Biosynthesis of commodity chemicals from oil palm empty fruit bunch lignin

Abstract

The present invention relates to metabolic engineering of microbial hosts for the synthesis of products from oil palm empty fruit bunches (OPEFB). In one embodiment, the genetically engineered microorganism is Escherichia coli, which contains a metabolic pathway consisting of nine enzymes (11 genes) to utilize depolymerized lignin, namely vanillin, p-coumaric acid, p-hydroxybenzaldehyde, vanillic acid, p-hydroxybenzoic acid, and ferulic acid, to produce β-ketoadipate, which can then be converted into important derivatives such as adipic acid and levulinic acid. The enzymes are feruloyl-CoA synthetase (fcs), enoyl-CoA hydratase (ech), vanillin dehydrogenase (vdh), vanillate O-demethylase (vanAB; vanA and vanB), p-hydroxybenzoate hydroxylase (pobA), protocatechuate 3,4-dioxygenase {pcaGH; pcaG and pcaH), 3-carboxy-cis,cis-muconate cycloisomerase (pcaB), 4-carboxymuconolactone decarboxylase (pcaC), and β-ketoadipate enol-lactone hydrolase (pcaD).

Description

Technical Field

[0001] The present invention relates to methods for metabolically engineering microbial hosts to synthesize chemicals from oil palm lignin. In particular, the present invention relates to producing adipic acid and levulinic acid from lignocellulosic biomass in engineered Escherichia coli, and also provides recombinant cells prepared using such methods. Background Art

[0002] Lignocellulosic biomass is the most abundant renewable resource [Vardon et al., Energy & Environmental Science 8: 617-628 (2015);Deng et al., Biochemical Engineering Journal 105: 16-26 (2016)]. In particular, oil palm empty fruit bunches (OPEFB) (a by-product of palm oil production) are abundant lignocellulosic biomass that are primarily burned for energy and are considered waste. It is estimated that 1.1 tons of OPEFB are produced per ton of oil palm, totaling 57 million tons per year [Murphy, Journal of Oil Palm Research 26: 1-24 (2014);Coral Medina et al., Bioresource Technology 194: 172-178 (2015)]. Given their large availability, OPEFB is an attractive renewable lignocellulose source that can be used as a raw material in biorefining for the production of products. OPEFB can be converted into fermentable sugars [Li et al., Biotechnology and Applied Biochemistry 61:426-431 (2014)] and lignin extractives [Mohamad Ibrahim et al., CLEAN-Soil, Air, Water 36:287-291 (2008)] through simple and cost-effective pretreatment using chemicals and heat.

[0003] Li et al. explored the use of fermentable sugars derived from OPEFB to support cell growth in 2014 [Li et al., Biotechnology and Applied Biochemistry 61:426-431 (2014)], who combined the use of dilute acid and whole fungal cell culture-catalyzed hydrolysis to extract fermentable sugars from OPEFB. Hemicellulose was first stripped from OPEFB using acid hydrolysis, and the remaining cellulose-lignin complex was converted to glucose by cellulase in whole fungal cell culture. OPEFB-derived sugars were then used as a carbon source to cultivate Escherichia coli (E. coli) as a proof of concept. Mohamad Ibraim et al. explored the use of OPEFB-derived lignin in 2008, where lignin was extracted from OPEFB using 20% ​​sulfuric acid, followed by nitrobenzene oxidation to decompose the lignin. This extraction method releases a plethora of depolymerized lignin compounds, particularly vanillin, p-coumaric acid, p-hydroxybenzaldehyde, vanillic acid, p-hydroxybenzoic acid, and ferulic acid [Xu et al., ChemSusChem 5:667-675 (2012)]. Vanillin and p-coumaric acid are the main degradation products, with concentrations of approximately 1800 ppm (1.8 g / L) and approximately 1000 ppm (1.0 g / L), respectively [Mohamad Ibrahim et al., CLEAN-Soil, Air, Water 36:287-291 (2008)]. These compounds are useful substrates for the production of important organic acids. In summary, the reported studies indicate that OPEFB derivatives can potentially be used in biorefining processes, and microbial cells can be engineered to convert aromatic compounds into chemicals while utilizing fermentable sugars for cell growth ( Figure 1 However, the effective use of depolymerized lignin is hampered by the need for fractionation processes for further valorization as a chemical. Currently, great efforts have been made to develop effective fractionation processes, but these expensive processes may potentially undermine the economic viability and practicality of OPEFB lignin.

[0004] Adipic acid is a high-demand chemical used as a lubricant and as a precursor for nylon 6,6, polyester polyols, and plasticizers [Vardon et al., Energy & Environmental Science 8:617-628 (2015)]. Adipic acid has a market capacity of 2.6 million tons per year [Polen et al., J Biotechnol 167:75-84 (2013)] and was worth $5.56 billion in 2016 [Research, Adipic Acid Market Size, Share & Trends Analysis Report by Application (Nylon 66 Fiber, Nylon 66 Resin, Polyurethane, Adipate Ester), By Region (APAC, North America, Europe, MEA, CSA), and Segment Forecasts, 2018-2024 (2018), worldwidewebdotgrandviewresearchdotcom / industry-analysis / adipic-acid-market].On the other hand, levulinic acid had a market value of US$164 million in 2020 [MarketWatch, Levulinic Acid Market Size 2020: Top Countries Data, Definition, Detailed Analysis of Current Industry Figures with Forecasts Growth By 2026(2020), worldwidewebdotmarketwatchdotcom / press-release / levulinic-acid-market-size-2020-top-countries-data-definition-detailed-analysis-of-current-industry-figures-with-forecasts-growth-by-2026-2020-07-13] and is a multi-purpose chemical used in industrial products such as resins, plasticizers, textiles, animal feed, coatings, and as an antifreeze agent [Ghorpade and Hanna, Cereals: Novel Uses and Processes, GM Campbell, C. Webb and SL Mckee. eds. (Boston, MA: Springer 433 US), 49-55 (1997)].

[0005] There is a need to improve the economics of utilizing OPEFB and its underutilized lignin fraction to produce chemicals. Summary of the Invention

[0006] Here, a microbial-based bioprocess was designed that directly utilizes unfractionated depolymerized OPEFB lignin as a substrate for chemical production ( Figure 1 To achieve this goal, the inventors engineered E. coli to have repurposed anabolic pathways to serve as a multi-substrate biocatalytic platform that can act on multiple depolymerized OPEFB lignin components and focus them on the formation of desired primary products.

[0007] Here, the inventors have identified and constructed a metabolic pathway consisting of 9 enzymes (11 genes) that enables E. coli to utilize all 6 components of depolymerized lignin to produce the versatile precursor molecule β-ketoadipate via the β-ketoadipate pathway [Wells and Ragauskas, Trends Biotechnol 30:627-637 (2012)] and subsequently convert this intermediate into various important derivatives, such as adipic acid (reduction) and levulinic acid (decarboxylation).

[0008] In order to further improve bioconversion and simplify bioprocess, Escherichia coli cells are engineered to have regulatory elements that act as genetic controllers based on dynamic sensors. In engineered cells, the expression of enzymatic pathway genes is usually controlled by an induction system, in which artificial inducers are used to activate enzyme expression. Although the use of such inducers is effective, it is less advantageous due to its high cost and high toxicity and the corresponding increased bioprocess complexity. For this reason, a two-layer genetic controller [Lo et al., Cell Syst 3: 133-143 (2016)] is used, which regulates enzyme expression and therefore regulates bioconversion based on the availability of nutrients and OPEFB lignin derivatives. This enables engineered Escherichia coli to autonomously activate the bioconversion process when substrate is available, without the need for additional inducers.

[0009] The biosynthesis of chemicals using this intermediate was also demonstrated, with up to 9.5 mg / L adipic acid and 455.57 mg / L levulinic acid produced from the reconstituted OPEFB lignin mixture under fermentor-controlled conditions.

[0010] The microbial host, E. coli MG 1655, was also subjected to strain optimization, in which native E. coli genes involved in competing metabolic pathways were systematically deleted to improve bioproduction yields. Deletion of sucCD and atoDA resulted in the greatest improvements in adipic acid production and levulinic acid production, respectively.

[0011] The present disclosure relates to a platform for the bioproduction of chemicals from OPEFB lignin using E. coli.

[0012] Broadly, the platform can use E. coli MG 1655 and includes:

[0013] (a) a complete heterologous metabolic pathway utilizing depolymerization of lignin (vanillin, p-coumaric acid, p-hydroxybenzaldehyde, vanillic acid, p-hydroxybenzoic acid, and ferulic acid) to produce the intermediate precursor β-ketoadipate; and / or

[0014] (b) a pathway for converting β-ketoadipate to adipic acid or levulinic acid; and / or

[0015] (c) a genetic controller that regulates the expression of a heterologous gene in the presence of a substrate (i.e., a hydroxycinnamic acid, such as ferulic acid and p-coumaric acid); and / or

[0016] (d) The sucCD and atoDA genes have been deleted to eliminate metabolic competition for the bioproduction pathway.

[0017] According to a first aspect, the present invention provides an isolated genetically engineered microorganism for producing β-ketoadipate from depolymerized lignin, wherein the microorganism has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprising heterologous β-ketoadipate pathway genes, namely, feruloyl-CoA synthetase (fcs), enoyl-CoA hydratase (ech), vanillin dehydrogenase (vdh), vanillate O-demethylase (vanAB; vanA and vanB), p-hydroxybenzoate hydroxylase (pobA), protocatechuate 3,4-dioxygenase (pcaGH; pcaG and pcaH), 3-carboxy-cis,cis-muconate cycloisomerase (pcaB), 4-carboxymuconolactone decarboxylase (pcaC), and β-ketoadipate enol-lactone hydrolase (pcaD), operably linked to at least one promoter, wherein the genetically engineered microorganism can convert depolymerized lignin into β-ketoadipate.

[0018] In some embodiments, the isolated genetically engineered microorganism further comprises:

[0019] (a) heterologous β-ketoadipate utilization genes, namely β-ketoadipate succinyl-CoA transferase (pcalJ; pcal and pcaJ), 3-hydroxyacyl-CoA dehydrogenase (paaH1), enoyl-CoA hydratase (ech), trans-enoyl-CoA reductase (ter), phosphotransbutyrylase (ptb), and butyrate kinase 1 (buk1), operably linked to at least one promoter, wherein the genetically engineered microorganism can convert β-ketoadipate to adipate, and / or

[0020] (b) a heterologous β-ketoadipate utilization gene, i.e., acetoacetate decarboxylase (adc), operably linked to at least one promoter, wherein the genetically engineered microorganism can convert β-ketoadipate into levulinic acid.

[0021] In some embodiments:

[0022] The fcs gene encodes the amino acid sequence shown in SEQ ID NO: 2; and / or

[0023] The ech gene encodes the amino acid sequence shown in SEQ ID NO: 4; and / or

[0024] The vdh gene encodes the amino acid sequence shown in SEQ ID NO: 6; and / or

[0025] The vanA gene encodes the amino acid sequence shown in SEQ ID NO: 8; and / or

[0026] The vanB gene encodes the amino acid sequence shown in SEQ ID NO: 10; and / or

[0027] The pobA gene encodes the amino acid sequence shown in SEQ ID NO: 12; and / or

[0028] The pcaH gene encodes the amino acid sequence shown in SEQ ID NO: 14; and / or

[0029] The pcaG gene encodes the amino acid sequence shown in SEQ ID NO: 16; and / or

[0030] The pcaB gene encodes the amino acid sequence shown in SEQ ID NO: 18; and / or

[0031] The pcaC gene encodes the amino acid sequence shown in SEQ ID NO: 20; and / or

[0032] The pcaD gene encodes the amino acid sequence shown in SEQ ID NO: 22; and / or

[0033] The ter gene encodes the amino acid sequence shown in SEQ ID NO: 24; and / or

[0034] The pcal gene encodes the amino acid sequence shown in SEQ ID NO: 26; and / or

[0035] The pcaJ gene encodes the amino acid sequence shown in SEQ ID NO: 28; and / or

[0036] The paaH1 gene encodes the amino acid sequence shown in SEQ ID NO: 30; and / or

[0037] The ech gene encodes the amino acid sequence shown in SEQ ID NO: 32; and / or

[0038] The ptb gene encodes the amino acid sequence shown in SEQ ID NO: 34; and / or

[0039] The buk1 gene encodes the amino acid sequence shown in SEQ ID NO: 36; and / or

[0040] The adc gene encodes the amino acid sequence shown in SEQ ID NO: 38.

[0041] The asterisk at the C-terminus of the sequence indicates a stop or termination codon.

[0042] In some embodiments:

[0043] The fcs gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 1; and / or

[0044] The ech gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 3; and / or

[0045] The vdh gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 5; and / or

[0046] The vanA gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 7; and / or

[0047] The vanB gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 9; and / or

[0048] The pobA gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 11; and / or

[0049] The pcaH gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 13; and / or

[0050] The pcaG gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 15; and / or

[0051] The pcaB gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 17; and / or

[0052] The pcaC gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 19; and / or

[0053] The pcaD gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 21; and / or

[0054] The ter gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 23; and / or

[0055] The pcal gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 25; and / or

[0056] The pcaJ gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 27; and / or

[0057] The paaH1 gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 29; and / or

[0058] The ech gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 31; and / or

[0059] The ptb gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 33; and / or

[0060] The buk1 gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 35; and / or

[0061] The adc gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 37.

[0062] It is understood that the specific pathway genes described herein can be replaced by related genes encoding enzymes with equivalent catalytic functions. It is also understood that the nucleic acid sequences of the genes used in the pathways of the present invention can be codon-optimized for specific engineered host cells.

[0063] In some embodiments, the at least one promoter is regulated by a heterologous genetic controller. In some embodiments, the at least one promoter is a constitutive promoter, such as T7. It should be understood that there are other promoters that may be suitable for use in the present invention.

[0064] In some embodiments, the heterologous genetic controller is pBAD or hydroxycinnamic acid (HA). In some embodiments, the pBAD controller comprises the nucleotide sequence shown in SEQ ID NO: 41. In some embodiments, the HA controller comprises the nucleotide sequence shown in SEQ ID NO: 42.

[0065] In some embodiments, the isolated genetically engineered microorganism according to any aspect of the present invention further comprises an inactivated endogenous succinyl-CoA synthetase gene, such as sucCD and / or an inactivated β-ketoadipyl-CoA thiolase gene, such as paaJ. In some embodiments, the sucCD gene encodes the amino acid sequences set forth in SEQ ID NO: 54 and SEQ ID NO: 56 (respectively, SucC and SucD). In some embodiments, the sucCD gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity, or 100% sequence identity to the polynucleotide sequences set forth in SEQ ID NO: 53 and SEQ ID NO: 55 (respectively, sucC and sucD). In some embodiments, the paaJ gene encodes the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the paaJ gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity, or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO:51.

[0066] In some embodiments, the isolated genetically engineered microorganism according to any aspect of the present invention further comprises an inactivated endogenous acyl-CoA:acetate / 3-ketoacid-CoA transferase gene, such as atoDA. In some embodiments, the atoDA gene encodes the amino acid sequences shown in SEQ ID NO:48 and SEQ ID NO:50 (AtoD and AtoA, respectively). In some embodiments, the atoDA gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity, or 100% sequence identity to the polynucleotide sequences shown in SEQ ID NO:47 and SEQ ID NO:49 (atoD and atoA, respectively).

[0067] In some embodiments, the isolated genetically engineered microorganism comprises bacteria or yeast, preferably bacteria, such as Escherichia coli. In some embodiments, the bacteria is Escherichia coli MG1655.

[0068] In some embodiments, the depolymerized lignin is from fibrous oil palm empty fruit bunches.

[0069] According to another aspect, the present invention provides use of an isolated genetically engineered microorganism according to any aspect of the present invention for producing adipic acid or for producing levulinic acid.

[0070] According to another aspect, the present invention provides a recombinant vector comprising heterologous β-ketoadipate pathway genes, namely, fcs, ech, vdh, vanAB (vanA and vanB), pobA, pcaGH (pcaG and pcaH), pcaB, pcaC and pcaD, and / or

[0071] Heterologous β-ketoadipate utilization genes operably linked to at least one promoter, namely, pcalJ (pcal and pcaJ), paaH1, ech, ter, ptb, and buk1, and / or

[0072] A heterologous beta-ketoadipate utilization gene, ie, adc, is operably linked to at least one promoter.

[0073] In some embodiments:

[0074] The fcs gene encodes the amino acid sequence shown in SEQ ID NO: 2; and / or

[0075] The ech gene encodes the amino acid sequence shown in SEQ ID NO: 4; and / or

[0076] The vdh gene encodes the amino acid sequence shown in SEQ ID NO: 6; and / or

[0077] The vanA gene encodes the amino acid sequence shown in SEQ ID NO: 8; and / or

[0078] The vanB gene encodes the amino acid sequence shown in SEQ ID NO: 10; and / or

[0079] The pobA gene encodes the amino acid sequence shown in SEQ ID NO: 12; and / or

[0080] The pcaH gene encodes the amino acid sequence shown in SEQ ID NO: 14; and / or

[0081] The pcaG gene encodes the amino acid sequence shown in SEQ ID NO: 16; and / or

[0082] The pcaB gene encodes the amino acid sequence shown in SEQ ID NO: 18; and / or

[0083] The pcaC gene encodes the amino acid sequence shown in SEQ ID NO: 20; and / or

[0084] The pcaD gene encodes the amino acid sequence shown in SEQ ID NO: 22; and / or

[0085] The ter gene encodes the amino acid sequence shown in SEQ ID NO: 24; and / or

[0086] The pcal gene encodes the amino acid sequence shown in SEQ ID NO: 26; and / or

[0087] The pcaJ gene encodes the amino acid sequence shown in SEQ ID NO: 28; and / or

[0088] The paaH1 gene encodes the amino acid sequence shown in SEQ ID NO: 30; and / or

[0089] The ech gene encodes the amino acid sequence shown in SEQ ID NO: 32; and / or

[0090] The ptb gene encodes the amino acid sequence shown in SEQ ID NO: 34; and / or

[0091] The buk1 gene encodes the amino acid sequence shown in SEQ ID NO: 36; and / or

[0092] The adc gene encodes the amino acid sequence shown in SEQ ID NO: 38.

[0093] In some embodiments:

[0094] The fcs gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 1; and / or

[0095] The ech gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 3; and / or

[0096] The vdh gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 5; and / or

[0097] The vanA gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 7; and / or

[0098] The vanB gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 9; and / or

[0099] The pobA gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 11; and / or

[0100] The pcaH gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 13; and / or

[0101] The pcaG gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 15; and / or

[0102] The pcaB gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 17; and / or

[0103] The pcaC gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 19; and / or

[0104] The pcaD gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 21; and / or

[0105] The ter gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 23; and / or

[0106] The pcal gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 25; and / or

[0107] The pcaJ gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 27; and / or

[0108] The paaH1 gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 29; and / or

[0109] The ech gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 31; and / or

[0110] The ptb gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 33; and / or

[0111] The buk1 gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 35; and / or

[0112] The adc gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence shown in SEQ ID NO: 37.

[0113] According to another aspect, the present invention provides a kit comprising the isolated genetically engineered microorganism according to any aspect of the present invention or the recombinant vector according to any aspect of the present invention.

[0114] According to another aspect, the present invention provides a method for producing β-ketoadipate from depolymerized lignin, the method comprising the step of culturing a plurality of genetically engineered microorganisms according to any aspect of the present invention under conditions where the β-ketoadipate is produced.

[0115] According to another aspect, the present invention provides a method for producing adipic acid from depolymerized lignin, the method comprising the step of culturing a plurality of genetically engineered microorganisms according to any aspect of the present invention under conditions whereby the adipic acid is produced.

[0116] According to another aspect, the present invention provides a method for producing levulinic acid from depolymerized lignin, the method comprising the step of culturing a plurality of genetically engineered microorganisms according to any aspect of the present invention under conditions that produce the levulinic acid.

[0117] In some embodiments, the method of any aspect of the invention further comprises isolating the product produced by the genetically engineered microorganism.

[0118] In some embodiments, the microorganism comprises bacteria, such as Escherichia coli, preferably Escherichia coli MG1655.

[0119] In some embodiments of the production methods of the present invention, the depolymerized lignin is derived from fibrous oil palm empty fruit bunches.

[0120] The platform E. coli strain enables direct utilization of depolymerized lignin mixtures without the need for fractionation into separate components. In addition, in some embodiments, the use of genetic controllers allows for autonomous induction of gene expression, which reduces the cost of commonly used artificial inducers (such as IPTG). The advantage of the platform is that it is customizable, wherein other pathways that can convert the precursor β-ketoadipate can be easily implemented for the target chemical. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 An overview of chemical production using oil palm empty fruit bunches (OPEFB) is shown. The purpose of this study is indicated by the dotted box. The composition of depolymerized lignin is reported in Mohamad Ibrahim et al., CLEAN-Soil, Air, Water 36:287-291 (2008); and the cultivation of biocatalytic cells is reported in Li et al., Biotechnology and Applied Biochemistry 61:426-431 (2014).

[0122] Figure 2 A- Figure 2 B shows the production of protocatechuate and β-ketoadipate. (A) The anabolic pathways involved in the conversion of OPEFB lignin derivatives converge on a single intermediate (protocatechuate) (1) and a linear precursor (β-ketoadipate) (2) required for the production of adipate and levulinic acid. Pathway genes (bold) and enzymes are fcs (feruloyl-CoA synthetase), ech (enoyl-CoA hydratase), vdh (vanillin dehydrogenase), vanAB (vanillate O-demethylase), pobA (p-hydroxybenzoate hydroxylase), pcaGH (protocatechuate 3,4-dioxygenase), pcaB (3-carboxy-cis,cis-muconate cycloisomerase), pcaC (4-carboxymuconolactone decarboxylase), and pcaD (β-ketoadipate enol-lactonhydrolase). (B) Production of protocatechuate using EFB lignin components as substrates (normalized to theoretical yield). FA, ferulic acid; Van, vanillin; VA, vanillic acid; P-Ca, p-coumaric acid; P-HB, p-hydroxybenzaldehyde; P-HA, p-hydroxybenzoic acid.

[0123] Figure 3 Schematic diagram of the genetic constructs used in this study. Constructs for the controllers a) hydroxycinnamic acid controller and b) L-arabinose controller are shown, using the plasmid backbone from pBbS8a. Constructs for OPEFB utilization and linearization c) protocatechuic acid production system and d) beta-ketoadipate production system are shown, using the plasmid backbone from pBbE8k. Constructs for organic acid production e) levulinic acid production system and f) adipic acid production system are shown, using the plasmid backbone from Pacyc.

[0124] Figure 4 A- Figure 4C shows the construction and validation of a novel metabolic pathway for the production of adipate using β-ketoadipate in E. coli. (A) Biosynthetic pathway for adipate in E. coli, with expression of pathway enzymes and Western blots showing products from each cell extract. (B) In vitro enzyme assay for β-ketoadipate succinyl-CoA transferase (PcaI and PcaJ), where β-ketoadipyl-CoA:Mg 2+ The formation of α-enoyl-CoA reductase (Ter) was measured at 305 nm and normalized to the control and shown as relative activity (au). A: pACYCDuet; I: Pcal; J: PcaJ; De: denatured at 85°C for 1 h before use. (C) Characterization of trans-enoyl-CoA reductase (Ter) activity by measuring the final production level of adipic acid. Ctrl: cell extracts from pACYCD and pBbE8K; egTer: egTer extract was used as Ter; tdTer: tdTer extract was used as Ter; De: denatured at 85°C for 1 h before use; ND: not detected.

[0125] Figure 5 A- Figure 5 C shows the production of chemicals from β-ketoadipate. (A) The anabolic pathway converts the linear precursor (β-ketoadipate) into levulinic acid (1. decarboxylation) and adipic acid (2. reduction). Native enzymes that could potentially compete with the pathway were identified in the working host strain Escherichia coli MG1655 and deleted in the strain. Pathway genes (in bold) and enzymes are pcaIJ (3-ketoacetate-CoA transferase), paaH1 (3-ketoadipyl-CoA reductase), ech* (enoyl-CoA hydratase), ter (2,3-dehydroadipyl-CoA reductase), ptb (phosphotransbutyrylase), buk1 (butyrate kinase 1), and adc (acetoacetate decarboxylase). ech* is an enoyl-CoA hydratase distinct from the enoyl-CoA hydratase used in the protocatechuate pathway. (B) Evaluation of adipic acid production in the deletion strain. The deleted genes are fadE (acyl-CoA dehydrogenase), fadD (long-chain fatty acid-CoA ligase), paaJ (β-ketoadipyl-CoA thiolase), and sucCD (succinyl-CoA synthetase). (C) The E. coli ΔatoDA strain was selected because it ensures metabolic flux toward levulinic acid production.

[0126] Figure 6 Shown are (A) an alignment of PcaI and AtoD, which share 60.4% sequence similarity across 235 residues, and (B) an alignment of PcaJ and AtoA amino acid sequences, which share 52.5% sequence similarity across 236 residues.

[0127] Figure 7 A- Figure 7 C shows controllers for enzyme regulation. (A) Genetic circuit controller system. The L-arabinose (arabinose inducible) controller system is compared to the hydroxycinnamic acid (lignin substrate inducible) controller system. The genetic controller activates the expression of T7 polymerase, which in turn controls the expression of enzymes required for bioconversion and depolymerization of lignin. (B) Bioconversion of p-coumarate to adipic acid using E. coli ΔsucCD with a controller system for regulating enzyme expression. (C) Bioconversion of p-coumarate to levulinic acid using E. coli ΔatoDA with a controller system for regulating enzyme expression.

[0128] Figure 8 A- Figure 8 C shows (A) the overall strategy for improving the biosynthesis of adipic acid or levulinic acid by engineered microorganisms using depolymerized EFB lignin derivatives. In a bioreactor, (B) the biosynthesis of adipic acid and levulinic acid and (C) the utilization of depolymerized EFB lignin derivatives were quantified. Figure 2 As outlined in the convergent pathway in

[15] , six aromatic compounds are converted to protocatechuic acid (PCA). pCA: p-coumaric acid, pHB: p-hydroxybenzaldehyde, pHA: p-hydroxybenzoic acid, FA: ferulic acid, Van: vanillin, VA: vanillic acid.

[0129] Figure 9 shows the cell growth at a given time point by equilibrium (expressed by OD 600 The production titer of adipic acid or levulinic acid was used to optimize the OPEFB lignin feed.

[0130] Figure 10 The plasmid map of S8a-controller-T7 RNAP is shown.

[0131] Figure 11 The plasmid map of E8k-BKA v3 C10 is shown.

[0132] Figure 12 The plasmid map of pACYC-adipate (tdTer) is shown.

[0133] Figure 13 The plasmid map of pACYC-T7p-adc (without lacO) is shown.

[0134] Figure 14 An engineered production pathway for adipic acid and levulinic acid from depolymerized OPEFB lignin according to the present invention is shown. DETAILED DESCRIPTION

[0135] definition

[0136] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0137] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0138] As used herein, the terms "comprising" or "including" should be interpreted as specifying the presence of the stated features, integers, steps or components mentioned, but do not exclude the presence or addition of one or more features, integers, steps or components or groups thereof. However, in the context of the present disclosure, the terms "comprising" or "including" also include "consisting of." Variants of the word "comprising" (such as "comprise" and "comprises") and variants of "including" (such as "include" and "includes") have correspondingly varied meanings.

[0139] The term "isolated" is defined herein as a component (e.g., a nucleic acid, peptide, or protein) that is substantially separated from, produced separately from, or purified from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins) in the cells of an organism in which the biological component naturally occurs. Isolated nucleic acids, peptides, and proteins therefore include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0140] As used herein, the terms "nucleotide," "nucleic acid," or "nucleic acid sequence" refer to an oligonucleotide, a polynucleotide, or any fragment thereof; to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or antisense strand; to peptide nucleic acid (PNA); or to any DNA-like or RNA-like material.

[0141] Several pathway enzymes contain two subunits encoded by two separate genes. As used herein, the two subunit genes may be referred to, for example, as sucCD, or alternatively, as sucC and sucD, respectively. Similarly, pathway enzymes may be referred to by their names, such as succinyl-CoA synthetase or SucCD. Throughout this disclosure, it will be understood that if an enzyme is to be inactivated, the inactivation may be achieved by various means, including, for example, deleting one or more genes encoding the enzyme subunits, or mutating the gene coding sequence to produce an inactive truncated or nonsense peptide.

[0142] As used herein, the term "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under appropriate conditions. For example, a control sequence that is "operably linked" to a protein coding sequence is connected to the protein coding sequence so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence. For example, when a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, operably linked DNA sequences are contiguous and, in the case where two protein coding regions need to be joined, are in the same reading frame.

[0143] As used herein, the term "amino acid" or "amino acid sequence" refers to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, as well as a naturally occurring or synthetic molecule. When "amino acid sequence" is described herein as referring to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms are not intended to limit the amino acid sequence to the complete native amino acid sequence associated with the described protein molecule.

[0144] As used herein, the terms "polypeptide", "peptide" or "protein" refer to one or more amino acid chains, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein the polypeptide or peptide may comprise multiple chains non-covalently and / or covalently linked together by peptide bonds (the multiple chains having the sequence of a natural protein (i.e., a protein produced by naturally occurring and, in particular, non-recombinant cells or by genetically engineered or recombinant cells)), and include molecules having the amino acid sequence of a natural protein or molecules having one or more amino acids deleted, added and / or substituted from the natural sequence. A "polypeptide", "peptide" or "protein" may comprise one (referred to as a "monomer") or multiple (referred to as a "multimer") amino acid chains.

[0145] For convenience, the bibliographical references mentioned in this specification are listed in the form of a list of references and attached at the end of the examples. The entire contents of such bibliographical references are incorporated herein by reference. Any discussion of the prior art is not an admission that the prior art is part of the common general knowledge in the field of the present invention.

[0146] The vector may comprise one or more catalytic enzyme nucleic acids in a form suitable for expressing the one or more nucleic acids in a host cell. Preferably, the recombinant expression vector comprises one or more regulatory sequences operably linked to one or more nucleic acid sequences to be expressed. The term "regulatory sequence" includes promoters, enhancers, ribosome binding sites and / or IRES elements and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct the constitutive expression of the nucleotide sequences disclosed in the Examples herein, such as the T7 promoter. The design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, etc. The expression vector of the present invention may be introduced into a host cell to produce a protein or polypeptide encoded by the nucleic acid as described herein, including a fusion protein or polypeptide (e.g., a catalytic enzyme protein).

[0147] The recombinant expression vectors of the present invention can be designed to catalyze the expression of enzyme proteins in prokaryotes or eukaryotic cells, more particularly in prokaryotes. For example, the polypeptides of the present invention can be expressed in bacteria (e.g., cyanobacteria) or yeast cells. Suitable host cells are further discussed in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California.

[0148] The methods described above utilize enzymes to catalyze a series of reactions. While these reactions can be performed individually or, more particularly, in combination of two or more, it is particularly preferred that all reactions be combined in a single pot to form a cascade reaction sequence that provides the product from the initial starting materials, thereby eliminating the need for intermediate isolation and potentially increasing the overall yield of the reaction sequence.

[0149] The engineered cells of the present invention further comprise inactivated genes to limit the host cell's utilization of intermediate compounds in other biosynthetic pathways and reduce the yield of the desired end product. The engineered cells can comprise inactivated endogenous succinyl-CoA synthetase genes, such as sucCD (sucC, SEQ ID NO: 53, and sucD, SEQ ID NO: 55) and / or inactivated β-ketoadipyl-CoA thiolase genes, such as paaJ (SEQ ID NO: 51) (if adipic acid is the intended product), or inactivated endogenous acyl-CoA:acetate / 3-ketoacid-CoA transferase genes, such as atoDA (atoD, SEQ ID NO: 47, and atoA, SEQ ID NO: 49) (if levulinic acid is the intended target product).

[0150] Having now generally described the invention, the same will be more readily understood by reference to the following examples which are provided by way of illustration and are not intended to be limiting of the invention.

[0151] Those skilled in the art will appreciate that the present invention can be practiced without undue experimentation according to the methods given herein. The methods, techniques, and chemicals are as described in the references given or in the protocols in standard biotechnology and molecular biology textbooks.

[0152] Example

[0153] Example 1

[0154] Materials and methods

[0155] Plasmid assembly

[0156] The plasmid backbone used in this study was the BglBrick vector [Lee et al., Journal of Biological Engineering 5:12 (2011)] pBbE8k and pBbE8a from the Joint BioEnergy Institute, USA. Cloning and modification of DNA segments (such as promoters, genes, and terminators) required the use of splicing overlap extension (SOE) technology [Heckman and Pease, Nature Protocols 2:924-932 (2007)]. Biological segments were PCR cloned from genomic templates of Pseudomonas putida KT2440 and Escherichia coli K-12 MG1655, or assembled using gene fragments (gBlocks) SOEs from Integrated DNA Technologies, USA. They were converted into BglBrick standards consisting of universal linkers (such as EcoRl, BglII, BamHI, and Xhol restriction sites) for assembly. The standard BglBrick assembly method described by Anderson et al. (2010) [Anderson et al., Journal of Biological Engineering 4:1 (2010)] was used to assemble Figure 3The genetic constructs listed in . The recombinant BglBrick plasmid is chemically transformed into Escherichia coli K-12 TOP10 (Invitrogen, the U.S.). First, the transformed E. coli strain is cultivated in Luria-Bertani (LB) broth at 37 ° C and 225 rpm and screened via colony PCR. Gene deletion is introduced using the previously described method [Datsenko and Wanner, PNAS USA 97: 6640-6645 (2000), which is incorporated herein by reference. The bacterial strains and plasmids used in this study are listed in Table 1.

[0157] Table 1

[0158]

[0159] 1 Lo et al. (2016). A Two-Layer Gene Circuit for Decoupling Cell Growth from Metabolite Production. Cell Syst 3:133-143.

[0160] Preparation of cell extracts for in vitro enzyme assays for validation of the adipate pathway.

[0161] E. coli BL21 (DE3) was transformed with each plasmid carrying one of the genes encoding PcaI, PcaJ, PaaH, Ech, egTer, tdTer, Ptb, or Buk1 in pBbE8k or pACYCDuet-1 (Novagen, Germany). Each gene was from Pseudomonas putida KT2440 (PcaI and PcaJ), Ralstonia eutropha (PaaH1), Ralstonia eutropha H16 (Ech), Euglena gracilis (egTer), Treponema denticola (TdTer), or Clostridium acetobutylicum (Ptb and Buk1). The seed culture of the transformant was prepared by cultivating overnight at 37°C and 225rpm in LB medium supplemented with appropriate antibiotics (30 μg / L kanamycin or 50 μg / L ampicillin). The seed culture was diluted 1:100 (v / v) into Terrific Broth medium supplemented with appropriate antibiotics (30 μg / L kanamycin or 50 μg / L ampicillin) and cultivated at 37°C and 225rpm. The diluted E. coli culture was heated to 400 ℃ with 0.1 mM IPTG at OD 600The culture was induced at 0.5-1.0 and incubated at 16°C and 225 rpm for 24 h. The culture was harvested and resuspended with 0.5 mL of lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM DTT and 10% (v / v) glycerol, pH 7.5, final concentration) and incubated with 1.5 mg / mL lysozyme at 25°C and 150 rpm for 1 h. After adding 0.1% Triton X-100 and 1x protease inhibitor (Promega), the culture was purified by FastPrep-24 TM The soluble fraction of the crude cell extract was prepared using 5G (MP Biomedicals) and acid-washed beads (≤106 μm) (Sigma-Aldrich) at 6.5 m / s and 45 s, followed by centrifugation at 4°C and 13,000 rpm for 10 min. Total protein in the soluble extract was manually quantified using Bradford reagent (Sigma-Aldrich). Overexpression of each gene was verified by SDS-PAGE.

[0162] In vitro enzyme assay of β-ketoadipate succinyl-CoA transferase

[0163] The activity of β-ketoadipate succinyl-CoA transferase (subunit PcaI and subunit PcaJ) was determined as previously described [MacLean et al., Appl Environ Microbiol 72:5403-5413 (2006)], which is incorporated herein by reference with slight modifications. Briefly, the reaction was started by adding a reaction mixture (200 mM Tris-HCl, 0.4 mM succinyl-CoA, 40 mM MgSO4, and 1 g / L β-ketoadipate, pH 8.0, final concentration) to an aliquot of the cell extract to a final volume of 0.1 mL. The β-ketoadipate:MgCl2+ was monitored at 305 nm and 30 ° C using a Biotek Synergy H1m microplate reader. 2+ The formation of lasted for 4 min.

[0164] In vitro adipate production

[0165] In vitro adipate production was performed as previously described [Yu et al., Biotechnol Bioeng 111:2580-2586 (2014)], which is incorporated herein by reference with the following modifications. Each cell extract of PcaI, PcaJ, PaaH1, Ech, Ter (egTer or tdTer), Ptb, and Buk1 (equivalent to 0.05 mg total protein) was added to a reaction mixture (50 mM potassium phosphate buffer, 0.4 mM succinyl-CoA, 4 mM NADH, 2 mM ADP, and 0.5 g / L β-ketoadipate, pH 7.0, final concentration) to a final volume of 0.2 mL and incubated at room temperature for 24 h. Subsequently, each sample was mixed to a volume of 0.5 mL with 0.2 mL 1 M HCl and an internal standard (1,14-tetradecanedioic acid) and vortexed for 30 s. After adding 0.5mL ethyl acetate, sample is fully vortexed for 1min, centrifuged for 1min at 13,000rpm subsequently.Then, aliquot 0.35mL ethyl acetate fraction and by using rotary evaporator to evaporate, be resuspended in 0.04mL ethyl acetate subsequently.By resuspended sample and N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) with 1:1 (v / v) ratio mixing, and at room temperature derivatize 24h.Use GC-MS to analyze the formation of adipic acid.

[0166] Shake flask adipic acid production for engineered host screening

[0167] Overnight seed cultures were diluted 1:100 (v / v) into 50 mL of M9 medium supplemented with 0.2% (w / v) glucose, 0.2% (w / v) casamino acids, and appropriate antibiotics (100 μg / L carbenicillin, 50 μg / L kanamycin, and 25 μg / L chloramphenicol) in 250 mL baffled flasks and incubated at 30°C and 225 rpm. 0.2% (w / v) L-arabinose was added to the culture medium at OD 600 The engineered E. coli culture was induced at 1.2-1.5, and then p-coumaric acid substrate was added to a final concentration of 0.1% (w / v). Samples were taken at 18 h and 36 h. The formation of adipic acid was analyzed by GC-MS.

[0168] Biotransformation via engineered cells carrying different controllers

[0169] The engineered E. coli MG1655 cells were first grown in M9 medium (supplemented with 0.2% (w / v) glucose and 0.2% (w / v) casamino acids) to an OD of 600 The inoculum was added to the shake flask (37°C, 225 rpm) to a final concentration of OD 6000.01, each flask contained 50 mL of M9 medium supplemented with 0.2% (w / v) glucose as a carbon source, 0.2% (w / v) casamino acids, and relevant lignin derivatives as substrates. p-Coumaric acid (Sigma-Aldrich, USA) was first dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10% (w / v), and then added to the M9 medium to a final concentration of 0.1% (w / v). After reaching OD 600 After 1.0, the L-arabinose system and the HA control system were induced with 0.2% (w / v) L-arabinose or 0.1% (w / v) p-coumarate, respectively. One milliliter of the biotransformation culture was extracted at each time point (18 h and 36 h) for GC-MS measurement.

[0170] Reconstitution of OPEFB-depolymerized lignin mixtures

[0171] OPEFB depolymerized lignin mixtures were reconstituted based on the identified aromatic compound concentrations reported in Mohamad Ibrahim et al. [Mohamad Ibrahim et al., CLEAN_-Soil, Air, Water 2=36:287-291 (2008)]. Briefly, individual compounds of OPEFB were prepared separately and then mixed together to give the final concentrations described in Table 2.

[0172] Table 2. Protocatechuic acid (PCA) production at 36 h using EFB lignin derivatives as substrates. The concentrations of substrates used represent the concentrations found in pretreated depolymerized EFB lignin.

[0173]

[0174]

[0175] All individual compounds were purchased from Sigma-Aldrich with a purity > 97% and prepared in DMSO at a concentration that limited DMSO to 1% (v / v) in the final lignin mixture solution. All stock solutions of the compound were kept at 4 ° C in aliquots before use. Ten milliliters of a lignin mixture were prepared in DMSO to include: 1.8 g vanillin (catalog number 94752), 1 g p-coumaric acid (≥98% (HPLC), catalog number C9008), 320 mg p-hydroxybenzaldehyde (4-hydroxybenzoic acid; ≥99%, catalog number 240141), 110 mg vanillic acid (4-hydroxy-3-methoxybenzoic acid; ≥97% (HPLC), catalog number 94770), 18 mg p-hydroxybenzoic acid (3,4-dihydroxybenzoic acid; ≥98%, catalog number 37580) and 13 mg ferulic acid (trans-ferulic acid; 99%, catalog number 128708). The solution was vortexed to ensure homogenization of the mixture. The mixture was diluted 100-fold in the reaction volume to yield a final substrate concentration and is referred to as "1 x OPEFB".

[0176] Biotransformation of engineered cells using OPEFB in batch culture.

[0177] In 5L working volume Batch fermentation is carried out in B-DCU II tabletop bioreactor (Sartorius Stedim) for bioconversion to produce adipic acid or levulinic acid.The temperature is maintained at 30 ℃, and by automatically adding acid (1M H2SO4) and alkaline solution (1M NaOH), pH is controlled at 7.0.With 10L / min continuous oxygen supply, and impeller speed is set to 400rpm to guarantee uniform aeration.In culture, defoamer (200 μ L) is added to prevent excessive foaming.The bacterial culture of related through engineering approaches Escherichia coli MG1655 cell is incubated overnight at 30 ℃, and is subsequently transferred to 1L and contains 3 kinds of antibiotics (100mg / L carbenicillin, 50mg / L kanamycin and 25mg / L chloramphenicol) in the fresh culture medium. For engineered E. coli fermentations using an L-arabinose control loop, substrate (OPEFB lignin mixture) and inducer (0.2% (w / v) L-arabinose) were added 4 h after inoculation when the culture reached late logarithmic phase. For engineered E. coli fermentations using an HA control loop, substrate was fed to the vessel immediately after inoculation. Aliquots of the samples were taken at 18 h, 36 h, and 42 h for further analysis using GC-MS and absorbance was measured at 600 nm. Batch fermentations were run in duplicate, and the results were reported as mean and standard deviation.

[0178] HPLC quantification

[0179] The quantification of ferulic acid, p-coumaric acid, vanillin, vanillic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid and protocatechuic acid is carried out using the scheme adopted by Barghini et al. [Barghini et al., Microbial Cell Factories 6:13 (2007)], which is incorporated herein by reference with modifications. First, the batch culture extracted by 0.4 mL was sterilized by filtration with a 0.22 μm filter (Sartorius Stedim, Germany) and then analyzed by an Agilent 1260 HPLC device equipped with an Inertsil ODS3 C18 reversed-phase column (250 mm in length, 4.6 mm in diameter and 5 μm in particle size) and a diode array detector (DAD). The compound in the filtered culture was eluted with an isocratic pressure of 150 bar, a mobile phase comprising 35% methanol and 1% acetic acid, and a flow rate of 1 mL / min. Detection was performed at UV wavelengths of 300 nm (ferulic acid, p-coumaric acid, vanillin, vanillic acid, p-hydroxybenzaldehyde) and 254 nm (p-hydroxybenzoic acid, protocatechuic acid), with a sample injection volume of 10 μl. The retention times of the samples were compared with those of purified standards (Sigma-Aldrich, USA) for identification and quantification.

[0180] Identification and quantification were performed by gas chromatography-mass spectrometry (GC-MS).

[0181] To extract organic acids (β-ketoadipate, adipic acid, and levulinic acid) for detection, 500 μL of 1 M HCl, 300 μL of ethyl acetate, and 100 μL of internal standard (1,14-tetradecanedioic acid) were added to 1 mL of cell culture sample. TM 5G and acid-washed beads (≤106 μm, run 4 times at 6.5 m / s and 1 min intervals) to disrupt the cells and centrifuge at 20,000×g for 10 min at 4° C. to separate the organic phase. The ethyl acetate extract was incubated overnight with a derivatizing agent (BSTFA with 1% trimethylchlorosilane (TCMS)) and then analyzed by gas-liquid chromatography (GC) using an Agilent 7890B GC system equipped with an HP-5MS column (Agilent) coupled to a mass spectrometer (Agilent 5977).

[0182] Example 2

[0183] Enzymatic pathways enabling utilization of OPEFB lignin

[0184] As a first step to convert depolymerized OPEFB lignin into chemicals, a 9-enzyme pathway ( Figure 2 A. Figure 3 d) and assembled in the workhorse of industrial biotechnology, E. coli K-12 MG1655. The E. coli K-12 MG1655 strain containing this metabolic pathway was examined for its ability to utilize all OPEFB lignin derivatives, convert them into a single compound, protocatechuic acid, and subsequently convert protocatechuic acid into the linear precursor β-ketoadipate.

[0185] First, a convergent pathway was constructed, which included feruloyl-CoA synthetase (Fcs), enoyl-CoA hydratase (Ech), vanillin dehydrogenase (Vdh), vanillate O-demethylase (VanAB), and p-hydroxybenzoate hydroxylase (PobA) ( Figure 3 C). To demonstrate the feasibility of the constructed pathway in a microbial host, we first verified the bioconversion of a single lignin substrate for protocatechuic acid production ( Figure 2 , Table 2), and subsequently verified the bioconversion of OPEFB lignin derivatives. The results demonstrated that the convergent pathway was able to utilize all OPEFB lignin derivatives—vanillic acid, ferulic acid, p-hydroxybenzaldehyde, p-coumaric acid, p-hydroxybenzoic acid, and vanillin (in descending order of conversion efficiency)—and convert them into a single intermediate molecule, protocatechuic acid. The most efficient conversions were observed with vanillic acid and p-coumaric acid, yielding approximately 100% and 70% of theoretical yields, respectively (Table 2).

[0186] When testing OPEFB lignin derivatives (prepared at naturally occurring ratios after pretreatment), up to 400 mg / L protocatechuic acid was detected, reaching 11.5% of the theoretical yield. The lower-than-expected yield was primarily due to the inefficient utilization of vanillin, where, despite its high initial concentration (1.8 g / L), only 2.7% of the molar conversion to protocatechuic acid was observed. Since high concentrations of vanillin have been reported to inhibit bacterial growth [Zaldivar et al., Biotechnology and Bioengineering 65:24-33 (1999)], a possible approach to improving vanillin utilization is to oxidize the depolymerized OPEFB lignin mixture, especially vanillin [Fargues et al., Chemical Engineering & Technology 19:127-136 (1996)] to a less toxic compound, vanillic acid, before feeding it to engineered cells. Since vanillic acid has been shown to be fully converted, this approach can improve the yield of bioproduction and reduce toxicity to host cells. However, these approaches were not fully explored in this study, as the aim of this study was to first confirm the feasibility of direct conversion of OPEFB lignin mixtures.

[0187] After the successful production of protocatechuic acid from OPEFB lignin derivatives, a dearomatization pathway involving protocatechuate 3,4-dioxygenase (PcaGH), 3-carboxy-cis,cis-muconate cycloisomerase (PcaB), 4-carboxymuconolactone decarboxylase (PcaC), and β-ketoadipate enol-lactonate hydrolase (PcaD) was demonstrated in subsequent experiments. Figure 2 ) works together with the organic acid production pathway.

[0188] Example 3

[0189] De novo organic acid production pathway starting from β-ketoadipate in Escherichia coli

[0190] Direct biosynthesis of adipic acid from carbon sources in Escherichia coli has been reported [Yu et al., Biotechnol Bioeng 111:2580-2586 (2014); Cheong et al., Nat Biotechnol 34:556-561 (2016); Zhao et al., Metabolic Engineering 47:254-262 (2018)], in which an artificial adipic acid synthesis pathway was constructed to convert glucose or glycerol into adipic acid. In a recent study, Niu et al. [Niu et al., Metabolic Engineering 59:151-161 (2020)] successfully demonstrated the production of adipic acid from β-ketoadipate in Pseudomonas putida KT2440. Adapted from these findings, an adipic acid production pathway was constructed and validated in Escherichia coli ( Figure 4 A). The constructed pathway utilizes β-ketoadipate by: (1) esterification with CoA by β-ketoadipate succinyl-CoA transferase (PcaIJ); (2) subsequent reduction of the 3-oxo group by 3-hydroxyacyl-CoA dehydrogenase (PaaH1), enoyl-CoA hydratase (Ech), and trans-enoyl-CoA reductase (Ter); and (3) removal of CoA to form adipate by phosphotransbutyrylase (Ptb) and butyrate kinase 1 (Buk1). To test this complete pathway, the six enzymes ( Figure 4 A) and subsequently characterized their activities. The in vitro enzyme activities for β-ketoadipate degradation and 3-oxo reduction to adipic acid were measured ( Figure 4 B. Figure 4C). We observed that 3-oxo reduction required screening for a suitable reductase, Ter, responsible for converting 2,3-dehydroadipyl-CoA to adipoyl-CoA. Adipate (1.18 mg / L) was detected only when Ter from Treponema denticola (TdTer) was used, whereas no adipate was detected when Ter from Euglena (EgTer) was used. Through this systematic in vitro enzyme characterization, we validated a novel enzymatic pathway from β-ketoadipate to adipate ( Figure 5 A).

[0191] Unlike the adipate pathway, levulinic acid production involves a single decarboxylation step starting from β-ketoadipate. This reaction is catalyzed by acetoacetate decarboxylase (Adc) from Clostridium acetobutylicum [Cheong et al., Nat Biotechnol 34:556-561 (2016)]. Under shake flask conditions, the level of levulinic acid exceeded 60 mg / L (100 mg / L) within 36 h of bioconversion. Figure 5 C).

[0192] Example 4

[0193] Host Engineering for Optimized Chemical Production

[0194] To promote the conversion of β-ketoadipate, the existing natural metabolic pathways in E. coli need to be repurposed to direct the reduction and decarboxylation pathways ( Figure 5 A). This involved using the E. coli K-12 MG1655 reference genome model in the EcoCyc database [Keseler et al., Nucleic Acids Research 45:D543-D550 (2016)] to search for potential native genes that might be able to divert intermediates or cofactors to other products. We hypothesized that native genes of E. coli could compete and negatively impact the designed pathway, i.e., fadE, fadED (fadE; SEQ ID NO:43 and fadD; SEQ ID NO:45), paaJ (SEQ ID NO:51), and sucCD (sucC; SEQ ID NO:53 and sucD; SEQ ID NO:55) for adipic acid production; and atoDA (atoD; SEQ ID NO:47 and atoA; SEQ ID NO:49) for levulinic acid production. We assessed the impact of each gene deletion based on the bioconversion of p-coumaric acid, and the amount produced was used as an indicator of the efficiency of the pathway repurposing ( Figure 5 B. Figure 5 C).

[0195] For adipate production, the acyl-CoA dehydrogenase (fadE) and long-chain fatty acid-CoA ligase (fadD) genes were targeted because these genes are involved in fatty acid metabolism and may potentially utilize a six-carbon dicarboxylic acid (adipic acid) for β-oxidation [Lennen et al., Biotechnol Bioeng 106: 193-202 (2010); Sathesh-Prabu and Lee, J Agric Food Chem 63: 8199-8208 (2015)]. This metabolism may potentially utilize a six-carbon dicarboxylic acid (adipic acid) for β-oxidation [Smit et al., Biotechnol Lett 27: 859-864 (2005)]. However, deletion of these genes did not significantly improve adipate production. With the focus on directing flux toward β-ketoadipyl-CoA, AtoDA (AtoD; SEQ ID NO:48 and AtoA; SEQ ID NO:50), which share approximately 50% amino acid sequence similarity with the engineered PcaIJ (Pcal; SEQ ID NO:26 and PcaJ; SEQ ID NO:28), were inactivated. However, the presence of the atoDA gene was found to play a key role in initiating this new pathway, as deletion completely abolished adipate production. Since succinyl-CoA is an important cofactor in the formation of β-ketoadipyl-CoA, sucCD (sucC; SEQ ID NO: 53 and sucD; SEQ ID NO: 55, which encode subunits of succinyl-CoA synthetase) was deleted to minimize the competitive conversion of succinyl-CoA to succinate [Birney et al., J Bacteriol 178: 2883-2889 (1996); Zhao et al., Metabolic Engineering 47: 254-262 (2018)]. β-ketoadipyl-CoA thiolase (paaJ; SEQ ID NO: 51) was also targeted for deletion due to its role in the reversible catalysis of β-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA [Yu et al., Biotechnol Bioeng 111: 2580-2586 (2014); Babu et al., Process Biochemistry 50: 2066-2071 (2015)]. Among the list of genes that could potentially shunt intermediates from the introduced reduction reaction, sucCD deletion resulted in the greatest improvement in adipic acid production. The sucCD mutant was able to convert the substrate with approximately 3-fold greater efficiency than the other mutants, as shown by the higher yield observed at the early time point (18 h) ( Figure 5 B).

[0196] For levulinic acid production, the atoDA gene from the acetoacetate degradation pathway in E. coli was targeted for deletion because acetoacetate decarboxylase (Adc) [Cheong et al., Nat Biotechnol 34:556-561 (2016)] is used to convert β-ketoadipate. The atoDA genes were targeted because they share >50% amino acid sequence similarity with the enzyme encoded by pcaIJ based on sequence alignment ( Figure 6 Based on the similarity, the deletion was expected to promote the expected decarboxylation. In fact, the atoDA deletion strain was able to promote an increase in levulinic acid by approximately 40% ( Figure 5 C).

[0197] In summary, the results of the host engineering approach showed that the host with sucCD deletion was suitable for adipic acid production, and the host with atoDA deletion was suitable for levulinic acid production. These two strains were used in subsequent downstream optimization experiments.

[0198] Example 5

[0199] Genetic controllers enabling autonomous OPEFB lignin-derived hydroxycinnamic acid-dependent regulation

[0200] During pathway validation and host engineering experiments, an inducible system based on L-arabinose [Guzman et al., J Bacteriol 177:4121-4130 (1995)] inducer was used to regulate the expression of pathway enzymes in a dose-dependent manner. The role of the genetic controller is to regulate downstream gene transcription through the expression of phage-based T7 polymerase ( Figure 7 A). Since the organic acid production pathway is long (15 enzymes for adipic acid production and 10 enzymes for levulinic acid production), in order to ensure good transcription of all genes, the genes are placed under a non-natural strong T7 promoter, which is recognized by the T7 polymerase to initiate downstream transcription. However, uncontrolled expression of the T7 polymerase may lead to overexpression of the target protein, which may increase the burden on the host cell [Kesik-Brodacka et al., Microbial Cell Factories 11: 109 (2012)]; therefore, expression must be regulated by a genetic controller that limits T7 polymerase transcription via external inputs (such as chemical inducers).

[0201] Although the L-arabinose controller (pBAD) is an effective genetic device, it requires an additional external resource, i.e., L-arabinose as an inducer, thus increasing the deployment cost of the biocatalytic cell. To improve the economic efficiency of OPEFB lignin utilization, we considered adopting the hydroxycinnamic acid (HA) controller system reported by Lo et al. in 2016 [Lo et al., Cell Syst 3:133-143 (2016)]. The HA controller system can be induced by HA (such as ferulic acid and p-coumaric acid), which is present in depolymerized OPEFB lignin.

[0202] For comparison, we tested both the L-arabinose (pBAD; SEQ ID NO: 41) controller and the HA (SEQ ID NO: 42) controller for adipic acid and levulinic acid production in optimized host strains (ΔsucCD and ΔatoDA, respectively) under shake flask conditions at 30°C and using p-coumaric acid (final concentration, 1 g / L) as a substrate (Table 1). The fermentation temperature was set to 30°C instead of the commonly used 37°C for two reasons: 1) less energy is required to maintain the lower temperature without affecting the growth of the engineered E. coli, and 2) the unstable compound β-ketoadipate may have a longer enzymatic conversion half-life at lower temperatures. The L-arabinose-induced controller performed better than the HA controller in terms of product yield: a 2-fold higher titer ( Figure 7 B. Figure 7 C) We hypothesize that the concentration of inducer L-arabinose used in the shake flask experiments (0.2% w / v) resulted in rapid overexpression of the enzyme within the given timeframe, resulting in higher bioconversion rates than the HA controller.

[0203] Example 6

[0204] Optimized host with hydroxycinnamic acid controller for efficient OPEFB lignin utilization and bioconversion

[0205] In an attempt to further improve yields, inherent problems faced by shake flask experiments that can affect the productivity of the microbial host, such as limited oxygen levels and uncontrolled pH, were overcome by using controlled bioreactors that can adjust these parameters during the fermentation process. OPEFB lignin conversion was performed using a bioreactor with oxygen (pO2) and pH sensors and their associated pumps to maintain these parameters at target values. Figure 8A). The concern about oxygen availability and the need for pH regulation are due to i) the need for oxygen for cell metabolic growth and dearomatization of protocatechuic acid and ii) CO2 production during adipic acid or levulinic acid production, which causes the pH in the cell culture to decrease over time. To further improve the bioconversion of the OPEFB lignin mixture by the engineered cells, the feed dosage to the bioreactor was optimized ( Figure 9 : 0.5x OPEFB lignin for levulinic acid conversion, and 0.375x OPEFB lignin for adipic acid conversion).

[0206] Under controlled conditions, the corresponding optimized host strains carrying the HA controller (ΔsucCD and ΔatoDA) performed similarly to, if not slightly better than, the strains carrying the L-arabinose controller: in the HA controller strain, approximately 1.8-fold higher titers of levulinic acid production were observed (455.7 mg / L vs. 253.5 mg / L per 1× OPEFB lignin at 36 h), and approximately 23% higher titers of adipic acid production (9.5 mg / L vs. 7.8 mg / L per 1× OPEFB lignin at 18 h) ( Figure 8 B). Given the modest production of adipic acid in our engineered cells, we sought to identify one or more potential rate-limiting steps in our anabolic pathway ( Figure 2 We quantified the expression of key substrates of the anabolic pathways ( Figure 8 C). This measurement showed a significant accumulation of vanillic acid in adipic acid-producing cells, indicating that the enzymatic conversion of vanillic acid is the main rate-limiting step. In levulinic acid-producing cells, p-coumaric acid and ferulic acid ( Figure 8 C), which represents the rate-limiting step in the anabolic pathway. This result suggests that if the above rate-limiting enzyme reaction is improved, the production of levulinic acid and adipic acid in our engineered cells can be further increased.

[0207] Summarize

[0208] In summary, this is the first report of cell-autonomous production of adipic acid and levulinic acid from an OPEFB lignin mixture without the need for upstream separation into individual derivatives prior to conversion and expensive chemical inducers. Here, we have demonstrated a method for producing adipic acid and levulinic acid from OPEFB lignin, primarily because both are industrially relevant chemicals that can be derived from the versatile dearomatization precursor β-ketoadipate.

[0209] In this study, we demonstrated the direct production of chemicals from unfractionated depolymerized OPEFB lignin using an engineered E. coli strain. E. coli was engineered to have three genetic modules for the following functions: 1. Genetic control for autonomous activation, 2. Conversion of depolymerized lignin derivatives to β-ketoadipate by pathway enzymes, and 3. Conversion of β-ketoadipate to chemicals by pathway enzymes. Figure 14 We demonstrated the production of adipic acid and levulinic acid using engineered Escherichia coli, producing up to 9.5 mg / L of adipic acid and 455.57 mg / L of levulinic acid from a reconstituted OPEFB lignin derivative under fermentor-controlled conditions. Our results demonstrate a simple, one-pot biosynthetic approach that can potentially be used to directly utilize agricultural waste derivatives for the production of chemicals.

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[0247] Zhao, M., Huang, D., Zhang, Sequence Listing <110> National University of Singapore <120> Biosynthesis of commodity chemicals from oil palm empty fruit bunch lignin <130> SP102194WO <150> SG10202002037R <151> 2020-03-05 <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 1884 <212> DNA <213> Artificial sequence <220> <223> FCS nucleotide sequence <400> 1 atgaataacg aagcccgctc agggtcgacc gaccctggcc aacgtccgcg ctaccgccag 60 gtggccatcg ggcatcccca ggtgcaggtc agtcacgtcg acgacgtgct gcgcatgcaa 120 cctgtcgagc cactggcgcc gctgccggcg cgcctgctcg agcgcctggt gcattgggcc 180 caggtgcgcc cggacaccac tttcatcgcg gcacgccagg cagacggtgc ctggcgttcg 240 atcagctacg tgcagatgct cgccgatgtg cgcaccatcg ccgccaactt gctaggactg 300 ggcctcagtg ccgagcgccc gctggcgctg ctttccggca acgacatcga acacctgcaa 360 atcgccctcg gcgccatgta tgccggtatt gcctattgcc cggtgtcgcc ggcctacgcg 420 ctgttgtcgc aagacttcgc caagttgcgc catgtctgcg aggtgctcac ccccggagtg 480 gtcttcgtca gcgacagcca gccgttccag cgcgccttcg aggcggtgct ggacgattcg 540 gtcggcgtga tcagcgtgcg tggccaggtc gcaggtcgcc cccatataag cttcgacagc 600 ctgttgcaac cgggtgacct ggcggcggcc gatgcggctt tcgccgccac cgggccggac 660 accatcgcca aattcctctt cacctcgggc tcgaccaagc tgcccaaggc ggtgatcacc 720 acccagcgca tgctgtgcgc caatcagcag atgcttctgc agacttttcc gacgttcgcc 780 gaggagccgc cggtgctggt ggactggctg ccgtggaacc acacgttcgg cggtagccac 840 aacctcggca tcgtgcttta caacgggggc agtttctacc tggacgccgg caagccgacc 900 ccgcaaggct tcgccgagac cttgcgcaat ttgcgcgaga tttcccccac ggcctacctc 960 accgtaccca agggctggga ggaactggtc aaggcactgg agcaggaccc cgcgctacgc 1020 gaggtgttct ttgcccgcat caagctgttc ttctttgccg ccgcaggcct gtcgcaaagc 1080 gtctgggacc ggctggaccg cattgccgag caacactgtg gcgaacgcat ccgcatgatg 1140 gccggccttg gcatgaccga agcctcgcca tcgtgcacct tcaccaccgg gcctttgtcg 1200 atggccggct atgtcgggct gccggcacct ggctgcgaag tgaagctggt gccggtgggc 1260 gacaagctcg aggcgcgctt ccgtggcccg catatcatgc cgggctactg gcgctcgccg 1320 cagcagaccg ccgaggcgtt cgacgaggag ggcttctact gttcgggcga cgcgttgaag 1380 ctggccgatg ccaggcagcc cgagcttggc ctgatgttcg atggccgtat cgctgaggac 1440 ttcaaacttt cgtccggggt attcgtcagt gtcgggccgc tgcgcaaccg cgcagtgctg 1500 gagggctcgc cttacgtaca ggacatcgtg gtcaccgcgc cggaccgtga atgcctgggc 1560 ctgctggtgt tcccgcgtct gcccgagtgt cggcgcctgg ccgggctggc agaggatgcc 1620 agcgatgcgc gggtgctggc caacgacacc gtgcgcagtt ggttcgctga ctggctggag 1680 cgcttgaacc gcgatgccca aggcaacgcc agccgtatcg aatggctgtc gctgctggcc 1740 gagccgccgt cgatcgacgc cggtgaaatc accgacaagg gctcgatcaa tcagcgcgcc 1800 gtgctgcagc ggcgcgccgc tcaggtcgag gcgctgtacc gtggcgaaga ccccgacgca 1860 ttgcacgcca aggtgcggcc ttaa 1884 <210> 2 <211> 627 <212> PRT [[ID=1十八]]<213> Artificial sequence <220> <223> Fcs amino acid sequence <400> 2 Met Asn Asn Glu Ala Arg Ser Gly Ser Thr Asp Pro Gly Gln Arg Pro 1 5 10 15 Arg Tyr Arg Gln Val Ala Ile Gly His Pro Gln Val Gln Val Ser His 20 25 30 Val Asp Asp Val Leu Arg Met Gln Pro Val Glu Pro Leu Ala Pro Leu 35 40 45 Pro Ala Arg Leu Leu Glu Arg Leu Val His Trp Ala Gln Val Arg Pro 50 55 60 It should be noted that in the translation of item , the Chinese "人工序列" is translated as "Artificial sequence". You can adjust it according to the actual requirements. If it is a specific professional term translation, it may need to be further optimized according to the relevant field's norms.Asp Thr Thr Phe Ile Ala Ala Arg Gln Ala Asp Gly Ala Trp Arg Ser 65 70 75 80 Ile Ser Tyr Val Gln Met Leu Ala Asp Val Arg Thr Ile Ala Ala Asn 85 90 95 Leu Leu Gly Leu Gly Leu Ser Ala Glu Arg Pro Leu Ala Leu Leu Ser 100 105 110 Gly Asn Asp Ile Glu His Leu Gln Ile Ala Leu Gly Ala Met Tyr Ala 115 120 125 Gly Ile Ala Tyr Cys Pro Val Ser Pro Ala Tyr Ala Leu Leu Ser Gln 130 135 140 Asp Phe Ala Lys Leu Arg His Val Cys Glu Val Leu Thr Pro Gly Val 145 150 155 160 Val Phe Val Ser Asp Ser Gln Pro Phe Gln Arg Ala Phe Glu Ala Val 165 170 175 Leu Asp Asp Ser Val Gly Val Ile Ser Val Arg Gly Gln Val Ala Gly 180 185 190 Arg Pro His Ile Ser Phe Asp Ser Leu Leu Gln Pro Gly Asp Leu Ala 195 200 205 Ala Ala Asp Ala Ala Phe Ala Ala Thr Gly Pro Asp Thr Ile Ala Lys 210 215 220 Phe Leu Phe Thr Ser Gly Ser Thr Lys Leu Pro Lys Ala Val Ile Thr 225 230 235 240 Thr Gln Arg Met Leu Cys Ala Asn Gln Gln Met Leu Leu Gln Thr Phe 245 250 255 Pro Thr Phe Ala Glu Glu Pro Pro Val Leu Val Asp Trp Leu Pro Trp 260 265 270 Asn His Thr Phe Gly Gly Ser His Asn Leu Gly Ile Val Leu Tyr Asn 275 280 285 Gly Gly Ser Phe Tyr Leu Asp Ala Gly Lys Pro Thr Pro Gln Gly Phe 290 295 300 Ala Glu Thr Leu Arg Asn Leu Arg Glu Ile Ser Pro Thr Ala Tyr Leu 305 310 315 320 Thr Val Pro Lys Gly Trp Glu Glu Leu Val Lys Ala Leu Glu Gln Asp 325 330 335 Pro Ala Leu Arg Glu Val Phe Phe Ala Arg Ile Lys Leu Phe Phe Phe 340 345 350 Ala Ala Ala Gly Leu Ser Gln Ser Val Trp Asp Arg Leu Asp Arg Ile 355 360 365 Ala Glu Gln His Cys Gly Glu Arg Ile Arg Met Met Ala Gly Leu Gly 370 375 380 Met Thr Glu Ala Ser Pro Ser Cys Thr Phe Thr Thr Gly Pro Leu Ser 385 390 395 400 Met Ala Gly Tyr Val Gly Leu Pro Ala Pro Gly Cys Glu Val Lys Leu 405 410 415 Val Pro Val Gly Asp Lys Leu Glu Ala Arg Phe Arg Gly Pro His Ile 420 425 430 Met Pro Gly Tyr Trp Arg Ser Pro Gln Gln Thr Ala Glu Ala Phe Asp 435 440 445 Glu Glu Gly Phe Tyr Cys Ser Gly Asp Ala Leu Lys Leu Ala Asp Ala 450 455 460 Arg Gln Pro Glu Leu Gly Leu Met Phe Asp Gly Arg Ile Ala Glu Asp 465 470 475 480 Phe Lys Leu Ser Ser Gly Val Phe Val Ser Val Gly Pro Leu Arg Asn 485 490 495 Arg Ala Val Leu Glu Gly Ser Pro Tyr Val Gln Asp Ile Val Val Thr 500 505 510 Ala Pro Asp Arg Glu Cys Leu Gly Leu Leu Val Phe Pro Arg Leu Pro 515 520 525 Glu Cys Arg Arg Leu Ala Gly Leu Ala Glu Asp Ala Ser Asp Ala Arg 530 535 540 Val Leu Ala Asn Asp Thr Val Arg Ser Trp Phe Ala Asp Trp Leu Glu 545 550 555 560 Arg Leu Asn Arg Asp Ala Gln Gly Asn Ala Ser Arg Ile Glu Trp Leu 565 570 575 Ser Leu Leu Ala Glu Pro Pro Ser Ile Asp Ala Gly Glu Ile Thr Asp 580 585 590 Lys Gly Ser Ile Asn Gln Arg Ala Val Leu Gln Arg Arg Ala Ala Gln 595 600 605 Val Glu Ala Leu Tyr Arg Gly Glu Asp Pro Asp Ala Leu His Ala Lys 610 615 620 Val Arg Pro 625 <210> 3 <211> 831 <212> DNA <213> Artificial Sequence <220> <223> ech nucleotide sequence <400> 3 atgagcaaat acgaaggccg ctggaccacc gtgaaggtcg aactggaagc gggcatcgcc 60[[ID=4']] tgggtgaccc tcaatcgccc ggaaaaacgc aatgccatga gccccaccct gaaccgggaa 120 atggtcgacg tgctggaaac ccttgagcag gacgctgacg ctggcgtgct ggtattgacc 180 ggtgccggcg agtcctggac cgccggcatg gacctgaagg agtacttccg cgaggtggac 240 gccggcccgg aaatcctcca ggaaaagatt cgtcgcgaag cctcgcaatg gcaatggaag 300 ttgctgcgtc tgtatgccaa accgaccatc gccatggtca acggctggtg cttcggcggc 360 ggcttcagcc cactggtggc atgcgacctg gcgatctgcg ccaacgaagc gaccttcggc 420 ctgtcggaaa tcaactgggg catcccgcct ggtaacctgg tcagcaaggc catggccgat 480 accgttggcc atcgtcagtc gctgtactac atcatgaccg gcaagacctt cgatggtcgc 540 aaggctgccg agatgggcct ggtgaacgac agtgtgccgc tggccgagct gcgtgaaacc 600 acccgcgagt tggcgctgaa cctgctggaa aagaacccgg tggtgctgcg tgccgcgaag 660 aatggcttca agcgttgccg cgagctgacc tgggaacaga acgaggacta cctctacgcc 720 aagctcgacc agtcgcgcct gctggacact accggcggcc gcgagcaggg catgaagcag 780 ttcctcgacg acaagagcat caagccaggc ctgcaggcct acaagcgcta a 831 <210> 4 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> Ech amino acid sequence <400> 4 Met Ser Lys Tyr Glu Gly Arg Trp Thr Thr Val Lys Val Glu Leu Glu 1 5 10 15 Ala Gly Ile Ala Trp Val Thr Leu Asn Arg Pro Glu Lys Arg Asn Ala 20 25 30 Met Ser Pro Thr Leu Asn Arg Glu Met Val Asp Val Leu Glu Thr Leu 35 40 45 Glu Gln Asp Ala Asp Ala Gly Val Leu Val Leu Thr Gly Ala Gly Glu 50 55 60 Ser Trp Thr Ala Gly Met Asp Leu Lys Glu Tyr Phe Arg Glu Val Asp 65 70 75 80 Ala Gly Pro Glu Ile Leu Gln Glu Lys Ile Arg Arg Glu Ala Ser Gln 85 90 95 Trp Gln Trp Lys Leu Leu Arg Leu Tyr Ala Lys Pro Thr Ile Ala Met 100 105 110 Val Asn Gly Trp Cys Phe Gly Gly Gly Phe Ser Pro Leu Val Ala Cys 115 120 125 Asp Leu Ala Ile Cys Ala Asn Glu Ala Thr Phe Gly Leu Ser Glu Ile 130 135 140 Asn Trp Gly Ile Pro Pro Gly Asn Leu Val Ser Lys Ala Met Ala Asp 145 150 155 160 Thr Val Gly His Arg Gln Ser Leu Tyr Tyr Ile Met Thr Gly Lys Thr 165 170 175 Phe Asp Gly Arg Lys Ala Ala Glu Met Gly Leu Val Asn Asp Ser Val 180 185 190 Pro Leu Ala Glu Leu Arg Glu Thr Thr Arg Glu Leu Ala Leu Asn Leu 195 200 205 Leu Glu Lys Asn Pro Val Val Leu Arg Ala Ala Lys Asn Gly Phe Lys 210 215 220 Arg Cys Arg Glu Leu Thr Trp Glu Gln Asn Glu Asp Tyr Leu Tyr Ala 225 230 235 240 Lys Leu Asp Gln Ser Arg Leu Leu Asp Thr Thr Gly Gly Arg Glu Gln 245 250 255 Gly Met Lys Gln Phe Leu Asp Asp Lys Ser Ile Lys Pro Gly Leu Gln 260 265 270 Ala Tyr Lys Arg 275 <210> 5 <211> 1449 <212> DNA <213> artificial sequence <220> <223> vdh sequence <400> 5 atgttgcagg tgcctttgct gattggcggg cagtcgcgcc ccgccagcga tggacgaacc 60 ttcgagcgct gtaacccggt gactggcgag gtggtgtcgc aggctgccgc cgccacactg 120 gccgatgccg atgccgcggt ggctgctgcc agcgcggcgt ttccggcctg ggccgccctg 180 gcaccgggcg agcggcgcag ccgcttgctg gcaggcgctg atctgttgca ggcgagggcc 240 gccgagttca tcgccgccgc cggtgaaacc ggggccatgg ccaactggta tggcttcaac 300 gtgaagttgg ccgccaacat gctgcgcgag gctgcagcca tgaccacgca gatcaccggt 360 gaagtgatcc cctcggacgt tcccggcagc ttcgcaatgg ccctgcgcgc gccctgcggc 420 gtggtgttgg gcatcgcacc gtggaacgcc ccggtgatac tggccacgcg tgccattgcc 480 atgccgctgg cctgcggcaa caccgtggtg ctcaaggcct cggagctgag cccggcggtc 540 catcggctga tcggccaggt gctccacgat gcaggcatcg gcgacggcgt ggtcaatgtc 600 atcagcaatg cgccgcagga tgccccccgcc atcgtcgagc ggctgatcgc caaccctgcg 660 gtacgccggg tcaacttcac cggttcgacg cacgtcgggc gcatcgtcgg cgaactggcg 720 gcccgccatc tcaagccggc cctgctcgaa ctgggcggca aggcaccttt gctggtgctc 780 gacgatgccg acctggacgc cacggtcgaa gcggcggcct tcggtgccta cttcaaccag 840 gggcaaatct gcatgtccac cgagcgcctt gtggtggaca gctgtattgc cgacgctttc 900 gtcgacaagc tggcggtgaa gatcgccggg ctgcgtgcag gtgatccgca agccagcacc 960 tcggtgctcg gctcgctggt cagcgcagcg gccggcgagc gcatcaaggc actgatcgac 1020 gatgccgtgg ccaagggcgc gcgcctggtc agcggcggcc agctggaagg cagcatcctg 1080 caaccgacct tgctcgacaa cgtcgatgcc agcatgcgcc tgtaccgcga ggagtccttc 1140 ggcccggtgg cggtggtact gcgcgccgaa ggcgacgaag ccttgctgca gctggccaac 1200 gactcggagt tcggtctgtc atcggccatt ttcagccgcg acaccagccg cgccctggcc 1260 ttggcccaac gggtggagtc gggtatctgc catatcaacg gcccgaccgt tcacgatgaa 1320 gcgcagatgc cgtttggcgg ggtcaagtcc agcggctatg gcagcttcgg cagccgcacg 1380 gccatcgatc agttcaccca gttgcgctgg gtcaccctcc agcacggccc gcgtcactat 1440 cccatctaa 1449 <210> 6 <211> 482 <212> PRT <213> artificial sequence <220> <223> Vdh amino acid sequence <400> 6 Met Leu Gln Val Pro Leu Leu Ile Gly Gly Gln Ser Arg Pro Ala Ser 1 5 10 15 Asp Gly Arg Thr Phe Glu Arg Cys Asn Pro Val Thr Gly Glu Val Val 20 25 30 Ser Gln Ala Ala Ala Ala Thr Leu Ala Asp Ala Asp Ala Ala Val Ala 35 40 45 Ala Ala Ser Ala Ala Phe Pro Ala Trp Ala Ala Leu Ala Pro Gly Glu 50 55 60 Arg Arg Ser Arg Leu Leu Ala Gly Ala Asp Leu Leu Gln Ala Arg Ala 65 70 75 80 Ala Glu Phe Ile Ala Ala Ala Gly Glu Thr Gly Ala Met Ala Asn Trp 85 90 95 Tyr Gly Phe Asn Val Lys Leu Ala Ala Asn Met Leu Arg Glu Ala Ala 100 105 110 Ala Met Thr Thr Gln Ile Thr Gly Glu Val Ile Pro Ser Asp Val Pro 115 120 125 Gly Ser Phe Ala Met Ala Leu Arg Ala Pro Cys Gly Val Val Leu Gly 130 135 140 Ile Ala Pro Trp Asn Ala Pro Val Ile Leu Ala Thr Arg Ala Ile Ala 145 150 155 160 Met Pro Leu Ala Cys Gly Asn Thr Val Val Leu Lys Ala Ser Glu Leu 165 170 175 Ser Pro Ala Val His Arg Leu Ile Gly Gln Val Leu His Asp Ala Gly 180 185 190 Ile Gly Asp Gly Val Val Asn Val Ile Ser Asn Ala Pro Gln Asp Ala 195 200 205 Pro Ala Ile Val Glu Arg Leu Ile Ala Asn Pro Ala Val Arg Arg Val 210 215 220 Asn Phe Thr Gly Ser Thr His Val Gly Arg Ile Val Gly Glu Leu Ala 225 230 235 240 Ala Arg His Leu Lys Pro Ala Leu Leu Glu Leu Gly Gly Lys Ala Pro 245 250 255 Leu Leu Val Leu Asp Asp Ala Asp Leu Asp Ala Thr Val Glu Ala Ala 260 265 270 Ala Phe Gly Ala Tyr Phe Asn Gln Gly Gln Ile Cys Met Ser Thr Glu 275 280 285 Arg Leu Val Val Asp Ser Cys Ile Ala Asp Ala Phe Val Asp Lys Leu 290 295 300 Ala Val Lys Ile Ala Gly Leu Arg Ala Gly Asp Pro Gln Ala Ser Thr 305 310 315 320 Ser Val Leu Gly Ser Leu Val Ser Ala Ala Ala Gly Glu Arg Ile Lys 325 330 335 Ala Leu Ile Asp Asp Ala Val Ala Lys Gly Ala Arg Leu Val Ser Gly 340 345 350 Gly Gln Leu Glu Gly Ser Ile Leu Gln Pro Thr Leu Leu Asp Asn Val 355 360 365 Asp Ala Ser Met Arg Leu Tyr Arg Glu Glu Ser Phe Gly Pro Val Ala 370 375 380 Val Val Leu Arg Ala Glu Gly Asp Glu Ala Leu Leu Gln Leu Ala Asn 385 390 395 400 Asp Ser Glu Phe Gly Leu Ser Ser Ala Ile Phe Ser Arg Asp Thr Ser 405 410 415 Arg Ala Leu Ala Leu Ala Gln Arg Val Glu Ser Gly Ile Cys His Ile 420 425 430 Asn Gly Pro Thr Val His Asp Glu Ala Gln Met Pro Phe Gly Gly Val 435 440 445 Lys Ser Ser Gly Tyr Gly Ser Phe Gly Ser Arg Thr Ala Ile Asp Gln 450 455 460 Phe Thr Gln Leu Arg Trp Val Thr Leu Gln His Gly Pro Arg His Tyr 465 470 475 480 Pro Ile <210> 7 <211> 1068 <212> DNA <213> Artificial sequence <220> <223> vanA nucleotide sequence <400> 7 atgtacccca aaaacacctg gtacgtcgcc tgcacccccg atgagatcgc caccaaaccc 60 ctgggccggc aaatctgcgg ggaaaaaatc gtgttctacc gcgcccgcga gaaccaagta 120 gccgccgtcg aggacttctg cccgcaccgc ggcgcaccgt tgtcgttggg ctatgtcgag 180 gacggcaacc tggtgtgcgg ctaccacggc ctggtgatgg gttgcgacgg caagaccgtg 240 tcgatgccgg gccaacgggt gcgtggcttc ccctgcaaca agacctttgc ggccgtcgag 300 cgctatggct tcatctgggt ctggcccggt gaccaggcgc aggccgaccc ggcgctgatt 360 ccgcatctgg aatgggcggt gagtgatgag tgggcctacg gcggcgggct gttccacatc 420 ggttgcgact accgcctgat gatcgacaac ctcatggacc tcacccatga aacctatgtg 480 cacgcctcca gcatcggcca gaaggagatc gacgaggcac cgccggtcac caccgtcacc 540 ggcgacgaag tggtcaccgc ccggcacatg gaaaacatca tggcgccacc gttctggcgc 600 atggccttgc gtggcaatgg cctggccgac gatgtaccag tggaccgctg gcaaatctgc 660 cgtttcaccc cacctagcca tgtgctgatc gaagtgggtg tagcgcatgc cggcaagggc 720 ggctaccacg ccgaggcaca gcataaggcg tcgagcatcg tggtcgactt catcacccct 780 gagagcgata cctctatctg gtacttctgg ggcatggcgc gcaacttcgc tgcgcacgac 840 cagaccctga ccgacaacat tcgtgagggc cagggcaaga ttttcagcga agacctggaa 900 atgctcgaac gccagcagca gaacctgctg gcccaccccg agcgcaactt gctgaagctg 960 aatatcgacg ccggcggcgt gcagtcacgc aaagtgctgg agcggatcat cgcccaagag 1020 cgtgcgccgc agccgcaact gatcgccacc agcgccaacc ctgcctga 1068 <210> 8 <211> 355 <212> PRT <213> Artificial Sequence <220> <223> VanA amino acid sequence <400> 8 Met Tyr Pro Lys Asn Thr Trp Tyr Val Ala Cys Thr Pro Asp Glu Ile 1 5 10 15 Ala Thr Lys Pro Leu Gly Arg Gln Ile Cys Gly Glu Lys Ile Val Phe 20 25 30 Tyr Arg Ala Arg Glu Asn Gln Val Ala Ala Val Glu Asp Phe Cys Pro 35 40 45 His Arg Gly Ala Pro Leu Ser Leu Gly Tyr Val Glu Asp Gly Asn Leu 50 55 60 Val Cys Gly Tyr His Gly Leu Val Met Gly Cys Asp Gly Lys Thr Val 65 70 75 80 Ser Met Pro Gly Gln Arg Val Arg Gly Phe Pro Cys Asn Lys Thr Phe 85 90 95 Ala Ala Val Glu Arg Tyr Gly Phe Ile Trp Val Trp Pro Gly Asp Gln 100 105 110 Ala Gln Ala Asp Pro Ala Leu Ile Pro His Leu Glu Trp Ala Val Ser 115 120 125 [[ID=B]] Asp Glu Trp Ala Tyr Gly Gly Gly Leu Phe His Ile Gly Cys Asp Tyr 130 135 140 Arg Leu Met Ile Asp Asn Leu Met Asp Leu Thr His Glu Thr Tyr Val 145 150 155 160 His Ala Ser Ser Ile Gly Gln Lys Glu Ile Asp Glu Ala Pro Pro Val 165 170 175 Thr Thr Val Thr Gly Asp Glu Val Val Thr Ala Arg His Met Glu Asn 180 185 190 Ile Met Ala Pro Pro Phe Trp Arg Met Ala Leu Arg Gly Asn Gly Leu 195 200 205 Ala Asp Asp Val Pro Val Asp Arg Trp Gln Ile Cys Arg Phe Thr Pro 210 215 220 Pro Ser His Val Leu Ile Glu Val Gly Val Ala His Ala Gly Lys Gly 225 230 235 240 Gly Tyr His Ala Glu Ala Gln His Lys Ala Ser Ser Ile Val Val Asp 245 250 255 Phe Ile Thr Pro Glu Ser Asp Thr Ser Ile Trp Tyr Phe Trp Gly Met 260 265 270 Ala Arg Asn Phe Ala Ala His Asp Gln Thr Leu Thr Asp Asn Ile Arg 275 280 285 Glu Gly Gln Gly Lys Ile Phe Ser Glu Asp Leu Glu Met Leu Glu Arg 290 295 300 Gln Gln Gln Asn Leu Leu Ala His Pro Glu Arg Asn Leu Leu Lys Leu 305 310 315 320 Asn Ile Asp Ala Gly Gly Val Gln Ser Arg Lys Val Leu Glu Arg Ile 325 330 335 Ile Ala Gln Glu Arg Ala Pro Gln Pro Gln Leu Ile Ala Thr Ser Ala 340 345 350 Asn Pro Ala 355 <210> 9 <211> 951 <212> DNA <213> Artificial sequence <220> <223> vanB nucleotide sequence <400> 9 atgatcgatg ccgtagtggt atcccgtaac gatgaagcgc agggtatctg cagcttcgag 60 ctggccgcgg cagatggcag cctgctgccg gcgttcagcg ccggcgccca tatcgacgtg 120 cacctgcccg acgggctggt gcgccagtat tcgctgtgca accaccccga agaacgccat 180 cgctatctga ttggcgtact caacgacccg gcttcgcggg gcggttctcg tagcctgcac 240 gaacaggtgc aagccggtgc ccggctgcgt atcagtgcgc cgcgcaacct gttcccgctg 300 gccgagggtg cgcagcgcag tttgctgttt gctggcggta tcggcattac cccaatcctg 360 tgcatggccg agcagctgtc cgacagcggc caggccttcg agctgcacta ctgtgcccgc 420 tccagcgagc gtgcggcgtt tgtcgagcgc atccgcagcg cgccgttcgc tgatcggctg 480 ttcgtgcatt ttgacgagca gccggaaacg gcgctggaca tcgcccaggt gctgggcaac 540 ccgcaagatg atgtgcacct gtatgtatgc gggcccggcg ggttcatgca gcatgtgctg 600 gacagcgcga aggggctggg ctggcaggag gccaacctgc accgcgagta cttcgccgca 660 gcaccggtgg atgccagcaa cgatggcagt ttcgcggtgc aggtgggcag cacgggacag 720 gtgttcgagg tgccagccga ccggaccgtg gtgcaggtgc tggaagagaa tggtatcgag 780 atcgccatgt cgtgcgagca gggtatttgc ggcacctgcc tgacacgcgt gctgcagggc 840 acaccggacc atcgcgatct gtttctcacc gaagaggaac aggccctgaa cgatcagttc 900 acgccctgct gctcgcgctc gaagacgccg ctgctggtgc tggacatctg a 951 <210> 10 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> VanB Amino Acid Sequence <400> 10 Met Ile Asp Ala Val Val Val Ser Arg Asn Asp Glu Ala Gln Gly Ile 1 5 10 15 Cys Ser Phe Glu Leu Ala Ala Ala Asp Gly Ser Leu Leu Pro Ala Phe 20 25 30 Ser Ala Gly Ala His Ile Asp Val His Leu Pro Asp Gly Leu Val Arg 35 40 45 Gln Tyr Ser Leu Cys Asn His Pro Glu Glu Arg His Arg Tyr Leu Ile 50 55 60 Gly Val Leu Asn Asp Pro Ala Ser Arg Gly Gly Ser Arg Ser Leu His 65 70 75 80 Glu Gln Val Gln Ala Gly Ala Arg Leu Arg Ile Ser Ala Pro Arg Asn 85 90 95 Leu Phe Pro Leu Ala Glu Gly Ala Gln Arg Ser Leu Leu Phe Ala Gly 100 105 110 Gly Ile Gly Ile Thr Pro Ile Leu Cys Met Ala Glu Gln Leu Ser Asp 115 120 125 Ser Gly Gln Ala Phe Glu Leu His Tyr Cys Ala Arg Ser Ser Glu Arg 130 135 140 Ala Ala Phe Val Glu Arg Ile Arg Ser Ala Pro Phe Ala Asp Arg Leu 145 150 155 160 Phe Val His Phe Asp Glu Gln Pro Glu Thr Ala Leu Asp Ile Ala Gln 165 170 175 Val Leu Gly Asn Pro Gln Asp Asp Val His Leu Tyr Val Cys Gly Pro 180 185 190 Gly Gly Phe Met Gln His Val Leu Asp Ser Ala Lys Gly Leu Gly Trp 195 200 205 Gln Glu Ala Asn Leu His Arg Glu Tyr Phe Ala Ala Ala Pro Val Asp 210 215 220 Ala Ser Asn Asp Gly Ser Phe Ala Val Gln Val Gly Ser Thr Gly Gln 225 230 235 240 Val Phe Glu Val Pro Ala Asp Arg Thr Val Val Gln Val Leu Glu Glu 245 250 255 Asn Gly Ile Glu Ile Ala Met Ser Cys Glu Gln Gly Ile Cys Gly Thr 260 265 270 Cys Leu Thr Arg Val Leu Gln Gly Thr Pro Asp His Arg Asp Leu Phe 275 280 285 Leu Thr Glu Glu Glu Gln Ala Leu Asn Asp Gln Phe Thr Pro Cys Cys 290 295 300 Ser Arg Ser Lys Thr Pro Leu Leu Val Leu Asp Ile 305 310 315 <210> 11 <211> 1188 <212> DNA <213> Artificial sequence <220> <223> pobA nucleotide sequence <400> 11 atgaaaactc aggttgcaat tattggtgca ggtccgtctg gcctgctgct gggccagctg 60 ctgcacaagg ccggtatcga taacatcatc gtcgaacgcc agactgccga gtacgtacta 120 ggccgcatcc gcgccggggt gctagagcaa ggcacggtcg acctgctgcg cgaggctggc 180 gtggccgagc gcatggaccg tgaaggcctg gtgcacgagg gggttgaact gctggttggc 240 gggcgccgcc agcgtctgga tctcaaagcc ctgaccggcg gcaagacggt gatggtctac 300 ggccagaccg aagtcacccg tgacctgatg caggcccgcg aagccagtgg tgcgccgatc 360 atttattcag ccgccaacgt tcagccgcat gaattgaaag gcgagaagcc ctacctgacg 420 ttcgaaaagg atggccgggt gcagcggatt gactgcgact atatcgccgg ctgcgacggc 480 ttccacggta tctcgcggca gagcatcccg gagggcgtgc tgaaacagta tgagcgggtt 540 tacccgtttg gctggctggg cctgctgtcg gacacaccgc cagtcaatca cgagttgatc 600 tacgcccacc atgagcgcgg tttcgcgttg tgtagccaac gctcgcaaac acgcagccgc 660 tactacctgc aggtaccttt gcaggatcgg gtcgaggagt ggtctgacga gcgtttctgg 720 gacgaactga aagcccgtct gcccgccgag gtggcggcgg acctggtcac aggccccgcg 780 ttggaaaaaa gtattgcgcc gctgcgtagc ctggtggtcg aacccatgca gtatggtcac 840 ctgttcctgg tgggggacgc ggcgcacatc gtccccccta cgggtgccaa aggccttaac900 ctggcggcct ccgacgtcaa ctacctgtac cgcattctgg tcaaggtgta ccgaaggg cgcgtcgacc tgcttgcgca atactcgccg ctggcactgc gccgcgtgtg gaagggcgag cgcttcagct ggttcatgac ccaactgctg catgacttcg gtagccacaa ggacgcctgg gaccagaga tgcaggaagc tgaccgcgag tacttcctga cctcgccggc gggcctggtg aacattgccg agaactatgt ggggctgccg ttcgaggaag ttgcctga <210> 12 <211> 395 <212> PRT <213> The snowstorm <220> <223> PobA snowflakes <400> 12 Met Lys Thr Gln Val Ala Ile Ile Gly Ala Gly Pro Ser Gly Leu Leu 1 5 10 15 Leu Gly Gln Leu Leu His Lys Ala Gly Ile Asp Asn Ile Ile Val Glu 20 25 30 Arg Gln Thr Ala Glu Tyr Val Leu Gly Arg Ile Arg Ala Gly Val Leu 35 40 45 Glu Gln Gly Thr Val Asp Leu Leu Arg Glu Ala Gly Val Ala Glu Arg 50 55 60 Met Asp Arg Glu Gly Leu Val His Glu Gly Val Glu Leu Leu Val Gly 65 70 75 80 Gly Arg Arg Gln Arg Leu Asp Leu Lys Ala Leu Thr Gly Gly Lys Thr 85 90 95 Val Met Val Tyr Gly Gln Thr Glu Val Thr Arg Asp Leu Met Gln Ala 100 105 110 Arg Glu Ala Ser Gly Ala Pro Ile Ile Tyr Ser Ala Ala Asn Val Gln 115 120 125 Pro His Glu Leu Lys Gly Glu Lys Pro Tyr Leu Thr Phe Glu Lys Asp 130 135 140 Gly Arg Val Gln Arg Ile Asp Cys Asp Tyr Ile Ala Gly Cys Asp Gly 145 150 155 160 Phe His Gly Ile Ser Arg Gln Ser Ile Pro Glu Gly Val Leu Lys Gln 165 170 175 Tyr Glu Arg Val Tyr Pro Phe Gly Trp Leu Gly Leu Leu Ser Asp Thr 180 185 190 Pro Pro Val Asn His Glu Leu Ile Tyr Ala His His Glu Arg Gly Phe 195 200 205 Ala Leu Cys Ser Gln Arg Ser Gln Thr Arg Ser Arg Tyr Tyr Leu Gln 210 215 220 Val Pro Leu Gln Asp Arg Val Glu Glu Trp Ser Asp Glu Arg Phe Trp 225 230 235 240 Asp Glu Leu Lys Ala Arg Leu Pro Ala Glu Val Ala Ala Asp Leu Val 245 250 255 Thr Gly Pro Ala Leu Glu Lys Ser Ile Ala Pro Leu Arg Ser Leu Val 260 265 270 Val Glu Pro Met Gln Tyr Gly His Leu Phe Leu Val Gly Asp Ala Ala 275 280 285 His Ile Val Pro Pro Thr Gly Ala Lys Gly Leu Asn Leu Ala Ala Ser 290 295 300 Asp Val Asn Tyr Leu Tyr Arg Ile Leu Val Lys Val Tyr His Glu Gly 305 310 315 320 Arg Val Asp Leu Leu Ala Gln Tyr Ser Pro Leu Ala Leu Arg Arg Val 325 330 335 Trp Lys Gly Glu Arg Phe Ser Trp Phe Met Thr Gln Leu Leu His Asp 340 345 350 Phe Gly Ser His Lys Asp Ala Trp Asp Gln Lys Met Gln Glu Ala Asp 355 360 365 Arg Glu Tyr Phe Leu Thr Ser Pro Ala Gly Leu Val Asn Ile Ala Glu 370 375 380 Asn Tyr Val Gly Leu Pro Phe Glu Glu Val Ala 385 390 395 <210> 13 <211> 720 <212> DNA <213> Artificial sequence <220> <223> pcaH nucleotide sequence <400> 13 atgcccgccc aggacaacag ccgcttcgtg atccgtgatc gcaactggca ccctaaagcc 60 cttacgcctg actacaagac ctccgttgcc cgctcgccgc gccaggcact ggtcagcatt 120 ccgcagtcga tcagcgaaac cactggtccg gacttttccc atctgggctt cggcgcccac 180 gaccatgacc tgctgctgaa cttcaataac ggtggcctgc ccattggcga gcgcatcatc 240 gtcgccggcc gtgtcgtcga ccagtacggc aagcctgtgc cgaacacttt ggtggagatg 300 tggcaagcca acgccggcgg ccgctatcgc cacaagaacg atcgctacct ggcgcccctg 360 gacccgaact tcggtggtgt tgggcggtgt ctgaccgacc gtgacggcta ttacagcttc 420 cgcaccatca agccgggccc gtacccatgg cgcaacggcc cgaacgactg gcgcccggcg 480 catatccact tcgccatcag cggcccatcg atcgccacca agctgatcac ccagttgtac 540 ttcgaaggtg acccgctgat cccgatgtgc ccgatcgtca agtcgatcgc caacccgcaa 600 gccgtgcagc agttgatcgc caagctcgac atgagcaacg ccaacccgat ggactgcctg 660 gcctaccgct ttgacatcgt gctgcgcggc cagcgcaaga cccacttcga aaactgctga 720 <210> 14 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> PcaH Amino Acid Sequence <400> 14 Met Pro Ala Gln Asp Asn Ser Arg Phe Val Ile Arg Asp Arg Asn Trp 1 5 10 15 [[ID=3)4]]His Pro Lys Ala Leu Thr Pro Asp Tyr Lys Thr Ser Val Ala Arg Ser 20 25 30 Pro Arg Gln Ala Leu Val Ser Ile Pro Gln Ser Ile Ser Glu Thr Thr 35 40 45 Gly Pro Asp Phe Ser His Leu Gly Phe Gly Ala His Asp His Asp Leu 50 55 60 Leu Leu Asn Phe Asn Asn Gly Gly Leu Pro Ile Gly Glu Arg Ile Ile 65 70 75 80 Val Ala Gly Arg Val Val Asp Gln Tyr Gly Lys Pro Val Pro Asn Thr 85 90 95 Leu Val Glu Met Trp Gln Ala Asn Ala Gly Gly Arg Tyr Arg His Lys 100 105 110 Asn Asp Arg Tyr Leu Ala Pro Leu Asp Pro Asn Phe Gly Gly Val Gly 115 120 125 Arg Cys Leu Thr Asp Arg Asp Gly Tyr Tyr Ser Phe Arg Thr Ile Lys 130 135 140 Pro Gly Pro Tyr Pro Trp Arg Asn Gly Pro Asn Asp Trp Arg Pro Ala 145 150 155 160 His Ile His Phe Ala Ile Ser Gly Pro Ser Ile Ala Thr Lys Leu Ile 165 170 175 Thr Gln Leu Tyr Phe Glu Gly Asp Pro Leu Ile Pro Met Cys Pro Ile 180 185 190 Val Lys Ser Ile Ala Asn Pro Gln Ala Val Gln Gln Leu Ile Ala Lys 195 200 205 Leu Asp Met Ser Asn Ala Asn Pro Met Asp Cys Leu Ala Tyr Arg Phe 210 215 220 Asp Ile Val Leu Arg Gly Gln Arg Lys Thr His Phe Glu Asn Cys 225 230 235 <210> 15 <211> 606 <212> DNA <213> Artificial Sequence <220> <223> pcaG nucleotide sequence <400> 15 atgccaatcg aactgctgcc ggaaacccct tcgcagactg ccggccccta cgtgcacatc 60 ggcctggccc tggaagccgc cggcaacccg acccgcgacc aggaaatctg gaactgcctg 120 gccaagccag acgccccggg cgagcacatt ctgctgatcg gccacgtata tgacggaaac 180 ggccacctgg tgcgcgactc gttcctggaa gtgtggcagg ccgacgccaa cggtgagtac 240 caggatgcct acaacctgga aaacgccttc aacagctttg gccgcacggc taccaccttc 300 gatgccggtg agtggacgct gcaaacggtc aagccgggtg tggtgaacaa cgctgctggc 360 gtgccgatgg cgccgcacat caacatcagc ctgtttgccc gtggcatcaa catccacctg 420 cacacgcgcc tgtatttcga tgatgaggcc caggccaatg ccaagtgccc ggtgctcaac 480 ctgatcgagc agccgcagcg gcgtgaaacc ttgattgcca agcgttgcga agtggatggg 540 aagacggcgt accgctttga tatccgcatt cagggggaag gggagaccgt cttcttcgac 600 ttctga 606 <210> 16 <211> 201 <212> PRT <213> Artificial sequence[[ID=!17]] <220> <223> Amino acid sequence of PcaG <400> 16 Met Pro Ile Glu Leu Leu Pro Glu Thr Pro Ser Gln Thr Ala Gly Pro 1 5 10 15 Tyr Val His Ile Gly Leu Ala Leu Glu Ala Ala Gly Asn Pro Thr Arg[[ID=2!9]] 20 25 30 Asp Gln Glu Ile Trp Asn Cys Leu Ala Lys Pro Asp Ala Pro Gly Glu 35 40 45 His Ile Leu Leu Ile Gly His Val Tyr Asp Gly Asn Gly His Leu Val 50 55 60 Arg Asp Ser Phe Leu Glu Val Trp Gln Ala Asp Ala Asn Gly Glu Tyr 65 70 75 80 It should be noted that there seems to be a small formatting issue in the original text where the tag "!17" and "2!9" are likely incorrect. I've translated them as they are but they might need to be corrected in the original source for proper interpretation.Gln Asp Ala Tyr Asn Leu Glu Asn Ala Phe Asn Ser Phe Gly Arg Thr 85 90 95 Ala Thr Thr Phe Asp Ala Gly Glu Trp Thr Leu Gln Thr Val Lys Pro 100 105 110 Gly Val Val Asn Asn Ala Ala Gly Val Pro Met Ala Pro His Ile Asn 115 120 125 Ile Ser Leu Phe Ala Arg Gly Ile Asn Ile His Leu His Thr Arg Leu 130 135 140 Tyr Phe Asp Asp Glu Ala Gln Ala Asn Ala Lys Cys Pro Val Leu Asn 145 150 155 160 Leu Ile Glu Gln Pro Gln Arg Arg Glu Thr Leu Ile Ala Lys Arg Cys 165 170 175 Glu Val Asp Gly Lys Thr Ala Tyr Arg Phe Asp Ile Arg Ile Gln Gly 180 185 190 Glu Gly Glu Thr Val Phe Phe Asp Phe 195 200 <210> 17 <211> 1353 <212> DNA <213> Artificial sequence <220> <223> pcaB nucleotide sequence <400> 17 [[ID=4 gaccgaggcc gcctgcaggg catgctggat ttcgaagccg cgcttgcccg agccgaagcc 120 tctgccggtt tggtcccgca cagcgcggta gcggccatcg aggcggcatg ccaggccgag 180 cgctatgacg ttggcgcgct ggccaatgcc atcgccaccg cgggcaactc ggccattccg 240 ctggtgaaag cgttgggcaa ggtgatcgcc accggcgtgc cagaggctga gcgctatgtg 300 caccttgggg ccaccagcca ggatgcgatg gataccggtc tggttctgca gctgcgcgat 360 gccctcgatt tgatcgaggc cgacctcggc aagctggccg ataccctgtc gcagcaggcc 420 ttgaagcacg ccgatacgcc cttggtgggt cgtacctggt tgcaacacgc caccccggtg 480 accctgggca tgaaactggc cggtgtactg ggtgctttga cccgccaccg tcagcgcctg 540 caggaactgc gcccgcgcct tctggtcctg cagttcggcg gtgcctcggg cagcctggcg 600 gcgctgggca gcaaggcgat gccggtggcc gaagcgctgg ccgaacagct caagctgacc 660 ctgcccgagc agccctggca cacccagcgc gaccgcctgg tggagtttgc ctcggtattg 720 ggcctggttg ccggcagcct gggcaagttc ggccgtgata tcagcttgct gatgcaaacc 780 gaggcggggg aggtgtttga gccttctgcg ccgggcaagg gtggttcttc gaccatgcca 840 cacaagcgca acccggtggg tgccgccgtg ttgatcggtg ccgcgacccg cgtgccgggc 900 ctgctgtcga cgctgttcgc agccatgcct caggagcacg aacgcagcct gggcctatgg 960 catgccgagt gggaaaccct gccggatatc tgctgcctgg tctctggcgc cctgcgccag 1020 gctcaagtga ttgccgaggg catggaggtg gatgccgcgc gcatgcgccg taacctcgac 1080 ctgacccaag gcctggtgct ggccgaagcg gtgagcatcg tcctcgccca gcgtctgggt 1140 cgcgaccgtg cccaccacct gctggaacaa tgctgccaac gcgcggtggc cgaacagcgg 1200 cacctgcgtg ccgtgctggg tgacgagccg caggtcagcg ccgagctgtc tggcgaagaa 1260 ctcgatcgcc tgctcgaccc tgcccattac ctgggccagg cccgcgtctg ggtggcgcgc 1320 gccgtgtccg aacatcaacg tttcactgcc tga 1353 <210> 18 <211> 450 <212> PRT <213> Synthetic Sequence <220> <223> PcaB Amino Acid Sequence <400> 18 Met Ser Asn Gln Leu Phe Asp Ala Tyr Phe Thr Ala Pro Ala Met Arg 1 5 10 15 Glu Ile Phe Ser Asp Arg Gly Arg Leu Gln Gly Met Leu Asp Phe Glu 20 25 30 Ala Ala Leu Ala Arg Ala Glu Ala Ser Ala Gly Leu Val Pro His Ser 35 40 45 Ala Val Ala Ala Ile Glu Ala Ala Cys Gln Ala Glu Arg Tyr Asp Val 50 55 60 Gly Ala Leu Ala Asn Ala Ile Ala Thr Ala Gly Asn Ser Ala Ile Pro 65 70 75 80 Leu Val Lys Ala Leu Gly Lys Val Ile Ala Thr Gly Val Pro Glu Ala 85 90 95 Glu Arg Tyr Val His Leu Gly Ala Thr Ser Gln Asp Ala Met Asp Thr 100 105 110 Gly Leu Val Leu Gln Leu Arg Asp Ala Leu Asp Leu Ile Glu Ala Asp 115 120 125 Leu Gly Lys Leu Ala Asp Thr Leu Ser Gln Gln Ala Leu Lys His Ala 130 135 140 Asp Thr Pro Leu Val Gly Arg Thr Trp Leu Gln His Ala Thr Pro Val 145 150 155 160 Thr Leu Gly Met Lys Leu Ala Gly Val Leu Gly Ala Leu Thr Arg His 165 170 175 Arg Gln Arg Leu Gln Glu Leu Arg Pro Arg Leu Leu Val Leu Gln Phe 180 185 190 Gly Gly Ala Ser Gly Ser Leu Ala Ala Leu Gly Ser Lys Ala Met Pro 195 200 205 Val Ala Glu Ala Leu Ala Glu Gln Leu Lys Leu Thr Leu Pro Glu Gln 210 215 220 Pro Trp His Thr Gln Arg Asp Arg Leu Val Glu Phe Ala Ser Val Leu 225 230 235 240 Gly Leu Val Ala Gly Ser Leu Gly Lys Phe Gly Arg Asp Ile Ser Leu 245 250 255 Leu Met Gln Thr Glu Ala Gly Glu Val Phe Glu Pro Ser Ala Pro Gly 260 265 270 Lys Gly Gly Ser Ser Thr Met Pro His Lys Arg Asn Pro Val Gly Ala 275 280 285 Ala Val Leu Ile Gly Ala Ala Thr Arg Val Pro Gly Leu Leu Ser Thr 290 295 300 Leu Phe Ala Ala Met Pro Gln Glu His Glu Arg Ser Leu Gly Leu Trp 305 310 315 320 His Ala Glu Trp Glu Thr Leu Pro Asp Ile Cys Cys Leu Val Ser Gly 325 330 335 Ala Leu Arg Gln Ala Gln Val Ile Ala Glu Gly Met Glu Val Asp Ala 340 345 350 Ala Arg Met Arg Arg Asn Leu Asp Leu Thr Gln Gly Leu Val Leu Ala 355 360 365 Glu Ala Val Ser Ile Val Leu Ala Gln Arg Leu Gly Arg Asp Arg Ala 370 375 380 His His Leu Leu Glu Gln Cys Cys Gln Arg Ala Val Ala Glu Gln Arg 385 390 395 400 His Leu Arg Ala Val Leu Gly Asp Glu Pro Gln Val Ser Ala Glu Leu 405 410 415 Ser Gly Glu Glu Leu Asp Arg Leu Leu Asp Pro Ala His Tyr Leu Gly 420 425 430 Gln Ala Arg Val Trp Val Ala Arg Ala Val Ser Glu His Gln Arg Phe 435 440 445 Thr Ala 450 <210> 19 <211> 393 <212> Ms <213> artificial sequence <220> <223> pcaC <400> 19 atggacgaga aacaacgtta cgacgctggc atgcaagtgc gccgcgcagt gctgggtgat 60 Met Asp Glu Lys Gln Arg Tyr Asp Ala Gly Met Gln Val Arg Arg Ala Val Leu Gly Asp gcccacgtgg accgcagcct ggagaagctc aacgacttca atggcgagtt ccaggaaatg 120 Ala His Val Asp Arg Ser Leu Glu Lys Leu Asn Asp Phe Asn Gly Glu Phe Gln Glu Met atcacccgcc acgcctgggg tgacatctgg acccgcccgg ggctgccgcg ccatacccgc 180 Ile Thr Arg His Ala Trp Gly Asp Ile Trp Thr Pro Pro Gly Ala Ala Ala His Thr Arg agcctgatca ccatcgccat gctgattggc atgaaccgca acgacgagct gaagctgcac 240 Ser Leu Ile Thr Ile Ala Met Leu Ile Gly Met Asn Arg Asn Asp Glu Leu Lys Leu His ctgcgtgcgg cggccaacaa tggcgtgacc cgcgacgaga tcaaggaagt gctgatgcag 300 Leu Arg Cys Arg Gly Gln Asn Gly Val Thr Arg Asp Glu Ile Arg Glu Val Leu Asp Gln agcgcgatct actgcggcat tccggcggcc aatgccacgt tccacctggc tgagtcggtg 360 Ser Arg Asp Leu Tyr Cys Gly Ile Pro Gly Gly Gln Met His Val Ser Thr Trp Leu Ser Val tgggatgaac ttggcgtaga gtctcgccag taa 393 Trp Asp Glu Leu Gly Val Arg Ser Ser Pro <210> 20 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> PcaC Amino Acid Sequence <400> 20<l Met Asp Glu Lys Gln Arg Tyr Asp Ala Gly Met Gln Val Arg Arg Ala 1 5 10 15 Val Leu Gly Asp Ala His Val Asp Arg Ser Leu Glu Lys Leu Asn Asp 20 25 30 Phe Asn Gly Glu Phe Gln Glu Met Ile Thr Arg His Ala Trp Gly Asp 35 40 45 Ile Trp Thr Arg Pro Gly Leu Pro Arg His Thr Arg Ser Leu Ile Thr 50 55 60 Ile Ala Met Leu Ile Gly Met Asn Arg Asn Asp Glu Leu Lys Leu His 65 70 75 80 Leu Arg Ala Ala Ala Asn Asn Gly Val Thr Arg Asp Glu Ile Lys Glu 85 90 95 Val Leu Met Gln Ser Ala Ile Tyr Cys Gly Ile Pro Ala Ala Asn Ala 100 105 110 Thr Phe His Leu Ala Glu Ser Val Trp Asp Glu Leu Gly Val Glu Ser 115 120 125 Arg Gln 130 <210> 21 <211> 792 <212> DNA <213> Artificial sequence <220> <223> pcaD nucleotide sequence <400> 21 atggcgcact tgcaactggc cgatggcgtt ttgaattacc agatcgatgg cccggatgac 60 gccccggtgc tggtcctgtc caactcgctg ggtaccgacc tgggcatgtg ggacacccag 120 attccgctct ggagtcagca cttccgggtg ctgcgctatg acacccgtgg tcacggcgca 180 tcgctggtca ctgaaggccc ttacagcatc gaacagctgg gccgcgacgt gctggccctg 240 ctcgatggcc tggacattca aaaggctcac ttcgtcggcc tgtcgatggg cggcctgatc 300 ctcgatggcc tggacattca aaaggctcac ttcgtcggcc tgtcgatggg cggcctgatc 300 ggccagtggc tgggtatcca tgcaggtgag cgcctgcaca gcctgaccct gtgcaacacg 360 ggccagtggc tgggtatcca tgcaggtgag cgcctgcaca gcctgaccct gtgcaacacg 360 gccgccaaga tcgccaatga cgaggtgtgg aacacccgta tcgacacggt actcaaaggc 420 gccgccaaga tcgccaatga cgaggtgtgg aacacccgta tcgacacggt actcaaaggc 420 ggccagcagg ccatggtcga cctgcgcgat gcctccatcg cccgctggtt caccccgggc 480 ggccagcagg ccatggtcga cctgcgcgat gcctccatcg cccgctggtt caccccgggc 480 tttgcccagg cgcaggcgga gcaggcccag cgtatctgcc agatgctggc gcaaaccagc 540 tttgcccagg cgcaggcgga gcaggcccag cgtatctgcc agatgctggc gcaaaccagc 540 ccgcaaggct acgcaggcaa ctgtgcagcg gtacgtgacg ctgattatcg tgagcaactg 600 ccgcaaggct acgcaggcaa ctgtgcagcg gtacgtgacg ctgattatcg tgagcaactg 600 ggccgcatcc aggtgcctgc gctgatcgtt gccggtaccc aagacgtggt taccacccct 660 ggccgcatcc aggtgcctgc gctgatcgtt gccggtaccc aagacgtggt taccacccct 660 gagcatggcc gcttcatgca ggccggtatc caaggtgccg agtacgtcga cttcccggcg 720 gagcatggcc gcttcatgca ggccggtatc caaggtgccg agtacgtcga cttcccggcg 720 gcgcacctgt ccaatgtcga gattggcgag gccttcagcc gccgcgtgct cgatttcctg 780 gcgcacctgt ccaatgtcga gattggcgag gccttcagcc gccgcgtgct cgatttcctg 780 ctggctcact ga 792 ctggctcact ga 792 <210> 22<210> 22 <211> 263<211> 263 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220> <223> PcaD amino acid sequence <223> PcaD amino acid sequence <400> 22 <400> 22 Met Ala His Leu Gln Leu Ala Asp Gly Val Leu Asn Tyr Gln Ile Asp Met Ala His Leu Gln Leu Ala Asp Gly Val Leu Asn Tyr Gln Ile Asp 1 5 10 15 Gly Pro Asp Asp Ala Pro Val Leu Val Leu Ser Asn Ser Leu Gly Thr 20 25 30 Asp Leu Gly Met Trp Asp Thr Gln Ile Pro Leu Trp Ser Gln His Phe 35 40 45 Arg Val Leu Arg Tyr Asp Thr Arg Gly His Gly Ala Ser Leu Val Thr 50 55 60 Glu Gly Pro Tyr Ser Ile Glu Gln Leu Gly Arg Asp Val Leu Ala Leu 65 70 75 80 Leu Asp Gly Leu Asp Ile Gln Lys Ala His Phe Val Gly Leu Ser Met 85 90 95 Gly Gly Leu Ile Gly Gln Trp Leu Gly Ile His Ala Gly Glu Arg Leu 100 105 110 His Ser Leu Thr Leu Cys Asn Thr Ala Ala Lys Ile Ala Asn Asp Glu 115 120 125 Val Trp Asn Thr Arg Ile Asp Thr Val Leu Lys Gly Gly Gln Gln Ala 130 135 140 Met Val Asp Leu Arg Asp Ala Ser Ile Ala Arg Trp Phe Thr Pro Gly 145 150 155 160 Phe Ala Gln Ala Gln Ala Glu Gln Ala Gln Arg Ile Cys Gln Met Leu 165 170 175 Ala Gln Thr Ser Pro Gln Gly Tyr Ala Gly Asn Cys Ala Ala Val Arg 180 185 190 Asp Ala Asp Tyr Arg Glu Gln Leu Gly Arg Ile Gln Val Pro Ala Leu 195 200 205 Ile Val Ala Gly Thr Gln Asp Val Val Thr Thr Pro Glu His Gly Arg 210 215 220 Phe Met Gln Ala Gly Ile Gln Gly Ala Glu Tyr Val Asp Phe Pro Ala 225 230 235 240 Ala His Leu Ser Asn Val Glu Ile Gly Glu Ala Phe Ser Arg Arg Val 245 250 255 Leu Asp Phe Leu Leu Ala His 260 <210> 23 <211> 1194 <212> DNA <213> Artificial sequence <220> <223> tdTer nucleotide sequence <400> 23 atgatcgtta aaccgatggt gcgcaataac atttgtctga atgcacatcc gcagggttgt 60 aaaaaaggtg ttgaagatca gatcgagtac accaaaaaac gtattacagc cgaagttaaa 120<​​gcaagccgta ttaccgcagc atttggttat ggcgcagcaa ccattggtgt tagctttgaa 240 aaagcaggta gcgaaaccaa atatggcacc cctggttggt atataacct ggcatttgat 300 gaagcagcaa aacgtgaagg tctgtatagc gttaccattg atggtgatgc atttagcgac 360 gaaattaaag cgcaggttat tgaagaggcc aaaaaaagg gcatcaaatt cgacctgatt 420 gttatagcc tggcaagtcc ggttcgtacc gatccggata ccggcatcat gcataaaagc 480 gttctgaaac cgtttggcaa aacctttacc ggcaaaaccg ttgatccgtt taccggtgaa 540 ctgaaagaaa ttagcgcaga accggcaaat gatgaagaag cagcagcaac cgttaaagtt 600 atgggtggtg aagattggga acgttggatt aaacagctga gcaaagaagg tctgctggaa 660 gaaggttgta ttaccctggc atatagttat attggtccgg aagcaaccca ggcactgtat 720 cgtaaaggca ccattggtaa agcaaaagaa catctggaag ccaccgcaca tcgtctgaat 780 aaagaaaatc cgagcattcg tgcatttgtg agcgttaata aaggtctggt tacccgtgca 840 agcgcagtga ttccggttat tccgctgtat ctggccagcc tgtttaaagt gatgaaagaa 900 aaaggtaacc acgaaggttg cattgagcag attacccgtc tgtatgcaga acgtctgtat 960 cgcaaagatg gcaccattcc ggtggatgaa gaaaatcgta ttcgtatcga tgattgggag 1020 cttgaagaag atgttcagaa agcagttagc gcactgatgg aaaaagtgac cggtgaaaat 1080 gcagaaagcc tgaccgatct ggcaggttat cgtcatgatt ttctggcaag taatggcttt 1140 gatgtggaag gcattaacta tgaagcagaa gtggaacgtt ttgaccgcat ctaa 1194 <210> 24 <211> 397 <212> PRT <213> Artificial Sequence <220> <223> TdTer Amino Acid Sequence <400> 24 Met Ile Val Lys Pro Met Val Arg Asn Asn Ile Cys Leu Asn Ala His 1 5 10 15 Pro Gln Gly Cys Lys Lys Gly Val Glu Asp Gln Ile Glu Tyr Thr Lys 20 25 30 Lys Arg Ile Thr Ala Glu Val Lys Ala Gly Ala Lys Ala Pro Lys Asn 35 40 45 Val Leu Val Leu Gly Cys Ser Asn Gly Tyr Gly Leu Ala Ser Arg Ile 50 55 60 Thr Ala Ala Phe Gly Tyr Gly Ala Ala Thr Ile Gly Val Ser Phe Glu 65 70 75 80 Lys Ala Gly Ser Glu Thr Lys Tyr Gly Thr Pro Gly Trp Tyr Asn Asn 85 90 95 Leu Ala Phe Asp Glu Ala Ala Lys Arg Glu Gly Leu Tyr Ser Val Thr 100 105 110 Ile Asp Gly Asp Ala Phe Ser Asp Glu Ile Lys Ala Gln Val Ile Glu 115 120 125 Glu Ala Lys Lys Lys Gly Ile Lys Phe Asp Leu Ile Val Tyr Ser Leu 130 135 140 Ala Ser Pro Val Arg Thr Asp Pro Asp Thr Gly Ile Met His Lys Ser 145 150 155 160 Val Leu Lys Pro Phe Gly Lys Thr Phe Thr Gly Lys Thr Val Asp Pro 165 170 175 Phe Thr Gly Glu Leu Lys Glu Ile Ser Ala Glu Pro Ala Asn Asp Glu 180 185 190 Glu Ala Ala Ala Thr Val Lys Val Met Gly Gly Glu Asp Trp Glu Arg 195 200 205 Trp Ile Lys Gln Leu Ser Lys Glu Gly Leu Leu Glu Glu Gly Cys Ile 210 215 220 Thr Leu Ala Tyr Ser Tyr Ile Gly Pro Glu Ala Thr Gln Ala Leu Tyr 225 230 235 240 Arg Lys Gly Thr Ile Gly Lys Ala Lys Glu His Leu Glu Ala Thr Ala 245 250 255 His Arg Leu Asn Lys Glu Asn Pro Ser Ile Arg Ala Phe Val Ser Val 260 265 270 Asn Lys Gly Leu Val Thr Arg Ala Ser Ala Val Ile Pro Val Ile Pro 275 280 285 Leu Tyr Leu Ala Ser Leu Phe Lys Val Met Lys Glu Lys Gly Asn His 290 295 300 Glu Gly Cys Ile Glu Gln Ile Thr Arg Leu Tyr Ala Glu Arg Leu Tyr 305 310 315 320 Arg Lys Asp Gly Thr Ile Pro Val Asp Glu Glu Asn Arg Ile Arg Ile 325 330 335 Asp Asp Trp Glu Leu Glu Glu Asp Val Gln Lys Ala Val Ser Ala Leu 340 345 350 Met Glu Lys Val Thr Gly Glu Asn Ala Glu Ser Leu Thr Asp Leu Ala 355 360 365 Gly Tyr Arg His Asp Phe Leu Ala Ser Asn Gly Phe Asp Val Glu Gly 370 375 380 Ile Asn Tyr Glu Ala Glu Val Glu Arg Phe Asp Arg Ile 385 390 395 <210> 25 <211> 696 <212> DNA <213> Artificial sequence <220> <223> pcaI nucleotide sequence <400> 25 atgatcaaca aaacctatga gagcattgca agcgcagttg aaggtattac cgatggtagc 60 accattatgg ttggtggttt tggcaccgca ggtatgccga gcgaactgat tgatggtctg 120 attgcaaccg gtgcacgtga tctgaccatt atttctaata atgccggtaa tggtgaaatt 180 ggtctggcag cactgctgat ggcaggtagc gttcgtaaag ttgtttgtag ctttccgcgt 240 cagagcgata gctatgtttt tgatgaactg tatcgcgcag gcaaaattga actggaagtt 300 gttccgcagg gtaatctggc agaacgtatt cgtgcagccg gtagcggtat tggtgcattt 360 tttagcccga ccggttatgg caccctgctg gccgaaggta aagaaacccg tgaaattgat 420 ggccgtatgt atgttctgga aatgccgctg catgccgatt ttgcactgat taaagcacat 480 aaaggtgatc gttggggcaa tctgacctat cgtaaagcag cacgcaattt tggtccgatt 540 atggcaatgg cagcaaaaac cgcaattgca caggttgatc aggttgttga actgggtgaa 600 ctggacccgg aacatattat cacaccgggt atttttgttc agcgtgttgt tgcagttacc 660 ggtgcagcag caagcagcat tgccaaagca gtttaa 696 <210> 26 <211> 231 <212> PRT <213> Artificial sequence <220> <223> PcaI amino acid sequence <400> 26 Met Ile Asn Lys Thr Tyr Glu Ser Ile Ala Ser Ala Val Glu Gly Ile 1 5 10 15 Thr Asp Gly Ser Thr Ile Met Val Gly Gly Phe Gly Thr Ala Gly Met 20 25 30 Pro Ser Glu Leu Ile Asp Gly Leu Ile Ala Thr Gly Ala Arg Asp Leu 35 40 45 Thr Ile Ile Ser Asn Asn Ala Gly Asn Gly Glu Ile Gly Leu Ala Ala 50 55 60 Leu Leu Met Ala Gly Ser Val Arg Lys Val Val Cys Ser Phe Pro Arg 65 70 75 80 Gln Ser Asp Ser Tyr Val Phe Asp Glu Leu Tyr Arg Ala Gly Lys Ile 85 90 95 Glu Leu Glu Val Val Pro Gln Gly Asn Leu Ala Glu Arg Ile Arg Ala 100 105 110 Ala Gly Ser Gly Ile Gly Ala Phe Phe Ser Pro Thr Gly Tyr Gly Thr 115 120 125 Leu Leu Ala Glu Gly Lys Glu Thr Arg Glu Ile Asp Gly Arg Met Tyr 130 135 140 Val Leu Glu Met Pro Leu His Ala Asp Phe Ala Leu Ile Lys Ala His 145 150 155 160 Lys Gly Asp Arg Trp Gly Asn Leu Thr Tyr Arg Lys Ala Ala Arg Asn 165 170 175 Phe Gly Pro Ile Met Ala Met Ala Ala Lys Thr Ala Ile Ala Gln Val 180 185 190 Asp Gln Val Val Glu Leu Gly Glu Leu Asp Pro Glu His Ile Ile Thr 195 200 205 Pro Gly Ile Phe Val Gln Arg Val Val Ala Val Thr Gly Ala Ala Ala 210 215 220 Ser Ser Ile Ala Lys Ala Val 225 230 <210> 27 <211> 642 <212> Ms <213> artificial sequence <220> <223> pcaJ <400> 27 atgaccatca ccaaaaaact gagccgtacc gaaatggcac agcgtgttgc agcagatatt 60 caagaaggtg catacgttaa tctgggtatt ggtgcaccga ccctggttgc aaattatctg 120 ggtgataaag aagtgtttct gcatagcgaa aatggtctgc tgggtatggg tccgagtccg 180 gcaccgggtg aagaggatga tgatctgatt aatgcaggta aacagcatgt taccctgctg 240 accggtggtg cattttttca tcatgcagat agctttagca tgatgcgtgg tggtcatctg 300 gatattgcag ttctgggtgc atttcaggtt agcgttaaag gtgatctggc aaattggcat 360 accggtgcag aaggtagcat tccggcagtt ggtggtgcaa tggatctggc caccggtgca 420 cgtcaggttt ttgttatgat ggatcatctg accaaaaccg gtgaaagcaa actggttccg 480 gaatgcacat atccgctgac cggcattgca tgtgttagcc gtatttatac cgatctggcc 540 gttctggaag ttacaccgga aggtctgaaa gttgttgaaa tttgtgccga tatcgatttc 600 gatgaactgc agaaactgag cggtgttccg ctgatcaaat aa 642 <210> 28 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of PcaJ <400> 28 Met Thr Ile Thr Lys Lys Leu Ser Arg Thr Glu Met Ala Gln Arg Val 1 5 10 15 Ala Ala Asp Ile Gln Glu Gly Ala Tyr Val Asn Leu Gly Ile Gly Ala 20 25 30 Pro Thr Leu Val Ala Asn Tyr Leu Gly Asp Lys Glu Val Phe Leu His 35 40 45 Ser Glu Asn Gly Leu Leu Gly Met Gly Pro Ser Pro Ala Pro Gly Glu 50 55 60 Glu Asp Asp Asp Leu Ile Asn Ala Gly Lys Gln His Val Thr Leu Leu 65 70 75 80 Thr Gly Gly Ala Phe Phe His His Ala Asp Ser Phe Ser Met Met Arg 85 90 95 Gly Gly His Leu Asp Ile Ala Val Leu Gly Ala Phe Gln Val Ser Val 100 105 1i0 Lys Gly Asp Leu Ala Asn Trp His Thr Gly Ala Glu Gly Ser Ile Pro l 115 120 125 Ala Val Gly Gly Ala Met Asp Leu Ala Thr Gly Ala Arg Gln Val Phe 130 135 140 Val Met Met Asp His Leu Thr Lys Thr Gly Glu Ser Lys Leu Val Pro 145 150 155 160 Glu Cys Thr Tyr Pro Leu Thr Gly Ile Ala Cys Val Ser Arg Ile Tyr 165 170 175 Thr Asp Leu Ala Val Leu Glu Val Thr Pro Glu Gly Leu Lys Val Val 180 185 190 Glu Ile Cys Ala Asp Ile Asp Phe Asp Glu Leu Gln Lys Leu Ser Gly 195 200 205 Val Pro Leu Ile Lys 210 <210> 29 <211> 855 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of paaH1 <400> 29 atgagcattc gtaccgttgg tattgttggt gcaggcacca tgggtaatgg tattgcacag 60 gcatgtgcag ttgttggtct gaatgttgtt atggtggata ttagtgatgc agccgttcag 120 aaaggtgttg caaccgttgc cagcagcctg gatcgtctga tcaaaaaaga aaaactgacc 180 gaagcagata aagcaagcgc actggcacgt attaaaggta gcaccagcta tgatgatctg 240 aaagcaaccg atattgttat tgaagcagcc accgaaaact atgacctgaa agtgaaaatc 300 ctgaaacaaa tcgatggtat cgtgggcgaa aatgttatta ttgcaagcaa taccagcagc 360 atcagcatta ccaaactggc agcagttacc agccgtgcag atcgttttat tggtatgcat 420 tttttcaatc cggttccggt tatggcactg gttgaactga ttcgtggcct gcagaccagc 480 gataccaccc atgcagcagt tgaagcactg agcaaacagc tgggtaaata tccgattacc 540 gtgaaaaatt caccgggttt tgttgtgaat cgtattctgt gtccgatgat caatgaagcc 600 ttttgtgttc tgggtgaagg tctggcaagt ccggaagaaa ttgatgaagg tatgaaactg 660 ggttgcaatc atccgattgg tccgctggca ctggcagata tgattggtct ggataccatg 720 ctggcagtta tggaagttct gtataccgaa tttgccgatc cgaaatatcg tcctgccatg 780 ctgatgcgtg aaatggttgc agcaggttat ctgggtcgta aaaccggtcg tggtgtttat 840 gtttatagca aataa 855 <210> 30 <211> 284 <212> PRT <213> Artificial sequence <220> <223> PaaH1 amino acid sequence <400> 30 Met Ser Ile Arg Thr Val Gly Ile Val Gly Ala Gly Thr Met Gly Asn 1 5 10 15 Gly Ile Ala Gln Ala Cys Ala Val Val Gly Leu Asn Val Val Met Val 20 25 30 Asp Ile Ser Asp Ala Ala Val Gln Lys Gly Val Ala Thr Val Ala Ser 35 40 45 Ser Leu Asp Arg Leu Ile Lys Lys Glu Lys Leu Thr Glu Ala Asp Lys 50 55 60 Ala Ser Ala Leu Ala Arg Ile Lys Gly Ser Thr Ser Tyr Asp Asp Leu 65 70 75 80 Lys Ala Thr Asp Ile Val Ile Glu Ala Ala Thr Glu Asn Tyr Asp Leu 85 90 95 Lys Val Lys Ile Leu Lys Gln Ile Asp Gly Ile Val Gly Glu Asn Val 100 105 110 Ile Ile Ala Ser Asn Thr Ser Ser Ile Ser Ile Thr Lys Leu Ala Ala 115 120 125 Val Thr Ser Arg Ala Asp Arg Phe Ile Gly Met His Phe Phe Asn Pro 130 135 140 Val Pro Val Met Ala Leu Val Glu Leu Ile Arg Gly Leu Gln Thr Ser 145 150 155 160 Asp Thr Thr His Ala Ala Val Glu Ala Leu Ser Lys Gln Leu Gly Lys 165 170 175 Tyr Pro Ile Thr Val Lys Asn Ser Pro Gly Phe Val Val Asn Arg Ile 180 185 190 Leu Cys Pro Met Ile Asn Glu Ala Phe Cys Val Leu Gly Glu Gly Leu 195 200 205 Ala Ser Pro Glu Glu Ile Asp Glu Gly Met Lys Leu Gly Cys Asn His 210 215 220 Pro Ile Gly Pro Leu Ala Leu Ala Asp Met Ile Gly Leu Asp Thr Met 225 230 235 240 Leu Ala Val Met Glu Val Leu Tyr Thr Glu Phe Ala Asp Pro Lys Tyr 245 250 255 Arg Pro Ala Met Leu Met Arg Glu Met Val Ala Ala Gly Tyr Leu Gly 260 265 270 Arg Lys Thr Gly Arg Gly Val Tyr Val Tyr Ser Lys 275 280 <210> 31 <211> 777 <212> DNA <213> Artificial sequence <220> <223> ech nucleotide sequence (adipate pathway) <400> 31 atgccgtatg aaaacattct ggttgaaacc cgtggtcgtg ttggtctggt taccctgaat 60 cgtccgaaag cactgaatgc cctgaatgat gcactgatgg atgaactggg tgcagcactg 120 accgcatttg atcaggatga aggtattggt gcaattgtta ttaccggtag cgaacgtgca 180 tttgcagccg gtgcagatat tggtatgatg gcaaaatata gcttcatgga cgtgtataaa 240 ggcgattata tcacccgtaa ttgggaaacc attcgcaaaa ttcgtaaacc ggttattgcc 300 ggtgttgcag gttatgcact gggtggtggt tgtgaactgg caatgatgtg tgatattatc 360 attgcagcag atagcgcgaa atttggtcag ccggaagtta aactgggcac catgcctggt 420 gcgggtggca cccaacgtct gcctcgtgca gttagcaaag caaaagcaat ggatctgtgt 480 ctgaccagcc gtatgatgga tgcagcagaa gcagaacgta gcggtctggt gagccgtgtt 540 gttccggcag ataaactgct ggatgaagtt ctggcagcag cagaaaccat tgcaggtttt 600 agcctgccgg ttgttatgat gattaaagaa agcgttaatg cagcctatga aaccaccctg 660 gcagaaggtg ttcattttga acgtcgtctg tttcatgcaa cctttgcaag cgaagatcag 720 aaagaaggta tggcagcatt tgttgaaaaa cgcagcccga attttcagca ccgttaa 777 <210> 32 <211> 258 <212> PRT <213> Artificial sequence <220> <223> Ech amino acid sequence (adipate pathway) <400> 32 Met Pro Tyr Glu Asn Ile Leu Val Glu Thr Arg Gly Arg Val Gly Leu 1 5 10 15 Val Thr Leu Asn Arg Pro Lys Ala Leu Asn Ala Leu Asn Asp Ala Leu 20 25 30 Met Asp Glu Leu Gly Ala Ala Leu Thr Ala Phe Asp Gln Asp Glu Gly 35 40 45 Ile Gly Ala Ile Val Ile Thr Gly Ser Glu Arg Ala Phe Ala Ala Gly 50 55 60 Ala Asp Ile Gly Met Met Ala Lys Tyr Ser Phe Met Asp Val Tyr Lys 65 70 75 80 Gly Asp Tyr Ile Thr Arg Asn Trp Glu Thr Ile Arg Lys Ile Arg Lys 85 90 95 Pro Val Ile Ala Gly Val Ala Gly Tyr Ala Leu Gly Gly Gly Cys Glu 100 105 110 Leu Ala Met Met Cys Asp Ile Ile Ile Ala Ala Asp Ser Ala Lys Phe 115 120 125 Gly Gln Pro Glu Val Lys Leu Gly Thr Met Pro Gly Ala Gly Gly Thr 130 135 140 Gln Arg Leu Pro Arg Ala Val Ser Lys Ala Lys Ala Met Asp Leu Cys 145 150 155 160 Leu Thr Ser Arg Met Met Asp Ala Ala Glu Ala Glu Arg Ser Gly Leu 165 170 175 Val Ser Arg Val Val Pro Ala Asp Lys Leu Leu Asp Glu Val Leu Ala 180 185 190 Ala Ala Glu Thr Ile Ala Gly Phe Ser Leu Pro Val Val Met Met Ile 195 200 205 Lys Glu Ser Val Asn Ala Ala Tyr Glu Thr Thr Leu Ala Glu Gly Val 210 215 220 His Phe Glu Arg Arg Leu Phe His Ala Thr Phe Ala Ser Glu Asp Gln 225 230 235 240 Lys Glu Gly Met Ala Ala Phe Val Glu Lys Arg Ser Pro Asn Phe Gln 245 250 255 His Arg <210> 33 <211> 906 <212> DNA <213> Artificial sequence <220> <223> PTB nucleotide sequence (adipate pathway) <400> 33 atgatcaaaa gcttcaacga gatcatcatg aaagtgaaaa gcaaagaaat gaaaaaagtt 60 gccgttgcag ttgcacagga tgaaccggtg ctggaagcag ttcgtgatgc caaaaaaaac 120 ggtattgcag atgcaattct ggtgggtgat catgatgaaa ttgttagcat tgccctgaaa 180 attggcatgg atgtgaacga ttttgaaatc gtgaatgagc cgaatgtgaa aaaagcagca 240 ctgaaagcag ttgaactggt tagcaccggt aaagcagata tggttatgaa aggtctggtt 300 aataccgcaa cctttctgcg tagcgttctg aataaagaag ttggtctgcg taccggtaaa 360 accatgagcc atgttgcagt ttttgaaacc gaaaaatttg atcgcctgct gtttctgacc 420 gatgttgcat ttaataccta tccggaactg aaagagaaaa tcgatatcgt taacaacagc 480 gtgaaagttg cacatgccat tggtattgaa aatccgaaag tggcaccgat ttgtgccgtt 540 gaagttatta atccgaaaat gccgagcacc ctggatgcag caatgctgag caaaatgagc 600 gatcgtggtc agattaaagg ttgtgttgtt gatggtccgc tggcactgga tattgcactg 660 agcgaagaag cagcacatca taaaggtgtt accggtgaag ttgcaggcaa agccgatatt 720 tttctgatgc cgaatattga aaccggcaac gtgatgtata aaaccctgac ctataccacc 780 gatagcaaaa atggtggtat tctggttggc accagcgcac cggttgttct gaccagccgt 840 gcagatagcc atgaaaccaa aatgaatagc attgcactgg cagcactggt tgcaggtaac 900 aaaataa 906 <210> 34 <211> 301 <212> PRT <213> artificial sequence <220> <223> Ptb amino acid sequence (acetate pathway) <400> 34 Met Ile Lys Ser Phe Asn Glu Ile Met Lys Val Lys Ser Lys Glu 1 5 10 15 Met Lys Lys Val Ala Val Ala Val Ala Gln Asp Glu Pro Val Leu Glu 20 25 30 Ala Val Arg Asp Ala Lys Lys Asn Gly Ile Ala Asp Ala Ile Leu Val 35 40 45 Gly Asp His Asp Glu Ile Val Ser Ile Ala Leu Lys Ile Gly Met Asp 50 55 60 Val Asn Asp Phe Glu Ile Val Asn Glu Pro Asn Val Lys Lys Ala Ala 65 70 75 80 Leu Lys Ala Val Glu Leu Val Ser Thr Gly Lys Ala Asp Met Val Met 85 90 95 Lys Gly Leu Val Asn Thr Ala Thr Phe Leu Arg Ser Val Leu Asn Lys 100 105 110 Glu Val Gly Leu Arg Thr Gly Lys Thr Met Ser His Val Ala Val Phe 115 120 125 Glu Thr Glu Lys Phe Asp Arg Leu Leu Phe Leu Thr Asp Val Ala Phe 130 135 140 Asn Thr Tyr Pro Glu Leu Lys Glu Lys Ile Asp Ile Val Asn Asn Ser 145 150 155 160 Val Lys Val Ala His Ala Ile Gly Ile Glu Asn Pro Lys Val Ala Pro 165 170 175 Ile Cys Ala Val Glu Val Ile Asn Pro Lys Met Pro Ser Thr Leu Asp 180 185 190 Ala Ala Met Leu Ser Lys Met Ser Asp Arg Gly Gln Ile Lys Gly Cys 195 200 205 Val Val Asp Gly Pro Leu Ala Leu Asp Ile Ala Leu Ser Glu Glu Ala 210 215 220 Ala His His Lys Gly Val Thr Gly Glu Val Ala Gly Lys Ala Asp Ile 225 230 235 240 Phe Leu Met Pro Asn Ile Glu Thr Gly Asn Val Met Tyr Lys Thr Leu 245 250 255 Thr Tyr Thr Thr Asp Ser Lys Asn Gly Gly Ile Leu Val Gly Thr Ser 260 265 270 Ala Pro Val Val Leu Thr Ser Arg Ala Asp Ser His Glu Thr Lys Met 275 280 285 Asn Ser Ile Ala Leu Ala Ala Leu Val Ala Gly Asn Lys 290 295 300 <210> 35 <211> 1068 <212> DNA <213> Artificial sequence <220> <223> buk1 nucleotide sequence (adipate pathway) <400> 35 atgtatcgcc tgctgattat caatccgggt agcaccagca ccaaaattgg tatttatgat 60 gatgagaaag aaatctttga aaaaaccctg cgtcatagcg cagaagaaat cgaaaaatac 120 aacaccatct tcgaccagtt tcagtttcgc aaaaatgtta ttctggatgc cctgaaagaa 180 gccaacattg aagttagcag cctgaatgca gttgttggtc gtggtggtct gctgaaaccg 240 attgttagcg gcacctatgc agttaatcag aaaatgctgg aagacctgaa agttggtgtt 300 cagggtcagc atgcaagcaa tctgggtggt attattgcaa acgaaatcgc caaagaaatt 360 aacgtgcctg cctatattgt tgatccggtt gttgttgatg aactggatga agtgagccgt 420 attagcggta tggcagatat tccgcgtaaa agcatttttc atgcgctgaa tcagaaagca 480 gttgcacgtc gttatgcaaa agaagtgggc aaaaatacg aggatctgaa tctgattgtt 540 gtgcacatgg gtggtggcac cagcgttggc acccataaag atggtcgtgt tattgaagtg 600 aataaccc tggatggtga aggtccgttt agtccggaac gtagcggtgg tgttccgatt 660 ggtgatctgg ttcgtctgtg ttttagcaac aatatacct acgaagaagt gatgaaaaaa 720 atcaacggta aaggtggtgt tgtgagctat ctgaatacca tcgattttaa agccgttgtg 780 gataaagcac tggaaggtga caaaaaatgt gccctgattt atgaagcctt tacctttcag 840 gttgcgaaag aaattggtaa atgtagcacc gttctgaaag gcaatgttga tgcaattatt 900 ctgaccggtg gtattgccta taatgaacac gtttgtaatg ccattgaaga tcgcgtgaaa 960 ttcattgcac cggttgttcg ttatggtggt gaagatgaac tgctggcact ggccgaaggt 1020 ggcctgcgtg ttctgcgtgg cgaagaaaaa gcaaaagaat acaaataa 1068 <210> 36 <211> 355 <212> PRT <213> Artificial sequence <220> <223> Buk1 amino acid sequence (adipate pathway) <400> 36 Met Tyr Arg Leu Leu Ile Ile Asn Pro Gly Ser Thr Ser Thr Lys Ile 1 5 10 15 Gly Ile Tyr Asp Asp Glu Lys Glu Ile Phe Glu Lys Thr Leu Arg His 20 25 30 Ser Ala Glu Glu Ile Glu Lys Tyr Asn Thr Ile Phe Asp Gln Phe Gln 35 40 45 Phe Arg Lys Asn Val Ile Leu Asp Ala Leu Lys Glu Ala Asn Ile Glu 50 55 60 Val Ser Ser Leu Asn Ala Val Val Gly Arg Gly Gly Leu Leu Lys Pro 65 70 75 80 Ile Val Ser Gly Thr Tyr Ala Val Asn Gln Lys Met Leu Glu Asp Leu 85 90 95 Lys Val Gly Val Gln Gly Gln His Ala Ser Asn Leu Gly Gly Ile Ile 100 105 110 Ala Asn Glu Ile Ala Lys Glu Ile Asn Val Pro Ala Tyr Ile Val Asp 115 120 125 Pro Val Val Val Asp Glu Leu Asp Glu Val Ser Arg Ile Ser Gly Met 130 135 140 Ala Asp Ile Pro Arg Lys Ser Ile Phe His Ala Leu Asn Gln Lys Ala 145 150 155 160 Val Ala Arg Arg Tyr Ala Lys Glu Val Gly Lys Lys Tyr Glu Asp Leu 165 170 175 Asn Leu Ile Val Val His Met Gly Gly Gly Thr Ser Val Gly Thr His 180 185 190 Lys Asp Gly Arg Val Ile Glu Val Asn Asn Thr Leu Asp Gly Glu Gly 195 200 205 Pro Phe Ser Pro Glu Arg Ser Gly Gly Val Pro Ile Gly Asp Leu Val 210 215 220 Arg Leu Cys Phe Ser Asn Lys Tyr Thr Tyr Glu Glu Val Met Lys Lys 225 230 235 240 Ile Asn Gly Lys Gly Gly Val Val Ser Tyr Leu Asn Thr Ile Asp Phe 245 250 255 Lys Ala Val Val Asp Lys Ala Leu Glu Gly Asp Lys Lys Cys Ala Leu 260 265 270 Ile Tyr Glu Ala Phe Thr Phe Gln Val Ala Lys Glu Ile Gly Lys Cys 275 280 285 Sir Thr Val Leu Lys Gly Asn Val Asp Ala Ile Ile Leu Thr Gly Gly 290 295 300 Ile Ala Tyr Asn Glu His Val Cys Asn Ala Ile Glu Asp Arg Val Lys 305 310 315 320 Phe Ile Ala Pro Val Val Arg Tyr Gly Gly Glu Asp Glu Leu Leu Ala 325 330 335 Leu Ala Glu Gly Gly Leu Arg Val Leu Arg Gly Glu Glu Lys Ala Lys 340 345 350 The Tyr Lys 355 <210> 37 <211> 738 <212> DNA <213> artificial sequence <220> <223> adc sequence <400> 37 atgttaaagg atgaagtaat taaacaaatt agcacgccat taacttgcc tgcatttcct 60 agagccct ataaatttca taatcgtgag tattttaaca ttgtatatcg tacagatatg 120 gatgcacttc gtaaagttgt gccagagcct ttagaaattg atgagccctt agtcaggttt 180 gaaattatgg caatgcatga tacgagtgga cttggttgtt atacagaaag cggacaggct 240 attcccgtaa gctttaatgg agttaaggga gattatcttc atatgatgta tttagataat 300 gagcctgcaa ttgcagtagg aagggaatta agtgcatatc ctaaaaagct cgggtatcca 360 aagctttttg tggattcaga tactttagta ggaactttag actatggaaa acttagagtt 420 gcgacagcta caatggggta caaacataaa gccttagatg ctaatgaagc aaaggatcaa 480 atttgtcgcc ctaattatat gttgaaaata atacccaatt atgatggaag ccctagaata 540 tgtgagctta taaatgcgaa aatcacagat gttaccgtac atgaagcttg gacaggacca 600 actcgactgc agttatttga tcacgctatg gcgccactta atgatttgcc agtaaaagag 660 attgtttcta gctctcacat tcttgcagat ataatattgc ctagagctga agttatatat 720 gattatctta agtaataa 738 <210> 38 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> Adc Amino Acid Sequence (Adipate Pathway) <400> 38 Met Leu Lys Asp Glu Val Ile Lys Gln Ile Ser Thr Pro Leu Thr Ser 1 5 10 15 Pro Ala Phe Pro Arg Gly Pro Tyr Lys Phe His Asn Arg Glu Tyr Phe 20 25 30 Asn Ile Val Tyr Arg Thr Asp Met Asp Ala Leu Arg Lys Val Val Pro 35 40 45 Glu Pro Leu Glu Ile Asp Glu Pro Leu Val Arg Phe Glu Ile Met Ala 50 55 60 Met His Asp Thr Ser Gly Leu Gly Cys Tyr Thr Glu Ser Gly Gln Ala 65 70 75 80 Ile Pro Val Ser Phe Asn Gly Val Lys Gly Asp Tyr Leu His Met Met 85 90 95 Tyr Leu Asp Asn Glu Pro Ala Ile Ala Val Gly Arg Glu Leu Ser Ala 100 105 110 Tyr Pro Lys Lys Leu Gly Tyr Pro Lys Leu Phe Val Asp Ser Asp Thr 115 120 125 Leu Val Gly Thr Leu Asp Tyr Gly Lys Leu Arg Val Ala Thr Ala Thr 130 135 140 Met Gly Tyr Lys His Lys Ala Leu Asp Ala Asn Glu Ala Lys Asp Gln 145 150 155 160 Ile Cys Arg Pro Asn Tyr Met Leu Lys Ile Ile Pro Asn Tyr Asp Gly 165 170 175 Ser Pro Arg Ile Cys Glu Leu Ile Asn Ala Lys Ile Thr Asp Val Thr 180 185 190 Val His Glu Ala Trp Thr Gly Pro Thr Arg Leu Gln Leu Phe Asp His 195 200 205 Ala Met Ala Pro Leu Asn Asp Leu Pro Val Lys Glu Ile Val Ser Ser 210 215 220 Ser His Ile Leu Ala Asp Ile Ile Leu Pro Arg Ala Glu Val Ile Tyr 225 230 235 240 Asp Tyr Leu Lys <210> 39 <211> 471 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of HA controller (UniProt ID: PP3359) <400> 39 atggctaggt ctgcccgtag taccgacgac gcttgcgtgg ctgctcctgt gggagaaggg 60 gtgcttgaag acttgatcgg ctacgccttg cgacgcgcgc aattgaagct gtttcagaac 120 cttattgccc ggctctcggc ccatgacctg cgcccggccc aattttccgc cctggcgatc 180 atcgaccaga accccgggct gatgcaggcc gacctggcgc gtgcgttggc aatcgacccc 240 ccgcaagtcg tgccaatgct gaacaaactg gaagagcgcg cgctggccgt gcgcgtgcgg 300 tgcaaaccgg acaagcgctc gtatgggatt ttcctgagca aatcgggcga ggccctgttg 360 aaggagttga agcacatcgc cgccgacagc gatcaccagg cgacatccaa cctctcggat 420 gacgaaagga ctgaactgtt gaggttattg aagaaaatct accgggactg a 471 <210> 40 <211> 156 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of HA controller (UniProt ID: PP3359) <400> 40 Met Ala Arg Ser Ala Arg Ser Thr Asp Asp Ala Cys Val Ala Ala Pro 1 5 10 15 Val Gly Glu Gly Val Leu Glu Asp Leu Ile Gly Tyr Ala Leu Arg Arg 20 25 30 Ala Gln Leu Lys Leu Phe Gln Asn Leu Ile Ala Arg Leu Ser Ala His​​​​​​​​​​​Pro Gln Val Val Pro Met Leu Asn Lys Leu Glu Glu Arg Ala Leu Ala 85 90 95 Val Arg Val Arg Cys Lys Pro Asp Lys Arg Ser Tyr Gly Ile Phe Leu 100 105 110 Ser Lys Ser Gly Glu Ala Leu Leu Lys Glu Leu Lys His Ile Ala Ala 115 120 125 Asp Ser Asp His Gln Ala Thr Ser Asn Leu Ser Asp Asp Glu Arg Thr 130 135 140 Glu Leu Leu Arg Leu Leu Lys Lys Ile Tyr Arg Asp 145 150 155 <210> 41 <211> 6175 <212> DNA <213> Artificial Sequence[[ID=2\7]] <220> <223> pBAD Controller <400> 41 ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct 60 cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc 120 aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg 180 gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag 240 atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt 300 gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg 360 tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt 420 tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa 480 caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa 540 tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg 600 gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa 660 cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg 720 accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa 780 atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga 840 gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt 900 cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg tcttttactg 960 gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt aacaaagcgg 1020 gaccaaagcc atgacaaaaa cgcgtaaacaa aagtgtctat aatcacggca gaaaagtcca 1080 cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat ccataagatt 1140 agcggattct acctgacgct ttttatcgca actctctact gtttctccat acccgtttt 1200 ttgggaattc aaaagatcta aagaggaaga aggatctatg aacacgatta acatcgctaa 1260 gaacgacttc tctgacatcg aactggctgc tatcccgttc aacactctgg ctgaccatta 1320 cggtgagcgt ttagctcgcg aacagttggc ccttgagcat gagtcttacg agatgggtga 1380 agcacgcttc cgcaagatgt ttgagcgtca acttaaagct ggtgaggttg cggataacgc 1440 tgccgccaag cctctcatca ctaccctact ccctaagatg attgcacgca tcaacgactg 1500 gtttgaggaa gtgaaagcta agcgcggcaa gcgcccgaca gccttccagt tcctgcaaga 1560 aatcaagccg gaagccgtag cgtacatcac cattaagacc actctggctt gcctaaccag 1620 tgctgacaat acaaccgttc aggctgtagc aagcgcaatc ggtcgggcca ttgaggacga 1680 ggctcgcttc ggtcgtatcc gtgaccttga agctaagcac ttcaagaaaa acgttgagga 1740 acaactcaac aagcgcgtag ggcacgtcta caagaaagca tttatgcaag ttgtcgaggc 1800 tgacatgctc tctaagggtc tactcggtgg cgaggcgtgg tcttcgtggc ataaggaaga 1860 ctctattcat gtaggagtac gctgcatcga gatgctcatt gagtcaaccg gaatggttag 1920 cttacaccgc caaaatgctg gcgtagtagg tcaagactct gagactatcg aactcgcacc 1980 tgaatacgct gaggctatcg caacccgtgc aggtgcgctg gctggcatct ctccgatgtt 2040 ccaaccttgc gtagttcctc ctaagccgtg gactggcatt actggtggtg gctattgggc 2100 taacggtcgt cgtcctctgg cgctggtgcg tactcacagt aagaaagcac tgatgcgcta 2160 cgaagacgtt tacatgcctg aggtgtacaa agcgattaac attgcgcaaa acaccgcatg 2220 gaaaatcaac aagaaagtcc tagcggtcgc caacgtaatc accaagtgga agcattgtcc 2280 ggtcgaggac atccctgcga ttgagcgtga agaactcccg atgaaaccgg aagacatcga 2340 catgaatcct gaggctctca ccgcgtggaa acgtgctgcc gctgctgtgt accgcaagga 2400 caaggctcgc aagtctcgcc gtatcagcct tgagttcatg cttgagcaag ccaataagtt 2460 tgctaaccat aaggccatct ggttccctta caacatggac tggcgcggtc gtgtttacgc 2520 tgtgtcaatg ttcaacccgc aaggtaacga tatgaccaaa ggactgctta cgctggcgaa 2580 aggtaaacca atcggtaagg aaggttacta ctggctgaaa atccacggtg caaactgtgc 2640 gggtgtcgat aaggttccgt tccctgagcg catcaagttc attgaggaaa accacgagaa 2700 catcatggct tgcgctaagt ctccactgga gaacacttgg tgggctgagc aagattctcc 2760 gttctgcttc cttgcgttct gctttgagta cgctggggta cagcaccacg gcctgagcta 2820 taactgctcc cttccgctgg cgtttgacgg gtcttgctct ggcatccagc acttctccgc 2880 gatgctccga gatgaggtag gtggtcgcgc ggttaacttg cttcctagtg aaaccgttca 2940 ggacatctac gggattgttg ctaagaaagt caacgagatt ctacaagcag acgcaatcaa 3000 tgggaccgat aacgaagtag ttaccgtgac cgatgagaac actggtgaaa tctctgagaa 3060 agtcaagctg ggcactaagg cactggctgg tcaatggctg gcttacggtg ttactcgcag 3120 tgtgactaag cgttcagtca tgacgctggc ttacgggtcc aaagagttcg gcttccgtca 3180 acaagtgctg gaagatacca ttcagccagc tattgattcc ggcaagggtc tgatgttcac 3240 tcagccgaat caggctgctg gatacatggc taagctgatt tgggaatctg tgagcgtgac 3300 ggtggtagct gcggttgaag caatgaactg gcttaagtct gctgctaagc tgctggctgc 3360 tgaggtcaaa gataagaaga ctggagagat tcttcgcaag cgttgcgctg tgcattgggt 3420 aactcctgat ggtttccctg tgtggcagga atacaagaag cctattcaga cgcgcttgaa 3480 cctgatgttc ctcggtcagt tccgcttaca gcctaccatt aacaccaaca aagatagcga 3540 gattgatgca cacaaacagg agtctggtat cgctcctaac tttgtacaca gccaagacgg 3600 tagccacctt cgtaagactg tagtgtgggc acacgagaag tacggaatcg aatcttttgc 3660 actgattcac gactccttcg gtaccattcc ggctgacgct gcgaacctgt tcaaagcagt 3720 gcgcgaaact atggttgaca catatgagtc ttgtgatgta ctggctgatt tctacgacca 3780 gttcgctgac cagttgcacg agtctcaatt ggacaaaatg ccagcacttc cggctaaagg 3840 taacttgaac ctccgtgaca tcttagagtc ggacttcgcg ttcgcgtaag gatctctgtt 3900 ctctaatgtt aactccccct aacctgttgc tttagttat catttcctgt ctcactttgc 3960 cttaataccc tacgttaaat gttactaatt tgttgctttt gatcaacaata agaaaacaat 4020 atgtcgcttt tgtgcgcatt tttcagaaat gtagatattt ttagattatg gctacgaaat 4080 gagcatcgcc atgtcaccct acatctcata agggatcttt taagaaggag atatacatat 4140 gaataacgaa gcccgctcag ggtcgaccga ccctggccaa cgtccgcgct accgccaggt 4200 ggccatcggg catccccagg tgcaggtcag tcacgtcgac gacgtgctgc gcatgcaacc 4260 tgtcgagcca ctggcgccgc tgccggcgcg cctgctcgag cgcctggtgc attgggccca 4320 ggtgcgcccg gacaccactt tcatcgcggc acgccaggca gacggtgcct ggcgttcgat 4380 cagctacgtg cagatgctcg ccgatgtgcg caccatcgcc gccaacttgc taggactggg 4440 cctcagtgcc gagcgcccgc tggcgctgct ttccggcaac gacatcgaac acctgcaaat 4500 cgccctcggc gccatgtatg ccggtattgc ctattgcccg gtgtcgccgg cctacgcgct 4560 gttgtcgcaa gacttcgcca agttgcgcca tgtctgcgag gtgctcaccc ccggagtggt 4620 cttcgtcagc gacagccagc cgttccagcg cgccttcgag gcggtgctgg acgattcggt 4680 cggcgtgatc agcgtgcgtg gccaggtcgc aggtcgcccc catataagct tcgacagcct 4740 gttgcaaccg ggtgacctgg cggcggccga tgcggctttc gccgccaccg ggccggacac 4800 catcgccaaa ttcctcttca cctcgggctc gaccaagctg cccaaggcgg tgatcaccac 4860 ccagcgcatg ctgtgcgcca atcagcagat gcttctgcag acttttccga cgttcgccga 4920 ggagccgccg gtgctggtgg actggctgcc gtggaaccac acgttcggcg gtagccacaa 4980 cctcggcatc gtgctttaca acgggggcag tttctacctg gacgccggca agccgacccc 5040 gcaaggcttc gccgagacct tgcgcaattt gcgcgagatt tcccccacgg cctacctcac 5100 cgtacccaag ggctgggagg aactggtcaa ggcactggag caggaccccg cgctacgcga 5160 ggtgttcttt gcccgcatca agctgttctt ctttgccgcc gcaggcctgt cgcaaagcgt 5220 ctgggaccgg ctggaccgca ttgccgagca acactgtggc gaacgcatcc gcatgatggc 5280 cggccttggc atgaccgaag cctcgccatc gtgcaccttc accaccgggc ctttgtcgat5340 ggccggctat gtcgggctgc cggcacctgg ctgcgaagtg aagctggtgc cggtgggcga 5400. caagctcgag gcgcgcttcc gtggcccgca tatcatgccg ggctactggc gctcgccgca 5460 gcagaccgcc gaggcgttcg acgaggaggg cttctactgt tcgggcgacg cgttgaagct 5520 ggccgatgcc aggcagcccg agcttggcct gatgttcgat ggccgtatcg ctgaggactt 5580 caaactttcg tccggggtat tcgtcagtgt cgggccgctg cgcaaccgcg cagtgctgga 5640 gggctcgcct tacgtacagg acatcgtggt caccgcgccg gaccgtgaat gcctgggcct 5700 gctggtgttc ccgcgtctgc ccgagtgtcg gcgcctggcc gggctggcag aggatgccag 5760 cgatgcgcgg gtgctggcca acgacaccgt gcgcagttgg ttcgctgact ggctggagcg 5820 cttgaaccgc gatgcccag gcaacgccag ccgtatcgaa tggctgtcgc tgctggccga gccgccgtcg atcgacgccg gtgaaatcac cgacaagggc tcgatcaatc agcgcgccgt 5940. gctgcagcgg cgcgccgctc aggtcgaggc gctgtaccgt ggcgaagacc ccgacgcatt 6000 gcacgccaag gtgcggcctt aaggatccaa actcgagtaa ggatctccag gcatcaaata 6060 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac 6120 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttata 6175 <210> 42 <211> 5147 <212> DNA <213> Artificial sequence <220> <223> HA controller <400> 42 ctcaggtttc atgctcctcg atcatgggta ataaagttac ctattttgcc tgtccttatg 60 cgattcggct agagaggttc tggaaaaagg cagcgcgcct aaccccagga caaagataaa 120 attgttaatg gttaattgac ataactaatt tgacccgtta gcgtggcccc atcacctcga 180 acaacggatc taaagaggag aaaggatcta tgaacacgat taacatcgct aagaacgact 240 tctctgacat cgaactggct gctatcccgt tcaacactct ggctgaccat tacggtgagc 300 gtttagctcg cgaacagttg gcccttgagc atgagtctta cgagatgggt gaagcacgct 360 tccgcaagat gtttgagcgt caacttaaag ctggtgaggt tgcggataac gctgccgcca 420 agcctctcat cactacccta ctccctaaga tgattgcacg catcaacgac tggtttgagg 480 aagtgaaagc taagcgcggc aagcgcccga cagccttcca gttcctgcaa gaaatcaagc 540 cggaagccgt agcgtacatc accattaaga ccactctggc ttgcctaacc agtgctgaca 600 atacaaccgt tcaggctgta gcaagcgcaa tcggtcgggc cattgaggac gaggctcgct 660 tcggtcgtat ccgtgacctt gaagctaagc acttcaagaa aaacgttgag gaacaactca 720 acaagcgcgt agggcacgtc tacaagaaag catttatgca agttgtcgag gctgacatgc 780 tctctaaggg tctactcggt ggcgaggcgt ggtcttcgtg gcataaggaa gactctattc 840 atgtaggagt acgctgcatc gagatgctca ttgagtcaac cggaatggtt agcttacacc 900 gccaaaatgc tggcgtagta ggtcaagact ctgagactat cgaactcgca cctgaatacg 960 ctgaggctat cgcaacccgt gcaggtgcgc tggctggcat ctctccgatg ttccaacctt 1020 gcgtagttcc tcctaagccg tggactggca ttactggtgg tggctattgg gctaacggtc 1080 gtcgtcctct ggcgctggtg cgtactcaca gtaagaaagc actgatgcgc tacgaagacg 1140 tttacatgcc tgaggtgtac aaagcgatta acattgcgca aaacaccgca tggaaaatca 1200 acaagaaagt cctagcggtc gccaacgtaa tcaccaagtg gaagcattgt ccggtcgagg 1260 acatccctgc gattgagcgt gaagaactcc cgatgaaacc ggagaagacatc gacatgaatc 1320 ctgaggctct caccgcgtgg aaacgtgctg ccgctgctgt gtaccgcaag gacaaggctc 1380 gcaagtctcg ccgtatcagc cttgagttca tgcttgagca agccaataag tttgctaacc 1440 ataaggccat ctggttccct tacaacatgg actggcgcgg tcgtgtttac gctgtgtcaa 1500 tgttcaaccc gcaaggtaac gatatgacca aggactgct tacgctggcg aaaggtaaac 1560 caatcggtaa ggaaggttac tactggctga aaatccacgg tgcaaactgt gcgggtgtcg 1620 ataaggttcc gttccctgag cgcatcaagt tcattgagga aaaccacgag aacatcatgg 1680 cttgcgctaa gtctccactg gagaacactt ggtgggctga gcaagattct ccgttctgct 1740 tccttgcgtt ctgctttgag tacgctgggg tacagcacca cggcctgagc tataactgct 1800 cccttccgct ggcgtttgac gggtcttgct ctggcatcca gcacttctcc gcgatgctcc 1860 gagatgaggt aggtggtcgc gcggttaact tgcttcctag tgaaaccgtt caggacatct 1920 acgggattgt tgctaagaaa gtcaacgaga ttctacaagc agacgcaatc aatgggaccg 1980 ataacgaagt agttaccgtg accgatgaga acactggtga aatctctgag aaagtcaagc 2040 tgggcactaa ggcactggct ggtcaatggc tggcttacgg tgttactcgc agtgtgacta 2100 agcgttcagt catgacgctg gcttacgggt ccaaagagtt cggcttccgt caacaagtgc 2160 tggagaatac cattcagcca gctattgatt ccggcaaggg tctgatgttc actcagccga 2220 atcaggctgc tggatacatg gctaagctga tttgggaatc tgtgagcgtg acggtggtag 2280 ctgcggttga agcaatgaac tggcttaagt ctgctgctaa gctgctggct gctgaggtca 2340 aagataagaa gactggagag attcttcgca agcgttgcgc tgtgcattgg gtaactcctg 2400 atggtttccc tgtgtggcag gaatacaaga agcctattca gacgcgcttg aacctgatgt 2460 tcctcggtca gttccgctta cagcctacca ttaacaccaa caaagatagc gagattgatg 2520 cacacaaaca ggagtctggt atcgctccta actttgtaca cagccaagac ggtagccacc 2580 ttcgtaagac tgtagtgtgg gcacacgaga agtacggaat cgaatcttttt gcactgattc 2640 acgactcctt cggtaccatt ccggctgacg ctgcgaacct gttcaaagca gtgcgcgaaa 2700 ctatggttga cacatatgag tcttgtgatg tactggctga tttctacgac cagttcgctg 2760 accagttgca cgagtctcaa ttggacaaaa tgccagcact tccggctaaa ggtaacttga 2820 acctccgtga catcttagag tcggacttcg cgtcgcgta aggatctctg ttctctaatg 2880 ttaactcccc ctaacctgtt gctttagtta ttcatttcct gtctcacttt gccttaatac 2940 cctacgttaa atgttactaa tttgttgctt ttgatcacaa taagaaaaca atatgcgct 3000 tttgtgcgca ttttcagaa atgtagatat ttttagatta tggctacgaa atgagcatcg 3060 ccatgtcacc ctacatctca taagggatct tttaagaagg agatatacat atgaataacg 3120 aagcccgctc agggtcgacc gaccctggcc aacgtccgcg ctaccgccag gtggccatcg 3180 ggcatcccca ggtgcaggtc agtcacgtcg acgacgtgct gcgcatgcaa cctgtcgagc 3240 cactggcgcc gctgccggcg cgcctgctcg agcgcctggt gcattgggcc caggtgcgcc 3300 cggacaccac tttcatcgcg gcacgccagg cagacggtgc ctggcgttcg atcagctacg 3360 tgcagatgct cgccgatgtg cgcaccatcg ccgccaactt gctaggactg ggcctcagtg 3420 ccgagcgccc gctggcgctg ctttccggca acgacatcga acacctgcaa atcgccctcg 3480 gcgccatgta tgccggtatt gcctattgcc cggtgtcgcc ggcctacgcg ctgttgtcgc 3540 aagacttcgc caagttgcgc catgtctgcg aggtgctcac ccccggagtg gtcttcgtca 3600 gcgacagcca gccgttccag cgcgccttcg aggcggtgct ggacgattcg gtcggcgtga 3660 tcagcgtgcg tggccaggtc gcaggtcgcc cccatataag cttcgacagc ctgttgcaac 3720 cgggtgacct ggcggcggcc gatgcggctt tcgccgccac cgggccggac accatcgcca 3780 aattcctctt cacctcgggc tcgaccaagc tgcccaaggc ggtgatcacc acccagcgca 3840 tgctgtgcgc caatcagcag atgcttctgc agacttttcc gacgttcgcc gaggagccgc 3900 cggtgctggt ggactggctg ccgtggaacc acacgttcgg cggtagccac aacctcggca 3960 tcgtgcttta caacgggggc agtttctacc tggacgccgg caagccgacc ccgcaaggct 4020 tcgccgagac cttgcgcaat ttgcgcgaga tttcccccac ggcctacctc accgtaccca 4080 agggctggga ggaactggtc aaggcactgg agcaggaccc cgcgctacgc gaggtgttct 4140 ttgcccgcat caagctgttc ttctttgccg ccgcaggcct gtcgcaaagc gtctgggacc 4200 ggctggaccg cattgccgag caacactgtg gcgaacgcat ccgcatgatg gccggccttg 4260 gcatgaccga agcctcgcca tcgtgcacct tcaccaccgg gcctttgtcg atggccggct 4320 atgtcgggct gccggcacct ggctgcgaag tgaagctggt gccggtgggc gacaagctcg 4380 aggcgcgctt ccgtggcccg catatcatgc cgggctactg gcgctcgccg cagcagaccg 4440 ccgaggcgtt cgacgaggag ggcttctact gttcgggcga cgcgttgaag ctggccgatg 4500 ccaggcagcc cgagcttggc ctgatgttcg atggccgtat cgctgaggac ttcaaacttt 4560 cgtccggggt attcgtcagt gtcgggccgc tgcgcaaccg cgcagtgctg gagggctcgc 4620 cttacgtaca ggacatcgtg gtcaccgcgc cggaccgtga atgcctgggc ctgctggtgt 4680 tcccgcgtct gcccgagtgt cggcgcctgg ccgggctggc agaggatgcc agcgatgcgc 4740 gggtgctggc caacgacacc gtgcgcagtt ggttcgctga ctggctggag cgcttgaacc 4800 gcgatgccca aggcaacgcc agccgtatcg aatggctgtc gctgctggcc gagccgccgt 4860 cgatcgacgc cggtgaaatc accgacaagg gctcgatcaa tcagcgcgcc gtgctgcagc 4920 ggcgcgccgc tcaggtcgag gcgctgtacc gtggcgaaga ccccgacgca ttgcacgcca 4980 aggtgcggcc ttaaggatcc aaactcgagt aaggatctcc aggcatcaaa taaaacgaaa 5040 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta 5100 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttata 5147 <210> 43 <211> 2445 <212> DNA <213> Artificial Sequence <220> <223> fadE nucleotide sequence <400> 43 atgatgattt tgagtattct cgctacggtt gtcctgctcg gcgcgttgtt ctatcaccgc 60 gtgagcttat ttatcagcag tctgattttg ctcgcctgga cagccgccct cggcgttgct 120 ggtctgtggt cggcgtgggt actggtgcct ctggccatta tcctcgtgcc atttaacttt 180 gcgcctatgc gtaagtcgat gatttccgcg ccggtatttc gcggtttccg taaggtgatg 240 ccgccgatgt cgcgcactga gaaagaagcg attgatgcgg gcaccacctg gtgggagggc 300 gacttgttcc agggcaagcc ggactggaaa aagctgcata actatccgca gccgcgcctg 360 accgccgaag agcaagcgtt tctcgacggc ccggtagaag aagcctgccg gatggcgaat 420 gatttccaga tcacccatga gctggcggat ctgccgccgg agttgtgggc gtaccttaaa 480 gagcatcgtt tcttcgcgat gatcatcaaa aaagagtacg gcgggctgga gttctcggct 540 tatgcccagt ctcgcgtgct gcaaaaactc tccggcgtga gcgggatcct ggcgattacc 600 gtcggcgtgc caaactcatt aggcccgggc gaactgttgc aacattacgg cactgacgag 660 cagaaagatc actatctgcc gcgtctggcg cgtggtcagg agatcccctg ctttgcactg 720 accagcccgg aagcgggttc cgatgcgggc gcgattccgg acaccgggat tgtctgcatg 780 ggcgaatggc agggccagca ggtgctgggg atgcgtctga cctggaacaa acgctacatt 840 acgctggcac cgattgcgac cgtgcttggg ctggcgttta aactctccga cccggaaaaa 900 ttatcggcg gtgcagaaga tttaggcatt acctgtgcgc tgatcccaac caccacgccg 960 ggcgtggaaa ttggtcgtcg ccacttcccg ctgaacgtac cgttccagaa cggaccgacg 1020 cgcggtaaag atgtcttcgt gccgatcgat tacatcatcg gcgggccgaa aatggccggg 1080 caaggctggc ggatgctggt ggagtgcctc tcggtaggcc gcggcatcac cctgccttcc 1140 aactcaaccg gcggcgtgaa atcggtagcg ctggcaaccg gcgcgtatgc tcacattcgc 1200 cgtcagttca aaatctctat tggtaagatg gaagggattg aagagccgct ggcgcgtatt 1260 gccggtaatg cctacgtgat ggatgctgcg gcatcgctga ttacctacgg cattatgctc 1320 ggcgaaaaac ctgccgtgct gtcggctatc gttaagtatc actgtaccca ccgcgggcag 1380 cagtcgatta ttgatgcgat ggatattacc ggcggtaaag gcattatgct cgggcaaagc 1440 aacttcctgg cgcgtgctta ccagggcgca ccgattgcca tcaccgttga aggggctaac 1500 attctgaccc gcagcatgat gatcttcgga caaggagcga ttcgttgcca tccgtacgtg 1560 ctggaagaga tggaagcggc gaacaat gacgtcaacg cgttcgataa actgttgttc 1620 aaacatatcg gtcacgtcgg tagcaacaaa gttcgcagct tctggctggg cctgacgcgc 1680 ggtttaacca gcagcacgcc aaccggcgat gccactaaac gctactatca gcacctgaac 1740 cgcctgagcg ccaacctcgc cctgctttct gatgtctcga tggcagtgct gggcggcagc 1800 ctgaaacgtc gcgagcgcat ctcggcccgt ctgggggata ttttaagcca gctctacctc 1860 gcctctgccg tgctgaagcg ttatgacgac gaaggccgta atgaagccga cctgccgctg 1920 gtgcactggg gcgtacaaga tgcgctgtat caggctgaac aggcgatgga tgatttactg 1980 caaaacttcc cgaaccgcgt ggttgccggg ctgctgaatg tggtgatctt cccgaccgga 2040 cgtcattatc tggcaccttc tgacaagctg gatcataaag tggcgaagat tttacaagtg 2100 ccgaacgcca cccgttcccg cattggtcgc ggtcagtacc tgacgccgag cgagcataat 2160 ccggttggct tgctggaaga ggcgctggtg gatgtgattg ccgccgaccc aattcatcag 2220 cggatctgta aagagctggg taaaaacctg ccgtttaccc gtctggatga actggcgcac 2280 aacgcgctgg tgaaggggct gattgataaa gatgaagccg ctattctggt gaaagctgaa 2340 gaaagccgtc tgcgcagtat taacgttgat gactttgatc cggaagagct ggcgacgaag 2400 ccggtaaagt tgccggagaa agtgcggaaa gttgaagccg cgtaa 2445 <210> 44 <211> 814 <212> PRT <213> Artificial sequence <220> <223> FadE amino acid sequence <400> 44 Met Met Ile Leu Ser Ile Leu Ala Thr Val Val Leu Leu Gly Ala Leu 1 5 10 15 Phe Tyr His Arg Val Ser Leu Phe Ile Ser Ser Leu Ile Leu Leu Ala 20 25 30 Trp Thr Ala Ala Leu Gly Val Ala Gly Leu Trp Ser Ala Trp Val Leu 35 40 45 Val Pro Leu Ala Ile Ile Leu Val Pro Phe Asn Phe Ala Pro Met Arg 50 55 60 Lys Ser Met Ile Ser Ala Pro Val Phe Arg Gly Phe Arg Lys Val Met 65 70 75 80 Pro Pro Met Ser Arg Thr Glu Lys Glu Ala Ile Asp Ala Gly Thr Thr 85 90 95 Trp Trp Glu Gly Asp Leu Phe Gln Gly Lys Pro Asp Trp Lys Lys Leu 100 105 110 His Asn Tyr Pro Gln Pro Arg Leu Thr Ala Glu Glu Gln Ala Phe Leu 115 120 125 Asp Gly Pro Val Glu Glu Ala Cys Arg Met Ala Asn Asp Phe Gln Ile 130 135 140 Thr His Glu Leu Ala Asp Leu Pro Pro Glu Leu Trp Ala Tyr Leu Lys 145 150 155 160 Glu His Arg Phe Phe Ala Met Ile Ile Lys Lys Glu Tyr Gly Gly Leu 165 170 175 Glu Phe Ser Ala Tyr Ala Gln Ser Arg Val Leu Gln Lys Leu Ser Gly 180 185 190 Val Ser Gly Ile Leu Ala Ile Thr Val Gly Val Pro Asn Ser Leu Gly 195 200 205 Pro Gly Glu Leu Leu Gln His Tyr Gly Thr Asp Glu Gln Lys Asp His 210 215 220 Tyr Leu Pro Arg Leu Ala Arg Gly Gln Glu Ile Pro Cys Phe Ala Leu 225 230 235 240 Thr Ser Pro Glu Ala Gly Ser Asp Ala Gly Ala Ile Pro Asp Thr Gly 245 250 255 Ile Val Cys Met Gly Glu Trp Gln Gly Gln Gln Val Leu Gly Met Arg 260 265 270 Leu Thr Trp Asn Lys Arg Tyr Ile Thr Leu Ala Pro Ile Ala Thr Val 275 280 285 Leu Gly Leu Ala Phe Lys Leu Ser Asp Pro Glu Lys Leu Leu Gly Gly 290 295 300 Ala Glu Asp Leu Gly Ile Thr Cys Ala Leu Ile Pro Thr Thr Thr Pro 305 310 315 320 Gly Val Glu Ile Gly Arg Arg His Phe Pro Leu Asn Val Pro Phe Gln 325 330 335 Asn Gly Pro Thr Arg Gly Lys Asp Val Phe Val Pro Ile Asp Tyr Ile 340 345 350 Ile Gly Gly Pro Lys Met Ala Gly Gln Gly Trp Arg Met Leu Val Glu 355 360 365 Cys Leu Ser Val Gly Arg Gly Ile Thr Leu Pro Ser Asn Ser Thr Gly 370 375 380 Gly Val Lys Ser Val Ala Leu Ala Thr Gly Ala Tyr Ala His Ile Arg 385 390 395 400 Arg Gln Phe Lys Ile Ser Ile Gly Lys Met Glu Gly Ile Glu Glu Pro 405 410 415 Leu Ala Arg Ile Ala Gly Asn Ala Tyr Val Met Asp Ala Ala Ala Ser 420 425 430 Leu Ile Thr Tyr Gly Ile Met Leu Gly Glu Lys Pro Ala Val Leu Ser 435 440 445 Ala Ile Val Lys Tyr His Cys Thr His Arg Gly Gln Gln Ser Ile Ile 450 455 460 Asp Ala Met Asp Ile Thr Gly Gly Lys Gly Ile Met Leu Gly Gln Ser 465 470 475 480 Asn Phe Leu Ala Arg Ala Tyr Gln Gly Ala Pro Ile Ala Ile Thr Val 485 490 495 Glu Gly Ala Asn Ile Leu Thr Arg Ser Met Met Ile Phe Gly Gln Gly 500 505 510 Ala Ile Arg Cys His Pro Tyr Val Leu Glu Glu Met Glu Ala Ala Lys 515 520 525 Asn Asn Asp Val Asn Ala Phe Asp Lys Leu Leu Phe Lys His Ile Gly 530 535 540 His Val Gly Ser Asn Lys Val Arg Ser Phe Trp Leu Gly Leu Thr Arg 545 550 555 560 Gly Leu Thr Ser Ser Thr Pro Thr Gly Asp Ala Thr Lys Arg Tyr Tyr 565 570 575 Gln His Leu Asn Arg Leu Ser Ala Asn Leu Ala Leu Leu Ser Asp Val 580 585 590 Ser Met Ala Val Leu Gly Gly Ser Leu Lys Arg Arg Glu Arg Ile Ser 595 600 605 Ala Arg Leu Gly Asp Ile Leu Ser Gln Leu Tyr Leu Ala Ser Ala Val 610 615 620 Leu Lys Arg Tyr Asp Asp Glu Gly Arg Asn Glu Ala Asp Leu Pro Leu 625 630 635 640 Val His Trp Gly Val Gln Asp Ala Leu Tyr Gln Ala Glu Gln Ala Met 645 650 655 Asp Asp Leu Leu Gln Asn Phe Pro Asn Arg Val Val Ala Gly Leu Leu 660 665 670 Asn Val Val Ile Phe Pro Thr Gly Arg His Tyr Leu Ala Pro Ser Asp 675 680 685 Lys Leu Asp His Lys Val Ala Lys Ile Leu Gln Val Pro Asn Ala Thr 690 695 700 Arg Ser Arg Ile Gly Arg Gly Gln Tyr Leu Thr Pro Ser Glu His Asn 705 710 715 720 Pro Val Gly Leu Leu Glu Glu Ala Leu Val Asp Val Ile Ala Ala Asp 725 730 735 Pro Ile His Gln Arg Ile Cys Lys Glu Leu Gly Lys Asn Leu Pro Phe 740 745 750 Thr Arg Leu Asp Glu Leu Ala His Asn Ala Leu Val Lys Gly Leu Ile 755 760 765 Asp Lys Asp Glu Ala Ala Ile Leu Val Lys Ala Glu Glu Ser Arg Leu 770 775 780 Arg Ser Ile Asn Val Asp Asp Phe Asp Pro Glu Glu Leu Ala Thr Lys 785 790 795 800 Pro Val Lys Leu Pro Glu Lys Val Arg Lys Val Glu Ala Ala 805 810 <210> 45 <211> 1686 <212> DNA <213> Artificial sequence <220> <223> fadD nucleotide sequence <400> 45 ttgaagaagg tttggcttaa ccgttatccc gcggacgttc cgacggagat caaccctgac 60 cgttatcaat ctctggtaga tatgtttgag cagtcggtcg cgcgctacgc cgatcaacct 120 gcgtttgtga atatggggga ggtaatgacc ttccgcaagc tggaagaacg cagtcgcgcg 180 tttgccgctt atttgcaaca agggttgggg ctgaagaaag gcgatcgcgt tgcgttgatg 240 atgcctaatt tattgcaata tccggtggcg ctgtttggca ttttgcgtgc cgggatgatc 300 gtcgtaaacg ttaacccgtt gtataccccg cgtgagcttg agcatcagct taacgatagc 360 ggcgcatcgg cgattgttat cgtgtctaac tttgctcaca cactggaaaa agtggttgat 420 aaaaccgccg ttcagcacgt aattctgacc cgtatgggcg atcagctatc tacggcaaaa 480 ggcacggtag tcaatttcgt tgttaaatac atcaagcgtt tggtgccgaa ataccatctg 540 ccagatgcca tttcatttcg tagcgcactg cataacggct accggatgca gtacgtcaaa 600 cccgaactgg tgccggaaga tttagctttt ctgcaataca ccggcggcac cactggtgtg 660 gcgaaaggcg cgatgctgac tcaccgcaat atgctggcga acctggaaca ggttaacgcg 720 acctatggtc cgctgttgca tccgggcaaa gagctggtgg tgacggcgct gccgctgtat 780 cacatttttg ccctgaccat taactgcctg ctgtttatcg aactgggtgg gcagaacctg 840 cttatcacta acccgcgcga tattccaggg ttggtaaaag agttagcgaa atatccgttt 900 accgctatca cgggcgttaa caccttgttc aatgcgttgc tgaacaataa agagttccag 960 cagctggatt tctccagtct gcatctttcc gcaggcggtg ggatgccagt gcagcaagtg 1020 gtggcagagc gttgggtgaa actgaccgga cagtatctgc tggaaggcta tggccttacc 1080 gagtgtgcgc cgctggtcag cgttaaccca tatgatattg attatcatag tggtagcatc 1140 ggtttgccgg tgccgtcgac ggaagccaaa ctggtggatg atgatgataa tgaagtacca 1200 ccaggtcaac cgggtgagct ttgtgtcaaa ggaccgcagg tgatgctggg ttactggcag 1260 cgtcccgatg ctaccgatga aatcatcaaa aatggctggt tacacaccgg cgacatcgcg 1320 gtaatggatg aagaaggatt cctgcgcatt gtcgatcgta aaaaagacat gattctggtt 1380 tccggtttta acgtctatcc caacgagatt gaagatgtcg tcatgcagca tcctggcgta 1440 caggaagtcg cggctgttgg cgtaccttcc ggctccagtg gtgaagcggt gaaaatcttc 1500 gtagtgaaaa aagatccatc gcttaccgaa gagtcactgg tgactttttg ccgccgtcag 1560 ctcacgggat acaaagtacc gaagctggtg gagtttcgtg atgagttacc gaaatctaac 1620 gtcggaaaaa ttttgcgacg agaattacgt gacgaagcgc gcggcaaagt ggacaataaa 1680 gcctga 1686 <210> 46 <211> 561 <212> PRT <213> artificial sequence <220> <223> FadD amino acid sequence <400> 46 Met Lys Lys Val Trp Leu Asn Arg Tyr Pro Ala Asp Val Pro Thr Glu 1 5 10 15 Ile Asn Pro Asp Arg Tyr Gln Ser Leu Val Asp Met Phe Glu Gln Ser 20 25 30 Val Ala Arg Tyr Ala Asp Gln Pro Ala Phe Val Asn Met Gly Glu Val 35 40 45 Met Thr Phe Arg Lys Leu Glu Glu Arg Ser Arg Ala Phe Ala Ala Tyr 50 55 60 Leu Gln Gln Gly Leu Gly Leu Lys Lys Gly Asp Arg Val Ala Leu Met 65 70 75 80 Met Pro Asn Leu Leu Gln Tyr Pro Val Ala Leu Phe Gly Ile Leu Arg 85 90 95 Ala Gly Met Ile Val Val Asn Val Asn Pro Leu Tyr Thr Pro Arg Glu 100 105 110 Leu Glu His Gln Leu Asn Asp Ser Gly Ala Ser Ala Ile Val Ile Val 115 120 125 Ser Asn Phe Ala His Thr Leu Glu Lys Val Val Asp Lys Thr Ala Val 130 135 140 Gln His Val Ile Leu Thr Arg Met Gly Asp Gln Leu Ser Thr Ala Lys 145 150 155 160 Gly Thr Val Val Asn Phe Val Val Lys Tyr Ile Lys Arg Leu Val Pro 165 170 175 Lys Tyr His Leu Pro Asp Ala Ile Ser Phe Arg Ser Ala Leu His Asn 180 185 190 Gly Tyr Arg Met Gln Tyr Val Lys Pro Glu Leu Val Pro Glu Asp Leu 195 200 205 Ala Phe Leu Gln Tyr Thr Gly Gly Thr Thr Gly Val Ala Lys Gly Ala 210 215 220 Met Leu Thr His Arg Asn Met Leu Ala Asn Leu Glu Gln Val Asn Ala 225 230 235 240 Thr Tyr Gly Pro Leu Leu His Pro Gly Lys Glu Leu Val Val Thr Ala 245 250 255 Leu Pro Leu Tyr His Ile Phe Ala Leu Thr Ile Asn Cys Leu Leu Phe 260 265 270 Ile Glu Leu Gly Gly Gln Asn Leu Leu Ile Thr Asn Pro Arg Asp Ile 275 280 285 Pro Gly Leu Val Lys Glu Leu Ala Lys Tyr Pro Phe Thr Ala Ile Thr 290 295 300 Gly Val Asn Thr Leu Phe Asn Ala Leu Leu Asn Asn Lys Glu Phe Gln 305 310 315 320 Gln Leu Asp Phe Ser Ser Leu His Leu Ser Ala Gly Gly Gly Met Pro 325 330 335 Val Gln Gln Val Val Ala Glu Arg Trp Val Lys Leu Thr Gly Gln Tyr 340 345 350 Leu Leu Glu Gly Tyr Gly Leu Thr Glu Cys Ala Pro Leu Val Ser Val 355 360 365 Asn Pro Tyr Asp Ile Asp Tyr His Ser Gly Ser Ile Gly Leu Pro Val 370 375 380 Pro Ser Thr Glu Ala Lys Leu Val Asp Asp Asp Asp Asn Glu Val Pro 385 390 395 400 Pro Gly Gln Pro Gly Glu Leu Cys Val Lys Gly Pro Gln Val Met Leu 405 410 415 Gly Tyr Trp Gln Arg Pro Asp Ala Thr Asp Glu Ile Ile Lys Asn Gly 420 425 430 Trp Leu His Thr Gly Asp Ile Ala Val Met Asp Glu Glu Gly Phe Leu 435 440 445 Arg Ile Val Asp Arg Lys Lys Asp Met Ile Leu Val Ser Gly Phe Asn 450 455 460 Val Tyr Pro Asn Glu Ile Glu Asp Val Val Met Gln His Pro Gly Val 465 470 475 480 Gln Glu Val Ala Ala Val Gly Val Pro Ser Gly Ser Ser Gly Glu Ala 485 490 495 Val Lys Ile Phe Val Val Lys Lys Asp Pro Ser Leu Thr Glu Glu Ser 500 505 510 Leu Val Thr Phe Cys Arg Arg Gln Leu Thr Gly Tyr Lys Val Pro Lys 515 520 525 Leu Val Glu Phe Arg Asp Glu Leu Pro Lys Ser Asn Val Gly Lys Ile 530 535 540 Leu Arg Arg Glu Leu Arg Asp Glu Ala Arg Gly Lys Val Asp Asn Lys 545 550 555 560 Ala [[ID=...]]<210> 47 <211> 663 <212> DNA <213> Artificial sequence <220> <223> atoD nucleotide sequence <400> 47 atgaaaacaa aattgatgac attacaagac gccaccggct tctttcgtga cggcatgacc 60 atcatggtgg gcggatttat ggggattggc actccatccc gcctggttga agcattactg 120 gaatctggtg ttcgcgacct gacattgata gccaatgata ccgcgtttgt tgataccggc 180 atcggtccgc tcatcgtcaa tggtcgagtc cgcaaagtga ttgcttcaca tatcggcacc 240 aacccggaaa caggtcggcg catgatatct ggtgagatgg acgtcgttct ggtgccgcaa 300 ggtacgctaa tcgagcaaat tcgctgtggt ggagctggac ttggtggttt tctcacccca 360 acgggtgtcg gcaccgtcgt agaggaaggc aaacagacac tgacactcga cggtaaaacc 420 tggctgctcg aacgcccact gcgcgccgac ctggcgctaa ttcgcgctca tcgttgcgac 480 acacttggca acctgaccta tcaacttagc gcccgcaact ttaaccccct gatagccctt 540 gcggctgata tcacgctggt agagccagat gaactggtcg aaaccggcga gctgcaacct 600 gaccatattg tcacccctgg tgccgttatc gaccacatca tcgtttcaca ggagagcaaa 660 taa 663 <210> 48 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> AtoD Amino Acid Sequence <400> 48 Met Lys Thr Lys Leu Met Thr Leu Gln Asp Ala Thr Gly Phe Phe Arg 1 5 10 15 Asp Gly Met Thr Ile Met Val Gly Gly Phe Met Gly Ile Gly Thr Pro 20 25 30 Ser Arg Leu Val Glu Ala Leu Leu Glu Ser Gly Val Arg Asp Leu Thr 35 40 45 Leu Ile Ala Asn Asp Thr Ala Phe Val Asp Thr Gly Ile Gly Pro Leu 50 55 60 Ile Val Asn Gly Arg Val Arg Lys Val Ile Ala Ser His Ile Gly Thr 65 70 75 80 Asn Pro Glu Thr Gly Arg Arg Met Ile Ser Gly Glu Met Asp Val Val 85 90 95 Leu Val Pro Gln Gly Thr Leu Ile Glu Gln Ile Arg Cys Gly Gly Ala 100 105 110 Gly Leu Gly Gly Phe Leu Thr Pro Thr Gly Val Gly Thr Val Val Glu 115 120 125 Glu Gly Lys Gln Thr Leu Thr Leu Asp Gly Lys Thr Trp Leu Leu Glu 130 135 140 Arg Pro Leu Arg Ala Asp Leu Ala Leu Ile Arg Ala His Arg Cys Asp 145 150 155 160 Thr Leu Gly Asn Leu Thr Tyr Gln Leu Ser Ala Arg Asn Phe Asn Pro 165 170 175 Leu Ile Ala Leu Ala Ala Asp Ile Thr Leu Val Glu Pro Asp Glu Leu 180 185 190 Val Glu Thr Gly Glu Leu Gln Pro Asp His Ile Val Thr Pro Gly Ala 195 200 205 Val Ile Asp His Ile Ile Val Ser Gln Glu Ser Lys 210 215 220 <210> 49 <211> 651 <212> DNA<000249�><213> Artificial sequence <220> <223> atoA nucleotide sequence <400> 49 atggatgcga aacaacgtat tgcgcgccgt gtggcgcaag agcttcgtga tggtgacatc 60 gttaacttag ggatcggttt acccacaatg gtcgccaatt atttaccgga gggtattcat 120 atcactctgc aatcggaaaa cggcttcctc ggtttaggcc cggtcacgac agcgcatcca 1⹮0 gatctggtga acgctggcgg gcaaccgtgc ggtgttttac ccggtgcagc catgtttgat 240 agcgccatgt catttgcgct aatccgtggc ggtcatattg atgcctgcgt gctcggcggt 300 It should be noted that there seems to be a small error in the original text where "<000249�>" should probably be " ". Also, in the translation of the nucleotide sequence part, it's presented in a way that might be more suitable for a technical or scientific context. If you have any specific requirements or need further adjustments, please let me know.ttgcaagtag acgaagaagc aaacctcgcg aactgggtag tgcctgggaa aatggtgccc 360 ggtatgggtg gcgcgatgga tctggtgacc gggtcgcgca aagtgatcat cgccatggaa 420 cattgcgcca aagatggttc agcaaaaatt ttgcgccgct gcaccatgcc actcactgcg 480 caacatgcgg tgcatatgct ggttactgaa ctggctgtct ttcgttttat tgacggcaaa 540 atgtggctca ccgaaattgc cgacgggtgt gatttagcca ccgtgcgtgc caaaacagaa 600 gctcggtttg aagtcgccgc cgatctgaat acgcaacggg gtgatttatg a 651 <210> 50 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> AtoA Amino Acid Sequence <400> 50 Met Asp Ala Lys Gln Arg Ile Ala Arg Arg Val Ala Gln Glu Leu Arg 1 5 10 15 Asp Gly Asp Ile Val Asn Leu Gly Ile Gly Leu Pro Thr Met Val Ala 20 25 30 Asn Tyr Leu Pro Glu Gly Ile His Ile Thr Leu Gln Ser Glu Asn Gly 35 40 45 Phe Leu Gly Leu Gly Pro Val Thr Thr Ala His Pro Asp Leu Val Asn 50 55 60 Ala Gly Gly Gln Pro Cys Gly Val Leu Pro Gly Ala Ala Met Phe Asp 65 70 75 80 Ser Ala Met Ser Phe Ala Leu Ile Arg Gly Gly His Ile Asp Ala Cys 85 90 95 Val Leu Gly Gly Leu Gln Val Asp Glu Glu Ala Asn Leu Ala Asn Trp 100 105 110 Val Val Pro Gly Lys Met Val Pro Gly Met Gly Gly Ala Met Asp Leu 115 120 125 Val Thr Gly Ser Arg Lys Val Ile Ile Ala Met Glu His Cys Ala Lys 130 135 140 Asp Gly Ser Ala Lys Ile Leu Arg Arg Cys Thr Met Pro Leu Thr Ala 145 150 155 160 Gln His Ala Val His Met Leu Val Thr Glu Leu Ala Val Phe Arg Phe 165 170 175 Ile Asp Gly Lys Met Trp Leu Thr Glu Ile Ala Asp Gly Cys Asp Leu 180 185 190 Ala Thr Val Arg Ala Lys Thr Glu Ala Arg Phe Glu Val Ala Ala Asp 195 200 205 Leu Asn Thr Gln Arg Gly Asp Leu 210 215 <210> 51 <211> 1206 <212> DNA <213> Artificial sequence <220> <223> paaJ nucleotide sequence <400> 51 atgcgtgaag cctttatttg tgacggaatt cgtacgccaa ttggtcgcta cggcggggca 60 ttatcaagtg ttcgggctga tgatctggct gctatccctt tgcgggaact gctggtgcga 120 aacccgcgtc tcgatgcgga gtgtatcgat gatgtgatcc tcggctgtgc taatcaggcg 180 ggagaagata accgtaacgt agcccggatg gcgactttac tggcggggct gccgcagagt 240 gtttccggca caaccattaa ccgcttgtgt ggttccgggc tggacgcact ggggtttgcc 300 gcacgggcga ttaaagcggg cgatggcgat ttgctgatcg ccggtggcgt ggagtcaatg 360 tcacgggcac cgtttgttat gggcaaggca gccagtgcat tttctcgtca ggctgagatg 420 ttcgatacca ctattggctg gcgatttgtg aacccgctca tggctcagca atttggaact 480 gacagcatgc cggaaacggc agagaatgta gctgaactgt taaaaatctc acgagaagat 540 caagatagtt ttgcgctacg cagtcagcaa cgtacggcaa aagcgcaatc ctcaggcatt 600 ctggctgagg agattgttcc ggttgtgttg aaaaacaaga aaggtgttgt aacagaaata 660 caacatgatg agcatctgcg cccggaaacg acgctggaac agttacgtgg gttaaaagca 720 ccatttcgtg ccaatggggt gattaccgca ggcaatgctt ccggggtgaa tgacggagcc 780 gctgcgttga ttattgccag tgaacagatg gcagcagcgc aaggactgac accgcgggcg 840 cgtatcgtag ccatggcaac cgccggggtg gaaccgcgcc tgatggggct tggtccggtg 900 cctgcaactc gccgggtgct ggaacgcgca gggctgagta ttcacgatat ggacgtgatt 960 gaactgaacg aagcgttcgc ggcccaggcg ttgggtgtac tacgcgaatt ggggctgcct 1020 gatgatgccc cacatgttaa ccccaacgga ggcgctatcg ccttaggcca tccgttggga 1080 atgagtggtg cccgcctggc actggctgcc agccatgagc tgcatcggcg taacggtcgt 1140 tacgcattgt gcaccatgtg catcggtgtc ggtcagggca tcgccatgat tctggagcgt 1200 gtttga 1206 <210> 52 <211> 401 <212> PRT <213> Artificial Sequence <220> <223> PaaJ Amino Acid Sequence <400> 52 Met Arg Glu Ala Phe Ile Cys Asp Gly Ile Arg Thr Pro Ile Gly Arg 1 5 10 15 Tyr Gly Gly Ala Leu Ser Ser Val Arg Ala Asp Asp Leu Ala Ala Ile 20 25 30 Pro Leu Arg Glu Leu Leu Val Arg Asn Pro Arg Leu Asp Ala Glu Cys 35 40 45 Ile Asp Asp Val Ile Leu Gly Cys Ala Asn Gln Ala Gly Glu Asp Asn 50 55 60 Arg Asn Val Ala Arg Met Ala Thr Leu Leu Ala Gly Leu Pro Gln Ser 65 70 75 80 Val Ser Gly Thr Thr Ile Asn Arg Leu Cys Gly Ser Gly Leu Asp Ala 85 90 95 Leu Gly Phe Ala Ala Arg Ala Ile Lys Ala Gly Asp Gly Asp Leu Leu 100 105 110 Ile Ala Gly Gly Val Glu Ser Met Ser Arg Ala Pro Phe Val Met Gly 115 120 125 Lys Ala Ala Ser Ala Phe Ser Arg Gln Ala Glu Met Phe Asp Thr Thr 130 135 140 Ile Gly Trp Arg Phe Val Asn Pro Leu Met Ala Gln Gln Phe Gly Thr 145 150 155 160 Asp Ser Met Pro Glu Thr Ala Glu Asn Val Ala Glu Leu Leu Lys Ile 165 170 175 Ser Arg Glu Asp Gln Asp Ser Phe Ala Leu Arg Ser Gln Gln Arg Thr 180 185 190 Ala Lys Ala Gln Ser Ser Gly Ile Leu Ala Glu Glu Ile Val Pro Val 195 200 205 Val Leu Lys Asn Lys Lys Gly Val Val Thr Glu Ile Gln His Asp Glu 210 215 220 His Leu Arg Pro Glu Thr Thr Leu Glu Gln Leu Arg Gly Leu Lys Ala 225 230 235 240 Pro Phe Arg Ala Asn Gly Val Ile Thr Ala Gly Asn Ala Ser Gly Val 245 250 255 Asn Asp Gly Ala Ala Ala Leu Ile Ile Ala Ser Glu Gln Met Ala Ala 260 265 270 Ala Gln Gly Leu Thr Pro Arg Ala Arg Ile Val Ala Met Ala Thr Ala 275 280 285 Gly Val Glu Pro Arg Leu Met Gly Leu Gly Pro Val Pro Ala Thr Arg 290 295 300 Arg Val Leu Glu Arg Ala Gly Leu Ser Ile His Asp Met Asp Val Ile 305 310 315 320 Glu Leu Asn Glu Ala Phe Ala Ala Gln Ala Leu Gly Val Leu Arg Glu 325 330 335 Leu Gly Leu Pro Asp Asp Ala Pro His Val Asn Pro Asn Gly Gly Ala 340 345 350 Ile Ala Leu Gly His Pro Leu Gly Met Ser Gly Ala Arg Leu Ala Leu 355 360 365 Ala Ala Ser His Glu Leu His Arg Arg Asn Gly Arg Tyr Ala Leu Cys 370 375 380 Thr Met Cys Ile Gly Val Gly Gln Gly Ile Ala Met Ile Leu Glu Arg 385 390 395 400 Val <210> 53 <211> 1167 <212> DNA <213> Artificial sequence <220> <223> sucC nucleotide sequence <400> 53 atgaacttac atgaatatca ggcaaaacaa ctttttgccc gctatggctt accagcaccg 60 gtgggttatg cctgtactac tccgcgcgaa gcagaagaag ccgcttcaaa aatcggtgcc 120 ggtccgtggg tagtgaaatg tcaggttcac gctggtggcc gcggtaaagc gggcggtgtg 180[[ID=J42]] aaagttgtaa acagcaaaga agacatccgt gcttttgcag aaaactggct gggcaagcgt 240 ctggtaacgt atcaaacaga tgccaatggc caaccggtta accagattct ggttgaagca 300 gcgaccgata tcgctaaaga gctgtatctc ggtgccgttg ttgaccgtag ttcccgtcgt 360 gtggtcttta tggcctccac cgaaggcggc gtggaaatcg aaaaagtggc ggaagaaact 420 ccgcacctga tccataaagt tgcgcttgat ccgctgactg gcccgatgcc gtatcaggga 480 cgcgagctgg cgttcaaact gggtctggaa ggtaaactgg ttcagcagtt caccaaaatc 540 ttcatgggcc tggcgaccat tttcctggag cgcgacctgg cgttgatcga aatcaacccg 600 ctggtcatca ccaaacaggg cgatctgatt tgcctcgacg gcaaactggg cgctgacggc 660 aacgcactgt tccgccagcc tgatctgcgc gaaatgcgtg accagtcgca ggaagatccg 720 cgtgaagcac aggctgcaca gtgggaactg aactacgttg cgctggacgg taacatcggt 780 tgtatggtta acggcgcagg tctggcgatg ggtacgatgg acatcgttaa actgcacggc 840 ggcgaaccgg ctaacttcct tgacgttggc ggcggcgcaa ccaaagaacg tgtaaccgaa 900 gcgttcaaaa tcatcctctc tgacgacaaa gtgaaagccg ttctggttaa catcttcggc 960 ggtatcgttc gttgcgacct gatcgctgac ggtatcatcg gcgcggtagc agaagtgggt 1020 gttaacgtac cggtcgtggt acgtctggaa ggtaacaacg ccgaactcgg cgcgaagaaa 1080 ctggctgaca gcggcctgaa tattattgca gcaaaaggtc tgacggatgc agctcagcag 1140 gttgttgccg cagtggaggg gaaataa 1167 <210> 54 <211> 388 <212> PRT <213> Artificial sequence <220> <223> SucC amino acid sequence <400> 54 Met Asn Leu His Glu Tyr Gln Ala Lys Gln Leu Phe Ala Arg Tyr Gly 1 5 10 15 Leu Pro Ala Pro Val Gly Tyr Ala Cys Thr Thr Pro Arg Glu Ala Glu 20 25 30 Glu Ala Ala Ser Lys Ile Gly Ala Gly Pro Trp Val Val Lys Cys Gln 35 40 45 Val His Ala Gly Gly Arg Gly Lys Ala Gly Gly Val Lys Val Val Asn 50 55 60 Ser Lys Glu Asp Ile Arg Ala Phe Ala Glu Asn Trp Leu Gly Lys Arg 65 70 75 80 Leu Val Thr Tyr Gln Thr Asp Ala Asn Gly Gln Pro Val Asn Gln Ile 85 90 95 Leu Val Glu Ala Ala Thr Asp Ile Ala Lys Glu Leu Tyr Leu Gly Ala 100 105 110 Val Val Asp Arg Ser Ser Arg Arg Val Val Phe Met Ala Ser Thr Glu 115 120 125 Gly Gly Val Glu Ile Glu Lys Val Ala Glu Glu Thr Pro His Leu Ile 130 135 140 His Lys Val Ala Leu Asp Pro Leu Thr Gly Pro Met Pro Tyr Gln Gly 145 150 155 160 Arg Glu Leu Ala Phe Lys Leu Gly Leu Glu Gly Lys Leu Val Gln Gln 165 170 175 Phe Thr Lys Ile Phe Met Gly Leu Ala Thr Ile Phe Leu Glu Arg Asp 180 185 190 Leu Ala Leu Ile Glu Ile Asn Pro Leu Val Ile Thr Lys Gln Gly Asp 195 200 205 Leu Ile Cys Leu Asp Gly Lys Leu Gly Ala Asp Gly Asn Ala Leu Phe 210 215 220 Arg Gln Pro Asp Leu Arg Glu Met Arg Asp Gln Ser Gln Glu Asp Pro 225 230 235 240 Arg Glu Ala Gln Ala Ala Gln Trp Glu Leu Asn Tyr Val Ala Leu Asp 245 250 255 Gly Asn Ile Gly Cys Met Val Asn Gly Ala Gly Leu Ala Met Gly Thr 260 265 270 Met Asp Ile Val Lys Leu His Gly Gly Glu Pro Ala Asn Phe Leu Asp 275 280 285 Val Gly Gly Gly Ala Thr Lys Glu Arg Val Thr Glu Ala Phe Lys Ile 290 295 300 Ile Leu Ser Asp Asp Lys Val Lys Ala Val Leu Val Asn Ile Phe Gly 305 310 315 320 Gly Ile Val Arg Cys Asp Leu Ile Ala Asp Gly Ile Ile Gly Ala Val 325 330 335 Ala Glu Val Gly Val Asn Val Pro Val Val Val Arg Leu Glu Gly Asn 340 345 350 Asn Ala Glu Leu Gly Ala Lys Lys Leu Ala Asp Ser Gly Leu Asn Ile 355 360 365 Ile Ala Ala Lys Gly Leu Thr Asp Ala Ala Gln Gln Val Val Ala Ala 370 375 380 Val Glu Gly Lys 385 <210> 55 <211> 870 <212> DNA <213> Artificial Sequence <220> <223> sucD nucleotide sequence <400> 55 atgtccattt taatcgataa aaacaccaag gttatctgcc agggctttac cggtagccag 60 gggactttcc actcagaaca ggccattgca tacggcacta aaatggttgg cggcgtaacc 120 ccaggtaaag gcggcaccac ccacctcggc ctgccggtgt tcaacaccgt gcgtgaagcc 180 gttgctgcca ctggcgctac cgcttctgtt atctacgtac cagcaccgtt ctgcaaagac 240 tccattctgg aagccatcga cgcaggcatc aaactgatta tcaccatcac tgaaggcatc 300 ccgacgctgg atatgctgac cgtgaaagtg aagctggatg aagcaggcgt tcgtatgatc 360 ggcccgaact gcccaggcgt tatcactccg ggtgaatgca aaatcggtat ccagcctggt 420 cacattcaca aaccgggtaa agtgggtatc gtttcccgtt ccggtacact gacctatgaa 480 gcggttaaac agaccacgga ttacggtttc ggtcagtcga cctgtgtcgg tatcggcggt 540 [[ID=2,8]]gacccgatcc cgggctctaa ctttatcgac attctcgaaa tgttcgaaaa agatccgcag 600 accgaagcga tcgtgatgat cggtgagatc ggcggtagcg ctgaagaaga agcagctgcg 660 tacatcaaag agcacgttac caagccagtt gtgggttaca tcgctggtgt gactgcgccg 720 aaaggcaaac gtatgggcca cgcgggtgcc atcattgccg gtgggaaagg gactgcggat 780 gagaaattcg ctgctctgga agccgcaggc gtgaaaaccg ttcgcagcct ggcggatatc 840 ggtgaagcac tgaaaactgt tctgaaataa 870 <210> 56 <211> 289 <212> PRT <213> Artificial Sequence <220> <223> SucD Amino Acid Sequence <400> 56 Met Ser Ile Leu Ile Asp Lys Asn Thr Lys Val Ile Cys Gln Gly Phe 1 5 10 15 Thr Gly Ser Gln Gly Thr Phe His Ser Glu Gln Ala Ile Ala Tyr Gly 20 25 30 Thr Lys Met Val Gly Gly Val Thr Pro Gly Lys Gly Gly Thr Thr His 35 40 45 Leu Gly Leu Pro Val Phe Asn Thr Val Arg Glu Ala Val Ala Ala Thr 50 55 60 Gly Ala Thr Ala Ser Val Ile Tyr Val Pro Ala Pro Phe Cys Lys Asp 65 70 75 80 Ser Ile Leu Glu Ala Ile Asp Ala Gly Ile Lys Leu Ile Ile Thr Ile 85 90 95 Thr Glu Gly Ile Pro Thr Leu Asp Met Leu Thr Val Lys Val Lys Leu 100 105 110 Asp Glu Ala Gly Val Arg Met Ile Gly Pro Asn Cys Pro Gly Val Ile 115 120 125 Thr Pro Gly Glu Cys Lys Ile Gly Ile Gln Pro Gly His Ile His Lys 130 135 140 Pro Gly Lys Val Gly Ile Val Ser Arg Ser Gly Thr Leu Thr Tyr Glu 145 150 155 160 Ala Val Lys Gln Thr Thr Asp Tyr Gly Phe Gly Gln Ser Thr Cys Val 165 170 175 Gly Ile Gly Gly Asp Pro Ile Pro Gly Ser Asn Phe Ile Asp Ile Leu 180 185 190 Glu Met Phe Glu Lys Asp Pro Gln Thr Glu Ala Ile Val Met Ile Gly 195 200 205 Glu Ile Gly Gly Ser Ala Glu Glu Glu Ala Ala Ala Tyr Ile Lys Glu 210 215 220 His Val Thr Lys Pro Val Val Gly Tyr Ile Ala Gly Val Thr Ala Pro 225 230 235 240 Lys Gly Lys Arg Met Gly His Ala Gly Ala Ile Ile Ala Gly Gly Lys 245 250 255 Gly Thr Ala Asp Glu Lys Phe Ala Ala Leu Glu Ala Ala Gly Val Lys 260 265 270 Thr Val Arg Ser Leu Ala Asp Ile Gly Glu Ala Leu Lys Thr Val Leu 275 280 285 Lys

Claims

1. An isolated genetically engineered Escherichia coli for producing β-ketoadipate from depolymerized lignin from fiber oil palm empty fruit bunches, wherein the Escherichia coli has been transformed with at least one polynucleotide molecule; the at least one polynucleotide molecule comprising: a)(i) heterologous β-ketoadipate pathway genes operably linked to at least one promoter, the genes being feruloyl-CoA synthetase (fcs), enoyl-CoA hydratase (ech), vanillin dehydrogenase (vdh), vanillate O-demethylase (vanAB; vanA and vanB), p-hydroxybenzoate hydroxylase (pobA), protocatechuate 3,4-dioxygenase (pcaGH; pcaG and pcaH), 3-carboxy-cis,cis-muconate cycloisomerase (pcaB), 4-carboxymuconolactone decarboxylase (pcaC), and β-ketoadipate enol-lactone hydrolase (pcaD), and a)(ii) heterologous β-ketoadipate utilization genes operably linked to at least one promoter, the genes being β-ketoadipate succinyl-CoA transferase (pcalJ; pcal and pcaJ), 3-hydroxyacyl-CoA dehydrogenase (paaH1), enoyl-CoA hydratase (ech), trans-enoyl-CoA reductase (ter), phosphotransbutyrylase (ptb), and butyrate kinase 1 (buk1), and a) (iii) a heterologous genetic controller that regulates the at least one promoter, wherein the heterologous genetic controller is pBAD or hydroxycinnamic acid (HA); or b) (i) heterologous β-ketoadipate pathway genes operably linked to at least one promoter, the genes being feruloyl-CoA synthetase (fcs), enoyl-CoA hydratase (ech), vanillin dehydrogenase (vdh), vanillate O-demethylase (vanAB; vanA and vanB), p-hydroxybenzoate hydroxylase (pobA), protocatechuate 3,4-dioxygenase (pcaGH; pcaG and pcaH), 3-carboxy-cis,cis-muconate cycloisomerase (pcaB), 4-carboxymuconolactone decarboxylase (pcaC), and β-ketoadipate enol-lactone hydrolase (pcaD), and b) (ii) a heterologous beta-ketoadipate utilization gene, i.e., acetoacetate decarboxylase (adc), operably linked to at least one promoter, and b) (iii) a heterologous genetic controller that regulates the at least one promoter, wherein the heterologous genetic controller is pBAD or hydroxycinnamic acid (HA); wherein the genetically engineered Escherichia coli of a) further comprises: an inactivated endogenous succinyl-CoA synthetase gene sucCD and / or an inactivated β-ketoadipyl-CoA thiolase gene paaJ; wherein the genetically engineered Escherichia coli of b) further comprises: an inactivated endogenous acyl-CoA:acetate / 3-ketoacid-CoA transferase gene atoDA; wherein the genetically engineered Escherichia coli of a) is capable of converting depolymerized lignin into adipic acid, and The genetically engineered Escherichia coli of b) is capable of converting depolymerized lignin into levulinic acid, wherein: The fcs gene encodes the amino acid sequence shown in SEQ ID NO: 2; The ech gene encodes the amino acid sequence shown in SEQ ID NO: 4; The vdh gene encodes the amino acid sequence shown in SEQ ID NO:6; The vanA gene encodes the amino acid sequence shown in SEQ ID NO: 8; The vanB gene encodes the amino acid sequence shown in SEQ ID NO: 10; The pobA gene encodes the amino acid sequence shown in SEQ ID NO: 12; The pcaH gene encodes the amino acid sequence shown in SEQ ID NO: 14; The pcaG gene encodes the amino acid sequence shown in SEQ ID NO: 16; The pcaB gene encodes the amino acid sequence shown in SEQ ID NO: 18; The pcaC gene encodes the amino acid sequence shown in SEQ ID NO: 20; The pcaD gene encodes the amino acid sequence shown in SEQ ID NO: 22; The ter gene encodes the amino acid sequence shown in SEQ ID NO: 24; The pcal gene encodes the amino acid sequence shown in SEQ ID NO: 26; The pcaJ gene encodes the amino acid sequence shown in SEQ ID NO: 28; The paaH1 gene encodes the amino acid sequence shown in SEQ ID NO: 30; The ech gene encodes the amino acid sequence shown in SEQ ID NO: 32; The ptb gene encodes the amino acid sequence shown in SEQ ID NO: 34; The buk1 gene encodes the amino acid sequence shown in SEQ ID NO: 36; and The adc gene encodes the amino acid sequence shown in SEQ ID NO:

38.

2. The isolated genetically engineered E. coli of claim 1, wherein the at least one promoter is the constitutive promoter T7.

3. The isolated genetically engineered Escherichia coli according to claim 1, wherein the heterologous genetic controller pBAD comprises the nucleic acid sequence shown in SEQ ID NO:41, or the hydroxycinnamic acid (HA) controller comprises the nucleic acid sequence shown in SEQ ID NO:

42.

4. The isolated genetically engineered Escherichia coli according to claim 1, wherein the sucCD gene encodes the amino acid sequence shown in SEQ ID NO: 54 and SEQ ID NO: 56, respectively, and / or the paaJ gene encodes the amino acid sequence shown in SEQ ID NO:

52.

5. The isolated genetically engineered Escherichia coli according to claim 1, wherein the atoDA gene encodes the amino acid sequences shown in SEQ ID NO: 48 and SEQ ID NO: 50, respectively. The isolated genetically engineered Escherichia coli according to claim 1 , wherein the Escherichia coli is Escherichia coli MG1655.

7. Use of the isolated genetically engineered Escherichia coli according to any one of claims 1 to 6 for producing adipic acid or for producing levulinic acid, wherein The genetically engineered Escherichia coli of a) is used to produce adipic acid, and the genetically engineered Escherichia coli of b) is used to produce levulinic acid.

8. A recombinant vector comprising heterologous β-ketoadipate pathway genes operably linked to at least one promoter, the genes being fcs, ech, vdh, vanAB (vanA and vanB), pobA, pcaGH (pcaG and pcaH), pcaB, pcaC, and pcaD, and heterologous β-ketoadipate utilization genes operably linked to at least one promoter, the genes being pcalJ (pcal and pcaJ), paaH1, ech, ter, ptb, and buk1; or A heterologous β-ketoadipate pathway gene operably linked to at least one promoter, the genes being fcs, ech, vdh, vanAB (vanA and vanB), pobA, pcaGH (pcaG and pcaH), pcaB, pcaC, and pcaD, and a heterologous β-ketoadipate utilization gene operably linked to at least one promoter, the gene being adc, wherein: The fcs gene encodes the amino acid sequence shown in SEQ ID NO: 2; The ech gene encodes the amino acid sequence shown in SEQ ID NO: 4; The vdh gene encodes the amino acid sequence shown in SEQ ID NO:6; The vanA gene encodes the amino acid sequence shown in SEQ ID NO: 8; The vanB gene encodes the amino acid sequence shown in SEQ ID NO: 10; The pobA gene encodes the amino acid sequence shown in SEQ ID NO: 12; The pcaH gene encodes the amino acid sequence shown in SEQ ID NO: 14; The pcaG gene encodes the amino acid sequence shown in SEQ ID NO: 16; The pcaB gene encodes the amino acid sequence shown in SEQ ID NO: 18; The pcaC gene encodes the amino acid sequence shown in SEQ ID NO: 20; The pcaD gene encodes the amino acid sequence shown in SEQ ID NO: 22; The ter gene encodes the amino acid sequence shown in SEQ ID NO: 24; The pcal gene encodes the amino acid sequence shown in SEQ ID NO: 26; The pcaJ gene encodes the amino acid sequence shown in SEQ ID NO: 28; The paaH1 gene encodes the amino acid sequence shown in SEQ ID NO: 30; The ech gene encodes the amino acid sequence shown in SEQ ID NO: 32; The ptb gene encodes the amino acid sequence shown in SEQ ID NO: 34; The buk1 gene encodes the amino acid sequence shown in SEQ ID NO: 36; and The adc gene encodes the amino acid sequence shown in SEQ ID NO:

38.

9. A kit comprising the isolated genetically engineered Escherichia coli according to any one of claims 1 to 6 or the recombinant vector according to claim 8.

10. A method for producing β-ketoadipate from depolymerized lignin from fiber oil palm empty fruit bunches, the method comprising the step of culturing a plurality of genetically engineered Escherichia coli according to claim 1 under conditions for producing the β-ketoadipate.

11. A method for producing adipic acid from depolymerized lignin from fiber oil palm empty fruit bunches, said method comprising the step of culturing a plurality of genetically engineered Escherichia coli according to claim 1a) under conditions that produce said adipic acid.

12. A method for producing levulinic acid from depolymerized lignin from fiber oil palm empty fruit bunches, the method comprising the step of culturing a plurality of genetically engineered Escherichia coli according to claim 1b) under conditions that produce the levulinic acid.

13. The method according to any one of claims 10 to 12, further comprising isolating the product produced by the genetically engineered E. coli.

14. The method according to any one of claims 10 to 12, wherein the Escherichia coli is Escherichia coli MG1655.

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