Patent Application: US-201514607624-A

Abstract:
systems , methods and microbes that allow the biological production of hydroxy fatty acids and dicarboxylic fatty acids are provided . specifically , hydroxy fatty acids and dicarboxylic fatty acids are produced by microbes that have been engineered to overexpress acyl acp thioesterase plus an alkane degration pathway , such as alkbgt or alkjh these can be in separate microbes or the same microbe , and separate microbes can be co - cultured or sequentially cultured . continuously fed systems transferring secreted fats from one microbial culture to another can also be used .

Description:
the invention generally includes microbes engineering to have te + as well as overexpressed alkane degradation proteins or genes . the invention also includes method of using said microbes , e . g ., to make fatty acid derivatives . we used the existing te + strains or vectors from our prior work herein . however , there are hundreds of available te genes and proteins , as well as hybrid te genes and proteins that can be used instead . the most extensively characterized alkane degradation pathway is that encoded on the oct plasmid of p . putida gpo1 , formerly identified as pseudomonas oleovorans gpo1 . the first enzyme of this pathway is an integral - membrane non - heme di - iron monooxygenase , named alkb , that hydroxylates alkanes at the terminal position alkb requires two soluble electron transfer proteins named rubredoxin ( alkg ) and rubredoxin reductase ( alkt ). there are now at least 60 alkb homologs known , and they show fairly high sequence diversity . alkj converts the alcohol to an aldehyde , and alkk converts the aldehyde to carboxyl , as shown in the reaction pathway below . further , we exemplified the system in e . coli , which is the workhorse of microbial engineering . however , this system can be engineered into any bacteria , including e . g ., bacillus subtilis , staphylococcus aureus , streptomyces peucetius , and the like . the cloning methods are the same , although species - specific promoters and codon optimization for a given species may be used to improve yields . in addition , the invention can be combined with other mutations to drive fat production . for example , at least one gene from the tricarboxylic acid cycle or glycolysis or both is inactivated to drive carbon in the direction of fat production . for example , in prior work an acp thioesterase was combined with deletions in native succ . these bacteria significantly increased overall fat levels . additionally , other genes can be added to further improve recoveries . for example , the co - expression of the alkl gene of p . putida encoding an outer membrane protein with so - far - unknown function increased the dodecanoic acid methyl ester oxygenation activity of recombinant e . coli 28 - fold ( julsing 2012 ). the construction of pbdtum3 and palkbgt were shown in fig1 . the 1 . 6 kb fatb2 of cuphea hookeriana plus tum3 promoter and rrnb terminator was amplified from previously constructed plasmid pkmch . the pcr fragments were digested by restriction enzymes , xbai and hindiii , and ligated to plasmid pbad33 , which was also digested with xbai and hindiii . the newly constructed pbdtum3 ( 7 . 0 kb ) expressed the heterologous mature acyl - atp thioesterase ( te ) of c . hookeriana ( aac72882 ). the synthesized alk operon including alkb , alkg and alkt genes from pseudomonas putida p1 was digested with saci and xbai , and ligated to plasmid ptrc99a which was also digested with saci and xbai . the newly constructed palkbgt ( 7 . 2 kb ) co - expresses the heterologous alkane - 1 - monooxygenase ( alkb ), rubredoxin ( alkg ) and rubredoxin reductase ( alkt ) of p . putida p1 . the heterologous alcohol dehydrogenase ( alkj ) and aldehyde dehydrogenase ( alkh ) of p . putida p1 were synthesized and cloned into ptrc99a to form palkjh ( 7 . 3 kb ) ( fig3 ). the metabolically engineered strain k272 , a ptsg mutant of e . coli strain k27 , was used in the initial experiments because this strain was an efficient host for short chain free fatty acid production . a single colony of strain k272 ( palkbgt ) was inoculated into 5 ml of luria - bertani ( lb ) and incubated in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the preculture was inoculated into a flask containing 40 ml of the culture medium with 1 % ( v / v ) inoculum . the culture medium contained : tryptone 10 g / l , yeast extract 5 g / l , nacl 5 g / l , glucose 15 g / l , octanoic acid 10 mm , ampicillin 100 μg / l , iptg 100 μm , ph 7 . 5 . shake flask experiment was performed at 30 ° c . with shaking at 250 rpm for 24 h . the samples were extracted by ethyl acetate , then dried under n 2 flow , and re - dissolved with 1 . 5 ml chloroform . the hydroxyoctanoic acid concentration was quantified by gc - fid system ( table 1 ). the next step was to combine this culture with a te + culture for conversion of secreted fatty acids to hydroxyoctanoic acid . the following experiment demonstrates this approach . strain k272 ( palkbgt ) and strain k272 ( pbdtum3 ) were inoculated into 5 ml of luria - bertani ( lb ) respectively and incubated in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the two precultures were simultaneously inoculated into a flask containing 40 ml of the culture medium with 1 % ( v / v ) inoculum . the co - culture medium contained : tryptone 10 g / l , yeast extract 5 g / l , nacl 5 g / l , glucose 15 g / l , ampicillin 100 μg / l , iptg 100 μm , ph 7 . 5 . shake flask experiment was performed at 30 ° c . with shaking at 250 rpm for 24 h . the samples were extracted by ethyl acetate , then dried under n 2 flow and re - dissolved with 1 . 5 ml chloroform . the hydroxyoctanoic acid concentration was quantified by gc - fid system ( table 2 ). these results indicated that co - culturing the two strains allows the first strain to make and secrete free fatty acids , which the second strain uses to make hydroxyoctanoic acid . note , there were no other hydroxyl fatty acid products , because the strain k272 ( pbdtum3 ) can only produce octanoic acid ( c8 fatty acid ). in the next experiment , we combined the two plasmids constructs into a single microbe , and again measured hydroxyoctanoic acid production . strain k272 ( palkbgt , pbdtum3 ) was inoculated into 5 ml of luria - bertani ( lb ) and incubated in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the preculture was inoculated into a flask containing 40 ml of the culture medium with 1 % ( v / v ) inoculum . the culture medium contained : tryptone 10 g / l , yeast extract 5 g / l , nacl 5 g / l , glucose 15 g / l , ampicillin 100 μg / l , iptg 100 μm , ph 7 . 5 . shake flask experiment was performed at 30 ° c . with shaking at 250 rpm for 24 h . the samples were extracted by ethyl acetate , then dried under n 2 flow and re - dissolved with 1 . 5 ml chloroform . the hydroxyoctanoic acid in the sample was identified by gc - ms system ( fig5 ) compared to the standard ( fig4 ). the hydroxyoctanoic acid concentration was quantified by gc - fid system ( table 3 ). in the next experiment , we combined the plasmid encoding proteins that convert free fatty acid to hydroxyl fatty acids ( alkbgt ) with the plasmid encoding proteins needed to convert the hydroxyl fatty acids to dicarboxylic acids ( alkjh ). in the first test , we co - cultured separate strains , each carrying a plasmid . additionally , we used a parent strain with a different background in order to show that the method is generally applicable , and not specific to the parent strains employed . the e . coli strain mg1655 ( genotype : f − lambda − ilvg − rfb - 50 rph - 1 ; serotype : or : h48 : k -) was used as host strain for each of the vectors . strain mg1655 ( palkbgt ) and strain mg1655 ( palkjh ) were inoculated into 5 ml of luria - bertani ( lb ) respectively and incubated in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the two precultures were inoculated into a flask containing 50 ml of the lb medium with 1 % ( v / v ) inoculum , respectively . 100 μm iptg are added in both of the cultures . after 6 hours incubation , these cells of two cultures are collected by centrifugation and re - suspended together in culture medium for 24 h and 48 h incubation . the co - culture medium contains : tryptone 10 g / l , yeast extract 5 g / l , nacl 5 g / l , octanoic acid 20 mm ( for use as a starting material ), ampicillin 100 μg / l , iptg 100 μm , ph 7 . 5 . shake flask experiment is performed at 30 ° c . with shaking at 250 rpm . the suberic acid was identified by lc - ms system as shown in fig6 and 7 . these results show that the co - culture produced the final product , suberic acid . therefore , the octanoic acid was converted to suberic acid using the two expression plasmids encoding the alkane degradation pathway . in the second co - culture test , we use hexanoic acid as the substrate , thus demonstrating applicability to other products . strains mg1655 ( palkbgt ) and mg1655 ( palkjh ) were cultured in the flasks containing 50 ml of the lb medium with 1 % ( v / v ) inoculum . iptg and ampicillin were added in the cultures . after 8 - 12 hours incubation , these cells were collected by centrifugation , washed by 0 . 95 % nacl solution and re - suspended together in culture medium for 24 h incubation . the co - culture medium contained hexanoic acid 20 mmol / l ( for use as a starting material ), ampicillin 100 μg / l , iptg 100 μmol / l , ph 7 . 5 . shake flask experiment is performed at 30 ° c . with shaking at 250 rpm . the 6 - hydroxyhexanoic acid was identified by lc - ms system as shown in fig8 . no dicarboxylate was detected , possibly due lack of uptake of the 6 - hydroxyhexanoic by the mg1655 ( palkjh ). we expect that issue would be eliminated in a single strain having all needed enzymes , or in a strain engineered to include a suitable transporter for the hydroxyl carboxylic acid . the work is still in progress to combine both alkbgt and palkjh + with a te + in the same strain , as the combined strains are expected to be simpler to use than a co - culture technique , but the preliminary results indicate that success is likely . further work will be needed to optimize the system , by e . g ., combining the various genes into a single plasmid or even a single operon and host strain manipulation to channel the carbon flux to the final product . in this prophetic experiment , we plan to convert hydroxyl fatty acid to dicarboxylic using dodecanoic acid as the substrate using a co - culture technique . strain mg1655 ( palkbgt ) and strain mg1655 ( palkjh ) are inoculated into 5 ml of luria - bertani ( lb ) respectively and incubated in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the two precultures are inoculated into a flask containing 50 ml of the lb medium with 1 % ( v / v ) inoculum , respectively . 100 μm iptg are added in both of the cultures . after 8 - 12 hours incubation , these cells of two cultures are collected by centrifugation and re - suspended together in culture medium for 24 h and 48 h incubation . the culture medium contains : tryptone 10 g / l , yeast extract 5 g / l , nacl 5 g / l , dodecanoic acid 20 mm as a starting material , ampicillin 100 μg / l , iptg 100 μm , ph 7 . 5 . shake flask experiment is performed at 30 ° c . with shaking at 250 rpm . the dodecanedioic acid can be identified by lc - ms system . in this prophetic experiment , we plan to put all the needed genes onto one plasmid and then into a single strain , thus avoiding co - culture methods . the operon of alkjh from p . putida p1 plus trc promoter and rrnb terminator is amplified from palkjh and cloned into palkgbt to form palkgbthj ( 9 . 3 k ). the newly constructed palkgbthj co - expresses the heterologous alkane - 1 - monooxygenase ( alkb ), rubredoxin ( alkg ) and rubredoxin reductase ( alkt ), alcohol dehydrogenase ( alkj ) and aldehyde dehydrogenase ( alkh ) of p . putida p1 . this plasmid can be introduced into e . g ., e . coli , b . subtilus , and the like . in this prophetic experiment , we plan to convert hydroxyl fatty acid to dicarboxylic using 8 - hydroxyoctanoic acid as the substrate . a single colony of strain k272 ( palkjh ) is inoculated into 5 ml of luria - bertani ( lb ) and incubates in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the preculture is inoculated into a flask containing 40 ml of the culture medium with 1 % ( v / v ) inoculum . the culture medium contains : tryptone 10 g / l , yeast extract 5 g / l , nacl 5 g / l , glucose 15 g / l , 8 - hydroxyoctanoic acid 10 mm , ampicillin 100 μg / l , iptg 100 μm , ph 7 . 5 . shake flask experiment is performed at 30 ° c . with shaking at 250 rpm for 24 h . the suberic acid concentration can be identified and quantified by lc - ms system . strain k272 ( pbdtum3 , palkbgt ) and strain k272 ( palkjh ) are inoculated into 5 ml of luria - bertani ( lb ) respectively and incubated in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the two precultures are inoculated into a flask containing 40 ml of the culture medium with 1 % ( v / v ) inoculum , respectively . after 6 - 8 hours incubation , these two cultures are combined together and incubated for 24 h . the culture medium contains : tryptone 10 g / l , yeast extract 5 g / l , nacl 5 g / l , glucose 15 g / l , ampicillin 100 μg / l , iptg 100 μm , ph 7 . 5 . shake flask experiment is performed at 30 ° c . with shaking at 250 rpm . the suberic acid concentration can be identified and quantified by gc - ms system . strain k272 ( pbdtum3 , palkbgtjh ) is inoculated into 5 ml of luria - bertani ( lb ) and incubated in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the preculture is inoculated into a flask containing 40 ml of the culture medium with 1 % ( v / v ) inoculum . the culture medium contains : tryptone 10 g / l , yeast extract 5 g / l , nacl 5 g / l , glucose 15 g / l , ampicillin 100 μg / l , iptg 100 μm , ph 7 . 5 . shake flask experiment is performed at 30 ° c . with shaking at 250 rpm for 24 h . the suberic acid concentration can be identified and quantified by gc - ms system . although the e . coli has become a new focus for fatty acid production and some breakthroughs have been made recently , the traditional oleaginous microorganisms like microalgae , fungi and yeast are well studied and can also be used as host cells to create the microbes herein described . thus , pbdtum3 and / or palkbgtjh or equivalent vectors are introduced into other bacteria , microalgae , fungi or yeast to take advantage of the well developed cell transformation and culturing methods generally available for such hosts . alternatively , the host selected may already have an overexpressed te . several cells containing overexpressed te proteins are already available , such as human and murine cells lines ( ishizuka 2004 ), simian cos cells ( soyombo 1997 ), yeast ( saerens 2006 ), arabidopsis thaliana ( voelker 1992 ), pseudomonas aeruginosa ( lee 2012 ), and various escherichae strains . thus , the addition of al kbgtjh in a suitable expression vector to such cells will be fairly simple , and positive results are expected . yeast may be a preferred host as large scale fermentation techniques are well developed for yeast . additionally , several commercially available yeast protein expression systems exist in organisms from the genera saccharomyces , pichia , kluyveromyces , hansenula and yarrowia . a growing number of engineered yeast strains are becoming available for protein expression . strains have been described that increase yield of secreted proteins , improve the performance of certain affinity tags , reduce proteolysis , define the composition of n - glycans , and permit non - native amino acids ( e . g . selenomethionine ) into proteins have been described . one yeast system that is commonly used for protein expression is kluyveromyces lactis . k . lactis and s . cerevisiae are the only two yeasts classified by the nih as host - vector i systems , an important biosafety designation , making these attractive hosts . for transfer into yeast , the vector would be exchanged , by moving the alkbgtjh coding region into a yeast expression vector , such as the pklac1 vector for k . lactis or pd1201 for s . cerevisiae . the following references are each incorporated herein in their entireties for all purposes . u . s . pat . no . 7 , 223 , 567 mutant e . coli strain with increased succinic acid production wo2013024114 biotechnological synthesis process of omega - functionalized carbon acids and carbon acid esters from simple carbon sources davis , m . s . ; cronan , j . e ., inhibition of escherichia coli acetyl coenzyme a carboxylase by acyl - 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