Transcriptional effects of CRP* expression in Escherichia coli.
BACKGROUND
Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) enables co-utilization of glucose and other sugars in E. coli. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.


RESULTS
The glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression.We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP+ ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes, among others, are consistent with these observations.


CONCLUSIONS
While the simplest model of CRP*-mediated gene expression assumes insensitivity to glucose (or cAMP), our results show that gene expression in the context of CRP* is very different from that of wild-type in the absence of glucose, and is influenced by the presence of glucose. Most of the transcription changes in response to CRP* expression are difficult to interpret in terms of possible systematic effects on metabolism. Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of crp* strains that extend beyond relief of catabolite repression.
Khankal
Reza
R
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA. khankal@engr.psu.edu
Chin
Jonathan W
JW
Ghosh
Debashis
D
Cirino
Patrick C
PC
Annu Rev Biochem. 1993;62:749-95
8394684
J Bacteriol. 1999 Feb;181(3):1030-4
9922271
Anal Biochem. 1997 Sep 5;251(2):153-7
9299010
J Bacteriol. 1975 Apr;122(1):338-40
164435
J Biotechnol. 2007 Mar 10;128(4):747-61
17331609
Biochim Biophys Acta. 2002 Sep 10;1555(1-3):122-7
12206903
Anal Biochem. 1973 Jun;53(2):452-8
4351948
Cell. 1985 Jul;41(3):745-51
2988785
J Bacteriol. 2004 Jun;186(11):3516-24
15150239
EMBO J. 1983;2(5):715-20
16453452
EMBO J. 1985 Dec 1;4(12):3329-32
3004951
Biochemistry. 2001 Nov 6;40(44):13177-87
11683626
Biotechnol Bioeng. 2009 May 1;103(1):148-61
19189409
Proc Natl Acad Sci U S A. 1982 Mar;79(5):1457-61
7041121
FEMS Microbiol Lett. 2001 Nov 13;204(2):247-52
11731130
J Bacteriol. 2008 Apr;190(7):2323-30
18223071
Biotechnol Bioeng. 2006 Dec 20;95(6):1167-76
16838379
J Biotechnol. 2008 Apr 30;134(3-4):246-52
18359531
Nat Protoc. 2009;4(1):44-57
19131956
Biotechnol Prog. 2004 Mar-Apr;20(2):403-11
15058984
J Bacteriol. 1983 Oct;156(1):81-8
6311808
Mol Syst Biol. 2006;2:2006.0008
16738554
J Biol Chem. 2004 Feb 20;279(8):6613-9
14660605
Nucleic Acids Res. 1995 Feb 25;23(4):599-605
7899079
J Biol Chem. 1970 Nov 25;245(22):5974-8
4320794
Curr Opin Biotechnol. 2000 Apr;11(2):187-98
10753763
Nucleic Acids Res. 2009 Jan;37(Database issue):D464-70
18974181
Mol Microbiol. 1998 Nov;30(3):487-98
9822815
Nucleic Acids Res. 1993 Apr 25;21(8):1827-35
8388097
Mol Syst Biol. 2006;2:2006.0007
16738553
J Bacteriol. 2008 Jun;190(11):3851-8
18375566
Microbiol Rev. 1992 Mar;56(1):100-22
1315922
Mol Microbiol. 1994 Sep;13(5):755-64
7815935
J Bacteriol. 1981 Dec;148(3):753-61
6273380
Genome Biol. 2003;4(5):P3
12734009
J Biol Chem. 1983 Mar 25;258(6):3750-8
6300054
J Biotechnol. 2004 Apr 8;109(1-2):21-30
15063611
J Biol Chem. 1995 Sep 15;270(37):21679-83
7665583
J Biol Chem. 1986 Dec 15;261(35):16332-9
3023348
J Bacteriol. 2009 Apr;191(7):2112-21
19181802
Microbiol Mol Biol Rev. 2005 Mar;69(1):12-50
15755952
Biochemistry. 2003 May 20;42(19):5555-65
12741811
Proc Natl Acad Sci U S A. 1970 Jun;66(2):480-7
4317918
Methods. 2001 Dec;25(4):402-8
11846609
Microbiol Rev. 1989 Mar;53(1):109-20
2651862
Res Microbiol. 2004 Mar;155(2):76-9
14990258
Mol Microbiol. 1999 Sep;33(6):1221-31
10510236
Biotechnol Bioeng. 2009 Jan 1;102(1):209-20
18698648
J Bacteriol. 2007 Oct;189(19):6891-900
17675376
Biotechnol Lett. 2008 Sep;30(9):1645-53
18414795
Nucleic Acids Res. 2004 Jan 1;32(Database issue):D277-80
14681412
Mol Microbiol. 1995 Jul;17(2):251-8
7494474
Mol Gen Genet. 1978 Sep 20;165(1):47-56
213702
Stat Appl Genet Mol Biol. 2004;3:Article3
16646809
Eur J Biochem. 2000 Oct;267(20):6102-9
11012661
J Biol Chem. 1995 May 19;270(20):11803-5
7744829