毕赤酵母表型
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Mut+ strains of P pastorzs are sensitive to high residual methanol concentrations
(2) In the presence of excess oxygen and methanol, formaldehyde, the
first product of methanol metabolism, rises to toxic levels and “pickles” the
cells (2-5). Thus, for successful cell growth and protein expression with Mut+
strains, it is critical to mamtam a low methanol concentration m the fermentor.
The advantage of Mut+ strains for expression relative to AOX-defective strains
is their much faster methanol growth (and foreign protein production) rate.
However, the concentration of methanol must be tightly controlled.
Muts Strains
MutS (ACLU-deletion) strains can be generated during transformation by
gene replacement events m which the AOXI gene is replaced by the expression
cassette (6) Alternatively, a I! pastorzs strain, such as KM7 1, which contams a
disrupted AOXl gene, can be utilized as host with expression vectors inserted
by smgle crossover type integration. As mentioned above, these strains grow
slowly on methanol because of their reliance on the AOX gene for alcohol
oxidase (7). The fermentation strategy for MutS strains is the same as with Mut+
strains, except for a lower methanol feed rate to mamtam methanol at a concentration
between 0.2 and 0.8% m the fermentor (7). An advantage of Mut” strains
is that the culture is not as sensitive to residual methanol m the fermentor media
relative to Mut+ strams and hence the process of scale-up can be easier.
An alternative induction strategy for MutS strains is to use a mixed
glycerol:methanol feed. Brierley et al. (I) mvestigated mixed feed ratios of 4: 1
and 2: 1 (g glycerol/h:g methanol/h). They also described a mixed feeding strategy
in which feeding was begun at a 2: 1 ratio, and gradually the proportion of
methanol m the feed was increased until methanol began to accumulate. The
maximum methanol uptake rate was achieved when a mixed feed ratio between
1: 1 and 1:3 was observed. This method of slowly increasing the proportion of
methanol during the mixed feed resulted m the highest recombinant protein
expression levels and shortened the mduction phase from 175 to 45 h. HowHigh
Cell-Density Fermentation 109
ever, mixed feeding at high rates can also result in the production of inhibitory
levels of ethanol (I).
Another induction feeding method for MutS strain fermentations is a batch
methanol mode in which a series of methanol additions are carried out over the
length of the induction period (s). The first addltlon of methanol 1s mltlated 30 mm
after exhaustion of glycerol from the first fed-batch phase.
Mut Strains
A Mut- strain of P pastorzs with dlsruptions m both the AOXI and AOX
genes can be utilized for expression (9). This strain 1s totally defective m alcohol
oxidase and therefore cannot utilize methanol at all. However, methanol
will still induce transcription of the AOXI promoter and expression of genes
regulated by the promoter (1
0). The Inability of the strain to grow on methanol
requires the use of an alternate carbon source, such as glycerol, for growth and
protein production. However, excess glycerol can cause represslon of the AOXI
promoter, thus affecting expression of foreign genes. The feeding strategy for
Mut expression strains 1s similar m concept to the mixed feed strategy for Muts
strains. Glycerol 1s fed under growth-hmltmg condltlons and methanol 1s mamtained
at 0.5% during the induction phase (10). It is important to monitor the methanol
level every 5-6 h and to perlodlcally add methanol to replace that lost to
evaporation from the fermentor. The optimum glycerol feed rate was determmed to
be 0.7 g glycerol/g of dry cell weight for maximum P-galactosidase protein production
with minrmal cell mass and ethanol production (10).
The followmg sections describe how to perform each of these fermentations.
In addition, we describe a moderate-density fermentation procedure for
Mut+ strains that does not require oxygen supplementatton.