Genetically modified organism and method for spinosyn production

Abstract

A genetically modified strain of Saccharopolyspora pogona to be used in a method for the production of high concentrations of spinosyns A Broadly, the disclosure relates to an improved method for spinosyn production and a genetically modified microbial host capable of increased production yields of spinosyns. The disclosure also relates to the modification of the bus gene cluster in S. pogona to convert S. pogona into a "classical" spinosyn A producing strain. The genetically modified S. pogona strain demonstrates an improvement in the production of spinosyn. The present disclosure also relates to a method of producing high titers of spinosyn A with an S. pogona host strain which has been genetically modified to convert its bus gene cluster into a "classical" spnA gene cluster capable of producing spinosyn A in S. pogona.

Description

GENETICALLY MODIFIED ORGANISM AND METHOD FOR SPINOSYN PRODUCTIONBACKGROUND

[0001] Spinosyns are natural products derived from bacteria which have a broad range of action against many insect pests. They are produced by several strains of the genus Saccharopolyspora. As insect control agents, they are used as insecticides for a variety of crops. Spinosyns are macrolide molecules (Figure 1) consisting of a 21 -carbon tetracyclic lactone and two deoxy-sugar moieties, forosamine and tri-O-methyl rhamnose, which are essential for the insecticidal activity of spinosyns. A commercially available product, Spinosad, is a mixture of spinosyn A and D, which differ from each other by the presence of an additional methyl group in the C6 position of the spinosyn D structure. Of importance to the following disclosure, Spinosad is composed primarily of spinosyn A.

[0002] Another group of compounds belonging to the spinosyns family are butenyl-spinosyns. These compounds are structurally similar to “classical” spinosyns, A and D, however they also possess a butenyl side chain at the C21 position of the lactone ring. Despite the high structural similarity of butenyl spinosyns to “classical” spinosyns, butenyl-spinosyn al, which is the predominant form produced, exhibits stronger insecticidal activity and possesses a broader insecticidal spectrum than Spinosad.

[0003] The mechanism of action of spinosyns is related to their binding of the GABA and nicotinic acetylcholine receptors on the membranes of insect neurons. This causes rapid excitation of the insect nervous system leading to muscle contractions, tremors, paralysis, and death.

[0004] Spinosyns and butenyl-spinosyns are secondary metabolites produced by two actinomycete strains Saccharopolyspora spinosa and Saccharopolyspora pogona, respectively. Since the early 1980’s when Saccharopolyspora spinosa and spinosyn were discovered and their insecticidal activity confirmed, many efforts have been made to increase production levels of spinosyns to match industrial production requirements. However, despite years of research and development activities with S. spinosa, big improvements in spinosyn production titers were seldom achieved, contributing to the current high market price of Spinosad as compared to other insecticides. Oneof the main obstacles causing such slow improvements is the lack of a simple and efficient genetic engineering system for S. spinosa (Rang et al., 2020).

[0005] In contrast, S. pogona, which produces butenyl-spinosyns and was discovered later, was not developed into industrial production strain, because butenyl spinosyns were not registered for commercial use. Other butenyl-spinosyn producing strains include Saccharopolyspora hattusasensis CR 35067 and Saccharopolyspora sp. ASAGF58 (Guo et al, 2020).

[0006] The biosynthetic gene cluster for spinosyn biosynthesis in S. spinosa has a size of approx. 80 kb and includes 23 genes (termed “spn” genes) encoding enzymes for spinosyn production. Five of the genes encode large polyketide synthases (PKS), four encode polyketide bridging genes, eight are related sugar biosynthesis genes, two glycosyltransferase genes and four sugar methylation genes.

[0007] The biosynthetic pathways and the corresponding genes of butenyl-spinosyn and spinosyn are highly similar but not identical. Initially, non-glycosylated intermediate, the aglycone, is biosynthesized and later rhamnose is attached as the first sugar. In addition, rhamnose is tri-O- methylated to yield the intermediate pseudoaglycone. In the final stage the second sugar, forosamine, is incorporated and active spinosyn molecules are generated.

[0008] The butenyl-spinosyn biosynthetic genes, termed also “bus” genes show strong similarity 91-97% to the homologous genes from the classical spinosyn “spn” gene cluster. Their protein sequences have a similarity of 81-97%. The major difference between both gene clusters is the presence of an additional 5301-bp sequence in the first PKS gene busA which does not have a counterpart in the spnA gene. This sequence encodes an additional polyketide synthase module lb (acetyltransferase (AT), dehydratase (DH), ketoreductase (KR) and acyl carrier protein (ACP) domains) and the ketosynthase (KS) domain of module la in the butenyl-spinosyn busA gene and is responsible for addition of two carbons in the C-21 tail of the spinosyn.

[0009] In principle, it is possible to modify the spinosyn or butenyl spinosyn biosynthetic genes. In fact, combinatorial modifications in PKS molecules were shown in the past to be a powerful tool to enrich the diversity of natural products. However, the success of targeted modifications is far from guaranteed as often the desired molecules are not produced or are produced at extremely low levels, impractical for industrial application.

[0010] The first Experiments of spinosyn gene cluster engineering were made by replacement of spinosyn PKS loading module with loading modules from erythromycin and avermectin gene clusters resulting in biosynthesis of several spinosyn derivatives when fed with different starter units (Sheehan et al., 2006). However, the main obstacle in generation of new improved spinosyn production strains remains the difficult genetic manipulation of S. spinosa.

[0011] One alternative approach for modification of the spinosyn gene cluster is cloning and transfer into a heterologous host in which modifications of biosynthetic genes are easier achieved. Such modification of the spn cluster was already performed with RedaP mediated linear-circular homologous recombination in E. coli after cloning of the entire cluster into a pBAC vector. The aim of the study was to construct a gene cluster encoding butenyl spinosyn biosynthesis by insertion of the additional 5301-bp region from the bus cluster into the spnA gene. The modified spn cluster with this “bus” insertion was transformed into a Streptomyces albus heterologous host and only low quantities of butenyl spinosyn were detected (Song et al., 2020). Most other studies which involved modification or relocation of PKS clusters into heterologous hosts also resulted in very low titers or absence of the target product.

[0012] In summary, production of spinosyn is slow and costly because the common spinosyn production strain, Saccharopolyspora spinosa, only produces low titers of spinosyn. Additionally, it is very difficult to modify and frequently results in unsuccessful genetic modifications. Further, even successful genetic modifications, such as with a bus insertion, still result in an S. spinosa strain having very low titers or an absence in production of spinosyn. Therefore, a new microbial host is needed that would be capable of producing high titers of spinosyn or butenyl spinosyn.

[0013] To address this issue, the present disclosure relates to an improved method for spinosyn production and a genetically modified microbial host capable of producing high titers of spinosyns.SUMMARY

[0014] The disclosure is related to the construction of a genetically modified microbial host capable of producing high concentrations of spinosyns and, specifically, a genetically modified Saccharopolyspora pagona strain engineered to produce butenyl-spinosyn. The drawback of the natural spinosyn production strain Saccharopolyspora spinosa is that it only produces low titers of spinosyn and is very difficult to genetically modify. Further, even successful geneticmodifications, such as with a bus insertion, result in an 5. spinosa strain having very low titers or an absence in production of spinosyn. Attempts at spinosyn gene cluster cloning and transfer into a heterologous host have also failed to produce high titers of spinosyn.

[0015] Until now, no one has considered whether it is possible to modify S. pogona for increased spinosyn production. The present disclosure relates to the modification of the bus gene cluster in S. pogona to convert S. pogona into a “classical” spinosyn A producing strain. The presently disclosed genetically modified S. pogona strain demonstrates an improvement in the production of spinosyn A and results in higher titers of this coveted insecticide. The present disclosure also relates to a method of producing high titers of spinosyn A with an S. pogona host strain which has been genetically modified to convert its bus gene cluster into a “classical” spnA gene cluster capable of producing spinosyn A in S. pogona. Therefore, the present disclosure presents an improvement over traditional methods and a genetically modified S. pogona capable of increased spinosyns production yields.

[0016] Therefore, based on the foregoing and continuing description, the subject invention in its various embodiments may comprise one or more of the following features in any non-mutually- exclusive combination:

[0017] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns, comprising a busA gene deletion;

[0018] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns, comprising a spnA gene insertion;

[0019] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns, comprising wherein one or more nucleotide sequences encode expression of enzymes to produce one or more spinosyns comprising an ethyl chain at a C21 position of a lactone ring;

[0020] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns, wherein the spnA gene is under control of a busA promoter region.

[0020] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns, wherein spinosyns comprising a butenyl chain at the C21 position of the lactone ring comprise no more than 0.05% total spinosyn produced;

[0020] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns, wherein a ratio of spinosyn A to spinosyn D production is larger than a ratio of spinosyn A to spinosyn D production in a wild-type Saccharopolyspora spinosa.

[0021] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising a portion of at least one nucleotide sequence encoding a busA promoter region (SEQ ID 41);

[0022] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising a portion of at least one nucleotide sequence encoding a spnA gene (Seq ID 33).

[0023] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising at least one nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 33.

[0024] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising at least one nucleotide sequence encoding a busA promoter region;

[0025] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising at least one nucleotide sequence downstream of the busA promoter region that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 33;

[0026] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising at least one nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 37;

[0027] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising at least one nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 39;

[0028] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising the one or more nucleotide sequences comprise a nucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID 38;

[0029] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns comprising the one or more nucleotide sequences comprise a nucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID 40;

[0030] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns wherein the one or more spinosyns comprises spinosyn A;

[0031] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns wherein the one or more spinosyns comprises spinosyn D;

[0032] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns wherein at least a portion of the busA gene has been replaced with at least a portion of the spnA gene.

[0033] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns wherein relative to wild type S. pogona, the one or more nucleotide sequences encoding expression of enzymes to produce one or more spinosyns does not encode at least one domain of wild type Saccharopolyspora pogona selected from the group consisting of acetyltransferase (AT), dehydratase (DH), ketoreductase (KR) and acyl carrier protein (ACP) of module lb and a ketosynthase (KS) of module la in a butenyl-spinosyn busA gene cluster of wild type Saccharopolyspora pogona.

[0034] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns wherein no greater than 9% of the one or more spinosyns is spinosyn D;

[0035] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns wherein one or more of the spinosyns comprises the chemical structure,wherein R is either a hydrogen or an alkyl group, the alkyl group having n carbons, wherein n is a number equal to or greater than 1 ;

[0036] A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns wherein R is a hydrogen group.

[0037] A method of making a Saccharopolyspora pogona cell comprising transforming a Saccharopolyspora pogona cell with one or more nucleotide sequences to effect a busA gene deletion;

[0038] A method of making a Saccharopolyspora pogona cell comprising transforming a Saccharopolyspora pogona cell with one or more nucleotide sequences to effect a spnA gene insertion;

[0039] A method of making a Saccharopolyspora pogona cell wherein one or more nucleotide sequences comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 33;

[0040] A method of making a Saccharopolyspora pogona cell wherein one or more nucleotide sequences comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 18;

[0041] A method of making a Saccharopolyspora pogona cell wherein one or more nucleotide sequences comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 21;

[0042] A method of making a Saccharopolyspora pogona cell wherein one or more nucleotide sequences comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to Seq ID 41;

[0001] An inoculant composition comprising a Saccharopolyspora pogona cell comprising a busA gene deletion;

[0002] An inoculant composition comprising a Saccharopolyspora pogona cell comprising a spnA gene insertion.

[0003] A method of producing spinosyn A with a Saccharopolyspora pogona cell comprising a spnA gene insertion;

[0004] A method of producing spinosyn A with a Saccharopolyspora pogona cell comprising a busA gene deletion;

[0005] A method of producing spinosyn D with a Saccharopolyspora pogona cell comprising a spnA gene insertion;

[0006] A method of producing spinosyn D with a Saccharopolyspora pogona cell comprising a busA gene deletion;

[0007] A Saccharopolyspora pogona cell genetically modified to produce butenyl-spinosyn, wherein said cell is engineered to express a spnA gene from Saccharopolyspora spinosa.

[0008] A Saccharopolyspora pogona cell genetically modified to produce butenyl-spinosyn, wherein one or more nucleotide sequences encode expression of enzymes to produce one or more spinosyns comprising an ethyl chain at a C21 position of a lactone ring;

[0009] A Saccharopolyspora pogona cell genetically modified to produce butenyl-spinosyn, wherein no greater than 9% of the one or more spinosyns is spinosyn D; and

[0010] A Saccharopolyspora pogona cell genetically modified to produce spinosyn, wherein no greater than 9% of one or more spinosyns is spinosyn D.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1. Structures of butenyl-spinosyn and spinosyn A. The difference at C21 position in butenyl-spinosyn is marked in a circle.

[0012] Figure 1.1 Structures of spinosyn A and D. The difference at C6 position is identified as R. Wherein R = H in Spinosyn A, and R = CH3 in Spinosyn D.

[0013] Figure 2. Comparison of spn and bus gene clusters. The first PKS gene busA contains the presence of an additional 5301 -bp sequence, blue, which does not have a counterpart in the spnA gene. This sequence encodes an additional polyketide synthase module, acetyltransferase (AT), dehydratase (DH), ketoreductase (KR), and acyl carrier protein (ACP) domains of module lb and the ketosynthase (KS) domain of module la in the butenyl-spinosyn busA gene and is responsible for addition of two carbons in the C-21 tail of the spinosyn.

[0014] Figure 3: Production of spinosyns by Saccharopolyspora pogona strains with shortened versions of busA gene.

[0015] Figure 4: Production of butenyl spinosyns and spinosyns by Saccharopolyspora pogona strains with replaced busA gene with spnA gene.

[0016] Figure 5: Production of butenyl spinosyns and spinosyns by Saccharopolyspora spinosa wild type strain NRRL18539 (spinosyn production control), Saccharopolyspora pogona strain NRRL30141 and strain SP19678 (expressing spnA gene instead busA).DETAILED SUMMARY

[0017] The present disclosure provides various embodiments, approaches, and strategies by way of Experiment only and are not meant to limit the scope of the disclosure. Certain aspects of each embodiment may be combined with aspects of other embodiments to yield further embodiments for producing spinosyn A in S. pogona.

[0018] The first attempt at reengineering of butenyl spinosyn producing genes into spinosyn A biosynthesis relied on deletion of the redundant 5301 -bp long fragment (“redundant fragment”) from the first biosynthetic gene busA in S. pogona (Experiment 1). In preparing the experimental plan, it was considered that PKS protein modules are tightly connected with specific linkers between them. To achieve functional deletion modification of the redundant fragment of the busA gene in S. pogona was performed by addition of two different variants of linkers. These linkers connected the modules and enabled adequate functionality of the modified BusA biosynthetic enzyme. While this attempt successfully achieved deletion modification of the redundant fragment, none of the obtained strains produced any spinosyn A, suggesting that S. pogona might not be a viable option for producing high concentrations of spinosyn A.

[0019] The second attempt, which was considered unlikely to succeed based on results from the first attempt, relied on the complete replacement of the busA gene from the bus gene cluster in S. pogona with homologous spnA gene from the spinosyn gene cluster of S. spinosa. Surprisingly, this approach was very successful, which was unexpected due to initial experimentation with S. pogona as a production strain. The spnA expressing S. pogona strain produced spinosyn A in concentrations around 50 mg / L in shake flask scale.

[0020] The present disclosure and related inventions have high commercial value and demonstrate that S. pogona is the heterologous strain with the highest potential for providing significant improvements in the commercial production of Spinosad, which is composed mainly of spinosyn A. The present disclosure demonstrates that replacing the busA gene of S. pogona ’s bus gene cluster with the spnA gene from S. spinosa spn gene cluster yields production of commercially useful spinosyn A without reduction in S. pogona ’s production capacity.

[0021] EXPERIMENTS

[0022] Experiment 1: Construction of plasmid DNA for deletion of the 5301-bp fragment of busA gene for spinosyn production

[0023] In order to modify busA gene for production of spinosyn A two plasmids containing different linkers highly recommended for smooth PKS production were constructed on the basis of suicide E. coli-Streptomyces shuttle vector.

[0024] The first step of the plasmid pSSP_72 (SEQ ID NO: 10) construction was PCR amplification of separate DNA fragments of the busA gene, performed using primer pair SEQ ID NO: 4 and SEQ ID 5 for the homologous region upstream of the 5301 bp fragment (SEQ ID NO: 6), and primer pair SEQ ID NO: 7 and SEQ ID NO: 8 for the homologous region downstream of the 5301 bp fragment (SEQ ID NO: 9) from the targeted deletion. The template for amplification of these DNA fragments was the isolated S. pogona NRRL 30141 genomic DNA. Genomic DNA was isolated from S. pogona culture using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) according to the protocol of the manufacturer. The primer set SEQ ID NO: 1 and SEQ ID NO: 2 was used for linearization of the suicide plasmid SEQ ID NO: 3. Primers SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8 were designed to contain overhanging sequences for HiFi assembly of amplified fragments into the circular plasmid.

[0025] For the second plasmid pSSP_73 (SEQ ID NO: 15) construction, primer pair SEQ ID NO: 4 and SEQ ID NO: 11 was used to amplify homologous region upstream of the 5301 bp fragment (SEQ ID NO: 12) while primer pair SEQ ID NO: 13 and SEQ ID NO: 8 resulted in homologous region downstream of the 5301 bp fragment SEQ ID NO: 14. Template for amplification of these two artificial DNA fragments was genomic DNA isolate from a S. pogona NRRL 30141 strain. Primers SEQ ID NO: 11 and SEQ ID NO: 13 were also designed to contain overhanging sequences that allow synthetized fragments to be assembled into a plasmid.

[0026] All fragments were amplified using Eppendorf cycler and Phusion polymerase (Thermo Fisher) in the buffer provided by the manufacturer and with the addition of 200 pM dNTPs, 0.5 pM of each primer, and approximately 10 ng of template in a final volume of 50 pl for 30 cycles using the PCR cycling conditions: 98°C for 30s, 30 cycles of (98°C for 30s, 70°C for 25s, 72°C for 40 / 60s), 72°C for 5 min, 10°C on hold.

[0027] PCR reaction products of each fragment were run on 0.8 % agarose gel, excised from the gel, and extracted from the gel by GeneJET Gel Extraction Kit (Thermo Fisher) according to the protocol provided by the manufacturer. PCR mixture for amplifying fragment SEQ ID NO: 3 (linear plasmid) was Dpnl digested prior electrophoresis.

[0028] Gel extracted fragments SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9 were further assembled into the plasmid using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs). Incubation was carried out according to the manufacturer’s instructions using Eppendorf cycler. HiFi assembly mixture containing assembled plasmid was used for transformation of electrocompetent DH 10(3 / coli cells. The transformation resulted in apramycin resistant colonies. E. coli culture of colony PCR confirmed transformant was used for plasmid isolation with GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). The correct assembly of DNA fragments into the plasmid pSSP_72 SEQ ID NO: 10 was additionally confirmed by sequencing.

[0029] Gel extracted fragments SEQ ID NO: 3, SEQ ID NO: 12 and SEQ ID NO: 14 were assembled into the plasmid pSSP_73 SEQ ID NO: 15. HiFi assembly mixture was used for transformation of electrocompetent DH1O0 E. coli cells and plasmid was isolated from overnight culture following the same procedure as described for pSSP_72 plasmid construction. Plasmid pSSP_73 was also confirmed by sequencing.

[0030] For the purpose of conjugational transfer of SEQ ID NO: 10 and SEQ ID NO: 15 into . pogona strain plasmids were first introduced into electrocompetent ETpUZ8002 E. coli cells using electroporation technique. Transformant obtained on 2TY agar plate supplemented with apramycin, chloramphenicol and kanamycin in concentrations of 100 pg / mL, 20 pg / mL and 50 pg / mL respectively was used for preparation of pre-culture further used in conjugational transfer with S. pogona strain. The presence of the correct plasmid DNA in conjugational E. coli strain ETpUZ8002 was previously confirmed with colony PCR.

[0031] Experiment 2: Construction of plasmid DNA for replacement of busA gene with spnA gene

[0032] Two different plasmids on the basis of suicide E.coli-Streptomyces shuttle vector (SEQ ID NO: 3) were constructed for the busA gene replacement with spnA fragment. The first, pSSP_79 (SEQ ID NO: 22), contains the homologous regions upstream and downstrean of the busA gene,while pSSP_80 (SEQ ID NO 32) also contains the spnA fragment between these two homologous regions.

[0033] Isolated genomic DNA from S. pogona strain was used as template for PCR amplification of homologous regions upstream and downstream of the busA gene. Primer pair SEQ ID NO: 16 and SEQ ID NO: 17 was designed for amplification of the 1355 base pairs long homologous region SEQ ID NO: 18 upstream of the busA gene while PCR product using primer pair SEQ ID NO: 19 and SEQ ID NO: 20 resulted in 1223 base pair long homologous region SEQ ID NO: 21 downstream of the busA gene. These two amplified fragments were assembled together with linearized plasmid (SEQ ID NO: 3) into the vector pSSP_79 (SEQ ID NO: 22). This construct was the basis for further construction of the final plasmid for busA gene replacement with a 7788 bp long spnA fragment (SEQ ID NO: 33).

[0034] For replacement of busA gene with spnA gene, isolated genomic DNA from Saccharopolyspora spinosa NRRL18539 was used for amplification of the 7788 bp long spnA fragment in two parts. S. spinosa genomic DNA was isolated from S. spinosa culture using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) according to the instructions of the manufacturer. First, part of spnA gene was amplified by a PCR reaction using primer pair SEQ ID NO: 26 and SEQ ID NO: 27 resulting in DNA fragment SEQ ID NO: 28 and the second part with primers SEQ ID NO: 29 and SEQ ID NO: 30 providing the DNA fragment SEQ ID NO: 31. For the shuttle vector backbone pSSP_79 SEQ ID NO: 22 was used. The plasmid was linearized by PCR reaction using primers SEQ ID NO: 23 and SEQ ID NO: 24. The PCR amplified linearized plasmid SEQ ID NO: 25 containing homologous regions for busA gene deletion was digested by Dpnl enzyme to remove any remaining template plasmid. This sequence together with both parts of spnA gene SEQ ID NO: 28 and SEQ ID NO: 31 was assembled into a complete shuttle vector for busA gene replacement with spnA gene pSSP_80 SEQ ID NO: 32.

[0035] For assembly of these three DNA fragments NEBuilder HiFi DNA Assembly Master Mix was used by the instruction of manufacturer. The assembled plasmid was further transformed into electrocompetent DH10|3 cells and isolated from E. coli overnight culture using GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) for plasmid isolation. Plasmid SEQ ID NO: 32 was further analyzed by digestion with Fast Digest restriction enzymes Xba I and Xho I (Fermentas). Finally, plasmid was confirmed by sequencing of all junction regions.

[0036] For the purpose of conjugational transfer into 5. pogona strain plasmid SEQ ID NO: 32 was introduced into ETpUZ8002 E. coli strain as described in Experiment 1.

[0037] Experiment 3: Selection of a starting strain

[0038] Saccharopolyspora pogona wild type strain NRRL 30141 and Saccharopolyspora spinosa NRRL18539 wild type strain were obtained from ARS culture collection.

[0039] Experiment 4: Conjugation of Saccharopolyspora pogona NRRL 30141 strainConjugation was performed following the description of the paper (Matsushima et al., 1994). Briefly, frozen vegetative stocks of the Saccharopolyspora pogona strain NRRL 30141 preserved in 20 % glycerol at -80°C were used to inoculate TS broth which was incubated at 30°C for 48h - 60h with shaking. The cultures were homogenized and re-inoculated into the same media. The cultures were further incubated at 30°C for 16h and again homogenized. Diluted culture (1 :4) in TS broth was additionally incubated at 30°C for 2 - 6 h. Cells were harvested by centrifugation and resuspended in TS broth.Table 1: Composition of tryptone soy (TS) broth medium

[0040] Medium was prepared according to the protocol provided by the manufacturer.E. coli strains containing either SEQ ID NO: 10, SEQ ID NO: 15, or SEQ ID NO: 33 constructs obtained as described in Experiments 1 and 2 were spread on 2TY agar plates containing antibiotics (apramycin 100 mg / L, kanamycin 25 mg / L, chloramphenicol 20 mg / L). Plates were incubated at 37°C for approximately 1 day. A single colony of each construct was inoculated in in 2TY liquid medium with appropriate antibiotics (apramycin 100 mg / L, kanamycin 25 mg / L, chloramphenicol 20 mg / L) and incubated overnight at 37°C. This culture was used for inoculation of fresh liquid 2TY medium with appropriate antibiotics and incubated at 37°C with shaking. The cells were harvested in early exponential phase and resuspended in fresh liquid 2TY medium.50 pL of Saccharopolyspora pogona culture was mixed with 50 pL of / / . coli cultures containing the individual construct. The mixture was spread on agar plates, incubated overnight and overlayedwith appropriate antibiotics (nalidixic acid and apramycin). Plates were further incubated at 30°C for 7-14 until exconjugants appeared.

[0041] We obtained 14 strains with SEQ ID NO 10 construct; 9 strains with SEQ ID NO 15; and 38 strains with SEQ ID NO 32. The presence of the integrated constructs in strains was confirmed with colony PCR.

[0042] Experiment

[0043] 5: Cultivation of Saccharopolyspora pogona strains in 48 deep well microtiter plates

[0044] All of the constructed strains obtained with conjugation, as described in Experiment 4, as well as control strains were cultivated using the following procedure. Vegetative stage medium was inoculated with 1 plug of the culture on sporulation agar medium per well in 48-deep well microtiter plates containing 1 mL of vegetative medium supplemented with 50 mg / L apramycin. The cultures were incubated at 30°C for 48h at 220 RPM with humidity controlled at 70%. The culture in the vegetative medium was used as seed culture which was then inoculated into the production medium 1. A 10 % inoculum was used (75 pL per 750 pL of production medium 1 in each well of 48- deep well microtiter plate). The cultures were incubated at 30°C up to 168h at 220 RPM with humidity control at 70%. The fermented cultures were sampled and analyzed as described in the Experiment 8. The titers of butenyl spinosyns were measured using the LC / MS.Table 3: Composition of vegetative (seed) medium

[0045] The ingredients of the vegetative medium (Table 3) were mixed and the pH was set to 7.The medium was autoclaved at 121 °C for 20 min.Table 4: Composition of production medium

[0046] The ingredients of the production medium 1 (Table 4) were mixed and the pH was set to 7. The medium was autoclaved at 121 °C for 30 min.

[0047] Experiment 6: Integration of introduced constructs

[0048] All of the strains obtained as described in Experiment 4 and having confirmed inactivation of butenyl spinosyn production in cultivation as described in Experiment 5, were submitted to subcultivations to enable integration of the introduced constructs into the genome. Strains obtained on sporulation agar medium supplemented with appropriate antibiotics obtained as described in Experiment 4 were repatched on sporulation agar medium supplemented with 50 mg / L apramycin. Plates were incubated at 30°C for 8 days and 1 plug of the culture was used for inoculation of 50 mL Falcon tubes containing 5 mL tryptone soy broth with 50 mg / L apramycin. The cultures were incubated at 30°C for 48 h at 220 RPM with humidity controlled at 70 % and homogenized. This culture was used to inoculate 50 mL Falcon tubes containing 5 mL tryptone soy broth (0.1%inoculum was used). The cultures were incubated at 30°C and 220 RPM with humidity controlled at 70% for 48h - 72h until good growth was observed.

[0049] Homogenization, filtration and inoculation in fresh tryptone soy broth was repeated 8 times in total. After each cultivation homogenized and filtered cultures were diluted in 0.9 % NaCl and dilutions spread on sporulation agar medium. Cultures were incubated at 30°C for 8 days. Grown single colonies were repatched on sporulation agar medium with and without addition of 50 mg / L apramycin.

[0050] For each construct tested, approximately 1,500 colonies were repatched. Cultures were incubated at 30°C for 8 days and the colonies that did not grow on sporulation agar medium supplemented with antibiotic but did grow on sporulation agar medium were selected for further testing (see table 7). One plug of culture from sporulation agar medium was used as inoculum of 50 mL Falcon tubes containing 5 mL tryptone soy broth supplemented with 10 g / L dextrose monohydrate. Cultures were incubated at 30°C for 48 h at 220 RPM with humidity control at 70 %. Cells were harvested by centrifugation; supernatant was discarded and cells resuspended in 20 % glycerol and stored at - 80°C.Table 5: Saccharopolyspora pogona strains with integrated constructs obtained by conjugation

[0051] Experiment 7 : Cultivation of Saccharopolyspora pogona strains in Erlenmeyer flasks

[0052] All of the strains obtained as described in Experiment 6 and control strains were cultivated according to the following procedure. For the vegetative stage 250 mL Erlenmeyer flasks were used containing 50 mL of seed medium. Seed medium was inoculated with 1% (0.5 mL per 50 mL of vegetative medium) of frozen vegetative stocks of the strains preserved in 20% glycerol at -80°C. The cultures were incubated at 30°C for 48h at 220 RPM with humidity controlled at 70%. Vegetative culture was again inoculated in the vegetative medium. A 10% inoculum was used (5 mL per 50 mL of vegetative medium in 250 mL Erlenmeyer flask). The cultures were incubated at 30°C for 48h at 220 RPM with humidity control at 70% and then 10% was used for inoculation of the production medium. The cultures were further incubated at 30°C up to 168h at 220 RPM with humidity controlled at 70 %. The fermented cultures were sampled and analyzed as described in the Experiment 8. The titers of butenyl spinosyns and spinosyns were measured using the HPLC UV detector and with LC / MS as described in Experiment 8.

[0053] Experiment 8: Analysis of butenyl spinosyns and spinosyns titers in fermentation broths

[0054] All of the strains that had been cultivated following the procedure described in Experiments 5 and 7 were analyzed according to the following procedure. For extraction of the metabolites, the fermented production medium was diluted 1: 1 with methanol. The samples were extracted for Ihat room temperature with constant agitation and then centrifuged. The samples were immediately analyzed by HPLC UV detector or stored at -20°C until analysis. If needed (detection of very low quantities of products) the samples were also analyzed by LC / MS.

[0055] The samples were analyzed on Thermo Accela 1000 HPLC instrument coupled with PDA detector. The method is based on Thermo Accucore Cl 8, 100x4.6 mm, 2.6 um particle size column, kept at 60°C, with mobile phase A - 100 mM ammonium acetate in water pH 4 and mobile phase B - acetonitrile, in isocratic elution, with conditions: 38 % A and 62 % B. Analytes spinosyn A, spinosyn D, butenyl spinosyn A and butenyl spinosyn D were observed at 254 nm.

[0056] For the detection of very low quantities of products the samples were also analyzed on Thermo Accela 1250 HPLC instrument coupled to Thermo TSQ Quantum Access MAX, MS / MS capable mass spectrometer. The method is based on Thermo Accucore Cl 8, 100x4.6 mm, 2.6 um particle size column, kept at 60°C, with mobile phase A - 100 mM ammonium acetate in water pH 4 and mobile phase B - acetonitrile, in gradient program, with starting conditions: 80% A, linear gradient increase in B% to 90% at 20 min, and 5 min stabilization to initial conditions, at 0.5 ml / min flow rate. Mass spectrometer was equipped with hESI ion source, operated in positive (+) mode, with spray voltage set at 4600 V, vaporizer temperature at 450°C, collision pressure at 1.0 torr and 40 V collision energy. Analytes spinosyn A, spinosyn D, butenyl spinosyn A and butenyl spinosyn D were observed in MRM mode with transitions from parent 733, 747, 759 and 775 m / z to daughter ion 142.2.

[0057] Experiment 9: Production of butenyl spinosyns and spinosyns by Saccharopolyspora pogona strains with shortened busA gene

[0058] Cultivation was performed as described in Experiment 7. The extraction and analysis by HPLC UV detector were performed as described in Experiment 8.

[0059] As seen in Figure 3, strains with shortened busA gene containing domain linker from SEQ ID NO: 10 (ABSP17563, ABSP17565) and domain linker from SEQ ID NO: 15 (ABSP17564, ABSP17566, ABSP17567, ABSP17568) were almost completely depleted, and no production of spinosyns was observed. From the results, it can be concluded that modified (shortened) busA gene(s) tested in this Experiment were inactive leading to disrupted spinosyns production. During PKS production, communication between domains and modules, mostly performed by linkers, isvery important. To date, this complex mechanism of communication during biosynthesis is not fully elucidated making these kinds of functional modifications difficult to be performed.

[0060] Experiment 10: Production of butenyl spinosyns and spinosyns by Saccharopolyspora pogona strains with replaced busA gene with spnA gene

[0061] The cultivation was performed as described in Experiment 7. The extraction and analysis were performed as described in Experiment 8.

[0062] As seen in Figure 4, two of the tested strains expressing spnA gene (SEQ ID NO 32) produced spinosyn A and no butenyl spinosyn in both tested media. Titers of spinosyn A were slightly lower compared to butenyl spinosyn titers in parent strain S. pogona NRRL30141.

[0063] Experiment 11: Re-evaluation of production of spinosyns by Saccharopolyspora pogona strain SP19678

[0064] Cultivation was performed as described in Experiment 7 by adding methyl oleate into the production medium for Saccharopolyspora spinosa NRRL18539 fermentation, which was used as control strain for spinosyn production. Saccharopolyspora pogona strain NRRL30141 is the origin strain for strain SP19678.

[0065] Extraction and analysis were performed as described in Experiment 8. As seen in Figure 5, Strain SP19678 had higher spinosyn production compared to NRRL18539 strain. Additionally, lower percentage of spinosyn D content was observed when cultivating SP19678 (8 - 9 %) compared to NRRL18539 (11 - 16 %).

[0066] Sequence Listings

Claims

CLAIMSWhat is claimed is:

1. A genetically modified Saccharopolyspora pogona cell engineered to produce spinosyns, comprising: a busA gene deletion; and a spnA gene insertion; wherein one or more nucleotide sequences encode expression of enzymes to produce one or more spinosyns comprising an ethyl chain at a C21 position of a lactone ring.

2. The Saccharopolyspora pogona cell of claim 1 , wherein the spnA gene is under control of a busA promoter region.

3. The Saccharopolyspora pogona cell of claim 1 , wherein: spinosyns comprising a butenyl chain at the C21 position of the lactone ring comprise no more than 0.05% total spinosyn produced.

4. The Saccharopolyspora pogona cell of claim 1, wherein: a ratio of spinosyn A to spinosyn D production is larger than a ratio of spinosyn A to spinosyn D production in a wild-type Saccharopolyspora spinosa.

5. The Saccharopolyspora pogona cell of claim 1, further comprising: a portion of at least one nucleotide sequence encoding a busA promoter region (SEQ ID 41); and a portion of at least one nucleotide sequence encoding a spnA gene (Seq ID 33).

6. The Saccharopolyspora pogona cell of claim 1, comprising: at least one nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 33.

7. The Saccharopolyspora pogona cell of claim 1, further comprising: at least one nucleotide sequence encoding a busA promoter region; and at least one nucleotide sequence downstream of the busA promoter region that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 33.

8. The Saccharopolyspora pogona cell of claim 1, further comprising: at least one nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 37.

9. The Saccharopolyspora pogona cell of claim 1, further comprising:at least one nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 39.

10. The Saccharopolyspora pogona cell of claim 1, further comprising: the one or more nucleotide sequences comprise a nucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID 38.

11. The Saccharopolyspora pogona cell of claim 1, further comprising: the one or more nucleotide sequences comprise a nucleotide sequence encoding an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID 40.

12. The Saccharopolyspora pogona cell of claim 1, wherein: the one or more spinosyns comprises spinosyn A.

13. The Saccharopolyspora pogona cell of claim 1, wherein: the one or more spinosyns comprises spinosyn D.

14. The Saccharopolyspora pogona cell of claim 1, wherein at least a portion of the busA gene has been replaced with at least a portion of the spnA gene.

15. The Saccharopolyspora pogona cell of claim 1, wherein relative to wild type S. pogona, the one or more nucleotide sequences encoding expression of enzymes to produce one or more spinosyns does not encode at least one domain of wild type Saccharopolyspora pogona selected from the group consisting of acetyltransferase (AT), dehydratase (DH), ketoreductase (KR) and acyl carrier protein (ACP) of module lb and a ketosynthase (KS) of module la in a butenyl- spinosyn busA gene cluster of wild type Saccharopolyspora pogona.

16. The Saccharopolyspora pogona cell of claim 1 , wherein no greater than 9% of the one or more spinosyns is spinosyn D.

17. The Saccharopolyspora pogona cell of claim 1 , wherein: one or more of the spinosyns comprises the chemical structure,wherein R is either a hydrogen or an alkyl group, the alkyl group having n carbons, wherein n is a number equal to or greater than 1.

18. The Saccharopolyspora pogona cell of claim 17, wherein R is a hydrogen group.

19. A method of making the Saccharopolyspora pogona cell of claim 1, comprising: transforming a Saccharopolyspora pogona cell with one or more nucleotide sequences to effect a busA gene deletion and a spnA gene insertion.

20. The method of claim 19, wherein: the one or more nucleotide sequences comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 33.

21. The method of claim 19, wherein: the one or more nucleotide sequences comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 18.

22. The method of claim 19, wherein: the one or more nucleotide sequences comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO 21.

23. The method of claim 19, wherein: the one or more nucleotide sequences comprise a nucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to Seq ID 41.

24. An inoculant composition comprising: the Saccharopolyspora pogona cell of claim 1.

25. A method of producing spinosyn A with the Saccharopolyspora pogona cell of claim 1.

26. A method of producing spinosyn D with the Saccharopolyspora pogona cell of claim 1.

27. A Saccharopolyspora pogona cell genetically modified to produce butenyl-spinosyn, wherein said cell is engineered to express a spnA gene from Saccharopolyspora spinosa.

28. The Saccharopolyspora pogona cell of claim 27, wherein one or more nucleotide sequences encode expression of enzymes to produce one or more spinosyns comprising an ethyl chain at a C21 position of a lactone ring; and wherein no greater than 9% of the one or more spinosyns is spinosyn D.

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