EA040778B1 - ADDING ARGININE TO INCREASE THE EFFICIENCY OF GAS FERMENTING ACETOGENES - Google Patents

Description

Cross-reference to related applications

This application claims priority over U.S. Provisional Applications No. 62/262886, filed December 3, 2015, and No. 62/282888, filed December 3, 2015, the entire contents of which are incorporated herein by reference in their entirety.

State of the art

About 10% of the world's energy and non-specific chemicals requirements are currently produced from renewable feedstocks, mostly using farmed sugars. However, increasing attention is being paid to the prospective use of non-food resources to achieve climate change goals. Gas fermentation offers a way to convert a wide range of inexpensive C1 industrial feedstocks such as industrial waste gases, syngas or reformed methane into chemicals and fuels. Considering this to be one of the first biochemical pathways to appear on Earth, the Wood-Ljungdahl metabolic pathway allows the acetogenic Clostridia to contribute these C1 gases to acetyl-CoA. Clostridium autoethanogenum in particular offers a robust and flexible platform for gas fermentation and is applied on an industrial scale. Gas fermentation with C. autoethanogenum is highly selective, resistant to contaminants, overcomes the refractivity of Fischer-Tropsch processes, and is economically viable even with low volume gas streams.

It is known that while acetogens are capable of producing many useful short chain chemicals, the production of longer chain carbon molecules for use in biodiesel or jet fuel is beyond the metabolic capacity of the acetogenic bacteria themselves due to the ATP-limiting process. According to Fast et al., although the Wood-Ljungdahl pathway is the best option for the production of acetate and ethanol, butanol fermentation is an ATP-limiting process, which makes the anaerobic production of butanol by the Wood-Ljungdahl pathway inefficient.

C. autoethanogenum initially produces acetate, ethanol, 2,3-butanediol (2,3-BDO), and lactate. If the energy hurdles are overcome, synthetic biology promises to expand the range of C. autoethanogenum products. Acetogenic bacteria are widely distributed in nature and play an important role in the global carbon cycle, but are thought to live at the thermodynamic edge of life.

The energetic characteristics of the Wood-Ljungdahl pathway of anaerobic acetogens are only beginning to take shape, but unlike the states of aerobic growth or glycolysis of sugar-fermenting organisms, ATP is not produced in the Wood-Ljungdahl pathway through substrate phosphorylation, and in fact one ATP molecule and a membrane gradient are actually needed to activate _CO2 (WO 2013/180584).

ATP generation via substrate phosphorylation can be used as a driving force for product synthesis, especially in ATP-limiting systems. In particular, acetogenic bacteria are known to live at the thermodynamic edge of life (Schuchmann, Nat. Rev. Microbiol., 12:809-821, 2014). Thus, all acetogenic microorganisms isolated to date have been described as producing acetate (Drake, Acetogenic Prokaryotes, Prokaryotes, 3rd edition, pp. 354-420, New York, NY, Springer, 2006; Liew et al., Understanding the _CO2 fixation pathway of Clostridium autethanogenum by targeted mutagenesis, mBio, 2016, 7:e00427-16) since acetate production enables the microorganism to directly generate ATP through substrate phosphorylation via Pta (phosphotransacetylase) (EC 2.3.1.8) and Ack (acetate kinase) ) (EC 2.7.2.1). The Pta-Ack system is very specific for the conversion of acetyl-CoA to acetate and does not use other acyl-CoAs. Although mechanisms such as membrane gradients and electron bifurcation enzymes associated with ion or proton translocation systems, such as the Rnf complex (Schuchmann, Nat. Rev. Microbiol., 12:809-821, 2014), conserve ATP in these microorganisms , direct generation of ATP remains critical to their survival. As a result, when heterologous pathways are introduced that do not generate ATP, acetate is produced as a by-product (Schiel-Bengelsdorf, FEBS Lett, 586:2191-2198, 2012).

The essence of the invention

The present invention provides a process for improving the fermentation efficiency of a gaseous C1-containing substrate in the production of a product, said process comprising

a) introducing a stream of gaseous C1-containing substrate into a bioreactor containing a culture of C1-fixing microorganism in a liquid nutrient medium; And

b) fermenting the culture to produce at least one product, wherein arginine is added to the culture as the sole source of nitrogen; and wherein the C1 fixing microorganism is a recombinant genetically modified microorganism containing an arginine metabolic pathway, and wherein the C1 fixing microorganism is Clostridium.

In one embodiment of the invention, said arginine metabolic pathway includes the arginine deiminase pathway and/or the arginine decarboxylase pathway, wherein the arginine deiminase pathway includes one or more enzymes selected from the group consisting of arginine

- 1 040778 deiminase (EC 3.5.3.6), ornithinecarbamoyltransferase (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2), and the arginine decarboxylase pathway includes one or more enzymes selected from the group consisting of arginine decarboxylase (EC 4.1. 1.19), agmatine deiminase (EC 3.5.3.12), putrescinecarbamoyltransferase (EC 2.1.3.6) and carbamate kinase (EC 2.7.2.2).

In one embodiment, said arginine metabolic pathway comprises one or more of an overexpressed endogenous enzyme, a mutated endogenous enzyme, or an exogenous enzyme.

In one of the embodiments of the invention, arginine is added to the culture in an amount exceeding the needs of the cells of the culture up to 1000 times.

In one of the embodiments of the invention, the concentration of arginine in the bioreactor is at least 20 mg/l.

In one of the embodiments of the invention, the requirement of the culture cells is 0.012 g of arginine per gram of cell biomass.

In one of the embodiments of the invention, the specified product is selected from the group consisting of ethanol, 2,3-butanediol, 1,3-butanediol, lactate, succinate, methyl ethyl ketone (MEK), butyrate, 2-butanol, 1,2-propanediol (1 ,2-PDO), 1-propanol, isopropanol (IPA), acetoin, isobutanol, isoprene, farnesene, bisabolene, pinene, limonene, acetone, 3-hydroxybutyrate, 2hydroxyisobutyric acid (2-HIBA), citramalate, butadiene, polylactic acid, 1-butanol, 3-hydroxypropionate (3-HP), benzoate, fatty acid ethyl esters, fatty acids and isobutylene.

In one embodiment, arginine is catabolized via the arginine metabolic pathway to produce ammonium, with the C1-fixing microorganism using the ammonium as a nitrogen source.

In one of the embodiments of the invention, the specified C1-fixing microorganism contains a destructive mutation in the arginine:ornithine transporter.

In one embodiment, the C1-fixing microorganism contains one or more genetic modifications in the arginine metabolic pathway, wherein the one or more genetic modifications are selected from the group consisting of (i) disruptive mutations of regulatory elements and (ii) replacement of operator binding sites. or native promoters to constitutive or synthetic promoters.

In one of the embodiments of the invention, the specified destructive mutation is a knockout of the arginine repressor argR.

In one embodiment of the invention, said replacement is a replacement of the arginine deiminase pathway operon promoter with a constitutive or synthetic promoter.

In some embodiments of this invention, arginine is added to the culture in an amount exceeding the needs of the cells from 2 to 1000 times, or from 2 to 800 times, or from 2 to 500 times, or from 2 to 100 times, or from 2 to 50 times, or 2 to 10 times, or 50 to 1000 times, or 50 to 800 times, or 50 to 600 times, or 50 to 500 times, or 50 to 300 times, or 50 to 200 times, or 50 to 100 times, or 100 to 1000 times, or 100 to 800 times, or 100 to 600 times, or 100 to 500 times, or 100 to 300 times, or 100 to 200 times.

In some embodiments of the invention, arginine is added to the culture such that the arginine concentration is maintained at at least 20 mg/l, or at least 100 mg/l, or at least 300 mg/l, or at least 500 mg/l, or at least 1 g/l, or at least 2 g/l, or at least 3 g/l, or at least 4 g/l, or at least 5 g/l, or at least 10 g/l, or at least 20 g/l. In some embodiments of this invention, the concentration of arginine is maintained at a level of from 20 mg/l to 20 g/l, or from 100 mg/l to 20 g/l, or from 500 mg/l to 20 g/l, or from 500 mg /l to 10 g/l, or from 1 to 10 g/l, or from 5 to 10 g/l, or from 5 to 20 g/l. In a certain embodiment of the invention, arginine is added to the culture such that the culture's arginine consumption is at least 20 mg arginine per gram cell dry weight, or at least 100 mg arginine per gram cell dry weight, or at least 1 g arginine per gram of cell dry weight, or at least 5 g of arginine per gram of cell dry weight, or at least 10 g of arginine per gram of cell dry weight. In some embodiments of the present invention, arginine is added to the culture such that the culture's arginine consumption is 20 mg to 20 g per gram of cell dry weight, or 100 mg to 20 g per gram of cell dry weight, or 1 to 10 g per grams dry weight of cells.

The need for cells in arginine for biomass synthesis can range from 0.012 to 24 g per gram of biomass. The requirement for arginine for biomass synthesis can be at least 0.012 g per gram of biomass, or at least 0.024 g per gram of biomass, up to 0.048 g per gram of biomass, or at least 0.120 g per gram of biomass, or at least 0. 24 g per gram of biomass, or at least 0.48 g per gram of biomass, or at least 1.2 g per gram of biomass, or at least 2.4 g per gram of biomass, or at least 4.8 g per gram of biomass, or at least 12 g per gram of biomass.

As a rule, arginine is added to the culture of the C1-fixing microorganism as a component

- 2 040778 liquid nutrient medium, which is continuously fed into the bioreactor. In other embodiments of this invention, arginine is added to the bioreactor independently of the liquid nutrient medium (for example, by continuous feeding or dosing).

Culture doubling time is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60% , or at least 70% when arginine is added to the culture in an amount exceeding the needs of the cells of the microorganism, compared to the culture when arginine is not added to the culture in an amount exceeding the needs of the cells of the culture.

The C1-fixing microorganism is an acetogenic carboxydotrophic microorganism. Examples of suitable C1-fixing microorganisms include Clostridium. In various embodiments of this invention, the microorganism is selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Clostridium ragsdalei, Clostridium coskatii.

In specific embodiments of this invention, the C1-fixing microorganism is Clostridium autoethanogenum or Clostridium ljungdahlii. In one particular embodiment of the present invention, the microorganism is Clostridium autoethanogenum. In particular embodiments of this invention, the bacterium has the identifying characteristics of the access number DSM10061, DSM19630, or DSM23693. These bacteria are deposited at the German Biological Material Resource Center (DSMZ), whose address is DSMZ GmbH Inhoffenstraβe, 7B, D-38124.

In some embodiments of this invention, the C1-fixing microorganism is selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium scatologenes, Clostridium drakei.

The concentration of arginine added to the culture can be increased to increase the production of at least one ATP-capable product. Typically, the rate of production of at least one ATP-hungry product is at least 1.5 times the rate of production of at least one ATP-hungry product when arginine is not added in excess of the needs of the culture cells. The production rate of at least one ATP-capacious product is at least 2 times greater, or at least 3 times greater, or at least 4 times greater, or at least 5 times greater, or at least 10 times more.

Generally, ATP-rich foods are defined as foods that have an immediate need for ATP in the metabolic pathway. Examples of ATP-intensive products (products that are characterized by a direct requirement of ATP for synthesis having an ATP-dependent reaction in said pathway) include, but are not limited to, products derived from the terpenoid/mevalonate pathway, including isoprene, farnesine, bisabolene and limonene, products derived from the fatty acid pathway, including fatty acids, fatty acid ethyl esters, or molecules such as 3-hydroxypropionate (3-HP) or isobutylene.

The concentration of arginine added to the culture can be increased to increase the production of at least one product selected from the group of products that do not directly require ATP, but also do not provide the same amount of ATP to acetyl-CoA as acetate production. In some embodiments of this invention, the concentration of arginine added to the culture is increased to increase the production of at least one product selected from the group consisting of acetone, IPA, 3-HB, 2-HIBA, 1,3-BDO, 2, 3-BDO, lactate, 1,2-PDO, 1-propanol, isobutanol, butryrate, butanol, citramalate, succinate and MEK.

The culture may produce a reduced amount of acetate compared to a culture to which arginine is not added in excess of the requirements of the cells. Thus, the culture may produce at least 10% less acetate, or at least 20% less acetate, or at least 30% less acetate, or at least 40% less acetate, or at least 50% less acetate. % less than acetate, or at least 60% less than acetate, or at least 80% less than acetate. In some embodiments of this invention, pure acetate is produced in an amount of less than 1 g/l acetate, less than 0.5 g/l acetate, less than 0.1 g/l acetate, less than 0.05 g/l acetate, or less than 0.01 g /l acetate.

In specific embodiments of this invention, the gaseous substrate is selected from the group consisting of CO, H ₂ and CO ₂ . In specific embodiments of this invention, arginine and gaseous substrate are taken up by the culture in a ratio of at least 2:1, or at least 1:1, or at least 1:2.

Brief description of graphic materials

In FIG. 1 is a graph showing the growth of C. autoethanogenum DSM10061 in a defined medium of 20 amino acids +5 g fructose/L.

In FIG. 2 is a graph showing data on consumption and production of amino

- 3 040778 acids for C. autoethanogenum DSM10061 growing on 20AK medium. The label indicates the OD when entering samples; the cysteine measurement for the first two samples was out of calibration range.

In FIG. 3 is a graph showing the growth of C. autoethanogenum DSM10061 on 14AK and 8AK media along with a comparison to 20AK media. The error bar is the standard deviation between duplicate cultures. 4x and 2x Mean a 4-fold and 2-fold increase in the concentration of AA compared to the 20AA medium, respectively.

In FIG. 4 is a graph showing the doubling time tD for C. autoethanogenum DSM10061 for 14AA and 8AA media compared to 20AA medium. tD - doubling time. The error bar is the standard deviation between duplicate cultures. 4x and 2x Mean a 4-fold and 2-fold increase in the concentration of AA compared to the 20AA medium, respectively.

In FIG. 5 is a graph showing amino acid intake and production data for C. autoethanogenum DSM10061 in 14AA 4x medium. 4x Indicates a 4-fold increase in the concentration of AA compared to 20AA medium. The label indicates the OD when introducing samples.

In FIG. 6 is a graph showing amino acid intake and production data for C. autoethanogenum DSM10061 in 8AA 2x medium. 2x Indicates a 2-fold increase in the concentration of AA compared to the medium 20AA. The label indicates the OD when introducing samples. The cysteine measurement for the first sample was out of calibration range.

In FIG. 7 is a graph showing growth data for C. autoethanogenum DSM10061 on 12AA and 4AA media. The error bar is the standard deviation between duplicate cultures.

In FIG. 8 is a graph showing amino acid intake and production data for C. autoethanogenum DSM10061 on 12AA medium. The label indicates the OD when introducing samples.

In FIG. 9 is a graph showing amino acid intake and production data for C. autoethanogenum DSM10061 in 4AA medium. The label indicates the OD when introducing samples. The μM unit does not apply to the 8.3 min peak.

In FIG. 10 is a graph showing growth data for C. autoethanogenum DSM10061 in 4AK medium using BugLab. BugUnit, ln raw data. The specific growth rate of 0.34 h-1 corresponds to tD ~2 h.

In FIG. 11 is a graph showing growth data for C. autoethanogenum DSM10061 in yeast-free PETC-MES medium with 5 g arginine/L+5 g fructose/L.

In FIG. 12 is a graph showing acetate production data for C. autoethanogenum DSM10061 in yeast-free PETC-MES medium with 5 g arginine/L+5 g fructose/L.

In FIG. 13 is a graph showing amino acid intake and production data for C. autoethanogenum DSM10061 in 5 g arginine/l+5 g fructose/l medium. The label indicates the OD when introducing samples. The μM unit does not apply to the 8.3 min peak.

In FIG. 14 is a graph showing growth data for C. autoethanogenum DSM10061 in yeast-free PETC-MES medium with 5 g arginine/L + 5 g fructose/L using BugLab. BugEd, ln raw data. The specific growth rate of 0.21 h-1 corresponds to tD ~3 h.

In FIG. 15 is a graph showing the difference in growth curve and doubling times between arginine and yeast extract in fructose bioreactor experiments.

In FIG. 16 is a graph showing the autotrophic growth of C. autoethanogenum DSM 23693 with (△) and without 1*1 arginine supplementation.

In FIG. 17 is a graph showing the autotrophic growth of C. autoethanogenum DSM 23693 with (△) and no addition of arginine, and the growth of arginine only in the absence of CO/CO2/H2 gas

In FIG. 18 is a log plot of the autotrophic growth of C. autoethanogenum DSM 23693 with and without (·) arginine supplementation.

In FIG. 19 is a graph showing acetate production during autotrophic growth of C. autoethanogenum DSM 23693 with (△) and without (·) arginine addition.

In FIG. 20 is a schematic representation of the arginine decarboxylase pathway.

In FIG. 21 is a schematic representation of the cleavage of L-arginine in the arginine deiminase pathway.

In FIG. 22A, 22B is a pie chart showing carbon flux for products.

In FIG. 23 shows the enzymes required for ornithine consumption in Clostridium sticklandii.

In FIG. 24 is a diagram showing the distribution of ATP production during heterotrophic growth of C. autoethanogenum on 4AA or ARG media with fructose.

Detailed description of the invention

Definitions and general information.

The term in excess of the needs of the cells refers to the addition of a component to a microorganism in an amount that is greater than the amount of the component required by the microorganism for biomass synthesis.

- 4 040778

The term ATP-capacious refers to a metabolite for the synthesis of which ATP (energy) is introduced at least one stage of its synthesis pathway (for example, in the presence of an ATP-dependent reaction in this pathway).

The term specific growth rate refers to the growth rate of cell biomass per cell biomass per hour.

The term doubling time refers to the time in hours when the cell biomass should double.

The term arginine metabolic pathway broadly refers to any pathway involved in the metabolism of arginine. Typically, the arginine metabolic pathway includes at least one of the arginine deiminase pathway and the arginine decarboxylase pathway.

The term genetic modification or genetic engineering broadly refers to the manipulation of the genome or nucleic acids of a microorganism. Similarly, the term genetically engineered refers to a microorganism containing a manipulated gene or nucleic acids. Methods for genetic modification include, for example, expression of heterologous genes, insertion or deletion of a gene or promoter, nucleic acid mutation, altered gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, and codon optimization.

The term recombinant indicates that the nucleic acid, protein, or microorganism is the product of genetic modification, engineering, or recombination. Generally, the term recombinant refers to a nucleic acid, protein, or microorganism that contains or is encoded by genetic material derived from multiple sources, such as two or more different strains or species of microorganisms. In this context, the term recombinant can also be used to describe a microorganism that includes a mutant nucleic acid or protein, including a mutated form of an endogenous nucleic acid or protein.

The term endogenous refers to a nucleic acid or protein that is present or expressed in the wild-type or parental microorganism from which the microorganism of the invention is derived. For example, an endogenous gene is a gene that is originally present in a wild-type microorganism or parental microorganism from which the microorganism of the present invention is derived. In one embodiment of the present invention, expression of an endogenous gene may be controlled by an exogenous regulatory element, such as an exogenous promoter.

The term exogenous refers to a nucleic acid or protein that is not present in the wild-type microorganism or parental microorganism from which the microorganism of the present invention is derived. In one embodiment of the present invention, the exogenous gene or enzyme may be derived from a heterologous (ie different) strain or species and introduced or expressed in the microorganism of the present invention. In another embodiment of this invention, an exogenous gene or enzyme can be artificially or recombinantly created and introduced or expressed in the microorganism according to this invention. Exogenous nucleic acids can be adapted to integrate into the genome of the microorganism of the invention, or to remain in an extrachromosomal state in the microorganism of the invention, for example, in a plasmid.

The term enzymatic activity or simply activity in a broad sense refers to enzymatic activity, including, but not limited to, the activity of the enzyme, the amount of the enzyme, or the availability of the enzyme to catalyze the reaction. Accordingly, the term increasing enzyme activity includes an increase in enzyme activity, an increase in the amount of enzyme, or an increase in the availability of an enzyme to catalyze a reaction. Similarly, the term decreasing enzyme activity includes a decrease in enzyme activity, a decrease in the amount of enzyme, or a decrease in the availability of an enzyme to catalyze a reaction.

The term mutated refers to a nucleic acid or protein that has been modified in the microorganism of the invention compared to the wild-type or parental microorganism from which the microorganism of the invention is derived. In one embodiment of the present invention, the mutation may be a deletion, insertion, or substitution in a gene encoding an enzyme. In another embodiment of the present invention, the mutation may be a deletion, insertion or substitution of one or more amino acids in the enzyme.

In particular, a destructive mutation is a mutation that reduces or eliminates (ie destroys) the expression or activity of a gene or enzyme. A destructive mutation may partially inactivate, completely inactivate, or remove a gene or enzyme. The destructive mutation may be a knockout mutation (KO). A disruptive mutation can be any mutation that reduces, prevents, or blocks the biosynthesis of a product produced by an enzyme. A disruptive mutation may include, for example, a mutation in a gene encoding an enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding an enzyme, the introduction of a nucleic acid producing a protein that reduces or inhibits

- 5 040778 enzyme activity or the introduction of a nucleic acid (eg antisense RNA, siRNA, CRISPR) or a protein that inhibits the expression of the enzyme. A destructive mutation can be introduced by any method known in the art.

The introduction of a destructive mutation results in the microorganism of the invention not producing the desired product, or essentially not producing the desired product, or producing a reduced amount of the desired product compared to the parent microorganism from which the microorganism of the invention is derived. For example, the microorganism according to this invention may not produce the desired product or produce the desired product in an amount of at least 1, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% less than compared to the parent microorganism. For example, the microorganism of this invention may produce less than 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0 g/L of the desired product.

The term codon optimization refers to the mutation of a nucleic acid, such as a gene, to optimize or improve translation of the nucleic acid in a particular strain or species. Codon optimization can result in faster translation rates or higher translation fidelity. In a preferred embodiment of the invention, the genes of the invention are a codon optimized for expression in Clostridium, in particular Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In yet another preferred embodiment of the present invention, the genes of the present invention are codons optimized for expression in Clostridium autoethanogenum LZ1561, which is deposited with the DSMZ under accession number DSM23693.

The term overexpressed refers to an increase in the expression of a nucleic acid or protein in a microorganism of the invention compared to the wild-type microorganism or parental microorganism from which the microorganism of the invention is derived. Overexpression can be achieved by any means known in the art, including modifying the copy number of a gene, the rate of gene transcription, the rate of translation of a gene, or the rate of enzymatic cleavage.

The term variants includes nucleic acids and proteins whose sequence differs from that of a reference nucleic acid and protein, such as the reference nucleic acid and protein sequence described in the prior art or exemplified herein. The invention may be practiced using nucleic acid or protein variants that perform essentially the same function as the reference nucleic acid or protein. For example, a protein variant may perform essentially the same function or catalyze essentially the same reaction as a reference protein. The gene variant may encode the same or substantially the same protein as the reference gene. The variant promoter may have substantially the same ability to drive the expression of one or more genes as the reference promoter.

Such nucleic acids or proteins may be referred to herein as functionally equivalent variants. By way of example, functionally equivalent nucleic acid variants may include allelic variants, gene fragments, mutated genes, polymorphisms, and the like. Homologous genes from other microorganisms are also examples of functionally equivalent variants. These include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii, or Clostridium Ijungdahlii, which are publicly available on websites such as Genbank or NCBI. Functionally equivalent variants also include nucleic acids whose sequence changes as a result of codon optimization for a particular microorganism. A functionally equivalent nucleic acid variant will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or more nucleic acid sequence identity (percent homology) with the reference nucleic acid. A functionally equivalent protein variant will preferably have at least about 70%, about 80%, about 85%, about 90%, about 95%, about 98% or more amino acid identity (percent homology) with the reference protein. The functional equivalence of a nucleic acid or protein variant can be assessed using any method known in the art.

Nucleic acids can be delivered to the microorganism of this invention using any method known in the art. For example, nucleic acids can be delivered as naked nucleic acids or can be combined with one or more agents such as liposomes. Nucleic acids can be DNA, RNA, cDNA, or combinations thereof, as appropriate. In some embodiments of the invention, restriction inhibitors may be used. Additional vectors may include plasmids, viruses, bacteriophages, cosmids, and artificial chromosomes. In a preferred embodiment of the present invention, the nucleic acids are delivered to the microorganism of the present invention using a plasmid. As an example, transformation (including transduction or transfection) can be achieved by electroporation, sonication, mediated transformation

- 6 040778 polyethylene glycol, chemical or natural competence, protoplast transformation, prophage induction or conjugation. In some embodiments of the invention having active restriction enzyme systems, it may be necessary to methylate the nucleic acid prior to introducing the nucleic acid into the microorganism.

In addition, nucleic acids can be designed to contain a regulatory element, such as a promoter, to increase or otherwise control the expression of a particular nucleic acid. The promoter may be a constitutive promoter or an inducible promoter. Ideally, the promoter is a Wood-Ljungdahl pathway promoter, a ferredoxin promoter, a pyruvate:ferredoxin oxidoreductase promoter, an Rnf complex operon promoter, an ATP synthase operon promoter, or a phosphotransanethylase/acetate kinase operon promoter.

A microorganism is a microscopic organism, especially a bacterium, archaea, virus, or fungus. Typically, the microorganism of this invention is a bacterium. In this context, the term microorganism should be used to refer to bacteria.

The parent microorganism is the microorganism used to generate the microorganism of this invention. The parent microorganism may be a naturally occurring microorganism (ie, a wild-type microorganism) or a microorganism that has been previously modified (ie, a mutant or recombinant microorganism). The microorganism of this invention can also be modified to express or overexpress one or more enzymes that were not expressed or overexpressed in the parent microorganism. Similarly, the microorganism of this invention can be modified to contain one or more genes that were not in the parent microorganism. The microorganism of this invention can also be modified to lack or lower expression of one or more enzymes that were expressed in the parent microorganism. In one embodiment of the invention, the parent microorganism is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment of the present invention, the parent microorganism is Clostridium autoethanogenum LZ1561, which was deposited June 7, 2010 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) located at Inhoffenstraβ 7B, D-38124 Braunschwieg, Germany, June 7, 2010 in accordance with the terms of the Budapest Treaty and received access number DSM23693.

The term derived from indicates that a nucleic acid, protein, or microorganism is modified or adapted from another (eg, parental or wild-type) nucleic acid, protein, or microorganism to produce a new nucleic acid, protein, or microorganism. Such modifications or adaptations typically include insertion, deletion, mutation, or substitution of nucleic acids or genes. As a rule, the microorganism according to this invention is derived from the parent microorganism. In one embodiment of this invention, the microorganism of this invention is derived from Clostridium autoethanogenum, Clostridium ljungdahlii or Clostridium ragsdalei. In a preferred embodiment of the present invention, the microorganism of the present invention is derived from Clostridium autoethanogenum LZ1561, which is deposited with the DSMZ under accession number DSM23693.

The microorganism of this invention can be further classified based on functional characteristics. For example, the microorganism of the present invention may be or may be derived from a C1-fixing microorganism, anaerobe, acetogen, ethanologen, carboxydotroph and/or methanotroph. The table below provides a representative list of microorganisms and their functional characteristics.

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Cl fixation Anaerobe Acetogen Ethanologen O They f οάτοϊ/ποχοράυ^ methanotroph Acetobacterium woodii + + + +/- ¹ - - - Alkalibaculum bacchii + + + + + + - Blautia product + + + - + + - B and tyri bacteri um m e thylotroph icum + + + + + + - Clostridium aceticum + + + - + + - Clostridium autoethanogenum + + + + + + - Clostridium carbonicivorans + + + + + + - Clostridium coskatii + + + + + + - Clostridium drakei + + + - + + - Clostridium formicoaceticum + + + - + + - Clostridium Ijungdahlii + + + + + + - Clostridium magnum + + + - + +/- ² - Clostridium ragsdalei + + + + + + - Clostridium scatologenes + + + - + + - Eubacterium limosum + + + - + + - Moorella thermautotrophica + + + + + + - Moorella thermoacetica (formerly Clostridium thermoaceticum) + + + 3 + + - Oxobacter pfennigii + + + - + + - Sporomus ovata + + + - + +/- ⁴ - Sporomusa silvacetica + + + - + +/- ⁵ - Sporomusa sphaeroides + + + - + +/- ⁶ - Thermoanaerobacter kiuvi + + + - + - -

¹ Acetobacterium woodi can produce ethanol from fructose, but not from gas.

² It has not been studied whether Clostridium magnum can grow on CO.

³ One strain of Moorella thermoacetica, Moorella sp. HUC22-1 is reported to produce ethanol from gas.

⁴ It has not been studied whether Sporomusa ovata can grow on CO.

⁵ It has not been studied whether Sporomusa silvacetica can grow on CO.

⁶ It has not been studied whether Sporomusa sphaeroides can grow on CO.

C1 refers to a molecule with one carbon, such as CO, CO ₂ , CH ₄ or CH ₃ OH. C1-oxygenate refers to a single carbon molecule that also contains at least one oxygen atom, such as CO, CO ₂ or CH ₃ OH. A C1 carbon source refers to a single carbon molecule that serves as a partial or sole carbon source for the microorganism of this invention. For example, the C1 carbon source may contain one or more of CO, CO ₂ , CH ₄ , CH ₃ OH, or CH ₂ O ₂ . Preferably, the C1 carbon source contains one or both of CO and CO ₂ . A C1 fixing microorganism is a microorganism that has the ability to produce one or more products from a C1 carbon source. Typically, the microorganism of this invention is a C1-fixing bacterium. In a preferred embodiment of the present invention, the microorganism of the present invention is derived from the C1-fixing microorganism indicated in the table above.

An anaerobe is a microorganism that does not require oxygen for growth. The anaerobe can react negatively or even die if oxygen is present above a certain threshold. As a rule, the microorganism according to this invention is an anaerobe. In a preferred embodiment of the present invention, the microorganism of the present invention is derived from an anaerobe indicated in the table above.

Acetogens are obligate anaerobic bacteria that use the WoodLjungdahl pathway as their primary energy conservation mechanism and for the synthesis of acetyl-CoA and acetyl-CoA derivatives such as acetate (Ragsdale, Biochim Biophys Acta, 1784:1873-1898, 2008). Acetogens use the acetyl-CoA pathway as (1) a mechanism for the reductive synthesis of acetyl-CoA from CO ₂ ;

(2) the energy conservation process associated with the acceptance of terminal electrons;

(3) the mechanism of fixation (assimilation) of CO ₂ in the synthesis of cellular carbon (Drake, Acetogenic pro-

- 8 040778 Karyotes, in Prokaryotes, 3rd edition, p. 354, New York, NY, 2006).

All natural acetogens are C1-fixing, anaerobic, autotrophic and non-methanotrophic. Typically, the microorganism of this invention is an acetogen. In a preferred embodiment of the present invention, the microorganism of the present invention is derived from the acetogen indicated in the table above.

An ethanologen is a microorganism that produces or is capable of producing ethanol. Typically, the microorganism of this invention is an ethanologen. In a preferred embodiment of this invention, the microorganism of this invention is derived from the ethanologen listed in the table above.

An autotroph is a microorganism that can grow in the absence of organic carbon. Instead, autotrophs use inorganic carbon sources such as CO and/or CO ₂ . As a rule, the microorganism according to this invention is an autotroph. In a preferred embodiment of the present invention, the microorganism of the present invention is derived from an autotroph listed in the table above.

A carboxydotroph is a microorganism capable of using CO as its sole carbon source. As a rule, the microorganism according to this invention is a carboxydotroph. In a preferred embodiment of the present invention, the microorganism of the present invention is derived from the carboxydotroph indicated in the table above.

A methanotroph is a microorganism capable of using methane as its sole source of carbon and energy. In some embodiments of this invention, the microorganism of this invention is derived from a methanotroph.

In a broader sense, the microorganism according to this invention may be derived from any genus or species listed in the table above.

In a preferred embodiment of the present invention, the microorganism of the present invention is derived from the Clostridia group, comprising the species Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei. These species were first described and characterized by Abrini, Arch. Microbiol., 161: 345-351, 1994 (Clostridium autoethanogenum), Tanner, Int. J. System Bacteriol., 43:232-236, 1993 (Clostridium ljungdahlii), and Huhnke, WO 2008/028055 (Clostridium ragsdalei).

These three species share many common characteristics. In particular, all these species are C1-fixing anaerobic, acetogenic, ethanologenic, and carboxydotrophic members of the Clostridium genus. These species share similar genotypes and phenotypes, as well as energy conservation pathways and enzymatic metabolism. Moreover, these species are grouped into group I clostrial rRNA homology with 16S rRNA DNA that is more than 99% identical, have a G+C DNA content of 22-30 mole %, are Gram positive, have similar morphology and size. (logarithmic cell growth between 0.5-0.7x3-µm), are mesophilic (grow optimally at 30-37°C), have similar pH ranges of 4-7.5 (with an optimal pH of 5.5-6), do not contain cytochromes and store energy through the Rnf complex. In addition, reduction of carboxylic acids to their respective alcohols has been proven for these species (Perez, Biotechnol. Bioeng., 110:1066-1077, 2012). Importantly, these species also show strong autotrophic growth on CO-containing gases, produce ethanol and acetate (or acetic acid) as the main fermentation products, and under certain conditions produce small amounts of 2,3-butanediol and lactic acid.

However, these three types also have a number of differences. These species have been isolated from various sources: Clostridium autoethanogenum from rabbit intestines, Clostridium ljungdahlii from chicken yard garbage, and Clostridium ragsdalei from freshwater pond sediment. These species differ in the use of various sugars (eg rhamnose, arabinose), acids (eg gluconate, citrate), amino acids (eg arginine, histidine) and other substrates (eg betaine, butanol). Moreover, these species differ in auxotrophy with respect to some vitamins (eg, thiamine, biotin). These species have differences in the nucleic and amino acid sequences of the genes and proteins of the Wood-Ljungdahl pathway, although the general organization and number of these genes and proteins turned out to be the same in all species of Korke, Curr. Opin. Biotechnol., 22:320-325, 2011).

Thus, in general, many characteristics of Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei are not specific to this species, but are rather general characteristics for this cluster of C1-fixing, anaerobic, acetogenic, ethanologenic, and carboxydotrophic members of the genus Clostridium. However, since these species are inherently different, genetic modification or manipulation of one of these species may not have the same effect in another of these species. For example, there may be differences in growth, functionality, or production of the product.

The microorganism of this invention may also be derived from an isolate or mutant of Clostridium autoethanogenum, Clostridium ljungdahlii or Clostridium ragsdalei, isolates and mutants of Clostridium autoethanogenum include JA1-1 (DSM10061) (Abrini, Arch. Microbiol., 161:345-351, 1994 ),

- 9 040778

LBS1560 (DSM19630) (WO 2009/064200) and LZ1561 (DSM23693). Clostridium ljungdahlii isolates and mutants include ATCC 49587 (Tanner, Int. J. Syst. Bacteriol., 43:232-236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (US 5593886) , C-01 (ATCC 55988) (US 6368819), O-52 (ATCC 55989) (US 6368819) and OTA-1 (Tirado-Acevedo, Bioethanol production from synthesis gas using Clostridium ljungdahlii, PhD thesis Sciences, North Carolina State University, 2010). Clostridium ragsdalei isolates and mutants include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).

The substrate refers to carbon and/or energy source for the microorganism according to this invention. Typically, the substrate is gaseous and contains a source of C1 carbon, such as CO, CO ₂ and/or CH ₄ . Preferably the substrate contains a source of C1-carbon or CO+CO2. The substrate may additionally contain other non-carbon components such as H ₂ , N 2 , or electrons.

Typically, the substrate contains at least some CO, such as 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mole % CO. The substrate may contain a CO range such as 20-80, 30-70 or 40-60 mole % CO. Preferably, the substrate contains 40-70 mol% CO (for example, steelmaking gas or blast furnace gas), 20-30 mol.% CO (for example, gas for the main oxygen-fed steelmaking furnace), or 15-45 mol.% CO (for example , synthesis gas). In some embodiments, implementation of this invention, the substrate may contain a relatively low amount of CO, such as 1-10 or 1-20 mol.% CO. Typically, the microorganism of the invention converts at least a portion of the CO in the substrate to a product. In some embodiments, implementation of this invention, the substrate does not contain or essentially does not contain (< 1 mol.%) CO.

The substrate may contain some H ₂ . For example, the substrate may contain 1, 2, 5, 10, 15, 20, or 30 mole % H ₂ . In some embodiments of this invention, the substrate may contain a relatively high amount of H ₂ , such as 60, 70, 80 or 90 mol.% H ₂ . In additional embodiments of this invention, the substrate does not contain or essentially does not contain (<1 mol.%) N ₂ .

The substrate may contain some CO2. For example, the substrate may contain 1-80 or 1-30 mol.% CO ₂ . In some embodiments of this invention, the substrate may contain less than 20, 15, 10 or 5 mol.% CO ₂ . In another embodiment of this invention, the substrate does not contain or essentially does not contain (< 1 mol.%) CO ₂ .

While the substrate is typically gaseous, the substrate can also be provided in alternative forms. For example, the substrate can be dissolved in a liquid saturated with CO-containing gas using a microbubble dispersion generator. As a further example, the substrate may be adsorbed onto a solid support.

The substrate and/or source of the C1 carbon may be an exhaust gas obtained as a by-product of an industrial process or obtained from some other source, such as from automobile exhaust or biomass gasification. In some embodiments of this invention, the industrial process is selected from the group consisting of the production of ferrous metal products, for example, steel production, non-ferrous metal production, oil refining processes, coal gasification, power generation, carbon black production, ammonia production, methanol production, and coke. In these embodiments, implementation of the present invention, the substrate and/or source of C1-carbon can be removed from the industrial process prior to its release into the atmosphere using any convenient method.

The substrate and/or source of the C1 carbon can be a synthesis gas, such as synthesis gas obtained from gasification of coal or refining process residues, gasification of biomass or lignocellulosic material, or reforming of natural gas. In another embodiment of the present invention, synthesis gas can be obtained from the gasification of municipal solid waste or industrial solid waste.

The composition of the substrate can have a significant impact on the efficiency and/or cost of the reaction. For example, the presence of oxygen (O2) can reduce the efficiency of an anaerobic fermentation process. Depending on the composition of the substrate, it may be desirable to treat, clean or filter the substrate to remove any undesirable contaminants such as toxins, undesirable components or dust particles and/or increase the concentration of the desired components.

The microorganism of this invention may be cultured to produce one or more products. For example, Clostridium autoethanogenum produces or can be engineered to produce ethanol (WO 2007/117157), acetate (WO 2007/117157), butanol (WO 2008/115080 and WO 2012/053905), butyrate (WO 2008/115080), 2, 3-butanediol (WO 2009/151342), lactate (WO 2011/112103), butene (WO 2012/024522), butadiene (WO 2012/024522), methyl ethyl ketone (2-butanone) (WO 2012/024522 and WO (WO 2012 /026833), acetone (WO 2012/115527), isopropanol (WO 2012/115527), lipids (WO 2013/036147), 3-hydroxypropionate (3-HP) (WO 2013/180581), isoprene (WO 2013/180584) , fatty acids (WO 2013/191567), 2-butanol (WO 2013/185123), 1,2-propanediol

- 10 040778 (WO 2014/0369152) and 1-propanol (WO 2014/0369152). In addition to one or more target products, the microorganism of this invention may also produce ethanol, acetate and/or 2,3-butanediol. In some embodiments of this invention, the microbial biomass itself may be considered a product.

A native product is a product that is produced by a non-genetically modified microorganism. For example, ethanol, acetate and 2,3-butanediol are native products of Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei. A non-native product is a product that is produced by a genetically modified microorganism but not produced by the non-genetically modified microorganism from which the genetically modified microorganism is derived.

Selectivity refers to the ratio of the production of the desired product to the production of all fermentation products that are produced by the microorganism. The microorganism of this invention can be designed to produce products with a certain selectivity or with a minimum selectivity. In one embodiment of this invention, the target product is at least 5, 10, 15, 20, 30, 50 or 75% of all fermentation products produced by the microorganism according to this invention. In one embodiment of this invention, the target product is at least 10% of all fermentation products produced by the microorganism of this invention, whereby the microorganism of this invention has a selectivity for the target product of at least 10%. In another embodiment of this invention, the target product is at least 30% of all fermentation products produced by the microorganism of this invention, whereby the microorganism of this invention has a selectivity for the target product of at least 30%.

Terms efficiency improvement, efficiency improvement, etc. include, but are not limited to, increasing specific growth rate, production rate or product volume, product volume per volume of substrate consumed, or product selectivity. Efficacy can be measured relative to the characteristics of the parent microorganism from which the microorganism of the present invention is derived.

The terms productivity or production rate represent the volumetric yield of the product. In continuous systems, the volumetric yield of the product is calculated as the ratio of the steady state concentration of the product and the settling time of the liquid. In batch systems, the volumetric yield of the product is calculated as the concentration and time required to obtain the specified concentration in the batch system. The volumetric yield of the product was recorded as g/l/day.

As a rule, the cultivation was carried out in a bioreactor. The term bioreactor includes a culture/fermentation device consisting of one or more vessels, racks, or tubing, e.g., agitated flow chemical reactor (FCRM), immobilized cell reactor (ICR), trickle bed reactor (ROS), bubble column, gas lift fermenter, static mixer, or other vessel or other device suitable for gas-liquid contact. In some embodiments of this invention, the bioreactor may comprise a first growth reactor and a second culture/fermentation reactor. The substrate may be placed in one or both of these reactors. In this context, the terms cultivation and fermentation are used interchangeably. These terms cover both the growth phase and the biosynthetic phase of the product during cultivation/fermentation.

Typically, the culture is usually maintained in an aqueous culture medium that contains the nutrients, vitamins and/or minerals necessary for the growth of the particular microorganism. Preferably, the aqueous culture medium is an anaerobic microbial growth medium, such as minimal anaerobic microbial growth medium. Suitable media are well known in the art.

Cultivation/fermentation is preferably carried out under suitable conditions for the production of the desired product. As a rule, cultivation/fermentation is carried out under anaerobic conditions. Reaction conditions considered include pressure (or partial pressure), temperature, gas flow rate, liquid flow rate, pH of the medium, redox potential of the medium, agitation rate (when using a stirred flow chemical reactor), inoculum level, maximum gas substrate concentrations to ensure that gas in the liquid phase does not become limiting, as well as maximum product concentrations to avoid product inhibition. In particular, the rate of introduction of the substrate can be controlled to ensure that the concentration of gas in the liquid phase does not become limiting, since the products can be consumed by the culture under gas limit conditions.

The operation of the bioreactor at elevated pressures makes it possible to increase the rate of transfer of the gas mass from the gas phase to the liquid phase. Accordingly, it is generally preferred to carry out the culture/fermentation at a pressure above atmospheric pressure. In addition, since this gas conversion rate depends in part on the retention time of the substrate, and the retention time determines the required volume of the bioreactor, the use of pressurized systems can

- 11 040778 significantly reduce the volume of the required bioreactor and, therefore, reduce the capital cost of equipment for cultivation/fermentation. This in turn means that the retention time, defined as the volume of liquid in the bioreactor divided by the inlet gas flow rate, can be reduced when the bioreactors are maintained at elevated pressure rather than at atmospheric pressure.

Optimum reaction conditions will depend in part on the particular microorganism used. However, in general, it is preferable to control the fermentation at a pressure above atmospheric pressure. In addition, since this gas conversion rate depends in part on the retention time of the substrate, and achieving the desired retention time in turn determines the volume of bioreactor required, the use of pressurized systems can significantly reduce the volume of bioreactor required and therefore reduce the capital cost of equipment for fermentation.

Target products can be separated or purified from the fermentation medium using any method or combination of methods known in the art, including, for example, fractional distillation, evaporation, evaporation through a semi-permeable membrane, stripping gas, phase separation and extraction fermentation, including, for example , liquid-liquid extraction. In some embodiments of this invention, the target products are recovered from the fermentation medium by continuously removing a portion of the medium from the bioreactor, separating the microbiological cells from the medium (conveniently by filtration), and recovering one or more target products from the medium. Alcohols and/or acetone can be recovered, for example, by distillation. Acids can be recovered, for example, by adsorption on activated carbon. Individual microbial cells are preferably returned to the bioreactor. The cell-free permeate remaining after removal of the desired products is also preferably returned to the bioreactor. Additional nutrients (such as B vitamins) can be added to the cell-free permeate to replenish the medium before it is returned to the bioreactor.

The term acid, acids, etc., when referring to the addition of acid to a culture or bioreactor in accordance with this invention, is widely used in this document and includes any monocarboxylic and dicarboxylic acids. Therefore, reference to the addition or production of equivalents should be used to include reference to the acid or mixture. For example, reference to the term acetate should be used to include acetic acid and vice versa. The ratio of molecular acid to carboxylate in the fermentation medium depends on the pH of the system. Examples of acids include acetic acid, propionic acid, n-butyric acid, n-pentanoic acid, n-hexanoic acid, and benzoic acid.

The present invention provides methods for improving fermentation efficiency. The inventors have found that the addition of certain amino acids in excess of the requirements of the cells to the microbial culture has a significant effect on the growth of the culture. In addition, the inventors have indicated that this excess addition of amino acids makes it possible to increase the production of fermentation products, especially fermentation products with high energy/ATP requirements.

The present invention provides methods for improving fermentation efficiency. The inventors have found that the addition of arginine in an amount exceeding the requirements of the cells to the microbial culture has a significant effect on the growth of the culture. In addition, the inventors have indicated that this excess addition of arginine makes it possible to increase the production of fermentation products, especially fermentation products with high energy/ATP requirements.

Without wishing to be bound by theory, it is believed that the growth rate enhancing effect of arginine supplementation is the result of ATP production from the catabolism of arginine by the microorganism. Arginine is catabolized by either the arginine deiminase (ADI) pathway or the arginine decarboxylase pathway.

Arginine is catabolized by arginine deiminase by the following mechanism:

Arginine ^- X ............... Xg ¹ _L

H ₂ o ammonium Phosphate L-orinitine ADP Ammonium n * ATP

The catabolism of arginine via the ADI pathway leads to the production of ammonium, CO2 and ATP.

The catabolism of arginine along the arginine decarboxylase pathway occurs according to the following mechanism:

Arginindecarb. oxidase

Putative agmatine deiminase

Putrescincarbamoyltransferase

... | Carbamos ✓'''''SG * lphosphate speA aguA and ίο _L 3.5.5.12 N-Carbamo

L-Arginine Agmatine _Ilputrescine

Ammonium

N

Phosphate PutrescineMDF^ _Carbama kinase

Ammonium ^ATP co.

2.7.2.2

The production of ATP obtained as a result of arginine catabolism makes it possible to increase the growth profile of the microorganism.

- 12 040778

Without wishing to be bound by theory, the authors believe that while arginine catabolism provides the microorganism's ATP requirement for its growth, arginine is not used as a carbon source by the microorganism, rather the carbon source used is the carbon component of the C1-containing gas.

Methods for microbial fermentation of C1 containing gaseous substrates to produce products such as ethanol and acetate are widely known in the art. Such methods provide a means for producing commercially viable fuels from industrial waste gases containing C1 carbon sources. Typically, these processes typically involve feeding a gaseous substrate containing, for example, CO2 into a bioreactor containing a culture of at least one acetogenic carboxydotrophic microorganism in a liquid nutrient medium. The gaseous substrate is subjected to anaerobic fermentation to produce alcohols, acids and mixtures thereof. The liquid nutrient medium used in the bioreactor typically contains various nutrients that support the growth of at least one acetogenic carboxydotrophic microorganism and are used in the metabolic pathways of one or more microorganisms to produce alcohols. Examples of such nutrients include MgCl, CaCl, KCl, H ₃ PO ₄ , Fe, Ni, Zn, Mn, B, W, Se, etc.

It is also known that Clostridium strains can be genetically engineered to produce a number of other beneficial products, including succinate, methyl ethyl ketone (MEK), 2-butanol, propanediol, 2-propanol, isopropanol, isoprene, acetoin, isobutanol, citramalate, butadiene, and also polylactic acid, acetone, butanol, isobutylene, 3-hydroxypropionate (3HP) and fatty acids.

Unexpectedly, the authors found the fact that increasing the concentration of arginine added to the microbial culture, increases the growth profile of the microorganism and increases the amount of ATP-rich products that can be produced by this culture.

One of the embodiments of the present invention includes adding arginine to the liquid nutrient medium, while the amount of arginine contained in the liquid nutrient medium exceeds the requirements of the C1-fixing microorganism cell. The addition of arginine in an amount exceeding the needs of the cell of the C1-fixing microorganism leads to an increase in the specific growth rate of the microbial culture. In some embodiments of the present invention, the specific growth rate of the C1-fixing microorganism is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70% compared to the microorganism without excess arginine.

The need for arginine of microorganism cells can be estimated by determining the amino acid composition of the biomass after hydrolysis of the biomass and analyzing the amino acids.

It has been demonstrated that by increasing the concentration of arginine in liquid nutrient media from a concentration equivalent to the requirement of cells to a concentration exceeding 1000 times (or more) the requirement for biomass synthesis, the doubling time of the microorganism decreased. In some embodiments of this invention, the doubling time of the culture obtained with an excess of arginine was reduced to 3.5 hours compared to the doubling time of 7.3 hours in a culture without excess arginine. Without wishing to be bound by theory, the authors also believe that increasing the concentration of arginine 2-80 times (or more) above the requirements of the cells increases the yield of ATP-rich fermentation products. In some embodiments of this invention, arginine is added to the culture in an amount exceeding the requirements for biomass synthesis from 2 to 1000 times, or from 2 to 800 times, or from 2 to 500 times, or from 2 to 100 times, or from 2 to 50 times , or 2 to 10 times, or 50 to 1000 times, or 50 to 800 times, or 50 to 600 times, or 50 to 500 times, or 50 to 300 times, or 50 to 200 times, or 50 to 100 times, or 100 to 1000 times, or 100 to 800 times, or 100 to 600 times, or 100 to 500 times, or 100 to 300 times, or 100 to 200 times.

With regard to the actual concentration, among the wide range of embodiments of the present invention presents an option in which the concentration of arginine in the liquid nutrient medium is from 20 mg/l to 20 g/l. In specific embodiments of this invention, the concentration of arginine is maintained at a level of at least 20 mg/l, or at least 100 mg/l, or at least 300 mg/l, or at least 500 mg/l, or at least 1 g/l, or at least 2 g/l, or at least 3 g/l, or at least 4 g/l, or at least 5 g/l, or at least 10 g/l, or at least 20 g/l. In some embodiments of this invention, the concentration of arginine is maintained at a level of from 20 mg/l to 20 g/l, or from 100 mg/l to 20 g/l, or from 500 mg/l to 20 g/l, or from 500 mg /l to 10 g/l, or from 1 to 10 g/l, or from 5 to 10 g/l, or from 5 to 20 g/l. In a certain embodiment of the invention, arginine is added to the culture such that the culture's arginine consumption is at least 20 mg arginine per gram cell dry weight, or at least 100 mg arginine per gram cell dry weight, or at least 1 g arginine per gram of cell dry weight, or at least 5 g of arginine per gram of cell dry weight, or at least 10 g of arginine per gram of cell dry weight. In some embodiments,

Arginine is added to the culture in such a way that the consumption of arginine by the culture is 20 mg to 20 g/g cell dry weight, or 100 mg to 20 g/g cell dry weight, or to 10 g/g dry weight of cells.

In some embodiments of this invention, at least 0.012 g of arginine is consumed by the culture to produce 1 g of biomass. In some embodiments of this invention, the need for cells in arginine for biomass synthesis is from 0.012 g/g biomass to 24 g/g biomass. In some embodiments of this invention, the requirement for arginine for biomass synthesis is at least 0.012 g/g biomass, or at least 0.024 g/g biomass, up to 0.048 g/g biomass, or at least 0.120 g/g biomass, or at least 0.24 g/g biomass, or at least 0.48 g/g biomass, or at least 1.2 g/g biomass, or at least 2.4 g/g biomass, or at least at least 4.8 g/g biomass, or at least 12 g/g biomass.

In some embodiments of this invention, increasing the concentration of arginine in the liquid nutrient medium from 2 to 80 times (or more) above the requirements of the cells increases the doubling time of the microorganism by at least 10%, or at least 20%, or at least 30 %, or at least 40%, or at least 50%, or at least 60%, or at least 70%.

When the concentration of arginine was increased to a concentration that was above the requirement of Clostridium autoethanogenum cells, the production of acetate by the microorganism was reduced. While not wishing to be bound by theory, the authors believe that the ability of Clostridium autoethanogenum to use arginine to produce ATP for growth negates the need for the microorganism to produce acetate for ATP uptake when arginine is added.

All acetate-producing acetogenic microorganisms have been described (Drake, Acetogenic Prokaryotes, in Prokaryotes, 3rd edition, pp. 354-420, New York, NY, Springer, 2006), since acetate production enables the microorganism to directly generate ATP by substrate phosphorylation via Pta (phosphotransacetylase) and Ack (phosphotransacetylase-acetate kinase). In particular, on an industrial scale, it is undesirable for microorganisms to produce acetate (or other organic acids required for the CoA transferase reaction) as a by-product, since acetate removes carbon from target products and thus affects the efficiency and volume of production of target products. In addition, acetate may be toxic to microorganisms and/or may serve as a substrate for the growth of contaminating microorganisms. Also, the presence of acetate makes it difficult to extract and separate the target products and control the fermentation conditions in favor of producing the target products.

The addition of arginine in excess of the cellular requirements of the Clostridium autoethanogenum culture resulted in a significant reduction in acetate production compared to a culture to which arginine was not added in excess of the cellular requirements. In addition, the authors demonstrated that the production of acetate is increased in a culture in which the source of arginine is completely depleted. In one embodiment of the invention, adding excess arginine to a microbial culture reduces acetate production by 20% or 30% or 40% or 50% or 60% or 70% or 80%.

The inventors have demonstrated that arginine is stoichiometrically converted to ornithine by being involved in the arginine deiminase pathway as the mechanism of arginine catabolism. This pathway converts arginine to ornithine, ammonium, ATP, and _CO2 . Increased growth will be facilitated by the supply of ATP and ammonium as a result of the breakdown of arginine. In one embodiment, the present invention provides a method for improving the stability of the fermentation process, wherein arginine is added to a microbial culture in the absence of an alternative nitrogen source.

It is well known in the art that nitrogen is required for the growth of a microorganism. Typically, nitrogen is added to the culture in the form of ammonium salt or ammonium hydroxide. As a rule, ammonia is obtained as a result of the Haber process, which is characterized by the following reaction:

Ν ₂ (γ) + 3H ₂ (g) 2ΝΗ ₃ (Γ) DN^ = -92 kJ mol-1

Currently, ammonia is produced mainly from natural gas and coal. In a typical process for producing ammonia from natural gas, hydrogen is supplied from natural gas and nitrogen is obtained from the atmosphere. Natural gas produces greenhouse gases, so while ammonia itself does not produce greenhouse gases, ammonia production does. It is desirable to find and use ammonia sources that are completely renewable. (http://www.agmrc.org/renewable_energy/renewable_energy/ammonia-as-a-transportation-fuel/). The cost of ammonium hydroxide (from 28.0 to 30.0 wt./wt.) is determined in the range of 9600 US dollars per 1000 kg.

Arginine (L-arginine) was first isolated from an extract of lupine sprouts in 1886. Later it was found that it is widely distributed in food and feed. Arginine can be obtained in a variety of ways, including protein hydrolysis, chemical synthesis, and microbiological synthesis. Most of the L-arginine is obtained by direct fermentation using renewable

- 14 040778 carbon sources (Jn.nutrition.org/content/134/10/2854S.full).

The identified path according to example 3 allows you to convert 1 mol of arginine to 3 mol of ammonia. Thus, arginine can provide an alternative source of nitrogen for bacteria, providing ammonia directly to the metabolism while maintaining the benefits described above. Since 1 molecule of arginine can be broken down into 3 molecules of ammonia, lower amounts required will result in significant cost savings as a result of the cheaper cost of arginine (99% nutritional value of arginine is about ~$17-18k/1000kg, while while the cost of 30% industrial ammonia is ~10-11 thousand US dollars / 1000 kg from Sigma Aldrich or Fisher) and will result in simplified handling. In addition, arginine can be obtained stable from biological sources, for example, by fermentation (T. Utagawa, J. Nutr., 2004, 134, 2854S-2857).

The arginine deiminase pathway is carried out through three enzymatic steps catalyzed by arginine deiminase (EC 3.5.3.6), carbomyltransferase (ornithinecarbomyltransferase, putrescinecarbomyltransferase) (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2). The corresponding enzymes have been identified in the genome of C. autoethanogenum.

In another aspect of the present invention, there is further provided a genetically engineered C1 fixing bacterium containing an optimized arginine deiminase pathway. In one embodiment, the invention provides a genetically engineered C1-fixing bacterium comprising one or more enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6), carbomyltransferase (ornithinecarbomyltransferase, putrescinecarbomyltransferase) (EC 2.1.3.3) and carbamate kinase (EC 2.1.3.3). EC 2.7.2.2), with each enzyme being an overexpressed endogenous enzyme, a mutated endogenous enzyme, or an exogenous enzyme. In specific embodiments of this invention, the C1-fixing bacterium is a Clostridium bacterium. In specific embodiments of this invention, the bacterium is Clostridium autoethanogenum.

Without wishing to be bound by theory, the authors believe that in order to increase the efficiency of the pathway, in particular if accumulation of intermediates such as citrulline is observed, the appropriate genes may be overexpressed by one of ordinary skill in the art, or genes from other sources may be introduced and heterologously expressed from using the methods described previously (WO 2012/053905, WO 2012/115527, WO 2013/180584).

The present invention further provides a method for producing at least one product from a substrate, said method comprising cultivating a genetically engineered C1-fixing bacterium containing one or more enzymes selected from the group consisting of arginine deiminase (EC 3.5.3.6) , carbomyltransferases (ornithinecarbomyltransferases, putrescinecarbomyltransferases) (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2), with each enzyme being an overexpressed endogenous enzyme, a mutated endogenous enzyme, or an exogenous enzyme.

In another aspect of the present invention, there is further provided a genetically engineered C1 fixing bacterium comprising one or more enzymes selected from the group consisting of ornithine racemase (EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12) and 2-amino-4-oxopentanoatethiolase. Without wishing to be bound by theory, the authors believe that any person skilled in the art can overexpress the corresponding genes or genes from other sources can be introduced and heterologously expressed using the methods described previously (WO 2012/053905, WO 2012/115527 and WO 2013-180584). The body can also be adapted and shaped to use ornithine more efficiently if it is adapted to arginine for dynamic growth. This is supported by the observed accumulation of ornithine.

The authors identified an arginine repressor that controls gene expression by binding to a palindromic operator sequence located at about 45 bp. above the start codon of arginine deiminase. The addition of arginine causes the repressor to unbind the operator sequence, allowing gene transcription in the direction of the 3' operator sequence. In one aspect of the present invention, a genetically engineered recombinant bacterium is provided comprising at least one heterologous gene, said heterologous gene being introduced in the direction of the 3'-argR binding operator sequence, wherein gene expression of the at least one heterologous gene can be activated by adding arginine.

The invention also provides a method for producing at least one product, said method comprising adding a carbon source to a culture containing a genetically engineered bacterium containing at least one heterologous gene, said heterologous gene being introduced in the 3'-argR direction. -binding operator sequence, providing the culture with arginine and fermenting the culture. In some embodiments of this invention, at least one heterologous gene is a heterologous gene in the biosynthetic pathway for said product.

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In one example, heterologous genes may encode a metabolic pathway that requires ATP to synthesize a product. For example, the mevalonate pathway is a heterologous pathway that converts acetyl-CoA to isopentenyl diphosphate at a cost of 3 moles of ATP equal to the cost of a mole of isopentenyl diphosphate. Using the described method, the expression of the heterologous mevalonate pathway can be activated by the addition of arginine, which will also provide ATP for the mevalonate pathway by cleaving arginine through the arginine deiminase pathway.

In another aspect, arginine can be used via the arginine decarboxylation pathway. Without wishing to be bound by theory, the authors believe that arginine can be decarboxylated to agmatine and CO2 by the enzyme arginine decarboxylase. Agmatine can then be converted to N-carbamoyl-putrescine by the enzyme agmatine deiminase, also producing ammonium. N-carbamoyl-putrescine plus phosphate can be converted to putrescine plus carbamoyl phosphate by the enzyme putrescinecarbamoyltransferase. Carbamoyl phosphate plus ADP is converted to ammonium + ATP + CO2 by carbamate kinase by the same mechanism as in the arginine deiminase pathway. The net yield of ammonium and ATP is that of the arginine deiminase pathway, but with two other intermediates (agmatine and N-carbamoyl-putrescine) and another by-product (putrescine). Putrescine is a by-product of greater value than ornithine or arginine and can be used as a feedstock for the production of various polymers including nylon-4,6 (Qian et al., 2009, Biotechnol. Bioeng, 104, pp. 651-662 ) and polyurethane.

In another aspect, the bacterium is modified to overexpress one or more endogenous genes, express another mutated endogenous gene, or heterologously express one or more exogenous genes encoding enzymes selected from the group consisting of ornithine racemase, ornithine aminomutase, subunit beta (OraE), ornithine aminomutase, alpha subunits (OraS), 2,4-diaminopentanoate dehydrogenase, 2,4-diaminopentanoate dehydrogenase, 2-amino-4-oxopentanoate thiolase, beta subunit, 2-amino-4-oxopentanoate thiolase, alpha subunit and functionally equivalent variants. Alternatively, the bacterium can be modified to express one or more exogenous genes selected from the group consisting of ornithine racemase, ornithine aminomutase, beta subunit (OraE), ornithine aminomutase, alpha subunit (OraS), 2,4-diaminopentanoate dehydrogenase, 2, 4-diaminopentanoate dehydrogenases, 2-amino-4-oxopentanoate thiolase, beta subunit, and 2-amino-4-oxopentanoate thiolase, alpha subunit. In preferred embodiments of this invention, the endogenous genes are derived from Clostridium sticklandii (FIG. 23). Without wishing to be bound by theory, the authors believe that when these genes are expressed, the results will lead to increased efficiency in the arginine deiminase pathway, especially when ornithine accumulation is observed in the parent strain. Alternatively, it is believed that the bacterium can be adapted and shaped to use ornithine more efficiently. In some embodiments of this invention, the bacterium is chosen to grow on arginine.

In another aspect, the bacterium contains one or more genetic modifications that disrupt the arginine:ornithine transporter. In one embodiment of the present invention, the genetic modification disrupts the expression of CAETHG3023 and CAETHG3024 (Genbank GeneID: 17336441 and 17336442, Genbank protein accession numbers: YP_008700482.1 and YP_008700483.1). Preferably, the genetic modification is a gene knockout mutation. Knockout of the arginine:ornithine transporter results in a decrease in the export of ornithine from the bacterial cell and promotes the metabolism of ornithine by the cell. In addition, the bacterium can be modified to express an alternative arginine transporter to import arginine without exporting ornithine. In alternative embodiments of the present invention, the bacterium can be modified to express an ornithine importer to allow recapture of excreted ornithine. Each of these approaches will increase the ability of the bacterium to metabolize ornithine.

Example 1 demonstrates the production of alanine from arginine by the microorganism C. autoethanogenum. In one aspect of the present invention, a method is provided for the production of one or more alanine-derived products, alanine-derived products, including 3-hydroxypropionate (3-HP) or acrylic acid. Acrylic acid is an important industrial product and is used in polymer flocculants, dispersants, coatings, paints, adhesives and binders for leather, paper and textiles with an estimated global demand of 5 Mt in 2014. 3-HP is a platform for acrylic acid, methylacrylic acid, acrylamide or 1,3-propanediol.

In one aspect of the present invention, there is provided a genetically engineered C1-fixing bacterium comprising at least one heterologous enzyme selected from the group consisting of enzymes for converting alanine to malonylsemialdehyde and 3-HP, enzymes for converting alanine to alanyl-CoA, enzymes for converting 3-HP to acrylyl-CoA, enzymes to convert alanyl-CoA to acrylyl-CoA, and enzymes to convert acrylyl-CoA to acrylate.

In specific embodiments of this invention, the C1-fixing microorganism is an acetogenic carboxydotrophic microorganism. Examples of suitable

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C1-fixing microorganisms include Clostridium, Moorella, Oxobacter, Peptostreptococcus, Acetobacterium, Eubacterium, or Butyribacterium. In various embodiments of this invention, the microorganism is selected from the group of microorganisms listed in table. 4.

Examples

The following examples further illustrate the invention, but of course should not be construed as limiting its scope in any way.

Table 1

PETC-MES medium without yeast extract

Component Quantity per 1 liter of medium nh ₄ ci 1 g KCI 0.1 g MgSO ₄ .7H ₂ O 0.2 g KN ₂ RO ₄ 0.2 g CoC1 ₂ 0.02 g 2-(/\/-morpholino)ethanesulfonic acid (MES) 20 g sodium acetate 0.1 g Fe (SO ₄ )x7H ₂ O 0.05 g Nitrilotriacetic acid 0.05 g Resazurin (mother solution 2 g/l) 0.5 ml Metal trace solution (see below) 10 ml Wolfe's vitamin solution (see below) 10 ml Reducing agent solution 1 (see below) 5 ml Reducing agent solution 2 (see below) 5 ml pH 5.6 Regulated with 4N NaOH Metal microimpurity solution per 1000 ml stock solution Nitrilotriacetic acid 2 g MnSO ₄ .H ₂ O. 1 g Fe (SO ₄ )x7H ₂ O 0.56 g _{CaCI2.6H2O _} 0.2 g _{ZnSO4.7H2O _} 0.2 g CuCI ₂ .2H ₂ O 0.02 g NaMoO ₄ .2H ₂ O 0.02 g Na ₂ SeO ₃ x5H ₂ O 0.03 g NiCl ₂ .6H ₂ O 0.02 g Na ₂ WO ₄ .2H ₂ O 0.02 g pH 7.6 Adjustable with 5M KOH Wolfe's vitamin solution per 1000 ml stock solution Biotin 2 mg Folic acid 2 mg Pyridoxine hydrochloride 10 mg Thiamine.HCI 5 mg Riboflavin 5 mg A nicotinic acid 5 mg Calcium U-(+)-pantothenate 5 mg Vitamin Βι ₂ 0.1 mg 4-aminobenzoic acid 5 mg Thioctic acid 5 mg Reducing agent 1 (storage under anaerobic conditions, in the dark) per 100 ml stock solution L-cysteine-HC! 4 g Reducing agent 2 (storage under anaerobic conditions, in the dark) per 100 ml stock solution NaOH 0.9 g (dissolve first) L-cysteine-HC! 4 g (dissolve after NaOH) Na ₂ S * 9 H ₂ O 4 g (dissolve after cysteine)

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table 2

Amino acid compositions with final concentrations in g/l

Amino acid 20AA 8AA 14AA 12AA 4AA Arginine

tryptophan 0.004 0.017 Tyrosine 0.069 Threonine 0.022 0.088 0.088 0.176 Valine 0.062 0.246 0.246 0.246 Proline 0.024 Alanine 0.029 Arginine 0.011 0.044 0.044 0.881 0.881 5,000 Aspartic acid 0.010 0.076 0.076 0.761 0.761 Asparagine 0.009 Cysteine 0.067 0.453 0.905 Histidine 0.011 0.042 0.042 0.849 0.849 Isoleucine 0.026 0.105 0.105 Glutam!nova acid 0.009 0.034 0.034 0.690 0.690 Glutamine 0.009 0.035 Glycine 0.033

Example 1 Identification of the preferred amino acids of acetonogenic C. autoethanogenum and increased growth rate with the addition of arginine.

This example demonstrates that arginine, histidine, aspartate, and glutamate are particularly preferred over other amino acids and are consumed at more than 80 times the amounts required for biomass synthesis by acetonogenic C. autoethanogenum DSM10061, and the addition of arginine to a specific medium allows C autoethanogenum grow with tD ~3 h, which is 3 times faster compared to the medium supplemented with yeast extract (YE).

Clostridium autoethanogenum DSM 10061 was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Inhoffenstraβe 7B, 38124 Braunschweig, Germany).

Yeast free PETC-MES medium was supplemented with various amino acid formulations: 20AA, 7AA, 8AA, 14AA, 12AA, 4AA and arginine (Table 2) to determine if any amino acids were growth supporting. Solutions were sterilized by filtration and made anoxic by N2 sparging.

First of all, 20AA medium was developed based on the amino acid composition of Clostridium acetobutylicum biomass (Lee et al. 2008) with final amino acid concentrations theoretically supporting the production of 1 g DCW/l biomass.

Growth studies were performed in serial cultivation in a volume of 50 ml of medium in 125 ml vials of serum using nitrogen in headspace with shaking (unless otherwise indicated) at 37°C. Optical density (OD) was measured at 600 nm with a reading of ~0.5 outside the anaerobic chamber compared to a fully oxidized environment as a reference.

Organic acids, fructose and amino acids were analyzed by HPLC. Organic acids, carbohydrates, and alcohols were quantitated by ion-exclusion chromatography using an Agilent 1200 HPLC system and an Agilent Hiplex H (300x7.7 mm, PL1170-6830) column with a guard column (SecurityGuard Carbo-H, Phenomenex PN: AJO - 4490). Typically, sugars and alcohols were monitored using a refractive index detector (Agilent RID, G1362A) set to positive polarity and an optical block temperature of 40°C, while organic acids were monitored at 210 nm (Agilent MWD, G1365B) and /or using a refractive index detector. 30 μl of sample was injected onto the column using an automatic dispenser (Agilent HiP-ALS, G1367B) and the column temperature was maintained at 65° C. using a thermostatted column compartment (Agilent TCC, G1316A). Analytes were eluted isocratically using 4 mM H2SO4 at 0.6 ml/min for 26 min. Fructose, sucrose and glucose were analyzed separately at a column temperature of 15°C and using high purity water (18.2 MP-cm) as the mobile phase and eluted isocratically at a rate of 0.4 ml/min for 21 min. Chromatograms were integrated using a ChemStation (Dietmair S., Timmins N.E., Gray P.P., Nielsen L.K., Kromer J.O., Toward a quantitative metabolomics of mammalian cells: development of a metabolite recovery protocol, Analytical biochemistry, 2010, 404:155-164). Soder

- 18 040778 Amino acid content was analyzed and quantified as previously described (R.B. McQualter, C. Bellasio, L.K. Gebbie, L.A. Petrasovits, R.W. Palfreyman, M.P. Hodson, M.R. Plan, D.M. Blackman, S.M. Brumbley, L.K. Nielsen, Plant. Biotechnol. J ., 2015).

Although the maximum OD achieved on 20AA medium was slightly lower (~20%) than on yeast extract, an equally rapid growth was observed (doubling time tDΞ9h, growth rate (μ=0.077) at the start of sampling (Fig. 1 ).

The analysis of AA unexpectedly demonstrated the preferential and rapid use of aspartate, glutamate, serine, histidine and threonine by AA media (Fig. 2). The reserves of arginine in the environment were quickly depleted. More importantly, serine and threonine were consumed at more than 6 times and aspartate, glutamate, histidine and arginine at more than 20 times what is needed for biomass production, indicating their use for purposes other than inclusion. into biomass. It is noteworthy that the accumulation of alanine instead of consumption was observed on both media during growth. Based on these results, the following media at 2-fold and 4-fold higher concentrations were designed to give a more minimal medium, faster growth, and higher OD: 14AA medium, by eliminating six AAs, did not consume at all or consumed slowly, and 8AA medium, by reducing additional amino acids.

In 14AA medium with a 4-fold increase in AA concentration, a higher maximum OD was achieved compared to growth on yeast extract (Fig. 3). It is important to note that the maximum OD achieved on 8AA medium corresponds to that of 20AA medium, demonstrating that a more minimal AA composition can support good growth. An increase in AA concentration had a positive effect on the doubling time: faster and equivalently faster growth was achieved on 14AA and 8AA media, respectively, compared to yeast extract and 20AA medium (Fig. 4).

The AA analysis confirmed the previous results and re-emphasized the importance of the AAs of aspartate, glutamate, histidine, and arginine (FIGS. 5 and 6). It is noteworthy that arginine was the most preferred amino acid, since it was already depleted at OD=0.24. Among other AKs, series and threonine were used more quickly. Again, significant accumulation of alanine was observed on both media. Of interest is the fact that ornithine was also produced. Aspartate, glutamate, histidine, and arginine were consumed in more than 10 times the amount needed for biomass production, indicating their use for energy generation.

Based on these results, the following two media were designed to obtain faster growth: 12AA medium by eliminating glutamine and tryptophan from 14AA medium and increasing the concentrations of serine, threonine and cysteine by 8 times, and a medium consisting of only 4 identified AAs: aspartate, glutamate , histidine and arginine - with an 80-fold increase in concentration compared to the 20AK medium (an increase of 2 and 20 times compared to the 14AK medium); 4AA medium containing only the 4 upper AAs of aspartate, glutamate, histidine and arginine with an 80-fold increase in concentrations compared to 20AA medium.

The 4AK medium design maintained a higher maximum OD compared to 12AK medium and was consistent with the maximum OD obtained with yeast extract and 20AK medium (FIG. 7). In both 4AA and 12AA media, rapid growth - tD ~2.5 h - was measured up to OD ~0.3, after which growth slowed down. It is important to note that the achieved tD ~2.5 h is 4 times faster compared to the addition of 1 g/l yeast extract, which is usually used.

Rapid initial growth followed by a slowdown is due to arginine depletion. It can be seen from the AA analysis of the 12AA medium that arginine is the preferred AA and is completely depleted at OD=0.37 (Fig. 8). These data also indicate that arginine strongly stimulated growth along with glutamate, aspartate, and histidine, maintaining a tD of ~2.5 h, which was measured in the OD range of 0.1 to 0.3, while growth slowed after depletion of arginine in the range of OP from 0.3 to 0.37. There was a simultaneous accumulation of ornithine. Again, a significant accumulation of alanine was found.

Growth retardation after arginine depletion was also seen in 4AK medium (FIG. 9). During the catabolism of arginine, in addition to ornithine, an accumulation of citrulline was also found. In addition to the accumulation of alanine for the first time, small amounts (~0.3 mM) of accumulated lysine and valine, as well as an unknown peak (retention time 8.3 min) showed an increase in peak areas.

Because the initial rapid growth was unexpected, only a couple of samples could be used to calculate tD. Therefore, the BugLab biomass monitoring device was attached to the vial of serum on 4AK medium in order to continuously monitor the increase in biomass until the arginine in the medium was depleted. This experiment confirmed the rapid rise on 4AK to arginine depletion, since tD was calculated as ~2 h (FIG. 10).

Even the addition of arginine resulted in very good growth with an equivalent high maximum OD on both 4AA medium and yeast extract. Growth of C. autoethanogenum DSM10061 in yeast-free PETC-MES medium with 5 g arginine/l + 5 g fructose/l resulted in rapid initial growth at an early stage (tD ~3 h) followed by slower growth in the second stage after arga depletion

- 19 040778 nina (tD ~40 h) (Fig. 11).

Surprisingly, it was found that during the initial phase of rapid growth formed a small amount of acetate. The production of acetate is again associated with growth in the second, slower growth phase (FIG. 12).

In FIG. 13 demonstrates the rapid use of arginine. In addition to the accumulation of alanine, small amounts (~0.5 mM) of lysine and valine were obtained similarly to 4AK medium. The same unknown peak (retention time 8.3 min) observed on 4AK medium showed an increase in peak area after arginine depletion.

In order to obtain a more accurate value of tD on the medium with arginine, the BugLab experiment was carried out to continuously monitor the increase in biomass to the consumption of arginine. This experiment confirmed the rapid rise to arginine consumption since the tD value was calculated as ~3 h (FIG. 14). The faster growth of arginine compared to the yeast extract is shown in FIG. 15.

Example 2. Enhancement of autotrophic growth with the addition of arginine.

This example demonstrates an increased specific growth rate and less acetate production for the acetogenic Clostridium autoethanogenum DSM 23693 when autotrophically grown with the addition of arginine.

Clostridium autoethanogenum DSM 23693 (derivative of Clostridium autoethanogenum DSM 10061; US Patent 2013/0217096) was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Inhoffenstraβe 7B, 38124 Braunschweig, Germany).

Growth was carried out in PETC-MES without yeast extract (Table 1) using standard anaerobic methods (Hungate, Meth. Microbiol., 3B:117-132, 1969; Wolfe, Adv. Microb. Physiol., 6:107-146 , 1971) and using a CO/CO2/H2 gas mixture (composition: 50% CO, 20% CO ₂ , 2% H ₂ , 28% N2) with a headspace pressure of 22 psi.

To study the effect of adding arginine, 5 g/l of arginine was added to the medium, and the culture growth and production of metabolites during autotrophic growth were compared with the control without arginine.

Growth was then analyzed by measuring optical density with a Thermo Genesys 20 spectrophotometer. Metabolites such as acetate (acetic acid), ethanol, 2,3-butanediol, or lactic acid were measured by high performance liquid chromatography (HPLC) on an Agilent LC with coefficient detection refraction (RI) at 35°C. Samples were prepared by diluting 400 μl with 100 μl of a solution of 5-sulfosalicylic acid (1% w/v in 1 M sulfuric acid) followed by 3 minutes centrifugation at 14,000 rpm; the supernatant was transferred into a glass vial for analysis. Separation was performed by injecting 10 µl onto an Alltech IOA-2000 column (150 mm x 6.5 mm x 8 µm) at 0.7 ml/min and 65° C. under isocratic conditions using 5 mM sulfuric acid mobile phase.

The experiment was performed in two biological replicates with triplicate cultures (n=3) for each condition in a volume of 40 ml of medium in 1 L Schott flasks at 37°C with orbital shaking (120 rpm, orbital shaking). In each case, the acetogenic strain C. autoethanogenum DSM 23693 was pre-cultured in PETC-MES without yeast extract to an OD of 600 nm=0.3 and one pre-culture was used for inoculation.

In the first experiment, the following conditions were met: PETC-MES without yeast extract + synthesis gas, 22 psi (original OD 600 nm = 0.005) and PETC-MES without yeast extract + synthesis gas, 22 psi + 5 g arginine/l (baseline OD 600 nm=0.005).

In a repeat experiment, the culture was also inoculated into medium supplemented with arginine, but without the addition of CO/CO2/H2 gas (100% N2, 22 psi as headspace). This experiment consisted of the following three conditions: PETC-MES without yeast extract + synthesis gas, 22 psi (original OD 600 nm = 0.03), PETC-MES without yeast extract + synthesis gas, 22 psi square inch+5 g arginine/L (original OD 600 nm=0.003) and PETC-MES without yeast extract+5 g arginine/L (original OD 600 nm=0.03). The arginine-containing medium was inoculated to a lower initial density than the arginine-free medium to reflect the unexpectedly increased specific growth rate with arginine observed in the first experiment.

In both experiments, cultures with arginine grew surprisingly fast (4 doublings in less than 15 h, corresponding to a doubling of time tD=3.5 h and specific growth rate μ=0.198) to an OD of 600 nm®0.8, while control growth was much slower with doubling time tD=7.3 h and specific growth rate μ=0.095 (FIGS. 16, 17). Therefore, the addition of arginine reduced the doubling time and increased the specific growth rate of the culture during autotrophic growth by more than 50%.

A log-transformed autotrophic growth plot of C. autoethanogenum clearly demonstrates the decrease in doubling time as a result of the addition of arginine (FIG. 18). The calculated doubling time was tD=7.3 h±0.2 for autotrophic growth without the addition of arginine and tD=3.5 h±0.2 with the addition of arginine, which is 52% less.

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Both with and without arginine, the culture reached the same final density during autotrophic growth, indicating that arginine was not used as a carbon source, but only increased the specific growth rate (Figs. 16, 17). This was also confirmed in a culture that was not supplied with gas; in cultures supplemented with arginine but without CO/CO2/H2 gas mixture, no growth was observed within 48 hours (FIG. 17). This supports the hypothesis that arginine is not used as a carbon source under these conditions, but rather CO/CO2 is used as a carbon source and ATP is provided from arginine metabolism.

The autotrophic growth of acetogens is usually associated with the formation of acetate (acetic acid), since the formation of acetate generates ATP through substrate phosphorylation in the acetate kinase reaction, which is necessary for growth. All acetogens isolated to date have been found to produce acetate. However, the formation of acetate is undesirable from the point of view of the process, since it removes carbon from the target products and is known to be toxic to microorganisms even at low concentrations of a few percent (J. Ballongue, E. Masion, J. Amine, H. Petitdemange, R. Gay, Appl. Microbiol. Biotechnol., 1987, 26, 568-573; G. Wang, D. I. Wang, Appl. Environ. Microbiol., 1984, 47, 294-8). In the growth medium supplemented with 5 g arginine/l, surprisingly no acetate production was observed until the growth pause at OD 600 nm-0.8 and only at this point was acetate produced (Fig. 19).

The results of this set of experiments confirm that C. autoethanogenum can use arginine to produce ATP for growth and therefore does not need to produce acetate when arginine is added.

Example 3 Identification and optimization of arginine utilization routes.

This example demonstrates how arginine provides additional ATP to the cell and can enter the central metabolism of acetogens.

As shown in Example 1, arginine is stoichiometrically converted to ornithine. Also, at some time points, the accumulation of citrullines was observed. Without wishing to be bound by theory, the authors believe that this observation implies the arginine deiminase pathway as an existing mechanism.

This pathway converts arginine to ornithine, ammonium, ATP and CO2. Increased growth will be facilitated by the supply of ATP and ammonium as a result of the breakdown of arginine. This supply of ATP also eliminates the need for acetogens to produce acetates.

|bargy _NIN/ ^ J,';., ..... ..... ..................CO,

H ₂ O ammonium Phosphate L-orinitine ADP Ammonium n ⁺ 2H ⁺ ATP

The arginine deiminase pathway is carried out via three enzymatic steps catalyzed by arginine deiminase (EC 3.5.3.6), ornithine carbomyl transferase, (putrescine carbomyltransferase) (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2). In the C. autoethanogenum genome, the corresponding enzymes (including two genes for the arginine/ornithine/enzyme transporter), ornithine carbomyltransferase, were identified, with carbamate kinase present in multiple copies (Table 3). Enzymes capable of catalyzing the same reactions are also present in other organisms, including many acetogens, including C. ljungdahlii, C. scatologenes, C. drakei, and Acetonema longum (Table 4). This pathway is also present in C. stricklandii or E. coli.

Table 3

Identified genes/enzymes of the arginine deiminase pathway in C. autoethanogenum

Name Nucleotide sequence (Access number, Genbank Gen ID, locus tag) Amino acid sequence (Access number, Genbank protein ID) arginine deiminase 17336439, CAETHG_3021 AGY77224 ornithinecarbomyltransferase 17336440,CAETHG_3022 17334022, CAETHG_0591 AGY77225 AGY74820 arginine/ornithine transporter 17336441, CAETHG3023 AGY77226 17336442,CAETHG_3024 AGY77227 carbamate kinase 17336443, CAETHG_3025 AGY77228 17333852, CAETHG_0421 AGY74650 17333876, CAETHG_0445 AGY74674 17337050, CAETHG 3632 AGY77835 17335507, CAETHG 2081 AGY76300

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Table 4

Identified genes/enzymes of the deiminase pathway in other organisms

Arginine deiminase organism Access number Clostridium Ijungdahlii DSM Clostridium scatologenes Clostridium drakei Propionispira raffinosivorans Acetonema longum Clostridium perfringens Clostridium sticklandii Clostridium cadaveris Clostridium colicanis Caldisalinibacter kiritimatiensis Caloranaerobacter azorensis Halothermothrix orenii Filifactor alocis Dethiosulfovibrio peptidovorans Aminomonas paucivorans Clostridiis alibacter paucivorans Thermanaerovibrio velox Thermanaerovibrio acidaminovorans Peptoniphilus indolicus ADK13995.1 AKA70116.1 WP_032076790.1 WP_026329241.1 WP_040292494.1 WP_003479447.1 WP_013361144.1 WP_051196258.1 WP_002599585.1 WP_006311966.1 WP_035161638.1 WP_012635610.1 WP_014262361.1 WP_005659654.1 WP_006299755.1 WP_026893703.1 WP_006582950.1 WP_012870198.1 WP_004823213.1 Borrelia hermsii Borrelia hermsii YBT Borrelia hermsii Borrelia hermsii HS1 Enterococcus phoeniculicola Vagococcus lutrae Borrelia hermsii Borrelia parkeri Brachyspira alvinipulli Borrelia persica Borrelia hispanica Borrelia coriaceae Borrelia turicatae Fervidicella metallireducens Carnobacterium divergens Borrelia crocidurae Borrelia duttonii Borrelia duttonii Borrelia crocidurae Thermobrachium celere Enterococcus faecalis Enterococcus faecalis CBRDO1 Borrelia recurrentis ALDO1 Enterococcus faecalis Caloramator australicus Atopobacter phocae Clostridium sulfidigenes Borrelia anserina Tetragenococcus halophilus Enterococcus haemoperoxidus Streptococcus marimammalium Tetragenococcus muriaticus Enterococcus gallinarum Clostridium dakarense Borrelia miyamotoi Thermoanaerobacterium aotearoense Streptococcus parauberis Enterococcus casseliflavus Clostridium botulinum Streptococcus porcinus Enterococcus casseliflavus Borrelia miyamotoi Thermoanaerobacterium xylanolyticum Streptococcus ictaluri Streptococcus pseudoporcinus WP_038443653.1 AHH12878.1 WP_043924507.1 AAX17338.1 WP_010766746.1 WP_023606773.1 WP_025400143.1 WP_025375819.1 WP_028331136.1 WP_038363688.1 WP_038359270.1 WP_025408405.1 WP_011772777.1 WP_035377985.1 WP_034570618.1 WP_038442698.1 WP_038366702.1 WP_012538581.1 WP_014696655.1 WP_018660497.1 WP_048941938.1 ESU74366.1 WP_012539214.1 WP_027308491.1 WP_002410400.1 WP_008907964.1 WP_025729241.1 WP_035132876.1 WP_025419989.1 WP_014125833.1 WP_010761089.1 WP_018369865.1 WP_038024314.1 WP_029486596.1 WP_042277345.1 WP_020955199.1 WP_014757694.1 WP_003104748.1 WP_010748983.1 WP_011987187.1 WP_003083226.1 WP_005230691.1 WP_025443390.1 WP_013786994.1 WP_008087965.1 WP_007892646.1 WP_017413713.1

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Clostridium tunisiense | Ornithinecarbomyltransferase organism Access number Clostridium ljungdahlii Propionispira raffinosivorans Clostridium scatologenes Acetonema longum Clostridium senegalense Clostridium argentinense Clostridium argentinense Candidatus Cloacimonas acidaminovorans Clostridium senegalense Clostridium botulinum Staphylococcus haemolyticus Clostridium tunisiense Staphylococcus Simula ns Clostridium sporogenes Staphylococcus haemolyticus Staphylococcus carnosus Staphylococcus epidermidis Staphylococcus carnosus Clostridium drakei Staphylococcus capitis Bacillus rubiinfantis Staphylococcus carnosus Staphylococcus aureus Lactobacillus rennin ! Staphylococcus microti Streptococcus agalactiae Lactobacillus parabrevis WP_013239445.1 WP_019552618.1 WP 046066002.1 WP_004092025.1 WP_010298035.1 WP_039635916.1 WP_039634993.1 WP_015423864.1 WP_010293963.1 WP_041345924.1 WP_053016198.1 WP_017413712.1 WP_023016208.1 WP_058009868.1 WP_053030082.1 WP_012664052.1 WP_049386361.1 WP_053464819.1 WP_032079337.1 WP_002452600.1 WP_042357582.1 WP_046100679.1 WP_031891031.1 WP_057873389.1 WP_044360832.1 WP_000793624.1 WP_020089358.1 Carbamate kinase organism Access number Clostridium scatologenes Clostridium drakei Clostridium argentinense Clostridium carbonicivorans Clostridium drakei Clostridium scatologenes Thermoanaerobacter thermocopriae Thermoanaerobacter matranii Thermoanaerobacter italicus Caldanaerobacter subterraneus Caldanaerobacter subterraneus WP_029954845.1 WP_032079746.1 WP_039633500.1 WP_007061333.1 WP_032075357.1 WP_029162276.1 WP_028991790.1 WP_013149923.1 WP_012994659.1 WP_022588163.1 WP_011024959.1

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Acetonema longum Thermoanaerobacter siderophilus Hypothetical protein [Propionispira raffinosivorans Thermoanaerobacter thermocopriae Thermoanaerobacter wiegelii Thermoanaerobacter kivui Thermoanaerobacterium thermosaccharolyticum Caldisalinibacter kiritimatiensis Thermoanaerobacterium thermosaccharolyticum Eubacterium nodatum Thermanaerovibrio velox Thermanaerovibrio acidaminovorans Aminiphilus circumscriptus Clostridium argentinense Anaerovorax odorimutans Thermoanaerobacterium xylanolyticum Clostridium aerotolerans Fervidicella metallireducens Clostridium argentinense Clostridium senegalense Clostridium sphenoides Clostridial bacterial oral taxon 876 Clostridium perfringens Clostridium sp. KNHs214 Clostridium perfringens Thermoanaerobacterium aotearoense Clostridium celercrescens Alkaliphilus metaliredigens Clostridium botulinum Clostridium algidicarnis Clostridium botulinum Clostridium drakei Clostridium sticklandii WP_004092029.1 WP_006569095.1 WP_019552620.1 WP_054644772.1 WP_014062398.1 WP_049684636.1 WP_013298601.1 WP_006311968.1 WP_015312281.1 WP_034819233.1 WP_006584132.1 WP_012869022.1 WP_026369063.1 WP_039635912.1 WP_027398659.1 WP_013787463.1 WP_026892136.1 WP_035377990.1 WP_039636739.1 WP_010298030.1 WP_054791734.1 WP_021654611.1 WP_025648248.1 WP_035294990.1 WP_003457589.1 WP_014759446.1 WP_038281265.1 WP_049765320.1 WP_024932088.1 WP_029453364.1 WP_012669533.1 WP_032077363.1 WP_013361146.1

To improve the efficiency of the pathway, in particular if accumulation of citrulline intermediates is observed, the corresponding genes can be overexpressed or genes from other sources can be introduced and heterologously expressed by any person skilled in the art using the methods described previously (US 2013/344547 , US 2013/330809, US 2013/323820, US 2013/224838, US 2013/224839, US 2011/256600, US 2011/236941).

Ornithine itself can be further converted to alanine, which has been shown to accumulate in the same manner as in Example 1. This conversion also generates additional NADP (H) reducing equivalents as well as another ammonia molecule and a key building block of acetyl-CoA from CoA. .

As illustrated in FIG. 23, the conversion of ornithine to alanine and acetyl-CoA occurs via enzymatic steps involving ornithine racemase (EC 5.1.1.12), ornithine aminomutase (EC 5.4.3.5), 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12) and 2-amino-4 -oxopentanoatethiolase (EC 2.3.1.B10). Relevant enzymes have been described, for example, from Clostridium sticklandii or environmental samples, with homologues identified in C. autoethanogenum (Table 5).

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Table 5

Identified ornithine genes/enzymes in the alanine and acetyl-CoA pathway from C. sticklandii or environmental samples and homologues of C. autoethanogenum

Name Nucleotide sequence (Access number, P) of a gene in Genbank Gen, locus tag) Amino acid sequence (Access number, P) of the protein in Genbank) ornithine racemase 9854830, CLOST1288 SVN21408 ornithine aminomutase, subunit beta (Ornithine) 9854831, CLOST1290 17333626, CAETHG0193 SVN21410 AGY74426 ornithine aminomutase, alpha subunit (OraS) 9856217, CLOST1291 SVN21411 2.4- diaminopentanoate dehydrogenase CU695246 CAQ42978.1 2-amino-4-oxopentanoatethiolase, beta subunit CU695248 CAQ42980.1 2-amino-4-oxopentanoatthiolase, alpha subunit CU695247 CAQ42979.1

To increase the efficiency of the pathway, in particular if ornithine accumulation is observed, the appropriate genes of C. autoethanogenum can be overexpressed by any person skilled in the art, or genes from C. sticklandii or environmental samples according to Table. 5 can be introduced and heterologously expressed using the methods described previously (WO 2012/053905, WO 2012/115527, WO 2013/180584). The body can also be adapted and shaped to use ornithine more efficiently if it is adapted to arginine for dynamic growth.

A knockout of the arginine:ornithine transporter (CAETHG3023-24) may also be required to obtain metabolic flux into the pathway to prevent ornithine from leaving the cell. Such knockouts can be carried out by any person skilled in the art using the methods described previously (WO 2012/053905, WO 2012/115527 and WO 2013/180584). You may also need to add an alternative arginine transporter. An alternative approach to knockout of the arginine:ornithine transporter would be to administer an ornithine importer, so ornithine could be further metabolized.

The identified pathways were also modeled in genome scale reconstruction to demonstrate the effect of metabolism and validate the identified pathways.

Metabolism reconstruction in a genome scale model for C. autoethanogenum was created based on published methods (L.-E. Quek, L.K. Nielsen, Genome Inform., 2008, 21, 89-100; C.G. de Oliveira Dal'Molin, L.-E. Quek, R. W. Palfreyman, S. M. Brumbley, L. K. Nielsen, Plant Physiol., 2010, 152, 579-89 C. Licona-Cassani, E. Marcellin, L.-E. Quek, S. Jacob, L. K. Nielsen, Antonie Van Leeuwenhoek , 2012, 102, 493-502.). The genome scale model core was reconstructed using the annotation backbone of the SEED model (CS. Henry, M. DeJongh, A.A. Best, P.M. Frybarger, B. Linsay, R.L. Stevens, Nat. Biotechnol., 2010, 28, 977-82). The reconstruction retained all response attributes from the SEED model, including unique responses, composite identifiers, and response reversibility. The model was gapped manually in Excel (Microsoft Corporation) for ease of annotation and commenting, in particular, central metabolism and the pathways of arginine use defined above were specially selected in manual mode. From this gene-oriented database, a representative 2D-oriented SBML (System Biology Markup Language, http://www.sbml.org) response was generated using a Java application (Oracle Corporation). Constraint-based reconstruction and analysis were performed using the COBRA toolkit (http://opencobra.sourceforge.net/) (J. Schellenberger, R. Que, R.M. T. Fleming, I. Thiele, J.D. Orth, A.M. Feist, D.C. Zielinski, A. Bordbar, N. E. Lewis, S. Rahmanian, J. Kang, D. R. Hyduke, B. O. Palsson, Nat. Protoc., 2011, 6, 1290-307). A set of scenarios for constraint-based modeling was run in the MATLAB environment. Metabolic flow balance analysis (Orth et al., 2010) was used to calculate key nutrients for growth, and implicit score analysis was performed to investigate the positive effect of amino acids (AA) on Clostridium autoethanogenum and determine which AAs have the greatest effect on ATP production. Metabolic flow balance analysis (FBA) was used to calculate key growth nutrients (Fan et al., 2014; Song et al., 2008); analyzed the effects of AA addition on target product synthesis (Licona-Cassani et al., 2012) and, in combination with latent cost analysis (Hillier and Lieberman, 2010), determined the substrates that had the greatest effect on ATO production (Teusink et al., 2006).

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Conventional hidden price analysis may not be correct with AK. If the latent cost analysis is done under autotrophy conditions (i.e., zero uptake), it will show the opportunity cost of AA synthesis, i.e. resources that could be freed up if this AC was added to the environment. The most expensive AA synthesis is not necessarily the best substrate for ATP production. For example, some cleavage path may not be available.

To overcome this problem, a latent price correction analysis was performed. For each of the 20 AAs, a maximum flow of 1.4 mmol/g DCW/h was available while maximizing the amount of biomass production. This flux represents the observed maximum specific fructose uptake rate by C. autoethanogenum DSM 10061 during preliminary growth experiments on standard PETC-MES medium (including yeast extract (YE)) and fructose. ACs with no splitting path cannot use the maximum allowable flow and therefore will have zero hidden cost. Latent price analysis revealed nine AAs: glutamine, histidine (HIS), cysteine (CYS), threonine, aspartate (ASP), arginine (ARG), glycine, serine, and glutamate (GLU) - out of the usual 20 that C. autoethanogenum should prefer because they lead to faster growth.

Model-based AA prediction was validated on 20AA medium for eight of the nine expected AAs (FIG. 2) except for glycine. Notably, the uptake of ASP, GLU, HIS and ARG per unit of biomass was more than 20 times higher than needed for biomass synthesis. AA uptake per gram of biomass was compared with the expected biomass concentration (cell requirement) based on measurements made in Clostridium acetobutylcium (Lee et al., 2008).

Surprisingly, it was found that the uptake of ASP, GLU, HIS and ARG was more than 120 times higher than required for biomass synthesis on the additionally developed 12AK medium, and that these amino acids can significantly accelerate growth. This indicates the potential involvement of ASP, GLU, HIS and ARG in energy generation. Of interest is the fact that concurrent with arginine consumption in the fast growth phase, an accumulation of ornithine was detected (FIG. 8), indicating a potential involvement of the arginine deiminase (ADI) pathway in providing cells with additional ATP. It should be noted that the rate of initially observed rapid growth (tD=2.5±0.1 h) is very close to the calculated increase with 20 AK (tD=2.9 h).

Of interest is the fact that lower acetate production per unit of biomass was observed in media containing ASP, GLU, HIS and ARG (4AK medium) (8.2±0.2 mmol/g DCW) and in media containing only arginine. (ARG medium) (6.7±0.7 mmol/g DCW) in which arginine was in excess compared to yeast extract medium (36.6 mmol/g DCW). Acetate production was greatly increased after arginine depletion (46.5 ± 11.2 and 34.5 ± 9.3 mmol/g DCW for 4AK medium and ARG medium, respectively), demonstrating that the ability to catabolize arginine greatly reduces the need to produce acetate by acetogen.

Heterotrophic cultures of C. autoethanogenum grown on 12AK medium accumulated ornithine simultaneously with arginine consumption (Fig. 8). The same was observed in bioreactor experiments with 4AK medium and arginine-only medium with a high proportion (about 60%) of consumed carbon excreted as ornithine (FIGS. 22A, 22B). In addition, a significant carbon flux to CO ₂ was detected, as well as a noticeable accumulation of citrulline. Other less significant by-products included acetate, alanine, and ethanol. In general, significant carbon fluxes to ornithine, CO ₂ , and citrulline indicate that arginine was catabolized via the ADI pathway, attributing its growth-enhancing effect to ATP supply (FIG. 21). In addition, the complete stoichiometric conversion of arginine to ornithine, CO ₂ , and citrulline suggests that arginine was metabolized strictly for energy production and not for biomass synthesis. This is consistent with the result showing no growth observed when arginine alone is added to yeast-free PETC-MES media containing Schott bottles under N2 gas pressure.

The involvement of the ADI pathway in promoting faster growth was predicted in primary simulations in silicone on 4AK medium and on arginine-only medium. The calculated total specific rate of ATP production (qA _TF ; mmol ATP/g DCW/h) was about 6 times higher under the latter conditions (SIM 4 and 5) compared to calculations for fructose (SIM 1) as the sole carbon source (Table .6). The same was observed when the model was limited to experimentally determined substrate uptake and product secretion rate (excluding citrulline and CO2) in bioreactor experiments on 4AA medium (SIM 6 and 9) and on arginine-only medium (SIM 12 and 15). with a qA _TF of approximately 3-4 times higher than calculated for a fructose-only medium. The observed faster growth (tD ~ 3-4 h) on media with AA compared to the calculated growth on media without the addition of AA (tD ~ 14 h) can be explained by a significantly higher level of energy production as a result of substrate phosphorylation during arginine catabolism (carbamate kinase pathway ADI) and indirectly by generating proton propulsion from NH4+ production and excretion. The latter is also useful in reducing the cost of the bioprocess due to the reduced need to neutralize the pH when adding NH4OH.

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Table 6

Model calculation for predicted specific growth rate, ATP production rate and products during heterotrophic growth with and without arginine supplementation

SIM 1 SIM 4 SIM 5 SIM 6 SIM 7 SIM 9 SIM 10 SIM 12 SIM 13 SIM 15 SIM 16 Substrate specific uptake rate (mmol/g DCW/h) Fructose 1.40 1.40 1.40 1.99 1.99 1.48 1.48 1.43 1.43 1.40 1.40 aspartate 0.00 1.40 0.00 0.76 0.76 0.85 0.85 0.00 0.00 0.00 0.00 Histidine 0.00 1.40 0.00 0.13 0.13 0.41 0.41 0.00 0.00 0.00 0.00 Glutamate 0.00 1.40 0.00 0.63 0.63 0.63 0.63 0.39 0.39 0.00 0.00 Cysteine 0.00 1.40 1.40 3.95 3.95 2.09 2.09 1.30 1.30 1.06 1.06 Arginine 0.00 10.00 10.00 19.11 19.11 13.10 13.10 10.22 10.22 11.99 11.99

Specific product release rate (mmol/g DCW/h) Acetate 3.15 35.97 28.15 2.03 55.01 1.97 37.78 1.38 28.47 1D4 31.77 ethanol 0.00 0.00 0.00 0.89 0.00 0.82 0.00 0.97 0.00 0.65 0.00 Carbon dioxide 0.11 9.06 5.38 22.51 13.75 15.58 9.82 11.45 6.56 12.48 7.16 Ornithine 0.00 0.00 0.00 19.10 0.00 12.71 0.00 9.49 0.00 11.02 0.00 Alanine 0.00 0.00 0.00 0.89 0.00 0.71 0.00 0.66 0.00 0.69 0.00 lactate 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 citrulline 0.00 0.00 0.00 1.05 0.00 0.81 0.00 0.45 0.00 0.46 0.00 2,3-butanediol 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2-oxobutarat 0.00 0.00 0.00 0.48 0.00 0.27 0.00 0.03 0.00 0.00 0.00 Formamide 0.00 1.38 0.00 0.11 0.09 0.39 0.38 0.00 0.00 0.00 0.00 Specific growth rate (h ¹ ) 0.05 0.28 0.17 0.25 0.54 0.24 0.43 0.19 0.31 0.19 0.32 Total specific rate of ATP production (mmol/g DCW/h) 6.80 45.57 38.55 34.81 72.08 23.82 49.11 18.92 37.70 20.83 42.40

The rapid growth and low acetate synthesis realized with arginine supplementation is of value to the biotechnology industry, as reducing carbon flux into the unwanted acetate by-product and obtaining additional ATP from alternative pathways is essential to expand the spectrum of acetogen products.

Large-scale genome models can be used to estimate intracellular metabolic fluxes and calculate carbon, redox, and energy balances by limiting the model to experimentally measured data (Bordbar et al., 2014; Dash et al., 2016; O'Brien et al., 2015) . A scale model of C. autoethanogenum genome metabolism similar to that described by Marcellin (Low Carbon Fuels and Waste Gas Chemicals A Systems Approach to Understanding Energy Metabolism in the Acetonogen Model, Green Chem, 2016) was used. Genome scale model analysis was performed for heterotrophic experiments on AA medium and on arginine-only medium by performing flow balance analytical calculations as described above. The model was further limited by the specific substrate uptake rate and product secretion rate (excluding citrulline and CO ₂ ), as well as the cell tD value. ATP dissipation maximization (i.e. unaccounted for ATP costs, see below) was used as an objective function for performing BPA (without taking into account energy maintenance costs).

Rapid growth on 4AA medium and ARG medium was provided by approximately 3-4 times higher q _ATP (29.3±5.5 and 19.9±1.0 mmol ATP/g DCW/h for 4AK medium and ARG medium, respectively ( SIM 6 and 9; and SIM 12 and 15 in Table 6) compared to 6.8 mmol ATP/g DCW/h calculated for growth on fructose alone (SIM 1). observed on 4AK medium compared to ARG medium is explained by a 2-fold higher specific flux due to the ATP-producing acetate kinase reaction (Fig. 24).However, on both media, ~53% of ATP was produced via the ADI pathway (Fig. 24), indicating that the catabolism of arginine is completely

- 27 040778 reorganized the energy exchange. It is of interest that although arginine intake favors many bacteria (Abdelal, 1979; Adamberg et al., 2006; Lahtvee et al., 2011; Mehmeti et al., 2011), its contribution to energy production is variable.

The predicted profile of the optimal flux during heterotrophic growth on fructose and AA was also analyzed. Restricting the model to experimentally measured substrate uptake rates and previously calculated unaccounted for ATP costs and maximizing for biomass yield, these calculations predicted faster growth (4AK medium tD=1.4±0.2 h, SIM 7 and 10; ARG medium tD=2.2±0.1 h, SIM 13 and 16) compared with the values obtained in the experiment (tD=2.8±0.1 h and tD=3.7±0.0 h, respectively) . Furthermore, in the models, ornithine produced during arginine catabolism was catabolized to GLU, demonstrating that further metabolization of ornithine via the ornithine pathway described above is possible. The predicted faster growth thus results from the catabolism of GLU to pyruvate via the methylaspartate pathway (as noted above) and its further conversion to acetyl-CoA and acetate, resulting in a decrease in Fd and ATP.

Example 4 Growth on a medium with arginine as the sole source of nitrogen.

This example demonstrates the replacement of ammonium as a source of nitrogen by arginine.

Nitrogen is an important nutrient for bacteria, and ammonium is commonly used in enzymatic reactions and as a source of nitrogen in media formulations for acetogens known from the literature (M. Kopke, C. Held, S. Hujer, H. Liesegang, A Wiezer, A. Wollherr, A. Ehrenreich, W. Liebl, G. Gottschalk, P. Durre, Proc. Natl. Acad. Sci., USA, 2010, 107, 13087-92; J. Mock, Y. Zheng, A. P. Mueller, S. Ly, L. Tran, S. Segovia, S. Nagaraju, M. Kopke, P. Durren, R. K. Thauer, J. Bacteriol, 2015, 197, 2965-2980; H. Richter, M. E. Martin, L. T. Angenent, Energies, 2013, 6, 3987-4000; J. L. Cotter, M. S. Chinn, A. M. Grunden, Bioprocess Biosyst. Eng., 2009, 32, 369-80; M. Straub, M. Demler, D. Weuster- Botz, P. Durre, J. Biotechnol., 2014.). However, ammonium production is dependent on abundant energy supplies, predominantly natural gas or liquefied petroleum gases (LPG) via the Haber-Bosch process (www.essentialchemicalindustry.org/chemicals/ammoinia.html) and hence ammonium is not a sustainable source. In addition, ammonium ions affect the pH during fermentation.

The identified path according to example 3 allows you to convert 1 mol of arginine to 3 mol of ammonia. Thus, arginine can provide an alternative source of nitrogen for bacteria, providing ammonia directly to the metabolism while maintaining the benefits described above. Since 1 molecule of arginine can be broken down into 3 molecules of ammonia, lower amounts required will result in significant cost savings as a result of the cheaper cost of arginine (99% nutritional value of arginine is about ~$17-18k/1000kg, while while the cost of 30% industrial ammonia is ~10-11 thousand US dollars/1000 kg from Sigma Aldrich or Fisher) and will result in simplified processing. In addition, arginine can be obtained stable from biological sources, for example, by fermentation (T. Utagawa, J. Nutr., 2004, 134, 2854S-2857).

To demonstrate that arginine can be used as the sole source of nitrogen for the acetogen Clostridium autoethanogenum DSM 23693, the organism was grown on yeast-free PETC-MES medium without the use of 1 g/l ammonium chloride (NH ₄ Cl) with 0.33 g/l non-synthetic monohydrochloride L-arginine (Sigma Aldrich; A6969).

Example 5 The addition of arginine results in an increase in the production of unnatural products from heterologous pathways, in particular ATP consumption pathways.

This example demonstrates that the addition of arginine increases the production of non-naturally occurring heterologous pathways.

The production of several non-naturally occurring has been demonstrated in acetogens from gas (WO 2012053905, WO 2012115527, WO 2013180584, WO 2013185123, WO 2013191567). Typically, some level of acetate is observed as a by-product, as the body produces ATP as a result of substrate phosphorylation via the acetate kinase reaction. This is particularly the case for pathways that require ATP, such as, but not limited to, the pathway for the production of isoprene or other terpenoids (the mevalonate pathway) or the pathway for the production of fatty acid derivatives such as biodiesel (via fatty acid biosynthesis). However, this also applies to other fermentation pathways that do not directly require ATP, but also do not provide the same amount of ATP per acetyl-CoA as acetate formation, for example, but not limited to, isopropanol, acetone, butanol production pathways (via the ABE pathway ).

The production of isopropanol from the gas was modeled using flow balance analysis (FBA) in the C. autoethanogenum genome scale model (as described in Example 3). Heterologous pathways were added to the model (Table 3) and modeling for the maximum theoretical yield was performed with and without arginine addition (Table 4).

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Table 7

heterologous pathways

Mevalonate pathway and isoprene synthase Name the equation thiolase 2 Acetyl = CoA <=> CoA + Acetoacetyl = CoA 3-HMS-CoA synthase (S) = 3 = hydroxy = 3 = methylglutaryl = CoA + CoA <=> Acetyl = CoA + acetoacetyl = CoA 3-HM6-CoA reductase (NADH) (R) = mevalonate + CoA + 2 NAD + <=> (S) = 3 = hydroxy = 3 = methylglutaryl = CoA + 2 NADH 3-HMO-CoA-reduct for-reductase (NADPH) (R) = mevalonate + CoA + 2 NADP + <=> (S) = 3 = hydroxy = 3 = methylglutaryl = CoA + 2 NADPH Mevalonate kinase (ATP) ATP + (R) = mevalonate => ADP + (R) = 5 = phosphomevalonate Mevalonate kinase (STR) CTP + (R) = mevalonate => CDP + (R) = 5 = phosphomevalonate Mevalonate kinase (GTP) GTP + (R) = mevalonate => GDP + (R) = 5 = phosphomevalonate Mevalonate kinase (UTP) UTP + (R) = mevalonate => UDP + (R) = 5 = phosphomevalonate phosphomevalonate kinase ATP + (R) = 5 = phosphomevalonate => ADP + (R) = 5 = diphosphomevalonate Diphosphomevalonate decarboxylase ATP + (R) = 5 = Diphosphomevalonate => ADP + orthophosphate + isopentenyl_diphosphate + CO2 Isopentenyl diphosphate Delta- isomerase Isopentenyl_diphosphate <=> Dimethylallyl_diphosphate Isoprene synthase Dimethylallyl_diphosphate => Isoprene + PP Isoprene transport Isoprene (cyt) => Isoprene (ext) isopropanol route Name the equation thiolase 2 Acetyl = CoA <=> CoA + Acetoacetyl = CoA CoA transferase Acetoacetyl = CoA + Acetate <=> Acetoacetate + Acetyl-CoA a cetoa ceta tde carboxy laza Acetoacetate => Acetone + CO2 secondary alcohol dehydrogenase Acetone + NADPH => Isopropanol isopropanol transport Isopropanol (cyt) => Isopropanol (ext)

Table 8

ABP-analysis of the maximum theoretical production volumes of target isoprene or isopropanol products from CO and CO2/H2 with and without arginine addition in mmol/g DW/h

target product SO 60 mmol CO/g DW/h Product Acetate ethanol Isopropanol 5.061008 0 2.408488 Isoprene 1.967815 2.209201 3.935631

target product CO + arginine 60 mmol CO + 2 mmol arginine/g DW/h Product Acetate ethanol Isopropanol 6.41679498 0 2.041474197 Isoprene 2.576176316 2.254853944 4.152352632

target product CO + arginine 48 mmol CO + 2 mmol arginine/g DW/h Product Acetate ethanol Isopropanol 5.404593388 0 1.559776585 Isoprene 2.182613259 1.813013818 3.365226517

- 29 040778

target product CO2/H2 60 mmol CO2/H2/g DW/h Product Acetate ethanol Isopropanol 5.00758257 0 2.408488 Isoprene 1.26220128 2.209201 3.935631

target product CO2/H2 + arginine 60 mmol CO2/H2 + 2 mmol arginine/g DW/h Product Acetate ethanol Isopropanol 6.361290323 0 2.041474 Isoprene 1.83911141 2.254854 4.152353

target product CO2/H2 + arginine 48 mmol CO2/H2 + 2 mmol arginine/g DW/h Product Acetate ethanol Isopropanol 5.359773808 0 1.559777 Isoprene 1.586671154 1.813014 3.365227

Modeling demonstrated that the addition of arginine can significantly increase the maximum theoretical production of the target compounds of isopropanol and isoprene on both CO and CO2/H2. Maximum production of isopropanol from CO can be significantly increased by more than 20% from 5.06 mmol/g DW/h to 6.42 mmol/g DW/h even with a small amount of arginine uptake (2 mM, 0.34 g/l) . For ATP using the mevalonate pathway to produce isoprene from CO, the maximum production can be increased even more significantly - by more than 30%: from 1.97 to 2.58 mmol/g DW/h. This effect is even more pronounced with production from CO2/H2, when the maximum production of isopropanol can be increased by more than 25%: from 5.0 to 6.36 mmol/g DW/h, and the production of isoprene by 45%: with 1.26 to 1.83 mmol/g DW/h. This clearly demonstrates that arginine can be used to increase the production of non-naturally occurring target molecules.

The simulation also shows that when 2 mM arginine is used together, production even increases with less CO uptake, demonstrating that the addition of arginine increases the efficiency of carbon to produce the target molecule.

Example 6 Use of the Arginine Repressor as a Genetic Switch to Stimulate the Expression of Heterologous Pathways.

This example demonstrates that arginine can be used as a genetic switch to stimulate the expression of heterologous pathways.

All genes in the identified arginine deiminase pathway of Example 3 were found to cluster together, and the ArgR arginine repressor was also found. Operator sequences (DNA sequences to which the argR protein binds to prevent transcription of the gene) have been identified in a number of microorganisms, including E. coli (Tian et al., 1992, J. Mol. Biol., 226, p. 387-397) , while the binding site is usually preserved on bacterial lines (Makarova et al., 2001, Genome Biol., 2, p. 1-8).

The arginine repressor controls gene expression by binding to a palindromic operator sequence located at about 45 bp. above the start codon of arginine deiminase. The addition of arginine causes the repressor to unbind the operator sequence, allowing gene transcription in the direction of the 3' operator sequence. In an example of a possible application, heterologous gene expression can be activated by the addition of arginine. Heterologous gene expression will be suppressed by the argR protein if an operator sequence binding argR is added to the left region of the heterologous gene operon. Thus, gene expression can be activated by the addition of arginine.

In one example, heterologous genes may encode a metabolic pathway that requires ATP to synthesize a product. For example, the mevalonate pathway is a heterologous pathway that converts acetyl-CoA to isopentenyl diphosphate at a cost of 3 moles of ATP equal to the cost of a mole of isopentenyl diphosphate. Using the described method, the expression of the heterologous mevalonate pathway can be activated by the addition of arginine, which will also provide ATP for the mevalonate pathway by cleaving arginine through the arginine deiminase pathway.

Example 7 Optimization of the efficiency of co-utilization of arginine with CO and/or H ₂ and/or CO ₂ gaseous substrates.

Deregulation may be required to achieve efficient sharing of arginine with CO and/or H ₂ and/or CO ₂ gaseous substrates. This can be achieved by

- 30 040778 knockout of the ArgR arginine repressor described above to eliminate the repression of the arginine deiminase pathway genes. Such knockouts can be carried out by any person skilled in the art using the methods described previously (WO 2012053905, WO 2012115527, WO 2013180584). However, removal of the arginine repressor will not allow upregulation of heterologous gene expression by the addition of arginine as described above. An alternative method would be to remove the above-described operator-binding sequences in the left side of the arginine deiminase pathway operon, or to replace the left side of the arginine deiminase pathway operon, including the operator sequence and promoter sequence, with a constitutive or synthetic promoter. Such modifications can be carried out by any person skilled in the art using the methods described previously (WO 2012053905, WO 2012115527, WO 2013180584). Suitable constitutive and synthetic promoters include, but are not limited to, the Pfdx ferredoxin promoter, Ppta or Ptet or PIPL12 acetate kinase promoter, which have been previously described (US 20160160223; Nagaraju et al., Clostridium autoethanogenum genome editing using CRISPR/Cas9, Biotechnol Biofuels., 2016, 9:219).

Similarly, promoter regions of genes responsible for the use of CO and/or H ₂ and/or CO ₂ , such as the Wood-Ljungdahl cassette or the Hyt operon (Brown et al., Comparison of single-molecule sequencing and hybrid approaches for final processing of the genome of Clostridium analysis of CRISPR systems in industrially significant, Clostridia. Biotechnology for Biofuels, 2014, 7:40), can be replaced by constitutive and synthetic promoters or appropriate regulators using knockout or knockdown. Those skilled in the art will be able to identify such regulators from transcriptomic data, as described in Example 10.

Example 8 Alternative Arginine Use and Putrescine Production.

This example demonstrates an alternative route for the use of arginine, which may result in the production of putrescine as a by-product.

Another possible use of arginine is through decarboxylation of arginine instead of deamination: arginine can be decarboxylated to agmatine and _CO2 by the enzyme arginine decarboxylase. Agmatine can then be converted to N-carbamoyl-putrescine by the enzyme agmatine deiminase, also producing ammonium. N-carbamoyl-putrescine plus phosphate can be converted to putrescine plus carbamoyl phosphate by the enzyme putrescinecarbamoyltransferase. Carbamoyl phosphate plus ADP is converted to ammonium + ATP + CO ₂ by carbamate kinase by the same mechanism as in the arginine deiminase pathway. The net yield of ammonium and ATP is the same as in the arginine deiminase pathway, but with two other intermediates (agmatine and N-carbamoylputrescine instead of citrulline) and another by-product (putrescine instead of ornithine).

Putrescine is a by-product of greater value than ornithine or arginine and can be used as a feedstock in the production of various polymers including nylon-4,6 (Qian et al., 2009, Biotechnol. Bioeng., 104, p. 651- 662) and polyurethane (Dahiyat et al., 1993, J. Biomater. Sci. Polym., ed, 4, p. 529-543).

Table 9

Gene names EU number Identifiers Arginine decarb oxylase EU 4.1.1.9 AGY76455, CAETHG 2244 Agmatine deiminase EU 3.5.3.12 AGY76293.1, CAETHG_2074 Putrescinecarbamoyltransferase EU 2.3.1.6 EKU86922, WP 003731415.1, AGY76301 Carbamate kinase EU 2.7.2.2 AGY77835, CAETHG_3632

Example 9 Alanine production and conversion to 3-HP or acrylic acid via heterologous pathways.

This example demonstrates the conversion of alanine to 3-hydroxypropionate or acrylic acid.

The production of alanine from arginine by the microorganism C. autoethanogenum was demonstrated in Example 1. Alanya can be further converted into high value 3-hydroxypropionate (3-HP) products or acrylic acid.

The conversion of alanine to acrylate has been shown in Clostridium propionicum (R.K. Dalai, M. Akedo, C.L. Cooney, A.J. Sinskey (1980), Microbial Acrylic Acid Production Pathway, Biosources Dig 2:89-97). Conversion can occur via malonyl semialdehyde and 3-HP or via alanyl-CoA. Both 3-HP and alanyl-CoA can be converted to acrylyl-CoA and further to acrylate. Isolated C. propionicum genes/enzymes can be introduced and heterologously expressed by any person skilled in the art using the methods described previously to produce 3-hydroxypropionate or acylate (WO 2012053905, WO 2012115527, WO 2013180584).

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Example 10 Transcriptometric Analysis

Transcriptometric analysis of bioreactor cultures of a biological duplicate of C. autoethanogenum growing heterotrophically on 4AK medium; see table. 2) was carried out using RNA sequencing. During the balanced phase of growth and uptake of all 4AAs, approximately 35 ml of a culture of 0.3 g DCW/l was harvested, granulated (5000xg for 3 min at 4°C) and resuspended in 5 ml RNAlater (Qiagen). The sample was stored at 4°C overnight, centrifuged (4000xg for 10 min at 4°C) and the pellet was stored at -80°C until further processing. The frozen beads were thawed, total RNA was extracted, and mRNA libraries were prepared as described in Marcellin et al., 2013. Sequencing was performed using a sequencer, Illumina Hiseq-2000.

Gene expression profiles on 4AK medium were compared with previously published RNA seq data. the same strain of C. autoethanogenum (DSM 10061) grown heterotrophically on a standard PETC-MES medium (including YE) (Marcellin et al., 2016). Sequential reads of both heterotrophic conditions were truncated to avoid read errors and then aligned/genome-matched using TopHat2 with (Kim et al., 2013) two mismatches allowed for read alignment. Transcript abundance was estimated using the Cufflinks FPKM function using upper quartile normalization. CuffDiff was used to evaluate differentially expressed transcripts and Cuffhorm was used to normalize data, a q-value below 0.05 (using a false positive rate; Benjamini and Hochberg, 1995) was used to determine significant changes in gene expression.

Transcriptometric analysis using RNA sequencing (RNA sequencing) was performed to further provide evidence of the involvement of the ADI pathway at the level of gene expression. For this purpose, bioreactor cultures of the biological duplicate of C. autoethanogenum, growing heterotrophically on 4AK medium, were selected during the balanced growth phase. This RNA-seq dataset compared with a previously published data set (Marcellin et al., 2016) of the same strain of C. autoethanogenum (DSM 10061) grown heterotrophically (fructose) on a standard YE-containing PETC-MES medium.

Transcriptometric analysis proved the role of the ADI pathway, as all ADI pathway genes were more than 500-fold upregulated (q-values <0.001) when cells were grown on PETC-MES medium supplemented with 4AA compared to YE. In addition, more than 380-fold upregulation (q-values <0.001) of putative arginine-ornithine antiporter genes (CAETHG_3023 and _3024) was observed. Data obtained by the authors from four genes associated with the breakdown of citrulline to carbamoyl phosphate and ornithine in C. autoethanogenum demonstrate that ornithine carbamoyl transferase (CAETHG_3022) is the protein that catalyzes the flux (645-fold upregulation, q<0.001) of ARG catabolism in C. autoethanogenum because its isoenzymes were either downregulated (CAETHG_0449 and _0591) or showed no change (CAETHG_2082) between compared conditions. Similarly, of the five genes associated with carbamoyl phosphate cleavage to CO ₂ , carbamate kinase CAETHG_3025 appears to be the flux-catalysing protein (623-fold upregulation, q<0.001), as its FPKM value is 1000-fold higher than the FPKM values for its isoenzymes.

Example 11. Optimization of the efficiency of the inclusion of arginine in the central metabolism.

Transcriptometric analysis revealed genes that in native metabolism determine the flow of arginine use and inclusion in the central metabolism in the form of ornithine carbamoyl transferase (CAETHG_3022) and carbamate kinase (CAETHG_3025). To improve efficiency, these genes can be overexpressed with potent or inducible promoters, such as, but not limited to, the Pfdx ferredoxin promoter, the Ppta or Ptet acetate kinase promoter, or PIPL12, which have been previously described (US 20160160223; Nagaraju et al., Genome Editing). Clostridium autoethanogenum using CRISPR/Cas9, Biotechnol Biofuels., 2016, 9:219).

All references, including publications, patent applications, and patents, cited in this specification are incorporated herein by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and are set forth in this specification in its entirety. its completeness. Reference to any prior art in this specification is not and should not be taken as an endorsement that the prior art is part of the known public knowledge of science in any country.

In the context of the description of this invention (especially in the context of the following claims), the terms in the singular refer to both the singular and the plural, unless otherwise indicated or clearly contrary to context. The terms consisting, having, including, and containing are to be construed as non-limiting (i.e., meaning including, but not limited to) unless otherwise indicated. The listing of ranges of values in this specification is solely for the purpose of making it easier to refer individually to each value within the range, unless otherwise indicated, and each individual value is included in this specification as if it were presented separately.

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Claims (11)

All of the methods described herein may be performed in any suitable order, unless otherwise indicated or clearly inconsistent with the context. Any examples or exemplification (eg, such as) herein are intended solely to better explain the invention and are not intended to limit the scope of the invention unless otherwise stated in the claims. No wording in this specification is to be understood as indicating any element omitted from the claims as being essential to the practice of the invention. This document describes the preferred embodiments of the present invention. Variations of these preferred embodiments of the invention may become apparent to those skilled in the art upon reading the above description. The authors assume that qualified specialists, if necessary, will apply embodiments of this invention that differ from the specific options described in this document. Accordingly, this invention includes all modifications and equivalents of the subject matter set forth in the claims appended herein, in accordance with applicable law. Moreover, any combination of the elements described above in all possible ways is included in the scope of this invention, unless otherwise indicated in this document or this clearly contradicts the context. CLAIM 1. A method for improving the efficiency of the fermentation of a gaseous C1-containing substrate when obtaining a product, while this method includes a) introducing a stream of gaseous C1-containing substrate into a bioreactor containing a culture of C1-fixing microorganism in a liquid nutrient medium; And b) fermenting the culture to produce at least one product, wherein arginine is added to the culture as the sole source of nitrogen, and wherein the C1 fixing microorganism is a recombinant genetically modified microorganism containing the arginine metabolic pathway, and wherein the C1 fixing microorganism is Clostridium. 2. The method according to claim 1, characterized in that said arginine metabolism pathway includes the arginine deiminase pathway and/or the arginine decarboxylase pathway, wherein the arginine deiminase pathway includes one or more enzymes selected from the group consisting of arginine deiminase (EC 3.5 .3.6), ornithinecarbamoyltransferase (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2), and the arginine decarboxylase pathway includes one or more enzymes selected from the group consisting of arginine decarboxylase (EC 4.1.1.19), agmatine deiminase (EC 3.5 .3.12), putrescinecarbamoyltransferase (EC 2.1.3.6) and carbamate kinase (EC 2.7.2.2). 3. The method of claim 2, wherein said arginine pathway comprises one or more of an overexpressed endogenous enzyme, a mutated endogenous enzyme, or an exogenous enzyme. 4. The method according to claim 1, characterized in that arginine is added to the culture in an amount exceeding the needs of the culture cells up to 1000 times. 5. The method according to claim 1, characterized in that the concentration of arginine in the bioreactor is at least 20 mg/l. 6. The method according to claim 1, characterized in that the requirement of the culture cells is 0.012 g of arginine per gram of cell biomass. 7. Process according to claim 1, characterized in that said product is selected from the group consisting of ethanol, 2,3-butanediol, 1,3-butanediol, lactate, succinate, methyl ethyl ketone (MEK), butyrate, 2-butanol, 1 ,2-propanediol (1,2-PDO), 1-propanol, isopropanol (IPA), acetoin, isobutanol, isoprene, farnesene, bisabolene, pinene, limonene, acetone, 3-hydroxybutyrate, 2-hydroxyisobutyric acid (2-HIBA) , citramalate, butadiene, polylactic acid, 1-butanol, 3-hydroxypropionate (3-HP), benzoate, fatty acid ethyl esters, fatty acids and isobutylene. 8. The method of claim 1 wherein arginine is catabolized via the arginine metabolic pathway to produce ammonium, wherein the C1-fixing microorganism uses ammonium as a nitrogen source. 9. The method of claim 1, wherein said C1-fixing microorganism contains a disruptive mutation in the arginine:ornithine transporter. 10. The method of claim 1, wherein the C1-fixing microorganism comprises one or more genetic modifications in the arginine metabolic pathway, wherein the one or more genetic modifications are selected from the group consisting of (i) disruptive mutation of regulatory elements and (ii) substitution operator binding sites or native promoters to constitutive or synthetic promoters. 11. The method of claim 10, wherein said destructive mutation is -