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Microbial Production of Rhamnolipids: Opportunities, Challenges and Strategies Huiqing Chong and Qingxin Li*

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Microbial Production of Rhamnolipids: Opportunities, Challenges and Strategies Huiqing Chong and Qingxin Li* Chong and Li Microb Cell Fact (2017) 16:137 DOI 10.1186/s12934-017-0753-2 Microbial Cell Factories REVIEW Open Access Microbial production of rhamnolipids: opportunities, challenges and strategies Huiqing Chong and Qingxin Li* Abstract Rhamnolipids are a class of biosurfactants which contain rhamnose as the sugar moiety linked to β-hydroxylated fatty acid chains. Rhamnolipids can be widely applied in many industries including petroleum, food, agriculture and bioremediation etc. Pseudomonas aeruginosa is still the most competent producer of rhamnolipids, but its patho- genicity may cause safety and health concerns during large-scale production and applications. Therefore, extensive studies have been carried out to explore safe and economical methods to produce rhamnolipids. Various metabolic engineering eforts have also been applied to either P. aeruginosa for improving its rhamnolipid production and diminishing its pathogenicity, or to other non-pathogenic strains by introducing the key genes for safe production of rhamnolipids. The three key enzymes for rhamnolipid biosynthesis, RhlA, RhlB and RhlC, are found almost exclu- sively in Pseudomonas sp. and Burkholderia sp., but have been successfully expressed in several non-pathogenic host bacteria to produce rhamnolipids in large scales. The composition of mono- and di-rhamnolipids can also be modi- fed through altering the expression levels of RhlB and RhlC. In addition, cell-free rhamnolipid synthesis by using the key enzymes and precursors from non-pathogenic sources is thought to not only eliminate pathogenic efects and simplify the downstream purifcation processes, but also to circumvent the complexity of quorum sensing sys- tem that regulates rhamnolipid biosynthesis. The pathogenicity of P. aeruginosa can also be reduced or eliminated through in vivo or in vitro enzymatic degradation of the toxins such as pyocyanin during rhamnolipid production. The rhamnolipid production cost can also be signifcantly reduced if rhamnolipid purifcation step can be bypassed, such as utilizing the fermentation broth or the rhamnolipid-producing strains directly in the industrial applications of rhamnolipids. Keywords: Rhamnolipids, Pseudomonas, fermentation, Metabolic engineering, Application Background may lead to environmental problems. Terefore, bio- Surfactants are amphipathic molecules with surface surfactants produced by microbial fermentation can be activities to reduce surface tensions. Tey are widely used to replace synthetic surfactants as environmental utilized as detergents, solubilizers or emulsifying agents friendly alternatives [4–6]. Te most attractive char- in many industrial felds such as petroleum, food, phar- acteristic of biosurfactant is that they are easily biode- maceutical and agricultural industries [1, 2]. Te total gradable and cause less toxic impact to the environment, worldwide production of surfactants is estimated to be while having similar properties to synthetic surfactants. over 15 million tons per year, and expected to increase Some biosurfactants are also tolerant to a wide range to over 24 million tons annually by 2020 [3]. Currently, of extreme conditions (low and high pH, high tempera- surfactants are mainly derived from petroleum prod- ture and high salinity) that are frequently encountered ucts chemically due to the low cost of production. How- in industrial processes, as well as several interesting ever, synthetic surfactants are non-biodegradable, which biological properties like antimicrobial activity [2, 7]. Biosurfactants can be classifed into several categories including glycolipids (rhamnolipids, sophorolipids, tre- *Correspondence: [email protected]‑star.edu.sg Institute of Chemical and Engineering Sciences, Agency for Science, halose lipids), lipopeptide (surfactin, iturin) and poly- Technology and Research, 1 Pesek Road, Jurong Island 627833, Singapore meric compounds (emulsan, alasan) [9]. Among them, © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chong and Li Microb Cell Fact (2017) 16:137 Page 2 of 12 rhamnolipids are the most extensively studied because concentration (CMC) is about 230 mg/l for rhamnolipid of their excellent physicochemical properties and abil- mixture with high proportion of congeners containing ity to reach high fermentation titers. Here we focus on unsaturated fatty acids [9], 5 mg/l for di-rhamnolipids reviewing the challenges and opportunities in microbial with C10 fatty acids, and 40 mg/l for mono-rhamnolipids production of rhamnolipids, as well as potential strate- with C10 fatty acids [2, 10]. gies to address these challenges. Rhamnolipid applications Rhamnolipids Rhamnolipids can be widely applied to many industrial Rhamnolipids are glycolipids containing a hydrophilic felds such as petroleum, bioremediation, agriculture, group, consisting of either one or two (l)-rhamnose mol- cosmetics, food processing and pharmaceuticals because ecules, with a glycosidic linkage to the hydrophobic group of their excellent surface activities and biological activi- made up of one or two β-hydroxy fatty acids (Fig. 1). ties [11, 12]. Particularly, rhamnolipids can be used in Microbial fermentation produces a diversity of rham- manufacturing fne chemicals and surface coatings, as nolipid congeners with variations in the chain length, food additives and as biological control agents [6]. Te degree of unsaturation for the fatty acid chains, and dif- applications have been described in detail in several ferences in the number of rhamnose molecules. Rham- reviews [13] and are summarized in Table 1. nolipids containing one and two rhamnose molecules are known as mono-rhamnolipids and di-rhamnolipids, Rhamnolipid‑producing strains respectively (Fig. 1). It is estimated that approximately Rhamnolipids are thought to be produced by hydro- 60 rhamnolipid congeners and homologues exist in the carbon-degrading microorganisms as the amphipathic fermentation broth, while the predominant rhamnolipid characteristics of rhamnolipids are essential for bacte- species and the concentrations of the congeners are ria to uptake and utilize the hydrophobic hydrocarbons dependent on the rhamnolipid-producing strains [8]. Te as the carbon source [14]. Some microorganisms pro- physicochemical properties of the rhamnolipids can be duce rhamnolipids only when hydrocarbons are used as greatly afected even with small changes in the compo- the carbon source [15]. Some rhamnolipid-producing sition of the congeners. For instance, the critical micelle strains can cause nosocomial infections, especially in individuals with reduced immunity because rhamnolip- ids may be important for the pathogenic efects [5]. Quite a few rhamnolipid-producing strains have been isolated and characterized (Table 2). Among the avail- able strains, the rhamnolipid yield of P. aeruginosa is still the highest. Rhamnolipid synthesis and regulation in P. aeruginosa Pseudomonas aeruginosa has been used as the model strain to understand the genes that are critical for rham- nolipid biosynthesis. Production of rhamnolipids in Pseudomonas involves several steps. Te precursors for rhamnolipid synthesis are the sugar (dTDP-l-rhamnose) and hydrophobic moieties such as 3-(3-hydroxyalkanoy- loxy)alkanoic acid (HAA). Te sugar moiety can be syn- thesized from d-glucose, while the hydrophobic moiety can be synthesized through the fatty acid synthesis path- way, starting with two-carbon units [16]. Most bacteria contain the required enzymes for synthesizing the pre- cursors in rhamnolipid biosynthesis but the enzymes involved in the synthesis of HAA, mono- and di-rham- nolipids are found almost exclusively in Pseudomonas sp. Fig. 1 Rhamnolipid production in Pseudomonas. The synthesis and Burkholderia sp. (Fig. 1). Synthesis of rhamnolipids pathway for rhamnolipids is indicated while the details for metabolic and their precursors only occurs upon induction of the pathway of glucose and synthesis of Acyl-CoA are not provided. RhlA, related gene products to express the key enzymes for the RhlB and RhlC are three enzymes essential for rhamnolipid synthesis rhamnolipid biosynthesis pathway. Chong and Li Microb Cell Fact (2017) 16:137 Page 3 of 12 Table 1 Applications of rhamnolipids Applications Examples References Oil recovery Microbial enhanced oil recovery (MEOR) [73] Increase amount of recoverable oil aided by rhamnolipid-producing microorganisms [2, 74] Bioremediation Bioremediation of petroleum at contaminated sites [46, 75–78] Addition of rhamnolipids improves solubility of hydrocarbons to facilitate degradation Bioremediation of heavy metal at contaminated lands or in water treatment plant; rhamnolipids can be [2, 11, 79] applied in foaming-surfactant technology to remove heavy metal contaminants Bioremediation of pesticides at agricultural felds; addition of rhamnolipids can enhance degradation of [11, 79] chemical pesticides Pest control As emulsifer, spreaders and dispersing agent in pesticide formulations [2, 79] As bio-pesticide against agricultural
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