Abstract
Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.
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- Bailey J. E. Host-vector interactions in Escherichia coli. Adv Biochem Eng Biotechnol. 1993;48:29–52. doi: 10.1007/BFb0007195. [DOI] [PubMed] [Google Scholar]
- Csonka L. N., Fraenkel D. G. Pathways of NADPH formation in Escherichia coli. J Biol Chem. 1977 May 25;252(10):3382–3391. [PubMed] [Google Scholar]
- Diesterhaft M. D., Freese E. Role of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme during growth and sporulation of Bacillus subtilis. J Biol Chem. 1973 Sep 10;248(17):6062–6070. [PubMed] [Google Scholar]
- Farmer I. S., Jones C. W. The effect of temperature on the molar growth yield and maintenance requirement of Escherichia coli W during aerobic growth in continuous culture. FEBS Lett. 1976 Sep 1;67(3):359–363. doi: 10.1016/0014-5793(76)80564-9. [DOI] [PubMed] [Google Scholar]
- Harwood C. R. Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol. 1992 Jul;10(7):247–256. doi: 10.1016/0167-7799(92)90233-l. [DOI] [PubMed] [Google Scholar]
- Heijnen J. J., Roels J. A., Stouthamer A. H. Application of balancing methods in modeling the penicillin fermentation. Biotechnol Bioeng. 1979 Dec;21(12):2175–2201. doi: 10.1002/bit.260211204. [DOI] [PubMed] [Google Scholar]
- Holms W. H. The central metabolic pathways of Escherichia coli: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate. Curr Top Cell Regul. 1986;28:69–105. doi: 10.1016/b978-0-12-152828-7.50004-4. [DOI] [PubMed] [Google Scholar]
- Kaneda T. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol Rev. 1991 Jun;55(2):288–302. doi: 10.1128/mr.55.2.288-302.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Katz J., Rognstad R. The labeling of pentose phosphate from glucose-14C and estimation of the rates of transaldolase, transketolase, the contribution of the pentose cycle, and ribose phosphate synthesis. Biochemistry. 1967 Jul;6(7):2227–2247. doi: 10.1021/bi00859a046. [DOI] [PubMed] [Google Scholar]
- Lopez J. M., Marks C. L., Freese E. The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis. Biochim Biophys Acta. 1979 Oct 4;587(2):238–252. doi: 10.1016/0304-4165(79)90357-x. [DOI] [PubMed] [Google Scholar]
- Mainzer S. E., Hempfling W. P. Effects of growth temperature on yield and maintenance during glucose-limited continuous culture of Escherichia coli. J Bacteriol. 1976 Apr;126(1):251–256. doi: 10.1128/jb.126.1.251-256.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manneberg M., Lahm H. W., Fountoulakis M. Oxidation of cysteine and methionine residues during acid hydrolysis of proteins in the presence of sodium azide. Anal Biochem. 1995 Jan 1;224(1):122–127. doi: 10.1006/abio.1995.1016. [DOI] [PubMed] [Google Scholar]
- Manneberg M., Lahm H. W., Fountoulakis M. Quantification of cysteine residues following oxidation to cysteic acid in the presence of sodium azide. Anal Biochem. 1995 Nov 1;231(2):349–353. doi: 10.1006/abio.1995.9988. [DOI] [PubMed] [Google Scholar]
- Model P., Rittenberg D. Measurement of the activity of the hexose monophosphate pathway of glucose metabolism with the use of [18O]glucose. Variations in its activity in Escherichia coli with growth conditions. Biochemistry. 1967 Jan;6(1):69–80. doi: 10.1021/bi00853a013. [DOI] [PubMed] [Google Scholar]
- Pirt S. J. The maintenance energy of bacteria in growing cultures. Proc R Soc Lond B Biol Sci. 1965 Oct 12;163(991):224–231. doi: 10.1098/rspb.1965.0069. [DOI] [PubMed] [Google Scholar]
- Russell J. B., Cook G. M. Energetics of bacterial growth: balance of anabolic and catabolic reactions. Microbiol Rev. 1995 Mar;59(1):48–62. doi: 10.1128/mr.59.1.48-62.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saito H., Shibata T., Ando T. Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Marburg. Mol Gen Genet. 1979 Feb 26;170(2):117–122. doi: 10.1007/BF00337785. [DOI] [PubMed] [Google Scholar]
- Santana M., Ionescu M. S., Vertes A., Longin R., Kunst F., Danchin A., Glaser P. Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants. J Bacteriol. 1994 Nov;176(22):6802–6811. doi: 10.1128/jb.176.22.6802-6811.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stouthamer A. H., Bettenhaussen C. Utilization of energy for growth and maintenance in continuous and batch cultures of microorganisms. A reevaluation of the method for the determination of ATP production by measuring molar growth yields. Biochim Biophys Acta. 1973 Feb 12;301(1):53–70. doi: 10.1016/0304-4173(73)90012-8. [DOI] [PubMed] [Google Scholar]
- Tempest D. W., Neijssel O. M. The status of YATP and maintenance energy as biologically interpretable phenomena. Annu Rev Microbiol. 1984;38:459–486. doi: 10.1146/annurev.mi.38.100184.002331. [DOI] [PubMed] [Google Scholar]
- Varma A., Palsson B. O. Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110. Appl Environ Microbiol. 1994 Oct;60(10):3724–3731. doi: 10.1128/aem.60.10.3724-3731.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Hollander J. A. Application of a metabolic balancing technique to the analysis of microbial fermentation data. Antonie Van Leeuwenhoek. 1991 Oct-Nov;60(3-4):275–292. doi: 10.1007/BF00430370. [DOI] [PubMed] [Google Scholar]