WO2009061477A1

WO2009061477A1 - Production of gamma-hydroxybutyrate

Info

Publication number: WO2009061477A1
Application number: PCT/US2008/012583
Authority: WO
Inventors: George N. Bennett; Thomas Bryce Causey; Mary Lou Harrison; Ka-Yiu San
Original assignee: Rice University
Priority date: 2007-11-06
Filing date: 2008-11-06
Publication date: 2009-05-14

Abstract

Cell strains for the production gamma-hydroxybutyrate (GHB) and methods of production GHB are disclosed.

Description

PRODUCTION OF GAMMA-HYDROXYBUTYRATE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims priority to U.S. Provisional Application No. 60/985,979, filed November 6, 2007 and which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] This invention may have been partially funded by grants from the United States

Government. Therefore, the U.S. government may have certain rights in the invention.

REFERENCE TO A COMPACT DISK APPENDIX

[0003] Not applicable.

BACKGROUND OF THE INVENTION

[0004] Gamma-hydroxybutyrate (GHB), also known as 4-hydroxybutyrate (4OHB) or sodium oxybate, is a naturally-occurring substance found in the central nervous system, wine, beef, small citrus fruits, and almost all animals in small amounts. It is sold under the brand name Xyrem® to treat cataplexy and excessive daytime sleepiness in patients with narcolepsy, and historically was used as a general anesthetic, to treat conditions such as insomnia, clinical depression, narcolepsy, and alcoholism, and to improve athletic performance. It is also used illegally as an intoxicant, and was put on Schedule I of the Controlled Substances Act in March 2000, although when sold as the approved drug Xyrem®, it is considered Schedule III. Thus, it is one of several drugs which is listed in multiple schedules.

[0005] Cells produce GHB by reduction of succinic semialdehyde. This enzyme appears to be induced by cAMP levels, meaning substances that elevate cAMP, such as forskolin and vinpocetine, may increase GHB synthesis and release. People with the disorder known as succinic semialdehyde dehydrogenase deficiency, also known as gamma-hydroxybutyric aciduria, have elevated levels of GHB in their urine, blood plasma and cerebrospinal fluid. [0006] GHB is also produced as a result of fermentation and so is found in small quantities in some beers and wines, particularly fruit wines. However, the amount of GHB found in wine is insignificant and is usually not sufficient to produce significant effects.

[0007] In addition to its medical uses, there are many commercial uses for GHB, particularly its various polymers and copolymers. Further, there are several related chemicals that are also useful.

[0008] 4-Hydroxybutaldehyde, for example, is a prodrug for GHB. However as with all aldehydes, this intermediate compound is caustic, strong-smelling and foul-tasting — actual use of this compound as an intoxicant is likely to be very unpleasant and result in severe nausea.

[0009] Another prodrug of GHB is the lactone gamma butyrolactone (GBL), formed by dehydration of 4-hydroxybutyrate. GBL is rapidly converted into GHB by lactamase in the blood, or can be chemically converted to GBH by adding sodium hydroxide (lye) in ethanol or water. GBL is a common solvent and reagent in chemistry and is used as an aroma compound, as a stain remover, as a superglue remover, as a paint stripper, and as a solvent in some wet aluminum electrolytic capacitors. GBL was produced at a level of 230,000 tons/yr in 2004. However, in addition to chemical synthesis routes, GBL can easily be made from gamma- hydroxybutyric acid (GHB) by removal of water or by distillation from such a mixture.

[0010] Yet another prodrug of GHB is 1,4-Butanediol, which is also used industrially as a solvent and in the manufacture of some types of plastics and fibers. 1,4-Butanediol is converted into GHB by the enzymes alcohol dehydrogenase and aldehyde dehydrogenase. 1,4- butanediol can also be used for the synthesis of GBL.

[001 1 ] Thus, GHB and related chemicals are both medically and industrially important.

Methods of synthesizing each of these chemicals exist, but the synthetic methods rely on toxic chemicals and sustainable, cleaner methods would be preferred. Therefore, microbial methods of making GHB and related chemicals would be of great benefit.

DISCLOSURE OF THE INVENTION

[0012] A novel approach was developed to form the widely used chemical intermediate gamma-hydroxybutyrate (GHB) by a microbial conversion from sugars. Engineered microorganisms are designed to produce GHB from standard media (glucose) or other carbon sources. The introduction of additional genes from other organisms into a suitably constructed genetically engineered host strain will allow this new metabolic pathway to operate, allowing the production of GHB and derivatives from renewable biomass.

[0013] Generally, two steps (not necessarily in that order) are taken to produce GHB in a microorganism. First, an appropriate host strain is generated by introducing disruptions in genes devoted to normal metabolic pathways which lead to undesired products such as acetate, lactate, and succinate. Second, genes involved in the formation of 4-hydroxybutyrate are introduced and expressed.

[0014] The microbial formation of 4-hydroxybutyrate from glucose involves conversion of the Krebs cycle intermediate α-ketoglutarate to succinate semialdehyde (SSA) followed by conversion of SSA to gamma hydroxybutyrate (GHB). Alternatively, excess glutamine can be converted to 4-aminobutyrate and then to SSA per the GABA shunt.

[0015] A number of genes that have been cloned and sequenced and can be employed in the invention are listed in Table 1. Additional enzymes and their genes can be found by homology or name search of databases such as GenBank:

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is E. coli metabolic pathways and possible gene deletions to increase GHB production.

[0017] FIG. 2 shows the conversion of glutamine or alpha-ketoglutarate to SSA to GHB.

[0018] FIG. 3 shows GBH and its prodrugs and enzymes involved in their interconversions.

[0019] FIG. 4 Variant TCA pathway showing conversion of alpha-ketoglutarate to succinic semialdehyde and then to succinate.

[0020] FIG. 5 shows 4-hydroxybutyrate formed in samples 3 and 4. The higher value in 4 shows the added contribution of the second plasmid to the formation of 4-hydroxybutyrate.

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] The abbreviations used herein are presented below:

[0022] The terms "operably associated" or "operably linked," as used herein, refer to functionally coupled nucleic acid sequences.

[0023] "Reduced activity" or "inactivation" is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species. Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, and the like.

[0024] "Increased activity" is defined herein to be greater than wild type activity, preferably above 125% increase, more preferably above 150% increase in protein activity as compared with an appropriate control species. Preferably, the activity is increased 100-500%.

Increased activity can be achieved by mutating the protein to produce a more active form, a more stable form, or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Increased activity can also be achieved by removing repressors, adding multiple copies of a gene to the cell, up-regulating an existing gene, adding an exogenous gene, and the like.

[0025] The term "exogenous" indicates that the protein or nucleic acid is a non-native molecule introduced from outside the organism or system, without regard to species of origin. For example, an exogenous peptide may be applied to the cell culture, an exogenous RNA may be expressed from a recombinant DNA transfected into a cell, or a native gene may be under the control of exogenous regulatory sequences.

[0026] As used herein "recombinant" is relating to, derived from, or containing genetically engineered material.

[0027] A gene or cDNA may be "optimized" for expression in E. coli, or other bacterial species using the codon bias for the species. Various nucleotides can encode a single peptide sequence. Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides which encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species. [0028] In calculating "% identity" the unaligned terminal portions of the query sequence are not included in the calculation. The identity is calculated over the entire length of the reference sequence, thus short local alignments with a query sequence are not relevant (e.g., % identity = number of aligned residues in the query sequence/length of reference sequence). Alignments are performed using BLAST homology alignment as described by Tatusova TA & Madden TL (1999) FEMS Microbiol. Lett. 174:247-250. The default parameters were used, except the filters were turned OFF. As of Jan. 1, 2001 the default parameters were as follows: BLASTN or BLASTP as appropriate; Matrix = none for BLASTN, BLOSUM62 for BLASTP; G Cost to open gap default = 5 for nucleotides, 1 1 for proteins; E Cost to extend gap [Integer] default = 2 for nucleotides, 1 for proteins; q Penalty for nucleotide mismatch [Integer] default = -3; r reward for nucleotide match [Integer] default = 1 ; e expect value [Real] default = 10; W word size [Integer] default = 1 1 for nucleotides, 3 for proteins; y Dropoff (X) for blast extensions in bits (default if zero) default = 20 for blastn, 7 for other programs; X dropoff value for gapped alignment (in bits) 30 for blastn, 15 for other programs; Z final X dropoff value for gapped alignment (in bits) 50 for blastn, 25 for other programs. This program is available online at NCBI™ (www.ncbi.nlm.nih.gov/BLAST/).

[0029] Common restriction enzymes and restriction sites are found at the NEB® (NEW

ENGLAND BIOLABS®) and INVITROGEN® websites. ATCC®, AMERICAN TYPE CULTURE COLLECTION™ has an extensive collection of cell strains that are publicly available and are incorporated herein by reference.

[0030] As is known in the art, the nomenclature of enyzmes is quite varied and inconsistent, especially in bacteria and depending on who first studied an enzyme and in what context. When an enzyme is referenced by name, all proteins that perform the same reaction are included therein, regardless of how that protein may be named. Thus, reference to "4- hydroxybutyrate dehydrogenase" in the claims is intended to include all proteins that can catalyze the same reaction, including proteins such as SSAR, AKR7A2, AKR7A4, and AKR7A5.

EXAMPLE 1: A-KETOGLUTARATE DECARBOXYLASE

[0031] Two enzymes are needed in bacteria to convert alpha-ketoglutarate to succinate semialdehyde to GHB and three to convert glutamine to 4-aminobutyrate to succinate semialdehyde to GHB. We will discuss the shortest pathway herein because fewer manipulations are needed, but either (or both) can be used.

[0032] The first enzyme is alpha-ketoglutarate decarboxylase, which converts α- ketoglutarate to succinate semialdehyde (SSA). It was originally identified from Mycobacterium tuberculosis as sue A (encoding Rv 1248c), but was later shown to be alpha-ketoglutarate decarboxylase (kgd) and produces succinic semialdehyde per the variation on the TCA cycle shown in Fig. 4.

[0033] We have investigated this enzyme and found it to have low activity. Therefore, we also identified other genes with significant homology from several other species that could be expected to also have good activity (Note: some of the genes are annotated as acd (acyl coA dehydrogenase), but are presumably should be kgd). Such kdh (or acd) genes were identified in Bradyrhizobium BTAiI, Bradythizobium japonicum USDAI lO, and Shewanella putrefaciens genomes, as follows:

NC_000962 {Mycobacterium tuberculosis H37Rv; kgd at nucleotide 1389357-1393052), see also NP 215764

NCJ304463 (Bradyrhizobium japonicum USDAlJO; kdg at nucleotide 488300-491257), see also NP 767092

NZ_ AALJO 1000007 (Bradyrhizobium BTAiI ctg66; acd at 32384-34174), see also ZP 00861434

NZ_ AALBO 1000065 (Shewanella putrefaciens CN-32 ctg99; acd at 5299-7089), see also ZP 00815645

Q7M266_EUGGR (Euglena gracilis) (EST of Euglena gracilis,ELL00002370)

NC_008380 (Mesorhizobium loti; 637481-639277) (NOT FOUND- says its Rhizobium leguminosarum)

INSDC:AY054980; INSDC: SME591784; UniProt:Q8RM51 ; UniProt:Q92S86; UniProt:Q8RM51 ; UniProt:Q92S86; YP_766200.1 ;

[0034] The polymerase chain reaction (PCR) was used to replicate each kgd (or acd) gene, which was then cloned into various bacterial plasmids. These plasmids can co-reside with other plasmids expressing genes for enzymes that convert SSA to GHB, or more than one gene could be placed on a plasmid or on the chromosome by known methods. For the proof of concept studies described herein, we have left single genes on plasmids for ease of manipulation.

[0035] To construct the plasmids, PCR forward and reverse primers were designed with specific restriction enzyme sites at both ends of the genes above. The PCR products were inserted separately into the plasmid pTrc99A. The inserted genes were then PCR amplified from the pTrc99A constructs along with the ptrC promoter to insert into the plasmid pDHK29, using new specific restriction sites. The result was two sets of plasmids bearing the two different kgd (or acd) genes — a pTrc99A-based set and a pDHK29-based set. These various plasmid constructs allow flexibility in combining genes for the α-ketoglutarate to SSA step with genes for the SSA to GHB step, below.

EXAMPLE 2: GAMMA HYDROXYBUTYRATE DEHYDROGENASE

[0036] The second required enzyme converts SSA to GHB and is known from several bacteria, including Clostridia, as well as from animal and plant sources. Clones of some of these have been tested for activity. Original genes or superior modified versions based on the general structure of these and selected for performance under the best growth conditions would be used in the production of a series of plasmids containing the various genes, as described above.

AF026947 human succinic semialdehyde reductase (SSAR) gamma hydroxy butyrate dehydrogenase (GHBD) at L36817 Ralstonia eutropha gamma hydroxy butyrate dehydrogenase at AY044183 Arabidopsis thaliana gamma hydroxy butyrate dehydrogenase at AAC41425 Alcaligenes eutrophus gamma hydroxy butyrate dehydrogenase at L21902 Clostridium kluyveri gamma hydroxy butyrate dehydrogenase at AJ250267 Clostridium aminobutyricum [0037] Plasmids containing genes from various species encoding proteins to convert alpha-ketoglutarate or glutamine to succinate semialdehyde and that convert succinate semialdehyde to gamma hydroxybutyrate will be combined in various combinations and tested for GHB production.

[0038] The best producers of GHB will be selected for further optimization.

Optimization can include codon optimization for genes of other species, culture condition optimization, serial passage and selection of best producers, and/or additional genetic modifications. Optimization may also include more permanent genetic construction, e.g., by moving the best genes into the bacterial chromosome, optimizing the promoters or by combining genes into a single artificial operon.

EXAMPLE 3: HOST CELLS

[0039] In a preferred embodiments, the host for effective GHB production would also have mutations in pathways that siphon intermediates away from the desired pathway or that produce detrimental products such as acetate. In E. coli mutations to reduce acetate formation (ackA-pta, ackA, or pta), to reduce ethanol formation (adhE), to reduce lactate formation (idhA), and to reduce conversion of α-ketoglutarate to succinate {sucA, ynel, gabD) would thus be desirable.

[0040] Thus, in a preferred embodiment, the host cell is the tetra-mutant ΔackA-pta (or

ΔackA or Apia), ΔadhE, ΔldhA, and ΔsucA and/or Δynel and/or ΔgabD.

[0041] In the proof of concept studies completed so far, we have made the bacterial strains listed in the following table. The table also provides an indication of the amount of GHB produced in each strain and shows that the concept is generally applicable to make GHB and related chemicals.

EXPERIMENT [0042] VNO4 is MG 1655 with deletions of adhE, ldhA, sucA (kgd), and atpFH genes.

The gbd gene for gamma hydroxy butyrate dehydrogenase from Ralstonia eutropha is inserted into the backbone of pGEX2T which is a Pharmacia product with a tac promoter, CoIEl origin, and ampicillin resistance. The pDHK29ptrcBjacd plasmid contains the acd gene for acyl-CoA dehydrogenase from Bradyrhizobium japonicum USDA 1 10 inserted along with a ptrc promoter into the backbone of pDHK29 which contains an RSF 1030 mutant origin and expresses resistance to kanamycin.

[0043] Initial cultures were started from glycerol stocks in 50 mis of Super Broth (SB), shaking at 37C with antibiotics as noted: 1 = VNO4 (Km^s)

2 = VNO4yneI (Km^s)

3 = VNO4yneI (Km^s) (pGEX2T-GHBD) with Ap¹⁰⁰

4 = VNO4>τje/ (Km^s) (pGEX2T-GHBD) (pDHK29ptrcBjacd) with Ap¹⁰⁰ and Cm³⁵

[0044] After overnight growth, cultures were expanded to final OD_OOO of 0.1 into 400 mis of fresh SB and shaken at 33-34C until they had reached an OD₆₀₀ of 1.5. Cultures were centrifuged and the pellets were resuspended to give a final OD_δoo of 15. Nine mis of cell suspension were aliquoted to each of three 250 ml flasks. A stock solution of concentrated glucose was added to all cell suspension aliquots for a final concentration of 3.7 mM glucose. A I M stock of IPTG was added to all aliquots for all samples for a final concentration of 1 mM IPTG. Stock solutions of antibiotics were added for samples 3 and 4, to the same final concentrations noted above. A stock solution of SSA was added to samples 3 and 4 to a final concentration of 18.4 mM.

[0045] All flasks were shaken aerobically at 37C. Aliquots of 1.5 ml suspension were taken at 1 hour; the cells were pelleted and 0.5 ml of supernatant was acidified with 10 ul of 50% H2SO4 for analysis by GC, which allowed for quantification 4OHB. The product found eluted at the position of the 4-hydroxybutyrate standard in two GC chromatography systems. The remaining supernatant was filtered and analyzed by HPLC, which allowed quantification of glucose, succinate and other TCA cycle -related metabolites.

Results of Experiment

„ , Relative

Sample _n . v Peak Area

3 13975

4 20870 SB blank 1070

Samples 1 and 2 showed no peak. [0046] Although various embodiments of the method and cell of the present invention have been illustrated in the accompanying figures and described in the above description, it will be understood that the invention is not limited to the embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the spirit of the invention as set forth herein.

The following references are incorporated by reference in their entirety:

1. Bravo DT, et al., Reliable, sensitive, rapid and quantitative enzyme-based assay for gamma-hydroxybutyric acid (GHB). J Forensic Sci. 2004 Mar;49(2):379-87.

2. Schaller, M., Schaffhauser, M., Sans, N., and Wermuth, B. (1999). Cloning and expression of succinic semialdehyde reductase from human brain. Eur. J. Biochem, 256: 1056-

1060.

3. Sohling, Brigitte and Gottschalk, Gerhard (1996). Molecular Analysis of the Anaerobic Succinate Degradation Pathway in Clostridium kluyveri. Journal of Bacteriology, 178: 871-880.

4. Tian, J., Bryk, R., Itoh, M., Suematsu, M., and Nathan, C. (2005). Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: Identification of α-ketoglutarate decarboxylase. PNAS,

102: 10670-10675.

5. Green LS, et ai, Catabolism of alpha-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum: evidence for an alternative tricarboxylic acid cycle. J Bacteriol. 2000 May; 182(10):2838-44. 6. Henne, A., et al., Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Applied and Environmental Microbiology, 65: 3901-3907 (1999).

7. Kaneko, T., Nakamura, Y., Sato, S., et. al. (2002). Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDAI lO. DNA Research, 9: 189-197.

8. Fuhrer T, et al., Computational prediction and experimental verification of the gene encoding the NAD+/NADP+-dependent succinate semialdehyde dehydrogenase in Escherichia coli. J. Bacteriol. 189(22):8073-8 (2007).

What is claimed is:

Claims

1. An engineered bacteria having increased activity of:

(a) 4-hydroxybutyrate dehydrogenase, and

(b) glutamine decarboxylase and 4-aminobutyrate aminotransferase, or alpha-ketoglutarate decarboxylase,

so that said bacteria produces more gamma hydroxybutyric acid than said bacteria without said increased activities.

2. The engineered bacteria of claim 1, further comprising decreased activity of 2-oxoglutarate dehydrogenase or aldehyde-dehydrogenase like protein or succinate semialdehyde dehydrogenase.

3. The engineered bacteria of claim 1 , further comprising decreased activity of succinate acetate kinase or phosphotransacetylase or both.

4. The engineered bacteria of claim 1 , further comprising decreased activity of lactate dehydrogenase.

5. The engineered bacteria of claim 1 , further comprising decreased activity of ethanol dehydrogenase.

6. The engineered bacteria of claim 1, comprising (AackA-pta or AackA or Apta) and ΔadhE and ΔldhA, and (ΔsucA or Δynel or ΔgabD).

7. The engineered bacteria of claim 1 , comprising AackA-pta, ΔadhE, ΔldhA.

8. The engineered bacteria of claim 1, comprising AackA-pta, ΔadhE, ΔldhA, ΔsucA.

9. The engineered bacteria of claim 1, comprising AackA-pta, ΔadhE, ΔldhA, Δynel.

10. The engineered bacteria of claim 1 , comprising AackA-pta, ΔadhE, ΔldhA, ΔgabD.

1 1. A method of producing gamma-hydroxybutyrate (GHB) comprising:

(a) culturing a recombinant bacteria of any of claims 1-10 to produce GHB, and

(b) isolating said GHB.