Resultado del objetivo específico 4

To determine if a recombinant strain was suitable for use in the studies, 200 mL shake flask cultures were carried out and the level o f expression o f recombinant ADH and total protein examined and compared to that o f natural bakers' yeast.

The experimental method for shake flask cultures o f E. coli strain WL2, S. cerevisiae

strain M CI and the ADH overexpressing strain is given in Sections 3.3.2.2, 3.3.3 and 3.3.4 respectively. Results for E. coli strain WL2 are shown in Table 4.1, results for

S. cerevisiae strain MCI are shown in Table 4.2 and results for the ADH overexpressing strain are shown in Table 4.3 overleaf. In all cases, typical results from the shake flask cultures are shown which were confirmed by repeated experiments. Typical levels o f ADH and total protein for bakers' yeast are shown in Table 4.4 overleaf to enable comparison to the recombinant strain results.

Sample Total Protein Concentration (m g.m L ‘) rgc-HLADH Specific Activity (U.mg*) Homogenised/Induced' 0.0165 0.84 Homogenised/Non-Induced' 0.0195 2.16 Sonicated/Induced' 0.0115 0.36 Sonicated/Non-Induced' 0.0165 0.18 Expected^ 0.0254 0.0129

' sample taken 20 h after inoculation ^ from Van de Goor-Kucerova (1992)

Table 4.1: Escherichia coli strain WL2 complex media shake fla sk results.

Sample Total Protein Concentration (m g.m L ‘) pg-ADH Specific Activity (U.mg*) Homogenised' 1.90 7.32

‘ sample taken 24 h after inoculation

Strain _{Concentration (mg.mL ‘)}Total Protein _{Activity (U.mg ')}ADH Specific Wild type' (SUB-63 no plasmid) 0.37 6.75 ADH overexpressor' (SUB-63 + PMW5-ADH2) 0.37 1 1 . 1 0

' maximum levels achieved, samples taken 55 h after inoculation

Table 4.3: AD H overexpressor defined media shake fia sk results.

Sample Total Protein Concentration (mg.mL') ADH Specific Activity (U.mg*) Fermented/Complex Media' 1.36 1.93 Fermented/Defined Media' 1.50 11.67

Packed Bakers' Yeast^ - 13

' sample taken 24 h after inoculation, data from Deghani (1996) ^ measured

Table 4.4: Typical levels o f AD H and total protein fo r bakers' yeast.

4.2.1 Escherichia coli strain WL2

The results for E. coli strain WL2 (Table 4.1) show that the total protein concentration in 200 mL shake flask cultures was between 45% and 77% o f the expected value, while the specific activity of recombinant horse liver alcohol dehydrogenase (rec-HLADH) was up to 167 times the expected value. The levels o f protein and rgc-HLADH in the sonicated samples were all lower than those in the homogenised samples. This was most likely due to damage to proteins and enzyme dénaturation during sonication. A comparison o f the values for the induced and non-induced homogenised samples shows that the non-induced sample had higher levels o f protein and rec-HLADH. This is surprising as it indicates that addition o f the IPTG inducer was not required to initiate expression o f the rec-HLADH.

The measured protein concentrations for E. coli strain WL2 were approximately 100 times lower than typical protein concentrations for bakers' yeast fermentations (Table 4.4). Rec-

HLADH specific activities were generally lower than typical values for ADH from bakers' yeast, the exception being the level in the homogenised/non-induced sample which was

1 . 1 2 times the level in a complex media bakers' yeast fermentation.

E. coli strain WL2 was found to be unsuitable for use in the downstream processing studies as it did not meet the required criteria. The low levels o f protein and rec-HLADH obtained indicate that further work is required to increase the cell density during culture in order to ensure that sufficient material for the downstream processing studies can be produced using available fermentation equipment. The low levels o f rec-HLADH activity achieved in most cases may also make accurate mass balancing difficult. Because expression o f the rec-HLADH appeared to occur before addition o f the ITPG inducer, the regulation o f rec-HLADH production would also need to be investigated to ensure a consistent product.

4.2.2 Saccharomyces cerevisiae strain MCI

A comparison o f the results for S. cerevisiae strain MCI (Table 4.2) and typical values for bakers' yeast (Table 4.4) shows that the total protein concentration in S. cerevisiae strain M C 1 200 mL shake flask cultures was 1.4 times the value for a complex media bakers' yeast fermentation and 1.27 times the value for a defined media bakers' yeast fermentation. The protein engineered alcohol dehydrogenase (pg-ADH) specific activity for S. cerevisiae

strain MCI was 3.8 times the value for a complex media bakers' yeast fermentation, 63% o f the value for a defined media bakers' yeast fermentation and 56% o f the value for packed bakers' yeast. The results obtained for S. cerevisiae strain MCI fulfilled the required criteria for use in the downstream processing studies. The levels o f protein and

pe-ADW were similar to those achieved by bakers' yeast and large scale fermentations would therefore be o f a similar scale to bakers' yeast fermentations used to produce material for the work o f Clarkson (1994). The strain was therefore selected for use in downstream processing studies.

4.2.3 ADH Overexpressor

A comparison o f results for the ADH overexpressor to those o f the wild type show approximately 2 fold overexpression o f ADH (Table 4.3). This was low compared to the expected overexpression o f 6 to 8 times (Section 1.6.2). Plasmid stability during shake flask culture remained high, with 78-106% o f cells retaining the plasmid PMW5-ADH2 (refer to Section 3.3.5.7 for method). The level o f overexpression achieved was more typical o f a standard strain o f S. cerevisiae containing the plasmid PMW5-ADH2, i.e. a strain without the ubiquitin gene deletion and suggested that there may have been some instability associated with the changes made in order to achieve the large overexpression. The maximum ADH specific activity during shake flask culture o f the ADH overexpressor was 6 times that typically achieved by bakers' yeast in complex media fermentations, 5% lower than that achieved in bakers' yeast defined media fermentations and 15% lower than that o f packed bakers' yeast. The viability o f the ADH overexpressing strain was also highly inconsistent, with cells taken from stocks failing to grow in many cases.

Due to the possible instability o f overexpression and inconsistent viability, the ADH overexpressor did not meet the required criteria and was not used in further downstream processing studies.