranei, have their complete genomes philes possible. Better performances for industrial applications will be sequenced. It is predictable that the number of sequenced genomes of achieved by engineered halophiles. Yet for long-term continuous halophiles will increase substantially in the coming years. The disclosure bioprocessing, currently used expression vectors (plasmids) are unsta- of genome information sequences of halophiles and systematic in silico ble. Vectors with stably inheriting mechanisms, i.e. a complementary analysis help better understand these promising microorganisms and plasmid harboring essential gene (Hägg et al., 2004), or chromosomal will provide wide applications of post-genomic development for new engineering approaches should be developed (Yin et al., 2014). Halo- pathway constructions and optimization using synthetic biology ap- philic microorganisms will soon become very useful hosts for low-cost proaches in halophiles for more industrial biotechnology applications production of chemicals, materials and biofuels. (Cai et al., 2011; Yue et al., 2014). Acknowledgements 5. Future prospects This collaborative research was ﬁnancially supported by 973 Basic Halophilic microorganisms are now well understood as producers Research Fund (Grant No. 2012CB725201) to GQC, National High Tech of polyhydroxyalkanoates (PHA), ectoines, halophilic hydrolases, 863 Grants (Grant No. 2012AA023102) to QW, a Zhicheng Grant

Table 3 Halomonas spp. with their whole genomes sequenced.

Organism Total sequence length (bp) Date of submission Reference

Halomonas elongata DSM 2581 4,061,296 20-MAY-2010 Schwibbert et al. (2011) Halomonas sp. TD01 4,091,699 20-JUN-2011 Cai et al. (2011) Halomonas sp. HAL1 4,351,324 07-OCT-2011 Lin et al. (2012) Halomonas boliviensis LC1 4,136,366 12-OCT-2011 Quillaguámn et al., direct submissiona Halomonas sp. GFAJ-1 3,624,896 30-NOV-2011 Phung et al. (2012) Halomonas sp. KM-1 4,992,811 25-JAN-2012 Kawata et al. (2012) Halomonas stevensii S18214 3,693,745 19-MAR-2012 Kim et al. (2012b) Halomonas smyrnensis AAD6 3,561,919 20-MAR-2012 Sogutcu et al. (2012) Halomonas jeotgali Hwa 2,847,098 27-SEP-2012 Bae et al., direct submissionb Halomonas titanicae BH1 5,339,792 07-JAN-2013 Sanchez-Porro et al. (2013) Halomonas zhanjiangensis DSM 21076 4,056,790 16-APR-2013 Kyrpides et al., direct submissionc Halomonas lutea DSM 23508 4,531,945 16-APR-2013 Kyrpides et al., direct submissiond Halomonas halocynthiae DSM 14573 2,874,234 02-JUL-2013 Kyrpides et al., direct submissione Halomonas anticariensis FP35 (DSM 16096) 5,017,289 02-JUL-2013 Kyrpides et al., direct submissionf Halomonas sp. BJGMM-B45 4,758,798 06-AUG-2013 Miao et al., direct submissiong Halomonas sp. PBN3 3,668,536 17-SEP-2013 Overholt et al. (2013)

a Source available at http://www.ncbi.nlm.nih.gov/nuccore/359393363. b Source available at http://www.ncbi.nlm.nih.gov/nuccore/NZ_AMQY00000000.1. c Source available at http://www.ncbi.nlm.nih.gov/nuccore/NZ_ARIT00000000.1. d Source available at http://www.ncbi.nlm.nih.gov/nuccore/NZ_ARKK00000000.1. e Source available at http://www.ncbi.nlm.nih.gov/nuccore/NZ_AUDZ00000000.1. f Source available at http://www.ncbi.nlm.nih.gov/nuccore/NZ_AUAB00000000.1. g Source available at http://www.ncbi.nlm.nih.gov/nuccore/NZ_AVBC00000000.1. 1440 J. Yin et al. / Biotechnology Advances 33 (2015) 1433–1442

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