Patent Application: US-201615095158-A

Abstract:
the present disclosure relates to an engineered microbe capable of improved productivity of fatty acid or fatty acid derivative . an nad + - dependent 3 - oxoacyl - acp reductase or nad + - dependent 3 - oxoacyl - coa reductase replaces or supplements the native nadp + - dependent 3 - oxoacyl - acp reductase so as to utilize the higher availability of nad + rather than nadp + in the cell . higher production , yield and titer of fatty acids are therefore obtained . such microbes can be combined with other mutations to further improve yield of fatty acids or fatty acid derivatives .

Description:
this disclosure provides the inventive concept of replacing or supplementing the nadp +- dependent enzyme in a type ii fatty acid synthesis pathway with an nad +- dependent enzyme so as to take advantage of the higher concentration of nadh / nad + in cells . in e . coli , as in many species , the 3 - oxoacyl - acp reductase is nadph - dependent , and is thus rate limiting , and adding an nadh - dependant reductase alleviates this bottleneck , allowing more fats to be made . to demonstrate the concept , we used previously constructed host strain e . coli strain , ml103 ( mg1655 , δfadd ) for fatty acid production with a deleted long - chain fatty acyl coenzyme a synthase gene fadd . the δfadd is not an essential component of the invention , although it does improve fatty acid accumulation . fadd is the first step in the fatty acid beta - oxidation pathway . it activates the fatty acid to acyl - coa before going into the beta - oxidation cycle , thus its deletion helps to conserve the fatty acids that are made . however , any enzyme in the beta - oxidation pathway can provide similar effects if reduced or knocked out . the base strain also contained a thioesterase ( te ) gene from ricinus communis , which functions to release free fats from the acp thus allow increased levels of free fatty acids to accumulate . te expression of some kind is needed to allow free fatty acid production . the host &# 39 ; s native te ( such as tesa and tesb ) is capable of providing some activity , but we have shown that overexpression of either endogenous or exogenous te significantly improves free fatty acids levels . furthermore , by tailoring the te gene used , we are able to influence the length of the free fatty acids produced . a plasmid carrying an acyl - acp thioesterase from ricinus communis ( acc no . : xm002515518 ) ( zhang et al ., 2011 ) and a nad + dependent 3 - oxoacyl - acp reductase from mycobacterium tuberculosis ( acc . no . : rv0242c ) is used as an example . two versions , one with the full length reductase ( named g1 ) and the other one with an omission of the first 16 amino acids ( named g2 ) of the 3 - oxoacyl - acp reductase were tested , because the first 16 amino acids were found to hinder the solubility of the recombinant protein ( dutta et al , 2011 ). the schematics of the plasmid constructs pxz18g1 and pxz18g2 are shown in fig2 and fig3 , respectively . by replacing or supplementing nadp + - dependent 3 - oxoacyl - acp reductase with nad + - dependent 3 - oxoacyl - acp reductase , the higher availability of nad + in the organism will facilitate synthesis of fatty acids and result in higher yield thereof , especially long - chain fatty acid of 14 or more carbons . this significantly increases the efficiency of fatty acid production and reduces the cost thereof . lb medium supplemented with approximately 15 g / l glycerol as a carbon source and 100 mg / l ampicillin for selection were used for culturing cells . isopropyl - β - d - thiogalactopyranoside ( iptg ) was added to the medium to a final concentration of 200 μm , thus inducing the expression of acyl - acp thioesterase and nad + dependent 3 - oxoacyl - acp reductase . a single colony of strain ( ml103 - 18 ( control ), ml103 - 18g1 ( nadh - dependent enzyme ) or ml103 - 18g2 ( truncated nadh - dependent enzyme ) was inoculated into 5 ml of luria - bertani ( lb ) and incubated in an orbital shaker operated at 250 rpm at 30 ° c . overnight . the pre - culture was inoculated into a flask containing 50 ml of the culture medium with 1 % ( v / v ) inoculum . the culture medium contained 50 ml lb and about 15 g / l of glycerol . shake flask experiments were performed at 30 ° c . with shaking at 250 rpm . samples were taken at four specific time points ( 0 , 24 , 48 and 72h ) to quantify the fatty acids produced and glycerol consumed . all experiments were carried out in triplicates . the results are shown in tables 1 and 2 . in summary , as shown in table 1 , both nad + dependent 3 - oxoacyl - acp reductase carrying strains , ml103 - 18g1 and ml103 - 18g2 , produced more fatty acids than that of the control strain ml103 - 18 ( table 1 ). at 72 hours , the ml103 - 18g1 and ml103 - 18g1 strains accumulated more than 34 % and 32 % of free fatty acids than that of the control strain ml103 - 18 , respectively ( table 1 ). in addition , both nad + dependent 3 - oxoacyl - acp reductase - carrying strains , ml103 - 18g1 and ml103 - 18g2 , gave higher yields than that of the control strain ml103 - 18 , more than 35 % at 72 hours ( table 1 ). both nad + dependent 3 - oxoacyl - acp reductase - carrying strains , ml103 - 18g1 and ml103 - 18g2 , also showed changes in the free fatty acid distribution as compared to that of the control strain ml103 - 18 ( table 2 ). the ml103 - 18g1 and ml103 - 18g2 strains accumulated more than 61 % and 68 % of c14 free fatty acids than that of the control strain ml103 - 18 , respectively ( table 2 ), but this is due to the substrate specificity of the added te gene . of course , the exit points can be modified by tailoring the exit point for the fatty acid elongation cycle ( see e . g ., wo2013096665 ). thus , by changing the added te gene , one can influence the fatty acid length . the ability of the nad + dependent 3 - oxoacyl - acp reductase to improve free fatty acid production in two pyridine nucleotide transhydrogenase mutant strains was also examined . the pyridine nucleotide transhydrogenases normally function to reoxidize nadph , according to the following : thus , deleting these would prevent the conversion of nadh to nadph . a strain wlk09 with the cytoplasmic transhydrogenase ( sth ) deactivated and the other strain wlk310 with the membrane bound transhydrogenase ( pntb ) deactivated were used . both strains also have the beta - oxidation pathway blocked by deleting the fadd gene to prevent the degradation of fatty acids produced in vivo . lb medium supplemented with approximately 15 g / l glycerol as a carbon source and 100 mg / l ampicillin for selection were used for culturing cells . iptg was added to a final concentration of 200 μm , thus inducing the expression of the added genes . a single colony of each strain ( ml309 - 18 , ml309 - 18g1 & amp ; ml309 - 18g2 or ml310 - 18 , ml310 - 18g1 or ml310 - 18g2 ) was inoculated into 5 ml of luria - bertani ( lb ) and treated as above . the results are shown in tables 3 and 4 below . again , both nad + dependent 3 - oxoacyl - acp reductase carrying strains , wlk309 - 18g2 and wlk310 - 18g2 , produced significantly more fatty acids and with higher yields than that of the corresponding control strains wlk309 - 18 and wlk310 - 18 ( table 3 ). at 72h , both nad + dependent 3 - oxoacyl - acp reductase carrying strains , wlk309 - 18g2 and wlk310 - 18g2 , produced 13 % and 30 % more fatty acids with 26 % and 30 % higher yield than that of the corresponding control strains wlk309 - 18 and wlk310 - 18 , respectively ( table 3 ). in addition , both nad + dependent 3 - oxoacyl - acp reductase carrying strains , wlk309 - 18g2 and wlk310 - 18g2 , produced more c14 fatty acids , 73 % and 164 %, than that of the corresponding control strains , respectively at 72 h ( table 4 ). therefore , these results demonstrate that nadph is a limiting factor , and it can be alleviated by the introduction of a nad - dependent 3 - oxoacyl - acp reductase . in addition , the introduction of a nad - dependent 3 - oxoacyl - acp reductase changes the composition of the fatty acids produced , yielding more c14 chain length fatty acid . the native nadph - dependant 3 - oxoacyl - acp reductase was believed to be an essential gene . therefore , we first tried to reduce its expression with antisense , so that some amount of gene / enzyme activity would remain . we made expression plasmids encoding 150 , 300 and 450 by antisense against the gene of nadph - dependant 3 - oxoacyl - acp reductase under the control of an iptg inducible promoter ( lacz ). we measured fatty acid levels at 24 and 48 hrs . see table 5 and 6 . surprisingly , those bacteria with reduced native nadph - dependant 3 - oxoacyl - acp reductase and added nadh - dependant 3 - oxoacyl - acp reductase made more fatty acids that those with wild type levels of expression of nadph - dependant 3 - oxoacyl - acp reductase and added nadh - dependant 3 - oxoacyl - acp reductase . we also knocked out the native nadph - dependant 3 - oxoacyl - acp reductase and added nadh - dependant 3 - oxoacyl - acp reductase , but these cells did less well that those cells with wild type nadph - dependant 3 - oxoacyl - acp reductase and added nadh - dependent 3 - oxoacyl - acp reductase ( data not shown ) possibly because the expression level of the added gene was not high enough . however , we expect that further fine - tuning of the nadh - dependent 3 - oxoacyl - acp reductase will improve the fatty acid production , and experiments are in progress to show this . we expect that combining the introduction of nadh - dependent 3 - oxoacyl - acp reductase with overexpressed udh + and / or pntab + will improve the fatty acid production because it will allow efficient usage of both nadh - dependent 3 - oxoacyl - acp reductase and the native nadph - dependent 3 - oxoacyl - acp reductase , and experiments are in progress to show this . our lab has made many engineered bacteria that produced increased amount of fatty acids , and many of those modifications are compatible herewith . direct strategies that have been tested and proven effective can be classified into two broad categories : i ) overexpression of enzymes catalyzing key steps in the fatty acid synthesis pathway , including endogenous or heterologous thioesterases ( te ), acetyl - coa carboxylase ( acc ), and acyl - coa ligases ( acl ); and ii ) deletion of enzymes involved in the β - oxidation pathway that degrades fatty acids , such as acyl - coa dehydrogenase ( fade ), acyl - coa synthetase ( fadd ), and a long - chain fatty acid outer membrane transporter ( fadl ). in one of the latest studies , efforts along this direction led to a titer of 5 . 1 g / l extracellular fatty acids and a yield of 4 . 1 % ( g per g glucose supplied ) in a fed - batch culture with online product extraction . the research efforts described above focused on local pathways directly related to fatty acids . however , modifications in distant pathways , such as glycolysis or tca cycle , can also improve fatty acid synthesis through redistribution of metabolite precursors towards fatty acid production . for example , the level of malonyl - coa , a precursor for fatty acids was improved 15 - fold through the deletion of acka - pta and adhe , together with the overexpression of acetyl - coa synthetase ( acs ). for another example , there are two other lactate dehydrogenases in e . coli encoded by ldha and lldd . knocking one or both out would block the formation of lactate from pyruvate and direct more carbon towards fatty acid biosynthesis . second , there is another acetate - producing pathway catalyzed by poxb encoded pyruvate oxidase . even though the amount of acetate was quite low after pta was deleted in the above study , further knockout of poxb would lead to complete elimination of this by - product . finally , it has been suggested that derepression of the glyoxylate bypass by iclr deletion alone cannot draw isocitrate from the tca cycle to the glyoxylate bypass because enzyme icda has a stronger affinity to isocitrate than enzymes acea and aceb . hence , to fully activate the glyoxylate bypass , icda may need to be knocked out in addition to iclr . these genetic combinations with the invention described herein will be explored in our future study , and the work is expected to proceed quickly as many base strains and / or expression plasmids are already available . high fat producing microbes can also be combined with genes that would allow the microbes to use less energy intensive food sources than glucose . for example , glycerol is a by - product of biodiesel production and is a very inexpensive food - source , and microbes can be altered to allow growth on glycerol . see murarka ( 2008 ). as another example , cellulosic food - sources are also readily available , and microbes have been engineered to secrete cellulose degrading enzymes and thus are able to grow or e . g ., switchgrass . bokinsky ( 2011 ). ultimately , the engineered microbes described herein may be combined with this additional type of engineering as the microbes are adapted for large scale production of fats or their derivatives . we predict that the inventive concept can be applied to other organisms having type ii fatty acid synthesis systems to achieve similar improvement of fatty acid production , as long as suitable nad + - dependent 3 - oxoacyl - acp reductase , native or engineered or exogenous , is available to replace or augment the native nadp + - dependent 3 - oxoacyl - acp reductase . as shown herein , there are thousands of such enzyme sequences that can be used when placed into a suitable expression vector for the chosen host species . if expression levels are low , the codon usage can be optimized for the species in question , as optimized codon charts are available for many species . further , the genes are fairly small , and complete synthesis of an optimized codon orf would be fairly quick and inexpensive . we expect that the higher availability of nad + than nadp + in such organisms will make the concept equally beneficial in these fasii organisms . examples of fasii organisms include most bacteria , algae and plants , including but not limited to escherichia , bacillus , lactobacillus , staphylococcus , salmonella , haemophilus , lemnoideae , chlamydomonas , chlorella , nannochloropsis . yeast mitochondria have fasii genes , as well . future experiments may test one of the microalgae or other bacteria , and we expect that improved production will be found on replacing or supplementing nadp - based enzymes with nadh - based enzymes . the above experiments can be repeated in bacillus subtilis . the same genes can be used , especially since bacillus has no significant codon bias . a protease - deficient strain like wb800n is preferably used for greater stability of heterologous protein . the e . coli - b . subtilis shuttle vector pmtlbs72 exhibiting full structural stability can be used to move the genes easily to a more suitable vector for bacillus . alternatively , two vectors pht01 and pht43 allow high - level expression of recombinant proteins within the cytoplasm . as yet another alternative , plasmids using the theta - mode of replication such as those derived from the natural plasmids pamβ1 and pbs72 can be used . several other suitable expression systems are available . since the fas genes are ubiquitous , the invention is predicted to function in bacillus . the inventors further tested the effect of co - overexpressing mtfabg ( fabg gene obtained from mycobacterium tuberculosis ) and fabz in order to improve fatty acid productivity . this is prompted by the observation of the following experiment with higher overexpression of mt fabg2 ( 3 - ketoacyl - acp reductase obtained from mycobacterium tuberculosis ). fabz is 3r - hydroxymyristoyl acp dehydratase , and as shown in fig1 b , it is involved in fatty acid synthesis . an experiment was designed to test if the expression of mt fabg is the limiting factor by cloning two copies of the mt fabg genes in the same plasmid ( the resulting plasmid is called pxz18dg2 , as shown in fig3 b ). fermentation experiments were performed with ml103 as the host carrying either the plasmid pxz18g2 or pxz18dg2 in glucose or glycerol supplemented lb medium . the results are shown in fig8 a - b . the values shown are the average of triplicate runs . unlike previous experimental observations that overexpression of a single copy of mt fabg gene increases fatty acid production , the double mt fabg did not show any improvement when comparing the strain ml103 ( pxz18g2 ) with ml103 ( pxz18dg2 ) in both glucose and glycerol . the decrease in fatty acid titer is more significant in glycerol than that of glucose . this reduction of fatty acid production in strains carrying pxz18dg2 suggests that too much β - ketoacyl reductase activity or nadh supply would lead to discoordination within the fatty acid elongation cycle . we hypothesize that increased β - ketoacyl reductase activity resulted in an accumulation of 3 - hydroxyacyl - acp and thus might cause feedback inhibition . it has been reported that acyl - acp intermediates might act as feedback inhibitors for fatty acid production . the inventors further tested the hypothesis of recovering the coordination among the reactions within the fatty acid elongation cycle by co - overexpressing mt fabg and fabz in order to improve fatty acid productivity . the results shown in fig9 support the hypothesis that fabg might not be the only limiting factor . the fatty acid production by the mt fabg and fabz double - overexpression strain increased by about 20 % when compared to the strain with mt fabg overexpression alone . co - overexpression of fabz , downstream of mt fabg , alleviates the imbalance caused by the overexpression of mt fabg alone , leading to an improved performance . the inventors also examined the effect of down regulation of the native e . coli nadph - dependent fabg ( ec fabg ) on fatty acid production . since nadh is more readily available in e . coli and that the nadh - dependent fabg ( mt fabg ) should be more efficient than the native ec fabg , we speculate that fatty acid production can be improved by increasing the relative ratio of the newly introduced mt fabg to that of the native ec fabg . we chose to use the anti - sense rna techniques to decrease the expression of ec fabg since fabg is an essential gene . the fermentation data showed that only the strain carry the plasmid with the longest anti - sense rna , pwl1tg2as1 , produced similar amount of fatty acid as control strain at 24 h , the other two strains carrying plasmids pwl1tg2as2 or pwl1tg2as3 were significantly lower ( fig1 ). these two strains however caught up at 48 h , but the final total fatty acid concentrations were similar to that of the control . at 48 h , the strain carrying pwl1tg2as1 produced about 3 . 9 g / l fatty acid , which is 16 . 5 % higher than that of the control . bacterial rnas are typically short lived , and some of the reports suggested that half - life is an important factor in anti - sense rna efficiency . it is therefore possible that the shorter sequences are more likely to be degraded quicker . as a result , only the longest anti - sense rna showed the positive improvement effect . the results also indicated that a system with mt fabg overexpression and native fabg down regulation provides the best performance . in order to better compare the effect of down - regulating the native ec fabg , a paired - termini ( pt ) design was used to stabilize the anti - sense rna . all three newly paired termini anti - sense rna constructs ( phwtas1 , phwtas2 , phwtas3 ) shared the same length and sequence as the earlier design , except being stabilized by the addition of a hairpin structure . a control plasmid phwtasc was also constructed using a dummy sequence to replace the anti - sense portion . fig1 shows the effect of anti - sense rnas expression , all three strains carrying the new anti - sense constructs showed similar improved fatty acid production over the control strain . the final titers are 3 . 59 , 3 . 72 and 3 . 76 g / l compared to the control of 2 . 74 g / l , which represent improvements of 31 . 1 %, 35 . 2 % and 36 . 8 %, respectively . the results further indicate that overexpression of a nad - dependent mt fabg and down - regulating the native nadph - dependent ecfabg is a practical approach to improve fatty acid production in e . coli . although work is still needed to scale up microalgae production for use in making biofuels , they are especially attractive as a source of fuel from an environmental standpoint because they consume carbon dioxide and can be grown on marginal land , using waste or salt water . indeed , ann ruffings group from sandia national laboratories has already engineered two strains of cyanobacteria to produce free fatty acid , and is working with a third . the cyanobacteria were chosen because fuel from engineered cyanobacteria is excreted outside the cell , in contrast to eukaryotic algae , in which fuel production occurs inside the cell . this greatly simplifies scale up , as the cyanobacteria continue to grow , while fats are skimmed from the top of the culture media . in addition , radakovitz has overexpressed two genes encoding acyl - acp thioesterase ( te ) of plant origin in p . tricornutum to produce medium - chain fatty acids in the oil fraction . these results provide adequate foundation for applying this invention to microalgae , such that the nadph dependent 3 - oxoacyl - acp reductase is supplemented or replaced with an nadh - dependent enzyme . further , significant advances in microalgal genomics have been achieved during the last decade . expressed sequence tag ( est ) databases have been established ; nuclear , mitochondrial , and chloroplast genomes from several microalgae have been sequenced ; and several more are being sequenced . historically , the green alga chlamydomonas reinhardtii has been the focus of most molecular and genetic physiological research . therefore , most of the tools for the expression of transgenes and gene knockdown have been developed for and are specific for this species . however , tools are now also being rapidly developed for diatoms and other algae that are of greater interest for industrial applications . additionally , successful genetic transformation has been reported for the green ( chlorophyta ), red ( rhodophyta ), and brown ( phaeophyta ) algae ; diatoms ; euglenids ; and dinoflagellates , although the efficiency of transformation seems to be strongly species dependent , and the method of transformation has to be carefully selected and optimized for each microalga , and the stability of expression improved through proper codon usage , the use of strong endogenous promoters , and inclusion of species - 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