WO2011153166A1 - Native nad-dependent gapdh replaced with nadp-dependent gapdh plus nadk - Google Patents

Abstract

This invention is metabolically engineered bacterial strains that provides increased intracellular NADPH availability for the purpose of increasing the yield and productivity of NADPH-dependent compounds. In the invention, native NAD-dependent GAPDH is replaced with NAPP-dependent GAPDH plus overexpressed NADK. Uses for the bacteria are also provided.

Description

NATIVE NAD-DEPENDENT GAPDH REPLACED WITH NADP- DEPENDENT GAPDH PLUS NADK

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Serial Number 61/350382, filed June 1, 2010 and incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

OR DEVELOPMENT

[0002] This invention was made with government support under

Grant No. 0828516 awarded by the National Science Foundation. The government has certain rights in the invention.

REFERENCE TO A COMPACT DISK APPENDIX

[0003] Not applicable.

FIELD OF THE INVENTION

[0004] This invention relates to metabolically engineered bacterial strains that provide greatly increased intracellular NADPH levels for the purpose of increasing the yield and productivity of NADPH-dependent compounds.

BACKGROUND OF THE INVENTION

[0005] Nicotinamide adenine dinucleotide (NAD) and its relative nicotinamide adenine dinucleotide phosphate (NADP) are two of the most important coenzymes in the cell. NADP is simply NAD with a third phosphate group attached as shown:

[0006] Because of the positive charge on the nitrogen atom in the nicotinamide ring (box), the oxidized forms of these important redox reagents are often depicted as NAD+ and NADP+ respectively.

[0007] In cells, most oxidations are accomplished by the removal of hydrogen atoms. Both of these coenzymes play crucial roles in this. Each molecule of NAD+ (or NADP+) can acquire two electrons; that is, be reduced by two electrons. However, only one proton accompanies the reduction. The other is liberated into the surrounding medium. For NAD, the reaction is thus:

[0008] NAD+ + 2H -> NADH + H+

[0009] NAD participates in many redox reactions in cells, including those in glycolysis and most of those in the citric acid cycle of cellular respiration.

[0010] NADP is the reducing agent produced by the light reactions of photosynthesis, consumed in the Calvin cycle of photosynthesis and used in many other anabolic reactions in both plants and animals. Because NADP is used in anabolic reactions, it is desired to increase its levels in order to drive bacteria towards the synthesis of desired compounds.

[0011] The purpose of this invention is to manipulate the E. coli intracellular NADPH availability, which in turn increases the yield and productivity of NADPH-dependent synthesis of compounds, such as the biodegradable polymer poly-3-hydroxybutyrate (PHB), sorbitol, alcohols, amino acids, lycopene terpenoids, flavanoids, carotenoids, mevalonate, purines, pyramidines, antibiotics, cholesterol, steroids, and the like. NADP dependant compounds include fatty acids of varying chain lengths, or lycopene, which can be used as an antioxidant or precursor to produce other carotenoids. Increased

NADP levels can also be useful for recycling some enzymes such as P450s, some of which require NADPH, and these can be active in drug metabolism and specialized oxidations. Additionally, chiral alcohols are products that often require reduction using NADPH. Thus, increasing NADP levels can be useful in a great many different contexts and applications.

SUMMARY OF THE INVENTION

[0012] We have developed a novel system to increase in vivo

NADPH availability through genetic engineering. The system consists of two parts.

[0013] First, we replaced a native NAD-dependent D- glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene, gap A, in E. coli with an NADP-dependent GAPDH gene, gapB, from Bacillus subtilis [1]. The GapB enzyme catalyzes the following reaction:

D-glyceraldehyde-3 -phosphate + phosphate + NADP⁺ = 1,3- diphosphateglycerate + NADPH

[0014] This approach provides an efficient source of NADPH, because for every mole of glucose passing through the glycolysis pathway, two moles of NADPH will be formed (see attached Figure). This is in sharp contrast to the native system where NADH will be formed.

[0015] Efficient as it is to convert NADP+ into NADPH, this approach is nonetheless limited by the low bioavailability of NADP+ inside the cell. To solve this problem, in the second part of the invention, we co-expressed gapB with an E. coli NAD kinase, encoded by nadK, which is an enzyme that converts NAD to NADP [2], providing an improved source of NADP for GapB. With both added components, cofactor availability and/or flux is greatly improved. [0016] Thus the invention generally relates to bacteria where native NAD dependant GAPDH is replaced with a NADP dependant GAPDH, and wherein an NAD kinase is overexpressed, and methods of using such bacteria to make various NADP dependant products.

[0017] As used herein "recombinant" is relating to, derived from, or containing genetically engineered material. In other words, the genome was intentionally manipulated in some way.

[0018] "Overexpression" or "overexpressed" is defined herein to be at least 150% of protein activity as compared with an appropriate control species. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or upregulating the endogenous gene, and the like. In a preferred embodiment, one or more expression vectors encoding the required protein(s) is/are added to the cell.

[0019] The terms "disruption" as used herein, refer to cell strains in which the native gene or promoter is mutated, deleted, interrupted, or down regulated in such a way as to decrease the activity of the gene at least 90%> over the wild type un-disrupted gene. A gene can be completely (100%) reduced by knockout or removal of the entire genomic DNA sequence. Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein.

[0020] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims or the specification means one or more than one, unless the context dictates otherwise.

[0021] The term "about" means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated. [0022] The use of the term "or" in the claims is used to mean

"and/or" unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[0023] The terms "comprise", "have", "include" and "contain" (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.

[0024] We have exemplified this system using E. coli and genes from Bacillus subtilis and E. coli. However, this was for convenience only because these genes were already available, and the invention can be used with any bacteria, since all bacteria have these enzymes and/or their equivalent. In fact, many such genes are already available in GenBank and other data bases and can readily be employed herein. Enteric bacteria, such as E. coli and others, may be preferred in some circumstances due to familiarity with large scale culture of such bacteria. DETAILED DESCRIPTION OF THE FIGURES

[0025] FIGURE 1 : Overexpression of GapB increases cyclohexanone monooxygenase (CHMO) consumption of cyclohexanone which requires NADPH as cofactor. Plasmid pTrc99a is modified to include CHMO, and placed into the AgapA, gapB+ bacteria. [0026] FIGURE 2: Overexpression of GapB increases lycopene production which requires a large amount of NADPH as cofactor (16 mole of NADPH for 1 mole of lycopene). pK19-lyco is a plasmid encoding the crtEBI operon, which is the lycopene biosynthesis operon from Erwinia herbicola.

[0027] FIGURE 3: Overexpression of both GapB and NADK increases yield of 2-chloropropionic acid formed by oxidization of 2- chloroacrylate by 2-haloacrylate dehydrogenase (CAA43) even more than just overexpression of GapB. pTrc99a-CAA43 is a plasmid encoding 2- haloacrylate dehydrogenase. DETAILED DESCRIPTION OF THE INVENTION

[0028] Exemplary genes/proteins that can be used in the invention are listed below, wherein the gene/protein actually used is indicated in bold. The table is by no means complete, but searching the databases with the relevant protein sequences will identify the relevant proteins in a large number of bacterial species, and alternatively, one can search by protein name. Each protein sequence is connected to a gene sequence, which can be used to generate either expression vectors or knockout vectors, etc. which can be used to manipulate the bacteria according to the claimed invention. As is apparent from the table, the proteins in question are remarkably conserved in a variety of species, thus the method is predicated to be usable in a great many species. In some species, there may be more than one gene that needs to be disrupted for optimal performance, although in most cases a single gene disruption should suffice.

Table 1 : Exemplary genes/proteins for use in the invention

Species Gene Protein % Identity Acc. No.

E. coli gapA glyceraldehyde 100% NC 000913

-3-phosphate NP_416293 dehydrogenase

A aka GAPDH,

NAD- dependant

Shigella flexneh gapA GAPDH 99% YP 688934.1

Salmonella gapA GAPDH 99% NP_456222.1 entehca

Citrobacter gapA GAPDH 97% YP_003364860.1 rodentium

Klebsiella gapA GAPDH 96% YP_002918985.1 pneumoniae

Providencia gapA GAPDH 91 % ZP_06123668.1 rettgeri

Erwinia gapA GAPDH 89% YP_003531334.1 amylovora

Bacillus subtilus gapB Glyceraldehyde 100% AAC00355

-3-phosphate NP_390780 dehydrogenase

2 aka GAPDH,

NADP

dependant

Bacillus gapB GAPDH 88% YP_001422195.1 amyloliquefacien

s

Bacillus gapl GAPDH 74% YP_002448147.1 | thuringiensis

Macrococcus gapB GAPDH 69% YP_002560757.1 caseolyticus Exiguobactehum Exig 21 GAPDH 66% YP_001814668. sibiricum 99

Costhdium gapC GAPDH 48% ADZ19759 acetobutylicum

Clostridium gap GADPH 63% YP_001786486.1 botulinum

E. coli nadK NAD Kinase, 100% AAC75664

aka NADK NP 417105

Citrobacter sp. CSAG NADK 99% ZP_0456001 1 .1

30 2 02408

Enterobacter Entcl 1 NADK or Acox 97% YP_003940658.1 | cloacae 10 kinase

Salmonella ppnK NADK 96% ZP_02347266.1 entehca

Cronobacter ppnk NADK 91 % YP_003211564.1 tuhcensis

EXAMPLE 1

[0029] The gapB gene was PCR amplified from Bacillus subtilis

168 genomic DNA and cloned into vector pDHC29 under the Plac promoter[6]. NADP+ dependent GAPDH activity of GapB was confirmed by spectrophotometrical analysis based on measurement of NADPH at OD 340 nm. The reaction mixture included 40 mM triethanolamine, 50 mM sodium biphosphate, 0.2 mM EDTA, 1 mM NADP+, 1 mM glyceraldehyde-3- phosphate and whole cell lysate. GapB showed higher activity in the presence of NADP than NAD, while the wild type control MG1655 pDHC29 preferred

NAD over NADP (Table 2).

[0030] To replace native E. coli GAPDH gene, gapA, with gapB, pDHC29-gapB was transformed in a gapA null mutant MG1655 gapA::Tn, which has a transposon insertion in PI promoter region of gapA and lost GapA activity [7]. This recombinant strain was later introduced with pTrc99a- CHMO, pK19-lyco or pTrc99a-CAA43 as exemplary test systems in order to confirm increased NADPH availability.

[0031] We have found that overexpression of NADP+- dependent GapB, or both GapB and NAD kinase, greatly increased NADPH availability in the cell, which in turn resulted in higher yield of NADPH dependent compounds. The level of cofactor can go up 10, 20 or more fold over the pre-modified bacteria.

[0032] To validate this novel system, the resulting recombinant

E. coli strains were tested in reporting systems that produce NADPH-dependent compounds.

[0033] The first reporting system was the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIB 9871. CHMO catalyzes Baeyer-Villiger (BV) reactions and oxidizes cyclohexanones into δ- and ε-caprolactones with high enantioselectivity (>95%) using NADPH as cofactor, and thereby gives a direct measurement of NADPH availability in the cell under aerobic conditions [3].

[0034] The second reporting system we used involved production of lycopene under aerobic conditions. Lycopene has been produced in E.coli using crtEBI operon from Erwinia herbicola [4] containing genes crtE, crtB and crtl, together with certain native genes. 16 moles of NADPH are required to produce 1 mole of lycopene.

[0035] The third reporting system we have developed is an anaerobic system which utilizes the 2-haloacrylate reductase CAA43 from soil bacterium Burkholderia sp. WS. This enzyme reduces 2-chloroacrylate to 2- chloropropionic acid while oxidizing NADPH to NADP+ [5]. μ

too^*

[0036] In the monooxygenase assay, we found that overexpression of GapB greatly increased consumption of cyclohexonone per mole of glucose used, indicating that in the presence of GapB, glucose generates more NADPH to be utilized in cyclohexonone oxidization (Figure 1).

[0037] Similarly in the lycopene assay, the GapB+ recombinant strain showed higher lycopene production than wild type strain with vector control (Figure 2).

[0038] When both GapB and NAD kinase were overexpressed, the recombinant strain showed a higher yield of 2-chlorolopropionic acid, which was reduced from 2-chloroacrylate by 2-haloacrylate dehydrogenase using NADPH as cofactor (Figure 3). The yeild was further improved over just the overexpression of GapB alone.

[0039] In summary, this invention established an in vivo system to increase NADPH availability in E. coli. By replacing its native NAD+- dependent gapA with NADP+-dependent GapB from B. subtilis, we were able to produce NADPH dependent compounds in E. coli in a higher yield. The increase was even more pronounced when NAD kinase was co-expressed along with gapB.

EXAMPLE 2

[0040] In addition to the bacteria described above, we also replaced E. coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum [8]. We constructed a recombinant E. coli strain by replacing the native NAD- dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The recombinant strain produces 2 mole of NADPH, instead of NADH, per mole of glucose consumed.

[0041] The effectiveness of the NADPH enhancing system was again tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

[0042] The NADK overexpression vector from above will be added to the base AgapA/gapC+ strain, and is predicted to further improve the base strain in a manner similar to that already shown in the original experiments (Figure 3). EXAMPLE 3

[0043] NADP and NADPH are used in a great many anabolic reactions, which are too numerous to be listed herein. However, a listing of NADP dependant enzymes can be found at en.wikipedia.org/wiki/Category:NADPH-dependent_enzymes (incorporated herein by reference). Of the hundereds of enzymes listed herein, some of the more important include limonene monooxygenases, hydroxybenzoate monooxygenases, steroid reductases, 6-methylsalicylic-acid synthase, acetoacetyl-CoA reductase, acyl-CoA dehydrogenase, alcohol dehydrogenases, aldehyde dehydrogenases, alkene monooxygenase, coA-glutathione reductase, D-lysopine dehydrogenase, D-nopaline dehydrogenase, estradiol dehydrogenases, farnesol dehydrogenase, fatty-acid synthase, formate dehydrogenase, formyltetrahydrofolate dehydrogenase, lovastatin nonaketide synthase, salicylate 1 -monooxygenase, and taxane hydroxylases. Thus, it is appaent that the bacteria of the invention can be used in a great many biosynthetic applications. [0044] The following references are incorporated by reference herein in their entirety.

[0045] 1. Fillinger, S., et al, Two glyceraldehyde-3 -phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium. J Biol Chem, 2000. 275(19): p. 14031-7.

[0046] 2. Kawai, S., et al., Molecular characterization of

Escherichia coli NAD kinase. Eur J Biochem, 2001. 268(15): p. 4359-65.

[0047] 3. Walton, A.Z. and J.D. Stewart, Understanding and improving NADPH-dependent reactions by nongrowing Escherichia coli cells. Biotechnol Prog, 2004. 20(2): p. 403-11.

[0048] 4. Alper, H., et al, Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metab Eng, 2005. 7(3): p. 155-64.

[0049] 5. Kurata, A., et al., 2-Haloacrylate reductase, a novel enzyme of the medium chain dehydrogenase/reductase superfamily that catalyzes the reduction of a carbon-carbon double bond of unsaturated organohalogen compounds. J Biol Chem, 2005. 280(21): p. 20286-91.

[0050] 6. Phillips, G.J., S.K. Park, and D. Huber, High copy number plasmids compatible with commonly used cloning vectors. Biotechniques, 2000. 28(3): p. 400-2, 404, 406 passim.

[0051] 7. Ganter, C. and A. Pluckthun, Glycine to alanine substitutions in helices of glyceraldehyde-3 -phosphate dehydrogenase: effects on stability. Biochemistry, 1990. 29(40): p. 9395-402.

[0052] 8. Martinez I, Zhu J, Lin H, Bennett GN, San KY, Replacing Escherichia coli NAD-dependent glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways, Metab Eng. 2008 Nov;10(6):352-9. Epub 2008 Sep 23.220

Claims

We claim:

1) A recombinant bacteria comprising i) a disrupted native NAD- dependent D-glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), ii) an overexpressed NADP-dependent GAPDH, and iii) an overexpressed NAD kinase.

3) The recombinant bacteria of claim 1, comprising a disrupted gapA gene, plus an overexpressed gapB or gapC gene, plus an overexpressed nadK gene.

4) The recombinant bacteria of claim 1 , comprising a deleted native gapA gene, plus one or more expression vectors comprising an overexpressed gene encoding NADP-dependent GAPDH and an overexpressed gene encoding a NAD kinase.

5) The recombinant bacteria of claim 1 that produces 10 fold more NADP+ than said bacteria without i) ii) and iii).

6) The recombinant bacteria of claim 1 that produces 20 fold more NADP+ than said bacteria without i) ii) and iii).

7) A recombinant E. coli, comprising i) a deleted native gapA gene, plus one or more expression vectors comprising ii) an overexpressed NADP- dependent GAPDH, and iii) an overexpressed NAD kinase.

8) The recombinant E. coli of claim 7, that produces 10 fold more NADP+ than said bacteria without i), ii) and iii).

9) The recombinant E. coli of claim 7, that produces 20 fold more NADP+ than said bacteria without i), ii) and iii).

10) A method of making a product that requires NADP in its synthesis, comprising culturing the bacteria of claims 1-9 in a culture media under conditions sufficient to make a product that requires NADP cofactor in its biosynthesis, and isolating said product from the culture media, or said bacteria, or both. 11) The method of claim 10, wherein the product is selected from the group consisting of amino acids, alcohols, fatty acids, poly-3-hydroxybutyrate, sorbitol, lycopene, flavanoids, carotenoids, mevalonate, glutamate, purines, pyramidines, antibiotics, cholesterol, and steroids.