Part:BBa_K3146007

RhlA

Required for rhamnolipid surfactant production. Supplies the acyl moieties for rhamnolipid biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the beta-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Catalyzes the formation of one molecule of beta-hydroxydecanoyl-beta-hydroxydecanoate from two molecules of beta-hydroxydecanoyl-ACP. Is the only enzyme required to generate the lipid component of rhamnolipid. Rhamnolipid production plays an important role in swarming motility.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 69
Illegal BamHI site found at 629
Illegal XhoI site found at 805
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 294
Illegal BsaI.rc site found at 478

Shewanella oneidensis strain MR-1 has been been extensively studied due to it’s extracellular electron transfer system(MTR pathway) under anaerobic conditions. We have assayed wether over expression of the following genes affected the Microbial Fuel Cells(MFC ) output following genes were encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP), the ex-tracytoplasmic function sigma factor RpoE, oxygen-responsive transcriptional regulator Fnr (EtrA) and the key gene responsible for rhamnolipids synthesis (rhlA).

All genes were Ligated to BBa_J23100 constitutive promoter, BBa_B0034 strong Ribosome Binding Site and transformed into Shewanella oneodensis in Psb3C1 vector as described in Shewanella Conjugation protocol. Ligations were performed using 3A assembly, Psb1C3 vector was used as Shewanella oneidensis Mr-1 is naturally resistant to Ampicillin. Cultures were grown in double antibiotic broth(100ng/ul of Chlorampheniucol and 100ng/ul Ampicillin) for 18 hours prior to inoculation of MFCs. 6 MFCs were set up as described in MFC protocol including control A Shewanella MR-1 without antibiotic, control B Shewanella Oneidensis MR-1 containing TPHP-PSB1C3(terephthalic acid permease channel) and 4 MFC’s test constructs supplemented with 100ng/ul of Chlorampheniucol and 100ng/ul Amphicillin for selection: CRP, EtrA, rhlA, RpoE. MFC out data was collected over 4 days.

Figure 1

Out of all constructs tested, over expression of oxygen-responsive transcriptional regulator Fnr EtrA showed most significant increase compared to both controls as can be seen in Figure 1. Interestingly, Control Shewanella Oneidensis containing TPHP-PSB1C3(terephthalic acid permease channel) showed significantly higher output compared to control as well as CRP, Rpoe and RhlA. Overexprerssion of RpoE and RhlA were not successful at increasing MFC output comparing to both controls and Overexpression of CRP in Shewanella produced higher output than one out of 2 controls.

This work could be build upon to study MTR pathway and anaerobic response in S. Oneidensis MR-1 and further increase efficiency of Microbial Fuel Cells as renewable energy source. Over-expression of anaerobic response transcription factors in S. Oneidensis could solve the problem of anaerobic requirements and oxygen leakage in Microbial fuel cells, however further tests are required to confirm functionality of the constructs in various conditions.

Usage by team Nanjing_NFLS 2020

Team Nanjing_NFLS 2020 use this part to increase the rhamnose synthesis and upregulate PYO level in Pseudomonas aeruginosa, which contributes to improvement of our efficiency of Microbial fuel cells (MFCs) this year. We added rhlA on the plasmid pBBR1MCS-5. Restriction endonuclease digestion result (Figure 1) showed the gene is successfully bind to the plasmid.

Figure 1. Identification of pBBR1MCS-5-phzM by restriction endonuclease digestion

We then tested the efficiency of the enzyme. First, different concentrations of rhamnose standard solution was prepared, and the absorbance was determined at 620 nm at different concentrations. Then the standard curve shown as y = 0.0065 x + 0.0081 (R2=0.9993), y for the absorbance of the sample, and x for the concentration of rhamnose in the corresponding sample(mg/L). According to the standard curve, rhamnose was 44.21% higher in engineered cells than that of the control group(Figure 2).

strain	rhamnose concentration (mg/L)
PAO1	409.80
PAO1-phzM	590.96

Figure.2 Rhamnose analysis in overexpression rhlA in P. aeruginosa PAO1

Contribution by team Nanjing_NFLS 2021

Team Nanjing_NFLS 2021 use this part to increase rhamnolipid synthesis and upregulate pyocyanine (an electron transfer mediator) production in order to improve microbial fuel cells (MFCs) efficiency and antibiotics degradation efficiency this year. We added rhlA on the plasmid pBBR1MCS-5. Restriction endonuclease digestion result showed the gene (Figure 1) is successfully bind to the plasmid.

Figure.1 Identification of pBBR1MCS-5-rhlA by restriction endonuclease digestion

Marker: DL5000; fragments digested by EcoR I and Hind III

We then tested rhamnolipids overproduction by the overexpression of rhlA. Ramnolipids production was analyzed with anthranone-H₂SO₄ method. As shown in the figure, after incubation for 5 days, the rhamnolipids produced by PAO1 is about 1.4 g/L, while it is about 2.0 g/L for PAO1-rhlA. The rhamnolipids production increased by 42.9%.

Strain	Rhamnolipids concentration (g/L)
PAO 1	1.4
PAO1-rhlA	2.0