In-vivo biosynthesis of homogentisate in recombinant E. coli

3.1.1. Overexpression of p-hydroxyphenylpyruvate dioxygenase (Hpd) from Pseudomonas putida in E. coli DH5/ pCAS2JF

p-Hydroxyphenylpyruvate dioxygenase (Hpd) (E.C. 1.13.11.27) converts p-Hydroxy-phenylpyruvate (p-HPP) via oxidative decarboxylation reaction into Homogentisic acid (HGA). It incorporates both atoms of molecular oxygen into a single substrate, one in the 2-hydroxyl and one in the carboxylate group as shown in figure 3.1. HGA is the aromatic precursor in the production of tocochromanol compounds.

Figure 3.1: Reaction scheme showing HGA production. Hpd catalyses the conversion of p-HPP into HGA in presence of molecular dioxygen releasing CO2 as by-product.

Wild type E. coli can produce p-HPP via the Shikimate pathway (see figure 1.8 in Chapter 1).

Hence, the first step in order to produce HGA in recombinant E. coli is, to clone hpd gene in an expression vector and study the overexpression in E. coli. Pseudomonas putida KT2440 genome sequence was published in 2002 (Nelson et. al. 2002). hpd gene from Pseudomonas putida KT2440 had been successfully cloned and expressed in E. coli (Moran 2004). Hpd overexpressed from Pseudomonas putida was also shown to be functionally active, by producing HGA in in-vivo recombinant E. coli (Moran 2004). Plasmid pCAS2JF was obtained after cloning hpd from chromosomal DNA of Pseudomonas putida KT2440 (chapter 2, table 2.2).

To study the overexpression of Hpd in E. coli, E. coli DH5was transformed (chemical transformation) with plasmid pCAS2JF, and as control, it was transformed with an empty

OH OH O

O

OH OH

OH O

O₂ CO₂ Hpd

(p-HPP) (HGA)

OH OH O

O

OH OH

OH O

O₂ CO₂ Hpd

(p-HPP) (HGA)

83

vector pJF119N. Cultivation was carried out in shaking flask at 30°C, in Luria Broth supplemented with 100 µg/ml of ampicillin i.e. LB - Amp100 medium. Cultures were induced with 1 mM IPTG (final conc.) at OD600nm of 0.8. Samples tested on SDS-PAGE can be seen in figure 3.2. After IPTG induction, a strong additional protein band of apparent size of 38 KDa (calculated based on relative mobility (Rf) method) appeared in case of E. coli DH5 / pCAS2JF sample (lane 1), which was absent in control (lane 3). No strong additional protein bands were seen in the same culture sample before IPTG induction, either in E. coli DH5a/pCAS2JF (lane 2) neither in E. coli DH5/pJF119N (lane 4). Hpd protein (358 amino acid residues) from Pseudomonas putida KT2440 has a calculated molecular mass of 40.04 KDa (calculated using ExPASy Proteomic server). The size of the additional protein band (apparent size of 38 kDa) seen on SDS-PAGE (shown by arrow) is slightly smaller than the calculated mass of 40.04 KDa. This may be due to the fact that hydrophobic proteins tend to migrate faster than the hydrophilic proteins (Shirai et. al. 2008). Hpd being relatively hydrophobic (Rüetschi et.al 1992) migrates faster than the protein marker at 39.2 kDa.

Hence, it can be concluded that the overexpressed protein in lane 1 corresponds to Hpd protein based on the molecular mass.

Table 3.3: Calculated properties of recombinant protein Hpd Protein Name

(Source)

Molecular size - M_w [kDa]

Isoelectric point - pI

Hpd

(Pseudomonas putida KT2440)

40.04 5.07

Mw and pI values were calculated using the ExPASy proteomic server.

Figure 3.2: SDS-PAGE (12 %) analysis of Hpd overexpression in E. coli DH5/pCAS2JF.

Shaking flask cultivation in LB-Amp100. The expected Hpd protein has molecular weight of 40.04 kDa. Strong protein band in lane 1 at approx. size of 38 kDa was seen. This band represents Hpd and is shown by arrow. Approx.

20 µg of protein samples were loaded in each lane. The description of samples loaded in each lane are as below,

1 – E. coli DH5α / pCAS2JF - 3 h after 1 mM IPTG 2 - E. coli DH5α / pCAS2JF – before IPTG

3 - E. coli DH5α / pJF119ΔN – 3 h after 1 mM IPTG 4 - E. coli DH5α / pJF119ΔN - before IPTG

M – Protein Marker

1 2 3 4 M

97.4 kDa 66.2 kDa 39.2 kDa

26.6 kDa 21.5 kDa 14.4 kDa 97.4 kDa 66.2 kDa 39.2 kDa

26.6 kDa 21.5 kDa 14.4 kDa

Hpd

1 2 3 4 M

97.4 kDa 66.2 kDa 39.2 kDa

26.6 kDa 21.5 kDa 14.4 kDa 97.4 kDa 66.2 kDa 39.2 kDa

26.6 kDa 21.5 kDa 14.4 kDa

1 2 3 4 M

97.4 kDa 66.2 kDa 39.2 kDa

26.6 kDa 21.5 kDa 14.4 kDa 97.4 kDa 66.2 kDa 39.2 kDa

26.6 kDa 21.5 kDa 14.4 kDa

Hpd

84

3.1.2. HGA production in shaking flask (100 RPM) in E. coli BW25113 lacZ⁺/ pCAS2JF After Hpd protein was overexpressed in E. coli DH5 / pCAS2JF, Hpd activity was further studied. E. coli DH5 / pCAS2JF was cultivated at 30 °C in shaking flask in LB medium supplemented with glycerol (2 % w/v) and 100 µg/ml ampicillin i.e. LB-Glycerol-Amp100. As control strain E. coli DH5/ pJF119N was cultivated. To check the activity of Hpd, cultures were induced with 1 mM IPTG (final conc.) at approx. OD600nm of 0.8. Culture supernatant samples and cell pellets were analysed (section 2.2.4.1) for HGA production using HPLC method tested previously for HGA standard. HGA is water soluble and the experimental solubility can go up to 850 mg/ml (human metabolome database www.hmdb.ca). Culture supernatants were injected to the HPLC (HPLC method see section 2.2.4.1) for HGA analysis. Approximately 2 mM HGA was produced in E. coli DH5/ pCAS2JF and no HGA was detected in the control strain E. coli DH5/pJF119N. Negligible amount of HGA (<

0.01 mM) was found in cell pellet of E. coli DH5/ pCAS2JF and not detected in control (Albermann et. al. 2008). After induction, the E. coli DH5/ pCAS2JF strain produced HGA as the major product in supernatant. After confirming that, E. coli DH5/pCAS2JF was able to produce HGA in shaking flask LB-Glycerol-Amp100, further cultivation was done in the production strain E. coli BW25113 lacZ⁺ as host strain. In the later stage, chromosomal integration of each individual gene was planned to be carried out in E. coli BW25113 lacZ⁺ strain. These chromosomal integrated strains (plasmid-free) would then be used as production strains. In order to compare the results of chromosomal integrated strain with strain carrying plasmid (plasmid-encoded), same host strain was essential. Hence, here for HGA production the host strain was changed from E. coli DH5 to E. coli BW25113 lacZ⁺.

E. coli BW25113 lacZ⁺ / pCAS2JF and E. coli BW25113 lacZ⁺/ pJF119N were cultivated in shaking flask in minimal medium (section 2.1.3) with glucose or glycerol (5 g/l each) as sole carbon and energy source and 100 µg/ml ampicillin. HPLC chromatogram for HGA analysis can be seen in figure 3.3, and the cultivation results of this experiment are shown in figure 3.4.

85

Figure 3.3: HPLC chromatogram showing HGA standard and HGA produced from E. coli BW25113/pCAS2JF.

(A) HGA standard. Single peak at retention time of 15.6 min with a UV spectrum (shown in inset) with a absorbance maximum at 290 nm.

(B) Supernatant sample from cultivation of E. coli BW25113 / pCAS2JF in MM with 27.8 mM glucose as carbon source. Major peak at 15.6 min (denoted by 1) was detected. This peak has a UVis spectrum (shown in inset) with absorbance maximum at 290 nm. Peak 1 was confirmed as HGA based on the identical retention time and maximum UV absorbance, at 290 nm.

Since the main aim in this sub-chapter was to produce high level of HGA and not to over-produce Hpd proteins, E. coli BW25113/pCAS2JF cultures were induced with 0.25 mM IPTG at OD_600nm between 0.7 and 0.8. HGA standard was analysed by HPLC which resulted in a major peak at retention time of 15.6 minutes seen in figure 3.3 A. This peak has a maximum UV absorption at 290 nm shown in the insets of the chromatogram. Analysis of supernatant samples from E. coli BW25113 / pCAS2JF in glucose and in glycerol resulted in a major peak at retention time of 15.6 min with a maximum UV absorption of 290 nm (seen in Fig.

3.3B). This was identical with the retention time and maximum UV absorption of the standard HGA. No HGA was detected in the control strain E. coli BW25113 / pJF119N.

Cell growth, glucose and glycerol concentrations can be seen in figure 3.4 A and 3.4 B.

(A)

(B)

HGA std.

Peak area [ mAU*min]

10 20 30 40

0 0 25 50 75 100

HGA std.

Peak area [ mAU*min]

10 20 30 40

0 0 25 50 75 100

Peak area [ mAU*min]

10 20 30 40

0 0 25 50 75 100

0 0 25 50 75 100

λ/ nm

200 300 400

Absorbance [ %]

290 nm 20

40 60 80 100

HGA std.

λ/ nm

200 300 400

Absorbance [ %]

290 nm 20

40 60 80 100

HGA std.

1

Time / min

Peak area [ mAU*min]

10 20 30 40

0 0 25 50 75

100 1

Time / min

Peak area [ mAU*min]

10 20 30 40

0 0 25 50 75 100

Peak area [ mAU*min]

10 20 30 40

0 0 25 50 75 100

0 0 25 50 75 100

λ/ nm

200 300 400

Absorbance [ %]

290 nm 1

20 40 60 80 100

λ/ nm

200 300 400

Absorbance [ %]

290 nm 1

20 40 60 80 100

200 300 400

Absorbance [ %]

290 nm 1

20 40 60 80 100

86

Figure 3.4: Cell growth curve and glucose/glycerol concentration in HGA production experiments. E. coli BW25113 strain was used as host strain carrying pJF119N (control) or pCAS2JF. Cultivations were carried out at 30 °C in shaking flask in minimal medium with different carbon sources. Cultures were induced with 0.25 mM IPTG (final conc.) at OD600nm of 0.8. (A) Shows cell density i.e. OD600nm and glucose conc. (as carbon, energy source) as funaction of time. (B) Shows cell density i.e. OD600nm and glycerol conc. (as carbon, energy source) as function of time.

Growth of E. coli BW25113 / pCAS2JF in MM-Glucose slowed after 11 h and ceased completely after 23 h (i.e. 19 h after induction). While growth in MM-Glycerol slowed down between 32 and 37 h and ceased completely after 37 h (i.e. 25 h after induction). In the control strain E. coli BW25113 / pJF119N, cell growth continued after induction to reach OD600nm of 4.2 and OD600nm of 5.6, in glucose and glycerol respectively (Figure 3.4). HGA production in E. coli BW25113 / pCAS2JF in glucose started earlier (7 - 11 h) compared to that in glycerol (11-23 h). Approx. 80 % of the HGA was produced between 23-32 h HGA in

pJF119N (OD) pCAS2JF (OD)

pCAS2JF (Glucose) pJF119N (Glucose)

pJF119N (OD) pCAS2JF (OD)

pCAS2JF (Glycerol) pJF119N (Glycerol) 0

2 4 6 8

0 10 20 30 40 50

Time [h]

OD600nm [-]; Glucose conc. [g/l]

0 2 4 6 8

0 10 20 30 40 50

Time [h]

OD600nm [-]; Glycerol conc. [g/l]

(A)

(B)

pJF119N (OD) pCAS2JF (OD)

pCAS2JF (Glucose) pJF119N (Glucose) pJF119N (OD) pCAS2JF (OD)

pCAS2JF (Glucose) pJF119N (Glucose)

pJF119N (OD) pCAS2JF (OD)

pCAS2JF (Glycerol) pJF119N (Glycerol) pJF119N (OD) pCAS2JF (OD)

pCAS2JF (Glycerol) pJF119N (Glycerol) 0

2 4 6 8

0 10 20 30 40 50

Time [h]

OD600nm [-]; Glucose conc. [g/l]

0 2 4 6 8

0 10 20 30 40 50

Time [h]

OD600nm [-]; Glycerol conc. [g/l]

(A)

(B)

87

both cultivations. HGA levels decreased (after 32.5 h), probably due to oxidation of HGA as a result of prolonged incubation time. No p-HPP was detected in any of the supernatant samples in E. coli BW25113 / pCAS2JF in. On the other side, neither HGA, nor p-HPP was detected in the supernatant samples of the control strain, E. coli BW25113 / pJF119N in glucose or in glycerol.

HGA can get easily oxidized and further polymerized to form brown ochronotic pigment (Gunsior et.al. 2004). Hence it was not possible to conclude the maximum HGA concentration which E. coli BW25113/pCAS2JF could produce in Glucose and in MM-Glycerol. No HGA peak was detected in the control strain E. coli BW25113 / pJF119N neither in MM-Glucose nor in MM-Glycerol. Finally, it can be concluded that HGA was produced using glucose or glycerol as sole carbon and energy sources, by using a low-copy number plasmid pCAS2JF in recombinant E. coli BW25113. Cell growth in HGA producing strains i.e. E. coli BW25113 / pCAS2JF (in MM-Glucose and MM-Glycerol) was impaired, when compared to the control strain. It could not be confirmed, whether the reason for the low OD_600nm in E. coli BW25113/pCAS2JF cultures was due to the toxicity of HGA, or the toxicity of oxidation product of HGA or the overexpressed Hpd proteins itself.

Figure 3.5A: HGA production in E. coli BW25113/pCAS2JF strain, in shaking flask in minimal medium with glucose (filled bars) and in minimal medium with glycerol (empty bars). No HGA was detected in the control strain E.

coli BW25113/pJF119N neither in glucose nor in glycerol as carbon source, and hence not shown in the figure.

E. coli BW25113 / pCAS2JF cultures started turning brown after 27h and 33 h respectively in Glucose and Glycerol containing medium. After centrifugation of culture samples (1 ml, 13000 RPM, 5 minutes), it was confirmed that the culture supernatant was brown in colour and cell pellet remained white/pale. Due to the brown colour of the culture supernatant, the cultures looked brown. Figure 3.5B shows the difference in supernatant colour in E. coli BW25113 / pCAS2JF and its control E. coli BW25113 / pJF119N in MM-Glucose. E. coli BW25113 / pCAS2JF culture supernatant (in MM-Glucose and MM-Glycerol) turned dark brown after 42 h. On the other side, the respective control strains i.e E. coli BW25113 /

0 200 400 600 800 1000 1200 1400

0 4 7 11 23 27 32.5 39 42

Time [h]

HGA [µg/]

Glucose Glycerol

88

pJF119N and E. coli BW25113 lacZ⁺ cultures didn’t turn brown during the entire cultivation time of 42 h.

Brown colour is assumed to be due to the oxidation of HGA (accumulated in the medium) to benzoquinonacetate and further polymerisation to form ochronotic pigment which is brown coloured.

Since no direct correlation between the brown pigment and HGA was available or could be established, it was difficult to calculate the highest concentration of HGA produced in each recombinant E. coli strain used in this study. E. coli BW25113 / pCAS2JF cultures in MM-Glucose resulted in 1213 ± 30 µg/l is one of the highest quantifiable HGA concentration reported so far in literature. Possibly, even higher HGA levels could have been reached during cultivations, if HGA oxidation was avoided.

Figure 3.5B: Colour of culture supernatant after 39 h of cultivation. (1) E. coli BW25113/pJF119N supernatant after 42 h.

(2) E. coli BW25113/pCAS2JF supernatant after 42 h. HGA oxidises and further polymerises to form brown ochronotic pigments (Gunsior et.al.2004). Similarly, HGA released in medium during HGA production in E. coli BW25113/pCAS2JF strain after prolonged cultivation (42 h, in shaking flasks at 30 °C in MM-Glucose medium) also probably oxidises and polymerises. This can be seen in right hand side of the figure marked by 2.

The control strain where no HGA is detected, the colour of the culture supernant stayed colourless, which is marked by 1.

1 2

1 2

89

3.2. In-vivo biosynthesis of Geranygeranyl Pyrophosphate (GGPP) in recombinant

Im Dokument Heterologous biosynthesis of 2-methyl-6-geranylgeranyl benzoquinone and &#948;-tocotrienol in recombinant Escherichia coli strains (Seite 82-89)