Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli

Jeremy J Minty; Ann A Lesnefsky; Fengming Lin; Yu Chen; Ted A Zaroff; Artur B Veloso; Bin Xie; Catie A McConnell; Rebecca J Ward; Donald R Schwartz; Jean-Marie Rouillard; Yuan Gao; Erdogan Gulari; Xiaoxia Nina Lin

doi:10.1186/1475-2859-10-18

Microbial Cell Factories

Table 1 Mutations discovered in genome resequencing of evolved isobutanol tolerant E. coli clones

From: Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli

Clone	Gene	Gene Description	Biological Process	Cellular Location	Genomic Coordinate	Nucleotide Change	Protein Change
G3.2/G3.6	phoQ	sensory histidine kinase in two-component regulatory system with PhoP	Signal transduction	Inner membrane	1197581	A→G	L209P
	*acrB*	*multidrug efflux system protein*	*Transport*	*Inner and outer membrane*	*480665*	*G→A*	*P988L*
	queA	S-adenosylmethionine:tRNA ribosyltransferase-isomerase (EC:5.-.-.-)	Translation	Cytoplasm	425270	A→G	N346D
	secA	preprotein translocase subunit, ATPase	Protein secretion	Inner membrane	108975	T→C	S233P
	cadA	lysine decarboxylase 1 (EC:4.1.1.18)	Amino acid metabolism	Cytoplasm	4363790	A→G	I686T
	groL	Cpn60 chaperonin GroEL, large subunit of GroESL	Protein folding	Cytoplasm	4378650	A→C	K132N
	mutL	methyl-directed mismatch repair protein	Mismatch repair	Cytoplasm	4405650	-19 bp	Frameshift
	pstC	phosphate transporter subunit	Transport	Inner membrane	3917582	T→C	D16G
	*rph*	*defective ribonuclease PH*	*RNA processing*	*Cytoplasm*	*3823229*	*+4:GGTC*	*Frameshift*
	yiaK	2,3-diketo-L-gulonate dehydrogenase, NADH-dependent (EC:1.1.1.-)	Carbohydrate metabolism	Cytoplasm	3750540	T→C	L193P
	gltD	glutamate synthase, 4Fe-4S protein, small subunit (EC:1.4.1.13)	Amino acid, Nitrogen metabolism	Cytoplasm	3367270	+1:G	Frameshift
	*mdh*	*malate dehydrogenase, NAD(P)-binding (EC:1.1.1.37)*	*Tricarboxylic acid cycle*	*Membrane peripheral*	*3390726*	*-1:C*	*Frameshift*
	nlpI	lipoprotein involved in cell division	Cell cycle	Inner membrane	3316213	T→C	Non-coding region; Possible effect on terminator before nlpI
	glnE	fused deadenylyltransferase/adenylyltransferase for glutamine	Nitrogen metabolism	Cytoplasm	3205272	IS186 insertion	Disruption
	hybB	predicted hydrogenase 2 cytochrome b type component	Electron transport chain	Inner membrane	3150318	A→G	V359A
	*gatZ*	*D-tagatose 1,6-bisphosphate aldolase 2, subunit (EC:4.1.2.40)*	*Carbohydrate metabolism*	*Cytoplasm*	*2182915*	*-1:C*	*Frameshift*
	yeeE	predicted inner membrane protein	-	Inner membrane	2092513	A→G	S333P
	lepB	leader peptidase (signal peptidase I) (EC:3.4.21.89)	Transport	Inner membrane	2711902	G→A	P213S
	hfq	HF-I, host factor for RNA phage Q β replication	Translation	Cytoplasm	4407505	-7:AGGAAAA	Non-coding region; Ribosome binding site deletion
	*marC*	*conserved protein; predicted transporter*	-	*Inner membrane*	*1625925*	*IS1 insertion*	*Disruption*
G3.266.7	groL	Cpn60 chaperonin GroEL, large subunit of GroESL	Protein folding	Cytoplasm	4378650	A→C	K132N
	*rph*	*defective ribonuclease PH*	*RNA processing*	*Cytoplasm*	*3823229*	*+4:GGTC*	*Frameshift*
	gltB-yhcE	-	-	-	-	-9.9 kb	ΔgltBDF, ΔyhcADE
	*mdh*	*malate dehydrogenase, NAD(P)-binding (EC:1.1.1.37)*	*Tricarboxylic acid cycle*	*Membrane peripheral*	*3390726*	*-1:C*	*Frameshift*
	glnE	fused deadenylyltransferase/adenylyltransferase for glutamine	Nitrogen metabolism	Cytoplasm	3205272	IS186 insertion	Disruption
	hfq	HF-I, host factor for RNA phage Q β replication	Translation	Cytoplasm	4407505	-7:AGGAAAA	Non-coding region; Ribosome binding site deletion
	ycfK	e14 prophage; predicted protein	-	-	1216432	-1688 bp	ΔycfK
	*marC*	*conserved protein; predicted transporter*	-	*Inner membrane*	*1625925*	*IS1 insertion*	*Disruption*
X3.5	*acrA*	*multidrug efflux system protein*	*Transport*	*Inner and outer membrane*	*483735*	*+1:A*	*Frameshift*
	rpsB	30S ribosomal subunit protein S2	Translation	Cytoplasm	190557	+1:A	Frameshift
	*rph*	*defective ribonuclease PH*	*RNA processing*	*Cytoplasm*		*-1:C*	*Frameshift*
	*mdh*	*malate dehydrogenase, NAD(P)-binding (EC:1.1.1.37)*	*Tricarboxylic acid cycle*	*Membrane peripheral*	*3390936*	*+5:AACCT*	*Frameshift*
	deaD	DEAD-box RNA helicase	Translation	Cytoplasm	3314027	+4:AGAC	Frameshift
	yfgO	predicted inner membrane protein	-	Inner membrane	2623022	C→T	G30D
	*gatC*	*galactitol-specific enzyme IIC component of PTS*	*Transport*	*Inner membrane*	*2180640*	*C→T*	*E290K*
	plsX	fatty acid/phospholipid synthesis protein	Fatty acid metabolism	Cytoplasm	1493514	A→G	E216G
	hrpA	ATP-dependent helicase	RNA processing	Cytoplasm	1493514	C→T	L1075L
	mdtJ-tqsA	MdtJ SMR protein; transporter of quorum signal AI-2	Transporter/Transporter	Inner membrane	1681114	IS5 insertion	Non-coding region; mdtJ and tqsA promoter region
	*marC*	*conserved protein; predicted transporter*	-	*Inner membrane*	*1626081*	*IS1 insertion*	*Disruption*

Clonal isolates from isobutanol tolerant E. coli EcNR1 populations were sequenced with the Illumina platform to identify mutations. Clones G3.2 and G3.6 were taken from the G3 end point population, which developed a mutator phenotype via a 19 bp deletion in mutL. These clones thus each have a large number of mutations, so for brevity we show here the subset of mutations shared between G3.2/G3.6 (full mutation lists available in Additional file 1). All mutations shown above were verified by Sanger sequencing. Mutation entries that are bold and italic denote loci that were mutated in all sequenced clones from end populations. The mutation positions are listed as absolute genomic coordinates in the E. coli EcNR1 reference sequence (available in Additional file 2). SNPs are indicated by base transition/transversion. Small insertions are indicated by a '+', with the size (number of bp) of the insertion and sequence of inserted bases. Small deletions are designated by '-' with a format similar to that for small insertions; for large deletions, the sequence of deleted bases is excluded. Transposons are indicated by the insertion sequence (IS) identity.

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