WO2015047202A2

WO2015047202A2 - Rhodobacter sphaeroides o.u.oo1 producing 5-aminolevulinic acid with a new metabolic pathway and 5-aminolevulinic acid production method

Info

Publication number: WO2015047202A2
Application number: PCT/TR2014/000346
Authority: WO
Inventors: Gokhan KARS
Original assignee: Kars Gokhan
Priority date: 2013-09-24
Filing date: 2014-09-19
Publication date: 2015-04-02
Also published as: WO2015047202A3

Abstract

The invention is about Rhodobacter sphaeroides O.U. 001 strain which can produce 5-aminolevulinic acid being a high value added product with important application areas in agriculture and biotechnology and it is about 5-ALA production method of this bacterium.

Description

DESCRIPTION

RHODOBACTER SPHAEROIDES O.U.001 PRODUCING 5-AMINOLEVULINIC ACID WITH A NEW METABOLIC PATHWAY AND 5-AMINOLEVULINIC ACID

PRODUCTION METHOD

TECHNICAL FIELD

The invention is about Rhodobacter sphaeroides O.U. 001 strain which can produce 5-aminoievuiinic acid (it will be denoted as 5-ALA thereafter) being a high value added product with important application areas in agriculture and biotechnology and it is about 5-ALA production method of this bacterium.

PRIOR ART

5-Aminolevulinic acid or 5-amino-4-oxo-pentanoic acid is used as a precursor for the synthesis of tetrapyrrole like porphyrin, vitamin B12 and chlorophyll in living things (certain bacteria, plants and mammals). 5-ALA having very important application areas in medicine, agriculture and biotechnology is a high value added product. However, the most important reason of its uncommon usage is that it cannot be produced in enough amounts within a cost-effective bioprocess. The chemical synthesis of it occurs with high cost since it includes many complex steps.

BRIEF EXPLANATION OF THE INVENTION

The invention is related with the production of 5-ALA being a high value added product with important applications areas in agriculture and biotechnology in a bioprocess using newly developed Rhodobacter sphaeroides O.U. 001 strain and it is related with newly developed Rhodobacter sphaeroides O.U. 001 strain.

LIST OF FIGURES

Figure 1 . The 5-ALA biosynthesis pathways.

Figure 2. The bacteria having C-5 pathway enzymes (genes) according to the data obtained from genome sequence databases.

Figure 3. The glutamyl-tRNA reductase gene obtained by Polymerase Chain

Reaction. DMSO concentrations are (1 -5) %3, %5, %7, %9 and %1 1 respectively. 6μΙ of each product was loaded. DNA ladder was loaded into the first well (SM0331 Fermentas, M).

Figure 4. The expected correct vector after the cloning process (a) and DNA fragments obtained by cutting vectors derived from selected five transformants with HincW (b). Based on these results, the 3^rd and 4^th colonies contain expected correct recombinant vector.

Figure 5. The expression analysis of glutamyl-tRNA reductase gene in

Rhodobacter Sphaeroides O.U.00. 1 : RT-, 2: RT+, 3: Positive control using recombinant pBBR1 MCS2-Glutamyl-tRNA reductase vector, M: DNA Ladder (100 bp).

THE DETAILED DESCRIPTION OF THE INVENTION

The biosynthesis of 5-ALA occurs in two different ways (Figure 1 ):

(1) Using succinyl-CoA and glycine (Shemin or C-4 pathway).

(2) Using glutamate (C-5 pathway).

In C-4 pathway, the key enzyme for the synthesis of 5-ALA from glycine and succinyl CoA is ALA synthetase (ALAS; succinyl-CoA:glycine C-succinyltransferase (decarboxylating); EC 2.3.1.37) [BEALE, 2006]. There are two genes, hemA and hemT, encoding ALA synthetase in Rhodobacter sphaeroides 2.4.1 . These genes encode the two isozymes of the ALA synthetase and while hemA is found on chromosome I, hemT is located on chromosome II [ZEILSTRA-RYALLS, 1996]. Based on these, it can be asserted that Rhodobacter sphaeroides O.U. 001 has also the genes coding for these enzymes and can produce 5-ALA using C-4 pathway.

The production of 5-ALA through C-5 pathway which is the second 5-ALA production pathway seen in living things occurs in three steps:

Glutamyl-t-RNA Glutamyl-t-RNA GSA amino-

Synthetase Reductase transferase

Glutamate _-_____^GIutamyl t-RNA Glutamate-l-semialdehyde(GSA) ALA

The bacteria like Escherichia coli, Caldicellulosiruptor saccharolyticus and Corynebacterium glutamicum possessing the three enzymes given above and the related genes synthesize ALA using C-5 pathway. When the porphyrin and chlorophyll metabolisms are examined using genome sequence databases, it is seen that certain bacteria synthesizing ALA using C-4 pathway have some enzymes of the C-5 pathway as well (Figure 2). One of these bacteria is Rhodobacter sphaeroides 2.4.1 strain.

It was observed that though Rhodobacter sphaeroides synthesize ALA using C-4 pathway, it has also the first (glutamyl-tRNA synthetase, RSP_0797, NCBI-GenelD: 3718415, [EC:6.1.1.17]) and the third (Glutamate-1-semialdehyde aminotransferase, RSP_1569, NCBI-GenelD: 3718596, [EC:5.4.3.8]) enzymes of the C-5 pathway. But, interestingly the second enzyme, glutamyl t-RNA reductase, is absent in this bacterium.

The gene coding for glutamyl t-RNA reductase enzyme (second enzyme) was taken from Rhodospirillum rubrum (DSM 467, ATCC 1 1170) by genetic engineering techniques and transferred to Rhodobacter sphaeroides O.U.001. In this way, in addition to C-4 pathway, C-5 pathway was enabled to function upon assembling all the genes of C-5 pathway. Consequently, a new and unique bacterial strain producing 5-ALA at different conditions was developed. This bacterial strain possessed the genes (enzymes) of both pathways and produced more 5-ALA. Since C-5 pathway was also active in this new bacterial strain in addition to C-4 pathway, more 5-ALA accumulation became possible. It will be possible to use 5- ALA in many areas upon high 5-ALA production. The stages of the development of this new bacterial strain are as follows:

1. The genomic DNA was obtained from Rhodospirillum rubrum (DSM 467, ATCC 11 170).

2. The glutamyl t-RNA reductase gene which is 181 1 bp long was synthesized by Polymerase Chain Reaction using designed primers (Figure 3). (Left primer: 5- GAATTCGTCACCACCGATCT-3; right primer: 5-GGCTCAGGTTCTCTTCCAAA-3).

3. The synthesized glutamyl t-RNA reductase gene was cloned into EcoRV site of pBBR MCS2 (5144 bp, Figure 4) [KOVACH, 1995].

4. Glutamyl t-RNA reductase gene cloned pBBR1 MCS2 was delivered to Rhodobacter sphaeroides O.U.001 by conjugation. 5. After performing Reverse-Transcriptase Polymerase Chain Reaction using designed primers (left: 5-GCGTGGAGATCTTTGGTCAT-3, right: 5- TTGACCTGCCCCAAAATATG-3), it was determined that glutamyl t-RNA reductase gene was transcribed in Rhodobacter sphaeroides O.U.001 (Figure 5). 6. The production of 5-ALA with Rhodobacter sphaeroides O.U.001 containing pBBR MCS2- glutamyl t-RNA reductase was performed. Sugar beet molasses was used as substrate. After the bacteria were inoculated into the medium prepared by using molasses at 27.5 g/L sugar concentration, the production of 5-ALA was performed under anaerobic conditions at 29 °C under light. 7. In the studies, Rhodobacter sphaeroides O.U.001 transferred with pBBR1 MCS2 which does not contain glutamyl t-RNA reductase gene was used as control.

8. According to the 5-ALA measurement results obtained by a colorimetric method [MAUZERALL, 1956], while Rhodobacter sphaeroides O.U.001 containing pBBR1 MCS2-glutamyl t-RNA reductase gene produced 16.52 mM 5-ALA, Rhodobacter sphaeroides O.U.001 transferred with pBBR1 MCS2 which does not contain glutamyl t-RNA reductase gene produced 7.93 mM 5-ALA. Based on the results, it was proven that C-5 pathway was enabled in addition to C-4 pathway after assembling all the genes of C-5 pathway by transferring glutamyl t-RNA reductase gene into Rhodobacter sphaeroides O.U.001 and thus more 5-ALA generation was achieved.

Claims

C L A I M S

1. Rhodobacter sphaeroides O.U. 001 strain producing 5-aminolevulinic acid through a new metabolic pathway. It was developed by transferring the gene coding for glutamyl t-RNA reductase enzyme taken from Rhodospirillum rubrum (DSM 467, ATCC 1 1 170) by genetic engineering techniques to Rhodobacter sphaeroides O.U.00 .

2. Rhodobacter sphaeroides O.U. 001 strain said in claim 1 , it has the characteristic of producing more 5-aminolevulinic acid due to the functioning of C-4 and C-5 pathways together.

3. The 5-ALA production method using Rhodobacter sphaeroides O.U.

001 which can produce 5-aminolevulinic acid through a new metabolic pathway and its features are as follows;

— The culture medium was prepared using sugar beet molasses at a sugar concentration of 27.5 g/L,

— After the bacteria were inoculated, cultures were incubated under anaerobic conditions at 29 °C under light,

— Levulinic acid was added to the culture at the 90^th h. at a concentration of 15 mM.

— At the 350^th h. of the growth, bacteria were removed from the culture liquid and the amount of 5-ALA was measured by a colorimetric method in this liquid,

The method is characterized by consisting of these steps.