Production of rhamnolipids-producing enzymes of <em>Pseudomonas</em> in <em>E. coli</em> and structural characterization

doi:10.1007/s11705-017-1637-z

Front. Chem. Sci. Eng.

2017, Vol. 11

Issue (1) : 133-138 https://doi.org/10.1007/s11705-017-1637-z

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Production of rhamnolipids-producing enzymes of Pseudomonas in E. coli and structural characterization

Kata Kiss,Wei Ting Ng,Qingxin Li(

)

Institute of Chemical & Engineering Sciences, Agency for Science, Technology and Research, Jurong Island 627833, Singapore

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Abstract

Rhamnolipids are a class of biosurfactants that have a great potential to be used in industries. Five proteins/enzymes, namely RhlA, RhlB, RhlC, RhlG and RhlI, are critical for the production of rhamnolipids in Pseudomonas aeruginosa. Four of the 5 proteins except RhlC were successfully over-expressed in E. coli and three of them (RhlA, RhlB and RhlI) were purified and obtained in milligram quantities. The purified proteins were shown to be folded in solution. Homology models were built for RhlA, RhlB and RhlI. These results lay a basis for further structural and functional characterization of these proteins in vitro to favor the construction of super strains for rhamnolipids production.

Keywords rhamnolipids Pseudomonas RhlA RhlB RhlI protein folding

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Corresponding Author(s): Qingxin Li

Just Accepted Date: 24 January 2017 Online First Date: 28 February 2017 Issue Date: 17 March 2017

Cite this article:

Kata Kiss,Wei Ting Ng,Qingxin Li. Production of rhamnolipids-producing enzymes of Pseudomonas in E. coli and structural characterization[J]. Front. Chem. Sci. Eng., 2017, 11(1): 133-138.

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https://academic.hep.com.cn/fcse/EN/10.1007/s11705-017-1637-z
https://academic.hep.com.cn/fcse/EN/Y2017/V11/I1/133

Fig.1 A pathway of rhamnolipids synthesis in Pseudomonas.

Acyl-CoA: acetyl-coenzyme; HAA: β-3-(3-hydroxyalkanoyloxy) alkanoic acid; ACP: acyl carrier protein; RhlA: β-3-(3-hydroxyalkanoyloxy) alkanoic acid synthetase; RhlB: rhamnosyltransferase 1; RhlC: rhamnosyltransferase 2

Fig.2 Small scale induction of rhamnolipids-producing related proteins of Pseudomonas in E. coli.

The protein induction was conducted as described in Materials and methods. The induced cells were harvested by centrifugation and mixed with 2×SDS-PAGE loading buffer. SDS-PAGE was used to analyze the protein induction. Clear induction band was observed by comparing with the reference sample which was prepared without adding IPTG. M. W: molecular weight standard for SDS-PAGE; Lane 1: none-induced cells; Lanes 2 to 6: inductions of RhlA, RhlB, RhlC, RhlI and RhlG, respectively

Fig.3 Purification of (A) RhlA, (B) RhlB and (C) Rhl I from E. coli.

M.W: molecular weight standard for SDS-PAGE; T: total cell lysate; L: sample that was mixed with Ni²⁺-NTA resin; E1 and E2: purified fractions from Ni²⁺-NTA resin; G1 and G2: purified fractions from gel filtration chromatography; D: Western blot analysis of purified proteins using an anti-his antibody. Lanes 1, 2 and 3: purified RhlA, RhlB and RhlI, respectively

Fig.4 Homology models and folding of purified proteins in solution.

(A) (B) and (C) homology models of RhlA, RhlB and RhlI, respectively. Structures from N to C-termini are shown in blue to red. (D), (E) ¹H NMR spectra of RhlA, RhlB and RhlI. Proton NMR for amide (D) and methyl (E) regions are shown. RhlA, B and I are shown in blue, red, and green, respectively. (F) ¹H-¹⁵N-HSQC spectrum of RhlI. All spectra were collected on a Bruker 600 MHz and processed using Topspin (2.1)

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