PRODUCTION OF ITACONIC ACID AND RELATED MOLECULES FROM AROMATIC COMPOUNDS
20190330665 ยท 2019-10-31
Inventors
- Joshua R. Elmore (Richland, WA, US)
- Jay Huenemann (Knoxville, TN, US)
- Davinia Salvachua (Golden, CO, US)
- Gregg T. Beckham (Golden, CO)
- Adam M. Guss (Knoxville, TN, US)
Cpc classification
C12N15/74
CHEMISTRY; METALLURGY
C12P7/46
CHEMISTRY; METALLURGY
International classification
C12N15/74
CHEMISTRY; METALLURGY
Abstract
This disclosure provides a genetically-modified bacterium from the genus Pseudomonas that produces itaconate or trans-aconitate. The disclosure further provides methods for producing itaconate or trans-aconitate using a genetically-modified bacterium from the genus Pseudomonas.
Claims
1. A genetically-modified bacterium from the genus Pseudomonas comprising an exogenous nucleic acid encoding an enzyme that uses cis-aconitate as a substrate.
2. The genetically-modified bacterium of claim 1, wherein the exogenous nucleic acid encodes a cis-aconitate decarboxylase.
3. The genetically-modified bacterium of claim 1, wherein the exogenous nucleic acid encodes an aconitate isomerase.
4. The genetically-modified bacterium of claim 3, wherein the bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate decarboxylase.
5. The genetically-modified bacterium of claim 3, wherein the bacterium does not have an exogenous nucleic acid encoding a trans-aconitate decarboxylase.
6. The genetically-modified bacterium of claim 1, wherein the bacterium comprises an exogenous nucleic acid encoding a cis-aconitate decarboxylase, an exogenous nucleic acid encoding an aconitate isomerase, and an exogenous nucleic acid encoding a trans-aconitate decarboxylase.
7. The genetically-modified bacterium of claim 1, wherein both the endogenous phaC1 gene and the endogenous phaC2 gene are inactivated in the bacterium.
8. The genetically-modified bacterium of claim 1, wherein the bacterium is grown on lignin or a breakdown product of lignin as a carbon source.
9. The genetically-modified bacterium of claim 1, wherein the breakdown product of lignin comprises p-coumaric acid, ferulic acid, or saccharides.
10. The genetically-modified bacterium of claim 1, wherein the bacterium is grown on an organic compound selected from the group consisting of an aromatic compound, a saccharide, an organic acid, and an alcohol.
11. The genetically-modified bacterium of claim 1, wherein the bacterium is grown on an organic compound selected from the group consisting glycerol, a diacid, a fatty acid, and benzoic acid.
12. The genetically-modified bacterium of claim 1, wherein the bacterium further comprises an exogenous nucleic acid encoding a citrate synthase.
13. The genetically-modified bacterium of claim 12, wherein the citrate synthase is a mutant enzyme that is immune to allosteric inhibition.
14. The genetically-modified bacterium of claim 1, wherein the level of isocitrate dehydrogenase in the bacterium is reduced compared to a non-genetically modified bacterium.
15. The genetically-modified bacterium of claim 14, wherein (i) the start codon of the isocitrate dehydrogenase gene is either GTG or TTG, (ii) the isocitrate dehydrogenase gene promoter comprises a mutation, (iii) the ribosome binding site of the isocitrate dehydrogenase gene transcript comprises a mutation, or (iv) the isocitrate dehydrogenase encoded by the isocitrate dehydrogenase gene comprises a protease recognition sequence.
16. The genetically-modified bacterium of claim 1, wherein the bacterium is selected from the group consisting of P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borborid, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. asplenii, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugate, P. fragi, P. lundensis, P. taetrolens, P. Antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. putida group, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthin, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophile, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. teessidea, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, P. taiwanensis.
17. The genetically-modified bacterium of claim 1, wherein the bacterium further comprises an exogenous nucleic acid encoding an itaconic acid efflux pump.
18. The genetically-modified bacterium of claim 5, wherein the bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate efflux pump.
19. A method for converting an organic compound to itaconic acid or trans-aconitate, the method comprising inoculating an aqueous solution containing said organic compound with a genetically-modified bacterium from the genus Pseudomonas, wherein the bacterium comprises an exogenous nucleic acid encoding an enzyme that uses cis-aconitate as a substrate.
20. The method of claim 19, wherein the organic compound is selected from aromatic compounds, saccharides, organic acids, and alcohols.
21. The method of claim 19, wherein the organic compound is a breakdown product of lignin produced during a lignin depolymerization process.
22. The method of claim 19, wherein the organic compound is selected from the group consisting of aromatic compounds, glycerol, diacids, fatty acids, and benzoic acid.
23. The method of claim 19, wherein the aqueous solution is a lignin depolymerization stream or derived from a lignin depolymerization stream.
24. The method of claim 19, wherein the lignin depolymerization stream contains p-coumaric acid, ferulic acid, and saccharides.
25. The method of claim 19, wherein the bacterium produces itaconate, and wherein the exogenous nucleic acid encodes a cis-aconitate decarboxylase.
26. The method of claim 19, wherein the exogenous nucleic acid encodes an aconitate isomerase.
27. The method of claim 26, wherein the bacterium produces itaconate, and wherein the bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate decarboxylase.
28. The method of claim 26, wherein the bacterium produces trans-itaconate, and wherein the bacterium does not have an exogenous nucleic acid encoding a trans-aconitate decarboxylase.
29. The method of claim 19, wherein both the endogenous phaC1 gene and the endogenous phaC2 gene are inactivated in the bacterium.
30. The method of claim 19, wherein the bacterium further comprises an exogenous nucleic acid encoding a citrate synthase.
31. The method of claim 30, wherein the citrate synthase is a mutant enzyme that is immune to allosteric inhibition.
32. The method of claim 19, wherein the level of isocitrate dehydrogenase in the bacterium is reduced compared to a non-genetically modified bacterium.
33. The genetically-modified bacterium of claim 32, wherein (i) the start codon of the isocitrate dehydrogenase gene is either GTG or TTG, (ii) the isocitrate dehydrogenase gene promoter comprises a mutation, (iii) the ribosome binding site of the isocitrate dehydrogenase gene transcript comprises a mutation, or (iv) the isocitrate dehydrogenase encoded by the isocitrate dehydrogenase gene comprises a protease recognition sequence.
34. The method of claim 19, wherein the bacterium is selected from the group consisting of P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borborid, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. asplenii, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugate, P. fragi, P. lundensis, P. taetrolens, P. Antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. putida group, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parajidva, P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthin, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophile, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. teessidea, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, P. taiwanensis.
35. The method of claim 19, wherein the bacterium further comprises an exogenous nucleic acid encoding an itaconic acid efflux pump.
36. The method of claim 28, wherein the bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate efflux pump.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0025] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
DETAILED DESCRIPTION OF THE DISCLOSURE
Definitions
[0034] As used herein, the term about refers to an approximately +/10% variation from a given value.
[0035] The term homolog means a gene related to a second gene by descent from a common ancestral DNA sequence, therefore, the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably at least 60%, even more preferably at least 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95, 97% or 99%). A homolog of a protein furthermore means that the function is equivalent to the function of the original protein.
[0036] The term cellulose (also lignocellulose or cellulosic substrate) refers to a structural material that comprises much of the mass of plants. Lignocellulose is composed mainly of carbohydrate polymers (cellulose, hemicelluloses) and an aromatic polymer (lignin).
[0037] As used herein, the term fermentation refers to the enzymatic and/or anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds such as alcohols. While fermentation may occur under anaerobic conditions, it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation may also occur under aerobic (e.g., in the presence of oxygen) or microaerobic conditions.
[0038] The term genetically engineered (or genetically modified) refers to a microorganism comprising a manipulated genome or nucleic acids.
[0039] Lignin, as used herein, refers to a complex polymer composed of monolignol subunits, primarily syringyl (S), guaiacyl (G) and p-hydroxyphenyl (H) monolignols, derived from sinapyl, coniferyl and p-coumaryl alcohols, respectively. Differences in the ratio of monolignols, and differences in expression and/or activity of lignin biosynthetic anabolic enzymes, create considerable variability in lignin structures, which differ between species, within species, within different tissues of a single plant and even within a single plant cell.
General Description
[0040] Disclosed herein are a genetically-modified bacterium from the genus Pseudomonas that can produce itaconic acid or trans-aconitate and methods of producing itaconic acid or trans-aconitate using the disclosed genetically-modified bacterium.
Genetically-Modified Bacterium
[0041] In some embodiments, the present disclosure is directed to a genetically-modified bacterium from the genus Pseudomonas comprising an exogenous nucleic acid encoding an enzyme that uses cis-aconitate as a substrate. In some embodiments, the genetically-modified bacterium is selected from the group consisting of P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borborid, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. asplenii, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugate, P. fragi, P. lundensis, P. taetrolens, P. Antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. putida group, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva, P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthin, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophile, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila. P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. teessidea, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, P. taiwanensis. In a specific embodiment, the bacterium is of the species P. putida.
[0042] In some embodiments, the exogenous nucleic acid sequence is codon optimized for the specific Pseudomonas strain used. The term codon-optimized refers to nucleic acid molecules that are modified based on the codon usage of the host species (herein the specific Pseudomonas strain used), but without altering the polypeptide sequence encoded by the nucleic acid.
[0043] In some embodiments, the genetically-modified bacterium comprises an exogenous nucleic acid encoding a cis-aconitate decarboxylase (cad) enzyme. In a specific embodiment, the cad enzyme is encoded by the cad1 gene from Aspergillus terreus having a protein sequence as shown by SEQ ID NO: 108, or a homolog thereof. In some embodiments, the expression of the cad1 gene is dynamically regulated. In some embodiments, the dynamic regulation of cad expression comprises limiting the expression to production phase. In some embodiments, the dynamic regulation of cad1 expression is achieved by an orthogonal RNA polymerase intermediary. In a specific embodiment, the orthogonal RNA polymerase intermediary is T7pol with a nitrogen-sensitive promoter. In a specific embodiment, the nitrogen-sensitive promoter comprises a sequence selected from SEQ ID NOs: 85-89.
[0044] In some embodiments, the genetically-modified bacterium comprises an exogenous nucleic acid encoding an aconitate isomerase enzyme. In a specific embodiment, the aconitate isomerase enzyme is encoded by the adi1 gene from Ustilago maydis having a protein sequence as shown by SEQ ID NO: 110, or a homolog thereof. In some embodiments, the exogenous nucleic acid further encodes a trans-aconitate decarboxylase enzyme. In a specific embodiment, the aconitate isomerase enzyme is encoded by the tad gene from Ustilago maydis having a protein sequence as shown by SEQ ID NO: 109, or a homolog thereof.
[0045] In some embodiments, the genetically-modified bacterium comprises an exogenous nucleic acid encoding a cis-aconitate decarboxylase (cad) enzyme, an exogenous nucleic acid encoding an aconitate isomerase, and an exogenous nucleic acid encoding a trans-aconitate decarboxylase as described above.
[0046] In some embodiments, a gene encoding for a poly-hydroxyalkonate synthase enzyme, or homolog thereof, is inactivated in the bacterium. In some embodiments, all poly-hydroxyalkonate synthase enzymes, or homologs thereof are inactivated in the bacterium. In a specific embodiment, the endogenous phaC1 gene and the endogenous phaC2 gene are inactivated in the bacterium.
[0047] In some embodiments, the inactivation of the poly-hydroxyalkonate synthase gene includes a deletion of the whole or a part of the gene such that no functional protein product is expressed (also known as gene knock out). The inactivation of a gene may include a deletion of the promoter or the coding region, in whole or in part, such that no functional protein product is expressed. In other embodiments, the inactivation of poly-hydroxyalkonate synthase includes introducing an inactivating mutation to the gene, such as an early STOP codon in the coding sequence of the gene, such that no functional protein product is expressed.
[0048] In some embodiments, gene inactivation is achieved using available gene targeting technologies in the art. Examples of gene targeting technologies include the Cre/Lox system (described in Khn, R., & M. Torres, R., Transgenesis Techniques: Principles and Protocols, (2002), 175-204.), homologous recombination (described in Capecchi, Mario R., Science (1989), 244: 1288-1292), and TALENs (described in Sommer et al., Chromosome Research (2015), 23: 43-55, and Cermak et al., Nucleic Acids Research (2011): gkr218.).
[0049] In one embodiment, poly-hydroxyalkonate synthase inactivation is achieved by a CRISPR/Cas system. CRISPR-Cas and similar gene targeting systems are well known in the art with reagents and protocols readily available. Exemplary genome editing protocols are described in Jennifer Doudna, and Prashant Mali, CRISPR-Cas: A Laboratory Manual (2016) (CSHL Press, ISBN: 978-1-621821-30-4) and Ran, F. Ann, et al. Nature Protocols (2013), 8 (11): 2281-2308.
[0050] In some embodiments, the genetically-modified bacterium further comprises an exogenous nucleic acid encoding a citrate synthase. In a specific embodiment, the citrate synthase enzyme is encoded by the gltA gene from E coli, or a homolog thereof. In some embodiments, the exogenously-expressed citrate synthase enzyme is a mutant enzyme that is immune to allosteric inhibition by intermediates expected to accumulate during production of itaconate, such as citrate.
[0051] In some embodiments, the level of endogenous isocitrate dehydrogenase in the genetically-modified bacterium is reduced compared to a non-genetically modified bacterium. In some embodiments, the level of endogenous isocitrate dehydrogenase is reduced because transcription or translation efficiency, or stability of the isocitrate dehydrogenase mRNA is decreased. In a specific embodiment, the start codon of the endogenous isocitrate dehydrogenase gene is either GTG or TTG instead of ATG. In some embodiments, the isocitrate dehydrogenase gene promoter comprises a mutation that decreases transcription efficiency. In some embodiments, the ribosome binding site of the isocitrate dehydrogenase gene transcript comprises a mutation that decreases the translation efficiency of the mRNA. In some embodiments, the level of endogenous isocitrate dehydrogenase is reduced because of a reduction in isocitrate dehydrogenase protein stability. In some embodiments, the isocitrate dehydrogenase protein encoded by the isocitrate dehydrogenase gene comprises a protease recognition sequence which renders it more likely to be degraded by cellular proteases.
[0052] In some embodiments, the genetically-modified bacterium is grown on an organic compound. In some embodiments, the organic compound is lignin, or a breakdown product of lignin (e.g., p-coumaric acid, ferulic acid, and saccharides). In some embodiments, the organic compound is selected from aromatic compounds, saccharides, organic acids, and alcohols. In some embodiments, the organic compound is a saccharide, not limited to a saccharide that Pseudomonas species can natively consume (e.g., glucose) but also one that the Pseudomonas species have been engineered to consume (e.g., xylose and arabinose). In some embodiments, the organic compound is an aromatic compound, and the aromatic compound comprises coumarate, ferulate, or benzoate. In some embodiments, the organic compound is an organic acid, and the organic compound comprises diacids (e.g., succinic acid), or fatty acids (e.g., acetic acid and octanoic acid), In some embodiments, the organic compound is a waste product from the production of biodiesel. In a specific embodiment, the waste product from the production of biodiesel is glycerol.
[0053] In some embodiments, the genetically-engineered bacterium further comprises an exogenous nucleic acid encoding an itaconic acid efflux pump. In some embodiments, the itaconic acid efflux pump is encoded by an itp1 gene. In a specific embodiment, the exogenous nucleic acid encodes an itp1 protein from Ustilago maydis having the sequence as shown in SEQ) ID NO: 111, or a homolog thereof. In some embodiments, the nucleic acid is codon optimized.
[0054] In some embodiments, the genetically-engineered bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate efflux pump. In some embodiments, the itaconic acid efflux pump is encoded by a tbrB gene. In a specific embodiment, the exogenous nucleic acid encodes a TbrB protein from Bacillus thuringiensus CT-43 having the sequence as shown in SEQ ID NO: 112, or a homolog thereof. In some embodiments, the nucleic acid is codon optimized.
Methods for Converting an Organic Compound to Itaconic Acid or Trans-Aconitate
[0055] Another aspect of the disclosure is directed to a method for converting an organic compound to itaconic acid or trans-aconitate, the method comprising inoculating an aqueous solution containing said organic compound with a genetically-modified bacterium from the genus Pseudomonas, wherein the bacterium comprises an exogenous nucleic acid encoding an enzyme that uses cis-aconitate as a substrate.
[0056] In some embodiments, the genetically-modified bacterium of the claimed method is grown on an organic compound. In some embodiments, the organic compound is lignin, or a breakdown product of lignin (e.g., p-coumaric acid, ferulic acid, and saccharides). In some embodiments, the organic compound is selected from aromatic compounds, saccharides, organic acids, and alcohols. In some embodiments, the organic compound is a saccharide, not limited to a saccharide that Pseudomonas species can natively consume (e.g., glucose) but also one that the Pseudomonas species have been engineered to consume (e.g., xylose and arabinose). In some embodiments, the organic compound is an aromatic compound, and the aromatic compound comprises coumarate, ferulate, or benzoate. In some embodiments, the organic compound is an organic acid, and the organic compound comprises diacids (e.g., succinic acid), or fatty acids (e.g., acetic acid and octanoic acid). In some embodiments, the organic compound is a waste product from the production of biodiesel. In a specific embodiment, the waste product from the production of biodiesel is glycerol.
[0057] In some embodiments, the genetically-modified bacterium of the claimed method is selected from the group consisting of P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borborid, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. asplenii, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugate, P. fragi, P. lundensis, P. taetrolens, P. Antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. putida group, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva, P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthin, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophile, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila. P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. teessidea, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, P. taiwanensis. In a specific embodiment, the bacterium is of the species P. putida.
[0058] In some embodiments, the exogenous nucleic acid sequence is codon optimized for the specific Pseudomonas strain used. The term codon-optimized refers to nucleic acid molecules that are modified based on the codon usage of the host species (herein the specific Pseudomonas strain used), but without altering the polypeptide sequence encoded by the nucleic acid.
[0059] In some embodiments, the genetically-modified bacterium of the claimed method comprises an exogenous nucleic acid encoding a cis-aconitate decarboxylase (cad) enzyme. In a specific embodiment, the cad enzyme is encoded by the cad1 gene from Aspergillus terreus having a protein sequence as shown by SEQ ID NO: 108, or a homolog thereof. In some embodiments, the expression of the cad/gene is dynamically regulated. In some embodiments, the dynamic regulation of cad1 expression comprises limiting the expression to production phase. In some embodiments, the dynamic regulation of cad1 expression is achieved by an orthogonal RNA polymerase intermediary. In a specific embodiment, the orthogonal RNA polymerase intermediary is T7pol with a nitrogen-sensitive promoter. In a specific embodiment, the nitrogen-sensitive promoter comprises a sequence selected from SEQ ID NOs: 85-89.
[0060] In some embodiments, the genetically-modified bacterium of the claimed method comprises an exogenous nucleic acid encoding an aconitate isomerase enzyme. In a specific embodiment, the aconitate isomerase enzyme is encoded by the adi1 gene from Ustilago maydis having a protein sequence as shown by SEQ ID NO: 110, or a homolog thereof. In some embodiments, the exogenous nucleic acid further encodes a trans-aconitate decarboxylase enzyme. In a specific embodiment, the aconitate isomerase enzyme is encoded by the tad1 gene from Ustilago maydis having a protein sequence as shown by SEQ ID NO: 109, or a homolog thereof.
[0061] In some embodiments, the genetically-modified bacterium of the claimed method comprises an exogenous nucleic acid encoding a cis-aconitate decarboxylase (cad) enzyme, an exogenous nucleic acid encoding an aconitate isomerase, and an exogenous nucleic acid encoding a trans-aconitate decarboxylase as described above.
[0062] In some embodiments, a gene encoding for a poly-hydroxyalkonate synthase enzyme, or homolog thereof, is inactivated in the bacterium. In some embodiments, all poly-hydroxyalkonate synthase enzymes, or homologs thereof, are inactivated in the bacterium. In a specific embodiment, the endogenous phaC1 gene and the endogenous phaC2 gene are inactivated in the bacterium.
[0063] In some embodiments, the inactivation of the poly-hydroxyalkonate synthase gene includes a deletion of the whole or a part of the gene such that no functional protein product is expressed (also known as gene knock out). The inactivation of a gene may include a deletion of the promoter or the coding region, in whole or in part, such that no functional protein product is expressed. In other embodiments, the inactivation of poly-hydroxyalkonate synthase includes introducing an inactivating mutation to the gene, such as an early STOP codon in the coding sequence of the gene, such that no functional protein product is expressed.
[0064] In some embodiments, gene inactivation is achieved using available gene targeting technologies in the art. Examples of gene targeting technologies include the Cre/Lox system (described in Khn, R., & M. Torres, R., Transgenesis Techniques: Principles and Protocols, (2002), 175-204.), homologous recombination (described in Capecchi, Mario R., Science (1989), 244: 1288-1292), and TALENs (described in Sommer et al., Chromosome Research (2015), 23: 43-55, and Cermak et al., Nucleic Acids Research (2011): gkr218.).
[0065] In one embodiment, poly-hydroxyalkonate synthase inactivation is achieved by a CRISPR/Cas system. CRISPR-Cas and similar gene targeting systems are well known in the art with reagents and protocols readily available. Exemplary genome editing protocols are described in Jennifer Doudna, and Prashant Mali, CRISPR-Cas: A Laboratory Manual (2016) (CSHL Press, ISBN: 978-1-621821-30-4) and Ran, F. Ann, et al. Nature Protocols (2013), 8 (1): 2281-2308, which are incorporated in their entireties.
[0066] In some embodiments, the genetically-modified bacterium further comprises an exogenous nucleic acid encoding an exogenous nucleic acid encoding a citrate synthase. In a specific embodiment, the citrate synthase enzyme is encoded by the gltA gene from E. coli, or a homolog thereof. In some embodiments, the exogenously-expressed citrate synthase enzyme is a mutant enzyme that is immune to allosteric inhibition by intermediates expected to accumulate during production of itaconate, such as citrate.
[0067] In some embodiments, the level of endogenous isocitrate dehydrogenase in the genetically-modified bacterium is reduced compared to a non-genetically modified bacterium. In some embodiments, the level of endogenous isocitrate dehydrogenase is reduced because transcription or translation efficiency, or stability of the isocitrate dehydrogenase mRNA is decreased. In a specific embodiment, the start codon of the endogenous isocitrate dehydrogenase gene is either GTG or TTG instead of ATG. In some embodiments, the isocitrate dehydrogenase gene promoter comprises a mutation that decreases transcription efficiency. In some embodiments, the ribosome binding site of the isocitrate dehydrogenase gene transcript comprises a mutation that decreases the translation efficiency of the mRNA. In some embodiments, the level of endogenous isocitrate dehydrogenase is reduced because of a reduction in isocitrate dehydrogenase protein stability. In some embodiments, the isocitrate dehydrogenase protein encoded by the isocitrate dehydrogenase gene comprises a protease recognition sequence which renders it more likely to be degraded by cellular proteases.
[0068] In some embodiments, the genetically-engineered bacterium further comprises an exogenous nucleic acid encoding an itaconic acid efflux pump. In some embodiments, the itaconic acid efflux pump is encoded by an itp1 gene. In a specific embodiment, the exogenous nucleic acid encodes an itp1 protein from Ustilago maydis having the sequence as shown in SEQ ID NO: 111, or a homolog thereof. In some embodiments, the nucleic acid is codon optimized.
[0069] In some embodiments, the genetically-engineered bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate efflux pump. In some embodiments, the itaconic acid efflux pump is encoded by a ThrB gene. In a specific embodiment, the exogenous nucleic acid encodes a TbrB protein from Bacillus thuringiensus CT-43 having the sequence as shown in SEQ ID NO: 112, or a homolog thereof. In some embodiments, the nucleic acid is codon optimized.
[0070] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0071] The present disclosure is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1: Materials and Methods
General Culture Conditions and Media
[0072]
TABLE-US-00001 TABLE 1 The bacterial strains used in this study: Strains Relevant Genotype NEB 5- Escherichia coli F proA.sup.+B.sup.+ lacI.sup.q (lacZ)M15 zzf::Tn10 (Tet.sup.R)/ alpha FIq fhuA2(argF-lacZ)U169 phoA glnV44 P80 lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 Epi400 Escherichia coli F.sup. mcrA (mrr-hsdRMS-mcrBC) 80dlacZM15 lacX74 recA1 endA1 araD139 (ara, leu)7697 galU galK .sup. rpsL (Str.sup.R) nupG trfA tonA pcnB4 dhfr QP15 Escherichia coli F proA.sup.+B.sup.+ lacI.sup.q (lacZ)M15 zzf::Tn10 (Tet.sup.R)/ mcrA (mrr-hsdRMS-mcrBC) 80dlacZM15 lacX74 recA1 endA1 araD139 (ara, leu)7697 galU galK .sup. rpsL (Str.sup.R) nupG trfA tonA pcnB4 dhfr BL21 (DE3) Escherichia coli F, ompT, hsdS.sub.B (r.sub.B, m.sub.B), dcm, gal, (DE3), pLysS pLysS, Cm.sup.r. JE90 Pseudomonas putida KT2440 hsdR::Bxb1int-attB JE2113 P. putida KT2440 hsdR::Bxb1int-attB ampC::lysY:PurtA:T7_RNAP JE3128 P. putida KT2440 hsdR::Bxb1int-attL:nptII:PT7:cadA:attR ampC::lysY:PurtA:T7_RNAP JE3215 P. putida KT2440 hsdR::Bxb1int-attB ampC::lysY:PurtA:T7_RNAP phaC1ZC2 JE3219 P. putida KT2440 hsdR::Bxb1int- attL:nptII:PT7:mNeonGreen:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 JE1622 P. putida KT2440 hsdR::Bxb1int-attB ampC::P.sub.PP2685:T7pol JE1626 P. putida KT2440 hsdR::Bxb1int-attB ampC::P.sub.PP2688:T7pol JE1629 P. putida KT2440 hsdR::Bxb1int-attB ampC::P.sub.urtA:T7pol JE1633 P. putida KT2440 hsdR::Bxb1int-attB ampC::P.sub.glnK:T7pol JE1651 P. putida KT2440 hsdR::Bxb1int- attL:nptII:P.sub.T7:mNeonGreen:attR ampC::P.sub.PP2685:T7pol JE1652 P. putida KT2440 hsdR::Bxb1int- attL:nptII:P.sub.T7:mNeonGreen:attR ampC::P.sub.PP2688:T7pol JE1653 P. putida KT2440 hsdR::Bxb1int- attL:nptII:P.sub.T7:mNeonGreen:attR ampC::P.sub.urtA:T7pol JE1654 P. putida KT2440 hsdR::Bxb1int- attL:nptII:P.sub.T7:mNeonGreen:attR ampC::P.sub.glnK:T7pol JE1655 P. putida KT2440 hsdR:Bxb1int- attL:nptII:mNeonGreen(promoterless):attR JE1657 P. putida KT2440 hsdR::Bxb1int- attL:nptII:P.sub.T7:mNeonGreen:attR JE2113 P. putida KT2440 hsdR::Bxb1int-attB ampC::lysY:P.sub.urtA:T7_RNAP JE2211 P. putida KT2440 hsdR::Bxb1int- attL:nptII:P.sub.tac:mNeonGreen:attR ampC::lysY:P.sub.urtA:T7pol JE2212 P. putida KT2440 hsdR::Bxb1int- attL:nptII:P.sub.T7:mNeonGreen:attR ampC::lysY:P.sub.urtA:T7pol JE3215 P. putida KT2440 hsdR::Bxb1int-attB ampC::lysY:P.sub.urtA:T7pol phaC.sub.1ZC.sub.2 JE3221 P. putida KT2440 hsdR::Bxb1int-attL:nptII:PT7:cadA:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 JE3659 P. putida KT2440 hsdR::Bxb1int-attL:nptII:PT7:tad1:adi1:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 JE3674 P. putida KT2440 hsdR::Bxb1int-attB ampC::lysY:PurtA:T7_RNAP phaC1ZC2 icdGTG:idhGTG JE3681 P. putida KT2440 hsdR::Bxb1int-attB ampC::lysY:PurtA:T7_RNAP phaC1ZC2 icdTTG:idhTTG JE3712 P. putida KT2440 hsdR::Bxb1int- attL:nptII:PT7:mNeonGreen:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 icdGYG:idhGYG JE3713 P. putida KT2440 hsdR::Bxb1int-attL:nptII:PT7:cadA:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 icdGTG:idhGTG JE3715 P. putida KT2440 hsdR::Bxb1int-attL:nptII:PT7:tad1:adi1:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 icdGTG:idhGYG JE3716 P. putida KT2440 hsdR::Bxb1int- attL:nptII:PT7:mNeonGreen:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 icdTTG:idhTTG JE3717 P. putida KT2440 hsdR::Bxb1int-attL:nptII:PT7:cadA:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 icdTTG:idhTTG JE3719 P. putida KT2440 hsdR::Bxb1int-attL:nptII:PT7:tad1:adi1:attR ampC::lysY:PurtA:T7_RNAP phaC1ZC2 icdTTG:idhTTG JE3899 P. putida KT2440 hsdR::Bxb1int-attL:nptII:PT7:adi1:attR ampC::lysY:PurtA:T7_RNAP AphaC1ZC2 icdTTG:idhTTG JE3729 P. putida KT2440 hsdR::Bxb1int-attL:nptII:P.sub.T7:mKate2:attR ampC::lysY:P.sub.urtA:T7pol JE3730 P. putida KT2440 hsdR::Bxb1int-attL:nptII:P.sub.T7.sub.
TABLE-US-00002 TABLE 2 Plasmids used in this study: Plasmids pJE382 pUC origin, nptII, sacB, mcs-lacZa pK18mobsacB pUC origin, nptII, sacB, Plac:mcs-lacZa pLysS p15A origin, cat, lysS pJE990 pUC origin, nptII, mNeonGreen (promoterless) pJE387 pK18mobsacB ampC pJE473 pJE382 phaC1ZC2 pJE1031 pJE382 ampC pJE1037 pJE382 ampC::PurtA:T7_RNAP pJE1040 pJE990 PT7:mNeonGreen pJE1180 pJE382 ampC::lysY:PglnK:T7_RNAP pJE1380 pJE990 PT7:cadA pJE1443 pJE990 PT7:tad1:adi1 pJE1444 pJE382 icdGTG:idhGTG pJE1445 pJE382 icdTTG.idhTTG pJE1383 pJE990 PT7:adi1
[0073] Routine cultivation of Escherichia coli for plasmid construction and maintenance was performed at 37 C. using LB (Miller) medium supplemented with 50 g/mL kanamycin sulfate and 15 g/L agar (for solid medium). All Pseudomonas putida cultures were incubated at 30 C., with shaking at 250 rpm for liquid cultures. LB (Miller) was used for routine Pseudomonas putida strain maintenance, competent cell preparations, and starter cultures. For itaconate production assay starter cultures, the media was supplemented with 50 g/mL kanamycin sulfate.
[0074] Modified M9 medium (M9*) with variable amounts of NH.sub.4Cl was utilized for shake flask experiments, growth rate assays, and fluorescent reporter assays (47.8 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl.sub.2, 18 M FeSO4, 1MME trace minerals, pH adjusted to 7 with KOH). 1000MME trace mineral stock solution contains per liter, 1 mL concentrated 1-HCl, 0.5 g Na.sub.4EDTA, 2 g FeCl.sub.3, 0.05 g each H.sub.3BO.sub.3, ZnClz, CuCl.sub.2.2H.sub.2O, MnCl.sub.2.4H.sub.2O, (NH.sub.4).sub.2MoO.sub.4, CoCl.sub.2.6H.sub.2O, NiCl.sub.2.6H.sub.2O. Unless otherwise noted, all M9* medium was supplemented with 20 mM p-coumarate (neutralized with NaOH) as a sole carbon source.
Production of Base-Catalyzed Depolymerized (BCD) Lignin (BCDL) and Depolymerized Lignin Media Preparation
[0075] In brief, dry solid material remaining from the enzymatic hydrolysis of pretreated corn stover (which follows the biorefinery process designed at NREL) was added as 10% (w/v) solids to a 2% NaOH solution and loaded into 200 mL stainless steel reactors. The reaction was carried out at 120 C. for 30 min. The sterile and solubilized material was neutralized with 4N H.sub.2SO.sub.4 and centrifuged at 8,000 rpm for 20 min in aseptic conditions. Then, the supernatant (90% v/v) was mixed with 10M9* salts (without any nitrogen source) and NH.sub.4Cl to generate M9*-BCDL medium supplemented with either 2 mM or 3 mM NH.sub.4Cl.
Plasmid & Pseudomonas Strain Construction
[0076] Phusion HF Polymerase (Thermo Scientific) and primers synthesized by Eurofins
[0077] Genomics were used in all PCR amplifications for plasmid construction. OneTaq (New England BiolabsNEB) was used for colony PCR. Plasmids were constructed by Gibson Assembly using NEBuilder HiFi DNA Assembly Master Mix (NEB) or ligation using T4 DNA ligase (NEB). Plasmids were transformed into either competent NEB 5-alpha FI.sup.q (NEB), Epi400 (Lucigen), or QP15 (Epi400 mated with NEB 5-alpha FI.sup.q to transfer the mini F plasmid to Epi400). Standard chemically competent Escherichia coli transformation protocols were used to construct plasmid host strains. Transformants were selected on LB (Miller) agar plates containing 50 pig/mL kanamycin sulfate for selection and incubated at 37 C., Template DNA was either synthesized by IDT or isolated from E. coli or P. putida KT2440 using Zymo Quick gDNA miniprep kit (Zymo Research). Zymoclean Gel DNA recovery kit (Zymo Research) was used for all DNA gel purifications. Plasmid DNA was purified from E. coli using GeneJet plasmid miniprep kit (ThermoScientific) or ZymoPURE plasmid midiprep kit (Zymo Research). Sequences of all plasmids were confirmed using Sanger sequencing performed by Eurofins Genomics. Plasmids used in this work are listed in Table 2.
[0078] P. putida JE90, a derivative of P. putida KT2440 where the restriction endonuclease hsdR has been replaced with the Bxb1-phage integrase and respective attB sequence (Elmore, J R., et al., Metabolic Eng. Comm., 5 (2017): 1-8), was used as a parent for all P. putida strains used in this study (Table 1). All genome modifications were performed using either the homologous recombination-based pK18mobsacB kanamycin resistance/sucrose sensitivity selection/counter-selection system (Marx, C J., BMC research notes 1.1 (2008): 1) as described in detail previously (Johnson, C W. et al., Metabolic Eng., 28 (2015): 240-247) or with the Bxb1-phage integrase system (Elmore, J R., et al., Metabolic Eng. Comm., 5 (2017): 1-8) with minor modifications to competent cell preparation procedures. These modifications cultivation overnight to stationary phase, rather than harvesting during exponential growth and all wash steps were performed at room temperature rather than at 4 C. Gene deletions and replacements were performed by homologous recombination, while integration of reporter and itaconate production pathway cassettes was performed with the Bxb1-phage integrase system. Primers used for screening P. putida strains for phaC1ZC2 deletion, ampC::T7_RNAP replacements, and icd/idh start codon swaps can be found below. Integration of pJE990-derivatives using the phage integrase system was confirmed by colony PCR using oligos oJE66 & oJE535.
Plasmid Construction Details
[0079] All enzymes used for plasmid construction were purchased from NEB.
[0080] For construction of pJE473 (SEQ ID NO: 91), homology arms to target deletion of phaC1ZC2 (PP_5003-5005) were amplified by PCR from wild-type P. putida genomic DNA using primer combinations oJE331/332 and oJE333/334, assembled into gel purified EcoRI/HindIII-linearized pJE382, and transformed into NEB 5-alpha FI.sup.Q. Resulting E. coli colonies were screened by colony for the presence of homology arms using primers oJE255/256. Candidates for pJE473 were purified from E. coli and sequenced using primers oJE255/256.
[0081] For construction of pJE1031 (SEQ ID) NO: 93), homology arms for the deletion of ampC (PP_2876) were amplified from pJE387 (SEQ ID NO: 90) using primer combination oJE92/608, assembled into gel purified EcoRI/HindIII-linearized pJE382, and transformed into NEB 5-alpha FI.sup.Q. Resulting E. coli colonies were screened by colony for the presence of homology arms using primers oJE255/256. Candidates for pJE1031 were purified from E. coli and sequenced using primers oJE255/256.
[0082] For construction of pJE1032 (SEQ ID NO: 94), pJE1033 (SEQ ID NO: 95), pJE1037 (SEQ ID NO: 96), and pJE1039 (SEQ ID NO: 97), promoter sequences containing 200-300 bp upstream of PP_2685, PP_2688, urtA (PP_4841), and glnK (PP_5234), respectively, were amplified from P. putida and assembled with T7 RNAP and a synthetic terminator sequence. The T7 RNA P polymerase and a downstream terminator was amplified from BL21(DE3) pLysS genomic DNA using oligos oJE625/626. A double terminator sequence for insulation of the construct was amplified from the T7_dbl_term gBlock using oJE627/628. Parts were assembled into BamHI/XbaI-linearized pJE1031, and transformed into NEB 5-alpha FI.sup.Q. Resulting E. coli colonies were screened by colony PCR using primers oJE177/178. Candidates for the plasmids were purified from E. coli and sequenced using oJE177/178/631/632/633.
[0083] For construction of the reporter plasmids the inventors annealed oligos containing desired promoter sequences and ligated the promoters into a promoterless mNeonGreen reporter plasmid, pJE990 (SEQ ID NO: 92). Plasmid pJE990 was linearized with BbsI. Promoter oligos pairs were phosphorylated with PNK (NEB) in T4 DNA ligase buffer, annealed by heating to 95 C. and cooling at 1 C./minute to room temperature. Annealed oligo sets oJE634/635, oJE97/98/133/134, oJE826/827, oJE828/829, oJE830/831, and oJE832/833 were ligated to BbsI-linearized pJE990 to construct plasmids pJE1040 (SEQ ID NO: 98), pJE1045, pJE1118, pJE1119, pJE1120, and pJE1121 respectively. Ligated DNA was transformed into NEB 5-alpha FIQ. Plasmids were isolated from transformant colonies and confirmed by sequencing with oJE535. For construction of mKate2 variant plasmids, mKate2 was amplified from the mKate2 gBlock using oligos oJE1724/1725 and digested with NdelI/XbaI. Plasmids pJE1040 and pJE1118-1121 were digested with NdeI/XbaI and ligated with NdeI/XbaI digested mKate2 gBlock to generate plasmids pJE1454-1458. Ligations were transformed into NEB 5-alpha FIQ, and candidates confirmed by sequencing of isolated plasmid DNA using oligos oJE535/536.
[0084] For construction of pJE1180 (SEQ ID NO: 99), the inventors amplified the cat and lysS genes from pLysS as two parts with primers designed to introduce the lysY mutation, assembled the resulting parts into SpeI-linearized pJE1040. Primers oJE817/818 and oJE819/820 were used to amplify the two parts. The resulting lysY/cat fragment was digested with SpeI and ligated into XbaI-linearized pJE1037, generating plasmid pJE1180.
[0085] For construction of pJE1380 (SEQ ID NO: 100), codon-optimized cadA from Aspergillus terreus was assembled into NdeI/XbaI-linearized pJE1040replacing mNeonGreen. The cadA gene was synthesized as gBlocks cadA_gBlock_1 & cadA_gBlock_2, gBlocks 1 & 2 were amplified using oligos oJE1408/1409 and oJE1410/1411, respectively. The assembly was transformed into NEB 5-alpha FI.sup.Q, and transformants were screened using oJE535/536. Plasmid DNA was isolated from PCR positive candidates, and sequenced using oJE535/536/1412.
[0086] For pJE1390, the cadA gene (encoding the cadA protein shown as SEQ ID NO: 108) from pJE1380 was excised using NdeI/XbaI, and ligated into NdeI/XbaI linearized pJE1045. The ligation was transformed into QP15, and transformants were screened by colony PCR using oligos oJE535/536. The assembly was transformed into NEB 5-alpha FI.sup.Q, and transformants were screened using oJE535/536. Plasmid DNA was isolated from PCR positive candidates, and sequenced using oJE535/536/1412.
[0087] For pJE1443 (SEQ ID) NO: 101), codon-optimized tad1 and adi1 genes from Ustilago maydis were assembled into AflIII/XbaI-linearized pJE1040replacing mNeonGreen and its RBS sequence. The tad1 and adi1 (SEQ ID NO: 107) genes were synthesized as gBlocks tad1 and adi1, which were amplified using primer combinations oJE1554/1547 and oJE1555/1548, respectively. The assembly was transformed into NEB 5-alpha FI.sup.Q, and transformants were screened using oJE535/536. Plasmid DNA was isolated from PCR positive candidates, and sequenced using oJE535/536/1559/1560/1561.
[0088] For pJE1483 (SEQ ID NO: 104), codon-optimized adi1 gene (SEQ ID NO: 107) used for pJE1443 was assembled into AflIII/XbaI-linearized pJE1040replacing mNeonGreen and its RIBS sequence. The adi1 sequence and its RIBS was amplified from pJE1443 using oligos oJE1760/1761. The assembly was transformed into NEB 5-alpha FI.sup.Q, and transformants were screened using oJE535/536. Plasmid DNA was isolated from PCR positive candidates, and sequenced using oJE535/536/1561.
[0089] For the construction of the icd/idh start codon swap plasmids pJE1444 (SEQ ID NO: 102) and pJE1445 (SEQ ID NO: 103), several PCR reactions were assembled containing homology arms for targeting, and mutations in the start codons (and RBS neutral mutations in the region between core RBS and start codon) of icd & idh. The homology arms for targeting insertion of the two plasmids into the icd idh locus were amplified using primer pairs oJE1564/1565 and oJE1568/1569 for both plasmids. The central fragment contained between the two homology arms, containing the various mutations, was amplified using oligos oJE1566/1567 for pJE1444 and oligos oJE1570/1571 for pJE1445. The parts were assembled into EcoRI/HindIII-linearized pJE382, transformed into NEB 5-alpha FI.sup.Q, and transformants were screened using oJE255/256. Plasmid DNA was isolated from PCR positive candidates, and sequenced using oJE1255/256/1572/1573.
TABLE-US-00003 TABLE3 Oligosusedinthedisclosure: Oligo Name OligoSequence(5-3) Purpose oJE255 attaatgcagctggcacgac(SEQIDNO:1) primersforscreeninginsertions intotheMCSofpJE382 oJE256 agctagatatcgccattcg(SEQIDNO:2) primersforscreeninginsertions intotheMCSofpJE382 oJE331 tagctcactcaggaaacagctatgacatgattac amplificationofhomologyarms gaattcGACCGAAAACATCGGTGC(SEQ toconstructionpJE473for IDNO:3) deletionofphaC1ZC2 oJE332 tcagcacgtaggtgcctTCTAGAgtctattgtaGG amplificationofhomologyarms ATCCTCTACGACGCTCCGTTG toconstructionpJE473for (SEQIDNO:4) deletionofphaC1ZC2 oJE333 aCAACGGAGCGTCGTAGAGGATCC amplificationofhomologyarms tacaatagacTCTAGAAGGCACCTACG toconstructionpJE473for TGCTG(SEQIDNO:5) deletionofphaC1ZC2 oJE334 ccagtcacgacgttgtaaaacgacggccagtgcca amplificationofhomologyarms agcttGCAGCCAAAACCGCAG(SEQID toconstructionpJE473for NO:6) deletionofphaC1ZC2 oJE335 cagtaccaggcattgctgaa(SEQIDNO:7) screeningdeletionofphaC1ZC2 (flanking) oJE336 gccaaggcagcagctaag(SEQIDNO:8) screeningdeletionofphaC1ZC2 (flanking) oJE337 TGGAGCIGAAGAACGIGTTG(SEQ screeningdeletionofphaC1ZC2 IDNO:9) (internaltophaG) oJE338 CTCGTCGACAAACAAAGCAA(SEQ screeningdeletionofphaC1ZC2 IDNO:10) (internaltophaG) oJE92 ccagtcacgacgttgtaaaacgacggccagtgcc amplificationofampCdeletion aagcttGTAACCACGGCCICACTGAA homologyarmsforconstruction (SEQIDNO:11) ofpJE1031 oJE608 tagctcactcaggaaacagctatgacatgattac amplificationofampCdeletion gaattcCTTGCCTCTGCCGGAAAC(SEQID homologyarmsforconstruction NO:12) ofpJE1031 oJE609 CTGTCGTTTTGTCCGACAATCAAC AmplifiesPP_2685promoter GCGAGCGttaggatccCATCGCCAGTG withoverlapstoconstruct ACAGACTG(SEQIDNO:13) pJE1032 oJE610 TGTCAGAGAAGTCGTTCTTAGCGA AmplifiesPP_2685promoter TGTTAATCGTGTTCATGCGGTTTC withoverlapstoconstruct CCTTGTGTIG(SEQIDNO:14) pJE1032 oJE611 CTGTCGTTTTGTCCGACAATCAAC AmplifiesPP_2688promoter GCGAGCGttaggatccGCCCGGGTCAA withoverlapstoconstruct AAGCCTTGTCAGAGAAGTCGTTCTT pJE1033 AGCGATGTT(SEQIDNO:15) oJE612 AATCGTGTTCATACCCACTCCTTG AmplifiesPP_2688promoter CCGCCGTT(SEQIDNO:16) withoverlapstoconstruct pJE1033 oJE619 CTGTCGTTTTGTCCGACAATCAAC AmplifiesPP_4841promoter GCGAGCCTttaggatccATGGCCTCGGG withoverlapstoconstruct GGCTGTTGTCAGAGAAGTCGTTCT pJE1037 TAGCGATGTT(SEQIDNO:17) oJE620 AATTCGTGTTCATGTGCTCTCTCCG AmplifiesPP_4841promoter CTGAGT(SEQIDNO:18) withoverlapstoconstruct pJE1037 oJE623 CTGTCCITTFIGTCCGACA,ATCAikC AmplifiesPP_5234promoter GCGAGCGttaggatccGCTGCGCACCG withoverlapstoconstruct AAATTG(SEQIDNO:19) pJE1039 oJE624 TGTCAGAGAAGTCGTTCTTAGCGA AmplifiesPP_5234promoter TGTTAATCGTGTTCATGAAACTCT withoverlapstoconstruct CTCCCGATTTGG(SEQIDNO:20) pJE1039 oJE625 ATGAACACGATTAACATCGCTAA amplificationofT7RNAPfor G(SEQIDNO:21) constructionofpJE1032,1033, 1037,1039 oJE626 GTAAAAKFTGCcATccCAACAGC amplificationofT7RNAPfor (SEQIDNO:22) constructionofpJE1032,1033, 1037,1039 oJE627 GAGCATCAATATGCAATGCTGTTG amplificationofdouble (SEQIDNO:22) terminatorfromDbl_term_T7 gBlockforconstructionof pJE1032,1033,1037,1039 oJE628 CGCTCAACGGACACGCT(SEQID amplificationofdouble NO:24) terminatorDbl_term_T7 gBlockforconstructionof pJE1.032,1033,1037,1039 oJE629 GACCATTACGGTGAGCGTTT(SEQ Amplifiesaninternalfragmentof IDNO:25) T7RNAPforPCRscreening oJE630 CGGGTTGAACATTGACACAG(SEQ Amplifiesaninternalfragment IDNO:26) ofT7RNAP oJE631 CTCAACAAGCGCGTAGG(SEQID Internalsequencingprimerfor NO:27) T7RNAPgene oJE632 GTTCATGCTTGAGCAAGCC(SEQ internalsequencingprimerfor IDNO:28) T7RNAPgene oJE633 GGTGTTACTCGCAGTGTGAC(SEQ InternalsequencingprimerforT7 IDNO:29) RNAPgene oJE634 gtctTAATACGACTCACTATAGGGA AnnealwithoJE634toconstruct GAGACCTGGAATTGTGAGCGGAT T7promoterforcloningof AACAATT(SEQIDNO:30) pJE1040 oJE635 taagAATTGTTATCCGCTICACAATFC AnnealwithoJE635toconstruct CAGGTCTCTCCCTATAGTGAGTCG T7promoterforcloningof TATTA(SEQIDNO:31) pJE1040 oJE535 GTTgctagcGTCGGGGTTTGTA(SEQ Screeningofgenomicintegration IDNO:32) ofpJE990/991anditsderivatives intoJE90derivativestrains,as wellasplasmidsequencing oJE536 aaaaccgcccagtctagctatcg Screeningofgenomic (SEQIDNO:33) integrationofpJE990/991andits derivativesintoJE90derivative strains,aswellasplasmid sequencing oJE93 GGCGTTGCTGGAAGAGTATT(SEQ flankingprimersforscreening IDNO:34) ampCdeletion oJE94 ACCACTGCCAGCAGAATTG(SEQ flankingprimersforscreening IDNO:35) ampCdeletion oJE546 gctgttgccatcgatcagt(SEQIDNO:36) amplifiesinternal851bp fragmentofampC.Usedfor screeningdeletion. oJE547 acgaccagttacaggccaag(SEQIDNO:37) amplifiesinternal851bp fragmentofampC.Usedfor screeningdeletion. oJE177 GGGAGACGGCTTCATCATG(SEQ Amplifiessequenceinsertedbtw IDNO:38) homologyarmsofpJE387/1031 oJE178 ATCACTGTATCCATCTTGTCATG Amplifiessequenceinsertedbtw (SEQIDNO:39) homologyarmsofpJE387/1031 oJE826 gtctTAKIACGACTCACTAtcaaggaaG cloningT7_C4promoterinto ACCTGGAATTGTGAGCGGATAAC pJE990 AATT(SEQIDNO:40) oJE827 taagAATTTGTTATCCGCTCACAATTC cloningT7_C4promoterinto CAGGTCttccttgaTAGTGAGTCGTAT pJE990 TA(SEQIDNO:41) oJE828 gtoTAATACGACTCACTAcggaagaaG cloningT7_H10promoterinto ACCIGGNATTGTGAGCGGATAAC pJE990 AATT(SEQIDNO:42) oJE829 taagAATTGTTATCCGCTCACAATIC cloningT7_H10promoterinto CAGGTCttcttccgTAGTGAGTCGTAT pJE990 TA(SEQIvDNO:43) oJE830 gtctTAATACGACTCACTAatactgaaGA cloningT7_H9promoterinto CCTGGAATTGTGAGCGGATAACA pJE990 ATT(SEQIDNO:44) oJE831 taagAATTTGTTATCCGCTCACAAVIC cloningT7_H9promoterinto CAGGTCttcagtatTAGTGAGTCGTAT pJE990 TA(SEQIDNO:45) oJE832 gtctTAATACGACTCACTAtttcggaaGA cloningT7_G6promoterinto CCTGGAATTGTGAGCGGATAACA pJE990 ATT(SEQIDNO:46) oJE833 taagAATTGTTATCCGCTCACAATTC cloningT7_G6promoterinto CAGGTCttccgaaaTAGTGAGTCGTAT pJE990 TA(SEQIDNO:47) oJE817 cccgaaaggggggcctatttcgttttggtcca amplifypartofpLysSfor ctagtCACTATCGACTACGCGATCATG constructionofpJE1110 (SEQIDNO:48) oJE818 GAAGGCGCTGGTCTTCGCGCCCAT amplifypartofpLysSfor CATGAGGTGGCGCCGTACGCTTGC constructionofpJE1110 CCTTCGTTCGAC(SEQIDNO:49) oJE819 TCTCCCACCAACGCTTAAGGTCGA amplifypartofpLysSfor ACGAAGGGCAAGCGTACGGCGCC constructionofpJE1110 ACCTCATGAT(SEQIDNO:50) oJE820 CAGGTCTCTCCCTATAGTGAGTCG amplifypartofpLysSfor TATTAagactactagtCCTGITGATACC constructionofpJE1110 GGGAAGC(SEQIDNO:51) oJE821 TCACGGACACCAACATTCTGAC sequencingofLysYfragmentof (SEQIDNO:52) pJE1180 oJE1408 GATAACAATTcttaagattaactcacacagga amplificationofcadAgBlocks gatatcat(SEQIDNO:53) forpJE1380construction oJE1409 CCTTTGGTAAACATTTTCAGAAAAC amplificationofcadAgBlocks C(SEQIDNO:54) forpJE1380construction oJE1410 GAACGCAGCTATGGGGGTTTTCTG amplificationofcadAgBlocks (SEQIDNO:55) forpJE1380construction oJE1411 AAGGCCCCCCGTTAGGGAGGCCT amplificationofcadAgBlocks TATTGTTCGTCtctagaTTAGACCAA forpJE1380construction GG(SEQIDNO:56) oJE1412 TGCATAGCGCAAGCATTGTG(SEQ sequencingofcadAinpJE1380 IDNO:57) oJE1547 ATTCTAGGCACTGCTGTACTGATA amplificationoftad1gBlockfor GGGTATTCACGCCGACGATGGAC assemblyofpJE1443 (SEQIDNO:58) oJE1548 CGTGTGTTGAGCCGTCCATCGTCG amplificationofadi1gBlockfor GCGTGAATACCCTATCAGTACAGC assemblyofpJE1443 AGTG(SEQIDNO:59) oJE1554 TGGAATTGTGAGCGGATAACAAT amplificationoftad1gBlockfor TcttaagGTagaTaAGAGCGGGTCATC assemblyofpJE1443 G(SEQIDNO:60) oJE1555 GTTAGGGAGGCCTTATTGTTCGTCt amplificationofadi1gBlockfor ctagaTCAGGACAAGCTCCGGTC assemblyofpJE1443 (SEQIDNO:61) oJE1559 AGCAACGGITGGATAGCATC(SEQ sequencingoftad1 IDNO:62) oJE1560 CAGGTCTTTCCCGATGCAAT(SEQ sequencingoftad1downstream IDNO:63) genes oJE1561 AACCGCATCCGTCCGATA(SEQ sequencingofadi1 IDNO:64) oJE1564 cactcaggaaacagctatgacatgattac amplificationofUPhomology gaattcgccgccatcaagcagtt armforpJE1444/1445 (SEQIDNO:65) construction oJE1565 ggataccagaaaatcaaggttccga(SEQID amplificationofUPhomology NO:66) armforpJE1444/1445 construction oJE1566 tcggaaccttgattttctggtatccCACC amplificationoficd/idhpromoter GAAgcactactccgctgtcg regionwithGTGstartcodonsfor (SEQIDNO:67) pJE1444construction oJE1567 tatagatgatcttggaacgggtgggCACg amplificationoficd/idh TTTgttaactactgtgtgctgagc promoterregionwithGTGstart (SEQIDNO:68) codonsforpJE1444construction oJE1568 cccacccgttccaagatcat(SEQIDNO:69) amplificationofDNhomology armforpJE1444/1445 construction oJE1569 cacgacgttgtaaaacgacggccagtgccaagct amplificationofDNhomology taacatgatcgggtcgga(SEQIDNO:70) armforpJE1444/1445 construction oJE1570 tcggaaccttgattactggtatccCAACGAAgca amplificationoficd/idhpromoter ctactccgctgtcg(SEQIDNO:71) regionwithTTGstartcodonsfor pJE1445construction oJE1571 tatagatgatcttggaacgggtgggCAAgTTTgtt amplificationoficd/idhpromoter aactctctgtgtgagagc(SEQIDNO:72) regionwithTTGstartcodonsfor pJE1445construction oJE1572 cgataccacataatcacgcac(SEQIDNO:73) sequencingofpJE1444/1445 oJE1573 ctctcgactttccgctca(SEQIDNO:74) sequencingofpJE1444/1445 oJE1574 ttttaggtatccCACCGAA(SEQIDNO: screeningforGTIGstartcodon 75) swapforicd/idhinP.putida oJE1575 gggtgggCACgTTT(SEQIDNO:76) screeningforGTGstartcodon swapforicd/idhinP.putida oJE1576 gatctggtatccCAACGAA(SEQIDNO: screeningforTTGstartcodon 77) swapforicd/idhinP.putida oJE1577 cgggtgggCAAgTTT(SEQIDNO:78) screeningforTTGstartcodonfor icd/idhinP.putida oJE1578 gattttctggtatcccatgct screeningforwild-typestart (SEQIDNO:79) codon oJE1579 gtgggcatgcgg(SEQIDNO:80) screeningforwild-typestart codon oJE1580 gtggcgatcacgtcgtact(SEQIDNO:81) screeningtoensurethatplasmid backboneisremovedfollowing startcodonswap oJE1581 aggaggtgatgcctttgtc(SEQIDNO:82) screeningtoensurethatplasmid backboneisremovedfollowing startcodonswap oJE1582 aggaatgatcggaggtcag(SEQIDNO:83) sequencingoficdpromoter region.UsewithoJE1581to amplifyregionforsequencing. oJE66 catgtagttgtaggcgtcttc screeningintegrationofpJE990- (SEQIDNO:84) derivativeplasmidsviatheBbx1- phageintegrasesystem
Growth Rate Analysis
[0090] LB medium was inoculated from glycerol stocks and incubated overnight at 30 C., 250 rpm for precultures. Cultures were washed twice by centrifugation (4000g for 10 minutes) and resuspension in equal volumes of 1M9 salts lacking NH.sub.4Cl to remove residual LB medium, and resuspended in volume 1M9 salts. Optical density (OD600) of resulting suspensions was measured using a 1 cm path length cuvette. Growth assays were performed with 600 L M9* medium supplemented with 20 mM p-coumarate and 20 mM NH.sub.4Cl in clear 48-well microtiter plates with an optically clear lid (Greiner Bio-One). All cultures were inoculated with washed cultures to an OD.sub.600 equivalent to 0.03 in a 1 cm pathlength cuvette. Plates were incubated at 30 C., fast shaking in an Epoch2 plate reader (Bio-Tek), with OD.sub.600 readings taken every 10 minutes. Exponential growth rates were determined using the CurveFitter software with data points in early mid-log phase. All growth rates were calculated from 3 replicate experiments.
Fluorescent Reporter Assays
[0091] Strains were revived from glycerol stocks in 5 mL LB with overnight incubation at 30 C., 250 rpm. 5 mL starter cultures in M9*+20 mM glucose+10 mM NH.sub.4Cl were inoculated with 1% of the recovery culture and similarly incubated. Coupled growth and fluorescence assays were performed with a Neo2SM (Bio-Tek) plate reader using 200 L/well of M9*+20 mM p-coumarate+2 (limiting) or 20 (replete) NH.sub.4Cl in black-walled, Clear flat-bottom, 96-well plates (Greiner Bio-One) with an optically clear lid. Plate cultures were inoculated with 0.5% inoculum from starter cultures, and incubated overnight at 30 C., fast shaking with OD.sub.600 and fluorescence (F.sub.510,530 for mNeonGreen and F.sub.588,633 for mKate2) measured every 10 minutes. Reporter expression per cell was estimated by dividing relative fluorescence units (RFU) by OD.sub.600 (as a proxy for cell number) for each time point and averaging those values for time points occurring during either exponential growth or stationary phase. Background absorbance and fluorescence readings from wells containing media blanks were averaged and subtracted from sample readings prior to analysis. Exponential phase was defined as time points where OD.sub.600 was between 0.039 and the OD.sub.600 curve inflection point, typically OD.sub.6000.2 (nitrogen limited) or 0.6 (nitrogen replete). Stationary phase was defined as time points starting 2 hours following end of exponential phase.
Shake Flask Experiments
[0092] Starter cultures were prepared as described for growth rate assays with the exception that 50 g/mL kanamycin sulfate was added to the medium. Starter cultures were inoculated to a final OD.sub.600 of 0.1 into 25 mL of M9* medium, supplemented with 20 mM p-coumarate and 2 mM NH.sub.4Cl, in a 125 mL erlenmeyer flask and incubated at 30 C., 250 rpm. Cultures were sampled periodically to measure growth by OD.sub.600, and analyte concentrations by high performance liquid chromatography (HPLC).
Analytical Techniques
[0093] For shake flask experiments, optical density at 600 nm (OD.sub.600) was measured using a spectrophotometer (Amersham, UltroSpec10). HPLC analysis for p-coumarate and organic acid detection was performed by injecting 20 L of 0.2 m filtered culture supernatant onto a Waters 1515 series system equipped with a Rezex RFQ-Fast Acid H+ (8%) column (Phenomenex) and a Micro-Guard Cation H.sup.+ cartridge (Bio-Rad). Samples were run with column at 60 C. using a mobile phase of 0.01 N sulfuric acid at a flow rate of 0.6 mL/min, with a refractive index detector and UV/Vis detector measuring A230 & A280 for analyte detection. Analytes were identified and quantified by comparing retention times and spectra with pure standards.
[0094] For shake flask experiments with M9*-BCDL, optical density at 600 nm (OD.sub.600) was measured with a Nanodrop (ThermoFisher Scientific) after diluting samples 6-fold. Uninoculated M9*-BCDL medium was used as a blank to subtract signal coming from components in the medium.
[0095] Itaconic acid quantitation in M9*-BCDL. Prior the analysis, a 0.1 mL. aliquot was taken from each sample and 0.9 mL of water were added to make a 10 dilution. Then, 34 L of 72% sulfuric acid were added to each diluted sample to decrease the pH below 2.0 and precipitate acid insoluble lignin. Samples were centrifuged, and the supernatant was filtered through a 0.2 M filter pore size. Itaconic acid quantification was performed on an Agilent 1100 series HPLC system, with a diode array detector (DAD) at 210 nm (Agilent Technologies). Analysis was performed by injecting 6 L of filtered culture supernatant onto a Phenomenex Rezex RFQ-Fast Acid H+ (8%) column with a cation H+guard cartridge (Bio-Rad Laboratories) at 85 C. using a mobile phase of 5 mM sulfuric acid at a flow rate of 1.0 mL/min.
[0096] Aromatic compounds quantitation in M9*-BCDL. Metabolite analysis in BCD was performed on an Agilent 1200 LC system (Agilent Technologies) equipped with a DAD. Each sample and standard was injected at a volume of 10 L onto a Phenomenex Luna C18(2) column 5 m, 4.6150 mm column (Phenomenex). The column temperature was maintained at 30 C. and the buffers used to separate the analytes of interest were A) 0.05% acetic acid in water and B) 0.05% acetic acid in acetonitrile. The chromatographic separation was carried out using a gradient of: initially starting at 1% B going to 50% B at 35 min before immediately switching to 99% B at 35.1 min, before equilibrium for a total run time of 47 min. The flow rate of the mobile phases was held constant at 0.6 mL/min. The same standards used in the BCDL experiments were also used to construct calibration curves, but between the ranges of 5-200 g/L. Three separate wavelengths from the DAD were used to identify and quantitate the analytes of interest. A wavelength of 210 nm and 225 nm was used for the analytes vanillic acid and 4-hydroxybenzoic acid. A wavelength of 325 nm was used for the analytes p-coumaric acid, and ferulic acid. A minimum of five calibration levels was used with an r.sup.2 coefficient of 0.995 or better for each analyte.
Transcriptional Profiling of P. putida
[0097] For the determination of NO.sub.3 induced promoters, strain JE1657, an engineered P. putida strain containing a Bxb1 phage integrases system for rapid genomic integration of DNA 3, and a PT7:mNeonGreen reporter cassette was used. JE1657 was cultured at 30 C in 50 mL MME mineral medium in a 250 mL erlenmeyer shake flask at 30 C., 250 rpm shaking and harvested mid-log (OD600=0.2) by centrifugation (16,000g, 2 minutes, 4 C.). Supernatants were quickly decanted, and cell pellets were frozen rapidly in liquid nitrogen prior to storage at 80 C. for storage prior to RNA isolation. Four samples were prepared for each condition for characterization of biosensor performance strain JE2212 under identical conditions.
[0098] Cell pellets were resuspended in TRIzol (ThermoFisher-Invitrogen, Waltham, Mass. USA) and processed according to the manufactures protocol for TRIzol reagent. In general, TRIzol was added to cell pellets and mixed by vortex and pipetting. Chloroform was then added and mixed and samples were centrifuged. After centrifugation the aqueous layer was removed and mixed 1:1 with 80% ethanol. The samples were then purified on a RNeasy column (Qiagen Hilden, Germany) following the manufactures protocol and the on-column DNase digestion. RNA was eluted off the column in 35 L RNAse free H20 (Qiagen, Hilden, Germany). RNA concentration was quantified using a Nanodrop 1000 instrument (ThermoScientific, Waltham, Mass.) and RNA quality was verified by obtaining RNA Integrity Numbers (RIN) using an RNA 6000 Nanochip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa. Clara, Calif.).
[0099] Ribosomal RNA was depleted from total RNA samples using a RiboZero rRNA Removal Kit (Epicentre-Illumina Inc. San Diego, Calif.) according to manufacturer's instructions. The depleted sample was purified on a RNA Clean & Concentrator-5 (Zymo Research, Irvine, Calif., USA) following the manufacturer's protocol, and then the depleted material was quantified using a Nanodrop 1000 and visualized on an Agilent 2100 Bioanalyzer instrument with a RNA 6000 Nanochip (Agilent Technologies, Santa Clara, Calif.). RNA depleted of ribosomal RNA was used as input material to synthesize cDNA libraries using a ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina-Epicentre, San Diego, Calif., USA) according to manufacturer's instructions and TruSeq compatible barcodes. Pooled barcoded libraries were sequenced in one direction for 50 bases (SE50) on an Illumina Hi-Seq2500 using v4 chemistry (Illumina Inc. San Diego, Calif.) and de-multiplexed as a sequencing service provided by The Genomic Services Lab at Hudson Alpha Institute for Biotechnology (HudsonAlpha, Huntsville, Ala.).
Differential Gene Expression Analysis
[0100] After Illumina sequencing, RNA-seq reads were mapped to modified versions of the P. putida KT2440 reference genome (NC_002947) containing the mutations found in JE1657 and JE2212 using the Geneious for RNA-seq mapping workflow. Read count per annotated gene was calculated for each treatment and replicate, as well as fragment per kilobase million (FPKM), a common normalization technique. The inventors then exported gene locus tags and raw read counts into tab-delimited files, one for each replicate. To calculate differential gene expression, R package DESeq was used which calculates log-fold change in expression and allows comparison between treatments using several replicates. There were three (JE2212 assay) or four (JE1657 assay) replicates per treatment, for a total of six or eight inputs per experiment.
Gene and Protein Sequences
[0101] SEQ ID) NO: 105: cadA gene (Codon-optimized for P. putida KT2440).
[0102] SEQ ID NO: 106: tad1 gene (Codon-optimized for P. putida KT2440).
[0103] SEQ ID NO: 107: adi1 gene (Codon-optimized for P. putida KT2440).
[0104] SEQ ID NO: 108: cadA protein (Organism: Aspergillus terreus).
[0105] SEQ ID NO: 109: tad1 protein (Organism: Ustilago maydis).
[0106] SEQ ID NO: 110: adi1 protein (Organism: Ustilago maydis).
[0107] SEQ ID NO: 111: itp1 (itaconate transporter) protein (Organism: Ustilago maydis)
[0108] SEQ ID NO: 112: TbrB (trans-aconitate transporter) protein (Organism: Bacillus thuringiensus CT-43)
Example 2: Dynamic Regulation Enables Two-Stage Production of Itaconate Production from Lignin-Derive Aromatics
[0109] The enzyme cis-aconitate decarboxylase produces itaconic acid (itaconate) by enzymatic decarboxylation of the TCA cycle intermediate cis-aconitate (
[0110] The inventors constructed an expression cassette containing codon optimized version of the ca A gene (SEQ ID NO: 105) under the control of the T7 promoter in a Bxb1 integrase target plasmid for rapid integration into the P. putida genome. This plasmid was integrated into the genome of P. putida JE2113 (Table 1), a host strain containing the P.sub.urtA:T7 RNAP:lysY+ cassette, generating strain JE3128. Itaconic acid production by JE3128 was assayed by shake flask cultivation with M9* medium supplemented with 20 mM p-coumarate, a model lignin-derived aromatic compound, and limiting amounts of nitrogen (2 mM N.sub.4Cl). With this strain and conditions, the inventors were able to detect production of itaconic acid, but the titer (23 mg/L) and molar yield (0.96% mol/mol) were low (Table 4).
TABLE-US-00004 TABLE 4 Production of itaconate from lignin-derived aromatics. Stationary Overall Phase Hosted Molar Molar Mass Production Yield Yield* Yield Titer Strain Pathway Relevant genotype (mol/mol) (mol/mol) (g/g) (g/L) JE3128 PT7:cad4 JE90P.sub.urtA:T7_RNAP:Pcat:lysY 0.01 n.d. 0.01 0.02 (cis) JE3221 PT7:cadA JE90P.sub.urtA:T7_RNAP:Pcat:lysY 0.09 0.18 0.07 0.22 (cis) phaC1ZC2 JE3659 PT7:tad1:adi1 JE90 0.23 0.39 0.19 0.57 (trans) P.sub.urtA:T7_RNAP:Pcat:lysY phaC1ZC2 JE3713 PT7:cadA JE90 0.29 0.79 0.23 0.72 (cis) P.sub.urtA:T7_RNAP:Pcat:lysY phaC1ZC2 icdGTG:idhGTG JE3715 PT7:tad1:adi1 JE90 0.43 1.02 0.34 1.09 (trans) P.sub.urtA:T7_RNAP:Pcat:lysY phaC1ZC2 icdGTG:idhGTG JE3717 PT7:cadA JE90 0.50 0.97 0.40 1.27 (cis) P.sub.urtA:T7_RNAP:Pcat:lysY phaC1ZC2 icdTTG:idhTTG JE3719 PT7:tad1:adi1 JE90 0.56 1.16 0.45 1.26 (trans) P.sub.urtA:T7_RNAP:Pcat:lysY phaC1ZC2 icdTTG:idhTTG
[0111] P. putida is well known to accumulate polyhydroxyalkanoates (PHA), a fatty acid-derived carbon storage polymer, from a variety of carbon sources, including lignin (Linger, J G., et al., PNAS 111.33 (2014): 12013-12018), in conditions where nitrogen is limited (Prieto, A. et al., Environmental Microbiology, 18.2 (2016): 341-357). Depending on the conditions, PHAs can accumulate to up to 8004 cell dry weight. As production of PHAs requires acetyl-CoA for production of fatty acid intermediates, it directly competes with itaconate production for acetyl-CoA (
Example 3: Metabolic Pathway Selection to Optimize Itaconate Production
[0112] To date, other than in organisms that natively produce itaconate, all attempts to engineer strains for itaconate production have focused on heterologous expression the cis-aconitate decarboxylase, or cis-pathway, from A. terreus. However, an alternate pathway for itaconate production was recently discovered in Ustilago maydis (Geiser et al., Microbial Biotech., 9.1 (2016): 116-126). This pathway, referred to here as the trans-pathway, proceeds through two steps. First, cis-aconitate is isomerized to the thermodynamically favorable isomer, trans-aconitate, by aconitate isomerase (adi1, P. putida KT2440 protein sequence SEQ ID NO: 110), which is subsequently decarboxylated by trans-aconitate decarboxylase (tad1, P. putida KT2440 protein sequence SEQ ID NO: 109) generating itaconate (
[0113] To test this hypothesis, the inventors constructed an expression cassette with the T7 promoter controlling expression of codon-optimized version of the tad1 & adi1 genes. The resulting plasmid was integrated into the genome of JE3215, generating strain JE3659. JE3659 was assayed for production of itaconate from p-coumarate under nitrogen-limited conditions. As hypothesized, utilization of the trans-pathway from U. maydis further increased both the titer (570 mg/L) and molar yield (23.39% mol/mol) (Table 4,
Example 4: Modulating TCA Cycle Flux Increases Itaconic Acid Yields and Titer
[0114] One of the most reliable methods to increase product formation in a chemical reaction is to increase substrate concentration. As an obligate aerobe, P. putida maintains robust TCA cycle activity for energy production. The inventors hypothesized that the increased substrate accumulation with the trans pathway was the determining factor for the increase itaconate yields of JE3659 (trans) relative to JE3221 (cis). Accordingly, it was predicted that increasing accumulation of cis-aconitate would significantly increase yields. Reducing the flux through isocitrate dehydrogenase (
[0115] As these mutations are predicted to increase substrate accumulation for both the trans- and cis-pathways, itaconate production was tested with both pathways in JE3674 and JE3681 host strains. The cis- and trans-pathways were integrated into JE3674, generating strains JE3713 (cis) and JE3715 (trans), and JE3681, generating strains JE3717 (cis) and JE3719 (trans). All 4 strains were assayed for production of itaconate from p-coumarate under nitrogen-limited conditions. As hypothesized, the mild reduction of isocitrate dehydrogenase activity induced by the GIG start codons significantly increased itaconate titers and overall yields (
Example 5: Production of Trans-Aconitate from Lignin-Derived Aromatics
[0116] Trans-aconitate, an intermediate in the production of itaconate with the trans-pathway, is a compound with potential industrial value as well. If production of trans-aconitate becomes commercially viable, there are uses for trans-aconitate in the production of materials such as plasticizers and building blocks for hyperbranched polyesters, among others. Given the robust itaconate production by the instant engineered P. putida strains, the inventors hypothesized that they might also be able to produce high yields of trans-aconitate from lignin-derived aromatics using a truncated version of the trans-pathway. To test this hypothesis, the inventors constructed an expression cassette with a truncated version of the itaconic acid production trans-pathway that lack the trans-aconitate decarboxylase gene tad1, and contains just the aconitate isomerase, adi1, under the control of the T7 promoter. This cassette was incorporated into strain JE3681, generating strain JE3899. JE3899 was tested for production of trans-aconitate form p-coumarate under nitrogen-limited conditions. After 72 hours most of the p-coumarate was consumed and 1.51 g/L trans-aconitate was produced (
Example 6: Modulating TCA Cycle Flux Increases Itaconic Acid Yields and Titer
[0117] As an obligate aerobe, P. putida maintains robust TCA cycle activity for energy production. The inventors hypothesized that reducing flux through isocitrate dehydrogenase (
[0118] To determine the impact of these mutations on itaconate production, the inventors integrated the Ptac:cadA cassette into both strains, generating strains JE4308 (icdGTG:idhGTG) and JE4307 (icdTTG:idhTITG), and assayed itaconate production from p-coumarate under nitrogen-limited and nitrogen-replete conditions. Slowing the TCA cycle was sufficient to allow detectable itaconate production under nitrogen-replete conditions, and further increased yields under nitrogen-limited conditions to 26.5% and 30.47% mol/mol with JE3708 and JE3707, respectively (
Example 7: Development of a Signal-Amplified Nitrogen-Limitation Biosensor for Dynamic Metabolic Control in Pseudomonas putida KT2440
[0119] By limiting its expression to production phase, dynamic regulation of the apparently toxic CadA protein could substantially improve itaconate production. Native regulatory systems are specifically tuned to provide expression sufficient for associated pathways which is often insufficient for heterologous pathways. Utilizing an orthogonal RNA polymerase intermediary, such as T7pol for dynamic regulation allows amplification of the original signal (
[0120] Here the inventors develop a biosensor that limits protein expression to production phase by controlling expression of T7pol with a nitrogen-sensitive promoter. Eleven candidate promoters were identified by comparing gene expression during growth on a good (NI-L) or poor (NO.sub.3) nitrogen source (Table 5).
Table 5: Differential expression of genes downstream potential nitrogen-sensitive promoters.
TABLE-US-00005 log.sub.2 fold change Locus Gene (NaNO.sub.3/ Base Tag Name NH.sub.4Cl) Mean Predicted gene function PP_1705 nirB 8.14 2029.14 nitrite reductase large subunit PP_2092 nasA 6.31 361.67 nitrate transporter PP_2094 nasS 2.79 51.23 nitrate binding protein PP_2685 4.44 320.51 Bacterial proteasome, beta subunit PP_2688 3.99 132.41 Circularly permuted ATP- grasp type 2 PP_2842 ureD 4.38 181.83 urease accessory protein PP_4053 treY 2.19 1151.28 maltooligosyl trehalose synthase PP_4841 urtA 4.37 455.68 urea ABC transporter substrate-binding protein PP_4842 urtB 4.56 72.42 urea ABC transporter permease PP_4845 urtE 3.77 67.21 ABC transporter ATP- binding protein PP_5234 glnK 1.45 8496.51 NRII(GlnL/NtrB) phosphatase activator
[0121] The inventors tested biosensors with four candidate promoters: P.sub.PP_2685, P.sub.PP_2688, P.sub.urtA, and P.sub.glnK. Candidate biosensors were integrated into the JE90 genome, replacing a -lactam resistance gene, ampC, and assayed for production of the fluorescent protein mNeonGreen under either nitrogen-replete or nitrogen-limited conditions (FIGS. 7B-7D). While the P.sub.glnK and P.sub.PP_2685 candidate biosensors were surprisingly nitrogen-agnostic, displaying constitutive mNeonGreen expression similar to the .sup.70 tac promoter (
[0122] While the initial P.sub.urtA biosensor variant allowed strong induced expression, basal expression in the presence of nitrogen was relatively high. To reduce basal T7pol activity the inventors constitutively expressed a catalytically-deactivated variant of T7 lysozyme (LysY) (U.S. Pat. No. 8,138,324), which allosterically inhibits T7pol activity (
[0123] Optimal pathway performance often requires tuning expression of individual proteins. Tuning expression can be achieved with promoter (Elmore et al., Metab Eng Commun 5, 1-8 (2017)) and/or ribosome binding site (RBS) (Salis et al., Nature Biotech. 27.10 (2009): 946) modifications. The inventors utilized a small library of T7 promoter variants (see Table 6) with the red fluorescent protein mKate2 to demonstrate ability to tune the magnitude of biosensor outputs. Unlike the .sup.70 tac promoter (
TABLE-US-00006 TABLE6 T7Promotervarianttesting. mKate2production Fold-induction T7Promoter (RFU/OD600) in Variant Nitrogen-limited N-limited Promoter Sequence Exponential Stationary stationaryphase Ptac 43755 1546 54287 572 1.24 0.03 (const.) PT7 taatacgactca 979 30 73036 2563 74.67 4.76 ctaTAGGGgaa (SEQID NO:85) PT7_C4 taatacgactca 95 24 12847 416 142.61 36.75 ctaTTCAAGgaa (SEQID NO:86) PT7_H10 taatacgactca 79 28 17782 301 262.27 1977 ctaCGGAAgaa (SEQID NO:87) PT7_H9 taatacgactca 91 9 14110 126 157.16 17 ctaATACTgaa (SEQID NO:88) PT7_G6 taatacgactca 74 38 817 12 15.92 12.22 ctaTTTCCTgaa (SEQID NO:89)
Example 8: Dynamic Regulation Improves Two-Stage Production of Itaconate Production from Lignin-Derived Aromatics
[0124] The inventors next sought to test whether dynamic regulation of cadA would improve itaconate production. For this, the inventors altered the isocitrate dehydrogenase start codons of JE2113, which contains P.sub.urtA:T7pol:lysY.sup.+ biosensor cassette, to generate strains JE3674 (icd.sup.GTG:idh.sup.GTG) and JE3681 (icd.sup.TTG:idh.sup.TTG). The inventors integrated a codon optimized copy of cadA (SEQ ID NO: 105) under the control of the T7 promoter into all three strains, and assayed production of itaconate from p-coumarate under nitrogen-limited conditions, Similar to previous shake flask experimentswith the exception of JE4307growth is complete with the first 24 hours, with some itaconate production occurring, likely after growth is completed. Strain JE3717 (P.sub.T7:cadA, icd.sup.TTG:idh.sup.TTG) achieved an itaconate yield of 510% mol/mol (
Example 9: Metabolic Pathway Selection to Optimize Itaconate Production
[0125] To date, other than in organisms that natively produce itaconate, attempts to engineer strains for itaconate production have focused on heterologous expression the cis-aconitate decarboxylase (termed here the cis-pathway) from A. terreus. However, an alternate pathway for itaconate production was recently discovered in Ustilago maydis (Geiser et al., Microbial Biotech., 9.1 (2016): 116-126). This pathway, referred to here as the trans-pathway, proceeds through two steps. First, cis-aconitate is isomerized to the thermodynamically favorable isomer, trans-aconitate, by aconitate isomerase (adi1), which is subsequently decarboxylated by trans-aconitate decarboxylase (tad1) generating itaconate (
[0126] Taken together, the inventors hypothesized that the trans-pathway would improve itaconate production relative to the cis-pathway by providing a thermodynamically favorable route to divert carbon flux from the TCA cycle. To test this hypothesis, the inventors integrated codon-optimized tad1 (SEQ) ID NO: 106) and adi1 (SEQ ID NO: 105) genes under the control of the T7 promoter into strains JE3674 and JE3681 and assayed the resulting strains JE3715 and JE3719, respectively, for itaconate production (
TABLE-US-00007 TABLE 7 Production of itaconic acid from p-coumaric acid Stationary Overall Phase Hosted Molar Molar Mass Production Relevant Parent Yield Yield* Yield Titer Strain Pathway Genotype (mol/mol) (mol/mol) (g/g) (g/L) JE4305 P.sub.tac:cadA JE90(Pseudomonas putida 0.04 0.1 0.03 0.02 (cis) KT2440 hsdR::Bxb1int- attB) JE4306 P.sub.tac:cadA JE90 phaC.sub.1ZC.sub.2 0.12 0.33 0.09 0.12 (cis) JE4308 P.sub.tac:cadA JE90 phaC.sub.1ZC.sub.2 0.27 0.72 0.21 0.34 (cis) icd.sup.GTG:idh.sup.GTG JE4307 P.sub.tac:cadA JE90 phaC.sub.1ZC.sub.2 0.3 n.d.** 0.24 0.75 (cis) icd.sup.TTG:idh.sup.TTG JE3221 P.sub.T7:cadA JE90 0.09 0.18 0.07 0.22 (cis) P.sub.urtA:T7pol:P.sub.cat:lysY phaC.sub.1ZC.sub.2 JE3713 P.sub.T7:cadA JE90 0.29 0.79 0.23 0.81 (cis) P.sub.urtA:T7pol:P.sub.cat:lysY phaC.sub.1ZC.sub.2 icd.sup.GTG:idh.sup.GTG JE3715 P.sub.T7:tad1:adi1 JE90 0.43 1.02 0.34 1.09 (trans) P.sub.urtA:T7pol:P.sub.cat:lysY phaC.sub.1ZC.sub.2 icd.sup.GTG:idh.sup.GTG JE3717 P.sub.T7:cadA JE90 0.5 0.97 0.4 1.27 (cis) P.sub.urtA:T7pol:P.sub.cat:lysY phaC.sub.1ZC.sub.2 icd.sup.TTG:idh.sup.TTG JE3719 P.sub.T7:tad1:adi1 JE90 0.56 1.16 0.45 1.26 (trans) P.sub.urtA:T7pol:P.sub.cat:lysY phaC.sub.1ZC.sub.2 icd.sup.TTG:idh.sup.TTG *Stationary phase molar yield was calculated using itaconate yield from 24 to 96 hour time points. **Not Determined.
Example 10: Production of Itaconate from Depolymerized Lignin
[0127] To test the viability of itaconate production from lignin, we assayed the ability of strain JE3715 to upgrade a depolymerized lignin stream produced from an industrially-relevant lignocellulose deconstruction process (Rodriguez, Acs Sustain Chem Eng 5, 8171-8180 (2017)) to itaconate. Base-catalyzed depolymerization of washed lignin was performed as described previously (Rodriguez, Acs Sustain Chem Eng 5, 8171-8180 (2017)), and the resulting liquor (BCDL) was diluted with concentrated modified M9 salts containing either 2 or 3 mM NH.sub.4Cl. This medium was analyzed and found to contain 1.74 g/L p-coumarate, 0.5 g/L ferulic acid (ferulate), trace amounts of other monomeric carbon sources, and residual higher molecular weight lignin. JE3715, chosen as a compromise between itaconate yield from coumarate and productivity, was inoculated into shake flasks containing the two media variants and assayed for itaconate production. Production of itaconic acid leveled off at 48 hours with titers between 1.4 and 1.43 g/L (
Example 11: Production of Itaconic Acid and Trans-Aconitate from Diverse Substrates
[0128] Tables 8 and 9 summarize embodiments where itaconic acid (Table 8) and trans-aconitate (Table 9) was produced from diverse substrates using genetically engineered Pseudomonas strains. It is noted that the AG4074 strain has an exogenous nucleic acid comprising the itp1 gene (encoding an efflux pump for itaconic acid), and the AG4116 strain has an exogenous nucleic acid comprising the thrB gene (efflux pump for trans-aconitate).
TABLE-US-00008 TABLE 8 Production of Itaconic Acid from diverse substrates. Engineered strains were cultured on substrates encompassing a variety organic compound classes. Samples were collected following 72 hours growth and the final titer (g/L) of either itaconic acid or trans-aconitate was determined via HPLC. Genotypes of the engineered species are as follows:AG4001 AG4001:PP_4740::Bxb1-attL:kanR:PT7:cadA:attR ampC::Pr_4841_T7_RNAP-lysY(+) phaC1/Z/C2 icd(A1T):idh(A1T) ged::araE-araCDABE fpvA:xylE-xylDXBC, AG4074:KT2440 hsdR::Bxb1attL-KanR:Plac: itp1:Pt7:tad1:adi1-attR ampC::Pr_ 4841_T7_RNAP-lysY(+) phaC1/Z/C2 icd(A1G):idh(A1G). Substrate (Concentration) Glucose Xylose Arabinose Coumarate Ferulate Benzoate Acetate Succinate Octanoate Glycerol Strain (20 mM) (20 mM) (20 mM) (20 mM) (20 mM) (20 mM) (30 mM) (30 mM) (15 mM) (40 mM) AG4001 0.970 0.359 0.475 N/A N/A N/A N/A N/A N/A N/A AG4074 NA N/A N/A 1.522 1.237 0.740 0.053 0.254 0.140 0.644 Sugar Non- Non- Aromatic Aromatic Aromatic Organic Organic Fatty Biodiesel native native Monomer Monomer through Acid Acid Acid Waste sugar sugar alternate pathway Substrate Class
TABLE-US-00009 TABLE 9 Production of Trans-aconitate from diverse substrates. Engineered strains were cultured on substrates encompassing a variety organic compound classes. Samples were collected following 72 hours growth and the final titer (g/L) of either itaconic acid or trans-aconitate was determined via HPLC. Genotypes of the engineered species are as follows:AG4003:PP_4740::Bxb1-attL:kanR:PT7:adi1:attR ampC::Pr_4841_T7_RNAP-lysY(+) phaC1/Z/C2 icd(A1T):idh(A1T) gcd::araE-araCDABE fpvA:xylE-xylDXBC, AG4116:KT2440 hsdR::Bxb1attL-KanR:Plac: tbrB:Pt7:adi1-attR ampC::Pr_4841_T7_RNAP-lysY(+) phaC1/Z/C2 icd(A1G):idh(A1G). Substrate (Concentration) Glucose Xylose Arabinose Coumarate Ferulate Benzoate Acetate Succinate Octanoate Glycerol Strain (20 mM) (20 mM) (20 mM) (20 mM) (20 mM) (20 mM) (30 mM) (30 mM) (15 mM) (40 mM) AG4003 0.0117 0.0128 0.0003 N/A N/A N/A N/A N/A N/A N/A AG4116 N/A N/A N/A 1.106 0.796 0.680 0.086 0.470 0.604 0.670 Sugar Non- Non- Aromatic Aromatic Aromatic Organic Organic Fatty Biodiesel native native Monomer Monomer through Acid Acid Acid Waste sugar sugar alternate pathway Substrate Class