Recombinant cells that highly express chromosomally-integrated heterologous genes

Abstract

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Description

[0001] This application is a continuation-in-part of co-pending application Ser. No. 07 / 352,062 (filed May 15, 1989), which is a continuation-in-part of application Ser. No. 07 / 239,099 (filed Aug. 31, 1988), now abandoned.[0002] Work relating to this invention was supported in part by Grant FG05-86ER3574 from the Office of Basic Energy Science, U.S. Department of Energy, and in part by Grant 88-37233-3987 from the Alcohol Fuels Program, U.S. Department of Agriculture. The U.S. Government has certain rights in the invention.BACKGROUND OF THE INVENTION [0003] The present invention relates to recombinant host cells that comprise a heterologous, polypeptide-encoding polynucleotide segment which is stably integrated into a chromosome and which is under control of an endogenous promoter. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enterobacterial cells within ...

AI Technical Summary

Benefits of technology

The present invention relates to a method for obtaining recombinant host cells that produce desired polypeptides at high levels. This is achieved by integrating a heterologous DNA segment containing the genes for the polypeptides into the host cell chromosome under the control of a strong promoter. The method also includes steps of selecting host cells with increased expression of the integrated DNA segment and mutating the host cell chromosome to further increase expression. The resulting recombinant host cells have stable expression of the integrated DNA segment and produce high levels of the desired polypeptides. The invention also provides a cell strain that contains a chromosome with a plurality of genes integrated into it. The method and cell strain can be used for efficient production of valuable products from biomass fermentation.

Problems solved by technology

They are relatively nontoxic under the conditions in which they are initially produced but become more toxic upon accumulation.

However, these are relatively expensive sources of biomass sugars and have competing value as foods.

For conventional ethanol-producing microorganisms, this biomass has limited commercial value at best, for instance, as a nutritional supplement, and therefore, represents inefficient utilization of the expensive sugar substrate.

No known organism in nature can rapidly and efficiently metabolize all of these sugars, particularly the pentoses, into ethanol or any other single product of value.

However, under anaerobic fermentation conditions, these organisms convert sugars to a mixture of soluble products, including small amounts of ethanol, that cannot be separated economically.

Thus, such enteric bacteria efficiently utilize the entire range of biomass-derived sugars but fail to produce a product of sufficient yield and uniformity to be commercially valuable.

Although Z. mobilis is nutritionally simple and capable of synthesizing amino acids, nucleotides and vitamins, the range of sugars metabolized by this organism is very limited and normally consists of glucose, fructose and sucrose.

Z. mobilis is incapable of growth without a fermentable sugar even in rich medium such as nutrient broth.

However, the exogenous genes were not completely stable because of the inherent instability of plasmids in the absence of selective pressure to ensure their retention in the host cell.

Due to plasmid incompatibilities, moreover, the use of a typical E. coli expression plasmid for the ethanol-production genes precludes the most convenient means for introduction into a basic commercial ethanol-producer strain of additional exogenous genes for production of other selected products, such as valuable proteins.

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