# Patent

## Title
Enhanced itaconic acid production in Aspergillus with increased LaeA expression

## Abstract

Fungi, such as Aspergillus niger, having a dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (Alg3) gene genetic inactivation, increased expression of a loss of aflR expression A (LaeA), or both, are described. In some examples, such mutants have several phenotypes, including an increased production of citric acid relative to the parental strain. Methods of using the disclosed fungi to make citric acid are also described, as are compositions and kits including the disclosed fungi. Further described are Aspergillus terreus fungi overexpressing the LaeA gene and the use of such fungi for the production of itaconic acid.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part of U.S. patent application Ser. No. 14/703,499, filed May 4, 2015, issued as U.S. Pat. No. 9,206,450 on Dec. 8, 2015, which is a continuation-in-part of U.S. application Ser. No. 13/691,396, filed Nov. 30, 2012, issued as U.S. Pat. No. 9,023,637 on May 5, 2015, which claims the benefit of U.S. Provisional Application No. 61/565,018, filed Nov. 30, 2011. The above-referenced applications are herein incorporated by reference in their entirety.


ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with government support under contract number DE-AC05-76RL01830 awarded by the U.S. Department of Energy. The government has certain rights in the invention.


FIELD
This disclosure concerns recombinant Aspergillus terreus fungi that are genetically enhanced to increase the expression levels of the loss of aflR expression A (LaeA) gene, which results in a significant increase in itaconic acid production. This disclosure further concerns methods of using these fungi to produce itaconic acid.
BACKGROUND
Filamentous fungi, such as Aspergillus niger, are well known for their industrial applications in protein and chemical productions. They are used to produce a wide variety of products ranging from human therapeutics, glycosyl hydrolases to specialty chemicals (Punt et al., Trends Biotechnol 20(5):200-206, 2002; Schuster et al., Appl Microbiol Biotechnol 59(4-5):426-435, 2002; Gerngross, Nat Biotechnol 22(11):1409-1414, 2004; Nevalainen et al., Trends Biotechnol 23(9):468-474, 2005; Sauer et al., Trends Biotechnol. 26(2):100-8, 2008; Magnuson and Lasure (2004). “Organic acid production by filamentous fungi.” Advances in fungal biotechnology for industry, agriculture, and medicine, pages 307-340). Some of industrial A. niger strains are capable of growing on solutions of glucose or sucrose in excess of 20% (w/v) and converting approximately 90% of the supplied carbohydrate to citric acid. These remarkable properties are the reason that A. niger has been used to produce citric acid for more 80 years and is currently the primary source of commercial citric acid production (Magnuson and Lasure (2004). “Organic acid production by filamentous fungi.” Advances in fungal biotechnology for industry, agriculture, and medicine, pages 307-340).
The maximum product output in fermentation processes is the result of optimal metabolic pathways and cellular formation, which are influenced by endogenous and exogenous factors. Cellular metabolisms are tightly controlled and highly interconnected, and are regulated spatially and temporally at different levels, such as transcription, post-transcription, translation, and post-translation. Therefore, different approaches have been explored to understand the regulatory mechanisms of metabolic processes and cellular formation for maximizing the product output in filamentous fungi. For example, comparative genomics was used to examine citric-acid-producing versus enzyme-producing A. niger strains (Andersen et al., Genome Res. 21(6): 885-97, 2011), proteomics was used to examine filamentous fungi related to enzymes or organic acid production (de Oliveira and de Graaff, Appl. Microbiol. Biotechnol. 89(2): 225-37, 2011), or combination of both genomics and proteomics were used to examine enzyme production (Jacobs et al., Fungal Genetics and Biology 46(1, Supplement):S141-S152, 2009). Although these studies examined the potential involvement of selected genes and proteins in optimizing production of organic acids or proteins in filamentous fungi, methods for altering the complex post-translation modifications (such as N-glycosylation of cellular proteins) for signal transduction, cellular formation and metabolism at different growth and development stages, which may affect product output, have not been examined.
Protein glycosylation is a ubiquitous and structurally diverse form of post translation modification, which occurs at all domains of life. More than two-thirds of eukaryotic proteins are predicted to be glycosylated (Apweiler et al., Biochim Biophys Acta 1473(1):4-8, 1999). N- and O-linked protein glycosylation are common types of protein glycosylation, occurring mainly on the asparagine (N) and serine/threonine (S/T) residues, respectively. N-linked glycosylation has been implicated in many biochemical and cellular processes, including protein secretion, stability and translocation, maintenance of cell structure, receptor-ligand interactions and cell signaling, cell-cell recognition, pathogen infection, and host defense in various organisms (Haltiwanger and Lowe, Ann. Rev. Biochem.73(1):491-537, 2004; Dellaporta et al., Plant Mol. Biol. Reporter 1(4):19-21, 1983; Nam et al., Biotech. Bioengineer. 100(6): 1178-1192, 2008; Trombetta and Parodi, Ann. Rev. Cell Dev. Biol. 19(1):649-676, 2003; Tsang et al., Fungal Genetics and Biology 46(1): S153-S160, 2009; Pang et al., Science, 333(6050):1761-4, 2011).
N-glycosylation is highly complex and has been extensively studied in mammalian systems (Yan and Lennarz, J. Biol. Chem. 280(5):3121, 2005; Silberstein and Gilmore, FASEB J. 10(8): 849, 1996; Kornfeld and Kornfeld, Annu. Rev. Biochem. 54:631-664, 2005; Kim et al., PLoS ONE 4(10): e7317, 2009, 2009) and yeast (Kukuruzinska et al., Annu. Rev. Biochem. 56(1):915-944, 1987). The protein N-glycosylation pathways in filamentous fungi have also been identified (Deshpande et al., Glycobiology 18(8):626-637, 2008; Geysens et al., Fungal Genetics and Biology 46(1, Supplement): S121-S140, 2009) on the basis of the known genomic sequences. Several genes involved in N-glycosylation have been studied in filamentous fungi (Kotz et al., PLoS ONE 5(12):e15729, 2010; Kainz et al., Appl Environ Microbiol 74(4):1076-86, 2008; Maras et al., J. Biotechnol. 77(2-3):255-63, 2000; Maddi and Free, Eukaryot Cell 9(11):1766-75, 2010; Bowman et al., Eukaryotic Cell 5(3):587-600, 2006). In these studies, the effects of gene deletion on N-linked glycan patterns formation, the cell wall formation, overall protein secretion and/or the phenotypic changes were demonstrated.
Alg3 is localized in the ER and catalyzes the initial transfer of a mannose residue from dolichol pyrophosphate-mannose to lipid-linked Man5GlcNAc2-PP-Dol on the ER luminal side. It is involved in the early N-glycan synthesis in eukaryotes for the assembly of a Glc3Man9GlcNAc2 core oligosaccharide that is linked to the lipid carrier dolichol pyrophosphate. The Alg3 gene and its functions have been identified and studied in S. cerevisiae, P. pastoris, T. brucei, A. thaliana, and human (Aebi et al., Glycobiol. 6(4):439-444, 1996; Korner et al., EMBO J. 18(23): 6816-6822, 1999; Davidson et al., Glycobiology 14(5):399-407, 2004; Manthri et al., Glycobiol. 18(5):367-83, 2008; Kajiura et al., Glycobiol. 20(6):736-51, 2010). In these studies, the Alg3 mutants exhibited a unique structural profile in the glycoproteins, such as Man3GlcNAc2, Man4GlcNAc2, Man5GlcNAc2, GlcMan5GlcNAc2, and Glc3Man5GlcNAc2, which affected the overall N-glycosylation by incomplete utilization of N-linked glycosites in glycoproteins. No obvious growth phenotype was observed in those Alg3Δ mutants of S. cerevisiae, P. pastoris, T. brucei, and plant except that the Alg3 defect in human caused severe diseases such as profound psychomotor delay, optic atrophy, acquired microcephaly, iris olobomas and hypsarrhythmia (Stibler et al., Neuropediatrics 26(5): 235-7, 1995; Sun et al., J. Clin. Endocrinol. Metab. 90(7):4371-5, 2005; Schollen et al., Eur. J. Med. Genet. 48(2):153-158, 2005, Kranz et al., Am. J. Med. Genet. 143A(13):1414-20, 2007; Denecke et al., Pediatr. Res. 58(2):248-53, 2005).
LaeA, a global regulator gene for the secondary metabolism, was first identified in A. nidulans through complementing the aflR deficient mutants (Bok and Keller, Eukaryot Cell 3:527-535, 2004). Deletion of LaeA gene inhibits the expression of secondary metabolic gene clusters, such as sterigmatocystin, penicillin, and lovastin, but has no effect on spore production in A. nidulans. The LaeA that was confirmed as a nuclear protein and a putative methyltransferase does not involve in gene clusters for nutrient utilization (Bok et al., Mol Microbiol 61:1636-45, 2006). Furthermore, the role of LaeA in secondary metabolism was confirmed in Aspergillus flavus and Aspergillus oryzae (Kale et al., Fungal Genet. Biol. 45:1422-9, 2008; Oda et al., Biosci Biotechnol Biochem 75:1832-4, 2011). Evidence indicates that LaeA reverses gene repression at the level of the heterochromatin state (Reyes-Dominguez et al., Molecular Microbiology 76:1376-86, 2010). LaeA is a component of the heterotrimeric VeA/VelB/LaeA protein complex (Bayram et al., Science Signalling 320:1504, 2008), which involves in the acetylation signal transduction for secondary metabolite production in A. nidulans (Soukup et al., Mol. Microbiol., 86(2):314-30, 2012). The veA/VelB/LaeA complex may coordinately respond to environmental cues (Ramamoorthy et al., Mol. Microbiol., 85(4):795-814, 2012) and has a role in fungal morphology (Calvo, Fungal Genetics and Biology 45:1053-61, 2008). LaeA may direct the formation of the VelB-VosA and VelB-VelA-LaeA complexes, control veA modification and protein levels, and be involved in light regulation of growth and development (Bayram et al., PLoS genetics 6: e1001226, 2010).
SUMMARY
Provided herein are isolated Aspergillus terreus fungi transformed with a heterologous nucleic acid molecule comprising an Aspergillus species gene. Also provided is a method of making itaconic acid by culturing an isolated A. terreus fungus transformed with a heterologous nucleic acid molecule comprising an Aspergillus species LaeA gene under conditions that permit the fungus to make itaconic acid. In some embodiments, the heterologous nucleic acid construct includes a heterologous LaeA gene, a heterologous promoter, a heterologous transcription terminator, and/or a heterologous selective marker gene. In non-limiting examples, the heterologous Aspergillus species LaeA gene is an A. fumigatus laeA gene, an A. nidulans laeA gene, an A. niger LaeA gene, or an A. oryzae laeA gene.
Although the current commercial conversion rate of carbohydrate to citric acid in A. niger is more than eighty to ninety percent, further improvement in production of citric acid and other metabolites is desirable. This disclosure describes the role of the dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase gene (α-1,3-mannosyltransferase, Alg3) on the spore germination, filamentous growth, sporulation, and production of citric acid in Aspergillus niger. In addition, the role of LaeA in citric acid production by its over-expression is shown, alone or in combination with an Alg3Δ mutant background.
Based on these observations, provided herein are isolated fungi (such as filamentous fungi) having a gene inactivation (also referred to herein as a gene deletion) of a dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (Alg3) gene (referred to herein as Alg3Δ strains), a gene enhancement (e.g., overexpression) of a LaeA gene (referred to herein as upregulated LaeA strains), or both. Any strain of fungi can be used, such as a filamentous fungi, for example Aspergillus niger (A. niger) or particular strains thereof (for example A. niger strain 11414 or 11414KusA). In particular examples, an Alg3Δ strain exhibits one or more of the following characteristics: slower growth on citric acid production (CAP) medium, complete medium (CM) or potato dextrose agar (PDA) medium; earlier spore germination and a higher germination rate in CAP medium; delayed spore germination in CM or PDA medium; reduced sporulation on complete medium; or combinations thereof. In some examples, such increases or decreases are relative to A. niger strain 11414KusA grown under the same conditions. The combination of Alg3Δ and over-expression of LaeA resulted in some improvement of sporulation on CM.
In particular examples, such Alg3Δ strains, up-regulated LaeA strains, or Alg3Δ-upregulated LaeA strains, produce more citric acid when grown in CAP medium, such as at least 20%, at least 50%, or at least 70% more than A. niger strain 11414KusA under identical growing conditions after at least 4 days, at least 5 days or at least 10 days. Thus, one strategy to increase citric acid production is to reduce the carbohydrate consumption for protein glycosylation and cellular formation, as altering protein glycosylation can augment the carbohydrate flux into citric acid production in A. niger. 
Also provided herein are compositions (such as fermentation broth) and kits that include a fungal Alg3Δ strain, up-regulated LaeA strain, or Alg3Δ-upregulated LaeA strain.
Also provided herein are methods of making citric acid using the disclosed fungal Alg3Δ strains, up-regulated LaeA strains, and Alg3Δ-upregulated LaeA strains. For example, such a method can include culturing an isolated Alg3Δ fungus, up-regulated LaeA fungus, or Alg3Δ-upregulated LaeA fungus, under conditions that permit the fungus to make citric acid, thereby making citric acid. For example, the Alg3Δ fungus, up-regulated LaeA fungus, or Alg3Δ-upregulated LaeA fungus, can be cultured in CAP medium. In some examples, the method further includes isolating the citric acid produced.
The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic drawing showing a restriction map of the 10.9 kb fragment containing the A. niger Alg3 gene.
FIG. 1B is a schematic drawing showing the introduction of the hyg-selective marker, which was flanked by the upstream and downstream DNA sequences of Alg3. Integration of the linear molecules by homologous recombination replaces Alg3 with hph in the chromosome.
FIG. 1C is a digital image of a Southern blot showing the genomic DNA hybridization of parent and Alg3Δ strains. One parent and two selected Alg3Δ strains are shown, which have the correct enzyme restriction pattern.
FIG. 2 is a series of digital images showing the Alg3Δ and parent 11414kusA strains grown on agar plates of complete medium (CM), potato dextrose (PDA), and minimal medium (MM) at 30° C. for 15 hours or 48 hours. Parent strain (panel A), Alg3Δ strain (panel B), and both parent and Alg3Δ strains (panel C) grown on complete medium plate. Parent strain (panel D), Alg3Δ strain (panel E), and both parent and Alg3Δ strains (panel F) were grown on PDA agar plates. Parent strain (panel G), Alg3Δ strain (panel H), and both parent and Alg3Δ strains (panel I) were grown on MM agar plates.
FIG. 3A is a series of digital images showing the spore germination of parent 11414kusA and Alg3Δ strains in liquid cultures of complete medium (CM), potato dextrose medium (PDA), and minimal medium (MM) at 30° C. for 7 hours. Panels A and B are germinated spores in CM liquid culture. Panels C and D are germinated spores in PDA liquid culture. Panels E and F are germinated spores in MM liquid culture. The left panels for the parent 11414kusA strain and the right panels for the Alg3Δ strain.
FIG. 3B provides graphs showing the time courses of the percentage of spore germination of parent 11414kusA and Alg3Δ strains grown in the liquid cultures of complete medium (CM; top graph), potato dextrose medium (PDA; middle graph), or minimal medium (MM; bottom graph). The solid filled cycle is the parent 11414kusA strain and open cycle is the Alg3Δ strain.
FIG. 4 shows a series of digital images (panels A and D) and stereo microscopy digital images (panels B, C, E and F) of parent and Alg3Δ strains grown on agar plates of complete medium (CM) and potato dextrose (PDA) at 30° C. for 4 days.
FIG. 5 provides stereo microscopy digital images parent 11414kusA and Alg3Δ strains grown on citric acid production (CAP) medium plates at different pH levels for 27 hours. The left panels for parent strain 11414kusA and right panels for the Alg3Δ strain.
FIGS. 6A and 6B show spore germination of parent 11414kusA and Alg3Δ strains in citric acid production (CAP) liquid medium. (FIG. 6A, top left and bottom left panel) Inverted microscopic images for parent 11414kusA strain. (FIG. 6A, top right and bottom right panel) Inverted microscopic images for Alg3Δ strain. (FIG. 6A, top panels) Strains grown in CAP liquid culture at 30° C. for 8 hours. (FIG. 6A, bottom panels) Strains grown in CAP liquid culture at 30° C. for 15 hours. (FIG. 6B) Time course of the spore germination rate (%) of parent and Alg3Δ strains grown in CAP liquid culture. The solid cycle for parent 11414kusA strain and open cycle for the Alg3Δ strain.
FIG. 7 is a graph showing the time course of citric acid production by parent 11414kusA and Alg3Δ strains in the liquid culture of citric acid production at 30° C. and 200 rmp.
FIGS. 8A and 8B show (FIG. 8A) a schematic diagram showing the construction of the transgene used to complement the Alg3Δ mutant, and (FIG. 8B) a is a graph showing the citric acid production by parent strain 11414kusA (kusA), Alg3Δ mutant (Alg3) and Alg3Δ mutant complemented with Alg3 gene (cAlg3) after growth at 30° C., 200 rpm for 12 days
FIG. 9 shows an alignment of Alg3 nucleic acid sequences from A. niger (top strand, nucleotides 1986-2518 of SEQ ID NO: 1) and A. oryzae (bottom strand, nucleotides 643-1175 of SEQ ID NO: 3).
FIGS. 10A and 10B show an alignment of Alg3 protein sequences from A. niger (SEQ ID NO: 2), A. nidulans (SEQ ID NO: 31), Fusarium oxysporum (SEQ ID NO: 32), Neurospora crassa (SEQ ID NO: 33), S. cerevisiae (SEQ ID NO: 34), Arabidopsis thaliana (SEQ ID NO: 35), and Homo sapiens (SEQ ID NO: 36). The signs at the top of the alignment show: ‘−’ the average weight of column pair exchanges is less than weight matrix mean value; ‘.’ is less than mean value plus one SD; ‘+’ is less than mean value plus two SD; and ‘*’ is more than mean value plus two SD.
FIG. 11 shows an alignment of Alg3 protein sequences from A. niger (top strand, amino acids 12-411 of SEQ ID NO: 2) and S. cerevisiae (bottom strand, amino acids 31-434 of SEQ ID NO: 34).
FIG. 12 shows an alignment of LaeA protein sequences from A. nidulans (top strand, amino acids 14-372 of SEQ ID NO: 41) and A. niger (bottom strand, amino acids 14-370 of SEQ ID NO: 59).
FIG. 13 is a schematic diagram showing the construction of pPTRpGPDALaeA plasmid vector. Both the glyceraldehyde 3-phosphate dehydrogenase (gpdA) promoter and LaeA (loss of aflR expression A) gene coding sequence of genomic DNA were isolated by overlap PCR from pAN7-1 plasmid vector and A. nidulans with additions of HindIII restriction enzyme sites at 5′-end of gpdA promoter and 3′-end of LaeA gene, confirmed by DNA sequence, and ligated into pPTR1 plasmid vector at HindIII restriction enzyme site.
FIG. 14 is a schematic illustrating a plasmid vector pRS426-LaeA, which contains the upstream region of pyrG gene of A. niger, the coding region of LaeA gene under the control of gpdA promoter and transcriptional terminator of trpC gene from A. nidulans, the pyrithiamine resistance (ptrA) gene from A. oryzae, and the downstream region of pyrG gene of A. niger. The unique restriction enzyme PmeI site was introduced at the both end of transgene expression fragment.
FIGS. 15A and 15B show the results of polymerase chain reaction (PCR) analysis of LaeA gene insertion in the transgenic A. niger genome of heterologous expression of A. nidulans LaeA gene. (FIG. 15A) PCR products of A. oryzae ptrA gene detected in selected single spore colony isolate (SCI) of LaeA gene transgenic mutants and parent kusA and Alg3Δ are control strains. (FIG. 15B) PCR products of transgene expression DNA fragment including the gpdA promoter, LaeA coding region and trpC gene transcriptional terminator. The SCI-1 to SCI-4 is the individual single spore colony of LaeA gene transgenic mutants and parent kusA and Alg3Δ are control strains. Lambda DNA marker is the restriction fragment of BstEII restriction enzyme.
FIG. 16 is a bar graph showing the results of citric acid production after 10 days in culture of parent strain (kusA), alg3Δ mutant (Alg3), and over-expression of LaeA gene in alg3Δ (LaeA-1 and LaeA-2) mutants. The data for each strain is the average of three replicates.
FIG. 17A is a schematic diagram showing the construction of the pBSK-LaeA deletion plasmid vector for A. niger. The DNA fragments of the 5′ and 3′ ends of A. niger LaeA (lack of aflR expression) gene and the hph (hygromycin phosphotransferase) expression cassette were isolated from A. niger genomic DNA (LaeA) or pCB1003 plasmid vector DNA by PCR with oligonucleotide pairs 1 and 2; 3 and 4; and 5 and 6. The PCR DNA fragments were assembled together by Gibson assembly cloning kit in pBSK backbone vector prepared by HindIII/PstI double digestion. The plasmid DNA fragment containing the LaeA gene deletion cassette was prepared by restriction enzyme digestion with restriction endonucleases of HindIII and XbaI and used for A. niger transformation.
FIG. 17B is a schematic diagram showing the construction of the pRS426-A. niger LaeA complementation plasmid vector for A. niger. The LaeA gene containing both its promoter and transcriptional terminator was isolated by PCR with oligonucleotide pair 9 and 10 from A. niger genomic DNA. The PCR fragment was cloned into the plasmid vector pRSB426-LaeA prepared by restriction endonuclease HindIII digestion and Klenow treatment with blunt-end ligation. The new plasmid DNA vector contains the upstream region of the pyrG gene of A. niger, the entire region of A. niger LaeA gene, the transcriptional terminator of the trpC gene from A. nidulans, the pyrithiamine resistance (ptrA) gene from A. oryzae, and the downstream region of the pyrG gene of A. niger. The unique restriction endonuclease PmeI site was introduced at both ends of the transgene expression fragment.
FIG. 18 shows a digital image of the results of PCR analysis of the LaeA gene deletion in the transgenic A. niger genome by homologous recombination at the pyrG locus. The PCR products corresponding to the 5′ end of the A. niger LaeA gene and part of the hph expression cassette were amplified with oligonucleotide pair 7 and 8, and were detected in selected single spore colony isolate (SCI) of LaeA gene deletion transgenic mutants.
FIG. 19 shows a digital image of the results of PCR analysis of the LaeA gene complementation (cLaeA) in the genetic background of LaeA deletion mutant or A. nidulans LaeA over-expression (LaeA) in the genetic background of 11414kusA of those transgenic A. niger genome by homologous recombination. PCR products of A. oryzae ptrA gene detected in selected single spore colony isolate (SCI) of LaeA gene transgenic mutants and parent kusA (negative) and plasmid DNA of pRS426-laeA (positive) represent control DNA.
FIG. 20 is a bar graph showing the results of citric acid production after 5 days in culture of the parent strain (kusA), the LaeAΔ mutant, A. niger LaeA gene complementation in the LaeAΔ mutant (cLaeAΔ), and over-expression of A. nidulans LaeA gene in the parent (KusA) strain. The data for each strain is the average of at least three biological replicates.
FIG. 21A is a diagram illustrating the LaeA deletion construct in which the LaeA coding region was replaced by the bacterial hygromycin B phosphotransferase (hph) gene. Oligonucleotides 1546, 108, 107 and 1553 (see Table 6) were used for LaeA gene deletion confirmation in A. terreus. 
FIG. 21B shows the genomic PCR confirmation of selected LaeA deletion mutants with oligonucleotide pair 1546/107 or 108/1553.
FIG. 22A is a diagram illustrating LaeA complementation in A. terreus LaeA deletion mutants. The complementation transgene fragment was targeted upstream of the LaeA gene and the bacterial bleomycin resistance gene (ble) was used for transgenic strain selection. The insertion of LaeA complementation was confirmed by oligonucleotide pair 1556/107.
FIG. 22B shows gel electrophoresis of genomic PCR product corresponding to ble and part of hph.
FIG. 23 is a graph showing itaconic acid production in selected strains of parent, LaeA deletion (LaeAΔ) and LaeA complementation (cLaeAΔ) mutants after 4 days of culture in itaconic acid production medium.
FIG. 24A shows a diagram of the transgene over-expression cassette of the A. nidulans LaeA gene under the control of the A. nidulans gpdA promoter and TrpC terminator with a locus target of 2.5 kb of the A. terreus LaeA gene. The pyrithiamine resistance gene (ptrA) of A. oryzae was used for transgene expression selection. Oligonucleotides 1563, 1198 and 1564 (see Table 6) were used for genomic PCR to confirm the gene insertion.
FIG. 24B shows gel electrophoresis of PCR products corresponding to the oligonucleotide pairs 1198/1564 and 1563/1564 in selected transgenic strains.
FIG. 25 is a bar graph showing itaconic acid production in selected A. terreus transgenic strains with the A. nidulans LaeA over-expression transgene after 5 days of culture in itaconic acid production medium.



SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on Oct. 19, 2015, 157 KB, which is incorporated by reference herein. In the accompanying sequence listing:
SEQ ID NOS: 1 and 2 are exemplary Alg3 nucleic acid and protein sequences, respectively, from A. niger. 
SEQ ID NOS: 3 and 4 are exemplary Alg3 nucleic acid and protein sequences, respectively, from A. oryzae. 
SEQ ID NOS: 5-30 show exemplary primer sequences.
SEQ ID NOS: 31-36 are exemplary Alg3 protein sequences from A. nidulans, Fusarium oxysporum, Arabidopsis thaliana, Neurospora crassa, S. cerevisiae, and Homo sapiens, respectively.
SEQ ID NO: 37 is an exemplary Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase (gpdA) promoter sequence.
SEQ ID NOS: 38 and 39 are exemplary forward and reverse primers, respectively, that can be used to isolate or amplify an A. nidulans gpdA promoter.
SEQ ID NOS: 40 and 41 are exemplary Aspergillus nidulans methyltransferase (LaeA) coding and protein sequences, respectively.
SEQ ID NOS: 42 and 43 are exemplary forward and reverse primers, respectively, that can be used to isolate or amplify an A. nidulans LaeA sequence.
SEQ ID NO: 44 is the nucleic acid sequence of the pGPDA-LaeA fragment described in FIG. 13 and Example 5.
SEQ ID NO: 45 is the upstream region of A. niger pyrG gene.
SEQ ID NO: 46 is the trpC transcriptional terminator of A. nidulans. 
SEQ ID NO: 47 is the pyrithiamine resistance gene (ptrA) of Aspergillus oryzae. 
SEQ ID NO: 48 is the downstream region of A. niger pyrG gene.
SEQ ID NOS: 49 and 50 are exemplary forward and reverse primers, respectively, that can be used to isolate or amplify an upstream region of A. niger pyrG.
SEQ ID NOS: 51 and 52 are exemplary forward and reverse primers, respectively, that can be used to isolate or amplify the trpC transcriptional terminator of A. nidulans. 
SEQ ID NOS: 53 and 54 are exemplary forward and reverse primers, respectively, that can be used to isolate or amplify ptrA of Aspergillus oryzae. 
SEQ ID NOS: 55 and 56 are exemplary forward and reverse primers, respectively, that can be used to isolate or a downstream region of A. niger pyrG.
SEQ ID NO: 57 is the nucleic acid sequence of the transgene fragment described in FIG. 13 and Example 6.
SEQ ID NOS: 58 and 59 are exemplary Aspergillus niger LaeA coding and protein sequences, respectively.
SEQ ID NO: 60 is the nucleic acid sequence of the transgene fragment used to complement the alg3Δ mutant with the original alg3 gene at pyrG locus.
SEQ ID NO: 61 is the nucleic acid sequence of the 5′ end of the A. niger LaeA gene.
SEQ ID NO: 62 is the nucleic acid sequence of the 3′ end of the A. niger LaeA gene.
SEQ ID NO: 63 is the nucleic acid sequence of the hph expression cassette.
SEQ ID NO: 64 is the nucleic acid sequence of the A. niger LaeA gene.
SEQ ID NO: 65 is the nucleic acid sequence of the LaeA deletion cassette.
SEQ ID NO: 66 is the nucleic acid sequence of the LaeA complementation construct.
SEQ ID NOs: 67-100 are oligonucleotide primers.
SEQ ID NO: 101 is the nucleotide sequence of the 1546/1548 fragment.
SEQ ID NO: 102 is the nucleotide sequence of the 1549/1550 fragment.
SEQ ID NO: 103 is the nucleotide sequence of the 1551/1553 fragment.
SEQ ID NO: 104 is the nucleotide sequence of the 1547/1552 fragment.
SEQ ID NO: 105 is the nucleotide sequence of the 1554/1555 fragment.
SEQ ID NO: 106 is the nucleotide sequence of the 1556/1557 fragment.
SEQ ID NO: 107 is the nucleotide sequence of the 1558/1559 fragment.
SEQ ID NO: 108 is the nucleotide sequence of the 1561/1562 fragment.
SEQ ID NO: 109 is the nucleotide sequence of the 1563/1564 fragment.
SEQ ID NO: 110 is the nucleotide sequence of the 1565/1566 fragment.
DETAILED DESCRIPTION
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All references and GENBANKTM Accession numbers mentioned herein are incorporated by reference (the sequence available on Nov. 30, 2011). The materials, methods, and examples are illustrative only and not intended to be limiting.
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:
Alg3 (dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase): Also known as asparagine-linked glycosylation 3 and α-1,3-mannosyltransferase. Alg3 encodes an enzyme which catalyzes the addition of the first dol-p-man derived mannose in an α-1,3 linkage to Man5GlcNAc2-PP-Dol. The term Alg3 (or Alg3) includes any Alg3 gene (such as a fungal Alg3 sequence), cDNA, mRNA, or protein, that is an Alg3 involved in catalyzing the addition of the first dol-p-man derived mannose in an α-1,3 linkage to Man5GlcNAc2-PP-Dol, and when genetically inactivated results in a fungus that has an ability to produce more citric acid than the parent strain (such as at least 20%, at least 30%, at least 50%, at least 60%, or at least 70% more than a parent strain under the same growing conditions).
Alg3 sequences are publicly available for many species of Aspergillus. For example, GENBANKTM Accession Nos: XM_001823992.2 and XP_001824044 disclose Aspergillus oryzae RIB40 Alg3 nucleic acid and protein sequences, respectively; GENBANKTM Accession Nos: XM_001398659.2 and XP_001398696.2 disclose Aspergillus niger CBS 513.88 Alg3 nucleic acid and protein sequences, respectively (SEQ ID NOS: 1 and 2); and GENBANKTM Accession Nos: XM_748359.1 and XP_753452 disclose Aspergillus fumigatus Af293 Alg3 nucleic acid and protein sequences, respectively. Additional exemplary Alg3 sequences are provided in SEQ ID NOS: 1-4 and 31-36. However, one skilled in the art will appreciate that in some examples, an Alg3 sequence can include variant sequences (such as allelic variants and homologs) that retain Alg3 activity but when genetically inactivated in Aspergillus results in a fungus that has an ability to produce more citric acid than the parent strain (such as at least 20%, at least 30%, at least 50%, at least 60%, or at least 70% more under the same growing conditions).
Aspergillus terreus: A fungus found in soil and vegetation. A. terreus is found worldwide, but is more prevalent in warmer climates, such as tropical and subtropical regions. This fungus is commonly used in industry to produce important organic acids, such as itaconic acid and cis-aconitic acid, and was the initial source for the drug mevinolin (lovastatin), a drug for lowering serum cholesterol (Bennett, “An Overview of the Genus Aspergillus,” pages 1-17, Aspergillus Molecular Biology and Genomics, edited by Masayuki Machida and Katsuyi Gomi, Caister, Academic Press, 2010).
Detectable: Capable of having an existence or presence ascertained. For example, production of citric acid is detectable if the signal generated is strong enough to be measurable.
Genetic enhancement or up-regulation: When used in reference to the expression of a nucleic acid molecule, such as a gene, refers to any process which results in an increase in production of a gene product. A gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Examples of processes that increase transcription include those that facilitate formation of a transcription initiation complex, those that increase transcription initiation rate, those that increase transcription elongation rate, those that increase processivity of transcription and those that relieve transcriptional repression (for example by blocking the binding of a transcriptional repressor). Gene up-regulation can include inhibition of repression as well as stimulation of expression above an existing level. Examples of processes that increase translation include those that increase translational initiation, those that increase translational elongation and those that increase mRNA stability. In one example, additional copies of genes are introduced into a cell in order to increase expression of that gene in the resulting transgenic cell.
Gene up-regulation includes any detectable increase in the production of a gene product. In certain examples, production of a gene product increases by at least 1.5-fold, at least 2-fold, or at least 5-fold), such as LaeA. For example, a genetic enhancement of a LaeA gene in Aspergillus (e.g., A. niger or A. terreus) results in an Aspergillus strain having increased levels of the LaeA protein relative to the parent strain, which can increase the ability of the fungus to produce more citric acid or itaconic acid. Genetic enhancement is also referred to herein as “enhancing or increasing expression.”
Genetic inactivation or down-regulation: When used in reference to the expression of a nucleic acid molecule, such as a gene, refers to any process which results in a decrease in production of a gene product. A gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Therefore, gene down-regulation or deactivation includes processes that decrease transcription of a gene or translation of mRNA.
For example, a mutation, such as a substitution, partial or complete deletion, insertion, or other variation, can be made to a gene sequence that significantly reduces (and in some cases eliminates) production of the gene product or renders the gene product substantially or completely non-functional. For example, a genetic inactivation of an Alg3 gene in Aspergillus (e.g., A. niger) results in Aspergillus having a non-functional or non-existent Alg3 protein, which results in an ability of the fungus to produce more citric acid. Genetic inactivation is also referred to herein as “functional deletion”.
Heterologous: Derived from separate genetic sources or species. For example, an LaeA gene that is heterologous to A. terreus is an LaeA gene from a species other than A. terreus. 
Isolated: To be significantly separated from other agents. An “isolated” biological component (such as a nucleic acid molecule or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component occurs, for example, other chromosomal and extra-chromosomal DNA and RNA, and proteins. Nucleic acid molecules and proteins which have been “isolated” include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized proteins and nucleic acids. Samples of isolated biological components include samples of the biological component wherein the biological component represents greater than 90% (for example, greater than 95%, such as greater than 98%) of the sample.
An “isolated” microorganism (such as an Alg3Δ strain of Aspergillus) has been substantially separated or purified away from microorganisms of different types, strains, or species. Microorganisms can be isolated by a variety of techniques, including serial dilution and culturing and resistance to certain chemicals.
Itaconic acid: An unsaturated dicarbonic acid; also known as 2-methylidenebutanedioic acid. Itaconic acid can be produced by the fermentation of carbohydrates using Aspergillus terreus. Itaconic acid can be used as a monomer for the production of a number of different products, including resins, plastics, acrylate latexes, paints and synthetic fibers.
Itaconic acid production medium: Media containing in grams per liter (g/l): glucose, 100; ammonium sulfate, 2.36; potassium phosphate dibase, 0.11; magnesium sulfate heptahydrate, 2.08; calcium chloride dihydrate, 0.13; sodium chloride, 0.074; copper sulfate pentahydrate 2×10−4; ferrous sulfate heptahydrate, 5.5×10−3; manganese chloride tetrahydrate, 7×10−4; and zinc sulfate heptahydrate, 1.3×10−3.
LaeA (loss of afiR expression A): LaeA encodes a protein which regulates secondary metabolite production in Aspergillus. The term LaeA (or LaeA) includes any LaeA gene (such as a fungal LaeA sequence), cDNA, mRNA, or protein, that is an LaeA involved in secondary metabolite production. In some embodiments, when LaeA expression is increased, for example in combination with a genetically inactivated Alg3 gene, it results in a fungus that has an ability to produce more citric acid than the parent strain (such as at least 20%, at least 30%, at least 40%, 50%, at least 60%, or at least 70% more than a parent strain under the same growing conditions). In some embodiments, when LaeA expression is increased, for example in A. terreus, it results in a fungus that has an ability to produce more itaconic acid than the parent strain (such as at least 20%, at least 30%, at least 40%, 50%, at least 60%, or at least 70% more than a parent strain under the same growing conditions).
LaeA sequences are publicly available for many species of Aspergillus. For example, GENBANKTM Accession Nos: AB267276 and BAF74528.1 disclose Aspergillus oryzae LaeA nucleic acid and protein sequences, respectively; GENBANKTM Accession No. EHA27020.1 discloses an exemplary Aspergillus niger ATCC1015 LaeA protein sequence, a parent strain of 11414kusA (other exemplary sequences are provided in SEQ ID NOS: 58 and 59); GENBANKTM Accession No: CBF88745 discloses an Aspergillus nidulans LaeA protein sequence; and GENBANKTM Accession Nos: AY422723 and AAR01218 disclose Aspergillus fumigatus LaeA nucleic acid and protein sequences, respectively. Additional exemplary LaeA sequences are provided in SEQ ID NOS: 40-41 and 58-59. However, one skilled in the art will appreciate that in some examples, an LaeA sequence can include variant sequences (such as allelic variants and homologs) that retain LaeA activity and when genetically up-regulated in Aspergillus (for example with addition of copy or in combination with Alg3Δ) results in a fungus that has an ability to produce more citric acid than the parent strain (such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% more under the same growing conditions).
Mutation: A change in a nucleic acid sequence (such as a gene sequence) or amino acid sequence, for example as compared to a nucleic acid or amino acid sequence present in a wild-type or native organism. In particular examples, a mutation is introduced into an Alg3 gene in Aspergillus. Mutations can occur spontaneously, or can be introduced, for example using molecular biology methods. In particular examples, a mutation includes one or more nucleotide substitutions, deletions, insertions, or combinations thereof. In particular examples, the presence of one or more mutations in a gene can significantly inactivate that gene.
Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence where the first nucleic acid sequence is joined in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Promoter: An array of nucleic acid sequences that governs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences to initiate gene transcription. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
Recombinant: A recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In particular examples, this artificial combination is accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques such as those described in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 3d ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. The term recombinant includes nucleic acid molecules that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid molecule.
Sequence identity/similarity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options can be set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to −1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq -i c:\seq1.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2.
To compare two amino acid sequences, the options of B12seq can be set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (e.g., 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (i.e., 1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (i.e., 15÷20*100=75).
For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). Homologs are typically characterized by possession of at least 70% sequence identity counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG. In addition, a manual alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, possess at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity determined by this method.
One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is possible that strongly significant homologs could be obtained that fall outside the ranges provided.
Transformed: A cell, such as a fungal cell, into which a nucleic acid molecule has been introduced, for example by molecular biology methods known in the art. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including, but not limited to transfection with viral vectors, conjugation, transformation with plasmid vectors, and introduction of naked DNA by chemical-mediated, electroporation, lipofection, and biolistic particle delivery.
Overview
Disclosed herein is the finding that overexpression of the LaeA gene in A. terreus results in a significant increase in production of itaconic acid. In particular, A. terreus transformed with a nucleic acid construct comprising an Aspergillus species LaeA gene produces significantly more itaconic acid relative to A. terreus that is not transformed with the construct.
Providing herein are isolated A. terreus fungi transformed with a heterologous nucleic acid construct comprising an Aspergillus species LaeA (loss of aflR expression A) gene. Expression of LaeA is increased in the transformed fungus compared to an A. terreus fungus that is not transformed with the heterologous nucleic acid construct.
In some embodiments, the heterologous nucleic acid construct comprises a heterologous LaeA gene, a heterologous promoter, a heterologous transcription terminator, a heterologous selective marker gene, or any combination thereof.
In some embodiments, the Aspergillus species LaeA gene is a homologous LaeA gene (i.e. an A. terreus LaeA gene). In other embodiments, the LaeA gene is a heterologous LaeA gene, such as an LaeA gene from A. nidulans, A. niger, A. oryzae, A. fumigatus or another Aspergillus species. In some examples, the A. nidulans LaeA gene encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 41. In non-limiting examples, the A. nidulans LaeA gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 41. In some examples, the A. niger LaeA gene encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 59. In non-limiting examples, the A. niger LaeA gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 59.
In some embodiments, the heterologous nucleic acid molecule comprises a promoter operably linked to an Aspergillus species LaeA gene, a transcription terminator and/or a selective marker gene. In particular examples, the promoter is a heterologous promoter, such as, but not limited to, the A. nidulans gpdA promoter, or any other promoter that is functional in Aspergillus (e.g., the actA, ubi4, Arsa-7, A-37, Brsa-109, gpdA, pyrG or tef1α promoters from A. niger). In other particular examples, the Aspergillus species LaeA gene is a heterologous LaeA gene, such as, but not limited to, an A. nidulans, A. niger, A. oryzae, or A. fumigatus LaeA gene. In other particular examples, the transcription terminator is a heterologous transcription terminator, such as, but not limited to, the A. nidulans TrpC transcription terminator. As an alternative to trpC, other transcriptional terminators can be used, such as promoters which include a transcriptional terminator (e.g., actA, ArsA7, Arsa-37, gpdA, pyrG, tef1α or ubi4). In yet other particular examples, the selective marker is a heterologous selective marker gene, such as a heterologous antibiotic resistance gene. In specific examples, selective marker gene is an A. oryzae pyrithiamine resistance (ptrA) gene. Other selective marker genes include, for example, the hygromycin B phosphotransferase (hph) gene, the bar gene (which confers glufosinate resistance), or bleomycin resistance gene (ble), or auxotrophic markers, such as pyrG.
In specific non-limiting examples, the heterologous nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 109.
Further provided herein is a method of making itaconic acid. In some embodiments, the includes culturing an isolated A. terreus fungus transformed with a heterologous nucleic acid molecule comprising an Aspergillus species LaeA gene under conditions that permit the fungus to make itaconic acid. Expression of LaeA is increased in the transformed fungus compared to an A. terreus fungus that is not transformed with the heterologous nucleic acid construct. In some embodiments, the fungus is cultured in itaconic acid production medium, which contains grams per liter (g/l): glucose, 100; ammonium sulfate, 2.36; potassium phosphate dibase, 0.11; magnesium sulfate heptahydrate, 2.08; calcium chloride dihydrate, 0.13; sodium chloride, 0.074; copper sulfate pentahydrate 2×10−4; ferrous sulfate heptahydrate, 5.5×10−3; manganese chloride tetrahydrate, 7×10−4; and zinc sulfate heptahydrate, 1.3×10−3.
In some embodiments of the methods of making itaconic acid, the heterologous nucleic acid construct comprises a heterologous LaeA gene, a heterologous promoter, a heterologous transcription terminator, a heterologous selective marker gene, or any combination thereof.
In some embodiments of the methods, the Aspergillus species LaeA gene is a homologous LaeA gene (i.e. an A. terreus LaeA gene). In other embodiments, the LaeA gene is a heterologous LaeA gene, such as an LaeA gene from A. nidulans, A. niger, A. oryzae, A. fumigatus or another Aspergillus species. In some examples, the A. nidulans LaeA gene encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 41. In non-limiting examples, the A. nidulans LaeA gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 41. In some examples, the A. niger LaeA gene encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 59. In non-limiting examples, the A. niger LaeA gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 59.
In some embodiments of the method, the heterologous nucleic acid molecule comprises a promoter operably linked to an Aspergillus species LaeA gene, a transcription terminator and/or a selective marker gene. In particular examples, the promoter is a heterologous promoter, such as, but not limited to, the A. nidulans gpdA promoter. In other particular examples, the Aspergillus species LaeA gene is a heterologous LaeA gene, such as, but not limited to, an A. nidulans, A. niger, A. oryzae, or A. fumigatus LaeA gene. In other particular examples, the transcription terminator is a heterologous transcription terminator, such as, but not limited to, the A. nidulans TrpC transcription terminator. In yet other particular examples, the selective marker is a heterologous selective marker gene, such as a heterologous antibiotic resistance gene. In specific examples, the selective marker gene is an A. oryzae pyrithiamine resistance (ptrA) gene. In specific non-limiting examples, the heterologous nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 109.
Also provided by the present disclosure are compositions that include isolated A. terreus fungi transformed with a heterologous nucleic acid construct. The compositions may further include growth medium. Further provided by the present disclosure are kits that include isolated A. terreus fungi transformed with a heterologous nucleic acid construct, such as a kit that includes a medium for culturing, storing, or growing the fungus, and/or antibiotics (for selection).
This disclosure also provides the first demonstration that genetic inactivation of Alg3, a gene involved in protein N-linked glycosylation, can result in substantial improvement of citric acid production in A. niger, while the total biomass is similar to the parent strain. The core oligosaccharide Glc3Man9GlcNAc2 is synthesized by a series of membrane-bound glycosyltransferases, which begins on the cytoplasmic side of the membrane of the endoplasmic reticulum (ER) and flips into the luminal side of the ER membrane to complete its synthesis. The lipid-linked core Glc3Man9GlcNAc2 is subsequently transferred to a nascent protein in the ER, where the glycoproteins are folded and then shuttled to the Golgi for additional, but divergent processing. The Alg3 gene encodes the enzyme α-1,3-mannosyltransferase that converts Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP on the ER membrane of the luminal side. Provided herein is a homolog of Saccharomyces cerevisiae Alg3 identified from Aspergillus niger (e.g., see SEQ ID NOS: 1 and 2).
It is shown herein that genetic inactivation of Alg3 in A. niger resulted in a significant reduction of growth on complete medium (CM) and potato dextrose agar medium (PDA), but no effect on minimal medium (MM). The Alg3 deletion also caused the substantial reduction in spore production of A. niger on CM, but no significant change on the PDA. When the spores were germinated in CM or PDA liquid culture medium, the Alg3Δ strain showed pronounced delay in spore germination. This growth phenotype is similar to the mutants with defects in signal transduction pathways observed in A. nidulans and A. niger (Fillinger et al., Mol. Microbiol. 44(4):1001-16, 2002; Saudohar et al., Microbiol. 148(8):2635-45, 2002; Xue et al., Eukaryot Cell 3(2):557-60, 2004). Deletion of pkaA, cycaA or schA/pkaA in A. nidulans substantially reduces its growth on CM medium plates and spore germination rate in MM liquid culture medium (Fillinger et al., Mol. Microbiol. 44(4):1001-1016, 2002) and similar growth phenotypes were observed in the strains with the deletion of pkaR, pkaC or double deletion of pkaR/pkaC in A. niger (Saudohar et al., Microbiol. 148(8):2635-2645, 2002). However, functional deletion of the MAP kinase SakA in A. fumigatus delays the spore germination in liquid CM, but stimulates spore germination in MM liquid medium (Xue et al., Eukaryot Cell 3(2): 557-560, 2004).
Furthermore, the Alg3 deletion reduced the overall growth on citric acid production (CAP) medium plates at different pHs. In contrast, the Alg3 deletion triggered early spore germination and substantially improved spore germination rate in CAP liquid culture medium. Citric acid production in CAP liquid culture medium was significantly improved in A. niger. When the alg3Δ mutant was complemented with the original alg3 gene at pyrG locus (FIG. 8A; SEQ ID NO: 60), its transcription levels was similar to parent strain with the cycle threshold (Ct) values about 23, while the Ct value for alg3Δ mutant was 30.8 in CAP liquid culture conditions, which was determined by real-time reverse-transcription PCR. Consequently, the citric acid production in the resulted complemented mutant strains was similar to the parent strain as shown in FIG. 8B. The results shown herein demonstrate the involvement of Alg3 on the growth and development and citric acid production in A. niger. 
It is proposed that inactivation of Alg3 influences the N-glycosylation of those proteins involving in signal transduction pathways. The N-glycosylation consensus sequence (N-glycosite) for N-glycosylation in those proteins from the signal transduction pathways was observed. Most of those proteins contained 1 to 7 N-glycosites, such as, 6 N-glycosites found in sskB (map kinase kinase), 7 in Ste11/SteC, 5 in acyA, 5 in rgsA, 6 in rgsC, 4 in gprA, 4 in pkaC2, 4 in flbA and 3 in Gβ. Comparison of these results with previous studies indicates that the effects of the Alg3 deletion on spore germination and growth may be regulated by altering the N-glycosylation in those proteins involved in signal transduction pathways in A. niger. 
When the Alg3Δ strain was grown on CM medium, spore production of Alg3Δ mutants was dramatically reduced as compared to the parent strain, while maintaining a similar level when grown on PDA medium. This phenotype of sporulation production may be influenced by both endogenous and exogenous factors. For example, protein glycosylation was greatly influenced by culture conditions in filamentous fungi, such as fully glycosylated Ce17A only isolated from MM culture medium (Stals et al., Glycobiology 14(8):725-737, 2004). In addition, higher amounts of proteases were secreted by the Alg3Δ strain than the parent in liquid MM culture supplemented 1 g/l yeast extract, which further influenced nutrient uptakes, cellular formation and overall N-glycosylation. This would alter the yield and N-glycosylation in G protein system in A. niger, where G protein signaling is crucial for detection of major environmental stimuli for food acquisition, asexual sporulation, and spore germination (Chang et al., Genetics 167(3):305, 2004; Li et al., Annu. Rev. Microbiol. 61:423-452, 2007).
The spores of parent strain germinated more slowly and had a lower germination rate than the Alg3Δ strain in CAP liquid culture medium, which contains limited nitrogen source (3.1 g/1 of NH4NO3), similar to MM. A similar phenotype was observed when the stress activating kinase, a MAP kinase, was deleted in A. fumigatus (SakAΔ strain) and grown in MM liquid culture medium (Xue et al., Eukaryot Cell 3(2): 557-560, 2004). The spore germination of SakAΔ strain was dramatically influenced by nitrogen sources. For example, similar rates of spore germination between parent and SakAΔ strains were observed on MM containing 10 mM NH4Cl or 10 mM Pro, while the spore germination rates of SakAΔ strain was much higher than the parent strain in the MM culture medium containing 10 mM NaNO3, NaNO2, or Phe. In addition, the CAP medium contains high level of glucose and low pH, which contributes additional stresses to A. niger growth. Although the Alg3Δ strain had earlier and higher germination in CAP medium, its biomass formation was less than the parent strain at early stages. The dried biomass yields for both parent and Alg3Δ strains were similar after growth in CAP medium for four and half days. However, more citric acid was produced by the Alg3Δ strain than the parent strain. This indicates more glucose was directly converted to citric acid by influence citric acid metabolism and reduction of glucose consumption for complex N-glycan formation and sequentially for other cellular metabolisms.
This disclosure also provides the first demonstration that genetic inactivation of Alg3, in combination with an increase in expression of the loss of aflR expression A (LaeA) gene, can result in substantial improvement of citric acid production in A. niger. It is proposed that increased expression of LaeA can also improve citric acid production in A. niger or other filamentous fungi. To increase expression of LaeA in fungal cells, a transgene was generated and expressed in A. niger as follows. The LaeA gene of A. nidulans was operably controlled by glyceraldehyde 3-phosphate dehydrogenase (gpdA) promoter and trpC transcriptional terminator (TtrpC) of A. nidulans. This chimeric gene was flanked with the upstream of A. niger pyrG gene, the pyrithiamine resistance (ptrA) gene of A. oryzae and the downstream of A. niger pyrG gene. The transgene expression fragment containing the chimeric gene was used to transform the protoplasts of alg3Δ mutants of A. niger. 
The present disclosure also describes the generation of an A. niger strain having a deletion of the LaeA gene (LaeAΔ), as well as an A. niger LaeAΔ strain complemented with a transgene encoding LaeA (c1LaeAΔ) at the pyrG locus. These strains were used to evaluate the role of LaeA expression on citric acid production in Aspergillus. The data disclosed herein demonstrates that deletion of the LaeA gene in A. niger (LaeAΔ) results in a loss of citric acid production in culture. When the original A. niger LaeA gene was used for complementation in the LaeAΔ mutant (cLaeAΔ) at the pyrG locus, citric acid production was partially recovered, which indicates the importance of the chromosomal location of LaeA gene. When A. nidulans LaeA was over-expressed in the A. niger parent strain, citric acid production was higher than the parent strain. These results indicate that LaeA enhances citric acid production in A. niger. 
In summary, the deletion of Alg3, increasing expression of LaeA, or both, can be used to increase citric acid production in fungi (such as filamentous fungi, e.g., A. niger). In addition, deletion of Alg3 alters the overall N-glycosylation and further influences the spore germination, filamentous growth, sporulation and other organic acid production in A. niger. 
Alg3Δ Fungi
The present disclosure provides isolated fungi having its Alg3 gene inactivated, wherein such inactivation results in increased citric acid production by the fungi. Such fungi are referred to herein as Alg3Δ fungi. It is disclosed herein that genetic inactivation of Alg3 results in Aspergillus fungi that can increase citric acid production as compared to Aspergillus having a native Alg3 sequence.
Contemplated herein are isolated fungi containing a genetic inactivation of a dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase gene (Alg3). Any fungus can be used, such as any genus or variety of Aspergillus. In particular examples, the disclosed Aspergillus fungus is A. niger, such as Aspergillus niger strain 11414 (American Type Culture Collection (ATCC) No. 11414; NRRL 2270); 1015 (ATCC No. 1015; NRRL 328, CBS 113.46); NRRL 3 (ATCC No. 9029, CBS 120.49, N400); NRRL 3122 (ATCC No. 22343); or 11414KusA-. In other specific examples, the Aspergillus is A. aculeatus, A. awamori, A. carbonarius, A. wentii, A. foetidus, A. oryzae, A. terreus, or A. fumigatus. 
In addition, any method for genetic inactivation can be used, as long as the expression of the gene is significantly reduced or eliminated, or the function of the expressed protein is significantly reduced or eliminated. In particular examples, the Alg3 gene is genetically inactivated by complete or partial deletion mutation or by insertional mutation. In some examples genetic inactivation need not be 100% genetic inactivation. In some embodiments, genetic inactivation refers to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% gene or protein inactivation. The term “reduced” or “decreased” as used herein with respect to a cell and a particular gene or protein activity refers to a lower level of activity than that measured in a comparable cell of the same species. For example, a particular fungi lacking Alg3 activity has reduced Alg3 activity if a comparable fungi not having an Alg3 genetic inactivation has detectable Alg3 activity.
Alg3 sequences are disclosed herein and others are publicly available, for example from GENBANKTM or EMBL. In some examples, the Alg3 gene functionally deleted encodes a protein having at least 80%, at least 90%, at least 95%, at least 97%, or at least 98% sequence identity to SEQ ID NO: 2, 4, 31, 32, 33, 34, 35, or 36. In some examples, the Alg3 gene functionally deleted comprises at least 80%, at least 90%, at least 95%, at least 97%, or at least 98% sequence identity to SEQ ID NO: 1 or 3 or nucleotides 1186-2582 of SEQ ID NO: 1.
The inactivation of Alg3 results in many phenotypes in the fungi. For example, Alg3Δ mutants can have one or more of the following phenotypes: slower growth on citric acid production (CAP) medium, earlier spore germination in CAP medium (for example germination in at least 3 hours, at least 4 hours, or at least 5 hours after inoculation, such as within 3 hours of inoculation), increased spore germination rate in CAP medium, increased citric acid production in CAP medium, slower growth on complete medium (CM) or potato dextrose (PDA) medium, delay initiation of spore germination in CM or PDA medium, reduced sporulation on CM, or combinations thereof.
Such changes (such as increases or decreases) can be relative to a fungi having a wild-type Alg3 gene, such as a parental strain (e.g., A. niger strain 11414KusA), grown under the same conditions as the Alg3Δ mutant. In some examples, an increased germination rate is germination of at least 20%, at least 25%, or at least 30% of the spores from an Alg3Δ fungus have germinated 8 hours after inoculation in CAP medium (such as 20% to 35%, such as 32%), as compared to no more than 20%, no more than 15%, or no more than 10% (such as 5 to 15%, or 10%) for A. niger strain 11414KusA. In some examples, an increased germination rate is germination of at least 80%, at least 85%, or at least 90% of the spores from an Alg3Δ fungus have germinated 15 hours after inoculation in CAP medium (such as 80% to 95%, such as 90%), as compared to no more than 60%, no more than 65%, or no more than 75% (such as 55 to 65%, or 60%) for A. niger strain 11414KusA. In some examples, increased citric acid production in CAP medium is an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, or at least 70%, by an Alg3Δ fungus as compared to A. niger strain 11414KusA. In some examples, reduced sporulation on complete medium is a reduction of sporulation by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%, (such as a 40% to 60% reduction) by an Alg34 fungus as compared to A. niger strain 11414KusA.
One skilled in the art will appreciate that additional genes can also be inactivated, wherein the additional genes may or may not provide additional enhancement of citric acid production to the fungus. In one example KusA (e.g., GENBANKTM Accession No. EF061656) is also genetically inactivated.
Also provided by the present disclosure are compositions that include isolated Alg3Δ fungi, such as a growth medium. Also provided by the present disclosure are kits that include isolated Alg3Δ fungi, such as a kit that includes a medium for culturing, storing, or growing the fungus. Exemplary mediums include solid medium (such as those containing agar, for example CM, PDA or MM) and liquid media (such as a fermentation broth, such as CM, MM, or CAP medium).
A. Methods of Functionally Deleting Genes
As used herein, an “inactivated” or “functionally deleted” gene means that the gene has been mutated, for example by insertion, deletion, or substitution (or combinations thereof) of one or more nucleotides such that the mutation substantially reduces (and in some cases abolishes) expression or biological activity of the encoded gene product. The mutation can act through affecting transcription or translation of the gene or its mRNA, or the mutation can affect the polypeptide product itself in such a way as to render it substantially inactive.
Genetic inactivation of one or more genes (which in some examples is also referred to as functional deletion) can be performed using any conventional method known in the art. In one example, a strain of Aspergillus is transformed with a vector which has the effect of down-regulating or otherwise inactivating an Alg3 gene. This can be done by mutating control elements such as promoters and the like which control gene expression, by mutating the coding region of the gene so that any protein expressed is substantially inactive, or by deleting the Alg3 gene entirely. For example, an Alg3 gene can be functionally deleted by complete or partial deletion mutation (for example by deleting a portion of the coding region of the gene) or by insertional mutation (for example by inserting a sequence of nucleotides into the coding region of the gene, such as a sequence of about 1-5000 nucleotides). Thus, the disclosure in some examples provides transformed fungi that include at least one exogenous nucleic acid molecule which genetically inactivates an Alg3 gene (such as a nucleic acid sequence encoding SEQ ID NO: 2 or 4). In one example, such a transformed cell produces more citric acid, for example relative to a comparable fungus with a native Alg3 sequence.
In particular examples, an insertional mutation includes introduction of a sequence that is in multiples of three bases (e.g., a sequence of 3, 9, 12, or 15 nucleotides) to reduce the possibility that the insertion will be polar on downstream genes. For example, insertion or deletion of even a single nucleotide that causes a frame shift in the open reading frame, which in turn can cause premature termination of the encoded Alg3 polypeptide or expression of a substantially inactive polypeptide. Mutations can also be generated through insertion of foreign gene sequences, for example the insertion of a gene encoding antibiotic resistance (such as hygromycin or bleomycin).
In one example, genetic inactivation is achieved by deletion of a portion of the coding region of the Alg3 gene. For example, some, most (such as at least 50%) or virtually the entire coding region can be deleted. In particular examples, about 5% to about 100% of the gene is deleted, such as at least 20% of the gene, at least 40% of the gene, at least 75% of the gene, or at least 90% of the Alg3 gene.
Deletion mutants can be constructed using any of a number of techniques known in the art. In one example, allelic exchange is employed to genetically inactivate one or more genes in Aspergillus. A specific example of such a method is described in Example 2 below.
In one example, a strategy using counterselectable markers can be employed which has been utilized to delete genes. For a review, see Reyrat et al. (Infec. Immun. 66:4011-4017, 1998). In this technique, a double selection strategy is employed wherein a plasmid is constructed encoding both a selectable and counterselectable marker, with flanking DNA sequences derived from both sides of the desired deletion. The selectable marker is used to select for fungi in which the plasmid has integrated into the genome in the appropriate location and manner. The counterselecteable marker is used to select for the very small percentage of fungi that have spontaneously eliminated the integrated plasmid. A fraction of these fungi will then contain only the desired deletion with no other foreign DNA present.
In another technique, the cre-lox system is used for site specific recombination of DNA (for example see Steiger et al., Appl. Environ. Microbiol. 77(1):114, 2011). The system includes 34 base pair lox sequences that are recognized by the bacterial cre recombinase gene. If the lox sites are present in the DNA in an appropriate orientation, DNA flanked by the lox sites will be excised by the cre recombinase, resulting in the deletion of all sequences except for one remaining copy of the lox sequence. Using standard recombination techniques, the targeted gene of interest (e.g., Alg3) can be deleted in the Aspergillus genome and to replace it with a selectable marker (for example a gene coding for kanamycin resistance) that is flanked by the lox sites. Transient expression (by electroporation of a suicide plasmid containing the cre gene under control of a promoter that functions in Aspergillus) of the cre recombinase should result in efficient elimination of the lox flanked marker. This process will produce a mutant containing the desired deletion mutation and one copy of the lox sequence.
In another method, an Alg3 gene sequence in the Aspergillus genome is replaced with a marker gene, such as green fluorescent protein, β-galactosidase, or luciferase. In this technique, DNA segments flanking a desired deletion are prepared by PCR and cloned into a suicide (non-replicating) vector for Aspergillus. An expression cassette, containing a promoter active in Aspergillus and the appropriate marker gene, is cloned between the flanking sequences. The plasmid is introduced into wild-type Aspergillus. Fungi that incorporate and express the marker gene are isolated and examined for the appropriate recombination event (replacement of the wild type Alg3 gene with the marker gene).
Thus, for example, a fungal cell can be engineered to have a disrupted Alg3 gene using common mutagenesis or knock-out technology. (Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Sterns, Cold Spring Harbor Press, 1998; Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97: 6640-5, 2000; and Dai et al., Appl. Environ. Microbiol. 70(4):2474-85, 2004). Alternatively, antisense technology can be used to reduce or eliminate the activity of Alg3. For example, a fungal cell can be engineered to contain a cDNA that encodes an antisense molecule that prevents Alg3 from being translated. The term “antisense molecule” encompasses any nucleic acid molecule or nucleic acid analog (e.g., peptide nucleic acids) that contains a sequence that corresponds to the coding strand of an endogenous Alg3 gene. An antisense molecule also can have flanking sequences (e.g., regulatory sequences). Thus, antisense molecules can be ribozymes or antisense oligonucleotides. A ribozyme can have any general structure including, without limitation, hairpin, hammerhead, or axehead structures, provided the molecule cleaves RNA. Further, gene silencing can be used to reduce the activity of Alg3.
B. Measuring Gene Inactivation
A fungus having an inactivated Alg3 gene can be identified using any method known in the art. For example, PCR and nucleic acid hybridization techniques, such as Northern and Southern analysis, can be used to confirm that a fungus has an inactivated Alg3 gene. Alternatively, real-time reverse transcription PCR (qRT-PCR) can be used for detection and quantification of targeted messenger RNA, such as mRNA of Alg3 gene in the parent and mutant strains as grown at the same culture conditions. Immunohistochemical and biochemical techniques can also be used to determine if a cell expresses Alg3 by detecting the expression of the Alg3 peptide encoded by Alga. For example, an antibody having specificity for Alg3 can be used to determine whether or not a particular fungus contains a functional nucleic acid encoding Alg3 protein. Further, biochemical techniques can be used to determine if a cell contains a particular gene inactivation by detecting a product produced as a result of the expression of the peptide. For example, structural determination of N-glycans excised from glycoproteins can indicate that a fungal cell contains an inactivated Alg3 gene. In addition, measurements of sporulation, germination, secondary metabolite production, and citric acid production can be measured using the methods described herein.
C. Measuring Citric Acid Production
Methods of determining whether a genetic inactivation of Alg3 in Aspergillus increases citric acid production, for example relative to the same strain with a native Alg3 sequence (such as a parental strain), are routine in the art. Although particular examples are disclosed herein, the methods are not limiting.
For example, production of citric acid by Aspergillus (such as an Alg3Δ strain) can be measured using a spectrophotometric assay. In one example citric acid production can be determined with an endpoint spectrophotometric enzyme assay (for example see, Bergmeyer, H. U. 1985. Metabolites 2: tri- and dicarboxylic acids, purines, pyrimidines and derivatives, coenzymes, inorganic compounds, p. 5-10. In Citric acids. VCH Publishers, Weinheim, Germany). Citric acid can also be measured by liquid chromatography (LC) or high-performance liquid chromatography (HPLC) methods.
D. Alg3 Sequences
Alg3 protein and nucleic acid sequences are publicly available and specific examples are provided herein. In addition, Alg3 sequences can be identified using routine molecular biology methods.
Examples of Alg3 nucleic acid sequences shown in SEQ ID NOS: 1 and 3. However, the disclosure also encompasses variants of SEQ ID NOS: 1 and 3 which retain the ability to encode an Alg3 protein. One skilled in the art will understand that variant Alg3 nucleic acid sequences can be inactivated. Variant sequences may contain a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions). In addition, the degeneracy of the code permits multiple nucleic acid sequences to encode the same protein. For example, FIG. 9 shows an alignment of Alg3 nucleic acid sequences from A. niger (nucleotides 1986-2518 of SEQ ID NO: 1) and A. oryzae (nucleotides 643-1175 of SEQ ID NO: 3), which permits one to identify nucleotides that can tolerate substitution (e.g., those that are not conserved between species) and those that may not (e.g., those that are conserved between species). Such nucleic acid molecules can share at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to any known Alg3 nucleic acid sequence, such as SEQ ID NO: 1 or 3 or nucleotides 1186-1306, 1393-1916 and 1989-2582 of SEQ ID NO: 1.
Examples of Alg3 protein sequences shown in SEQ ID NOS: 2, 4, 31, 32, 33, 34, 35, and 36. However, the disclosure also encompasses variants SEQ ID NOS: 2, 4, 31, 32, 33, 34, 35, and 36 which retain Alg3 activity. One skilled in the art will understand that variant Alg3 enzyme sequences can be inactivated. Variant sequences may contain a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions). Such polypeptides share at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to an Alg3 sequence, such as SEQ ID NO: 2, 4, 31, 32, 33, 34, 35, or 36.
Variant sequences can be identified, for example by aligning known Alg3 sequences. For example, FIGS. 10A and 10B show the alignment of seven different Alg3 sequences from different organisms. In addition, FIG. 11 shows a detailed alignment of Alg3 protein sequences from A. niger (amino acids 12-411 of SEQ ID NO: 2) and S. cerevisiae, indicating amino acids that are identical, conserved (+) or not conserved (space). Based on these alignments, variants of Alg3 sequences can be identified. For example, amino acid residues that are conserved between organisms are ones that should not be substituted (such as amino acids M50, T70, Y81, Q100 and D150 based on the numbering for A. niger), while amino acid residues that are not conserved between organisms are ones likely to tolerate substitution (such as amino acids R8, L160, 5395 and N405 based on the numbering for A. niger). Similarly, amino acid positions in FIGS. 10A and 10B indicated with different amino acids at the same position are ones likely to tolerate substitution, while positions with the same amino acid (*) are not.
In some examples, an Alg3 sequence that is to be genetically inactivated encodes or includes one or more conservative amino acid substitutions. A conservative amino acid substitution is a substitution of one amino acid (such as one found in a native sequence) for another amino acid having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting peptide. In one example, an Alg3 sequence (such as any of SEQ ID NOS: 2, 4, 31, 32, 33, 34, 35, or 36) includes one or more amino acid substitutions (for example at 1, 2, 5 or 10 residues). Examples of amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val. Further information about conservative substitutions can be found in, among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7, 1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al., (Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5, 1988), WO 00/67796 (Curd et al.) and in standard textbooks of genetics and molecular biology.
The Alg3 gene inactivated in a fungus, in particular examples, includes a sequence that encodes an Alg3 protein having at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to an Alg3 sequence, such as SEQ ID NO: 2, 4, 31, 32, 33, 34, 35, or 36, wherein the protein can catalyze the addition of the first dol-p-man derived mannose in an α-1,3 linkage to Man5GlcNAc2-PP-Dol. In a specific example, the Alg3 gene inactivated in a fungus encodes an Alg3 protein shown in SEQ ID NO: 2, 4, 31, 32, 33, 34, 35, or 36.
The Alg3 gene that is to be inactivated in a fungus, in particular examples, includes a sequence having at least at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to an Alg3 nucleic acid sequence, such as SEQ ID NO: 1 or 3 or nucleotides 1186-1306, 1393-1916 and 1989-2582 of SEQ ID NO: 1, and encode an Alg3 protein that can catalyze the addition of the first dol-p-man derived mannose in an α-1,3 linkage to Man5GlcNAc2-PP-Dol. In a specific example, the Alg3 gene inactivated in a fungus is shown in SEQ ID NO: 2 or 4.
One skilled in the art will appreciate that additional Alg3 sequences can be identified using any method such as those described herein. For example, Alg3 nucleic acid molecules that encode an Alg3 protein can be identified and obtained using common molecular cloning or chemical nucleic acid synthesis procedures and techniques, including PCR. In addition, standard nucleic acid sequencing techniques and software programs that translate nucleic acid sequences into amino acid sequences based on the genetic code can be used to determine whether or not a particular nucleic acid has any sequence homology with known Alg3 sequences. Sequence alignment software such as MEGALIGN (DNASTAR, Madison, Wis., 1997) can be used to compare various sequences.
In addition, nucleic acid hybridization techniques can be used to identify and obtain a nucleic acid molecule that encodes an Alg3 protein. Briefly, any known Alg3 nucleic acid molecule, or fragment thereof, can be used as a probe to identify similar nucleic acid molecules by hybridization under conditions of moderate to high stringency. Such similar nucleic acid molecules then can be isolated, sequenced, and analyzed to determine whether the encoded protein is an Alg3 protein.
Any method can be used to introduce an exogenous nucleic acid molecule into a fungal cell, for example to genetically inactivate Alg3. For example, chemical mediated-protoplast transformation, electroporation, Agrobacterium-mediated transformation, fusion of protoplasts, and biolistic delivery are common methods for introducing nucleic acid into fungal cells. (See, e.g., Ito et al., J. Bacterol. 153:163-8, 1983; Durrens et al., Curr. Genet. 18:7-12, 1990; Sambrook et al., Molecular cloning: A laboratory manual, Cold Spring Harbour Laboratory Press, New York, USA, third edition, 2001; and Becker and Guarente, Methods in Enzymology 194:182-7, 1991. An exogenous nucleic acid molecule contained within a particular cell of the disclosure can be maintained within that cell in any form. For example, exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episomal state. That is, a cell can be a stable or transient transformant.
Fungi with Increased LaeA Expression or Increased LaeA Expression and Alg3 Deletion
The present disclosure provides isolated fungi having increased LaeA expression, wherein such increased expression or activity (for example in combination with an Alg3 functional inactivation, Alg3Δ) results in increased citric acid production by the fungi. Such fungi are referred to herein as increased LaeA fungal strains. It is disclosed herein that increased expression of LaeA (for example in combination with genetic inactivation of Alg3, Alg3Δ) results in Aspergillus fungi that can increase citric acid production as compared to Aspergillus having native levels of expression.
Contemplated herein are isolated fungi having increased LaeA activity/expression, for example in combination with a genetic inactivation of Alg3. Any fungus can be used, such as any genus or variety of Aspergillus. In particular examples, the Aspergillus fungus is A. niger, such as Aspergillus niger strain 11414 (American Type Culture Collection (ATCC) No. 11414; NRRL 2270); 1015 (ATCC No. 1015; NRRL 328, CBS 113.46); NRRL 3 (ATCC No. 9029, CBS 120.49, N400); NRRL 3122 (ATCC No. 22343); or 11414KusA-. In other specific examples, the Aspergillus is A. aculeatus, A. awamori, A. carbonarius, A. wentii, A. foetidus, A. fumigatus, A. oryzae, or A. terreus. 
The present disclosure also provides isolated A. terreus fungi having increased LaeA expression, wherein such increased expression or activity results in increased itaconic acid production by the fungi. It is disclosed herein that increased expression of LaeA results in A. terreus fungi that can increase itaconic acid production as compared to A. terreus having native levels of LaeA expression. Contemplated herein are isolated A. terreus fungi transformed with a nucleic acid construct comprising an Aspergillus species LaeA gene. The LaeA gene can be from any species of Aspergillus. In some examples, the LaeA gene is an A. niger, A. nidulans, A. aculeatus, A. awamori, A. carbonarius, A. wentii, A. foetidus, A. fumigatus, A. oryzae, or A. terreus LaeA gene.
Any method for genetic enhancement or up-regulation can be used, as long as the expression of the gene and/or gene product is significantly increased, or the function of the expressed protein is significantly increased. In particular examples, LaeA gene expression is up-regulated by transformation of the fungi with one or more copies of a LaeA coding or genomic sequence (which can be a native or non-native LaeA sequence). In some embodiments, up-regulation refers to an increase in gene or protein expression of at least 20%, at least 40%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500%, for example relative to the parental fungal strain without the additional copies of an LaeA gene. The term “increased” or “up-regulated” as used herein with respect to a cell and a particular gene or protein activity refers to a higher level of activity than that measured in a comparable cell of the same species. For example, a particular fungi having increased or up-regulated LaeA activity has increased LaeA activity if a comparable fungi having native LaeA activity has less detectable LaeA activity (for example as measured by gene or protein expression).
LaeA sequences are disclosed herein and others are publicly available, for example from GENBANKTM or EMBL. In some examples, the LaeA gene upregulated encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 41 or SEQ ID NO: 59. In some examples, the LaeA gene upregulated comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 40 (e.g., nt 1-236 and 367-1252 of SEQ ID NO: 41) or SEQ ID NO: 58 (e.g., nt 1-230 and 373-1267 of SEQ ID NO: 58).
Increasing LaeA activity (for example in combination with genetic inactivation of Alg3) results in many phenotypes in the fungi. For example, such recombinant fungi exhibit increased citric acid production in CAP medium. Such increases can be relative to a fungi having a native or wild-type level of LaeA (or LaeA and Alg3) gene or protein expression, such as a parental strain (e.g., A. niger strain 11414KusA), grown under the same conditions as the fungi with increased LaeA activity (or increased LaeA activity and decreased Alg3 activity). In some examples, increased citric acid production in CAP medium is an increase of at least 20%, at least 30%, at least 50%, at least 60%, at least 65%, or at least 70%, by such a recombinant fungus as compared to A. niger strain 11414KusA. In some examples, recombinant fungi with increased LaeA activity (for example in combination with genetic inactivation of Alg3) have increased sporulation relative to A. niger Alg3Δ on MM, accumulate red color pigments to A. niger strain 11414KusA on complete medium, or both. In other examples, increasing LaeA activity in A. terreus fungi results in an increase in itaconic acid production in itaconic acid production media. Such increases can be relative to A. terreus fungi having a native or wild-type level of LaeA gene or protein expression, such as a parental strain A. terreus strain grown under the same conditions as the fungi with increased LaeA activity. In some examples, increased itaconic acid production is an increase of at least 20%, at least 30%, at least 50%, at least 60%, at least 65%, or at least 70%, as compared to a parental strain.
One skilled in the art will appreciate that additional genes can also be inactivated or upregulated, wherein the additional genes may or may not provide additional enhancement of citric acid production or itaconic acid production to the fungus. In one example KusA (e.g., GENBANKTM Accession No. EF061656) is also genetically inactivated.
Also provided by the present disclosure are compositions that include isolated LaeA up-regulated fungi, such as a growth medium. Also provided by the present disclosure are kits that include isolated LaeA up-regulated fungi, such as a kit that includes a medium for culturing, storing, or growing the fungus. Exemplary mediums include solid medium (such as those containing agar, for example CM, PDA or MM) and liquid media (such as a fermentation broth, such as CM, MM, or CAP medium).
A. Methods of Up-Regulating Gene and/or Protein Expression
As used herein, an “activated” or “up-regulated” gene means that expression of the gene or gene product (e.g., protein) has been up-regulated, for example by introduction of additional copies of the appropriate gene or coding sequence into the fungus (or other common molecular biology methods), such that the introduce nucleic acid sequence is expressed, resulting in increased expression or biological activity of the encoded gene product.
Increasing expression of one or more genes (which in some examples is also referred to as up-regulation) can be performed using any conventional method known in the art. In one example, a strain of Aspergillus is transformed with a vector which has the effect of up-regulating or otherwise activating a LaeA gene (such as a native or non-native LaeA gene). This can be done by introducing one or more LaeA coding sequences (such as a gene sequence), whose expression is controlled by elements such as promoters and the like which control gene expression, by introducing a nucleic acid sequence which itself (or its encoded protein) can increase LaeA protein activity in the fungus, or by introducing another molecule (such as a protein or antibody) increases LaeA protein activity in the fungus. For example, a LaeA gene can be up-regulated by introduction of a vector that includes one or more LaeA sequences (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LaeA sequences or copies of such sequences) into the desired fungus. In some examples, such LaeA sequences are from different fungal species, can be multiple copies from a single species, or combinations thereof, such as LaeA sequences from at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different fungal species. In some examples, the LaeA sequence(s) introduced into the fungus is optimized for codon usage. Thus, the disclosure in some examples provides transformed fungi that include at least one exogenous nucleic acid molecule which includes a LaeA gene or coding sequence (such as a nucleic acid sequence encoding SEQ ID NOs: 41 or 59), for example in combination with Alg3Δ. In one example, such transformed cells produce more citric acid, for example relative to a comparable fungus with a native LaeA sequence (or a native LaeA sequence combined with a native Alg3 sequence).
In another technique, the cre-lox system is used for site specific recombination of DNA (for example see Steiger et al., Appl. Environ. Microbiol. 77(1):114, 2011). The system includes 34 base pair lox sequences that are recognized by the bacterial cre recombinase gene. If the lox sites are present in the DNA in an appropriate orientation, DNA flanked by the lox sites will be excised by the cre recombinase, resulting in the deletion of all sequences except for one remaining copy of the lox sequence. Using standard recombination techniques, the targeted gene of interest (e.g., LaeA) can be deleted in the Aspergillus genome and replaced with one or more copies of a non-native LaeA sequence (for example in A. niger, replacing one or both A. niger LaeA sequences with one or more, or combination of, LaeA sequences from A. nidulans, A. flavus, fusarium oxysperorum, penicillium chrysogenum, which have high secondary metabolite production) flanked by the lox sites. Transient expression (by electroporation of a suicide plasmid containing the cre gene under control of a promoter that functions in Aspergillus) of the cre recombinase should result in efficient elimination of the lox flanked marker. This process will produce a fungus containing the desired insertion mutation and one copy of the lox sequence.
In one example, one or more LaeA genes are introduced into fugal cells by chemical mediated proteoplast transformation in combination of yeast-gap repairing method for transgene expression construction.
In one example, a transgene is generated and expressed in the desired fungal cell, such as an Alg3Δ cell, to increase LaeA expression. For example, such a transgene can include a LaeA genomic or cDNA sequence (such as one having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to any known LaeA sequence, such as SEQ ID NO: 40 or 58), for example operably linked to a promoter, such as a glyceraldehyde 3-phosphate dehydrogenase (gpdA) promoter or other promoter, such as one that has high activity in CAP culture medium, for example a polyubiquitin promoter, Arsa-7, and A-37 from A. niger. In one example, the promoter has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37. In one example, the promoter comprises or consists of the sequence shown in SEQ ID NO: 37. In some examples, the transgene further includes pyrG upstream and downstream sequences (for example that are at the 5′- and 3′-end, respectively, of the transgene). The pyrG gene in A. niger is mutated and has lost its original functions. Thus, other non-essential gene loci can be used as long as it is not influenced by the native neighbor genes. In one example, the pyrG upstream and downstream sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45 and 48, respectively. In one example, the pyrG upstream and downstream sequences comprise or consist of the sequence shown in SEQ ID NO: 45 or 48, respectively. In some examples, the transgene further includes a trpC transcriptional terminator sequence of A. nidulans, for example downstream of the LaeA sequence. As an alternative to trpC, other transcriptional terminators can be used, such as promoters which include a transcriptional terminators (e.g., ArsA7, Arsa-37, polyubiquitin (ubi4)). In one example, the trpC transcriptional terminator has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46. In one example, the trpC transcriptional terminator comprises or consists of the sequence shown in SEQ ID NO: 46. In some examples, the transgene further includes a ptrA sequence, for example downstream of the trpC transcriptional terminator sequence. As an alternative to ptrA, the bleomycin gene or bar gene can be used. In one example, the ptrA sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 47. In one example, the ptrA sequence comprises or consists of the sequence shown in SEQ ID NO: 47. In one example, the transgene comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44 or 57. In one example, the transgene comprises or consists of the sequence shown in SEQ ID NO: 44 or 57.
Thus, for example, a fungal cell can be engineered to have increased copies of LaeA using common recombinant technology methods.
B. Measuring Gene Activation or Up-Regulation
A fungus having an activated or up-regulated LaeA gene can be identified using any method known in the art. For example, PCR and nucleic acid hybridization techniques, such as Northern, RT-PCR, and Southern analysis, can be used to confirm that a fungus has an up-regulated LaeA gene, such as an increase in the LaeA copy number. Immunohistochemical and biochemical techniques can also be used to determine if a cell expresses LaeA by detecting the expression of the LaeA peptide encoded by LaeA. For example, an antibody having specificity for LaeA can be used to determine whether or not a particular fungus has increased LaeA protein expression. Further, biochemical techniques can be used to determine if a cell has increased LaeA expression by detecting a product produced as a result of the expression of the peptide. For example, measurement of secondary metabolites can indicate that a fungal cell contains an up-regulated LaeA gene. In addition, measurements of citric acid production can be measured using the methods described herein.
C. Measuring Citric Acid Production
Methods of determining whether a genetic up-regulation of LaeA (alone or in combination with inactivation of Alg3) in Aspergillus increases citric acid production, for example relative to the same strain with a native LaeA sequence, Alg3 sequence, or both (such as a parental strain), are routine in the art. Although particular examples are disclosed herein (see above and in the examples below), the methods are not limiting.
D. Measuring Itaconic Acid Production
Methods of determining whether a genetic up-regulation (e.g. overexpression) of LaeA in Aspergillus terreus increases itaconic acid production, for example relative to the same strain that does not overexpress LaeA (such as a parental strain), are routine in the art. Although particular examples are disclosed herein (see Example 14), the methods are not limiting. For example, production of itaconic acid by A. terreus can be performed as previously described by Dickman (Analytical Chem 24: 1064-1066, 1952), or using the modified method described in Example 14.
E. LaeA Sequences
LaeA protein and nucleic acid sequences are publicly available and specific examples are provided herein. In addition, LaeA sequences can be identified using routine molecular biology methods.
Examples of LaeA nucleic acid sequences shown in SEQ ID NOs: 40 and 58. However, the disclosure also encompasses variants of SEQ ID NOs: 40 and 58 (such as the coding regions nt 1-236 and 367-1252 of SEQ ID NO: 41 and nt 1-230 and 373-1267 of SEQ ID NO: 58) which retain the ability to encode a LaeA protein. One skilled in the art will understand that variant LaeA nucleic acid sequences can be used to increase expression of LaeA. Variant sequences may contain a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions). In addition, the degeneracy of the code permits multiple nucleic acid sequences to encode the same protein. Thus, in one example, a LaeA sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any known LaeA sequence, such as SEQ ID NO: 40 or SEQ ID NO: 58 (such as the coding regions nt 1-236 and 367-1252 of SEQ ID NO: 41 and nt 1-230 and 373-1267 of SEQ ID NO: 58) can be expressed in a fungal cell to increase LaeA expression in the fungal cell.
For example, FIG. 12 shows an alignment of LaeA protein sequences from A. nidulans (aa 14-372 of SEQ ID NO: 41) and A. niger (aa 14-370 of SEQ ID NO: 59), which permits one to identify amino acids that can tolerate substitution (e.g., those that are not conserved between species) and those that may not (e.g., those that are conserved between species). Based on these alignments, variants of LaeA sequences can be identified. For example, amino acid residues that are conserved between organisms are ones that should not be substituted (such as amino acids S16, M42, and P52 based on the numbering for A. nidulans), while amino acid residues that are not conserved between organisms are ones likely to tolerate substitution (such as amino acids T15, S44, and N325 based on the numbering for A. nidulans). Similarly, amino acid positions in FIG. 12 indicated with a space are ones likely to tolerate substitution, while positions with the same amino acid are not.
Such protein molecules can share at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any known LaeA nucleic acid sequence, such as SEQ ID NOs: 41 and 59, and such variants can be used to increase LaeA activity in a fungal cell. One skilled in the art will understand that variant LaeA enzyme sequences can be used to increase LaeA activity in a fungal cell. Variant sequences may contain a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions).
In some examples, a LaeA sequence whose expression is to be up-regulated encodes or includes one or more conservative amino acid substitutions. A conservative amino acid substitution is a substitution of one amino acid (such as one found in a native sequence) for another amino acid having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting peptide. In one example, a LaeA sequence (such as any of SEQ ID NOs: 41 and 59) includes one or more amino acid substitutions (for example at 1, 2, 5 or 10 residues). Examples of amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include those discussed above for Alg3.
The LaeA gene up-regulated in a fungus, in particular examples, includes a sequence that encodes a LaeA protein having at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an LaeA sequence, such as SEQ ID NO: 41 or SEQ ID NO: 59, wherein the protein can regulate secondary metabolite production in Aspergillus. In a specific example, the LaeA gene up-regulated in a fungus encodes a LaeA protein shown in SEQ ID NO: 41 or 59.
The LaeA gene up-regulated in a fungus, in particular examples, includes a sequence having at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a LaeA nucleic acid sequence, such as SEQ ID NO: 40 or SEQ ID NO: 58 (or to the coding regions nt 1-236 and 367-1252 of SEQ ID NO: 41 or nt 1-230 and 373-1267 of SEQ ID NO: 58), and encodes a LaeA protein which can regulate secondary metabolite production in Aspergillus. In a specific example, the LaeA gene upregulated in a fungus is shown in SEQ ID NO: 40 or SEQ ID NO: 58 (or includes the coding regions nt 1-236 and 367-1252 of SEQ ID NO: 41 or nt 1-230 and 373-1267 of SEQ ID NO: 58).
One skilled in the art will appreciate that additional LaeA sequences can be identified and obtained using any method such as those described herein. For example, LaeA nucleic acid molecules that encode a LaeA protein can be identified and obtained using common molecular cloning or chemical nucleic acid synthesis procedures and techniques, including PCR. In addition, standard nucleic acid sequencing techniques and software programs that translate nucleic acid sequences into amino acid sequences based on the genetic code can be used to determine whether or not a particular nucleic acid has any sequence homology with known LaeA sequences. Sequence alignment software such as MEGALIGN (DNASTAR, Madison, Wis., 1997) can be used to compare various sequences.
In addition, nucleic acid hybridization techniques can be used to identify and obtain a nucleic acid molecule that encodes a LaeA protein. Briefly, any known LaeA nucleic acid molecule, or fragment thereof, can be used as a probe to identify similar nucleic acid molecules by hybridization under conditions of moderate to high stringency. Such similar nucleic acid molecules then can be isolated, sequenced, and analyzed to determine whether the encoded protein is a LaeA protein. The gene specific oligonucleotide pair can also be designed, synthesized and used for real-time RT-PCR to quantify the LaeA gene transcription level.
Any method can be used to introduce an exogenous nucleic acid molecule into a fungal cell, for example to genetically enhance LaeA expression. For example, chemical mediated-protoplast transformation, electroporation, Agrobacterium-mediated transformation, fusion of protoplasts, and biolistic delivery are common methods for introducing nucleic acid into fungal cells. (See, e.g., Ito et al., J. Bacterol. 153:163-8, 1983; Durrens et al., Curr. Genet. 18:7-12, 1990; Sambrook et al., Molecular cloning: A laboratory manual, Cold Spring Harbour Laboratory Press, New York, USA, second edition, 1989; and Becker and Guarente, Methods in Enzymology 194:182-7, 1991). An exogenous nucleic acid molecule contained within a particular cell of the disclosure can be maintained within that cell in any form. For example, exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episomal state. That is, a cell can be a stable or transient transformant.
F. LaeA Deletion and Complementation
Disclosed herein is a LaeA deletion cassette for generating a fungus, such an Aspergillus species fungus, having a deletion in the LaeA gene. A schematic of the LaeA deletion cassette is shown in FIG. 17A. To generate the deletion cassette, DNA fragments comprising the 5′ and 3′ ends of the A. niger LaeA gene (SEQ ID NOs: 61 and 62, respectively) were isolated from A. niger genomic DNA and the hph expression cassette (SEQ ID NO: 63) (hph) was isolated from pCB1003 plasmid vector DNA by PCR. The PCR DNA fragments were assembled in the pBSK backbone vector to produce the LaeA deletion cassette (SEQ ID NO: 65).
Provided herein is a nucleic acid molecule comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 65. In some embodiments, the nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 65. Further provided is an isolated fungus transformed with a nucleic acid molecule comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 65. In some embodiments, the isolated fungus is transformed with a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 65. In some embodiments, the fungus is a species of Aspergillus, such as A. niger. 
Further provided herein are isolated fungi (such as filamentous fungi) having a gene inactivation (also referred to as a gene deletion) of a LaeA gene. Any strain of fungi can be used, such as a filamentous fungi, for example A. niger or particular strains thereof (for example A. niger strain 11414 or 11414KusA). In particular examples, the LaeA gene is genetically inactivated by complete or partial deletion mutation or by insertional mutation. In some examples genetic inactivation need not be 100% genetic inactivation. In some embodiments, genetic inactivation refers to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% gene or protein inactivation. The term “reduced” or “decreased” as used herein with respect to a cell and a particular gene or protein activity refers to a lower level of activity than that measured in a comparable cell of the same species. For example, a particular fungi lacking LaeA activity has reduced LaeA activity if a comparable fungi not having an LaeA genetic inactivation has detectable LaeA activity. In some embodiments, the isolated fungi having a gene inactivation are generated using the LaeA deletion construct set forth herein as SEQ ID NO: 65.
Also provided herein is a LaeA complementation construct for complementing expression of LaeA in fungi having a deleted LaeA gene. A schematic of the LaeA complementation construct is shown in FIG. 17B. To generate the complementation construct, the LaeA gene containing both its promoter and transcriptional terminator was isolated by PCR from A. niger genomic DNA. The PCR fragment was cloned into the plasmid vector pRSB426-LaeA. The new plasmid DNA vector contained the upstream region of the pyrG gene of A. niger, the entire region of A. niger LaeA gene, the transcriptional terminator of the trpC gene from A. nidulans, the pyrithiamine resistance (ptrA) gene from A. oryzae, and the downstream region of the pyrG gene of A. niger (SEQ ID NO: 66).
Provided herein is a nucleic acid molecule comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 66. In some embodiments, the nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 66. Further provided is an isolated fungus transformed with a nucleic acid molecule comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 66. In some embodiments, the isolated fungus is transformed with a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 66. In some embodiments, the fungus is a species of Aspergillus, such as A. niger. 
Further provided herein are isolated fungi (such as filamentous fungi) having a gene inactivation (also referred to as a gene deletion) of a LaeA gene and further transformed by a nucleic acid construct comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 66. In some embodiments, the isolated fungus is transformed with a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 66. In some embodiments, the fungus is a species of Aspergillus, such as A. niger. Any strain of fungi can be used, such as a filamentous fungi, for example A. niger or particular strains thereof (for example A. niger strain 11414 or 11414KusA).
Production of Citric Acid Using Alg3Δ Mutants, Fungi with Increased LaeA Expression, or Both
The fungi provided herein, namely Alg3Δ fungi, up-regulated LaeA fungi, and fungi with both Alg3Δ and up-regulated LaeA, can be used to produce citric acid, as well as derivatives thereof such as hydroxycitric acid (for example for medical applications). Such fungi can be from any species, such as Aspergillus or Rhizopus cells. For example, the disclosure provides methods of making citric acid, which can include culturing Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, under conditions that permit the fungus to make citric acid, for example in CAP medium.
Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) combines a pleasant taste with low toxicity and palatability and is a ubiquitous food additive. It is also able to complex heavy metal ions, like iron and copper, and is therefore applied in the stabilization of oils and fats or ascorbinic acid during metal ion-catalyzed oxidation reactions. Consequently, it is today one of the bulk products produced by fermentation, most of which occurs with the fungus Aspergillus niger, although a small portion is also produced by fermentation with yeast, such as Candida oleophila and Candida lipolytica. 
Citric acid production generally requires a unique combination of several unusual nutrient conditions (e.g., excessive concentrations of carbon source, H+, and dissolved oxygen, or suboptimal concentrations of certain trace metals and phosphate), which synergistically influence the yield of citric acid. Table 1 below shows the environmental parameters that influence citric acid accumulation.







TABLE 1







Parameters that influence citric acid accumulation by A. niger








Parameter
Requirement for citric acid accumulation





Carbon source concentration
Higher than 50 g/l


Carbon source type
Enable rapid catabolism


Nitrogen source
Consumption leads to some decrease in



pH


Phosphate concentration
Suboptimal


Aeration
In excess


Trace metal ions
Limiting, especially Mn2+


pH
Below pH 3









Methods of making citric acid, which can include culturing Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, under conditions that permit the fungus to make citric acid, are provided. In general, the culture media and/or culture conditions can be such that the fungi grow to an adequate density and produce citric acid efficiently. In one example the Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, are cultured or grown in a liquid medium that includes sucrose and/or glucose as the carbon source, for example at a concentration of at least 50 g/liter, such as at least 100 g/l, or at least 140 g/l. Thus, a fungus within the scope of the disclosure in some examples can utilize a variety of carbon sources. In one example the Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, are cultured or grown in a liquid medium that includes a very small amount of manganese, such as less than 100 parts per billion (ppb), less than 50 ppb, less than 20 ppb, less than 15 ppb, for example 5 ppb to 15 ppb or 10 ppb to 15 ppb, such as 5, 10, 13, 15 or 20 ppb. In one example the Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, are cultured or grown in a liquid medium having an initial pH of less than 3, such as less than 2.5, for example about pH 1.8 to 3, 1.8 to 2.5, 1.8 to 2.2, 1.9 to 2.1, for example pH 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9. In some examples the Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, are cultured or grown in a liquid medium at about 25 to 35° C. (such as 28 to 32° C., or 30° C.) with rotation of 180 to 300 rpm.
In a specific example, the Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, are grown in citric acid production (CAP) medium. In a specific example, the CAP medium includes 140 g of glucose/liter, 3.1 g of NH4NO3/liter, 0.15 g of KH2PO4/liter, 0.15 g of NaCl/liter, 2.2 g of MgSO4 7H2O/liter, 6.6 mg of ZnSO4 7H2O/liter, and 0.1 mg of FeCl3/liter adjusted to about pH 2 with 4 M H2SO4. Cations can be removed from the glucose solution by ion exchange on Dowex 50W-X8, 100/200-mesh, H cation exchange resin (Fisher Scientific, Pittsburgh, Pa.) prior to adding the other nutrient components. The manganese concentration in the medium can be adjusted by the addition of appropriate volumes of a stock solution of MnCl2 4H2O (10 mM). In one example, the manganese concentration is less than 50 ppb, such as less than 20 ppb, for example 5 to 15 ppb, such as 10 ppb.
Methods of culturing Aspergillus to enable citric acid production are well known in the art. In one example, the fungi are grown in culture containers (such as baffled flasks, and in some examples are silanized (5% solution of dichlorodimethylsilane in heptane (Sigma, St. Louis, Mo.)). The Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, provided herein can be grown in CAP media containing low amounts of Mn2+(e.g., 10 ppb) at 30° C. with rotation (e.g., 200 to 250 rpm) for at least 3 days (e.g., 3 to 7 days). Each culture container is inoculated with spores (such as at least 106 spores/ml) and incubated for at least 12 or at least 15 hours at 30° C. and 200 to 250 rpm to obtain properly pelleted morphology.
In one example, the Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, produce more citric acid than a corresponding fungus with wild-type Alga. In specific examples, the Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA, produce at least 25 g/1 of citric acid (for example at least 30 g/l, at least 32 g/l, at least 35 g/l, at least 40 g/l, at least 42 g/l, at least 45 g/l, at least 50 g/l, at least 52 g/1 or at least 55 g/l), for example after at least 4 days (such as at least 5 days, at least 6 days, at least 7 days, at least 8 days, or at least 10 days, such as after 4 to 6 days, 8 to 10 days, or 4 to 5 days) when grown in CAP medium at 30° C. with 200 rpm shaking.
In some examples, the method further includes isolating the citric acid made by the Alg3Δ fungi, up-regulated LaeA fungi, or fungi with both Alg3Δ and up-regulated LaeA. Once produced, any method can be used to isolate the citric acid. For example, common separation techniques can be used to remove the fungal biomass from the culture medium, and common isolation procedures (e.g., filtration, distillation, precipitation, electrodialysis, and ion-exchange procedures) can be used to obtain the citric acid from the broth (such as a fungi-free broth). In addition, the citric acid can be isolated from the culture medium after the citric acid production phase has been terminated.
Example 1
Materials and Methods
This example describes methods used in the experiments described in Examples 2-5 below.
Strains and Media.
The Escherichia coli strains Top10 and Saccharomyces cerevisiae strain YVH10 were used as hosts for routine cloning and gap repair experiments. A. niger strain ATCC 11414 (American Type Culture Collection, Rockville, Md.), was grown on potato dextrose agar plates (PDA) and complete medium (CM) agar plates at 30° C. for culture maintenance and spore preparation, respectively. The mutant strain Aspergillus niger 11414KusA was generated by the deletion of kusA in A. niger strain 11414 by the replacement with A. fumigatus pyrG gene, which encodes the ortholog of the ku70 protein that involves in the non-homologous end joining pathway of DNA repair for the integration of a DNA fragment into the genome in other eukaryotes, and was confirmed by Southern blotting analysis. The 11414kusA strain with high rate of homologous replacement was mainly used as a parent strain. The cultures on PDA or complete medium (CM) agar plates were incubated for four days at 30° C. and the spores were harvested by washing with sterile 0.8% Tween 80 (polyoxyethylenesorbitan monooleate). The CM medium contains 20 g of D-glucose/liter, 5 g yeast extract/liter, 2 g trypticase peptone/liter, 1 g casamino acids/liter, 6 g NaNO3/liter, 0.52 g KCl/liter, 0.52 g MgSO4.7H2O/liter, 1.52 g KH2PO4/liter, 36.7 mg ZnSO4.7H2O/liter, 18.3 mg H3BO3/liter, 8.3 mg MnCl2.4H2O/liter, 8.3 mg FeSO47H2O, 2.8 mg CoCl2.6H2O/liter, 2.7 mg CuSO4.5H2O/liter, 2.5 mg Na2MoO4.2H2O/liter, 83.3 mg Na2EDTA/liter, 1 mg biotin/liter, 1 mg pyridoxin/liter, 1 mg thiamine/liter, 1 mg riboflavin/liter, 1 mg p-aminobenzoic acid/liter and 1 mg nicotinic acid/liter. The PDA medium contains 4 g/liter potato starch and 20 g/liter dextrose. Conidia were enumerated with a hemacytometer. Aliquots of the resulting spore suspension (1×109 spores/ml) were used to inoculate baffled-flask liquid cultures. The citric acid production (CAP) medium contained 140 g/l of glucose, 3.1 g/l NH4NO3, 0.15 g/l KH2PO4, 0.15 g/l NaCl, 2.2 g/l MgSO4.7H2O, 6.6 mg/l ZnSO4.7H2O, and 0.1 mg/l FeCl3 adjusted to pH 2.1 with 4 M H2SO4. Cations were removed from the glucose solution by ion-exchange on Dowex 50W-X8, 100-200 mesh, H cation exchange resin (Fisher Scientific, Pittsburgh, Pa.) prior to adding the other nutrient components.
Culture Methods.
Glass baffled-flasks of 250 ml or 1000 ml were silanized by rinsing in a 5% solution of dichlorodimethylsilane in heptane (Sigma, St. Louis, Mo.) to minimize leaching of metals. For citric acid production tests, 1×106 spores/ml of parent or mutant strains were grown in 80 ml CAP media containing 10 ppb Mn2+ in 250 ml baffled flasks or 220 ml CAP media in 1000 ml baffled flasks at 30° C. and 200 rpm. Samples for citric acid analysis were taken at intervals. The biomass of transgenic clones and parent stain were prepared from 2 ml CM station cultures with proper antibiotics and grown in 16×125 mm glass culture-tubes at 30° C. without shaking. The biomass formed on the surface of the culture medium was collected, frozen immediately in liquid nitrogen and dried in the lyophilizer.
Dried Biomass Measurement.
After proper cultivation, the cell mass from citric acid production culture was collected by centrifugation at room temperature and 4500×g for 5 min in Sorvall floor centrifuge with swinging-bucket rotor. The cell mass was then transferred onto the Whatman Grade No 1 filter paper or left in centrifuge tubes for freeze-drying. The biomass was then dried in high temperature oven at 80° C. or freeze-dried in the lyophilizer. Prior to being used, the centrifuge tube or Whatman filter paper was weighted and re-weighted after the biomass was completely dried.
Total Genomic DNA Isolation for PCR and Southern Blotting Analysis.
Total genomic DNA was isolated from A. niger according to the SDS extraction method described previously by Dellaporta et al. (Plant Molecular Biology Reporter 1(4):19-21, 1983) with some modifications. Briefly, fungal biomass from 2 ml station cultures was looped and transferred into a 1.5 ml microcentrifuge tube. A needle size hole on the cap was punched with 18 gauge needle. The tube was immediately frozen in liquid N2 for 5 minutes and biomass in the tube was dried in a VirTis benchtop manifold freeze dryer (SP Scientific, Gardiner, N.Y.) overnight. The dried biomass and two 3.5 mm diameter glass beads were transferred into the 2 ml polypropylene microvial, where biomass was pulverized into fine power with Mini-Beadbeater-8 (Bio Spec Products Inc., Bartlesville, Okla.) for one minute. Then, 500 μl of 60° C. extraction buffer and 80 μl of 15% SDS were added into the microcentrifuge tube and incubated at 65° C. for at least 30 minutes with occasionally swirling to mix. Two-hundred microliters of 5M potassium acetate was added, mixed and incubated on ice for 30 minutes. The supernatant was collected by centrifugation at 12,000 g for 10 minutes at 4° C. and transferred into the new microcentrifuge tube. The total nucleic acids were precipitated with 780 μl of 2-propanol for 30 minutes at −20° C. and centrifuged at 12,000 g for 10 minutes. The nucleic acids were re-suspended in 200 μl 50TE buffer containing 2 RNase (10 μg/μl stock solution) and incubated in Eppendorf thermomixer at 50° C. and 500 rpm for 30 minutes. The proteins and cell debris was removed by being added and well mixed 20 μl 3M sodium acetate and equal volume of phenol:chloroform and centrifuged at 15,000 g for 5 minutes. The supernatant was transferred to new DNase-free microcentrifuge tube containing 220 μl of 2-propanol, mixed well and incubated at room temperature for 5 minutes. The genomic DNA was pelleted by centrifugation at 15,000 g for 10 minutes and washed with 500 μl of 70% ethanol. The genomic DNA was re-suspended in 80 μl 10 mM TrisHCl (pH8.0) buffer and determined with Qubit fluorometer (Invitrogen, Carisbad, Calif.). One microgram of total genomic DNA was digested with restriction endonuclease BamH and SacII. The genomic DNA fragments were separated in 1% agarose gel electrophoretically and transferred onto the zeta-probe membrane (BioRad) with alkaline capillary transfer method. A 3.8 kb genomic DNA fragment containing the Alg3 sequence was used for preparation of the biotin-labeled probe. The genomic DNA in Zeta-probe membrane was hybridized with the biotin-labeled probe overnight in 60° C. hybridization oven. The genomic DNA on hybridized membrane was visualized with North2South chemiluminescent detection kit (Pierce Protein Research Products, Rockford, Ill.).
Spore Production and Germination.
The spore production on the PDA or CM agar plates described above was excised with plastic closures of culture tubes in 27 mm diameter and transferred into the 50 ml centrifuge tubes containing 25 ml 0.8% tween 80. The spores were released from the agar surface by scraping with plastic loops and vortexed with vortex mixer at top speed. The spores were diluted properly and enumerated with a hemacytometer. The spore production in a unit area (cm2) was determined. For spore germination, 1×105 spores per well were added into each well of 24 well Schwarz sensoplate and incubated in the microscopic incubator with temperature control at 30° C. The spore germination was automatically imaged hourly for 24 hours through the Olympus inverted system microscope (Olympus America Inc., Center Valley, Pa., USA). The spore germination was visualized with Adobe Photoshop CSS (San Jose, Calif.) and counted manually.
Citric Acid Measurements.
Citric acid concentrations were determined with an end-point spectrophotometric enzyme assay as described in the instruction from the manufacturer (R-Biopharm AG/Roche, Darmstadt, Germany) with a proper dilution.
Table 2 shows oligonucleotides used in the methods.







TABLE 2







Oligonucleotides











Product




size


Name
Sequence
(kb)










Alg3Δ construction









Alg3-ForScr
CGGTTTCCCTTCAGTTTCCAGT (SEQ ID NO: 5)



 


Alg3-1
GTAACGCCAGGGTTTTCCCAGTCACGACGTCATAACTTCTCTCCCCTCC
1.06



(SEQ ID NO: 6)



 


Alg3-2
ATCCACTTAACGTTACTGAAATCTCCAACTTCATGGACACACACAGACC




(SEQ ID NO: 7)



 


Hph-F
GGTCTGTGTGTGTCCATGAAGTTGGAGATTTCAGTAACGTTAAGTGGAT
1.49



(SEQ ID NO: 8)



 


Hph-R
GCTACTACTGATCCCTCTGCGTCGGAGACAGAAGATGATATTGAAGGAG
1.03



(SEQ ID NO: 9)



 


Alg3-3
CTCCTTCAATATCATCTTCTGTCTCCGACGCAGAGGGATCAGTAGTAGC




(SEQ ID NO: 10)



 


Alg3-4
GCGGATAACAATTTCACACAGGAAACAGCCGTGAGAGGTTTGTAGTACG




(SEQ ID NO: 11)



 


Alg3-RevScr
AAGCTGAGAGCGACATCTTCA (SEQ ID NO: 12)



 


hyg-RevScr
GTACTTCTACACAGCCATCGGTCCA (SEQ ID NO: 13)



 


hyg-ForScr
GTACTTCTACACAGCCATCGGTCCA (SEQ ID NO: 14)



 







Alg3Δ + Alg3 construction









pryGScr
TCTGCTGTCTTGCATGAGGTCCTT (SEQ ID NO: 15)



 


pyrGScr
Agcgtaggacaaggtcgtctctgt (SEQ ID NO: 16)
2.34


 


5-pyrG5F
GTAACGCCAGGGTTTTCCCAGTCACGACGtttaaacATGCATCATTCTC




CCGCTTTGT (SEQ ID NO: 17)
1.69


 


5-pyrG3R
agaaagagtcaccggtcacGacatcgccaatcacctcaatcac
1.47



(SEQ ID NO: 18)
1.47


 


ble5F
gtgattgaggtgattggcgatgtCgtgaccggtgactctttct
1.23



(SEQ ID NO: 19)



 


Ble3R
TCCAACCTTGTAGCAACCAAAGCTTCGAGCGTCCCAAAACCT 




(SEQ ID NO: 20)



 


Alg3-5F1
AGGTTTTGGGACGCTCGAAGCTTTGGTTGCTACAAGGTTGGA




(SEQ ID NO: 21)



 


Alg3-3R1
TCAAGTAGAGCACAGCAAATAGTATCTGA (SEQ ID NO: 22)



 


Alg3-5F2
TCAGATACTATTTGCTGTGCTCTACTTGA (SEQ ID NO: 23)



 


Alg3-3R2
ttgatccttgtgccacaccaTCCTACGTGGTCATCGATACCA 




(SEQ ID NO: 24)



 


3-pyrG5F
TGGTATCGATGACCACGTAGGAtggtgtggcacaaggatcaa 




(SEQ ID NO: 25)



 


3-pyrG3R
GCGGATAACAATTTCACACAGGAAACAGCgtttaaactgtgccagtcaa




ttgtccgaagt (SEQ ID NO: 26)



 


Alg3Seq-1
TACAGACGCGTGTACGCATGT (SEQ ID NO: 27)



 


Alg3seq-2
TGCTATTGTCCACAGATACCGAGA (SEQ ID NO: 28)



 


Alg3seq-3
GAGCTAACCAGACAGTTCATGT (SEQ ID NO: 29)



 


Alg3seq-4
Tcgtcgtaccgcattgatcct(SEQ ID NO: 30)









Example 2
Genetic Inactivation of Alg3 in A. niger 
This example describes methods used to genetically clone and then inactivate Alg3 in A. niger strain 11414KusA. Based on these teachings, one skilled in the art will appreciate that Alg3 can be similarly inactivated in other strains of Aspergillus. 
Alg3 has been identified and characterized in Arabidopsis thaliana, Homo sapiens, Pichia pastoris, Trypanosoma brucei and Saccharomyces cerevisiae (see for example Korner et al., EMBO J. 18(23): 6816-6822, 1999; Davidson et al., Glycobiology 14(5):399-407, 2004; Manthri et al., Glycobiology 18(5): 367-383, 2008; Kajiura et al., Glycobiology 20(6):736-751, 2010). A database search based on the amino acid sequence of S. cerevisiae Alg3 identified a putative α-1,3-mannosyltransferase gene in JGI (DOE Joint Genome Institute)-A. niger genome database (jgilAspni5|42720). The A. niger Alg3 gene contains two introns and its 1400 bp open reading frame (nt 1186-1306, 1393-1916 and 1989-2582 of SEQ ID NO: 1) encodes a protein consisting of 413 amino acids (SEQ ID NO: 2), which contains one potential N-glycosite at the amino acid position 374. The predicted Alg3 amino acid sequence has 39% sequence identity to the S. cerevisiae Alg3.
Alg3 was functionally inactivated in A. niger using a gene deletion vector constructed by yeast gap repairing approach. The 5′- and 3′-end of the hygromycin marker (hph) gene was flanked with about 1 kb upstream and downstream fragments of Alg3 coding region that were isolated by PCR from A. niger genomic DNA. The DNA sequence of the upstream and downstream fragments was confirmed by DNA sequencing analysis. The Alg3 in A. niger was deleted by homologous replacement with hygromycin marker (hph) gene in the kusA deletion background of A. niger, where the kusA gene, encoding the ortholog of the Ku70 protein in other eukaryotes, was deleted for dramatically improved homologous integration efficiency. FIGS. 1A and 1B show the predicted restriction enzyme digestion patterns of genomic DNA of the parent and mutant strains with BamHI and SacII. FIG. 1C shows the Southern blotting analysis of the digested genomic DNA of parent and mutant strains. The results confirm that the Alg3 coding region in A. niger was replaced by the hygromycin selection marker gene (hph) in the Alg3Δ strains.
Example 3
Effects of Alg3 Deletion on A. niger Growth and Development
This example describes methods used to determine the effect genetically inactivating Alg3 in A. niger. 
It was previously demonstrated that the deletion of Alg3 in different organisms causes underglycosylation, but no obvious phenotype changes were observed at the selected culture condition in those studies (Aebi et al., Glycobiology 6(4):439-444, 1996; Korner et al., EMBO J. 18(23): 6816-6822, 1999; Davidson et al., Glycobiology 14(5):399-407, 2004; Manthri et al., Glycobiology 18(5): 367-383, 2008; Kajiura et al., Glycobiology 20(6):736-751, 2010).
The effects of the Alg3 deletion were examined on CM, PDA and MM plates. As exhibited in FIGS. 2A-I, the Alg3Δ strain grew much slower than the parent strain when grown on either CM or PDA medium plate, but there was no significant difference between the Alg3Δ mutants and parent strain when grown on the MM medium plate. When both Alg3Δ strain and parent strain were grown in the liquid culture of CM and PDA, the initiation of spore germination of Alg3Δ strain was pronouncedly delayed (FIG. 3A), but the spore germination rate was not affected by the Alg3 deletion (FIG. 3B). These results demonstrate that deletion of Alg3 has significant effects on A. niger growth on nutrient rich media.
The effects of Alg3 deletion on spore production on both CM and PDA plates were also examined. The Alg3 deletion had a substantial reduction of sporulation on CM medium plate, while no obvious difference was exhibited on PDA plate (FIG. 4). The spore production at given area was enumerated with hemocytometer (Table 3). The average spore production of Alg3Δ was 2.64×107 spores/cm2, about 40% of parent strain (6.44×107 spores/cm2) on CM medium plates, while average spore production per a square millimeter was similar between Alg3Δ mutant (7.72×107 spores/cm2) and parent strains (7.89×107 spores/cm2) on PDA medium plates.







TABLE 3







Average spore production in PDA and CM media plates (×107 sp/cm{circumflex over ( )}2).


This was averaged from four cuts and 3 replicate counting.











Strain
CM pates
PDA







11414-kusA
6.44 ± 1.24
7.72 ± 1.78



Alg3Δ
2.64 ± 0.49
7.89 ± 1.18










Example 4
Effects of Alg3 Deletion on Spore Germination, Growth and Citric Acid Production
This example describes methods used to measure spore germination, growth, and citric acid production in the Alg3 Δ A. niger strain generated in Example 1. Based on these teachings, one skilled in the art will appreciate that spore germination, growth, and citric acid production can be similarly measured in other Alg3Δ strains of Aspergillus. 
A. niger strain ATCC11414 is a strain developed for industrial production of citric acid. A. niger morphology plays a role in citric acid production. The fungal morphology affect overall molecular regulation in response to the endogenous and exogenous factors, which include the regulations of transcription, post-transcription, translation and post-translation. Therefore, the effects of Alg3 deletion on A. niger growth on CAP agar plates at different pHs or in CAP liquid culture conditions was determined.
FIG. 5 shows the effects of Alg3 deletion in fungi grown on CAP medium plates at different pH conditions after 28 hours in culture. When the Alg3Δ strain was grown on the CAP medium plates at pH 1.8, its growth was relatively slower than the parent strain, where its colonies were much smaller than the parent strain. At pH 2.1, the Alg3Δ strain formed a less tight pellet than that of parent strain. Growth of the Alg3Δ strain on the CAP medium at pH 4.5 and pH 7.0 was affected more profoundly than that of parent strain, where the Alg3Δ strain formed thinner layer of biomass on the agar plates than that of parent strain. These results show that the Alg3 deletion reduces the normal growth of A. niger at different levels on CAP culture medium plates at different pHs.
The spore germination of the Alg3Δ and parent strains in CAP liquid culture medium was also examined by using automated microscopic imaging, enumerating the germination manually in a same visual unit area, and expressing spore germination as a percentage of total spores at the same visual unit area. FIG. 6 shows the different dynamics of spore germination between the Alg3Δ and parent strains. The spore germination of Alg3Δ strain began as early as 3 hours after spore inoculation, while the parent strain was not initiated until 6 hours after spore inoculation. After 8 hours inoculation, more than 32% spores of Alg3Δ mutant germinated, while only 10% of parent strain spores did (FIG. 6A, top panels; and FIG. 6B). After 15 hours growth in CAP liquid medium, more than 90% spores of Alg3Δ strain germinated, while only 50% spores of parent strain did (FIG. 6A, bottom panels; and FIG. 6B). After 24 hours of growth in the liquid culture, about 75% spores of parent strain germinated, while the Alg3Δ strain achieved 94% of germination rate (FIG. 6B). The Alg3 deletion leads to earlier germination and a higher germination rate than parent strain.
The effect of Alg3 deletion on citric acid production was determined in CAP flask cultures. FIG. 7 shows the time course of citric acid production in CAP liquid medium. The yield of citric acid production was similar between the Alg3Δ mutant and parent strain in 2 days culture. After 3 days culture, the average citric acid production by Alg3Δ mutants was 8.8 g/1 citric acid, while the parent strain only produced 5.8 g/l. After 4.5 days of culture, the parent strain accumulated 18.8 g/1 citric acid and the Alg3Δ mutant produced 33.3 g/1 citric acid (more than 70% higher than the parent strain). Thus, the Alg3 deletion substantially improves the citric acid production in A. niger. 
The effect of Alg3 deletion on citric acid production was also examined by complementation of its original gene into the alg3Δ mutant. FIG. 8B shows the citric acid production in CAP liquid medium after 10 days of culture. The yield of citric acid production was similar between the alg3Δ complemented (cAlg3Δ) mutant and parent strain, but much lower than the alg3Δ mutant in 10 days culture. After 10 days culture, the average citric acid production by Alg3Δ mutants was 46.1 g/1 citric acid, while the parent and calg3Δ strain only produced 34.8 and 29.4 g/l, respectively.
Example 5
pPTRpGPDALaeA Plasmid Vector Construction
This example describes methods used to generate the pPTRpGPDALaeA plasmid vector (FIG. 13). One skilled in the art will appreciate that although gpdA and LaeA sequences were used from Aspergillus nidulans, one skilled in the art will appreciate that variants of these sequences can be used in the fungi and methods provided herein, such as gpdA and LaeA sequences from other Aspergillus species. In one example, a gpdA sequence having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 37 is used in the fungi and methods provided herein.
The Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase (gpdA) promoter (SEQ ID NO: 37) was isolated from the pAN8-1 plasmid DNA using the primer set gpdA5F/gpdA3R (gpdA5F: CGCAGATCTC AAGCTGTAAG GATTTCGGCA SEQ ID NO: 38; gpdA3R: CACCGGGCCC ATCTCAAACA TTGTGATGTC TGCTCAAGCG SEQ ID NO: 39) and the LaeA coding sequence of genomic DNA from A. nidulans (SEQ ID NO: 40) obtained by PCR using LaeA5F/LaeA3R (LaeA5F: CGCTTGAGCA GACATCACAA TGTTTGAGAT GGGCCCGGTG; SEQ ID NO: 42; LaeA3R: CGCAGATCTG AGGATTATGA GAAGGGAGC; SEQ ID NO: 43).
The DNA fragment of pGPDA and LaeA was filled together by overlap PCR and a HindIII restriction enzyme site was introduced at both 5′- and 3′-end of the DNA fragment. The DNA fragment (SEQ ID NO: 44) and pPTR1 plasmid DNA were cut with Hind III and ligated together by a quick DNA ligation kit at 25° C. for 30 min. The ligated plasmid DNA was transferred into the Top10 E. coli competent cells by lithium acetate mediated transformation. The transformed bacterial colonies were screened for the DNA fragment insertion by PCR with the primers gpdA5F (SEQ ID NO: 38) and LaeA3R (SEQ ID NO: 43). The plasmid DNA for the selected transformed colonies was prepared for restriction enzyme confirmation and further expression vector construction.
Example 6
pRS426-LaeA Vector Construction
This example describes methods used to generate a transgene containing A. niger LaeA (FIG. 14).
PCR was performed to isolate DNA fragments of A. niger pyrG upstream region (SEQ ID NO: 45), trpC transcriptional terminator of A. nidulans (SEQ ID NO: 46), pyrithiamine resistance gene (ptrA) of Aspergillus oryzae (SEQ ID NO: 47), and A. niger pyrG downstream region (SEQ ID NO: 48), using primers pyrGU5F/PTRU3R (pyrGU5F: GTAACGCCAG GGTTTTCCCA GTCACGACGT TTAAACATGC ATCATTCTCC CGCTTTGT, SEQ ID NO: 49; pyrGU3R: TGCCGAAATC CTTACAGCTT GAAGCTTCAT CGCCAATCAC CTCAATCAC, SEQ ID NO: 50), Trp5F/Trp3R (Trp5F: AGCTCCCTTC TCATAATCCT CAAGCTTGGA CCGATGGCTG TGTAGAAGT, SEQ ID NO: 51; Trp3R: CGTAATCAAT TGCCCGTCTG TCAGAGAGCG GATTCCTCAG TCTCGT; SEQ ID NO: 52), PTR5F/PTR3R (PTR5F: ACGAGACTGA GGAATCCGCT CTCTGACAGA CGGGCAATTG ATTACG, SEQ ID NO: 53; PTR3R: ACAGCAGTGC TTATCTGCGA TGACGAGCCG CTCTTGCATC TTTGT, SEQ ID NO: 54) and PyrGD5F/PTRD3R (pyrGD5F: ACAAAGATGC AAGAGCGGCT CGTCATCGCA GATAAGCACT GCTGT; SEQ ID NO: 55, pyrGD3R: TGAGACGCTG TTTCACCGAG TACATCGCCA ATCACCTCAA TCAC, SEQ ID NO: 56), respectively.
As shown in FIG. 14, the DNA fragment of pGDPALaeA (SEQ ID NO: 44) was isolated from pPTRpGDPALaeA (FIG. 13) by HindIII digestion. The yeast gap repairing vector pRS426 was double digested with restriction enzyme HindIII and XhoI. Hundred nanograms of each DNA fragment generated from PCR (i.e., SEQ ID NOs: 45-56) or restriction enzyme digestions were used for S. cerevisiae transformation. The gap repairing plasmid DNA in the total S. cerevisiae genomic DNA was isolated by transferred into the Top10 E. coli cells.
The transformed plasmid DNA was confirmed by PCR and digested with PmeI. The PmeI DNA fragment (SEQ ID NO: 57) was used for A. niger transformation.
One skilled in the art will appreciate that although the pyrG upstream and downstream sequences, trpC transcriptional terminator sequence, and ptrA sequence used were from particular organisms, one skilled in the art will appreciate that variants of these sequences can be used in the fungi and methods provided herein, such as those from other Aspergillus species. In one example, pyrG upstream and downstream sequences having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 45 and 48 are used in the fungi and methods provided herein. In one example, a trpC transcriptional terminator sequence having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 46 is used in the fungi and methods provided herein. In one example, a ptrA sequence having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 47 is used in the fungi and methods provided herein.
Example 7
Expression of pGDPALaeA-Containing Transgene in A. niger Alg3Δ
This example describes methods used to introduce pGPDALaeA (SEQ ID NO: 57) into A. niger. 
The originally transformed A. niger colonies were picked from the minimal medium plates with 0.1 μg/ml pyrithiamine hydrobromide selection on minimal medium agar plates without thiamine supplementation. The single spore colonies were picked for spore production after the initial transformant spores were grown on the same selection medium plates. The biomass was harvested from the single spore colony isolates grown in minimal medium with pyrithiamine selection and dried in the VirTis bench top freeze dryer. The genomic DNA was prepared for PCR confirmation of pGDPALaeA insertion in transgenic A. niger. As shown in FIG. 15A, the primer set of PTR5F (SEQ ID NO: 53) and PTR3R (SEQ ID NO: 54) was used to confirm the presence of A. oryzae pyrithiamine resistance gene (ptrA) in transgenic A. niger with the expected size of 2 kb PCR DNA fragment. As shown in FIG. 15B, the primer set LaeA5F (SEQ ID NO: 42) and TRP3R (SEQ ID NO: 52) was used to demonstrate that the transgene A. nidulans LaeA was under the control of gdpA promoter and trpC transcriptional terminator of A. nidulans with the expected 3.4 kb PCR fragment size. The genomic DNA of parent strain and the plasmid DNA of transgene vector carrying the pGDPALaeA fragment were used for negative and positive references.
Example 8
Increased Production of Citric Acid
This example describes methods used to demonstrate that citric acid production was increased in the presence of increased expression of LaeA, alone or in combination with deletion of Alg3.
Citric acid was produced as described in Example 1.
As shown in FIG. 16, of citric acid production was increased in the Alg3Δ mutant (Alg3), and in the mutants over-expressing LaeA in Alg3Δ (LaeA-1 and LaeA-2), as compared to the parent strain (kusA).
Example 9
pBSK-LaeA a Plasmid Vector Construction
This example describes methods used to generate the pBSK-LaeAΔ plasmid vector (FIG. 17A). The 5′ (SEQ ID NO: 61) and 3′ (SEQ ID NO: 62) ends of the Aspergillus niger LaeA gene were isolated from A. niger genomic DNA by PCR using oligonucleotide sets D1 & D2, and D5 & D6 (see Table 4 below). The hph expression cassette was isolated from plasmid vector DNA of pCB1003 (SEQ ID NO: 63) by PCR using the oligonucleotide sets D3 & D4. The DNA fragments were assembled together into the backbone plasmid vector of pBSK linearized with restriction endonucleases of both HindIII and PstI using the Gibson assembly cloning kit. The assembled plasmid DNA was transferred into the Top10 E. coli competent cells by lithium acetate mediated transformation. The transformed bacterial colonies were screened for the DNA fragment insertion by restriction endonuclease digestion of PvuII and XhoI. The plasmid DNA for the transformed colonies was prepared and digested with endonucleases of HindIII and XbaI for further LaeA deletion in A. niger.







TABLE 4







Oligonucleotide Primers for A. niger LaeA Deletion









Name
Sequence
SEQ ID NO:





D1
gtcgacggtatcgataAGCTTCAAGACAGC
67



GGCTGCAA



 


D2
gccgaccgTGGTAGAGATACAGGGGTTC
68


 


D3
ctctaccaCGGTCGGCATCTACTCTATTC
69


 


D4
gcgactgaGCTGGAGCTAGTGGAGGT
70


 


D5
gctccagcTCAGTCGCATCTTTCACTG
71


 


D6
atcccccgggctgcaTGGTAGGCTGTCT
72



CAAGG



 


SC7
ACTCGCAGCAGAGATGCCATCT
73


 


SC8
CGTTATGTTTATCGGCACTTTGCAT
74









Example 10
pRS426-A. niger LaeA Complementation Plasmid Vector Construction
This example describes methods used to generate the pBSK-LaeAΔ plasmid vector (FIG. 17B). The entire LaeA gene (2.34 kb; SEQ ID NO: 64) containing 740 bp of promoter region and 330 bp of transcriptional terminator region was isolated from A. niger genomic DNA by PCR using oligonucleotide set CP9 & CP10 (see Table 5 below). The DNA fragment was ligated into the plasmid DNA of pRS426-A. nidulans LaeA over-expression vector at a HindIII restriction endonuclease site after the pGPDALaeA fragment in the vector was removed by agarose gel separation. The assembled plasmid DNA was transferred into the Top10 E. coli competent cells by lithium acetate mediated transformation. The transformed bacterial colonies were screened for the DNA fragment insertion by restriction endonuclease digestion of BamHI and XhoI. The plasmid DNA for the transformed colonies was prepared and digested with PmeI restriction endonuclease for A. niger LaeA expression at pyrG locus to complement the LaeA deletion in A. niger LaeAΔ mutants.







TABLE 5







Oligonucleotide Primers for A. niger LaeA 


Complementation









Name
Sequence
SEQ ID NO:





CP9
aggtgattggcgatgaTGCCGTTCAGC
75



TGTCTGC



 


CP10
acagccatcggtccaTCTCTCCTCGTA
76



ACGCCTG



 


SC11
ggatagaatcgggtgccgctgatct
77


 


SC12
gagaaccatggcaccgaaggt
78









Example 11
Deletion of A. niger LaeA Gene in A. niger 
This example describes methods used to introduce the HindIII/XbaI DNA fragment contain the LaeA deletion cassette (SEQ ID NO: 65) into A. niger. 
The originally transformed A. niger colonies were picked from the minimal medium plates with 100 μg/ml Hygromycin B selection on minimal medium agar plates. The single spore colonies were picked for spore production after the initial transformant spores were grown on the same selection medium plates. The biomass was harvested from the single spore colony isolates grown in complete medium with Hygromycin B selection and dried in the VirTis bench top freeze dryer. The genomic DNA was prepared for PCR confirmation of the replacement of LaeA gene coding region in transgenic A. niger. As shown in FIG. 18, the primer set SC7 and SC8 (see Table 4) was used to confirm the hph replacement of the LaeA gene in transgenic A. niger with the expected 2 kb PCR DNA fragment. The genomic DNA of parent strain was used for negative and positive references.
Example 12
Expression of pGDPALaeA-Containing Transgene in A. niger 
This example describes methods used to introduce the A. niger LaeA gene into the pyrG locus in the A. niger LaeAΔ mutant to complement the loss of LaeA function with PmeI DNA fragment (SEQ ID NO: 66).
The originally transformed A. niger colonies were picked from the minimal medium plates with 0.1 μg/ml pyrithiamine hydrobromide selection on minimal medium agar plates without thiamine supplementation. The single spore colonies were picked for spore production after the initial transformant spores were grown on the same selection medium plates. The biomass was harvested from the single spore colony isolates grown in minimal medium with pyrithiamine selection and dried in the VirTis bench top freeze dryer. The genomic DNA was prepared for PCR confirmation of LaeA gene insertion in transgenic A. niger. As shown in FIG. 19, the primer set of PTR5F (SEQ ID NO: 53) and PTR3R (SEQ ID NO: 54) was used to confirm the presence of A. oryzae pyrithiamine resistance gene (ptrA) in transgenic A. niger with the expected size of 2 kb PCR DNA fragment. The genomic DNA of the parent strain and the plasmid DNA of the transgene vector carrying the A. oryzae pyrithiamine resistance gene (ptrA) fragment were used for negative and positive references.
Example 13
The Effects of LaeA Gene Deletion and Complementation and Over-Expression of A. nidulans LaeA Gene on Citric Acid Production
This example describes methods used to demonstrate that citric acid production in A. niger mutant strains was significantly influenced by perturbing the LaeA gene expression in A. niger. At least three individual clones per mutant strain were selected for spore production on complete medium plates at 30° C. for 4 days. The spores were counted with a hemocytometer. Citric acid production culture was initiated by inoculation with a 1×106 spore/ml of 75 ml citric acid production culture medium in a 250 ml baffled Erlenmeyer glass flask siliconized with a 5% solution of dichlorodimethylsilane in hexane solvent. The culture was maintained at 30° C. and 220 rpm in shaker incubator for 5 days. One microliter of culture was harvested and briefly spun down in a microcentrifuge at full speed. The supernatants of cultures were diluted 200-fold with dH2O prior to citric acid measurement. The citric acid in the supernatant was quantified with the citric acid assay kit from R-Biopharm AG. The detailed procedure for the citric acid assay was performed essentially according to the manufacturer's description. The results in FIG. 20 show that deletion of the LaeA gene in A. niger (LaeAΔ) led to loss of citric acid production in citric acid production culture. When the original A. niger LaeA gene was used for complementation in the LaeAΔ mutant (cLaeAΔ) at the pyrG locus, the citric acid production was partially recovered, which indicates the importance of chromosome location of LaeA gene. When A. nidulans LaeA was over-expressed in the A. niger parent strain (LaeA), the citric acid production was higher than the parent strain. This indicates that the LaeA is involved in citric acid production in A. niger. 
Example 14
Enhancement of Itaconic Production by LaeA Complementation A. terreus Growth Conditions
All strains were maintained on potato dextrose agar (PDA), which is composed of 39 g/L of Difco Potato Dextrose Agar (BD, USA). Spore inocula were grown on PDA and harvested after 5 days. The transformant was selected for either hygromycin or bleomycin resistance on minimum media (MM, 50 ml 20× nitrate salts, 1 ml 1000× trace elements, 1 ml 1000× vitamin stock, 10 g glucose, pH 6.5, 100 μg/ml hygromycin or 250 μg/ml bleomycin). The 20×NO3 salts contains in one liter H2O: Na2NO3, 120 g; KCL, 10.4 g; MgSO4.7H2O, 10.4 g; KH2PO4, 30.4 g. The 1000× vitamin solution (yellow) contains in 100 ml H2O: biotin, 0.01 g; pyridoxine-HCl, 0.01 g; thiamine-HCl, 0.01 g; riboflavin, 0.01 g; para-amino benzoic acid, 0.01 g; and nicotinic acid (or niacin), 0.01 g, filtered and stored at 4° C. The 1000× trace elements contains per 100 ml H2O: ZnSO4.7H2O, 2.2 g; H3BO3, 1.1 g; MnCl2.4H2O, 0.5 g; FeSO4.7H2O, 0.5 g; CoCl2.6H2O, 0.17 g; CuSO4.5H2O, 0.16 g; Na2MoO4.2H2O, 0.15 g; Na2EDTA, 5 g, which is added in the order, boil and cool to room temperature and adjusted the pH to 6.5 with KOH. For pyrithiamine marker selection, the thiamine-HCl was eliminated from minimum media.
About 1×106 spores/ml were used to inoculate 50 ml of itaconic acid production media in 250 ml glass flask. The itaconic acid production medium essentially followed the description of Riscaldati et al. (J Biotechnol 83:219-230, 2000). Itaconic acid production medium contains in grams per liter (g/l): glucose, 100; ammonium sulfate, 2.36; potassium phosphate dibase, 0.11; magnesium sulfate heptahydrate, 2.08; calcium chloride dihydrate, 0.13; sodium chloride, 0.074; copper sulfate pentahydrate 2×10−4; ferrous sulfate heptahydrate, 5.5×10−3; manganese chloride tetrahydrate, 7×10−4; and zinc sulfate heptahydrate, 1.3×10−3. The flask cultures were shaken at 30° C. and 150 rpm. At least three biological replicates for each strain were used for each experiment.
Itaconic Acid Measurement
The itaconic acid method followed the previous description by Dickman (Analytical Chem 24: 1064-1066, 1952) with some modification. The reaction volume was scaled down to 1.4 ml for each reaction. Ten to 250 μl of culture supernatant or itaconic acid standard dissolved in dH2O were used. The proper amount of culture supernatant or itaconic acid standard was added into 690 to 930 μl dH2O in 1.5 ml microcentrifuge tube. Then, 60 μl of freshly prepared metaphosphoric acid (0.425 g/ml) was added into the tube, mixed well, and incubated at 4° C. for 10 minutes. Next, 400 μl of freshly prepared potassium permanganate (5 mM) from stock solution (100 mM) was added, mixed well and incubated in the dark at room temperature for 10 minutes. Finally, the absorbency of the reaction mixture was determined at 530 nm spectrophotometrically.
LaeA Deletion Construct
The DNA fragments corresponding to the upstream and downstream regions of the LaeA gene in A. terreus and hph were isolated with oligonucleotide pairs 1546/1548, 1551/1553 and 1549/1550, respectively (see Table 6 for oligonucleotide sequences). These three DNA fragments (SEQ ID NOs: 89-91) were fused together by double-joint PCR (Yu, et al., Fungal Genetics and Biology 41: 973-981, 2004). The whole LaeA deletion fragment was isolated by PCR with oligonucleotide pair 1547/1552 (see Table 6 and SEQ ID NO: 104). FIG. 21A depicts the LaeA deletion construct. FIG. 21B shows genomic PCR confirmation of selected LaeA deletion mutants with oligonucleotide pair 1546/107 or 108/1553.
LaeA Complementation Construct
The LaeA complementation construct was prepared for targeting upstream of the LaeA locus in A. terreus using the Gibson Assembly method. The DNA fragments of the whole LaeA gene, ble selection marker gene, and hph gene used for gene deletion were isolated by PCR with oligonucleotide pairs 1554/1555, 1556/15557, and 1558/1559, respectively (see Table 6). In order to isolate the whole transgene cassette, an XbaI restriction endonuclease site was introduced into the upstream fragment of 1554/1555. The three DNA fragments (SEQ ID NOs: 107-109) were then assembled into the cloning vector of pBluescript SK(−). FIG. 22A is a diagram illustrating the LaeA complementation constructs. The insertion of LaeA complementation was confirmed by oligonucleotide pair 1556/107. FIG. 22B shows gel electrophoresis of genomic PCR products corresponding to ble and part of hph.
LaeA Over-Expression Construct
The Aspergillus nidulans LaeA gene under the control of A. nidulans gpdA promoter and TrpC transcription terminator, and the selection marker gene of pyrithiamine resistance gene (ptrA) of Aspergillus oryzae, were isolated from the transgene expression construct used for A. niger transformation (as described in Example 6) with oligonucleotide pair 1563 and 1564 (Table 6). Due to the extremely high homologous replacement rate in A. terreus, the laeA overexpression transgene cassette was designed to replace the 127 bp genomic DNA fragment of the non-gene region located at 1571 bp upstream in A. terreus laeA coding gene sequence. The corresponding DNA fragments franked to the both ends of LaeA expression cassette for homologous replacement were isolated by PCR with oligonucleotide pairs 1561/1562 and 1565/1566 (Table 6). The three DNA fragments (SEQ ID NOs: 96-98) were fused together in pBluescript SK(−) by Gibson Assembly. The restriction endonuclease site for PmeI was introduced into the 5′ end of the upstream region fragment of 1561/1562 (SEQ ID NO: 96) and the 3′ end of the downstream region fragment of 1565/1566 (SEQ ID NO: 110), which enables the isolation of whole transgene expression construct. FIG. 24A is a diagram of the transgene over-expression cassette of the A. nidulans LaeA gene. Oligonucleotides 1563, 1198 and 1564 (see Table 6) were used for genomic PCR to confirm the gene insertion. FIG. 24B shows gel electrophoresis of PCR products corresponding to the oligonucleotide pairs 1198/1564 and 1563/1564 in selected transgenic strains.
A. terreus Protoplast Isolation and Transformation
A. terreus (106 conidia/ml) was inoculated into 100 ml of complete media containing in g/1: D-glucose, 10; peptone, 2; yeast extract, 1; casamino acids, 1; 20× nitrate salts, 50 ml; trace elements, 1 ml; vitamin solution, 1 ml (pH6.5) or MM without thiamine-HCl. The inoculated culture was shaken at 150 rpm and 30° C. for 16-22 hours. The mycelia were harvested by filtering the culture through Miracloth and rinsed well with sterile dH2O. The mycelia (mass of approximately several bean sizes) were digested with 20 ml of 20 mg/ml lysing enzyme (L1412, Sigma) that was dissolved in osmotic washing buffer (0.5 M KCl, 10 mM sodium phosphate, pH 5.8) for approximately 2-3 hours at 80 rpm and 30° C. Protoplasts were filtered through sterile miracloth into a 50 ml sterile centrifuge tube and centrifuged for 10 minutes at 1000×g and 4° C. Protoplasts were washed twice with 20 ml washing solution (0.6M KCl, 0.1 M Tris-HCl, pH 7.0) and once in 10 ml conditioning solution (0.6M KCl, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5).
For transformation, 1-2 μg DNA was added to 2×107 protoplasts in 0.1 ml conditioning solution. After addition of 25 μl PEG solution, the protoplasts were incubated on ice for 20 minutes. An additional 500 μl PEG solution was carefully added, mixed and incubated on ice for additional 5 minutes. Finally, 1 ml cold conditioning solution was added and mixed by inverting the tube several times. About 300-500 μl of transformed protoplasts were pipetted into 12 ml melted MM agar (MM+0.6M KCl+1.5% agar+antibiotics) tubes at 50° C. The transformed protoplasts were mixed well by inverting the tubes 3-4 times and poured directly onto the 100 mm petri dishes. The petri dishes were incubated overnight at 30° C. An additional 8 ml of regular MM agar with proper antibiotics was overlaid and further incubated at 30° C. for approximately 3-4 days. The transformed colonies were picked and further grown on 2 ml MM agar gel for spore production.







TABLE 6







Oligonucleotides









Oligo
DNA sequence
SEQ ID NO:





 107
GTACTTCTACACAGCCATCGGTCCA
79


 


 108
CGTTATGTTTATCGGCACTTTGCAT
80


 


1546
ACAGGTACTTCCATCTTGTACTGGT
81


 


1547
TCtcctccaacgtccgatct
82


 


1548
acctccactagctccagcaagccgaacaga
83



ggtaaagacga



 


1549
tcgtctttacctctgttcggcttgctggag
84



ctagtggaggtca



 


1550
taccaacgtgcgaccatttTCTcggtcggc
85



atctactctattcct



 


1551
aggaatagagtagatgccgaccgAGAaaat
86



ggtcgcacgttggta



 


1552
AAGCGTCTCTTTCCTGGGTCTT
87


 


1553
TGCCAGTTCTGTTGGACATCTCT
88


 


1554
tcgaggtcgacggtatcgatatctagaACA
89



GGTACTTCCATCTTGTAC



 


1555
ggtcactgtTCCTGGCAGCTGACATTG
90


 


1556
ctgccaggaACAGTGACCGGTGACTCT
91


 


1557
aagcagcagatACGACCGTTGATCTGCTTG
92


 


1558
acggtcgtATCTGCTGCTTGGTGCAC
93


 


1559
actagtggatcccccgggctgcagCGGTCG
94



GCATCTACTCTATTC



 


1561
cgaggtcgacggtatcgataGTTTAAACCT
95



CCCAGGTACCGACTAAC



 


1562
ctcaatcacaGATCATGTTTGGGTGGGTTC
96


 


1563
aaacatgatcTGTGATTGAGGTGATTGGCG
97


 


1564
ctctgtgcctACAGCAGTGCTTATCTGC
98



GATG



 


1565
gcactgctgtAGGCACAGAGTAACAGGTAG
99



GTAGACAG



 


1566
agtggatcccccgggctgcaGTTTAAACTC
100 



CGCACCACGAAAGCAACT










Results

Itaconic acid production in selected LaeA deletion (LaeAΔ) and LaeA complementation (cLaeAΔ) mutants of A. terreus was tested after four days of culture in itaconic acid production medium. The parental A. terreus strain was also tested as a control. As shown in FIG. 23, deletion of LaeA significantly decreased the production of itaconic acid. However, complementation of the LaeA gene in A. terreus enabled itaconic acid production to similar levels observed in the A. terreus parent.
An additional study was performed to test the effect of overexpression of the LaeA gene in A. terreus. FIG. 25 is a graph showing itaconic acid production in selected A. terreus transgenic strains having the A. nidulans LaeA over-expression transgene following five days of culture in itaconic acid production medium. Also shown in FIG. 25 is the percentage of itaconic acid production relative to the parent strain. The results demonstrated that overexpression of LaeA in A. terreus led to a significant increase in itaconic acid production relative to itaconic acid production in the parental strain.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

## Claims

1. An isolated Aspergillus terreus fungus transformed with a heterologous nucleic acid construct comprising an Aspergillus species LaeA (loss of aflR expression A) gene, wherein the LaeA gene encodes a LaeA protein comprising an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 59 and the heterologous nucleic acid construct is inserted upstream of the coding region of the endogenous LaeA gene, and wherein expression of the Aspergillus species LaeA gene is increased in the transformed fungus compared to an A. terreus fungus that is not transformed with the heterologous nucleic acid construct.

2. The isolated A. terreus fungus of claim 1, wherein the heterologous nucleic acid construct further comprises a heterologous promoter, a heterologous transcription terminator, a heterologous selective marker gene, or any combination thereof.

3. The isolated A. terreus fungus of claim 1, wherein the Aspergillus species LaeA gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 41.

4. The isolated A. terreus fungus of claim 1, wherein the Aspergillus species LaeA gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 59.

5. The isolated A. terreus fungus of claim 1, wherein the heterologous nucleic acid construct comprises a promoter operably linked to the Aspergillus species LaeA gene, a transcription terminator and a selective marker gene.

6. The isolated A. terreus fungus of claim 5, wherein:
the promoter comprises the A. nidulans gpdA promoter;
the Aspergillus species LaeA gene is an A. nidulans LaeA gene;
the transcription terminator comprises the A. nidulans TrpC transcription terminator; or
the selective marker gene comprises the A. oryzae pyrithiamine resistance (ptrA) gene.

7. The isolated A. terreus fungus of claim 6, wherein the heterologous nucleic acid construct comprises the nucleotide sequence of SEQ ID NO: 109.

8. A method of making itaconic acid, comprising culturing the isolated A. terreus fungus of claim 1 under conditions that permit the fungus to make itaconic acid, thereby making itaconic acid.

9. The method of claim 8, wherein the fungus is cultured in itaconic acid production media.

10. The method of claim 8, wherein the heterologous nucleic acid construct further comprises a heterologous promoter, a heterologous transcription terminator, a heterologous selective maker gene, or any combination thereof.

11. The method of claim 10, wherein the Aspergillus species LaeA gene in the heterologous nucleic acid construct is an A. nidulans or an A. niger LaeA gene.

12. The method of claim 9, wherein the Aspergillus species LaeA gene encodes a protein having an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 59.

13. The method of claim 12, wherein the Aspergillus species LaeA gene encodes a protein having the amino acid sequence of SEQ ID NO: 41 or the amino acid sequence of SEQ ID NO: 59.

14. The method of claim 8, wherein the heterologous nucleic acid construct comprises a promoter operably linked to the Aspergillus species LaeA gene, a transcription terminator and a selective marker gene.

15. The method of claim 14, wherein:
the promoter comprises the A. nidulans gpdA promoter;
the Aspergillus species LaeA gene is an A. nidulans or an A. niger LaeA gene;
the transcription terminator comprises the A. nidulans TrpC transcription terminator; or
the selective marker gene comprises the A. oryzae pyrithiamine resistance (ptrA) gene.

16. The method of claim 15, wherein the heterologous nucleic acid construct comprises the nucleotide sequence of SEQ ID NO: 109.