The present invention relates to genetically modified bacteria and methods of optimizing genetically modified bacteria for the production of a metabolite.

The present invention relates to genetically modified bacteria and methods of optimizing genetically modified bacteria for the production of a metabolite.

The invention provides non-natural microbial organisms containing enzymatic pathways and/or metabolic modifications for enhancing carbon flux through acetyl-CoA, or oxaloacetate and acetyl-CoA. Embodiments of the invention include microbial organisms having a pathway to acetyl-CoA and oxaloacetate that includes phosphoketolase (a PK pathway). The organisms also have either (i) a genetic modification that enhances the activity of the non-phosphotransferase system (non-PTS) for sugar uptake, and/or (ii) a genetic modification(s) to the organism's electron transport chain (ETC) that enhances efficiency of ATP production, that enhances availability of reducing equivalents or both. The microbial organisms can optionally include (iii) a genetic modification that maintains, attenuates, or eliminates the activity of a phosphotransferase system (PTS) for sugar uptake. The enhanced carbon flux through acetyl-CoA and oxaloacetate can be used for production of a bioderived compound, and the microbial organisms can further include a pathway capable of producing the bioderived compound.

The invention provides non-natural microbial organisms containing enzymatic pathways and/or metabolic modifications for enhancing carbon flux through acetyl-CoA, or oxaloacetate and acetyl-CoA. Embodiments of the invention include microbial organisms having a pathway to acetyl-CoA and oxaloacetate that includes phosphoketolase (a PK pathway). The organisms also have either (i) a genetic modification that enhances the activity of the non-phosphotransferase system (non-PTS) for sugar uptake, and/or (ii) a genetic modification(s) to the organism's electron transport chain (ETC) that enhances efficiency of ATP production, that enhances availability of reducing equivalents or both. The microbial organisms can optionally include (iii) a genetic modification that maintains, attenuates, or eliminates the activity of a phosphotransferase system (PTS) for sugar uptake. The enhanced carbon flux through acetyl-CoA and oxaloacetate can be used for production of a bioderived compound, and the microbial organisms can further include a pathway capable of producing the bioderived compound.

The disclosure provides genetically engineered C1-fixing microorganisms capable of producing nanobodies. Additionally, the disclosure provides engineered microorganisms comprising one or more disrupted genes to strategically divert carbon flux away from nonessential or undesirable products towards products and/or co-products of interest. The disclosure enables co-production of useful chemicals from gaseous substrates.

The aim of the invention is to provide a method for biotechnological production of carboxylic acids, in which the acid-forming micro-organisms are cultured in an unsterile manner in a submerged phase containing waste water containing all carbon and nutrient medium components necessary for the production of the carboxylic acid, which method avoids the disadvantages of known methods and enables high product concentrations and productivity while at the same time the resources of water and power are being conserved. This aim is achieved, according to the invention, in that micro-organisms are used that are cultured under unsterile conditions in a culture medium containing waste water with the addition of carbon-rich compounds.

The present invention provides for a method to increase the free lipoic acid production in an isolated genetically engineered bacteria or yeast cell. The method involves culturing in a cysteine supplemented culture medium the engineered bacteria or yeast that is transformed with a recombinant expression vector encoding polynucleotide molecules that results in the overexpression of the following genes that are linked to at least one promoter: (1) substrate protein (e.g. Gcv3p); (2) octanoyltransferase or lipoyl synthase; (3) cofactor S-adenosyl methionine synthase; and (4) lipoamidase. The invention also relates to the engineered bacteria or yeast cell thereof.

The present invention provides for a method to increase the free lipoic acid production in an isolated genetically engineered bacteria or yeast cell. The method involves culturing in a cysteine supplemented culture medium the engineered bacteria or yeast that is transformed with a recombinant expression vector encoding polynucleotide molecules that results in the overexpression of the following genes that are linked to at least one promoter: (1) substrate protein (e.g. Gcv3p); (2) octanoyltransferase or lipoyl synthase; (3) cofactor S-adenosyl methionine synthase; and (4) lipoamidase. The invention also relates to the engineered bacteria or yeast cell thereof.

It has been found that wild-type ferulic acid decarboxylase derived from Saccharomyces has high catalytic activity for the production of unsaturated hydrocarbon compounds. Further, it has been found that in the ferulic acid decarboxylase, substitute of the amino acid at position 398 with glutamine, methionine, asparagine, phenylalanine, histidine, or threonine more improves the catalytic activity, making it possible to provide a method capable of producing an unsaturated hydrocarbon compound such as butadiene with high productivity, and an enzyme used in the method.

Provided herein are genetically modified host cells, compositions, and methods for improved production of artemisinic acid. The host cells are genetically modified to contain a heterologous nucleic acid that expresses novel and optimized variants of amorpha-4,11-diene 12-monooxygenase. Also provided herein are methods for screening for variants of cytochrome p450 enzymes that have increased enzymatic activity relative to a parental control enzyme.