Limiting an essential nutrient has a profound impact on microbial growth. The notion of growth under limited conditions was first described using simple Monod kinetics proposed in the 1940s. Different operational modes (chemostat, fed-batch processes) were soon developed to address questions related to microbial physiology and cell maintenance and to enhance product formation. With more recent developments of metabolic engineering and systems biology, as well as high-throughput approaches, the focus of current engineers and applied microbiologists has shifted from these fundamental biochemical processes.
Nitrogen occurs throughout cells, in proteins, nucleotides, and many metabolites. Nitrogen as ammonium is assimilated in E. coli and most bacteria and yeast into glutamine and glutamate [100][49], which are the primary intracellular nitrogen donors. In bacteria, 88% of the cellular nitrogen is derived from glutamate, while 12% is derived from glutamine [101][50]. Glutamate is the most abundant metabolite in E. coli, accounting for about 40% of the total metabolite concentration [102][51]. In many bacteria such as E. coli, two ammonium-assimilating pathways are available, a NADPH-dependent glutamate dehydrogenase and a high-affinity glutamate synthase (glutamine oxoglutarate aminotransferase, GOGAT)/glutamine synthase. Glutamate dehydrogenases generally have high values of KM for ammonium, so that during N-limited growth, glutamine synthase expression is elevated to maintain sufficient glutamate [103][52]. Dynamic N starvation in E. coli and S. cerevisiae growing on glucose depletes glutamine and to a lesser extent glutamate while α-ketoglutarate increases markedly and can even be excreted [65,104][47][53]. Accumulation of α-ketoglutarate occurs in cyanobacteria also, a signal which upregulates nitrogen assimilation via the global regulator NtcA and other regulators [105,106][54][55]. In yeast, the concentration of tryptophan, which relies on glutamine for its synthesis, decreases, while phenylalanine and tyrosine, which rely on glutamate for nitrogen, do not change, resulting in an accumulation of phenylpyruvate and phenylethanol, a quorum-sensing signal [107][56]. Accumulated α-ketoglutarate in E. coli noncompetitively and cooperatively inhibits EI of the PTS [108][57], citrate synthase [109][58], and PEP synthetase [110][59]. Furthermore, sudden nitrogen availability in N-starved wild-type E. coli induces a decrease in α-ketoglutarate and rapid increase in glucose uptake rate, while in a PTS-deficient strain with elevated galactose permease, glucose uptake is insensitive to N-availability [108][57]. In general, S. cerevisiae N-limitation leads to depletion of intracellular amino acids, particularly at low dilution rates [104][53]. Because intracellular glutamine and arginine concentration correlates strongly with dilution rate, these compounds likely control growth in N-limited S. cerevisiae [104][53]. Synechocystis also accumulate α-ketoglutarate [111][60] under N-starvation as well as glycogen, which is associated with the induction of the glgX gene [112,113,114][61][62][63]. E. coli has a high protein turnover under N-limited conditions compared to C-limited or P-limited conditions [115][64].
Sulfur is present in the amino acids methionine and cysteine, and important metabolites such as S-adenosylmethionine, Coenzyme A (CoA), and lipoic acid. In most media, S is supplied as the sulfate ion, and thus, S-limitation is often studied specifically as sulfate-limitation.
Unsurprisingly, S-limited growth leads to increased transcription of proteins encoding for the uptake of sulfur, such as in S. cerevisiae [141][71], E. coli [184][74]. Under S-limitation or S-starvation, several organisms preferentially express proteins having a low sulfur content [10,141,185,186][71][75][76][77]. For example, under S-limitation, S. cerevisiae upregulates by as great as 50-fold the PDC6 transcript expressing a protein with only 6 sulfur-containing amino acids compared to isozymes PDC1 and PDC5 having 17–18 sulfur-containing amino acids [141][71], while K. aerogenes maintains lower protein content in the cell wall under S-limitation compared to other nutrient-limited conditions, and this remaining protein contains a low sulfur content [187][78]. The ABC transporter sulfate-binding proteins of Salmonella typhimurium and E. coli responsible for sulfate uptake in a low S environment themselves contain no sulfur [188][79]. When Pseudomonas putida encounters S-depletion, it replaces proteins having high S content with proteins having lower amounts of cysteine and methionine [86][80]. K. aerogenes also excretes proteins lacking S when grown under S-limited conditions on glucose [161][81]. This phenomenon has been observed in many microbes, including cyanobacteria [186][77]. In transitioning between S-limitation and S-enrichment, cells must synthesize new RNA, whereas under C-limitation, the cells use previously synthesized translation machinery, including inactive ribosomes [156,189][72][82]. In E. coli, S-starvation resulted in a 2.8-fold greater glucose uptake rate than N-starvation, and 40% greater glucose uptake rate than P-starvation, but about 70% less than the glucose uptake rate observed during Mg-starvation [190][83]. In B. subtilis, S-starvation resulted in identical glucose uptake rates as observed during N-, P-starved conditions [190][83]. S-limitation in E. coli led to secretion of pyruvate (yield of 0.33 g/g), succinate (0.11 g/g), and acetate (0.10 g/g) despite the aerobic conditions [190][83]. S-limited chemostat cultures of K. aerogenes also secreted more pyruvate than N-, P- or C-limited growth on glucose [161][81], while S-limited chemostat cultures of K. aerogenes (formerly Aerobacter aerogenes) at high growth rate (0.42 h−1) showed pyruvate and 2-oxoglutarate accumulation [191][84]. These observations may be explained by comparing a typical protein (~3% S content by mass) to the sulfur-containing cofactors CoA-SH (4.2% S), lipoic acid (31.1% S), and thiamine pyrophosphate (7.5% S). Each one of these cofactors is a component of the subsequent pyruvate/2-oxoglutarate dehydrogenase step, and the limitation of these cofactors would likely limit metabolic conversion of pyruvate under S-limited conditions [191][84]. Further evidence identifies lipoic acid as the predominant limiting factor [192][85]. Interestingly, in contrast to growth on glucose which generated pyruvate and acetate, acetate was the only significant product when K. aerogenes was grown on glycerol, mannitol or lactate under S-limited conditions [161][81]. S-limitation appears to favor plasmid stability compared to C-, N- or P-limitation, particularly at high growth rate [193][86]. This observation was attributed to the fact that among these four nutrients, only S is not a constituent of nucleic acids. S-starvation resulted in the greatest mevalonate yield from glucose (0.6 mol/mol) compared to Mg-, P- or N-starvation [194,195][87][88]. S-starvation (referred to as MgSO4 limitation) increased limonene formation by E. coli [196][89]. Similarly, lipid formation elevated when the oleaginous yeast Rhodosporidium toruloides became starved for S [197][90]. The fluxes through the TCA cycle and toward acetate formation were suppressed by S-starvation, and the pentose phosphate pathway appears to be the principal route for NADPH generation [194][87].Consistent with the theme of cells conserving limited resources, Fe-limitation causes cells to reduce their reliance on pathways which contain significant Fe, such as the tricarboxylic acid cycle (aconitase, fumarate hydratase, succinate dehydrogenase) and the proton-pumping components of the electron transport chain [71,231,232][101][102][103]. This realignment of metabolism has physiological consequences. For example, under Fe-limiting aerobic conditions, E. coli generates acetate at a yield of 0.25 g/g at a dilution rate of 0.4 h−1, but accumulates predominantly lactate (yield of 0.60 g/g) at 0.1 h−1 [71][101]. Elevated lactate under the most severe steady-state Fe-limitation provides cells a means to oxidize NADH when the Fe-requiring Nuo complex is curtailed, while an increased glucose uptake rate serves to meet ATP demand [71][101]. Lactate formation is also observed for Staphylococcus aureus exposed to Fe-starvation [233][104]. The reduction in activity of TCA cycle enzymes and accumulation of NADH encourages the formation of ethyl acetate by Kluyveromyces marxianus [234][105] and Candida utilis [235][106]. Because of the restricted capacity of the electron transport chain subject to Fe-limited conditions, cells essentially behave like they are encountering anaerobic conditions. For example, E. coli accumulates acetate and formate under Fe-limited steady-state conditions, attains a 60% greater glycolytic flux, and reduces by a factor of 5 the fraction of glucose entering the TCA cycle [236][107]. More generally, Fe-limitation impacts oxygenation and redox state because numerous electron-carrying enzymes contain Fe-S clusters, and many Fe-containing enzymes or pathways are constrained under Fe-limitation. For example, the nitrogenase enzyme system contains significant Fe [237][108], and Fe-limitation thus severely reduces nitrogen fixation by Azotobacter vinelandii [238][109]. Notably, lactic acid bacteria, which lack cytochrome and show generally high tolerance to peroxide, do not require iron for growth at all [239,240][110][111].