To study the behavior of an E. coli
ethanologen in ACSH, we constructed strain GLBRCE1. GLBRCE1 was derived from the well-studied E. coli
K-12 strain MG1655 (12
) and was similar in design to the E. coli
W ethanologen KO11 (64
). Unlike strain KO11, GLBRCE1 contained multiple sources of the Z. mobilis
PET cassette: a single chromosomal copy in which expression of Z. mobilis pdc
were driven by the pflB
promoters (similar to KO11), and additional copies transcribed from a lac
promoter in the low-copy-number plasmid pJGG2 (29
). GLBRCE1 also contained deletions of the genes encoding lactate dehydrogenase (ldhA
) and acetate kinase (ackA
) (A; also, see Materials and Methods). GLBRCE1 was grown in a hydrolysate derived from ammonia-pretreated corn stover (ACSH; see Materials and Methods) that contained ~60 g glucose/liter (~330 mM), ~30 g xylose/liter (~200 mM), lesser amounts (<35 mM) of arabinose, galactose, mannose, rhamnose, and fucose, and amino acids (from 50 to 700 μM) and other nutrients and minerals (see Tables S1, S2, S4, and S5 in the supplemental material). To understand the metabolic changes that occur when E. coli
K-12 uses ACSH as a carbon and energy source anaerobically (and thus inform the design of strains to ferment ACSH efficiently), we correlated changes in global gene expression with the rates of growth, sugar uptake, and production of end products.
Fig 1 Genotype and growth of GLBRCE1 in ACSH. (A) Engineered genetic alterations in GLBRCE1 and pathways affected by those changes. A red X indicates a deleted gene. The dashed line indicates the reactions catalyzed by the products of the Z. mobilis pdc and (more ...)
GLBRCE1 grew robustly under anaerobic conditions in ACSH (μ = 0.37 h−1), 2.5-fold faster than in minimal salt 1% glucose medium (GMM; μ = 0.17 h−1) (B and C; ). Despite the high concentration of available glucose in the hydrolysate, only ~10% of the glucose was consumed during a relatively short exponential phase (<6 h). An additional ~15% of glucose was consumed during a protracted transition phase (from 6 to 22 h) that followed and during which growth slowed gradually (B). Complete growth arrest occurred upon entry into stationary phase (22 h), but the cells remained metabolically active and continued assimilating the remaining glucose, albeit at lower rates per cell than observed during the exponential and transition phases (B; ). Disruption of the genes for acetate kinase, lactate dehydrogenase, and pyruvate formate lyase were not responsible for growth arrest, because growth of the parental MG1655 strain containing pJGG2 underwent a similar metabolically active growth arrest in ACSH (data not shown). Furthermore, derivatives of GLBRCE1 containing just the vector or lacking any plasmid also stopped growing in ACSH prior to glucose depletion (data not shown). Growth arrest and the metabolically active stationary phase were of particular interest because they direct carbon to ethanol rather than to cell growth. To investigate these states and the multiphasic behavior of E. coli in ACSH generally, we used gene expression profiling coupled with substrate and end product measurements to examine how the physiology of E. coli changed in the different growth phases.
Estimated rates of growth, uptake, and production by GLBRCE1
The global patterns of gene expression of GLBRCE1 in ACSH were dynamic but highly reproducible during the initial 52 h of the experiment (r2 > 0.95), as illustrated by expression heat maps of the individual replicates (). After ~70 h, the replicate patterns diverged, with a dramatic change in expression pattern in replicate 1 being evident at 70 h. This change corresponded to the time of glucose depletion, which occurred earlier in replicate 1 than replicate 2 (~70 h versus ~100 h). Thus, we restricted our analysis of gene expression changes to times before 70 h and used the growth rates and expression heat maps to group samples into exponential, transition, and stationary phases (). We describe key aspects of gene expression in each growth phase below.
Fig 2 Global gene expression patterns of GLBRCE1. The heatmap of global gene expression changes in GLBRCE1 during growth in ACSH, SynH, and GMM reveals patterns used to group samples for analysis (38 columns; 4,240 genes per sample). For ACSH bioreplicates (more ...) Distribution of carbon and energy sources in hydrolysates.
Despite the unusual growth behavior of the strain, glucose was fermented predominantly to ethanol (B), as expected from the strain design. The rate of glucose uptake relative to cell density was maximal during exponential phase (13.6 mmol · unit of OD600−1 · h−1) () and then decreased by a factor of 3.7 in transition phase and by a further factor of 2 in stationary phase (). The expression of the genes encoding enzymes in the glycolytic pathway and glucose transporters remained relatively high through the transition and stationary phases (see Fig. S3A in the supplemental material). The rate of ethanol production was generally correlated with glucose consumption, showing a high rate of production during exponential phase and a reduced rate during the transition and stationary phases even though transcript profiling indicated that expression of the PET cassette genes pdc and adhB remained high throughout (see Fig. S3A). Overall net ethanol yield was 58 to 67% of the theoretical maximum, but this measure underestimated actual ethanol production, because some ethanol evaporated during fermentation. Ethanol evaporation at ~2 mM h−1 was evident after glucose depletion (A); this estimate was verified by detection of similar ethanol evaporation from uninoculated, identically sparged bioreactors containing 5% ethanol (data not shown).
Although glucose was ultimately consumed in its entirety, only a small amount of xylose was consumed over the course of the experiment (≤10%) (B; also, see Tables S4 and S5 in the supplemental material). This result is expected with the GLBRCE1 design, which was not optimized for xylose utilization, and was consistent with the observed low expression of xylose utilization genes (xylE
, and xylAB
) (; also, see Fig S3B in the supplemental material). Low expression of xylose genes likely reflected repression by arabinose-bound AraC (24
), since ACSH contains 33 mM arabinose (see Table S1 in the supplemental material). Further, inducer exclusion by PtsG activated by glucose transport may also limit xylose uptake. Lesser amounts of mannose, fructose, galactose, and arabinose were also present in hydrolysate and represented minor pathways of carbon utilization (see Tables S1 and S5 in the supplemental material). Other potential substrates, such as acetate and glycerol, were not utilized as carbon sources by the ethanologen (see Tables S4 and S5 in the supplemental material).
Genes of interest exhibiting changes in expression in different phases in ACSH
Small amounts of the anaerobic respiratory substrate formate (11 mM) and the electron acceptor nitrate (~270 μM) were also present in ACSH (see Tables S1 and S2 in the supplemental material) but were rapidly consumed in exponential phase; these changes suggested that some anaerobic respiration occurred during initial cell growth. In accordance with the rapid loss of nitrate from the medium and the known nitrate-dependent expression of napFDAGHBC
(encoding the Nap periplasmic nitrate reductase and formate dehydrogenase-N, respectively), expression of these operons decreased 2- to 4-fold as cells entered transition phase (). It is unlikely that this limited anaerobic respiration contributed significantly to total ATP synthesis during the exponential phase, because glucose uptake rates were ~7-fold greater than formate uptake rates (), and there was insufficient nitrate present to respire all of the formate. Rather, we hypothesize that most formate was likely oxidized to CO2
via formate hydrogenlyase, consistent with maximal expression of the genes (fdhF
) encoding these enzymes early in exponential phase (; also, see Table S6 in the supplemental material). Expression of genes encoding trimethylamine N
-oxide reductase [torCAD
; specifically inducible by trimethylamine N
)] was highly upregulated compared to GMM-grown cells throughout all growth phases (; also, see Table S6 in the supplemental material), suggesting that the electron acceptor trimethylamine N
-oxide is also present in ACSH.
In addition to ethanol, the other significant and expected end product of ACSH fermentation by GLBRCE1 was succinate (), which accumulated to 62 to 76 mM (see Tables S4 and S5 in the supplemental material). During anaerobic growth, succinate is produced from oxaloacetate (OAA) by reversal of carbon flux through the tricarboxylic acid (TCA) cycle (A) (37
). However, not all the succinate appeared to derive from glucose, because the aggregate rates of ethanol and succinate production during exponential and stationary phases exceeded what could be theoretically produced based only on the glucose uptake rate (). We investigated this question by testing specific gene deletions and found that, even though deletion of frdA
eliminated succinate production, deletion of ppc
, which encodes the phosphoenolpyruvate (PEP) carboxylase responsible for converting PEP to oxaloacetate (A), eliminated only 50% of succinate production (data not shown). This result confirmed the idea that cells must utilize another succinate precursor in ACSH. Although a possible source would be aspartate or asparagine, which can be deaminated to oxaloacetate or fumarate, neither was present at a high enough concentration to contribute significantly to succinate production (C; also, see Table S1 in the supplemental material). An alternative source of succinate could be malate, which was present at 9 mM in ACSH and consumed (see Table S5 in the supplemental material). Additionally, citrate was a likely succinate precursor, based on the exceptionally high expression of the citrate-inducible citCDEFXG
operon, which encodes citrate lyase (A; ). Although difficult to quantify, citrate was detectable in ACSH (data not shown). We conclude that succinate was derived both from glucose and from alternate carbon sources, like malate and citrate, present in ACSH.
Fig 3 Intracellular ATP concentrations and extracellular amino acid concentrations during anaerobic growth of GLBRCE1 in ACSH. (A) Mean intracellular ATP concentration measured during growth in ACSH in relation to cell density (same as in ). Error bars (more ...)
Fig 4 Expression profiling of strain GLBRCE1 during growth in ACSH, SynH, and GMM. (A) Gene expression levels are plotted as a function of fold change (log2) and P value. Downregulated genes are represented by negative values. Genes with significantly different (more ...)
Genes exhibiting the greatest changes in expression in different growth phases in ACSH
Depletion of amino acids from ACSH during growth.
Free amino acids remain in corn stover after AFEX treatment (47
) and are a logical contributor to cell growth. To determine if amino acids contributed to growth of ethanologenic E. coli
in ACSH, we quantified the levels of the amino acids in the medium over the course of fermentation (B and C). We detected significant concentrations of alanine, aspartate, glutamate, phenylalanine, glycine, histidine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, and tyrosine, but isoleucine, methionine, tryptophan, and cysteine were not detected by the assay we used, possibly due to interfering compounds in ACSH. During growth of the ethanologen, the concentrations of most amino acids decreased. However, the patterns of changes in amino acid concentrations were disparate and fell roughly into four groups. The first group (serine, asparagine, and histidine) was depleted from the medium in the first 3 to 6 h of incubation. A second group (glutamate, threonine, lysine, aspartate, and glutamine) was depleted later in the fermentation, after 10 to 14 h of incubation. The third group (valine, tyrosine, arginine, and leucine) remained detectable into stationary phase. Finally, a fourth group either remained nearly constant (glycine) or increased in concentration (phenylalanine, proline and alanine) over the course of the fermentation, suggesting net synthesis of these amino acids by the ethanologen.
Expression levels of amino acid biosynthetic genes were broadly consistent with the patterns of amino acid depletion from ACSH. For example, expression of serAC
, and aspC
genes increased after approximately 8 to 12 h of growth, approximately the point at which these amino acids became undetectable (). In addition, gltB
, and glnA
, which encode enzymes for glutamate and glutamine biosynthesis, were upregulated when the cognate amino acids became depleted (early in transition phase; 8 to 14 h). Coincident with the upregulation of gltB
, we observed increased expression of glnK
and amtB. glnK
encodes a PII protein that regulates Ntr gene expression when nitrogen is limiting (13
). Upregulation of genes involved in nitrogen utilization likely represented a metabolic switch that occurred when the cells transitioned from use of glutamate and glutamine as a nitrogen source to synthesis of glutamate and glutamine from inorganic nitrogen sources, such as ammonia.
Transcriptional profiling indicated the initial presence of most acids in ACSH, including methionine, tryptophan, and isoleucine that escaped detection by direct assay. However, cysteine appeared to be absent in ACSH, as cysZCANHGWUDIPJ, which encode enzymes for cysteine biosynthesis, were expressed at levels similar to those observed in GMM even during exponential phase in ACSH (; also, see Table S6 in the supplemental material). In contrast, the trp and met operons were initially repressed in ACSH and reached GMM levels only in transition phase (; also, see Table S6); this pattern indicated that tryptophan and methionine were present in ACSH but were rapidly consumed. Expression of the biosynthetic genes for two amino acids diverged from this overall trend. Genes for biosynthesis of alanine (alaA, ilvE, and avtA) and proline (proAB) were expressed at high levels throughout the fermentation (; also, see Table S6), even though extracellular alanine and proline levels were high and increased over time (B).
K-12 encodes catabolic systems for l
-glutamate, glycine, l
-cysteine, and l
). With the exception of aspA
, which encodes aspartate lyase, an enzyme that can degrade aspartate to fumarate, and ansB
, which encodes asparaginase II and is involved in the degradation of asparagine to aspartate, expression of genes associated with amino acid degradation were expressed at low levels during growth in ACSH (; also, see Table S6). Thus, most amino acids present in ACSH were likely used as precursors for protein synthesis, rather than being catabolized for use as carbon sources.
Effect of amino acids on growth of the ethanologen in hydrolysate.
Since the entry of the ethanologen into the transition and stationary phases correlated with the depletion of subsets of amino acids (), we tested whether the slowing of E. coli growth in ACSH resulted from decreases in amino acids. We first tested supplementation of amino acids to three times the concentrations measured in ACSH (see Fig. S1 in the supplemental material), along with methionine (300 μM), isoleucine (700 μM), tryptophan (150 μM), and cysteine (50 μM). Increasing amino acid concentrations delayed entry into stationary phase, increased final cell density, and increased growth rate during transition phase (). We concluded that amino acids significantly impact E. coli growth dynamics in ACSH.
Fig 5 Growth of GLBRCE1 in ACSH, SynH, and GMM in the presence and absence of amino acid supplements. Growth of GLBRCE1 in GMM, SynH, and ACSH was compared to growth in SynH and ACSH containing amino acids at concentrations three times those measured in ACSH (more ...)
We hypothesized two possible ways amino acids could improve growth. First, amino acids could provide osmolytes or osmolyte precursors to mitigate the osmotic stress caused by the concentrated hydrolysate medium. Second, amino acids could reduce the energetic load of amino acid biosynthesis, which could increase the energy available to combat stresses associated with growth in ACSH. To test these hypotheses, we compared gene expression in ACSH to that in synthetic, chemically defined medium.
Comparative transcriptomics suggests physiological roles of amino acids during growth in hydrolysate.
GLBRCE1 grew at a uniform rate in GMM and entered stationary phase precipitously when glucose was exhausted (data not shown), without the apparent transition phase that was evident in ACSH and SynH (). When grown in ACSH or SynH, GLBRCE1 exhibited similar exponential, transition, and stationary phases and reached comparable final cell densities before growth arrest (). In addition, the strain produced similar amounts of ethanol (450 mM, SynH; 370 mM, ACSH), grew at similar exponential rates (0.37 h−1, ACSH; 0.29 h−1, SynH), assimilated glucose similarly (13 mM · unit of OD600 · h−1, SynH; 13.6 mM · unit of OD600 · h−1, ACSH) but exhibited somewhat different patterns of amino acid depletion (; also, see Tables S3 and S4 and Fig. S1 in the supplemental material). During stationary phase, the glucose uptake rate was slightly higher in SynH than in ACSH (2.6 versus 1.9 mM · unit of OD600 · h−1, respectively) (), which led to glucose being consumed more quickly in SynH than in ACSH (compare Tables S3 and S4 in the supplemental material). In addition, more xylose was consumed in SynH (; also, see Tables S3 and S4). Strikingly, proline was rapidly depleted from SynH, in contrast to its accumulation in ACSH (see Fig. S1). Given that proline is a major osmoprotectant in E. coli, this led us to consider how osmotic stress may differ in ACSH and SynH.
Cells growing in concentrated lignocellulose hydrolysates are expected to experience osmotic stress due to the high sugar concentration (~10%). The sugars and other solutes generate osmolality near one mol/kg, which decreases ~20 to 30% during fermentation (initial to final osmolality of 1.44 to 0.97 mol/kg for ACSH and 0.97 to 0.75 mol/kg for SynH). Such high solute levels are known to reduce growth rate, reduce ethanol yield, and inhibit xylose utilization by E. coli
Despite the slightly lower osmotic strength of SynH, osmotic stress responses were stronger in SynH than in ACSH. In SynH, genes typically induced by osmotic stress were upregulated relative to their expression in GMM. These included proVWX
, which encode transporters for the osmoprotectants glycine betaine and proline (9
, which encode enzymes for synthesis of the osmoprotectant trehalose (32
), and betABT
, encoding enzymes for glycine betaine synthesis and transport (~4-, ~3-, ~5-, and ~2-fold upregulation in SynH for proVWX
, and betABT
, respectively [B; ]). To test whether growth in SynH depended on osmoprotectants, we measured final cell ODs for cultures incubated in SynH lacking amino acids and supplemented with different amino acids (). Without supplementation, GLBRCE1 did not grow, but proline, glycine betaine, or high concentrations of some amino acid combinations could restore growth (; also, see Fig. S2 in the supplemental material). We conclude that alleviation of osmotic stress is a crucial contribution of amino acids to growth of GLBRCE1 in SynH.
Fig 6 Effect of amino acids on growth of GLBRCE1 in SynH (black) or ACSH (gray). The final OD600 of cells grown anaerobically in SynH with or without amino acids (AA) or glycine betaine was measured, and the values from replicate cultures are plotted relative (more ...)
However, gene expression patterns and supplementation revealed a somewhat different picture in ACSH. During exponential phase in ACSH, proVWX, proP, otsAB, and betABT exhibited less induction or even repression relative to GMM (1.3-, −1.5-, 2.1-, and 2-fold, respectively [B; ]). Lower expression of proVWX and proP was consistent with the lack of proline depletion from ACSH (C). As the cells entered stationary phase in ACSH, expression of proP and betAB increased, suggestive of elevated osmotic stress. Overall, despite the high osmolality of ACSH, the ethanologen exhibited only modest expression of genes associated with osmotic stress.
One explanation for the initially modest osmotic stress response in ACSH could be that components of ACSH not present in SynH provide alternative means for cells to mitigate the osmotic stress. Indeed, our NMR analysis of ACSH revealed that glycine betaine is present at 0.7 mM, a concentration previously shown to mitigate osmotic stress (83
). Furthermore, carnitine (0.2 mM), a known osmoprotectant transported by the ProP transporter, and choline (0.7 mM), which can be converted to glycine betaine under some conditions (77
), were also detected (see Tables S1 and S5 in the supplemental material). We conclude that ACSH contains compounds that can be exploited to mitigate the osmotic stress of the ethanologen.
Acetate and acetamide stress.
Plant cell walls contain many acetyl groups on sugars, which are released as acetamide by AFEX pretreatment and may be converted to acetate during hydrolysate preparation. NMR analysis of ACSH revealed 75 mM acetamide and 33 mM acetate, which increased to 41 mM acetate with concomitant decrease in acetamide by the end of the fermentation (see Table S5 in the supplemental material). Although the effect of acetamide on cell physiology is not well characterized, acetate at concentrations greater than 8 mM is reported to reduce the growth rate of E. coli
and to be potentiated by increased osmolality (5
). Our analysis of cells grown in ACSH versus SynH identified altered expression for approximately one-third of the genes previously linked to acetate stress (5
), including ACSH-dependent upregulation of the hdeA
, and slp
genes (). The limited overlap of our data set of regulated genes with previous studies may result from differences in growth conditions, in that the previous studies were carried out under aerobic conditions and with a nonethanologenic strain of E. coli
. However, the similarities in growth rate between cells grown in ACSH and SynH indicate that the acetate and acetamide in ACSH did not greatly impair cell growth.
Growth of the ethanologen in ACSH or SynH yielded 15 to 22 g ethanol/liter (330 to 480 mM). Previous results showed that exogenously supplied ethanol at more than 20 g/liter decreases the growth rate of E. coli
; Keating et al., submitted). Further, ethanol sensitivity can directly impact ethanol yield during fermentation of E. coli
). To determine if the ethanologen experienced ethanol stress during growth in ACSH, we compared gene expression patterns in exponential-, transition-, and stationary-phase ethanologen cells versus cells of the nonethanologenic strain MG1655 (C). We reasoned that changes in gene expression as ethanol accumulated in transition and stationary phase could identify ethanol stress responses. Indeed, we found that several genes previously associated with ethanol stress were upregulated in the ethanologen (16
; Keating et al., submitted). Specifically, expression of pspABCDE
, encoding phage shock proteins, and ibpAB
, encoding heat shock proteins, increased over the course of the fermentation in both ACSH and SynH media, with sorbitol utilization genes increasing in ACSH as well (C; ). Although differences observed in the osmotic stress response between the two media may account for the differences in the sorbitol response, the similarity of expression in SynH and ACSH of pspABCDE
suggests a common cause, the most obvious of which is ethanol stress. Thus, our results support the idea that ethanol stress contributes to growth inhibition in ACSH.
Hydrolysates prepared from lignocellulose have previously been reported to contain compounds (collectively referred to as lignotoxins) that affect ethanol productivity, overall growth rate, as well as glucose and xylose consumption (48
). These compounds include aromatic carboxylates, like ferulate, coumarate, and salicylate, and aldehydes, like furfural and vanillin. Comparison of gene expression in cells grown in ACSH versus SynH revealed upregulation of diverse stress responses predicted to be associated with lignotoxins, including upregulation of genes associated with small molecule efflux and detoxification of metals and other toxic compounds (D).
Efflux pumps were a prominent class of upregulated genes in comparisons of cells grown in ACSH and SynH. Examples of these pathways include the aaeAB
genes, which encode an efflux system associated with tolerance to aromatic hydroxylates, such as p
-hydroxybenzoate and cinnamic acid (85
), and the emrAB
genes, which encode an efflux system reported to confer resistance to hydrophobic aromatic compounds such as carbonyl cyanide, m
-chlorophenylhydrazone, tetrachlorosalicylanilide and nalidixic acid (53
). Some multidrug resistance (MDR) efflux pumps also were upregulated in ACSH, many of which have been reported to be involved in resistance to hydrophobic compounds and detergents. Upregulated MDR genes include acrB
, and mdtABCGIJM
. The acrB
gene products combine with AcrA and TolC to form MDR systems involved in efflux of aminoglycoside antibiotics and polymyxin B, steroid acids such as bile salts, and sodium dodecyl sulfate (SDS), whereas the mdtABCGIJM
gene products are involved in resistance to bile salts, aminocoumarins and other aromatic antibiotics, and SDS (41
Interestingly, expression of the acrAB
genes is regulated by marA
gene, which encodes a global regulator that affects tolerance to antibiotics, organic solvents, and oxidative stress agents (3
). Expression of the marAB
genes is controlled by the MarR repressor, whose function is antagonized by compounds like salicylate, cinnamate, and ferulate (21
; Tang et al., unpublished) found in plant cell wall hydrolysates like ACSH. Thus, the MarABR regulatory system appears to be a key point of control for genes associated with lignotoxin stress and a possible point of engineering for generating stress-tolerant organisms.
Finally, stress responses associated with heavy metals and other toxic compounds were found to be upregulated during growth of the ethanologen in ACSH. Examples of these systems include the cusCFBA
genes, which encode a permease involved in export of copper and silver ions (27
), and arsRBC
, which have been reported to be involved in resistance to arsenite, arsenate, and antimonite (17
). Although we were unable to determine the concentration of arsenite or related compounds, the concentrations of copper and other heavy metals in hydrolysate were significantly below what has been reported to cause growth inhibition (see Table S2 in the supplemental material). However, these previous experiments were not carried out in concentrated complex media like hydrolysate or anaerobically, and it seems likely that the toxicity of these compounds could synergize with other ACSH-induced stresses to affect cell physiology.