Method of synthesizing proteins from methanol

The invention disclosed provides a method for synthesizing proteins by means of strains of microorganisms improved as regards their performances in using methanol.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method of synthesizing new improved 
proteins from a methanol containing culture, by means of methylotrophic 
microorganisms selected as glycine resistant mutants. 
2. Description of the Prior Art 
Numerous attempts have been made in the prior art to prepare methylotrophic 
microorganisms using methanol by way of serine (2-amino-3-hydroxypropionic 
acid). Those microorganisms which have been isolated by the conventional 
methods such as repeated picking out of the microorganisms from a medium 
using methanol as the sole source of carbon, have kinetic performances 
which are frequently mediocre. 
In methods of producing proteins from unicellular organisms, the strain of 
microorganisms is of considerable importance. For a given carbon 
substrate, the biological values of the product, such as absence of 
toxicity, the degree of nutritional value, or the like, depend upon the 
particular strain. Also, the economics of continuous industrial production 
of the proteins are highly dependent upon the particular strain so it is 
important that the selection be performed with certainty and care. A most 
important value of a particular strain would appear to be the kinetic 
performances of the strain which dictates the extent of the scale of the 
industrial installation and, hence, the capital investment required. 
Although the methods of isolating strains by continuous selection make it 
possible to isolate the performing strains, these methods are, 
nevertheless, protracted since it takes at least one month of 
experimentation in a continuous fermenter. The methods are delicate and 
the results hazardous. On the other hand, only an enrichment of 
microorganism is achieved in this way and it is essential to isolate the 
best performing microorganism from the complex flora being processed. 
Some of the prior art attempts are described in the literature, more 
particularly, in the following texts: W. Harder, M. M. Atwood, J. R. 
Quayle, J. Gen. Microbiol. (1973) 78 155-163, Asthana et Coll. Biotechnol, 
Bioeng. (1971) 13, 923 and (1971) 8, 923, M. Reuss et Coll. Eur. J. Appl. 
Microbiol. (1975) 1, 295-305, Whittenbury et al. J. Gen. Microbiol. 
(1970), 61 205, Hirsh and Conti Archiv. Fin Mikrobiologie (1964) 43; 358, 
Ogata et Coll. J. Ferment. Technol. (1970) 48, 470. 
Applicants have discovered that, among the strains which metabolize 
methanol by way of serine, the resistance to glycine habitually involves 
an increased utilization of methanol. 
It has now been found that, by practice of the present invention proteins 
may be prepared from methanol with improved strains which are 
characterized as glycine-resistant methylotrophic strains. 
SUMMARY OF THE INVENTION 
Generally stated, the present method provides a method for synthesizing 
proteins by culturing a strain of microorganisms in a methanol containing 
medium. The harvested cellulor suspension from the culture is diluted and 
then disposed in a glycine-containing culture from which glycine-resistant 
mutants are recovered. The glycine-resistant mutants are then cultured and 
recovered. 
An object of the present invention is to synthesize proteins with strains 
of microorganisms which are characterized as glycine-resistant 
methylotrophic strains. 
Another object of the present invention is to provide a method of 
improvement for methylotrophic strains, the strains being disposed in a 
glycine-containing culture from which may be recovered glycine-resistant 
mutants. 
Other objects, advantages and features of the present invention will become 
more apparent from the following detailed description. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
By practice of the present method, it is possible to use for the 
manufacture of proteins from methanol, an improved strain of any one of 
the strains exhibiting the characteristic of utilization of methanol by 
way of serine. The improvement of said methylotrophic strain consists, on 
the one hand, in an increase in the affinity for methanol and, on the 
other hand, in a reduction of the generation time on methanol carbon 
substrate. 
The increase in affinity for methanol may be determined by its apparent 
constant affinity for methanol (Ks) determined in parts per million (ppm) 
which may be measured by gas phase chromatography. 
Practice of the present method makes it possible to use improved strains of 
methylotrophic microorganisms using methanol metabolically known as 
serine. The improvement of the strains, making their kinetic performances 
compatible with a continuous method of industrial production of proteins 
from methanol, by means of the present invention can be applied to a large 
number of microorganisms such as, non-limitatively, microorganisms of the 
following types: Bacillus, Micrococcus, Arthrobacter, Flavobacterium, 
Pseudomonas, Aeromonas, Methylomonas, Rhodopseudomonas, Methylococcus, 
Klebsiella, Rhodospirillum, Rhodomicrobium, Hyphomicrobium, Acinetobacter, 
Achromobacter, Moraxella, and the like, by way of example. 
The improved methylotrophic strains presently prepared are distinguished 
from the wild strains from which they issue, by their resistance to the 
effect of glycine. 
The improved methylotrophic strains remain insensitive to the action of 
glycine for concentrations of amino acid at least ten times higher than 
those which cause a beginning in the inhibition of the use of the methanol 
substrate (increase in Ks) in the wild strain. 
The improved methylotrophic strain, by practice of the present invention, 
can grow on a medium composed of methanol substrate and glycine, whereas 
the corresponding wild strain is incapable of growing in the presence of 
these concentrations of glycine. Desirably, the concentration of the 
glycine in the medium is about 0.5% to 2% by weight. 
The method of the invention consists in selecting glycine-resistant mutants 
from optional methylotrophic strains, and the recovered mutants can then 
be cultured. The culture of the strain is found to possess improved 
characteristics in the following way: 
The cells are harvested, preferably in the exponential stage of growth, 
then spread on a selection sieve comprising a solid medium for 
methylotrophic microorganisms, plus about 0.5% to 2% by weight glycine, 
either directly, or after the preliminary action of a physical or chemical 
mutagenic agent. 
All the strains thus selected exhibit the character of resistance to 
glycine in concentrations from about 0.5% to 2%. 
The improved strains are inoculated into a culture medium which contains 
mineral salts, nitrogen in mineral form and methanol in concentrations 
variable from 0.1 to 2% (weight/volume). The methanol, sterilized by 
filtration, is added to the autoclaved medium heated to 120.degree. C. for 
30 minutes. The pH should be adjusted between 6 and 8. 
The culture is performed in sterile flasks, with sufficient agitation such 
that the aeration is not limiting, and at temperatures which may vary from 
about 25.degree. C. to 45.degree. C. 
The cells may be harvested by centrifugation. The measurement of the rate 
of specific growth is made by measuring the increase in the dry weight of 
cells per unit of volume as a function of the time. The concentration of 
the methanol in the culture medium is measured by gas phase 
chromatography. 
The strains obtained by the present method can be used not only for the 
synthesis of proteins from methanol, but also for the biological 
purification of an effluent from methanol, the possible reduction of 
methanol in fermented drinks, and, in general, in any fermentation 
involving methanol as a carbon substrate.

The following non-limiting examples are presented to illustrate practice of 
the present invention: 
EXAMPLE 1 
A strain identified as being of the genus Pseudomonas stuzeri is cultured 
on the following medium: 
______________________________________ 
Culture Medium (M.sub.1) 
Ingredient Amount 
______________________________________ 
NH.sub.4 Cl 3 g. 
Na.sub.2 HPO.sub.4 3 g. 
KH.sub.2 PO.sub.4 0.5 g. 
CaCl.sub.2 0.01 g. 
FeSO.sub.4 0.001 g. 
ZnCl.sub.2 0.001 g. 
MnSO.sub.4 0.001 g. 
MgSO.sub.4 0.5 g. 
CH.sub.3 OH 5 g. 
Water Quantity 1 liter 
Sufficient 
to Prepare 
H is adjusted to 6.8 
______________________________________ 
The culture temperature is 30.degree. C. and the generation time of this 
strain is 120 minutes. The apparent constant affinity for methanol (Ks) is 
determined to be 1090 ppm. 
The effective production of biomass at the end of three days' culture is 
0.30 grams per 1 gram of methanol. 
A cellular suspension is harvested from this culture and is diluted in 
normal saline solution so as to produce about 10.sup.8 to 10.sup.9 cells 
per ml., before being spread on a medium (M.sub.1) prepared with 2.5% by 
weight agar-agar and made up with 2% by weight glycine. 
The petri dishes are placed in the oven at 30.degree. C. The appearance of 
resistant mutants is manifested at the end of a few days. The colonies are 
sampled and seeded into liquid medium (M.sub.1) in order to test their 
methylotrophic characters. 
Several glycine-resistant mutants are obtained. The study of the kinetic 
performances of one of those mutants yielded the following results on the 
M.sub.1 culture medium: 
______________________________________ 
Generation Time 100 minutes 
Ks Methanol 20 ppm 
______________________________________ 
The effective production of biomass at the end of three days' culture is 
0.40 g. per 1 g. of methanol. 
EXAMPLE 2 
Other mutants of the strain recovered in the procedure of Example 1 and 
resistant to 2% glycine were isolated with a preliminary treatment of the 
cellular suspension with 2% ethylmethane sulphonate for 2 hours at 
30.degree. C. at pH 7.4. The same improvements were found as in the mutant 
of Example 1, namely reduction in the generation time and above all great 
reduction of the Ks: 
______________________________________ 
(I) Wild Strain (II) Mutant Strain 
______________________________________ 
Generation 
120 minutes Generation Time 
88 minutes 
Time 
Ks Methanol 
1090 ppm Ks Methanol 
20 ppm 
______________________________________ 
EXAMPLE 3 
The treatment employed in the procedure of Example 1 is repeated using a 
strain of Pseudomonas aeruginosa methylotrophus. The mutants resistant to 
glycine are isolated on the selection sieve (medium M.sub.1 +0.5% 
glycine). 
The kinetic performances of these new strains (on M.sub.1 medium) are 
definitely improved compared to the wild strains, the results of which 
follow: 
______________________________________ 
(I) Wild Strain (II) Mutant Strain 
______________________________________ 
Generation 
53/4 Hours Generation Time 
31/2 Hours 
Ks Methanol 
830 ppm Ks Methanol 
100 ppm 
______________________________________ 
EXAMPLE 4 
The treatment employed in the procedure of Example 1 is repeated using a 
strain of Microccus varians methylotrophus. It is, thus, possible to 
isolate strains exhibiting improved performances on methanol compared to 
the wild strain, the results of which follow: 
______________________________________ 
(I) Wild Strain (II) Mutant Strain 
______________________________________ 
Generation 
41/3 Hours Generation Time 
31/2 Hours 
Ks Methanol 
450 ppm Ks Methanol 
150 ppm 
______________________________________ 
Inasmuch as many changes and variations in detail are possible and which 
are readily apparent to those skilled in the art within the scope of the 
present invention, it is intended that the description thereof is 
illustrative rather than limiting.