# Patent

## Title
Method of construction of recombinant organisms using multiple genes co-integration

## Abstract

Provided is a method of simultaneous co-integration of multiple nucleic acid sequences into a microbial organism comprising culturing a microbial organism to be transformed until a growth phase is reached in which a random integration is facilitated; and transforming the microbial organism by mixing the cultured microbial organism with a sample to be co-integrated into the microbial organism with, wherein the sample comprises more than one nucleic acid sequence, and the products generated from the method.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of Korean Patent Application No. 10-2013-0023575, filed on Mar. 5, 2013 in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: One 38,086 bytes ASCII (Text) file named “716221_ST25.TXT,” created on Mar. 3, 2014.
BACKGROUND OF THE INVENTION
1. Field
The present disclosure relates to methods of simultaneously transforming various types of nucleic acid sequences (e.g., genes) efficiently.
2. Description of the Related Art
Much biomaterials research is currently underway. Specifically, research into the production of industrially useful materials using microbes (i.e., microbial organisms) is in progress. However, microbial organisms do not only produce desired metabolites. When specific metabolites are produced in excess, growth of the microbial organisms may be inhibited, and the microbial organisms may no longer produce the desired metabolites or may produce undesirable by-products.
To overcome such limitations, research is underway to develop microbial organisms that may uniquely produce the desired metabolites. However, preparing transformed microbial organisms by transforming all metabolic pathways of the microbial organism requires a long period of time and great effort.
Specifically, the greater the number of nucleic acid sequences to be transformed, the greater the number of processes that are needed during the transformation. Each process is laborious and requires a long period of time, and gene transfer efficiency decreases. Accordingly, there is a need for improvement in gene transfer methods.
Microbial growth occurs regularly for a certain period of time, during which the microbes grow exponentially. However, after a certain period of exponential growth, the microbes show a slowed growth rate. When a population of the microbes is shown as a function of time, a typical S shaped curve may be obtained. A graph illustrating a number of the microbe population as a function of time is referred to as a growth curve of the microbes. The growth curve may be divided into four phases.
The first phase is a lag phase. The lag phase is a period in which the microbes adjust to a new medium. When the microbes are inoculated and cultured in the new medium, chemical changes such as an activation of enzymes and an increase in cell components such as RNAs occur. During the lag phase, the amount of RNA in cells increases substantially, the size of the microbes increases and respiratory activity of the microbes is high.
The second phase is a log phase. During the log phase, the microbes that passed through the lag phase grow actively. The log phase is a period in which the microbes grow logarithmically. As physiological functions of the microbes increase during the log phase, the microbes begin to excrete enzymes and become sensitive to physical and chemical treatments. Also, generation time and the size of the microbes are uniform during the log phase and the growth rate is determined according to environmental factors such as nutritive conditions, pH, temperature, and oxygen partial pressure of the medium.
The third phase is a stationary phase. The number of the microbes becomes constant after the log phase, the highest number of the microbes is maintained, and the microbes excrete enzymes, during this phase. Also during the stationary phase, some cells die while others grow, thereby maintaining a constant state where a number of dying microbes and a number of proliferating microbes are almost the same. Also, as the overcrowding of microbes causes depletion of nutrients, accumulation of metabolites of the microbes, changes in pH of the medium, and a depletion of the oxygen supply, the environment becomes unsuitable for growth of the microbes, and thus, the number of the microbes does not increase.
The last phase is a death phase. This is the final phase of culturing where the number of the microbes decreases. The primary reasons for death include dissolution of cells due to a decomposition caused by nuclease or protease, denaturing of enzymatic proteins, and autolysis of microbes killed by enzymes.
The microbes may be transformed by using microbes in an initial phase of the log phase, which is a period when the cells are most active and OD600 value is between about 0.5 to about 1.0. When the microbes subject to the transformation and the conditions of the transformation are controlled, an increase in the efficiency of the transformation may be observed.
BRIEF SUMMARY OF THE INVENTION
Provided are methods of efficiently preparing genetically recombined microbes (i.e. microbial organisms) by simultaneously co-integrating multiple nucleic acid sequences (e.g., genes) into a microbial organism.
Additional aspects will be set forth in part in the description which follows and, in part, will be apparent from the description, or may be learned by practice of the presented embodiments.



BRIEF DESCRIPTION OF THE DRAWINGS
These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIGS. 1A to 1C illustrate a system for preparing a recombinant strain, respectively. FIG. 1A illustrates a method of transformation by using a sequential gene integration. FIG. 1B illustrates a method of transformation by using a multiple gene expression vector. FIG. 1C illustrates a method of transformation by using co-transformation of multiple vectors;
FIG. 2 illustrates a summary of simultaneous co-integration of multiple nucleic acid sequences (e.g., genes) using a non-homologous end joining (NHEJ) system;
FIG. 3 illustrates an expression vector for expressing nucleic acid sequences within K. marxianus; and
FIG. 4 illustrates a comparison between a sequential transformation method, which is a conventional technology, and a simultaneous co-integration of multiple nucleic acid sequences (i.e., Multiple Genes Co-Integration) according to an embodiment. In the FIG. 4, the labels on the X axis represent day post integration and the horizontal bars represent the period of performing each experiment.



DETAILED DESCRIPTION OF THE INVENTION
According to an aspect of the present disclosure, there is provided a method for the simultaneous co-integration of multiple nucleic acid sequences including culturing a microbial organism to be transformed until a phase in which a random integration is initiated; and transforming the microbial organism by mixing the cultured organism with a sample to be co-integrated into the microbial organism, wherein the sample comprises more than one nucleic acid sequence.
A method for the simultaneous co-integration of multiple nucleic acid sequences is as follows.
First, the method includes culturing a microbial organism to be transformed until a growth phase is reached in which a random integration is facilitated. Here, the phase in which the random integration is facilitated may vary depending on the microbial organism cultured.
An embodiment of the phase in which the random integration is facilitated may be in a state where the microbial organism has a cell density greater than its cell density during its log phase of growth. Here, the state in which the cell density is greater than the cell density of the log phase may have an optical density at 600 nm (OD600) of about 2.0 to about 10.0. Also, the OD600 may be about 3.5 to about 8.0. Also, the OD600 may be about 3.5 to about 6.0, and may be about 4.0 to about 5.0. However, if the OD600 is a value selected from the latter half of the log phase, or from a stationary phase, the OD600 in a flask may be suitably selected between 3.0 and 8.0. Also, the OD600 may be suitably selected according to the chosen type of microbial organism.
The microbes to be transformed may be a strain having a non-homologous end joining (NHEJ) as a dominant system. The term “a non-homologous end joining as a dominant system” means that NHEJ is more dominant than homologous recombination as DNA repair system in a microbial organism. Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. Here, the strain having the NHEJ as a dominant system may be non-conventional yeast, fungi, or mammalian cells. The term “non-conventional yeast” refers to yeast species with outstanding characteristics investigated for biotechnology and research, not to S. cerevisiae. The non-conventional yeast may be Kluyveromyces, Hansenula, or Kluyveromyces marxianus. K. marxianus is an acid resistant yeast that may produce organic acids with high efficiency at low acidity and thus, K. marxianus may be used as a microbial organism suitable for transformations for industrialization.
Hereinafter, provided is a process for preparing a sample to be co-integrated into the microbes, the sample including more than one nucleic acid sequence.
The sample may contain multiple nucleic acid sequences to be integrated into the microbe. For example, the sample may comprise 2 to 10 different nucleic acid sequences, or 3 to 6 different nucleic acid sequences. However, the sequences may be suitably selected as needed. Also, the nucleic acid sequences may comprise coding sequences for genes of interest. They may also be DNA fragments. As disclosed here, the nucleic acid sequences to be co-integrated may be multiple copies of the same nucleic acid sequence or encoding the same gene product, or different nucleic sequences (e.g., encoding different gene products). Also, the nucleic acid sequences may encode an enzyme participating in a metabolic pathway of the microbial organism, and the nucleic acid sequences may encode a homologous or a heterologous protein expression enzyme. Also, the nucleic acid sequences may be related to a synthesis of organic acids. Also, the nucleic acid sequences may be related to a saccharification.
Also, the nucleic acid sequences to be included in the sample may be linear genes obtained by inserting the genes into vectors and excising an autonomously replicating sequence (ARS) in the vectors by a restriction enzyme. The restriction enzyme removes the ARS sequence existing in the vector, and enables the ARS sequence to be used as a linear gene. The types of the restriction enzyme used may vary depending on the genes to be co-integrated. The restriction enzyme may be PfoI, HindIII, XhoI, BgIII, KpnI, PacI, and/or AatII; however, the restriction enzyme is not limited thereto.
The term “vector” as used herein refers to a DNA product including a DNA sequence operably linked to a suitable regulatory sequence for expressing DNA in a suitable host. Vectors such as a plasmid vector, a bacteriophage vector, or a cosmid vector, and preferably the plasmid vector may be used. Also, the nucleic acid sequences may be connected to an operable promoter.
Thereafter, provided is a phase for mixing the prepared sample and the cultured microbial organism, thereby transforming the microbial organism.
Here, the transformation process may involve the simultaneous co-integration of multiple nucleic acid sequences. This involves a simultaneous co-integration of multiple nucleic acid sequences into a microbial organism during one co-integration process by using an NHEJ system. Such a process is referred to as multiple genes co-integration technology.
Conventionally, co-integrating multiple nucleic acid sequences into a genome of a strain using a multiple co-transformation method requires using plasmids having different selectable markers and the strain must also be manipulated to use the selectable markers. FIGS. 1A to 1C illustrate the conventional system for preparing a recombinant strain, respectively. FIG. 1A illustrates a method of transformation by using a sequential gene integration. FIG. 1B illustrates a method of transformation by using a multiple gene expression vector. FIG. 1C illustrates a method of transformation by using co-transformation of multiple vectors. The conventional method is limited by the number of usable selectable markers.
However, the simultaneous co-integration of multiple nucleic acid sequences using the method disclosed herein is not limited by the number of selectable markers. It also takes the same period of time for co-integrating the nucleic acid sequences into the genome regardless of the number of the genes. The period of time for co-integrating the genes into the genome is about 6 days, which is a substantially shortened period of time compared to that of the multiple co-transformation method. Further, the genes may be repeatedly inserted by using a cassette including a repeat sequence where a marker pop-out is possible. The term “marker pop-out” refers to removing or deleting marker gene. FIG. 4 illustrates a comparison between a sequential transformation method, which is a conventional technology, and a simultaneous co-integration of multiple nucleic acid sequences according to an embodiment
The simultaneous co-integration of multiple nucleic acid sequences using the method disclosed in the embodiment may use numerous selectable markers. For instance, one to three selectable markers or one selectable marker may be used. The repeat sequence where the marker pop-out is possible may be included.
According to another aspect, there are provided transformed microbial organisms prepared by the method including various types of different genes.
The microbial organisms to be transformed may be a strain having an NHEJ as a dominant system. Here, the strain having the NHEJ as a dominant system may be non-conventional yeast, fungi, or mammalian cells. The non-conventional yeast may be Kluyveromyces, Hansenula, and preferably Kluyveromyces marxianus. K. marxianus is an acid resistant yeast and may be used as the microbial organism for transformation for industrialization, because the yeast may produce organic acids with high efficiency at low acidity.
Also, the nucleic acid sequence may be one or more nucleic acid sequence. Further, the genes may be 2 different or 10 different nucleic acid sequences, and may be 3 different or 6 different nucleic acid sequences. Also, the nucleic acid sequences may encode an enzyme participating in a metabolic pathway of the microbial organism, and the nucleic acid sequences may encode homologous or heterologous protein expression enzymes. Also, the nucleic acid sequences may be related to a synthesis of organic acids. Also, the nucleic acid sequences may be related to a saccharification.
In one embodiment of the present disclosure, there is provided a method for the simultaneous co-integration of multiple nucleic acid sequences including culturing a microbial organism to be transformed until the optical density at 600 nm (OD600) is at least about 2.0; and transforming the microbial organisms by mixing the cultured organism with a sample to be co-integrated into them, wherein the sample comprises more than one nucleic acid sequence and the microbial organism has a stronger Non-homologous end joining (NHEJ) than homologous recombination as DNA repair system.
The OD600 may be about 2.0 to about 10.0, about 2.5 to about 8.0, about 3.0 to about 8.0, about 3.0 to about 7.0, 3.0 to about 6.5, about 3.0 to about 6.0, about 3.0 to about 5.5 or about 3.0 to about 5.0. The microbial organism to be transformed may be a competent cell. The term “competent cell” refers to a cell which has the ability to take up and replicate an exogenous nucleic acid. In one example, co-integration efficiency of competent cell prepared when OD600 was about 3.5 for 6 genes was 100%, while that of competent cell prepared when OD600 was about 1.0 was 28%.
Another aspect relates to a method of preparing metabolites by using the transformed microbial organism. The method of preparing the metabolites by using the transformed microbial organism may include culturing the transformed microbial organism; and retrieving the metabolites from a culture medium.
A method of preparing the metabolites is as follows.
First, provided is a process of culturing the transformed microbial organism prepared by the simultaneous co-integration of multiple nucleic acid sequences.
The term “metabolites” as used herein refers to all materials produced by metabolic reactions of the microbial organism. The metabolites may be intermediate products of the metabolic reactions of the microbial organism or final products of the metabolic reactions of the microbial organism. Examples of the metabolites include succinic acid, lactic acid, 1,4-butanediol (1,4-BDO), 3-hydroxypropionate, and the like, but are not limited thereto.
The microbial organism may be cultured in carbon sources usable by the microbial organism such as monosaccharides, disaccharides, or polysaccharides. Specifically, glucose, fructose, mannose, or galactose may be used. Also, nitrogen sources usable by the microbial organism may be organic nitrogen compounds, or inorganic nitrogen compounds. Specifically, the nitrogen sources may be amino acids, amides, amines, nitrates, or ammonium salts. Oxygen conditions for culturing the microbial organism may be an aerobic condition of a normal oxygen partial pressure, a low oxygen condition including about 0.1% to about 10% of oxygen in atmosphere, or an anaerobic condition without oxygen.
Thereafter, provided is a process of retrieving the metabolites from the culture medium.
The metabolites may be succinic acid, 3-hydropropionate (3HP), and lactic acid. The metabolites may be retrieved through, for example, filtering.
Unless defined otherwise, all technical and scientific terms used in the present specification have the same meanings as generally understood by one of ordinary skill in the art. Generally, nomenclatures used in the present specification are well known and commonly used in the art.
Reference will now be made in detail to embodiments. The following embodiments are for illustrative purposes only and it is apparent to one of ordinary skill in the art that the scope of the present invention is not limited by the embodiments. Specifically, the embodiments only show a method using K. marxianus as a model system; however, it is apparent to one of ordinary skill in the art that the embodiments also apply to other microbial organism than K. marxianus based on the present specification.
EXAMPLE 1
Preparing a Gene Expression Vector
To prepare a gene expression vector that may be co-integrated into a uracil (Ura) auxotrophic K. marxianus strain, a replication origin, an autonomously replicating sequence (ARS), and a centromere of K. marxianus were amplified by a polymerase chain reaction (PCR) to prepare amplified products, and the amplified products were co-inserted into a pRS306 vector purchased from ATCC to prepare a K. marxianus-E. coli shuttle vector. A GPD promoter and a CYC terminator gene were inserted into the shuttle vector (pJSKM316-GPD, SEQ No. 1; FIG. 3). Sacharomyces cerevisiae ALD6 (SEQ No. 2), S. cerevisiae ACS2 (SEQ No. 3), and K. marxianus ACC (SEQ No. 4) genes were enabled to be expressed in K. marxianus by the GPD promoter and the CYC terminator. FIG. 3 illustrates an expression vector for expressing nucleic acid sequences within K. marxianus. 
EXAMPLE 2
Co-Integrating Genes into K. marxianus 
2-1. Preparing Competent Cells by Using K. marxianus Strain
The Ura auxotrophic K. marxianus strain was streaked in a Glycerol stock on a YPD plate and cultured at a temperature of 37° C. for 2 days. One colony was inoculated in 100 ml of YPD culture medium, and cultured at a temperature of 37° C. at 250 rpm overnight. The colony was cultured until OD600 reached about 3.0 to about 5.0, and the same was centrifuged at 5,000 rpm for 5 minutes to separate cells. After washing the cells with 60 ml of sterilized water, 10 ml of lithium acetate buffer (100 mM lithium acetate, 10 mM Tris-Cl pH 7.5, 10 mM DTT, and 0.6M Sorbitol) was added thereto, and the cells were maintained at room temperature for 30 minutes. Thereafter, the cells were washed three times by using 10 ml of 1M sorbitol, and resuspended by using 1 ml of 1M sorbitol, divided the cells into 100 ul, and maintained the cells at a temperature of −80° C. and used.
2-2. Preparing DNA Sample for Transformation
pJSKM316-GPD_inBGL1 (intracellular beta-glucosidase), pJSKM316-GPD_exBGL1 (extracellular beta-glucosidase), pJSKM316-GPD_EG1 (endoglucanase), pJSKM316-GPD_CBH1 (cellobiohydrolase), and pJSKM316-GPD_CBH2 (cellobiohydrolase II) plasmids were individually treated with PacI restriction enzyme respectively to excise ARS1/CEN from the plasmid, and then electrophoresed in an agarose gel. Linear DNA fragments other than 800 bps of ARS were each extracted from the agarose gel.
2-3. Multiple Co-Integration of Various Genes in K. marxianus Strain
300 ng of each linear DNA fragment were transformed into the K. marxianus competent cells (such that the total DNA volume is less than 10 ul) by combining the linear DNA fragments with the cells. Thereafter, the K. marxianus competent cells were reacted in ice for 20 minutes and then electroporated (MicroPulser Electorporator, available from Bio-Rad). Thereafter, the K. marxianus competent cells were resuspended by using 1 ml of YPDS (YPD+1M sorbitol) and cultured at a temperature of 37° C. for 2 hours at 250 rpm, spread on synthethic media (6.7 g/L Yeast nitrogen base w/o amino acid, 1.92 g/L Synthetic drop-out mix w/o uracil, 20 g/L glucose, and 20 g/L agar), and cultured at a temperature of 37° C. for 36 hours.
2-4. Confirming Genes Co-Integrated Into K. marxianus Strain
After co-integrating the genes, the K. marxianus competent cells were spread on minimal media having no uridine and cultured at a temperature of 37° C. for 2 days. After extracting genomic DNA from a single colony, gene co-integration was confirmed through PCR, and K. marxianus strain where all of ALD6, ACS1, and ACC genes were co-integrated was obtained. To amplify each gene, primers of Table 1 were used. Also, a PCR process involved treating the K. marxianus competent cells for 2 minutes at a temperature of 95° C., and then repeating for 30 times a thermal denaturation for 30 seconds at a temperature of 95° C., an annealing for 30 seconds at a temperature of 55° C., and an elongation for 3 minutes at a temperature of 72° C. to prepare a reaction product, and the reaction product was reacted for 54 minutes at a temperature of 72° C.









TABLE 1





Amplified materials
Sequences
SEQ No.


















ALD6
Forward-direction
5′-cattatcaatactcgccatttcaaag-3′
SEQ No. 5



primer




 



Reverse-direction
5′-ttacaacttaattctgacagcttttac-3′
SEQ No. 6



primer




 


ACS2
Forward-direction
5′-cattatcaatactcgccatttcaaag-3′
SEQ No. 7



primer




 



Reverse-direction
5′-ttatttcttttgttggttaagaattggg-3′
SEQ No. 8



primer




 


ACC
Forward-direction
5′-cattatcaatactcgccatttcaaag-3′
SEQ No. 9



primer




 



Reverse-direction
5′-tcattccaaagccttttgaagcttttctttg-3′
SEQ No. 10



primer









EXAMPLE 3
Co-Integration Efficiency According to OD Values of Strains
As shown in Example 2, dhaB1 (SEQ No. 11), dhaB2 (SEQ No. 12), dhaB3 (SEQ No. 13), gdrA (SEQ No. 14), gdrB (SEQ No. 15), aldH (SEQ No. 16), and KGSadh (SEQ No. 17) were inserted into a vector, respectively, and the vectors were excised by using a restriction enzyme to prepare a sample including the genes for transformation. Thereafter, the genes were co-integrated into K. marxianus competent cells. Also, primers in Table 2 were used to confirm the co-integration of the genes. Table 3 shows the co-integration efficiency and strain preparation time of K. marxianus when OD600 is 1 (Exp.1) and 3.5 (Exp.2). Specifically, Exp. 1 is relates to competent cell cultured until an optical density of 600 nm (OD600) of the cell is 1 and Exp. 2 is relates to competent cell cultured until an OD600 of the cell is 3.5.









TABLE 2





Amplified materials
Sequences
SEQ No.


















dhaB1
Forward-
5′-ATT GTG GAG TTG GAT GGT AAG CGT-
SEQ No. 18



direction primer
3′



 



Reverse-
5′-CCA AGT CAA GCA TGG AGG CAA TCA
SEQ No. 19



direction primer
A-3′



 


dhaB2
Forward-
5′-GGA GCT AAT CGC TGG TGT TGA AGA-
SEQ No. 20



direction primer
3′



 



Reverse-
5′-CCA AGT CGA TAT GTA AGG TGA CAG
SEQ No. 21



direction primer
GT-3′



 


dhaB3
Forward-
5′-CTA TGA GAG TTC AGG ATT ACC CAC
SEQ No. 22



direction primer
T-3′



 



Reverse-
5′-CCC TTT CTT AGC TTA TGT CTT TGT
SEQ No. 23



direction primer
TGG T-3′



 


gdrA
Forward-
5′-GAG GGT TGG ATT GTT TTG ATC GAT
SEQ No. 24



direction primer
GA-3′



 



Reverse-
5′-CCT CTA ATA TCT CTG ACA GGA GCA-
SEQ No. 25



direction primer
3′



 


gdrB
Forward-
5′-TCT TTC CCC TCC AGG TGT CAG GT-3′
SEQ No. 26



direction primer




 



Reverse-
5′-CGG ACA ATG GCA AAA CCT TCA CCA-
SEQ No. 27



direction primer
3′



 


aldH
Forward-
5′-CTG ACT TGA TGG AAG CCC ACG CT-3′
SEQ No. 28



direction primer




 



Reverse-
5′-GGA ACC TGT GAA GGC AAT AGC GTC
SEQ No. 29



direction primer
A-3′



 


KGSadh
Forward-
5′-CGA TGC CAT TGC TCA ACT TAT GAC
SEQ No. 30



direction primer
TCA-3′



 



Reverse-
5′-CCA AAC CAA TCA CGC CAG CTG GA-3′
SEQ No. 31



direction primer



















TABLE 3







Exp. 1*
Exp. 2*


















Host
KM36907
KM36907


Expression vector
pJSKM-316
pJSKM-316


Promoter
GPD promoter
GPD promoter


Terminator
CYC1 terminator
CYC1 terminator


Number of genes
6 types*
6 types*



(dhaB1, B2, B3,
(dhaB1, B2, B3,



gdrA, gdrB, aldH)
gdrA, gdrB, KGSadh)


Gene co-integration
28.8%
100%


efficiency


Strain preparation
6 days
5 days


time





*Exp. 1 shows 6-gene co-integration efficiency and co-integrated strain preparation time of K. marxianus when OD600 is 1.


*Exp. 2 shows 6-gene co-integration efficiency and co-integrated strain preparation time of K. marxianus when OD600 is 3.5.


*dhaB1 (SEQ No. 11), dhaB2 (SEQ No. 12), dhaB3 (SEQ No. 13), gdrA (SEQ No. 14), gdrB (SEQ No. 15), aldH (SEQ No. 16), KGSadh (SEQ No. 17).






When the method of the embodiments is used, genes may be more efficiently co-integrated into microbial organism to prepare a recombinant yeast strain than the conventional method. When the multiple genes co-integration technology is used, the strain preparation time of the new technology was lower by 4 times (6 days according to an embodiment) than strain preparation time of the conventional technology (27 days). Accordingly, by using the efficient multiple genes co-integration technology, various types of genes may be co-integrated into the microbial organism more effectively.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. A method of simultaneous co-integration of multiple nucleic acid sequences into a genome of a microbial organism comprising:
culturing a microbial organism to be transformed until a growth phase is reached in which a random integration is facilitated, wherein during the growth phase in which random integration is facilitated, the microbial organism has a cell density greater than its cell density during its log phase of growth, and wherein the growth phase is a stationary phase; and
transforming the microbial organism by mixing the cultured microbial organism with a sample to be co-integrated into the microbial organism with, wherein the sample comprises multiple nucleic acid fragments encoding different gene products, wherein the microbial organism to be transformed is a strain having a dominant non-homologous end joining (NHEJ) system, wherein the multiple nucleic acid fragments are linear nucleic acid fragments comprising a promoter, an open reading frame encoding a gene product, and a terminator, and the gene product is at least part of an enzyme participating in a metabolic pathway of the microbial organism,
and the metabolic pathway is related to a synthesis of one or more organic acids.

2. The method of claim 1, wherein during the growth phase in which the random integration is initiated, the microbial organism has an optical density at 600 nm (OD600) of about 2.0 to about 10.0.

3. The method of claim 2, wherein the OD600 is about 3.5 to about 6.0.

4. The method of claim 1, wherein the microbial organism to be transformed is a non-conventional yeast, or fungi cell.

5. The method of claim 1, wherein the microbial organism to be transformed is K. marxianus.

6. The method of claim 1, wherein the sample comprises 2 to 10 of the multiple nucleic acid fragments encoding different gene products.

7. The method of claim 6, wherein the sample comprises 3 to 6 of the multiple nucleic acid fragments encoding different gene products.

8. The method of claim 1 further comprising co-integrating multiple copies of a nucleic acid sequence into the microbial organism.