Background

With the global climate change caused by burning fossil fuels and the growing demand for energy [1, 2], sustainable bioenergy has drawn great attentions [3]. Currently, bioethanol, an environmental-friendly renewable liquid biofuel, has been intensively studied as one of the most promising alternatives to fossil fuels [4, 5]. However, bioethanol has been produced primarily from food crops with high content of sugar and starch thus far, which would compete with the food supply and could potentially lead to a global food crisis.

Lignocellulosic materials, derived mainly from agriculture wastes or forestry residues, are the most abundant, low-cost, and promising feedstocks for bioethanol production [6, 7]. However, these biomass resources are naturally recalcitrant, which require deconstruction processes such as size reduction and pretreatment to breakdown the rigid biomass structure to release fermentable sugars for subsequent microbial fermentation [8, 9]. Among different pretreatment methods, acid pretreatment is a prevailing strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, such as xylose and arabinose, and is usually followed by a subsequent enzymatic hydrolysis step to release hexoses from the cellulose, which can be used as substrates for biochemical production. The acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment and consequently impedes cell growth resulting in reduced ethanol titer and productivity [10, 11].

To minimize the detrimental effect of acidic pH on microbes, acid-pretreated liquor must be neutralized by high-cost processes such as extra chemical addition before microbial fermentation, especially in large industrial scales [12], whereas a natural acidic-pH condition of acid-pretreated liquor provides an opportunity to effectively prevent the potential bacterial contamination and makes the open (non-sterilized) fermentation applicable [13, 14]. It is reported that ethanol production under the non-sterilized condition can save 30 ~ 40% energy consumption and make the process simpler [15]. Hence, it will be ideal to develop more acidic-pH-tolerant strains for ethanol production, which has been developed in species, such as Escherichia coli [16, 17] and yeast [12, 18, 19].

Zymomonas mobilis is a facultative anaerobic and natural ethanologenic bacterium with desirable industrial biocatalyst characteristics, such as a highly specific rate of sugar uptake, high ethanol yield, no oxygen requirement for cell growth and ethanol fermentation, and a relatively low biomass production during fermentation [20, 21]. In addition, Z. mobilis has a generally regarded as safe (GRAS) status [22, 23]. Up until now, many different stress-tolerant strains of Z. mobilis have been constructed with enhanced tolerance to acetate [24, 25], furfural [26, 27], and hydrolysate [28, 29]. However, acidic-pH conditions are still a challenge for Z. mobilis using lignocellulosic feedstock hydrolyzed by acid as the substrate. For example, Z. mobilis NS-7 is an acid-tolerant strain developed by nitrosoguanidine (NTG) mutation and acid medium selection, which can ferment at an acidic pH of 4.5 under non-sterilized condition without being contaminated [15]. Z. mobilis GZNS1 is another mutant strain evolved by culturing at pH 4.0 condition that could produce ethanol from acidic kitchen garbage [14]. An increased acid tolerance was also observed in Z. mobilis recombinant strain carrying Pbp (proton-buffering peptide, Pbp) from E. coli [30].

In addition, some genomic variants relevant to acid tolerance in Z. mobilis have been identified. For example, the acetate-tolerant phenotype in AcR mutant may be due to the over-expression of ZMO0119 encoding Na+/H+ antiporter resulting from a 1.5-kb deletion in AcR mutant [24, 25]. And single nucleotide variants (SNVs) in genes ZMO0056 and ZMO0589, which encode a glutamine-fructose-6-phosphate aminotransferase and a DNA repair protein RadA, respectively, have been characterized to likely contribute to acid tolerance in mutant stains developed by a multi-round atmospheric and room temperature plasma (mARTP) mutagenesis [56].

Moreover, a mutation (A to G) was also found in the intergenic region between ZMO1432 and ZMO1433 in mutant 3.6M (Table 2), which is in the upstream of the promoter region of ZMO1432 predicted by BPROM [57]. As shown in the RNA-Seq results, the expression of the whole operon encoding an RND efflux system consisted of ZMO1432, ZMO1431, ZMO1430 and ZMO1429 was significantly upregulated at acidic pH 3.8 in two mutant strains compared with ZM4, and 3.6M had the highest expression level among these strains (Additional file 1: Table S1, Additional file 3: Table S2). The mutation in the intergenic region in mutant 3.6M could help upregulate the expression of downstream genes, since the expression of these genes was also upregulated under pH 6.2 in 3.6M compared with ZM4 (Additional file 3: Table S2). Combining these mutations and transcriptomic results, the RND efflux pump may play a crucial role in acidic-pH resistance in mutant strains.

The last common mutation shared in both mutant strains was within oxyR gene (ZMO1733). OxyR is a LysR family transcriptional regulator consisting of an N-terminal DNA-binding domain (DBD) and a C-terminal regulatory domain (RD), which controls the OxyR regulon consisting of almost 40 genes that can help protect cells from oxidative stress [58]. The T7K mutation in OxyR was in the N-terminal of LysR-type helix–turn–helix (HTH) DNA-binding domain (PS50931, 6-63 aa), which likely changes the binding affinity of HTH with its target DNA sequence due to the amino acid change from threonine with short side chain to lysine with long side chain (Table 2). Our RNA-Seq results showed that several genes involved in reactive oxygen species (ROS) detoxification possibly regulated by OxyR, such as ZMO0918 (catalase) and ZMO1060 (superoxide dismutase), were significantly upregulated in all strains, especially in ZM4 at pH 3.8 compared to pH 6.2, while ZMO1211 (glutathione reductase) was significantly upregulated at pH 3.8 only in wild-type ZM4 (Additional file 3: Table S2). Since acidic pH could induce a secondary oxidative stress and the acid tolerance response overlaps with the oxidative stress response [59, 73]. F1Fo ATP synthase (F1Fo ATPase) can utilize the proton gradient for ATP synthesis; it can also reverse and hydrolyze ATP to pump H+ out to maintain intracellular pH homeostasis [74, 75]. For example, genes encoding F1Fo ATPase in S. mutans were upregulated at acidic pH to help resist acid stress [76]. Another study indicated that when respiration was impeded, F1Fo ATPase hydrolyzed ATP to pump protons and contributed to the intracellular neutral condition maintaining the essential mitochondrial membrane potential [77]. Our results demonstrated that 7 genes encoding F1Fo ATP synthase (ZMO0239, ZMO0240, ZMO0241, ZMO0667, ZMO0668, ZMO0669, ZMO0671) and another gene encoding F1Fo ATP synthase assembly protein (ZMO2005) were significantly upregulated at pH 3.8 compared to pH 6.2 for the mutant strain 3.6M (Fig. 5f; Additional file 3: Table S2). Since the cellular respiration process was uncoupled with cell growth in Z. mobilis [78], and the ATP generation was majorly from glycolysis whose activity was increased as discussed above, the upregulation of F1Fo ATPase genes may possibly help pump H+ out from the cytoplasm through consuming ATP.

Furthermore, proton translocation was suggested to result in an alkalization of the intracellular medium in Z. mobilis at pH 6.5 during the respiration by transferring the H+ out of cytoplasm [79]. Two genes related to the respiration chain for transferring electrons to oxygen, ZMO0012 and ZMO0568, were downregulated significantly; and six other genes, ZMO0956–ZMO0958, ZMO0961, ZMO1253 and ZMO1255, were reduced more than 1.5 times in ZM4 at pH 3.8 compared with pH 6.2 (Fig. 5f; Additional file 3: Table S2). In addition, six genes encoding Rnf complex (ZMO1809–ZMO1814) and an assembly gene (ZMO1808) were also downregulated at pH 3.8 compared with pH 6.2 in ZM4 but not in mutant strains (Fig. 5f; Additional file 3: Table S2). The Rnf complex is required for the electron transfer to nitrogenase during nitrogen fixation with proton excretion in Rhodobacter capsulatus [80]. Furthermore, the gene ZMO0456 encoding the ferredoxin, which is the electron acceptor from NADH and electron donor for nitrogenase, was also downregulated at acidic pH 3.8 compared with neutral pH 6.2 in ZM4 (Fig. 5f; Additional file 3: Table S2). The downregulation of genes associated with the electron transfer chain at the acidic-pH condition in wild-type ZM4 could make the excretion of protons against proton gradient from cytoplasm difficult, leading to growth inhibition. In contrast, the expression of these genes in the mutant background was not significantly downregulated at acidic pH 3.8 compared with neutral pH 6.2. Instead, they were upregulated compared with ZM4 at pH 3.8 (Fig. 5f; Additional file 3: Table S2). These results indicated that mutants could maintain relatively high proton transportation capacity against acidic-pH conditions.

Proton consumption and alkaline compound production for enhanced acidic-pH resistance

Biosynthesis of branched-chain amino acids (BCAAs) was reported to reduce H+ concentration in the cytoplasm by consuming proton or producing ammonia [64]. Two genes involved in the conversion of isoleucine from threonine in Z. mobilis (ZMO0687 and ZMO0115) were significantly upregulated in mutants 3.5M and 3.6M compared with ZM4 at pH 3.8 (Fig. 5G; Additional file 3: Table S2).

In addition, gene ZMO0296 encoding adenosine deaminase (Ada) to convert adenosine into inosine with ammonia production was significantly upregulated at pH 3.8 in 3.6M strains compared with ZM4 (Fig. 5g; Additional file 3: Table S2). The expression of ZMO1207 gene encoding nitrilase (Nit, EC 3.5.5.1) that catalyzes the substrate containing cyano group to ammonia was also upregulated at pH 3.8 in mutant strain 3.6M compared with ZM4 (Fig. 5g; Additional file 3: Table S2). At acidic pH conditions, ammonia could react with protons to produce the ammonium ion [81], which indicated that mutant strain 3.6M possessed greater capacity than mutant strain 3.5M and ZM4 to neutralize the intracellular pH by proton-consuming and alkali-producing reactions resulting in enhanced acidic-pH resistance.

However, the cytoplasmic pH homeostasis is connected with the proton motive force (PMF), which consists of two components of a transmembrane pH gradient (ΔpH) and a transmembrane electrical potential (Δψ) maintaining intercellular negative relative to outside [81]. The production of NH4+ from NH3 and proton thus will result in excess intracellular positive charges while reducing the ΔpH, which could destroy the PMF and impair cellular functions. To balance the excess intracellular positive charges, exporting NH3 and NH4+ by an ammonium transporter would avoid excessive positive charges hyperpolarizing the cell membrane [81]. Our RNA-Seq results showed that the transcriptional level of ammonium transporter encoded by ZMO0346 was upregulated significantly in both mutant strains compared with ZM4 (Fig. 5g; Additional file 3: Table S2), which may help transport NH3 and NH4+ outside the cell and ensure normal PMF function on the membrane. Moreover, it was reported that the conversion of CO2 to HCO3− by carbonate anhydrase (CA) also contributed to acid–base equilibrium in H. pylori [21, 81]. It is interesting that the transcriptional level of ZMO1133 encoding carbonate anhydrase was significantly upregulated at pH 3.8 compared to that at pH 6.2 in all strains (Fig. 5g; Additional file 3: Table S2). Since Z. mobilis can consume sugars and produce CO2 efficiently [82], CO2/HCO3− could also be involved in kee** acid–base equilibrium at acidic-pH conditions.

Reduced energy consumption on macromolecular repair for enhanced acidic pH tolerance of mutant strains

Cell membrane, proteins, and DNA would be damaged when bacteria are cultured in acidic environments. To reduce the damage, the expression of repair and defense proteins such as DnaK, RecA, UvrA, IrrE, and AP endonuclease could be increased to protect the macromolecules from the damage [64, 75, 83]. Our results showed that the transcription level of ZMO0660 (dnaK) together with its co-chaperone ZMO1690 (dnaJ) as well as ZMO1588 (uvrA) and its subunit ZMO0362 (uvrB) were upregulated in ZM4 at acidic pH 3.8 than at neutral pH 6.2. Moreover, the expression level of Clp protease complex, ZMO0405 (clpA), ZMO0948 (clpP), ZMO0949 (clpX) and ZMO1424 (clpB) involved in protein remodeling and reactivation [64, 84], altered similarly as ZMO0660 (Fig. 5h; Additional file 3: Table S2). These results demonstrated that it is necessary to enhance the expression of these proteins in order to protect DNA and protein from damage in acidic cytoplasm.

However, the expression level of these genes was down-regulated at pH 3.8 in mutants compared to ZM4, except for gene recA, which had no significant changes at different pH conditions in any strains. In addition, the transcriptional level of ZMO1929, which encodes GroEL protein and is important during adaptation to acid [64], was downregulated at pH 3.8 in mutant strains compared to ZM4 (Fig. 5h; Additional file 3: Table S2). The deficient in HtrA, a surface protease involved in the degradation of aberrant proteins, reduced the ability of the mutant strain to endure acidic conditions [85], which demonstrated that this protein is important for cells to defend acid conditions.

The phenomenon that the expression of macromolecular repair genes that are indispensable for acid resistance was upregulated at acidic pH only in wild-type ZM4 background indicated that a great demand on these proteins is needed for ZM4 to survive at acidic-pH conditions, while the downregulation of these genes in mutant backgrounds compared with ZM4 suggested that acidic-pH-tolerant mutants may acquire the capability to manage defense responses without triggering abrupt augmented macromolecular repair activities and thus conserve energy for cell growth instead.

Genetic confirmation of genes associated with acidic-pH resistance in Z. mobilis ZM4

To evaluate the impact of candidate genes associated with acidic-pH resistance identified through our genomic and transcriptomic studies as discussed above, six plasmids containing candidate operons were constructed based on the shuttle vector pEZ15Asp with Ptet as the promoter [93]. Candidate strains containing correct plasmid construct were identified by colony PCR, and confirmed by Sanger sequencing (Tsingke, China). Cell growth of these strains was evaluated at different pHs (3.6, 4.0, 6.0) in RMG5 medium using Bioscreen C. Tetracycline was added at concentrations of 0.4 μg/mL and 0.8 μg/mL to induce genes expression.