Random mutagenesis

Thermus sp. ATN1 cells were treated with N-methyl-N´-nitro-nitroso-guanidine (MNNG or NTG) for chemical mutagenesis. Survivors were selectively enriched by cultivation on media containing penicillin and n-hexadecane as sole carbon source.

2.4.1.1 NTG treatment

A modification of the procedure described by Adelberg et al. (1965) was used. Cells were grown overnight in 100 ml DSMZ 74 medium at 65 °C. 4 ml of this culture were used for cell harvesting by centrifugation at 9000 rpm for 15 minutes. Cells were re-suspended in 1 ml 20 mM potassium phosphate buffer pH 6.5 containing NTG in concentrations ranging from 0 to 160 µg/ml. These preparations were incubated 40 minutes at 70°C and 350 rpm in a thermomixer. After cooling down, cells were pelleted at 9000 rpm for 2 minutes and were washed 3 times with 1 ml 10 mM potassium phosphate buffer containing 0.85 %w NaCl, then they were re-suspended in 1 ml 20

mM potassium phosphate buffer and finally dilutions were prepared and volumes of 50 µl were spread on DSMZ74 Gelrite plates. Plates were incubated for 72 h at 65 °C. NTG concentrations rendering survivor rates between 1 and 10 % were selected for mutagenesis in combination with mutant selective enrichment.

2.4.1.2 Penicillin enrichment

On the basis that penicillin can kill only growing cells by inhibiting the cross-linking of peptidogly-can polymers essential for the structural integrity of the cell wall, a mutant enrichment step with Penicillin V followed the NTG treatment (Fitzgerald et al., 1975).

Conditions for the mutagenic treatment prior enrichment were similar to those reported for oth-er type of mutations caused by NTG treatment in Thoth-ermus thoth-ermophilus, studied by Kobashi et al.

(1999). Cells treated with 20 and 40 µg/mL NTG were enriched over one, two or three penicillin enrichment cycles, plated and selected for a preliminary phenotype screening procedure.

Mutants were incubated in minimal medium with n-hexadecane as sole carbon source and peni-cillin concentrations of 250 U/mL and 2500 U/mL were tested (values in literature ranged from 100 up to 4000 U/mL for this type of treatment). Time of treatment for one enrichment cycle corresponded to 12 h (1 generation time of the wild type strain in n-eicosane, as reported by Otto, 2001). Optimally penicillin was added at a concentration of 250 U/mL for the treatment, but only after a pre-enrichment starvation time of 6 to 8 h (to permit cessation of growth of the desired mutants, Herdman et al., 1980). Enrichment was stopped by ice chilling followed by cen-trifugation, cell washing and re-suspension in complex medium (DSMZ74). After 3 h of incuba-tion, dilutions were prepared and spread on DSMZ74 Gelrite plates. Repeated penicillin enrich-ment steps before plating were executed to improve mutant yield.

2.4.1.3 Random mutants screening

The approach to screen for disruption of the long-chain alkane metabolic pathway in the random mutants included 2 stages:

2.4.1.3.1 Plate screening

Transferring complex-media-growing colonies to mineral medium plates supplemented with long-chain n-alkanes and intermediate expected metabolites (fatty alcohol and fatty acids) as single carbon sources.

Mutants on DSMZ74 Gelrite plates were replicated on mineral medium plates with 1.13 g/L n-hexadecane or n-octadecane as sole carbon source (5 mM equivalent). Replicas on 0.37 g/L 1-dodecanol (2mM) and 1.28 g/L hexadecanoic acid (5 mM) were also tested. Incubation was at 65

°C for 6 to 8 days. As Thermus sp. ATN1 colonies grow flat and almost colorless on mineral medi-um, a colony staining method using 0.1% Coomassie Brilliant Blue R stain was used.

The staining solution was poured on the plate and left for 45 seconds, then the solution was poured off followed by a rinsing step with (4:5:1) water/methanol/acetic acid during 1 minute.

After pouring the rinsing solution off the plates were inverted on a paper towel to dry them. Af-ter colony staining, it was possible to differentiate mutants still growing on the n-alkanes from those showing no apparent growth, the later were further studied.

It was assumed that cells growing on the long-chain n-alkane had no mutations that could lead to terminal oxidized products. The same assumption was made for colonies not growing in the al-kane but able to grow on fatty alcohols, since for this case mutation(s) would have occurred at the first step of the metabolic pathway (possibly a damaged monooxygenase system). In contrast cells able to grow in the fatty acid (fatty acid auxotrophs) but not able to grow on the alkane nor on the alcohol were considered as positive mutants and with potential to produce interesting intermediates (table 2.8).

Table 2.8 - Possible disruptions of the long-chain n-alkanes metabolic pathway of Thermus sp.

ATN1 that could be detected with the proposed phenotype screening approach.Only mutant types 3 and 4 (not selectable with the screening strategy) are interesting for further study within the scope of this thesis.

Growth in Substrate as Sole Carbon

Source

Possible Pathway Disruption

Possible Products from Long-chain n-Alkanes Bioconversion

(Growing on a different Car-bon Source)

Mutant Type Long-chain n-Alkane Fatty Alcohol Fatty Acid None Monooxygenase Alcohol Dehydrogenase Aldehyde Dehydrogenase β-Oxidation Fatty Alcohol α,ω - Long-chain Diols Fatty Acids α,ω - Long-chain Dioic Acids

1 2 3 4

2.4.1.3.2 Liquid culture screening

A subsequent screening procedure for mutants selected from plate screening was performed in liquid mineral medium containing the same substrates and concentrations as used in solid media plates. Experiments were carried out in 15 mL Falcon tubes containing 5 mL medium incubated at 70°C and 120 rpm. Incubation for each set of cultures ran for several days. Mutants showing growth in liquid mineral media with n-hexadecane as sole carbon source were discarded as pos-sible candidates for n-alkane bioconversion.