RU1389297C - Method of structural and replicating stabilization of expressed recombinant plasmids in microorganism populations - Google Patents

Abstract

The invention relates to the microbiological industry and genetic engineering, in particular to the experimental and laboratory production of useful compounds with the help of microorganisms containing roccinate plasmids, due to which the synthesis of popshes is performed. A method has been created that ensures the stability of expression of recombinant plasmids. it goes without saying that in order to preserve the genetic structure of these plasmids, microorganisms are cultured under conditions of non-simulated growth maintained from the outside. 2 tab.

Description

The invention relates to the microbiological industry and to genetic engineering, in particular to the industrial, experimental laboratory production of useful compounds using microorganisms containing recombinant plzmids, through which useful products are synthesized.

A method that ensures the stability of the expression of recombinant plasmids is that, to preserve the genetic structure of these plasmids, microorganisms are cultured under conditions of externally supported unlimited growth, ..

PRI me R 1, a strain of methylotrophic yeast is cultivated. Hansenula polymorpha DL-1 leu 2, unable to synthesize leucine carrying the recombinant plasmid pL2, with the Ieu2 gene, which provides leucine synthesis in a chemostat mode on a medium without leucine. The inoculum is grown in a 500 ml flask in which 200 ml of HE medium of the following composition (mg / l) is placed: KH2PO / 1000, MgSO / i -71420 500; NH4C 800: FeS04 TNGO 16; CaCl2. 6H20.0.66; ZnS04 biao 0.18; CuS04 5H20 0.16; MnS04 5H20 0.66; CoCl2 6H20 0.18; biotin 0.1; T11amine 0.4; methanol 5000. Non-g (continuous cultivation was carried out in a chehodat at,, 0; p02 30% of saturation. The set pH value was maintained with an ammonia solution. An- (anoslenm of the stationary chemostatic cultivation mode was judged by establishing constant optical values culture density, titrant consumption rate and dissolved oxygen concentration, as well as residual methanol content.

During the chemostat cultivation of the strain H. polymorpha DL-1 Ieu2 containing recombinant p azmide pL2. we performed in two independent experiments at flow rates (volumetric lazy breaks) of 0.05 h and 0.13 h, which revealed that under these conditions of limited cultivation, the fraction of plasmid cells in the population increased; see trained recombinant plasmids undergo significant structural changes. In this case, a significant increase in the molecular weight of plasmid pL2 is from 8.2 Md, xapaiaepHO is the original, to 45-60 Md for recombinant plasmids isolated from cells that underwent chemostatic cultivation.

Thus, control experiments demonstrate that chemostatic

cultivation of microorganisms containing recombinant plasmids leads to the fact that cells with a modified structure of recombinant plasmids accumulate in the fermenter, which is completely consistent with the results obtained previously by researchers.This means that chemostat cultivation of microorganisms containing recombinant plasmids

plasmids, does not ensure the conservation of the genetic structure of recombinant plasmids and cannot be used in practice (Table 1).

P r and m 8 r 2. The strain of metmotrophic

The yeast H.poiymorpha DL-1 Ieu2 carrying the recombinant plasmid pL2 is cultured under pH-auxostat conditions. The inoculum is prepared as described in example 1, for uninterrupted cultivation use

Wednesday 1E (example 1) and a fermentation unit. In order to ensure cultivation in the pH-auxostat mode, the components of the nutrient medium are supplied at the same volume rate to two

pneumatic metering pumps (dispensers), which are synchronously controlled by pulses of the automatic correction system. pH, The working volume of the dispensers is calibrated by direct measurements of the dose of the nutrient medium with the built-in burette according to the operating instructions of the fermenter. The calibration is monitored during the experiment. The flow rate is determined by the pulse counter,

fed to metering pumps in a given amount of time, usually within 1 hour

As a result of pH-a.oxostatic cultivation of a strain of H.poiymorpha DL-1 Ieu2 containing a recombinant plasmid

pL2, it is established that the number of plasmid cells throughout the cultivation is 60-70%, and all tested clones that have passed 1.652 generations contain recombinant plasmids identical to the initial one (Table 2). The flow rate at the end of cultivation 0.17-0.18 hours

PRI me R 3. Cultivate a strain of Hansenula poiymorpha DL-1 Ga2, bearing

recombinant plasmid pL2, in the mode of pH-aucrostat on 11 E medium containing as: the only carbon source and anergy of 5000 mg / l ethanol. Under these conditions, the fraction of plasmid cells in a population of

it is 60-70% throughout the cultivation and the vast majority of plasmid cells (90-100%) that have passed 1, S, and BO generations contain recombinant plasmids with an unchanged structure

(table 2). The flow rate established with during growth is 0.27 hours

Example 4 A strain of Hansenula polymorpha OL-1 (eu2 carrying the recombinant plasmid pL2. Was cultured under pH auxostat mode as described in Example 2, medium 11 E containing 5000 mg / l glucose as a substrate. Throughout

0

cultivation of plasmid cells is 60-80% and plasmid cells isolated by 1.5. 95 generations, the vast majority (90-100%) contain recombinant plasmids. their structure is identical to the original (Table 2). The flow rate at the end of the cultivation is 0.41 hours

Table

The instability of the strain H. polymorpha DL-1 teu2 containing the recombinant plasmid pL2 in a chemostatic mode of cultivation

Note: In Tables 1 and 2, when determining the number of plasmid Klet population, a sample of 500-600 cells was studied. When determining changes in the structure of plschamids, 10 yeast clones were obtained from the above generations of chemostat cultivation.

.Table 2

Stability of H. poiymorpha DL-1 ieu2 strain containing a recombinant plasmid

pL2. with pH auxostatic cultivation