Screening of xylonic acid utilizing microorganisms
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.
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 selected for further investigation. The 16S rRNA gene of this strain was sequenced and has been submitted to GenBank with the accession number of MG779638. The dendrogram of strain 1 and some related strains are shown in Supplementary Figure 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 identification
1H and 13C NMR spectral data of the presumed glycolic acid sample compared to the spectra of a standard glycolic acid (Sodium salt commercial product) are given in Supplementary Figure 2 A & B. 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.
HPLC chromatograms and GC chromatograms of ethylene glycol are given in Supplementary Figure 2 C & D. The retention times of standard ethylene glycol and sample were both 12.2 min for HPLC and both 8.2 min for GC analysis. These results confirmed that ethylene glycol was the presumed metabolite.
Carbon sources utilization ability of E. cloacae S1
To determine the range of carbon sources that can be utilised by E. cloacae S1 the strain was cultured in flasks with M9 medium using either xylonic acid, xylose, glucose, gluconic acid, 2-ketogluconic acid or glycerol as the sole carbon source and the metabolites detected are listed in Supplementary Table1. 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.
Gene recombination method development
Red recombinase assisted gene replacement of E. cloacae was developed as shown in the Method section based on the method we developed in K. pneumoniae . pIJ790 is a plasmid that contains the red recombinase genes and used in E. coli for gene recombination . However, this plasmid could not be used directly for gene recombination in E. cloacae. pSARI is a low copy number plasmid containing a temperature-inducible promoter and kanamycin resistance gene. pSARI can be transferred into E. cloacae and was used for red recombinase meditated gene manipulations. 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 forglycolaldehyde synthesis from xylonic acid
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 . yjhG, yagF, yjhH and yagE 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 (93% identities) and yjhH (93% identities) were found. The two genes were located nearby in the yjh operon (Figure 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 Figure 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. 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.
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 four strains were determined, and the results are presented in Figure 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 Figure 2). 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 Figure 2). 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 fromglycolaldehyde
YqhD, a NADPH-dependent aldehyde reductase, was shown to catalyze the conversion of glycolaldehyde to ethylene glycol in E. coli . Homologous gene of yqhD was amplified from E. cloacae S1. yqhD of E. cloacae S1 was 81% identical to that of E. coli W3110 suggesting that it also uses NADPH as cofactor. The ethylene glycol dehydrogenase activity of purified 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±0.003 and 0.13±0.005 U/mgP, respectively. Whereas the activity of purified YqhD was 0.004±0.0005 and 0.175±0.003 U/mgP of that using NADH or NADPH as the cofactor respectively. These results confirmed 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 an 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 Figure 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 fromglycolaldehyde
aldA encoding an aldehyde dehydrogenase that is responsible for glycolic acid synthesis from glycolaldehyde in E. coli . 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 gene products were obtained from the lysate of these strains and analyzed for their glycolaldehyde dehydrogenase activities. The cell lysate of E. cloacae S1 was used as a control for the glycolaldehyde dehydrogenase activity assay. The results are shown in Supplementary Table2.
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. Among 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 Figure 5.
The cell growth of these three strains was similar. Glycolic acid and ethylene glycol synthesized by E. cloacae ΔbetB were 1.7 g/L and 6.2 g/L respectively, which were slightly decreased compared with that of the wild-type strain, the latter synthesized 2.1g/L of glycolic acid and 7.2 g/L of ethylene glycol. However, glycolic acid and ethylene glycol synthesized by E. cloacae+betB were 1.5 g/L and 4.6 g/L, thus slightly decreased compared with levels of 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 controlled 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 Figure 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, and culture pH was kept constant at pH 6.5. Fermentation results of E. cloacae S1 at different agitation rates are presented in Figure 7.
Cells growth showed a positive correlation with agitation rate with cells grown at 600 rpm and 800 rpm gave the highest cell densities (OD 19.9 and 20.4 respectively), 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 (3.8 g/Lh), 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 agitation rate 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 Figure 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 Figure 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 of 1.2 g/Lh 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 cultivation. 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.