Described herein are plasmid addiction systems comprising a host cell comprising one or more inactivated host cell essential genes; and a plasmid comprising one or more plasmid essential genes operably linked to a constitutively active promoter. Also described herein are metabolism-based plasmid addiction systems (PAS) comprising a host cell and a plasmid operably linked to a constitutively active promoter for producing value-based products (e.g., 1-butanol) and methods of generating PASs in microorganisms and producing 1-butanol from a PAS in the absence of antibiotics and/or co-inducers.

The present disclosure relates to a genetically modified microorganism satisfying some of predetermined conditions. The predetermined conditions include: (I) succinate dehydrogenase activity or fumarate reductase activity being reduced or inactivated relative to a wild-type microorganism; (II) lactate dehydrogenase activity being reduced or inactivated relative to the wild-type microorganism; (III) the genetically modified microorganism having modified phosphoenolpyruvate carboxylase activity showing resistance to feedback inhibition by aspartic acid in wild-type phosphoenolpyruvate carboxylase activity, or exogenous phosphoenolpyruvate carboxylase activity having higher resistance to feedback inhibition by aspartic acid than that of the wild-type phosphoenolpyruvate carboxylase activity shown by the wild-type microorganism; and (IV) pyruvate:quinone oxidoreductase being reduced or inactivated relative to the wild-type microorganism.

The disclosure discloses construction and application of an engineered strain of E. coli for producing malic acid by fixing CO.sub.2, and belongs to the field of fermentation. The engineered strain is obtained by performing genetic engineering transformation on Escherichia coli MG1655; the genetic engineering transformation includes knocking out a fumarate reductase gene, a fumarase gene, a lactate dehydrogenase gene and an alcohol dehydrogenase gene and freely overexpressing a formate dehydrogenase, an acetyl coenzyme A synthetase, an acylated acetaldehyde dehydrogenase, a formaldehyde lyase, a dihydroxyacetone kinase, a malic enzyme and a phosphite oxidoreductase to obtain a strain GH0407. The strain is used for producing malic acid by fermentation, anaerobic fermentation is performed for 72 hours with CO.sub.2 and glucose as a co-substrate, the production of malic acid reaches 39 g/L, the yield is 1.53 mol/mol, and accumulation of malic acid in the original strain is not achieved.

A butanol expression cassette includes a butanol production related genes and a fermentation regulatory element. The fermentation regulatory element controls the expression of the butanol production related gene and locates upstream of the butanol production related gene. The fermentation regulatory element includes a promoter, a ribosome binding site and a transcription factor binding site of a fermentation gene. A fermentation in which the fermentation regulatory element involves includes an acetic acid fermentation, an alcohol fermentation, a succinic acid fermentation or a lactic acid fermentation, the butanol production related gene is not the fermentation gene or a gene of an upstream product of the fermentation in which the fermentation gene involves. The present invention provides a recombinant plasmid formed by cloning the butanol expression cassettes in the expression vector. The present invention also provides a butanol production related gene expression method to express butanol production related gene by using recombinant plasmid.

The disclosure discloses construction and application of an engineered strain of E. coli for producing malic acid by fixing CO.sub.2, and belongs to the field of fermentation. The engineered strain is obtained by performing genetic engineering transformation on Escherichia coli MG1655; the genetic engineering transformation includes knocking out a fumarate reductase gene, a fumarase gene, a lactate dehydrogenase gene and an alcohol dehydrogenase gene and freely overexpressing a formate dehydrogenase, an acetyl coenzyme A synthetase, an acylated acetaldehyde dehydrogenase, a formaldehyde lyase, a dihydroxyacetone kinase, a malic enzyme and a phosphite oxidoreductase to obtain a strain GH0407. The strain is used for producing malic acid by fermentation, anaerobic fermentation is performed for 72 hours with CO.sub.2 and glucose as a co-substrate, the production of malic acid reaches 39 g/L, the yield is 1.53 mol/mol, and accumulation of malic acid in the original strain is not achieved.

A butanol expression cassette includes a butanol production related genes and a fermentation regulatory element. The fermentation regulatory element controls the expression of the butanol production related gene and locates upstream of the butanol production related gene. The fermentation regulatory element includes a promoter, a ribosome binding site and a transcription factor binding site of a fermentation gene. A fermentation in which the fermentation regulatory element involves includes an acetic acid fermentation, an alcohol fermentation, a succinic acid fermentation or a lactic acid fermentation, the butanol production related gene is not the fermentation gene or a gene of an upstream product of the fermentation in which the fermentation gene involves. The present invention provides a recombinant plasmid formed by cloning the butanol expression cassettes in the expression vector. The present invention also provides a butanol production related gene expression method to express butanol production related gene by using recombinant plasmid.