Applied Microbiology and Biotechnology
Title: Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha
H16
Running title: Identification and analysis of an extracellular lipase from Ralstonia eutropha
H16
Jingnan Lu1, Christopher J. Brigham2, ChoKyun Rha3, Anthony J. Sinskey2, 4, 5*
Department of Chemistry1, Department of Biology29 , Biomaterials Science & Engineering
Laboratory3, Division of Health Sciences and Technology4, Engineering Systems Division5,
Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts
02139, USA
* Corresponding author. Mailing address: Bldg. 68-370, Department of Biology,
Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA.
Phone: (617) 253-6721. Fax: (617) 253-8550. E-mail: asinskey@mit.edu
Online Resources:
Online Resource 1: List of primers used in this study.
Name
Sequencea
ΔA3742 upstream F
5’-CATAGAGCTCTACCTGAAGGCCAGCGGC-3’
ΔA3742 upstream R
5’-AGACTTAATTAAGTTCACGGGCGCTCTCCCT-3’
ΔA3742 downstream F
5’-CACATTAATTAAGTAAATCCCCGCCGGCGCAA-3’
ΔA3742 downstream R
5’-AGTATCTAGATGCCGGTAGCCGGTCTCAGA-3’
ΔA3742 diagF
5’-CGGCAAGGATGAGTTCGA-3’
ΔA3742 diagR
5’-CGCGTTGATGAAATGGGTG-3’
ΔA1323 upstream F
5’-GCATTCTAGACGCTCAAGATCCTGACCACGCGTG-3’
ΔA1323 upstream R
5’-CTTTGGGCGATGCAGCGCGGCTACACCCGGCCGCCGCCGCGCCGGC-3’
ΔA1323 downstream F
5’-GCCGGCGCGGCGGCGGCCGCGGTGTAGCCGCGCTGCATCGCCCAAAG3’
ΔA1323 downstream R
5’-GCCGGGATCCGCAACCCGCCATGGACCATCCAC-3’
ΔA1323 diagF
5’-GGACCGGCTGGTGTACGACGATATC-3’
ΔA1323 diagR
5’-GCCTGCGCCAACGCCGCACCGATGTC-3’
A1323 F
5’-GCATGGATCCGTGTACTGGCTGACGATGCCG-3’
A1323 R
5’-GCATGAGCTCCTATCGTGCCTGCCGCCGCGC-3’
a
Restriction sites are underlined
Online Resource 2: Palm oil emulsification by H16, Re2313 (lipA deletion), and Re2318 (lipB
deletion) after 24 h of growth.
Online Resource 3: SDS-PAGE gel of extracellular protein in the concentrated supernatant of: H16
(WT, Lane 1); H16/pCJB201 (lipA overexpression, Lane 2); Re2313 (ΔlipA, Lane 3); Re2318 (ΔlipB,
Lane 4). PL: BenchMarkTM Protein Ladder (Invitrogen). Supernatants (100 mL total volume) of R.
eutropha strains, grown on 1% palm oil at a culture time of 24 h, were lyophilized and concentrated to
1 mL. Concentrations of proteins in the concentrated supernatant determined by Bradford assay were
as follows: H16, 640 µg/ml; H16/CJB201, 850 µg/ml; Re2313, 425 µg/ml; Re2318, 385 µg/ml. The
concentrated supernatants are stained with Coomassie brilliant blue. 15 µl of concentrated supernatant
and protein ladder were individually loaded onto the gel. Protein ladder molecular weight indicated in
kDa. LipA has molecular weight of 38.6 kDa.
Online Resource 4: Palm oil emulsification by H16/pBBR1MCS-2 (WT) vs. H16/pCJB201 (lipA
overexpression) 5 h following inoculation.
Online Resource 5: Nucleotide sequence and amino acid sequences of LipA. The conserved catalytic
triad Ser, His, and Asp residues were bolded in red. The cysteine residues were in italicized green.
ATGGAGCACAGCGGACACCACGCGTGCGGACGACGCACGCCCGCGCTACGCGAGCAGAAGGCCGGCAAGGCCTTGCGCAGGCTCGCAGGCACAGCCACGC
M E H S G H H A C G R R T P A L R E Q K A G K A L R R L A G T A T L
10
20
30
40
50
60
70
80
90
< 100
TGGTCGCAGCGGCCATGCTGGCGCAGCCAGCACCAGCCCTGGCCGCGAGCGGCGACTACGCGAAGACGCGTTACCCGATCGTGCTGGTCCACGGACTGAC
V A A A M L A Q P A P A L A A S G D Y A K T R Y P I V L V H G L T
110
120
130
140
150
160
170
180
190
< 200
CGGCGCGGCCAGGATGGGTGGTGTGCTCGACTACTGGTATGGCATCCCCGAAGTGCTGCGGGCCAACGGCGCGCAAGTCTATGTGGCCACGGTGCCGTCG
G A A R M G G V L D Y W Y G I P E V L R A N G A Q V Y V A T V P S
210
220
230
240
250
260
270
280
290
< 300
TTCAACAGCGACGAAGAGCGCGCCCTGGCGCTGCAGGCCTATGTGCGCGCGGTCAAGCTGGAAAGCGGCGCAGACAAGGTCAACCTGATCGGCCACAGCC
F N S D E E R A L A L Q A Y V R A V K L E S G A D K V N L I G H S Q
310
320
330
340
350
360
370
380
390
< 400
AGGGCGGGCCGACCTCGCGCATGCTGGCGGCGATGTCGCCGCAGGACGTGGCCTCGGTCACCACCATCGGCAGCCCGCATCGCGGCAGCGAAGTGGCCGA
G G P T S R M L A A M S P Q D V A S V T T I G S P H R G S E V A D
410
420
430
440
450
460
470
480
490
< 500
CACCGTGCTGGACCTGATCAATGGCGTCAACAGTATTCCGATCGCCGGGCCGGTACTCGTCGGCATCATCCAGGGCGTGCTCGATACAGTGGGCTGGTTC
T V L D L I N G V N S I P I A G P V L V G I I Q G V L D T V G W F
510
520
530
540
550
560
570
580
590
< 600
AACGGCATCGGCAACGGGCAGGCGCTGGACCAGGATGCGCTGGCCTCGCTCAAGATCCTGACCACGCGTGGCGCCGCCGAGCAGAATGCGCGCCTCGATG
N G I G N G Q A L D Q D A L A S L K I L T T R G A A E Q N A R L D A
610
620
630
640
650
660
670
680
690
< 700
CCACGCTGGTGCCCGGCACGAAATCGGCACTGGGGCCGGATTGCAATACTGCTGGCGCGGTGTCCGAGCAACGGCAGGCGCGCGATGCCGCGGGCAACTT
T L V P G T K S A L G P D C N T A G A V S E Q R Q A R D A A G N F
710
720
730
740
750
760
770
780
790
< 800
CGTCACCCATACCCAGGCCGCGTATTCCTGGACCGGGCAGGGTGGGCCGTTCAGCCTGCTGCGCTCCAACCTGCTGGATCCGACCACGGTGATGATGTCG
V T H T Q A A Y S W T G Q G G P F S L L R S N L L D P T T V M M S
810
820
830
840
850
860
870
880
890
< 900
GCGTCGGCCGGGCTGATGGACCTGAAGGGCGCCGGCCCCAATGACGGGCTGGTCTCGGTCTGCAGCAGCAAGTGGGGCCGGGTGCTGGCCACGGGCTACT
A S A G L M D L K G A G P N D G L V S V C S S K W G R V L A T G Y Y
910
920
930
940
950
960
970
980
990
< 1000
ACTGGAACCATCTCGACGAGGTGAACCAGATGGCCGGCCTGTACCAGGACGTCGATCCGCGCACGGTCATTCTCAGCCACGCCAACCGGCTGCGCAATGA
W N H L D E V N Q M A G L Y Q D V D P R T V I L S H A N R L R N D
1010
1020
1030
1040
1050
1060
1070
1080
1090
< 1100
CCAGCTCTGA
Q L *
< 1110