NL2017636B1

NL2017636B1 - Methods for increasing production of carbapenem antibiotics and derivatives in bacteria

Info

Publication number: NL2017636B1
Application number: NL2017636A
Authority: NL
Inventors: Edward Bokinsky Gregory; Shomar Monges Helena
Original assignee: Univ Delft Tech
Priority date: 2016-10-18
Filing date: 2016-10-18
Publication date: 2018-04-26
Also published as: WO2018074916A1

Abstract

The present invention is in the field of production of carbapenem antibiotics by microorganisms, specifically bacteria. The present invention relates to an improved method for producing high levels of antibiotics, and specifically carbapenem antibiotics, a method for producing an engineered microorganism capable of producing the antibiotic and said microorganism, and a method for improving antibiotic production with an engineered microorganism.

Description

Methods for increasing production of carbapenem antibiotics and derivatives in bacteria

FIELD OF THE INVENTION

The present invention is in the field of production of carbapenem antibiotics by microorganisms, specifically bacteria .

BACKGROUND OF THE INVENTION

The present invention is in the field of production of antibiotics by microorganisms, specifically bacteria.

An antibiotic relates to an agent that either kills or inhibits the growth of a microorganism. At present most modern antibacterials are semisynthetic modifications of various naturally occurring compounds. There are many classes of antibiotic compounds. Some compounds are still isolated from living organisms, such as aminoglycosides, whereas many others are produced by chemical synthesis. Following screening of antibacterials against a wide range of bacteria, production of the active compounds is often carried in strongly aerobic conditions .

Antibacterial compounds may be classified on the basis of their origin into natural, semisynthetic, and synthetic. Another classification system is based on biological activity; in this classification, antibacterials are divided into two broad groups according to their biological effect on microorganisms: Bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth.

Antibiotics have many medical uses, such as treatment of a bacterial infection, of a protozoan infection, immunomod-ulation, and non-operative resource for patients who have non-complicated acute appendicitis. Also prevention of infection may be considered, such as of a surgical wound, and dental antibiotic prophylaxis.

The use of antibiotics to treat and cure infectious disease has removed one of the major causes of death to the human population. Recently, the antibiotic arsenal is losing its effectiveness, as resistant bacteria are beginning to spread, making infections that only a decade ago would be considered trivial often fatal. Furthermore, healthcare costs are rising worldwide. Any technology that might alleviate the upward pressure on healthcare costs would save lives.

Antibacterial antibiotics may also be classified based on their mechanism of action, chemical structure, or spectrum of activity; typically they target a bacterial function or growth process, such as a bacterial cell wall, and a cell membrane, and interference with essential bacterial enzymes. Bactericidal aminoglycosides may target protein synthesis (macrolides, lincosamides and tetracycline but are typically not a bacteriostatic. Further detailed categorization may be done .

In general a problem with present antibiotics is that microorganisms become resistant. In view thereof, and also in view of other treatments, novel antibiotics may need to be developed. While novel antibiotics have been discovered, the microbial producers are often species that are either difficult or impossible to culture in large-scale fermentations. This presents a severe hindrance to producing the antibiotics on an industrial scale, which is needed if the antibiotics are to find medical applications. Hence, alternative production methods, may be required to develop and produce antibiotics at an increased speed and frequency.

Various chemical classes of antibiotics exist, such as the beta lactam class. Members of this beta lactam class are carbapenems, penicillins and cephalosporins. An example of a carbapenem is thienamycin, a naturally derived product of Streptomyces cattleya.

In general, production of antibiotics and precursors thereof by microorganisms normally not producing the antibiotic is inherently cumbersome, as increased levels of antibiotics inside the microorganism kill the microorganism. So despite successes in identifying metabolic pathways for antibiotic production, improvements have only been established towards non-toxic or slightly toxic intermediate products.

Antibiotics may be produced within engineered bacteria; however, some antibiotics cannot be made commercially by these engineered bacteria because the host is not amenable to genetic engineering or industrial cultivation processes. In an alternative natural producers can be mutated or otherwise manipulated, but alterations to natural producers may not be sufficient to achieve industrially-relevant titers or productivity. In these instances it would be advantageous to make the antibiotic within a cultivation-friendly host such as Escherichia coli or Bacillus subtilis. However, these hosts are susceptible to antibiotics, limiting their application to antibiotic production.

Antibiotics are therefore not normally produced by susceptible hosts because they are toxic to the hosts, and this toxicity would impede production to high titers. It is therefore a problem to make antibiotics in bacterial species that are highly susceptible to the antibiotic. This problem has not been addressed yet to the knowledge of the inventors. Typically production of such antibiotic compounds is achieved in native producers, which have natural mechanisms to resist antibiotics. Even these native hosts do not produce large amounts as they are rarely totally resistant to their own products. In principle these natural mechanisms could be replicated in engineered strains. However, there is no guarantee these mechanisms would work in alternative production hosts such as E. coli.

Recently advances have been made in identifying potential production routes, such as for kynamycin.

Some documents recite carbapenem production routes.

For instance US2013/0065878 A1 recites cell-free systems for generating carbapenems. US5,871,922 A recites genes involved in the biosynthetic pathway of carbapenem, comprising: a) at least one of the genes carA, carB, carC, carD, carE, carF, carG, carH, b) DNA capable of hybridizing to any of the genes defined in a) and capable of functioning as such genes in the biosynthetic pathway of a carbapenem, c) DNA which is a) or b) above by virtue of the degeneracy of the genetic code. Polypeptides encoded by such DNA.

The present invention therefore relates to an improved method for producing high levels of antibiotics, and specifically carbapenem antibiotics, which solve one or more of the above problems and drawbacks of the prior art, providing reliable results, without jeopardizing functionality and advantages .

SUMMARY OF THE INVENTION

The present invention relates to an improved method of production of an engineered microorganism, capable of (3S,5S)-carbapenam production, according to claim 1.

The present invention enables production of antibiotics that are considered immediately toxic to a microorganism's cell, such as carbapenem antibiotics, which are derived from a common metabolite (3S,5S)-carbapenam. The present class of antibiotics (carbapenems) cannot yet be industrially produced via a biological process, and is produced using chemical synthesis, which greatly raises its cost to medical systems worldwide. A benefit of the present methods is the development of a process for producing an expensive class of antibiotics via inexpensive microbial synthesis. The development of such processes has caused antibiotic costs to dramatically plummet for other beta-lactam antibiotics. A further benefit is that the ease of genetic modifications enables the antibiotic production pathway to be readily modifiable by the addition or removal of other genes, enabling the production of derivatives the present antibiotics that evade resistance mechanisms. The term "gene" also refers to homologues thereof, and derivatives thereof. It is noted that this would not overcome the problem of resistance; it would however expand the repertoire of available drugs.

The present method improves the production of precursors of the carbapenem class of antibiotics, which are used extensively in the clinic; in an example Escherichia coli is manipulated such that production of beta-lactam antibiotics are made possible while avoiding cell lysis, which would normally cease antibiotic production.

Inventors have engineered the metabolism of Escherichia coli to produce a precursor to clinically-relevant antibiotics known as carbapenems. In an example inventors cloned genes from Pectobacterium carotovora species that produces a simple carbapenem, known as "Car" (l-carbapen-2-em-3-carboxylic acid). In addition inventors implemented several modifications to the strain that together have resulted in a 25-fold increase in total Car production. Several of these improvements are applicable for production of other carbapenem compounds, such as thienamycin and their derivatives.

Inventors also used several new approaches for generating antibiotics while preserving the metabolism of the production host, without which, productivity is considered to be severely limited by the toxicity of the antibiotic compound. These are approaches that are found generally useful for production of antibiotics in vulnerable species.

Inventors have found a way to produce sufficient Car to lyse the producing cells, thus providing a platform to test approaches to circumvent beta-lactam toxicity to cells that enable production of antibiotics to continue over time. The have combined feedback-resistant ProBA enzymes within a living system, resulting in increases in carboxymethylproline and antibiotic production. In addition inventors used a method for increasing malonyl-CoA concentrations to improve carboxymethylproline production. The also used timed iron feeding to delay Car production until a sufficient amount of biomass had been accumulated. Withholding iron (Fe) is found to render iron-dependent CarC enzyme inactive. Iron is added only once sufficient numbers of cells have accumulated in the growth medium: this leads to a 3.5-fold increase in final Car levels. Inventors inhibit lysis caused by beta-lactam antibiotics by inhibiting fatty acid synthesis. In addition, CarE is used specifically to improve CarC activity.

In a first step of the present engineering method at least one nucleotide sequence comprising genes carB, carA, and optionally carE and/or carC, encoded thereon, is provided. Subsequently a step of expressing the genes encoded on the at least one nucleotide sequence in the microorganism is performed, and finally a step of culturing the microorganism in a medium. Therewith at least one nucleotide sequence comprising genetic codes is provided to the microorganism, which enables the microorganism to produce (3S,5S)-carbapenam.

In an exemplary biochemical production route of a microorganism a precursor molecule l-pyrroline-5-carboxylate (P5C) is converted into carboxymethyl proline. Thereto the CarB gene is provided into the present microorganism. Typically as a co-enzyme malonyl-CoA is required for this conversion. Next the carboxymethyl proline is converted into the precursor (3S,5S)-carbapenam. Thereto the CarA gene is provided into the present microorganism. Typically ATP is required for this conversion .

In an example the carbapenem antibiotics are derived from a common precursor, (3S,5S)-carbapenam. Examples thereof are mentioned below. For instance, for thienamycin further genes thnL, thnP, or thnK from Streptomyces cattleya are added.

In a further step 3S,5S)-carbapenam may be converted into carbapenem. Thereto the carC and/or carE gene is/are provided into the present microorganism. It is noted that in an aqueous environment carbapenem is typically unstable.

The present invention now makes it possible to produce antibiotics in bacterial species that are highly susceptible to the antibiotic, and thus are not normally produced by these susceptible hosts like E. coli, because they are toxic to the hosts, and this toxicity would impede production to high titers. The present method avoids exposure to the antibiotic to some extent while the cell remains vulnerable (during growth, and expression of antibiotic production enzymes). The present method helps surmount this barrier, which enables in principle production of several classes of antibiotics within hosts rather than native producers. This not only leads to lower production costs for antibiotics, but more importantly, it enables rapid development of production methods for novel antibiotics .

It is noted that according to the knowledge of the inventor the present problem has not even been addressed. As mentioned, typically production of antibiotic compounds is achieved in native producers, which have natural mechanisms to resist antibiotics. These natural mechanisms could in principle be replicated in engineered strains. However, these natural mechanisms may be overwhelmed (even in native producers) at high concentrations achieved of antibiotics or toxic precursors during production in an industrial fermentation. Furthermore there is no guarantee these mechanisms would work in alternative susceptible production hosts such as E. coli.

The present inventor has found that production of antibiotics in genetically-tractable and fast-growing species can be much faster and much cheaper than production in native strains, as genetic manipulation tools have been established. Growth is very rapid (enabling the quick production of large quantities of biomass), and there is much experience with using e.g. E. coli in industrial fermentation, indicating that boundary conditions per se for growth of E. coli are well known. In another aspect the production of antibiotics in E. coli enables a much quicker development of antibiotic production pathways after the discovery of the genes responsible for their production. This also enables a rapid diversification of antibiotics using biochemical diverse synthesis.

For better understanding of the biosynthesis routes, engineering of microorganisms, and details thereof, reference can be made to the a presentation by H. Shomar et al, 3rd Synthetic Biology Congress, London, United Kingdom. October 20, 2016, and a to be published paper by G. Bokinsky on the same topic, of which the contents are incorporated by reference.

In a second aspect the present invention relates to a microorganism, such as E.coli, S.cerevisiae and Bacillus sub-tilis, obtainable by the engineering method according to the invention .

In a third aspect the present invention relates to a method of producing a carbapenem compound, comprising the step of providing the microorganism of the present invention.

In a fourth aspect the present invention relates to a method for improving antibiotic production with an engineered microorganism, such as the microorganism of the present invention. Typically microorganisms, such as E. coli, rely on Fe (or Fe ions) when growing. It has now been found, which is unexpected in view of the Fe dependency, that if Fe is withheld for a period of time, starting when growth of the culture is initiated by inoculation, until sufficient cell biomass has been generated, such as during at least 0.5 hour in an initial biomass production stage, preferably during at least 1 hour, such as at least two hours, cell lysis can be prevented and production of the antibiotic is increased significantly. The amount of cell biomass may be considered sufficient at 5xl08-5x 109 cells per ml, which can be measured by optical density such as at 600 nm. A concentration of Fe in a medium is pref erably 10~6-10~4 mole/1 Fe, more preferably 5*10-6-5*10~5 mole/1, such as 10-5-3*10“5 mole/1. Likewise production of antibiotic is increased if cell growth is in at least one stage during production of the antibiotic inhibited.

In an example thereof the present antibiotic is produced in so-called growth-arrested cells; the cells are put in circumstances where amino acid starvation occurs. It is considered that non-growing cells show increased resistance to antibiotics. A method for creating non-growing cells that are still capable of producing antibiotics is to withhold a nutrient needed for growth, which nutrient is not critical for antibiotic production, such as amino acids. The removal of ProC is an example of this approach. ProC knockout cells are unable to make proline, and thus cannot grow without exogenously-provided proline. When a culture runs out of proline, growth will arrest as a consequence, and the cells will cease to grow; these cells are found to become more resistant to antibiotics (especially beta-lactam antibiotics, such as car-bapenems). Thus, cells can be grown in the presence of limiting proline, such that the cells will cease to grow, but continue to consume other nutrients available (e.g. glutamate, glucose, ammonia) which will be used to produce the carbapenem antibiotics (or other antibiotics). The proline concentration may be tuned to attain a desired amount of biomass (as biomass production is considered impossible without proline). The time at which cells are triggered to produce carbapenem antibiotics (as expressing the Car pathway requires proline, but activity does not) may also be tuned. This is also supported by timing Fe supplementation (i.e. by withholding Fe until proline is exhausted, ensuring no Car is produced until the cell is protected against Car by proline starvation). Thus production may be split into two phases: a growth phase, during which the cells use exogenous proline to produce biomass and express the Car pathway, and an antibiotic production phase, during which the cells have consumed all the available proline and are producing the Car antibiotic in a growth-arrested state that renders them resistant to the toxicity of the antibiotic.

Proline may be supplemented during the production phase. A phenomenon that has been observed so far is that the productivity (rate of amino acid production) of the growth-arrested cells during amino acid starvation decays over several hours, due to unknown factors. Supplementation of the limiting amino acid can restore productivity once again. Addition of very small amounts of proline during production phase is considered sufficient to restore productivity. Next to the present approach with proline (wherein the ProC knockout is present, limitation of other amino acids or nutrients whose lack causes immunity to beta-lactam antibiotics, is envisaged. This includes, but is not limited to, phenylalanine, tryptophan, tyrosine, leucine, isoleucine, valine, serine, glycine, alanine, and glutamine.

This approach is considered better than other triggers for growth arrest, as amino acid limitation can be readily engineered by proper formulation of growth medium used in fermentation, and does not rely on the expression of growth-arresting proteins (e.g. HipA, RelA), which depend upon the addition of expensive chemical inducers, and the maintenance of plasmids encoding the growth-arresting proteins.

In a fifth aspect the present invention relates to an antibiotic obtainable by the present invention. In an exemplary embodiment the antibiotic product may further comprises residual products of the production method. These residual products may provide further advantages, such as inherently a mixture of antibiotics may be produced, making the mixture (or cocktail) more effective.

Thereby the present invention provides a solution to one or more of the above mentioned problems and drawbacks.

Advantages of the present description are detailed throughout the description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in a first aspect to an engineering method according to claim 1.

In an exemplary embodiment of the present engineering method the microorganism is selected from fungi and bacteria, such as from Enterobacteriales, such as Escherichia, such as Escherichia coli, Bacillaceae, such as a Bacillus, such as Bacillus subtilis, and Fungi, such as Ascomycota and Basidiomy-cota, such as Saccharomycetes, such as S.cerevisiae. These genera and species have been found especially suited for engineering genes thereof.

In an exemplary embodiment of the present engineering method the at least one nucleotide sequence additionally comprises proA and proB. In an initial step of the present syntheses glutamate (Glu) may be converted into glutamyl-5-phosphate (G5P). Thereto the ProB gene is provided into the present microorganism in order to support said conversion. In a further step of the present syntheses glutamyl-5-phosphate (G5P) may be converted into L-glutamate-5-semialdehyde (GSH). Thereto the ProA gene is provided into the present microorganism in order to support said conversion. GSH may than release a water molecule in order to convert to P5C in an equilibrium reaction.

In an exemplary embodiment of the present engineering method a glutamate kinase enzyme is co-expressed, and the glutamate kinase has been mutated to relieve feedback inhibition by proline, such as wherein the proB gene has been mutated to proB*. As such it has been found that yields of antibiotics can be increased. One proB mutation that has been shown to relieve feedback inhibition by proline is I69E, though other mutations that relieve feedback inhibition, such as N134D, K145A, may also increase yields of antibiotic.

In an exemplary embodiment of the present engineering method the genes carB, carA, and optionally at least one of carE, carC, proA, proB, and proB*, are arranged in at least one operon, preferably an operon in a plasmid, preferably in the same plasmid. Likewise the genes and operon may be incorporated in a chromosome of the microorganism, especially for production on industrial scale. As such it has been found easier to incorporate said genes into the microorganism. The optional genes may support the yield of the present antibiotic.

In an exemplary embodiment of the present engineering method the at least one nucleotide sequence further comprises at least one of genes carD, carF, carG, and carH, optionally arranged in the present operon. These further genes can typically be found in microorganisms, such as P. carotovorum or Streptomyces cattleya, and may be associated with production of carbapenem or precursors thereof.

In an exemplary embodiment of the present engineering method the proC gene has been removed. As such conversion of P5C to proline is significantly reduced and thereby antibiotic yield is increased.

In an exemplary embodiment of the present engineering method the at least one nucleotide sequence is extracted from P. carotovorum or Streptomyces cattleya. That is it is identified, isolated and extracted, as well as treated further, such as by PCR. Likewise the nucleotide sequence is produced by chemical synthesis.

In general for the engineering of microorganisms, as described above, the DNA of the microbial species, or likewise a combination of species, can be changed. After changing the DNA in an initial stage the microorganism is cultured in order to obtain a population. Said population may be used for further purposes, such as producing an antibiotic.

In a second aspect the present invention relates to a microorganism, such as E.coli, S.cerevisiae and Bacillus sub-tilis, obtainable by an engineering method according to the invention .

In a third aspect the present invention relates to a method of producing a carbapenem compound, comprising the step of providing the microorganism of claim 8, culturing the microorganism, and thereby producing at least one of a carbapenem (l-carbapen-2-em-3-carboxylic acid) precursor, (3S,5S) carbapenam, and carbapenem.

In an exemplary embodiment of the present production method the carbapenem compound is a carbapenem antibiotic, such as azabicyclo[3.2.0]hept-2-ene-2-carboxylic acids, such as 7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acids, such as thienamycin ((5R,6S)-3-[(2-Aminoethyl)thio]-6-[(1R)-1-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid), imipenem (5R,6S)-6-[(1R)-1-hydroxyethyl]-3-([2-[(iminomethyl) amino]ethyl}thio)-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, meropenem 4R,5S,6S)-3-(((3S,5S)-5-(Dimethylcarbamoyl)pyrrolidin-3-yl)thio)-6-((R)-1-hydroxy-ethyl )-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, ertapenem (4R,5S,6S)-3-[(3S,5S)-5-[(3-carboxyphenyl) carbamoyl]pyrrolidin-3-yl]sulfanyl-6-(1- hydroxyethyl)-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, doripenem (4R,5S,6S) -6-(1-Hydroxyethyl)-4-methyl-7-oxo-3-(((5 S)-5-((sulfamoylamino)methyl)pyrrolidin-3-yl)thio)-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, panipenem/betamipron (5R,6S)-3-{ [(3 S)-1-ethanimidoylpyrroli-din-3-yl]sulfanyl}- 6-[(1R)-1-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, biapenem (4R,5S,6S)-3-(6,7-dihydro-5H- pyrazolo[1,2-a][ 1,2,4]triazol-8-ium-6-ylsulfanyl) - 6-(1-hydroxyethyl)- 4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2- ene-2-carboxylate, razupenem (4R, 5S, 6S)-6-((R)-1-hydroxyethyl)-4-methyl-3-((4-((S)-5-methyl-2,5-dihydro-lH-pyrrol-3-yl)thiazol-2-yl)thio)-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, tebipenem (4R,5S,6S)-(Pivaloyloxy)methyl 3-((1-(4,5-dihydrothiazol-2-yl)azetidin-3-yl)thio)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate, lenapenem, and tomopenem ((4R,5S,6S)-3-[ (3S,5S)-5-[ (3S)-3-[[2-(diamino-methylideneamino)acetyl]amino]pyrrolidine-l-carbonyl]-1-methylpyrrolidin-3-yl]sulfanyl-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid) , or a derivative thereof, or an analogue thereof. Hence a large variety of antibiotics can be produced.

In an exemplary embodiment of the present production method Fe is withheld for a period of time, starting when growth of the culture is initiated by inoculation, until sufficient cell biomass has been generated, such as during at least 0.5 hour during bacterial growth, preferably during at least 1 hour, such as at least two hours, therewith preventing cell lysis, wherein the medium comprises 10~6-10~4 mole/1 Fe, more preferably 5*10~6-5*10”5 mole/1, such as 10~5-3*10~5 mole/1. The amount of cell biomass may be considered sufficient at 5x108-5x 109 cells per ml, which can be measured by optical density such as at 600 nm. Surprisingly and unexpectedly, as typically microorganisms as E. coli rely on Fe in their biosynthesis, reducing an amount of Fe in e.g. the growth medium in an initial stadium of antibiotic production, significantly increase an amount of carbapenem produced by about a factor 3-5. Likewise, when Fe is withheld fully hardly any carbapenem is produced, showing the dependency of the pro duction pathway activity within the microorganism on Fe during antibiotic production.

In an exemplary embodiment of the present production method cell growth is in at least one antibiotic production stage inhibited. It has been found that such inhibition enables antibiotic production to continue for longer, such as about 50-200% longer.

In an exemplary embodiment of the present production method a 10-fold improvement in production of Car is found by co-expressing the gene CarE (from the original operon).

In an example a several-fold improvement in production of Car is found by increasing precursor supply by coexpression feedback-resistant mutants of ProBA, which are not inhibited by proline and thus are found to supply the Car pathway with a higher concentration of precursor molecule P5C.

In an exemplary embodiment of the present production method an improvement is achieved in the precursor pool car-boxymethylproline by increasing the precursor malonyl-CoA.

In an exemplary embodiment of the present production method an improvement is achieved by preventing cell lysis when the antibiotic is produced; this is considered to prevent the antibiotic from halting production, thereby improving car-bapenam production.

In an exemplary embodiment of the present production method an improvement is achieved by a method for preventing cell lysis before sufficient biomass is generated: by withholding iron (Fe), CarC is considered to be kept inactive until a sufficient amount of cells have accumulated. This may prevent antibiotic accumulation, which may lead to cell lysis and prevent the accumulation of sufficient biomass that could enable high-titer production. Once enough biomass has accumulated, addition of iron is found to trigger Car production. Inventors have identified an optimum time for iron addition for maximum benefit.

In an exemplary embodiment the present production method comprises at least one further biological or chemical synthesis step of producing an antibiotic; i.e. one may start with the present biosynthesis and complete a full synthesis, towards a desired molecule, with at least one further biologi cal or chemical synthesis step. The present method may be considered to deliver an intermediate product in such a case.

By splitting the present method in various steps also alternative biosynthesis routes become directly available, starting from more common steps. Such makes the present method very versatile.

The above characteristics of the host cell provide specific advantages for the production of specific antibotics, such as carbapenems.

In a fifth aspect the present invention relates to an antibiotic product obtained by the present method.

In an exemplary embodiment the product further comprises residual products of the production method. These residual products may provide further advantages, such as inherently a mixture of antibiotics may be produced, making the mixture (or cocktail) more effective.

The one or more of the above examples and embodiments may be combined, falling within the scope of the invention.

FIGURES

Fig. 1: Car biosynthesis pathway.

Fig. 2: diagram of examples of the present invention su perimposed on the Car pathway.

Fig. 3: production of Car.

Fig. 4: Effect of Fe delay on Car production.

Fig. 5a,b: Operon construct.

Fig. 6a,b shows the chemical structures of carboxymethyl-proline (CMP) and hCar, respectively.

DETAILED DESCRIPTION OF THE FIGURES

Fig. 1: Integration of the Car synthesis pathway from P. carotovorum with E. coli proline synthesis pathway. The production of the carbapenam intermediate is considered relevant to synthetic pathways of other carbapenem antibiotics (side paths). The figure has been detailed through the description .

Fig. 2 shows an overview of steps modified that result in increases of Car or carboxymethylproline production.

In section 1 a relief of inhibition of ProBA by proline is achieved. In section 2 intracellular malonyl-CoA concentra tions are increased. Such can e.g. be done by inhibiting a fatty acid pathway, such as by timed starvation of the microorganism and/or by removing fatty acid synthesis enzymes from the microorganism. In section 3 CarC activity is regenerated with CarE. This is found to be Fe dependent. In section 4 ProC is removed to decrease competition for P5C. Therewith genes are expressed, a high cell mass is obtained, CarE/CarC are/can be activated, and production is increased (Fe).

Fig. 3 shows the production of Car per cell after 24 hours. Influences of various boundary conditions are studied. With Care, CarA, and CarB (CAB) and tricine added small amounts of Car were produced. With CarC, CarB, CarA, CarD and CarE (CBADE) and with CarC, CarB, CarA, and CarE (CBAE), i.e. with CarE, the production of Car significantly increased. Mutant ProBA* further increased the production of Car.

Figure 4: Effect of Fe delay on Car production. When no Fe was supplied hardly or no Car was produced (bottom line). Under conditions when Fe was supplied production gradually picked up over time (middle line). Unexpectedly, when supply of Fe was delayed for some time, initial production of Car stayed low, but when Fe was supplied production of Car increased rapidly. The vertical axis shows total Car production, whereas the horizontal axis shows time (hours).

Fig. 5a shows the naturally-occurring operon encoding enzymes for Car production as found in P. carotovorum. Therein three enzymes, CarA, CarB, and CarC, are found necessary for the biosynthesis of Car. The enzymes CarD and CarE are found to increase the production of Car.

Fig. 5b shows an exemplary embodiment of the present artificial, engineered, operon for E.coli. It is preferably encoded on a plasmid, preferably on the same plasmid; an alternative to the plasmid is a chromosome. Therein the genes proB*, proA, carA, carB, carC, and carE, are shown. ProB* is a mutant of E. coli glutamate kinase enzyme ProB in which feedback inhibition has been removed. ProA is E. coli glutamate semi-aldehyde dehydrogenase. The genes are turned on c.q. off at the same time.

The invention is further detailed by the accompanying example, which is exemplary and explanatory of nature and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.

EXAMPLE

Example of an experiment for carbapenem production.

Genetic manipulations.

The specific nucleotide sequences as used are provided in the appendices to this application.

Growth conditions.

Production of Car in E. coli from the present synthetic operon, is achieved using the following growth conditions .

Medium composition

Car production is carried out in a MOPS minimal medium, which composition was based on the standard recipe described in http: / /www. genome. wi sc . edu/resources/protocols/mopsmlnimal. htm.l. A 10X MOPS salts solution without iron was prepared according to the recipe above, except for the addition of FeS04 salts. The Car production medium was prepared using the iron-depleted MOPS salts, supplemented with 0.4% glucose, 28.5mM NH4C1 and 0,5% potassium glutamate. lOx MOPS-Fe Salts (1 L) A one litre solution was prepared having 83.72 g MOPS, 7.17 g Tricine, 29.2 g NaCl, 0.11 g MgCl2-7H20, and 28 pL of 20 g CaCl2. MCM-Fe (500 mL) A 500 ml solution was prepared having 50 mL 10X MOPS-Fe salts, 20 mL 10% glucose, 7.5 mL 1.9 M NH4CI, 5 mL K2HPO4, 1 mL K2SO4, 50 mL 5% potassium glutamate, and 366.5 mL millipore H20.

Culture conditions and iron supplementation

Freshly transformed Car producing strains are incubated overnight in a minimal medium without iron (supplemented with the required antibiotics) at 37°C and 250 rpm, the day prior the experiment.

Car production is carried out by inoculating 25mL of medium from the overnight cultures, and with a normalized ini tial cell density (monitored as optical density measured at 600 nm) of 0.06. Cultures are incubated at 37°C and 250 rpm in 250mL Erlenmeyer flasks to an OD600 of 0.5-0.6 followed by induction of the Car pathway with IPTG (0.25mM final concentration) . This high density was not found if the cultures are grown in the presence of iron, due to the leaky expression of the Car pathway. Simultaneously, a sterile solution of 28mg/mL FeSCy (-0.18 M/l) is freshly prepared and diluted 10-fold (-0.018 M/l); 25yL of the diluted iron sulfate solution is added at time of induction to each culture. The presence of Fe2+ ions (-1.8 *10“5 M/l) initiates the catalytic activity of the enzyme CarC.

Validation

To detect and quantify the levels of production of the intermediate CMP (carboxymethyl-proline) and carbapenem, an LC-MS analytical method was developed, based on HILIC chromatography. It is considered that as the end product carbapenem is chemically unstable, its hydrolyzed form hCar is detected.

Metabolite concentrations in the culture supernatant were measured using liquid chromatography/mass spectrometry (LC/MS). Samples of the producing cultures were taken at different time points and immediately centrifuged at maximum speed for 3 minutes. For each sample, the supernatant was collected and stored at -20°C until measurement. Prior analysis, 5yL of supernatant was transferred into a clean Eppendorf containing 195yL ACN with 0,1% formic acid. This is to ensure that the total amount of carbapenem present in samples would be hydrolyzed and converted to hCar, and to ensure optimal conditions for the chromatography. For measurement of CMP and hCar, liquid chromatography separation was conducted at 30°C with an Agilent ZORBAX HILIC Plus column (100-mm length, 2.1-mm internal diameter, 3.5 ym particle size) using a LC-MS system (Agilent) consisting of a binary pump (G1312B), an autosampler (G7167A), a temperature-controlled column compartment (G1316A), and a triple quadrupole mass spectrometer (G6460C) equipped with a standard ESI source. For each measurement 2yL of injection volume was used. The mobile phase was composed of 25mM ammonium formate (solvent A) and 100% ace tonitrile (solvent B). The metabolites were separated with a gradient from 95% to 60% of solvent B for 5 minutes at a flow rate of 0.5 ml/min, a subsequent gradient from 60% to 50% for 2 minutes at a flow rate from 0.5 mL/min to 0.6 mL/min was carried, 50% to 95% solvent B for 2 minutes at 0.7mL/min, followed by a hold at 95% solvent B for 2 min at a flow of 0.5mL/min.

Peaks were analyzed by mass spectrometry using ESI ionization in MRM mode. The precursor ions analyzed for each compound was determined by mass calculation based on the chemical formula. Biological controls were used to confirm that the peaks obtained (and the resulting product ions used for quantification and qualitative analysis) were exclusively present in the presence of the exogenous enzymes responsible for their synthesis and corresponding substrates.

Com- Formula Mass Pre- Pro- Dwell Frag- Collision Polarity Reten- pound cursor duct- mentor Energy(V) tion ____ion__ions______Time_ hCar C7H9N04 171.05 170 126 60 90 8 Negative 2.75 min _____100____8___ CMP C7HhN04 173.07 174.1 128 60 90 12 Positive 4.946 min ____ 114 __|_J2___

Table 1: analytical results.

The analytical results show that Car was produced in detectable amounts.

For the purpose of searching prior art the following section is added, representing a translation of the claims in English: 1. Method for the production of an engineered microorganism, capable of (3S,5S)-carbapenam production, comprising the steps of providing a microorganism, providing at least one nucleotide sequence comprising genes carB, carA, and optionally carE and/or carC, encoded thereon, expressing the genes encoded on the at least one nucleotide sequence in the microorganism, and culturing the microorganism in a medium. 2. Method according to claim 1, wherein the microorganism is selected from fungi and bacteria, such as from En-terobacteriales, such as Escherichia, such as Escherichia coli, Bacillaceae, such as a Bacillus, such as Bacillus sub-tilis, and Fungi, such as Ascomycota and Basidiomycota, such as Saccharomycetes, such as S.cerevisiae. 3. Method according to claim 1 or claim 2, wherein the at least one nucleotide sequence additionally comprises proA and proB. 4. Method according to claim 3, wherein a glutamate kinase enzyme is co-expressed, and the glutamate kinase has been mutated to relieve feedback inhibition by proline, such as wherein the proB gene has been mutated to proB*. 5. Method according to any of the preceding claims, wherein the genes carB, carA, and optionally at least one of carE, carC, proA, proB, and proB*, are arranged in at least one operon, preferably a plasmid operon. 6. Method according to any of the preceding claims, wherein the at least one nucleotide sequence further comprises at least one of genes carD, carF, carG, and carH, optionally arranged in the operon of claim 4. 7. Method according to any of the preceding claims, wherein the proC gene has been removed. 8. Method according to any of the preceding claims, wherein the at least one nucleotide sequence is extracted from P. carotovorum or Streptomyces cattleya. 9. Microorganism, such as E.coli, S.cerevisiae and Bacillus subtilis, obtainable by a method according to any of the preceding claims. 10. Method of producing a carbapenem compound, comprising the step of providing the microorganism of claim 8, culturing the microorganism, and thereby producing at least one of a carbapenem (l-carbapen-2-em-3-carboxylic acid) precursor, (3S,5S) carbapenam, and carbapenem. 11. Method according to claim 10, wherein the carbapenem compound is a carbapenem antibiotic, such as azabicy-clo[3.2.0]hept-2-ene-2-carboxylic acids, such as 7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acids, such as thienamycin ((5R,6S)-3-[(2-Aminoethyl)thio]-6-[(1R)-1-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid), imipenem (5R,6S)-6-[(1R)-1-hydroxyethyl]-3-([2- [(iminomethyl)amino]ethyl}thio)-7-oxo-l-azabicyclo[3.2.0]hept- 2-ene-2-carboxylic acid, meropenem 4R,5S,6S)-3-(((3S,5S)-5-(Dimethylcarbamoyl)pyrrolidin-3-yl)thio)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, ertapenem (4R,5S,6S)—3—[(3S,5S)—5—[(3— carboxyphenyl) carbamoyl]pyrrolidin-3-yl]sulfanyl-6-(1-hydroxyethyl)-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, doripenem (4R,5S,6S)-6-(1-Hydroxyethyl)-4-methyl-7-oxo-3-(((5 S)-5-((sulfamoylamino)methyl)pyrrolidin-3-yl)thio)-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, panipenem/betamipron (5R,6S)-3-{ [(3 S)-1-ethanimidoyl-pyrrolidin-3-yl]sulfanyl}- 6-[(1R)-1-hydroxyethyl]-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, biapenem (4R,5S,6S)-3-(6,7-dihydro-5H- pyrazolo[1,2-a] [1,2,4]triazol-8-ium-6-ylsulfanyl) - 6-(1-hydroxyethyl)- 4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2- ene-2-carboxylate, razupenem (4R,5S,6S)-6-((R)-1-hydroxyethyl)-4-methyl-3-((4-((S)-5-methyl-2,5-dihydro-lH-pyrrol-3-yl)thiazol-2-yl)thio)-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, tebipenem (4R,5S,6S)-(Pivaloyloxy)methyl 3-((1-(4,5-dihydrothiazol-2-yl)azetidin-3-yl)thio)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate, lenapenem, and tomopenem ((4R, 5S,6S)-3-[(3S,5S)-5-[(3S)-3-[[2-(diaminomethyl-ideneamino)acetyl]amino]pyrrolidine-l-carbonyl]-1-methyl-pyrrolidin-3-yl]sulfanyl-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid), or a derivative thereof, or an analogue thereof. 12. Method according to any of claims 10-11, wherein Fe is withheld for a period of time, starting when growth of the culture is initiated by inoculation, until sufficient cell biomass has been generated, therewith preventing cell lysis, wherein the medium comprises 10~6-10~4 mole/1 Fe, more preferably 5*10”6-5*10”5 mole/1, such as 10~5-3*10”5 mole/1. 13. Method according to any of claims 10-12, wherein cell growth is in at least one antibiotic production stage inhibited. 14. Method according to any of claims 10-13, comprising at least one further biological or chemical synthesis step of producing an antibiotic. 15. Method for improving antibiotic production with an engineered microorganism, such as the microorganism of claim 9, wherein Fe is withheld for a period of time, starting when growth of a culture is initiated by inoculation, until sufficient cell biomass has been generated, therewith preventing cell lysis, wherein the medium comprises 10“6-10“4 mole/1 Fe, more preferably 5*1(Γ6-5*10”5 mole/1, such as 10”5-3*10”5 mole/1, and/or wherein cell growth is in at least one antibiotic production stage inhibited.

Claims

A method for producing a engineered microorganism (3S, 5S) can produce carbapenam comprising the steps of providing a microorganism, providing at least one nucleotide sequence comprising genes carB, carA, and optionally carE and / or carC encoded thereon, expressing the genes encoded on the at least one nucleotide sequence in the microorganism, and culturing the microorganism in a medium.

The method of claim 1, wherein the microorganism is selected from fungi and bacteria, such as from Terobacteriales, such as Escherichia, such as Escherichia coli, Bacillaceae, such as a Bacillus, such as Bacillus subtilis, and fungi, such as Ascomycota and Basidiomycota, such as Saccharo mycetes, such as S. cerevisiae.

The method of claim 1 or claim 2, wherein the at least one nucleotide sequence additionally comprises prauw and proB.

The method of claim 3, wherein a glutamate kinase enzyme is co-expressed, and the glumatate kinase is mutated to alleviate feedback inhibition by pro-line, such as wherein the proB-qen is mutated to proB *.

A method according to any one of the preceding claims, wherein the genes carB, carA, and optionally at least one of carE, carC, proA, proB, and proB * provided in at least one operon, preferably a plasmid operon.

The method of any one of the preceding claims, wherein the at least one nucleotide sequence further comprises at least one of the genes carD, carF, carG, and carH, optionally applied to the operon of claim 4.

The method of any one of the preceding claims, wherein the proC gene is deleted.

The method of any one of the preceding claims, wherein the at least one nucleotide sequence is extracted from P. carotovorum or Streptomyces cattleya.

Microorganisms, such as E. coli, S. cerevisiae and Bacillus subtilis, obtainable by a method according to any one of the preceding claims.

A method for manufacturing a carbismatic compound, comprising the step of providing the microorganism according to claim 8, cultivating the microorganism, and thereby producing at least one of a carbapenem (1-carbapenem -2-em-3-carboxylic acid) precursor, (3S, 5S) carbapenam, and carbapenem.

The method of claim 10, wherein the carbapenem compound is a carbapenem antibiotic, such as azabi-cyclo- [3.2.0] hept-2-en-2-carboxylic acids, such as 7-oxo-1-azabi-cyclo- [3.2. O] hept-2-en-2-carboxylic acids, such as thienamycin- ((5R, 6S) -3 - [(2-aminoethyl) thio] -6 - [(IR) -1-hydroxyethyl] -7-oxo-1 - azabicyclo [3.2.0] hept-2-en-2-carboxylic acid), imipenem (5R, 6S) -6 - [(IR) -1-hydroxyethyl] -3 - ({2 - [(iminomethyl) amino] - ethyl} -thio) -7-oxo-1-azabicyclo [3.2.0] hept-2-en-2-carboxylic acid, mer-openem 4R, 5S, 6S) -3 - (((3S, 5S) -5- (Dimethylcarbamoyl) pyrrolidin-3-yl) thio) -6 - ((R) -1-hydroxyethyl) -4-methyl-7-oxo-1-azabi-cyclo [3.2.0] hept-2-en-2- carboxylic acid, ertapenem (4R, 5S, 6S) -3 - [(3S, 5S) -5 - [(3-carboxyphenyl) carbamoyl] pyrrolidin-3-yl] sulfanyl-6- (1-hydroxyethyl) -4- methyl 7-oxo-1-azabicyclo [3.2.0] hept-2-en-2-carboxylic acid, doripenem (4R, 5S, 6S) -6- (1-hydroxyethyl) -4-methyl-7-oxo-3 - (((5 S) -5 - ((sulfamoylamino) methyl) pyrrolidin-3-yl) thio) -1-azabicyclo [3.2.0] hept-2-en-2-carboxylic acid, pan-penem / betamiprone (5R 6S) -3 - {[(3S) -1-eth animidoylpyrrolidin-3-yl] sulfanyl} -6 - [(IR) -1-hydroxyethyl] -7-oxo-1-azabicyclo- [3.2.0] hept-2-en-2-carboxylic acid, biapenem (4R, 5S, 6S) -3- (6,7-dihydro-5 H -pyrazolo [1,2-a] [1,2,4] triazole-8-ium-6-ylsulfanyl) -6- (1-hydroxyethyl) -4- methyl 7-oxo-1-azabicyclo [3.2.0] hept-2-en-2-carboxylate, razupenem (4R, 5S, 6S) -6 - ((R) -1-hydroxyethyl) -4-methyl-3- ( (4 - ((S) -5-methyl-2,5-dihydro-1 H -pyrrol-3-yl) -thiazol-2-yl) -thio) -7-oxo-1-azabicyclo [3.2.0] hept- 2-en-2-carboxylic acid, tebipenem (4R, 5S, 6S) - (pivaloyloxy) methyl3 - ((1- (4,5-dihydrothiazol-2-yl) azetidin-3-yl) thio) -6 - (( R) -1-hydroxyethyl) -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-en-2-carboxylate, lenapenemen / tomopenem ((4R, 5S, 6S) -3 - [( 3S, 5S) -5 - [(3S) -3 - [[2- (diaminomethylideneamino) acetyl] amino] pyrrolidine-1-carbonyl] -1-methyl-pyrrolidin-3yl] sulfanyl-6 - [(IR) - 1-hydroxyethyl] -4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-en-2-carboxylic acid), or a derivative thereof, or an analogue thereof.

A method according to any of claims 10-11, wherein Fe is remembered for a period starting when growth of a culture is started by inoculation, until sufficient cell biomass is generated, thereby preventing cell lysis, wherein the medium is 10 "-6- 10 "4 moles / 1 Fe, more preferably 5 * 10" 6-5 * 10 "5 moles / 1, such as 10-5-3 * 10" 5 moles / 1.

The method of any one of claims 10-12, wherein the cell growth is inhibited in at least one antibiotic production phase.

A method according to any of claims 10-13, comprising at least one further biological or chemical synthesis step of producing an antibiotic.

A method for improving antibiotic production with a manipulated microorganism, such as the microorganism according to claim 9, wherein Fe is retained for a period starting when the growth of a culture is started by inoculation until sufficient cell biomass has been generated, thereby preventing cell lysis, the medium being 10 ~ 6-1CT4 mol / l Fe, more preferably 5 * 10 ~ 6-5 * 10 "5 mol / l, such as 10" 5-3 * 10 "5 mol / l 1, and / or wherein cell growth is inhibited in at least one antibiotic production phase.