Screening of xylonic acid utilizing microorganisms and strain identification
Xylonic acid utilizing microorganisms were enriched from soil samples and 4 colonies with different morphologies were isolated from LB agar plates and cultured in flasks. E. coli W3110 was also cultured at the same time as a control. Fermentation results of these strains are shown in Table 1.
Table 1
Metabolites produced by xylonic acid utilizing microorganisms tested.
Strains | Residual xylonic acid (g/L) | Metabolites (g/L) |
Ethylene glycol | Glycolic acid | Acetic acid |
1 | 0 | 11.1 | 3.1 | 0 |
2 | 27.5 | 0 | 0 | 0 |
3 | 28.0 | 0 | 0 | 0 |
4 | 37.9 | 0 | 0 | 0 |
W3110 | 10.1 | 3.3 | 1.9 | 2.5 |
Xylonic acid was consumed by isolated strains (1–3) and E. coli W3110, but not by strain 4. Of the xylonic acid utilizing strains, no known metabolites were detected in the broth of strains 2 and 3. For strain 1 and E. coli W3110, ethylene glycol (assumed) and glycolic acid (assumed) were the major metabolites. The identification of ethylene glycol and glycolic acid are shown in the following section. Acetic acid was found as a metabolite of E. coli W3110, but not for any of the other strains.
Strain 1 has a higher ethylene glycol and glycolic acid productivity and yields than E. coli W3110. This strain was identified by the 16S rRNA gene. The sequence has been submitted to GenBank with the accession number of MG779638. This gene sequence was blasted in the NCBI, and a dendrogram was then composed to elucidate evolutionary relationships between strain 1 and some related strains (see the Supplementary Fig. 1). Based on 16S rRNA gene sequence and the dendrogram, strain 1 was tentatively identified as E. cloacae, and named E. cloacae S1. The genome of this strain was subsequently sequenced and the raw sequence has been submitted to GenBank with the accession numbers of VSZU00000000. This strain was used for further investigation.
Ethylene glycol and glycolic acid were the major metabolites of E. cloacae S1 using xylonic acid as the carbon source. The strain was also grown using xylose, glucose, gluconic acid, 2-ketogluconic acid and glycerol as the sole carbon source and the metabolites detected are listed in Supplementary Table 2.
Ethylene glycol and glycolic acid were the main metabolites of E. cloacae S1 using xylonic acid as the sole carbon source. However, the two chemicals were not synthesized by this strain using any of the other carbon sources tested. 2,3-Butanediol and acetic acid were the major metabolites using xylose and 2-ketogluconic acid as the sole carbon sources, respectively. Acetoin and 2,3-butanediol were the major metabolites using glycerol as the sole carbon. When using glucose or gluconic acid as the sole carbon source, acetic acid, acetoin, and 2,3-butanediol were all synthesized by this strain.
Ethylene glycol and glycolic acid identification
1H and 13C NMR spectral data of glycolic acid sample compared to the spectra of a standard glycolic acid (Sodium salt commercial product) are given in Supplementary Fig. 2.
1H NMR chemical shift for CH2 of glycolic acid was 3.83 and 4.00 ppm for sample and standard, respectively. 13C NMR chemical shifts of glycolic acid were 179.52 (C1), 60.95 (C2) ppm for the sample, and 176.14 (C1), 59.12 (C2) ppm for the standard. The NMR data of the sample correlated well with the standard glycolic acid data. From this comparison, it was concluded that the compound was glycolic acid.
Retention times of standard ethylene glycol and sample were all 12.2 min for HPLC and all 8.2 min for GC analysis. These results confirmed that ethylene glycol was the presumed metabolite.
Gene recombination method development
Gene recombination using linear DNA with 39 and 40 nt homologous extensions that was directly amplified from plasmid pIJ778 was tried first. However, no colonies were obtained on selection plates. So linear DNA with 500 bp of homologous regions was used for gene recombination in E. cloacae. Commonly, 100 colonies were obtained in a single recombination experiment using this method.
Identification of genes responsible for glycolaldehyde synthesis from xylonic acid
Glycolaldehyde is an intermediate of the Dahms pathway. Ethylene glycol, and glycolic acid are all synthesized directly from glycolaldehyde. There are two D-xylonic acid dehydratases (YjhG, YagF) catalyzing the conversion of xylonic acid to 2-dehydro-3-deoxy-D-pentonate, and two 2-dehydro-3-deoxy-D-pentonate aldolases (YjhH, YagE) that catalyze the conversion of 2-dehydro-3-deoxy-D-pentonate to glycolaldehyde in E. coli [2]. yjhG, yagF, yjhH and yagE gene sequences of E. coli were blasted against the NCBI database and the genome of E. cloacae S1 to find the homologous genes of E. cloacae. However, only homologues of yjhG and yjhH were found. The two genes were located nearby in the yjh operon (Fig. 1, A). Beside, this operon contains genes of yhcH, yagG, xyl and iclR, which encoding a beta subunit of beta-galactosidase, a sugar transporter, a beta-D-xylosidase, and a regulatory gene, respectively. yjhG and yjhH were knocked out individually to generate mutant strains of E. cloacae ΔyjhG and E. cloacae ΔyjhH, respectively.
Physiological characteristics of these strains were determined by culturing them in M9 medium with xylonic acid or xylose as the sole carbon source, and results are presented in Fig. 2.
Growing with xylose as the sole carbon source 2.2, 1.2 and 1.5 g/L of 2,3-butanediol and 1.1, 2.3 and 1.8 g/L acetic acid were synthesized after 24 h culture by E. cloacae S1, E. cloacae ΔyjhG and E. cloacae ΔyjhH, respectively. There was not any distinct differences between these strains for xylose utilization. Thus it appears that YjhG and YjhH are not directly involved in xylose metabolism.
Using xylonic acid as the sole carbon source, 2.1 g/L ethylene glycol and 0.7 g/L glycolic acid were synthesized by E. cloacae S1. However, E. cloacae ΔyjhG and E. cloacae ΔyjhH were unable to grow in this medium, and no metabolites were synthesized. These results indicated that yjhG and yjhH encoding D-xylonic acid dehydratase and 2-dehydro-3-deoxy-D-pentonate aldolase respectively were responsible for glycolaldehyde synthesis from xylonic acid, and these two enzymes seem to have no isoenzymes in this strain.
The roles of other genes in the yjh operon on xylose and xylonic acid catabolism
As yjhG and yjhH are responsible for xylonic acid catabolism it was suspected that other genes in the same operon might also be related to xylose or xylonic acid catabolism. iclR, yhcH, yagG, and xyL were disrupted individually to obtain strains E. cloacae ΔiclR, E. cloacae ΔyhcH, E. cloacae ΔyagG and E. cloacae ΔxyL, respectively. Physiological characteristics of these 4 strains were determined, and the results are presented in Fig. 3.
Xylose was used by E. cloacae ΔiclR, E. cloacae ΔyhcH, E. cloacae ΔyagG, and E. cloacae ΔxyL, and 2.2–2.3 g/L of 2,3-butanediol were produced by these strains. The cell growth and 2,3-butanediol synthesis were comparable to that of E. cloacae S1 (shown in Fig. 3). Xylonic acid was used by all these strains, and 0.3–0.5 g/L of glycolic acid and 1.9–2.4 g/L of ethylene glycol were synthesized by these strains. Also, these titers were similar to that of E. cloacae S1 (shown in Fig. 3). On the whole, the fermentation results showed that there were no distinct differences between the wild strain and these mutants regarding xylose and xylonic acid utilization.
Identification of genes responsible for ethylene glycol synthesis from glycolaldehyde
YqhD, a NADPH-dependent aldehyde reductase, was shown to catalyze the conversion of glycolaldehyde to ethylene glycol in E. coli [2]. E. cloacae contains a homologous gene called yqhD, and this gene was amplified from E. cloacae S1. yqhD was cloned into a pet 28a plasmid and over-expressed in E. coli. YqhD was obtained from the cell lysate. The ethylene glycol dehydrogenase activity of YqhD and the cell lysate of E. cloacae S1 were assayed using either NADH or NADPH as cofactor.
Ethylene glycol dehydrogenase activities of cell lysate of E. cloacae S1 using NADH or NADPH as cofactor were 0.006 and 0.13 U/mgP, respectively. Whereas the activity of purified YqhD was 0.004 and 0.175 U/mgP that using NADH or NADPH as the cofactor respectively. These results indicated that the ethylene glycol dehydrogenase in E. cloacae S1 uses NADPH as the cofactor, and YqhD of E. cloacae S1 is an ethylene glycol dehydrogenase.
To further investigate the in vivo function of yqhD in ethylene glycol formation, yqhD was knocked out and also a YqhD over-expressing strain was constructed. E. cloacae S1, E. cloacae ΔyqhD and E. cloacae + yqhD were cultured in flasks for ethylene glycol production. Fermentation medium was used, and the results are presented in Fig. 4.
Xylonic acid was exhausted by E. cloacae S1 after 18 h of culture, and 8.3 g/L ethylene glycol and 2.1 g/L of glycolic acid were produced. Xylonic acid utilization by E. cloacae ΔyqhD was much slower, however, ethylene glycol synthesis ability was not totally lost; the strain still produced 1.6 g/L of ethylene glycol. Similar to ethylene glycol, glycolic acid synthesized by E. cloacae ΔyqhD was decreased to 0.1 g/L. The final levels of ethylene glycol and glycolic acid produced by E. cloacae + yqhD were only slightly lower compared to that of the wild-type strain. These results indicate YqhD is responsible for the conversion of glycolaldehyde to ethylene glycol in vivo. However, other ethylene glycol dehydrogenase isoenzymes exist in the cell that could explain the small quantities of ethylene glycol synthesized by the deletion mutant.
Identification of genes responsible for glycolic acid synthesis from glycolaldehyde
aldA was identified as coding for an aldehyde dehydrogenase for glycolic acid synthesis from glycolaldehyde in E. coli [2]. However, no homologous genes of aldA were found in the genome of E. cloacae S1. aldB, betB, ad1, and ad2 that are presumed to be aldehyde dehydrogenases or putative aldehyde dehydrogenases in the genome of E. cloacae were cloned and over-expressed in E. coli to obtain E. coli BL21/aldB, E. coli BL21/betB, E. coli BL21/ad1, and E. coli BL21/ad2. Purified enzymes of these genes were obtained from the lysate of these strains and analyzed for their glycolaldehyde dehydrogenase activities. The results are shown in Supplementary Table 3. The cell lysate of E. cloacae S1 was used as a control for the glycolaldehyde dehydrogenase activity assay.
Glycolaldehyde dehydrogenase activity of cell lysate of E. cloacae S1 using NAD as cofactor was 0.0021 U/mgP. While no activity was measured using NADP as the cofactor. For the purified enzymes, only BetB showed a distinct glycolaldehyde dehydrogenase activity of 0.21 U/mgP when using NAD as the cofactor. All other enzymes exhibited a very low level of glycolaldehyde dehydrogenase activity using NAD as the cofactor. When using NADP as the cofactor, all these selected enzymes showed a very low level of activity. These results indicate that BetB might be responsible for glycolic acid formation from glycolaldehyde.
To further investigate the role of BetB in the glycolic acid formation from glycolaldehyde, a gene knock- out strain E. cloacae ΔbetB and an over-expression strain E. cloacae + betB were constructed. These strains were cultured in flasks for ethylene glycol production, and fermentation results are shown in Fig. 5.
The cell growth of these three strains was similar. Glycolic acid and ethylene glycol synthesis by E. cloacae ΔbetB were slightly decreased compared with that of the wild-type strain. However, glycolic acid and ethylene glycol synthesized by E. cloacae + betB was a bit decreased compared with wild type strain and E. cloacae ΔbetB.
Culture parameters optimization
E. cloacae S1 was batch cultured in 5L stirred tank bioreactors for ethylene glycol and glycolic acid production. The culture pH was kept stable at 6.0, 6.5 7.0 and 7.5, respectively. Agitation rate was maintained at 500 rpm, and cell growth and metabolites produced are presented in Fig. 6.
After 6 hours of lag phase, cells started to grow and reached the exponential phase after about 10–12 hours. Xylonic acid was not used by cells until cell density reached about OD 7. Cells could grow in the whole experimental culture pH range with cells at pH 6.5 had the fastest growth rate, whereas cells grown at pH 7.5 had the lowest growth rate. The effect of culture pH on cell growth, xylonic acid consumption, ethylene glycol, and glycolic acid production showed a similar trend with the pH 6.5 culture showing fastest utilization of xylonic acid in parallel with the fastest production of ethylene glycol and glycolic acid. Thus pH 6.5 was selected as the optimal culture pH.
Oxygen supplementation is a key parameter for cell growth and product synthesis. The agitation rate was set at 200, 400, 600 and 800 rpm to give micro-aerobic condition at the lowest rate to fully aerobic conditions at the highest rate. Fermentation results of E. cloacae S1 at different agitation rates are presented in Fig. 7, culture pH was kept constant at pH 6.5.
Cells growth showed a positive correlation with agitation rate with cells grown at 600 rpm and 800 rpm gave the highest cell densities, and those at 200 rpm had the lowest cell density (OD 8.0 ). The trend of xylonic acid consumption was similar to that of cell growth, with cells grown at 600 rpm gave the fastest xylonic acid consumption rate, and those grown 200 rpm had the lowest xylonic acid consumption rate (0.9 g/lh). Ethylene glycol and glycolic acid production were positively correlated to agitation rate from 200 to 600 rpm. However, the product synthesis was strictly inhibited at the condition of 800 rpm agitation. Thus, medium level of oxygen supply appears to favor both ethylene glycol and glycolic acid synthesis, and therefore 600 rpm was selected as the optimal agitation condition.
3.9 Ethylene glycol production in fed-batch fermentation
E. cloacae S1 was cultured in a 5L stirred tank bioreactor, and xylonic acid was fed in the process using bolus additions. Fermentation results are presented in Fig. 8.
Similar to the batch fermentations, xylonic acid was quickly consumed after cells reached the exponential phase. After 15 h of batch culture, xylonic acid was fed for the first time, and 8 bolus additions of xylonic acid were made in total as shown in Fig. 8B. The highest cell density of 16.4 (OD) was reached at 21 h; after that cell density started to decrease. Ethylene glycol had a high production rate from about 10 h to 30 h, and then the productivity decreased. The trend of glycolic acid synthesis was similar to that of ethylene glycol production. In total, 34.1 g/L ethylene glycol and 13.2 g/L glycolic acid were produced after 46 h of culture. The molecular conversion ratio calculated was 0.217 mol/mol for glycolic acid and 0.772 mol/mol for ethylene glycol, and the total conversion ratio reached 0.989 mol/mol xylonic acid.