CLIAMS:1. A method for in vivo targeted random mutagenesis in at least one target host comprising the following steps: i) introducing a nucleic acid construct encoding an error-prone DNA polymerase in one or more organelles of at least one target host; and ii) growing at least one target host under conducive conditions to obtain positive transformants; wherein the target host is a microorganism.

2. The method of claim 1, wherein said positive transformants are further subjected to Single-Cell Fluorescence Activated Cell Sorting (FACS) to separate cells and growing these separated cells to use these cells selectively to obtain the traits of interest (TOIs).

3. The method of claim 1, wherein said organelle is selected from the group comprising chloroplast, nucleus and mitochondria of the microorganism.

4. The method of claim 1, wherein the error-prone DNA polymerase is controllably expressed in one or more organelles in the isolated positive transformants to obtain a mutant host comprising traits of interest.

5. The method of claim 1, in which the nucleic acid construct comprises i) an origin of replication; ii) a gene sequence encoding the error-prone DNA polymerase for random mutation; iii) an inducible promoter to control the expression of the error-prone DNA polymerase; iv) a genetic marker; and an organelle-specific localization marker.

6. The method as claimed in any one of the preceding claims, in which the construct comprises nucleotide motifs for stable maintenance and/or propagation of the nucleic acid construct.

7. The method as claimed in any one of the preceding claims, wherein the inducible promoter is a Cyc6 promoter or an Hsp-70 promoter; and the genetic marker comprises green fluorescent protein (GFP) and an antibiotic resistance gene.

8. The method as claimed in any one of the preceding claims, wherein the target hosts are grown in the presence of at least one selection agent, preferably an antibiotic.

9. A microorganism that includes a nucleic acid construct encoding an error prone DNA polymerase for in vivo targeted random mutagenesis.

10. A kit for in vivo targeted mutagenesis in an organism including a nucleic acid construct comprising: i) an origin of replication; ii) a gene sequence encoding an error-prone DNA polymerase for random mutation; iii) an inducible promoter to control the expression of an error-prone DNA polymerase; iv) a genetic marker for selecting the positive transformants in the target host; and an organelle-specific localization marker. ,TagSPECI:This application is a patent of addition with respect to Indian Patent Application No. 649/MUM/2013 dated 04.03.2013, the entire contents of which, are specifically incorporated herein by reference.
FIELD
The present disclosure relates to a method for in vivo localized targeted random mutagenesis in an organism using error-prone DNA polymerases. The present disclosure also relates to a microorganism that includes a nucleic acid construct encoding an error prone DNA polymerase for in vivo targeted random mutagenesis. The present invention also relates to a kit for in vivo targeted mutagenesis in an organism.

BACKGROUND
Random mutagenesis is a powerful approach to genetically improve an organism (such as a microorganism) for the traits of interest (TOIs) without any prior genetic knowledge of the organism. Suitable examples of the TOIs may include, but are not limited to lipid production traits and photosynthesis traits.

Typically, random mutagenesis is achieved by exposing the cells of an organism either to mutagenic chemicals or to high energy radiation. Consequently, in any of the aforementioned techniques, the deoxyribonucleic acid (DNA) sequence of an organism is randomly altered to an extent depending on the amount of exposure received by the cells. Subsequently, viable mutants are screened for improved TOIs.

However, the aforesaid method of random mutagenesis results in only a very few advantageous mutants in a pool of millions. Therefore, a critical challenge for every random mutagenesis driven strain improvement technique is to identify and isolate a tiny fraction of the advantageous mutants from a large pool of mutants. Since, the mutagenesis occurs randomly, most of the mutations occur at such sites in the genome that may not be metabolically connected to the TOIs. Accordingly, such mutagenesis remains unsuccessful in view of the objective of achieving an improved trait of interest in a target organism.

Therefore, to achieve a larger fraction of the population with improved TOIs, random mutagenesis restricted only to the genes involved in certain metabolic pathways, may be more efficient. For example, to increase the efficiency of the ribosome, random mutagenesis of only the targeted genes corresponding to the ribosomal RNAs (rRNAs) and the ribosomal proteins (rProteins) may be more effective than random mutagenesis of the entire genome.

The state of the art describes several methods for introducing random mutations in a target organism. Various conventional methods have been developed that involve the use of different types of error-prone DNA polymerases (EPDPs) to introduce random mutations in one or more specific genes, in vitro or in vivo. Error-prone DNA polymerases result in higher rate of errors/ mis-incorporation of nucleotides compared to housekeeping DNA polymerases while acting on undamaged DNA templates.

The conventional random mutagenesis methods involve mutation of the whole genome and/ or mere optimization of error-prone polymerase mutants under regulated culture conditions to achieve an elevated frequency of mutagenesis. Further, these methods involving the use of either chemicals or high energy radiation are not effective in targeting only a specific set of genes as it is impossible to restrict either radiation-induced random mutagenesis or chemical-induced random mutagenesis to a localized set of genes for example, an organelle-specific genome/ set of genes.

Consequently, these methods may not be proficient for achieving improved traits of interest (TOIs) in organisms, specifically in microorganisms such as algae, wherein a localized mutation may be more efficient for improved TOIs. Effectively restricting random mutagenesis to a localized set such as an organelle-specific set of genes without affecting other genes in an organism has remained a major challenge. Accordingly, there exists a need for an efficient method for in vivo localized targeted random mutagenesis using suitable error-prone DNA polymerases.

OBJECTS
Some of the objects of the present disclosure which at least one embodiment is adapted to provide, are described herein below:

It is an object of the present disclosure to provide an efficient method for in vivo targeted random mutagenesis in an organism for obtaining traits of interest (TOI).

It is still another object of the present disclosure is to provide an efficient method for in vivo targeted random mutagenesis in a microorganism to localize the expression/function of an error-prone DNA polymerase to one or more specific regions in the microorganism, particularly organelles, or nucleus without affecting the other regions.

It is yet another object of the present disclosure to provide error-prone DNA polymerases which are efficient in mediating in vivo localized targeted random mutagenesis in the organism.

It is still another object of the present disclosure to provide a microorganism including which includes a nucleic acid construct comprising an error-prone DNA polymerase for mediating in vivo localized targeted random mutagenesis.

It is still another object of the present disclosure to provide a kit for in vivo targeted random mutagenesis in the organism.

Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY

In accordance with one aspect of the present disclosure, there is provided a method for in vivo targeted random mutagenesis in at least one target host, said method comprises the following steps: introducing a nucleic acid construct encoding an error-prone DNA polymerase in one or more organelles of at least one target host; and growing at least one target host under conducive conditions to obtain positive transformants; wherein the target host is a microorganism. The positive transformants are further subjected to Single-Cell Fluorescence Activated Cell Sorting (FACS) to separate cells and growing these separated cells to use these cells selectively to obtain the traits of interest (TOIs).

In accordance with another aspect of the present disclosure there is provided a microorganism that includes without limitation, a nucleic acid construct encoding an error prone DNA polymerase for in vivo targeted random mutagenesis.

In accordance with still another aspect of the present disclosure there is provided a kit for in vivo targeted mutagenesis in a microorganism including a nucleic acid construct comprising: an origin of replication from where the replication is initiated; an error-prone DNA polymerase for random mutation; an inducible promoter to control the initiation and the extent (length) /intensity of the expression of an error-prone DNA polymerase in the target host, and a genetic marker for selecting the positive transformants. The kit may optionally include a target host strain. The kit may further include a selective agent.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING

The disclosure will now be explained in relation to the non-limiting accompanying drawings, in which:
Figure 1 illustrates a sequence listing (protein sequence) of DNA polymerase kappa (κ) of Chlamydomonas reinhardtii.
DETAILED DESCRIPTION

The co-pending patent application No. 649/MUM/2013 overcomes the drawbacks associated with known random mutagenesis methods. Conventional random mutagenesis methods generally lead to mutation of the whole genome and/ or the mere optimization of the error-prone polymerase mutants under regulated culture conditions. The latter may lead to an elevation in the frequency of mutagenesis, however, mutagenesis at specific target sites is hard to achieve by the means of most of the afore-stated conventional methods. Further, use of chemicals or high energy radiations for achieving random mutagenesis is also ineffective in targeting only a specific set of genes such as an organelle-specific genome/ set of genes.

In view of the above drawbacks associated with the conventional methods of random mutagenesis, the inventors of the present disclosure have envisaged an efficient method for in vivo localized targeted random mutagenesis by employing an error prone DNA polymerase, which effectively targets and causes a specific set of genes in the genome of a microorganism to mutate, without affecting the other genes. This method is effective to target one or more organelles or nucleus in a microorganism, particularly in a photosynthetic microorganism. The present disclosure also provides a microorganism including a nucleic acid construct encoding an error prone DNA polymerase for in vivo localized targeted random mutagenesis.

The present disclosure provides an efficient method employing an error-prone DNA polymerase for in vivo localized targeted random mutagenesis in an organism.
In accordance with an aspect of the present disclosure, there is provided a method for in vivo targeted random mutagenesis. The method is described herein below:

Initially, an error-prone DNA polymerase is extracted from a microorganism such as but not limited to Chlamydomonas and a nucleic acid construct encoding this error-prone DNA polymerase is introduced in one or more organelles in the target host. Alternatively, a nucleic acid construct encoding an error-prone DNA polymerase is introduced directly in the target host.

The nucleic acid construct according to the present disclosure may comprise an origin of replication from where the replication is initiated; an error-prone DNA polymerase for random mutation; an inducible promoter to control the initiation and the extent (length) /intensity of the expression of an error-prone DNA polymerase in the target host and a genetic marker for selecting positive transformants. Alternatively, the nucleic acid construct further comprises an organelle-specific localization marker.

In addition, other nucleotide motifs as required for stable maintenance and/ or propagation of the nucleic acid construct may also be included in the nucleic acid construct.

An error-prone DNA polymerase could include an error-prone DNA polymerase of an organism other than the host or a recombinant error-prone DNA polymerase. An error-prone DNA polymerase according to the present disclosure includes but is not limited to Chlamydomonas DNA polymerase kappa (κ), modified Chlamydomonas DNA polymerase kappa (κ), human pol κ and DinB. An error-prone DNA polymerase, preferably, according to the present disclosure is Chlamydomonas DNA polymerase kappa (κ). In accordance with an exemplary embodiment of the present disclosure, DNA polymerase kappa (κ) of Chlamydomonas reinhardtii is the error-prone DNA polymerase, the sequence listing of which is provided in Figure 1. Specifically, the modified Chlamydomonas DNA polymerase kappa (κ) is a Y-family polymerase of algal origin and exhibits a high degree of sequence conservation with DNA polymerase kappa. Further, pol κ is yet another Y-family polymerase that is error-prone while copying undamaged template.

The inducible promoter may be an externally controllable promoter, such as but not limited to Cyc6 and Hsp-70 promoter, which can be controlled by changing concentration of small molecules such as copper/ nickel molecules in the growth media.
The genetic marker includes but is not limited to a green fluorescent protein (GFP), an antibiotic resistance gene and the like. In the embodiment where the genetic marker is an antibiotic resistance gene, at least one selection agent is incorporated in the growth media for screening. The selection agent according to the present disclosure includes but is not limited to an antibiotic from any source.

An organelle-specific localization marker according to the present disclosure includes but is not limited to a chloroplast marker, a mitochondria marker and a nuclear marker. An organelle-specific localization marker according to the present disclosure enables the shuttling of the over-expressed error-prone DNA polymerase into a specific organelle while targeting genes therein for random mutation. The genes of interest include but are not limited to genes corresponding to one or more metabolic pathways, preferably, lipid production genes and photosynthetic genes. The chloroplast specific marker, for example, may be derived from sequences of chloroplast protein coding genes, such as PsaD. The attachment of an organelle-specific localization marker to the suitable error-prone DNA polymerase, enables shuttling of the over expressed error-prone DNA polymerase into a specific sub-cellular organelle for functioning within the organelle while targeting genes therein for random mutation. A suitable example of such a sub-cellular organelle is a chloroplast, wherein the mutation in the target genes of the chloroplast by the error-prone DNA polymerase allows for obtaining desired traits such as improved lipid synthesis. Therefore, according to the method of the present disclosure, the function of error-prone DNA polymerase is effectively limited to the chloroplast-specific genes leaving the nuclear and other organeller genes unaffected.

The nucleic acid construct, preferably, is a transformation plasmid vector suitable for organelle specific transformation of target hosts. Examples of such vectors include, but are not limited to, pChlamy and modified forms thereof.

An organelle according to the present disclosure includes but is not limited to chloroplast, mitochondria and nucleus.
The target host includes but is not limited to algae, fungi, other plant and animal cells and the like. In accordance with the present disclosure, the preferable target host is microalgae. A suitable microalgae when used as a target host according to the present disclosure is Chlamydomonas reinhardtii. In one embodiment, the target hosts are microorganisms. In another embodiment, the target hosts are photosynthetic microorganisms.

In the next step, the target hosts are grown under conditions that selectively enhance the growth of the positive transformants.

Then, the positive transformants are isolated. The error-prone DNA polymerase is expressed in the specific organelle in the isolated positive transformants to cause localized mutations in the organelle to obtain mutant hosts comprising improved traits of interest. According to the present disclosure, an organelle specific transformation with error-prone DNA polymerase may be performed for random mutation of organelle specific genes. Examples include chloroplast specific transformation, wherein random mutation of genes responsible for lipid synthesis may be performed. Accordingly, the mutations in such lipid synthetic genes by error-prone DNA polymerase allow generation of improved lipid synthesis.

Alternatively, the error-prone DNA polymerase is expressed in the isolated positive transformants which is directed via organelle specific localization marker to shuttle into the organelle and cause localized mutations in the genes of interest to obtain mutant hosts comprising improved traits of interest. Accordingly, the function of the error-prone DNA polymerase provided with an organelle-specific localization marker may only be limited to the specific organelle. The over-expression of the error-prone DNA polymerase results in the escalation of localized random mutagenesis in the specific organelle. The over-expressed error prone DNA polymerase according to the present disclosure is directed via organelle-specific localization marker to shuttle into the desired organelle or may be by direct transformation into the organelle by organelle-specific transformation.
The naturally present traits of interest in the host, according to the present disclosure comprise one or more metabolic pathways which include, but are not limited to, a lipid production pathway and a photosynthetic pathway. The error-prone DNA polymerase according to the present disclosure is expressed under, but not restricted to, the control of an inducible promoter. The method of the present disclosure facilitates a regulated, over-expression of error-prone DNA polymerase but not restricted to such. The activity of an error-prone DNA polymerase within the host (i.e., dosage) may be modulated by controlling the extent (length) and/ or intensity of the induction of expression of the error-prone DNA polymerase.

Therefore, according to the method of the present disclosure, either the expression or the function of the error prone DNA polymerase is limited to a specific organelle so that mutagenesis only occurs in the genome of that organelle, leaving the rest of the genome unaffected.

The method according to the present disclosure serves as a tool for targeted random mutagenesis for yielding more mutations in a specific set of genes of interest compared to the rest of the genes. Random mutagenesis, for example, of one or more strains of a specific organism for improved lipid production and photosynthesis can be achieved by the method of the present disclosure by targeting specific set of genes corresponding to the lipid production and photosynthesis.

The isolated positive transformants are finally grown in a medium such as but not limited to Tris Phosphate Acetate medium (TAP) medium. These cells are then subjected to Single-Cell Fluorescence Activated Cell Sorting (FACS). In FACS cells with differing fluorescence are sorted out as single cells and can be re-grown into single populations. Wild type samples are taken as reference to set right voltages on Flow Cytometer. 10, 000 total events are recorded for each sample. Each sample is recorded three times. In accordance with the present disclosure, Auto Fluorescence is measured in PerCP channel. This measures the amount of chlorophyll that is obtained in Chlamydomonas cells, as they are photosynthetic microorganisms. The cells are first gated on the basis of size, then singlet’s populations were gated and on it chlorophyll positive cells are gated. In the present disclosure, low and high chlorophyll positive cells are sorted. The results of the samples on FACS are depicted in Table-1 below:

Table-1: Result of samples on FACS
Phenotype Vector DPK transformant 1 DPK transformant 2
High chlorophyll cells 1 40 26
Low chlorophyll cells 44 88 120
(The values are averaged for three, the initial O.D. are the same to start with)

As illustrated in in Table-1, in the vector transformed (Chlamydomonas reinhardtii transformed with pChlamy_1 vector) shows less number of high chlorophyll and low chlorophyll containing cells. In contrast, Chlamydomonas reinhardtii transformed with DNA pol Kappa has higher number of high chlorophyll and low chlorophyll containing cells.
From the above data it is seen that DNA pol Kappa is able to mutagenize Chlamydomonas selectively in such a way that the population now contains cells having high or low chlorophyll in them and these cells can be separated to separately generate a population containing high chlorophyll cells and a population containing low chlorophyll cells.
The high chlorophyll containing cells will have a higher productivity, especially in low light conditions.
The low chlorophyll containing cells having pale green appearance will have high productivity in high light conditions.

In accordance with yet another aspect of the present disclosure, there is provided an organism including but not limiting to a microorganism that includes without limitation, a nucleic acid construct encoding an error prone DNA polymerase for in vivo targeted random mutagenesis.

In accordance with still another aspect of the present disclosure there is provided a kit for in vivo targeted mutagenesis in a microorganism, including a nucleic acid construct comprising: an origin of replication from where the replication is initiated; an error-prone DNA polymerase for random mutation; an inducible promoter to control the initiation and the extent (length) /intensity of the expression of an error-prone DNA polymerase in the target host, and a genetic marker for selecting the positive transformants. The kit may optionally include a target host strain. The kit may further include a selection agent.

In addition, other nucleotide motifs as required for stable maintenance and/ or propagation of the nucleic acid construct may also be included in the nucleic acid construct.

Using the method of the present disclosure a yield of 20-30g/m2/day can be obtained in the transformed Chlamydomonas as compared to 15g/m2/day yield obtained using conventional method.

These high biomass producing cells can be especially important for the extraction of oil and other high value products from algae.

The method of the present disclosure can also be applied to generate algal cells having higher lipid content by utilizing the principle of Nile Red staining. High Nile Red positive cells would be indicative of high lipid. Thus, the method of the present disclosure can also be used to generate algal cells having higher lipid content. Algal cells having 30 to 40% lipid content can be obtained using the method of the present disclosure as compared to 20% lipid content obtained using the conventional methods.

TECHNICAL ADVANCEMENT:

The technical advancements offered by the present disclosure are as follows:
• The method of the present disclosure provides for efficient localized targeted random mutations resulting in mutant strains with improved traits.
• The method of the present disclosure localizes the mutations on the traits of interest thus escaping the need for mutating the whole genome.
• The method of the present disclosure provides mutant strains, such as algae mutant strains, that exhibit enhanced traits such as enhanced biomass production.

The embodiments as described herein above, and various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the description. Descriptions of well-known aspects, components and molecular biology techniques are omitted so as to not unnecessarily obscure the embodiments herein.

The foregoing description of specific embodiments will so fully reveal the general nature of the embodiments herein, that others can, by applying current knowledge, readily modify and/or adapt for various applications of such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Further, it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Having described and illustrated the principles of the present disclosure with reference to the described embodiments, it will be recognized that the described embodiments can be modified in arrangement and detail without departing from the scope of such principles.

While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.