single - cell microorganisms 
 ( scm ) constitute an emerging alternative 
 to source high - value lipids for a series of growing markets demanding 
 low - cost , high - quality alternatives .
scm as a broad class display 
 a series of advantages when compared to plants and animals as lipid 
 sources .
in addition to being more genetically accessible , scm are 
 capable of producing greater diversity and storing higher percentages 
 of lipids .
therefore , their productivity per volume and energy input 
 can be up to 5 or 6 times that of plants and even more when compared 
 to animal sources .
additionally , single - cell microorganism 
 architecture and circuitry tend to be more straightforward than those 
 in plant or animal cells .
for example , there are fewer organelle compartments 
 in fungal cells than in plant cells , and 
 some microalgal metabolic pathways ( e.g. , fatty acid synthesis ) are 
 encoded by single - copy genes , whereas plants have multiple genes encoding 
 proteins with redundant enzymatic activities .
scm can be readily grown under controlled conditions , and the key 
 metabolic and biochemical questions can be answered more simply and 
 quickly than in complex multicellular systems . in principle
, scm can 
 achieve greater sustainability to alleviate the increasing problem 
 of sourcing oils for both the fuel and human consumption markets , 
 thus mitigating the continuous increase in commodity oil prices .
microbial 
 lipids can also become sources of safe and clean biomaterials ( e.g. , 
 biosurfactants ) at reduced costs and continuous availability .
a complete mechanistic understanding of cellular machinery is the 
 key to moving from principle to practice in lipid production .
the 
 purpose of this paper is to provide an overview of the current knowledge 
 of the different cellular and biochemical mechanisms involved in neutral 
 and polymeric lipid accumulation within four main single - cell microbial 
 groups : yeasts , microalgae , bacteria , and archaea .
special emphasis 
 is given to the production of triacylglycerols ( tag ) and polyhydroxyalkanoates 
 ( pha ) as the hallmark lipids accumulated in eukaryotes and prokaryotes , 
 respectively .
insights are provided on the mechanistic differences 
 existing in each group during the transformation of substrates ( e.g. , 
 acetyl coenzyme a ) , formation of intermediate pools ( e.g. , fatty acyl 
 chains ) , allocation to intracellular lipid pools ( e.g. , polar lipid 
 pools or neutral lipid pools ) , and the architecture of lipid accumulation 
 storage ( e.g. , lipid droplets ) . in microorganisms , different 
 types of neutral lipids are accumulated 
 by different types of scm .
for example , in oleaginous yeast , tag and/or 
 sterol esters ( se ) are the primary neutral lipids accumulated inside 
 the cell , whereas in specific types of 
 bacteria and archaea polyhydroxyalkanoates ( pha ) , and to a lesser 
 extent tag and wax esters , are preferentially stored . 

these lipids are currently viewed as valuable potential biofuel sources , 
 high - value compounds in the food and pharmaceutic industries , building 
 blocks for biomaterials , potential tools to treat chronic diseases , 
 and natural alternatives for the production of oleochemicals , among 
 many other uses .
neutral lipids follow distinct cellular accumulation 
 strategies compared to polar lipids , such as phospholipids ( pl ) , galactolipids , 
 and sulfolipids among others . however , in most cases , both classes 
 share key biosynthetic steps .
depending on cell needs , the cell 
 is capable of transforming polar 
 lipids into neutral lipids and vice versa due to the different pools 
 present in strategic cellular locations .
for instance , polar lipids 
 are pooled and may be mobilized from the different cell membranes . 

key membranes involved in the accumulation of lipids include the plasma 
 membrane in both eukaryotes and prokaryotes , and in the case of eukaryotes 
 the endoplasmic reticulum ( er ) , peroxisome , mitochondrial , plastidial 
 ( and their variants , such as apicoplastidial ) , or even thylakoid membranes , 
 depending on the organism .
key intermediates , such as acetyl coenzyme 
 a ( ac - coa ) , malonyl coenzyme a ( mal - coa ) , acyl coenzyme a ( acyl - coa ) , 
 and glycerol-3-phosphate ( g3p ) are also pooled in strategic cellular 
 locations such as the cytosol , plastidial stroma , and mitochondrial 
 intermembrane space .
the distribution of the different biosynthetic 
 enzymes plays a key role in achieving the correct lipid traffic toward 
 accumulation .
the accumulation of intracellular neutral lipid is a 
 highly reductive process , requiring more nadph and atp than the storage 
 of glucose and glucose derivatives .
accumulation 
 of lipids is a biological process that fulfills several 
 roles in microorganisms .
storage of lipids contributes to cell growth , 
 cell division , stress response , and as energy storage for survival . in some cases ,
some scm have the capacity to 
 accumulate lipids > 20% of their weight when exposed to an environmental 
 stress , such as the lack of a key nutrient .
when 
 growing on limiting concentrations of a key nutrient ( typically 
 nitrogen ) and with sufficient or excess carbon sources , oscm will 
 stop their replication processes and utilize the available carbon 
 to synthesize and store it in the way of reduced lipids . in the case 
 of tag and se ,
oscm shift their metabolic machinery to generate a pool 
 of ac - coa , as well as nadph , which is the reducing power driving fatty 
 acid ( fa ) synthesis forward the strategies vary 
 among different classes of oscm , and details will be discussed in 
 the following sections . in the second stage , ac - coa is carboxylated 
 to produce mal - coa , transferred to acyl carrier protein ( acp ) , and 
 further transformed into an acyl acp by sequential turns within the 
 fatty acid synthase ( fas ) .
there are two types of fas : type i is an 
 integrated enzyme complex ( common in eukaryotic cytoplasm ) , and type 
 ii is a dissociated version in which all subunits are independent 
 ( common in prokaryotes and in organelles of prokaryotic origin in 
 primary and secondary endosymbionts , such as plastids and mitochondria ) . 

type i can be subdivided in type ia , present in fungi ( 66 complex in the cytoplasm ) , and type ib , 
 present in animals ( as cytoplasmic 2 dimers ) . for 
 reviews describing type
i fas and type ii fas , the reader is referred 
 to the works of schweizer and lu et al .
, depending on the cell s needs and the 
 type of scm , the acyl chain might end in a specific type of lipid 
 pool , such as membrane lipid ( coupling the acyl chain to g3p , ac - coa , 
 or other glycerol - based backbone to form pl or other types of polar 
 lipid ) or can eventually be incorporated into a neutral lipid droplet 
 core ( via the construction of tag or se molecules ) .
the nature , location , 
 and dynamics of this stage vary among species and are explained more 
 in detail in the following sections . in this stage , further elongation 
 and desaturation of the acyl chains may also occur , and these phenomena 
 vary throughout the different domains of life . finally , the fourth 
 stage involves the formation of intracellular lipid droplets ( ld ) . 

variations occur depending on the organism and the environmental conditions . in the case of prokaryotes ,
accumulation of tag or se is less common . 
 in some members of the gram - positive actinomycete group , such as mycobacterium , rhodococcus , nocardia , dietzia , and streptomyces , tag
interestingly , bacterial tag biosynthesis 
 occurs frequently in the environment because actinomycetes are the 
 most abundant microorganisms in soil .
accumulation of tag in gram - negative bacteria has been reported 
 only in species from the genus acinetobacter , but only as a minor component of the neutral accumulated lipid . 

wax esters are the main compound stored in these species . for the rest of the gram - negative bacteria ,
however , several 
 studies have successfully transformed model bacteria such as escherichia coli by inserting the required genes 
 to become oleaginous , with remarkable success .
in addition , 
 tag have been reported as major components of the neutral lipid of 
 the cyanobacterium nostoc commune , 
 although no indications were obtained for the storage of lipid inclusions 
 in the cells . in the case of archaea ,
it is more common in prokaryotes to accumulate pha , comprising 
 specialized lipids such as poly(3-hydroxybutyrate ) ( p3hb ) , polyhydroxyvalerate 
 ( phv ) , or copolymers such as poly(3-hydroxybutyrate - co-3-hydroxyvalerate ) ( phbv ) .
pha possess thermoplastic and elastomeric 
 properties and are recyclable materials that can be easily degraded 
 into carbon dioxide and water .
the most promising are as 
 film formation , paper coating , foils 
 and diaphragms , and multiple - use packages , 
 because the melted polymers have low viscosity , permitting the injection 
 molding of objects with thin walls .
the end product is very hard and 
 can be used at temperatures from 30 to 120 c .
chemical structures of three different types of pha , synthesized 
 and accumulated in certain prokaryotes .
the first stage involves the generation of a suitable 
 cytoplasmic ac - coa pool .
the second one involves the synthesis of 
 the hydroxyalkanoate ( ha ) monomer or monomers , which can change depending 
 on the species and substrates .
the third stage consists of polymerizing 
 or copolymerizing the monomers into pha chains , and the fourth one 
 involves the formation of the intracellular pha ld . in both 
 prokaryotes and eukaryotes , accumulation of neutral lipids 
 results in the formation of intracellular fat bodies typically called 
 lipid droplets ( ld ) .
ld are also called lipid bodies , fat bodies , 
 or adiposomes , and in the case of plants they have also been termed 
 oleosomes , spherosomes , or plastoglobules , 
 the latter if their location is inside the plastid . for the present discussion , the term lipid droplet will 
 be used . in most of the eukaryotes ld are intracellular sphere - like 
 particles composed of a core of neutral lipids , such as tag or se 
 or both , coated by a monolayer of polar lipids , mainly pl , and decorated 
 with a series of proteins .
the polar heads face the cytoplasm , and the nonpolar tails face 
 the inside , where the neutral lipids reside .
the inserted proteins 
 within the polar lipid monolayer play functional , structural , and 
 regulatory roles in the life cycle of ld . in the case 
 of polyhydroxyalkanoate - accumulating prokaryotes , they form ld , which 
 are also called lipid granules or carbonosomes . in some higher organisms ( e.g. , humans ) , lipid droplet interaction 
 with other cellular organelles , and their dynamics
is closely related 
 to progression of metabolic diseases , such as obesity , fatty liver , 
 type 2 diabetes mellitus , and atherosclerosis .
therefore , it has become apparent that ld are not 
 just a static 
 storage compartment in the cell , but rather a dynamic organelle that 
 interacts with other organelles within the cell and actively participates 
 in the intricate cellular processing symphony , to the point that some authors consider ld as specialized organelles 
 in cells . as described in later 
 sections ,
strategies for lipid accumulation 
 in scm vary among the different types of scm , and it is important 
 to understand these differences to improve and/or create productive 
 strategies that can become economically as well as technologically 
 feasible for the generation of biofuels as well as nutritional lipid 
 metabolites such as long - chain polyunsaturated fatty acids ( lc - pufa ) .
thus , they are capable of metabolizing carbon from simple 
 sugars or from other simple compounds such as glycerol .
when oleaginous 
 yeasts encounter an environmental stress , such as a limiting nutrient 
 ( e.g. , nitrogen ) , they shift their metabolic machinery to stop synthesizing 
 proteins and nucleic acids and , thus , begin to allocate the available 
 carbon in the form of reduced lipids .
other examples of limiting nutrients 
 can be phosphorus or magnesium . in practice 
 to date
, nitrogen has been extensively used because it seems to yield 
 higher lipid accumulation when compared to other nutrients .
the carbon to nitrogen ratio is a useful tool 
 to construct nitrogen - rich or nitrogen - depleted media .
for example , 
 it has been shown that for yeasts , poor lipid accumulation occurs 
 in media in which the carbon to nitrogen ( c : n ) ratio is < 20 , whereas 
 ideal lipid production occurs in a c : n ratio range of 3080 . 

the optimal c : n ratio for lipid accumulation varies greatly with the 
 microbial species , strain , and carbon sources present in the growth 
 medium . in yeasts , oleaginicity depends 
 on the ability to produce ac - coa , the necessary precursor of fatty 
 acids , in an effective manner .
the conversion 
 of 1 mol of ac - coa to fatty acids requires the formation 
 of 2 mol of nadph , which constitutes the reducing power necessary 
 to drive the reaction forward .
the metabolic steps for lipid 
 accumulation in yeast can be divided into four main stages , as mentioned 
 before ( see figure 2 ) .
the diagram also includes the connection between fatty 
 acid synthesis , triacylglycerol synthesis , and lipid droplet formation . 

abbreviations : me , malic enzyme ( cytosolic ) ; me 2 , malic enzyme ( er 
 membrane , responsible for fatty acid desaturation ) ; acl , atp : citrate 
 lyase ; i d , isocitrate dehydrogenase ; nld , nascent lipid droplet ; ld , 
 lipid droplet ; er , endoplasmic reticulum ; lpa , lysophosphatidic acid ; 
 pa , phosphatidic acid , fas i , fatty acid synthase i ; dag , diacylglycerol ; 
 tag , triacylglycerol ; ampd , amp deaminase .
the cell responds 
 by activating amp deaminase and inducing an acute decrease of cellular 
 amp content . in mitochondria of oleaginous species ,
the decrease in 
 cellular amp levels causes isocitrate dehydrogenase ( idh ) activity 
 to decrease or even stop . as a consequence , 
 the production of -ketoglurarate drops and the tricarboxylic 
 cycle ( tca ) is dramatically reduced or even stopped .
to reverse this situation , 
 aconitase transforms isocitrate back to citrate ( cit ) , leading to 
 an accumulation of cit in mitochondria .
cit is transported by an antiport 
 protein , known as citrate / malate translocase ( cmt ) , from the mitochondria 
 to the cytoplasm . in the cytoplasm ,
cit is cleaved to form oxaloacetate 
 and ac - coa , by atp : citrate lyase ( acl ) .
cytosolic malate is converted 
 to pyruvate by malic enzyme ( me ) , encoded by me1 . 

this reaction generates nadph and is coupled to a 
 series of parallel reactions as well .
the first is the carboxylation 
 of pyruvate to form oxaloacetate by pyruvate carboxylase inside mitochondria . 

this nadph is the reducing power required to convert ac - coa into fatty 
 acids .
thus , pyruvate carboxylase , md , and me are known as the transhydrogenation 
 machinery .
the resulting pyruvate completes 
 the cycle and goes inside the mitochondria ( figure 2 ) .
me is present in all fungi , but its role in supplying nadph 
 for de novo lipogenesis is preponderant in oleaginous species .
it has been found that , upon nitrogen limitation , 
 me changes from isoform d to isoform e , which supplies nadph for de 
 novo lipogenesis .
ac - coa is 
 converted by acetyl - coa carboxylase ( acc ) to mal - coa , which is then 
 used to synthesize fatty acids . in yeast , cytosolic acc
the mitochondrial version closely resembles in both 
 its molecular mass and amino acid sequence the cytoplasmic one .
acc uses biotin as a cofactor to transfer co2 to ac - coa in a two - step process .
it is a trifunctional enzyme , 
 harboring a biotin carboxyl carrier protein domain , a biotin 
carboxylase 
 domain ( where co2 binds to biotin ) , and a carboxyl - transferase 
 domain ( where co2 is transferred to ac - coa to yield mal - coa ) .
the fatty acyl chain is built in a type i cytosolic 
 fatty acid synthase ( fas i ) .
it is an 66 complex encoded by two genes , fas1 (  
 subunit ) and fas2 (  subunit ) .
it is suggested to contain 6 equivalent sites of fa synthesis 
 with a total of 42 catalytic domains , organized in a ring - like structure .
however , variations to this architecture exist 
 and probably may affect the performance in de novo lipogenesis .
for example , ac - coa is always the basis of fa biosynthesis 
 and is the substrate of -ketoacyl acp synthase ( ksa ) in bacteria .
the sequence of reactions in yeast fas ii is 
 condensation of ac - coa with mal - coa ( via ks ) , reduction ( via ketoacyl 
 reductase , kr ) , dehydration ( via a dehydratase , dh ) and further reduction 
 ( via enoyl reductase ear ) in a repetitive manner until palmitoyl - acp 
 is formed .
release of the acyl moiety is a key step that differs from 
 species and is currently a subject of intense research .
yeasts employ 
 malonyl - palmitoyl transacylase ( mpt ) to transfer the acyl chain from 
 acyl acp to acyl - coa . in algae
, the acyl chain can 
 be released in three ways , depending on the cell s needs .
first , 
 it can be hydrolyzed from acp as a free fatty acid by means of a thioesterase 
 ( te ) .
second , it can be transferred either to g3p or monoacylglycerol-3-phosphate 
 ( mag3p ) through an acyltransferase ( at ) in the chloroplast .
lastly , if the acyl chain is 
 destined to leave the plastid , it can also be released in the acyl - coa 
 form by acyl - coenzyme a synthetases ( acs ) .
it 
 has been found that acs play a key role in regulating in each compartment 
 the internal acyl - coa pools by esterification of fa to coa .
the localization 
 of the pools is maintained due to acyl - coa s not being able 
 to cross the intracellular membranes .
the final fatty acid composition 
 of different types of microalgae is in great part dependent on the 
 activity of these enzymes .
for example , microalgal chain length specific 
 te have been reported , which can release acyl moieties of specific 
 length ( e.g. , c 12:0 or even c 8:0 ) .
this type of fatty acid is more 
 suitable for the production of gasoline and jet fuel .
in contrast , gram - positive bacteria first form acyl - phosphate 
 from acyl - coa ( via plsx ) , which is then transferred onto g3p by plsy . 

gram - negative species use plsb acyltransferase only to load an acyl 
 group directly onto g3p from acyl acp . in yeast , acyl - coas released from cytosolic fas 
 i system
there , a series of esterifications 
 to a g3p backbone occur , also known as the kennedy pathway .
the first 
 step consists of esterifying the acyl moiety from acyl - coa to g3p 
 or dihydroxyacetone 3-phosphate via glycerol-3-phosphate acyl transferase 
 ( g3pat ) , on the sn-1 position .
g3pat is encoded in yeast by gat1 and ayr1 , when the acceptor is g3p 
 or dihydroxyacetone 3-phosphate , respectively .
gat1 does not exhibit a particular preference for acyl - coa , 
 whereas ayr1 prefers palmitoyl - coa , thus defining fa composition . 

a second acyl moiety can be attached on the sn-2 position to generate 
 phosphatidate ( pa ) , catalyzed by acyl - coa : lysophosphatidic acyltransferase 
 ( lpaat ) .
pa plays a key role in the regulation of acc1 , fas1 , and fas2 genes , as it is 
 involved in an autoregulatory loop directed by the concentrations 
 of inositol and choline in the cytosol and pa in the er membrane .
expression of upstream activating sequence ( uasino)-operated 
 genes changes in parallel to pa , inositol , and choline concentrations . 
 in the case when pa is going to be transformed into tag
first , the phosphate group can be directly hydrolyzed by 
 a phosphatidate phosphatase ( pap ) to create dag .
the 
 second one involves synthesizing cytidine diphosphate dag ( cdp - dag ) 
 from pa , catalyzed by cdp - dag kinase , encoded by cds1 .
cdp - dag is the precursor of the different 
 pl in the er membrane .
it thus stays as either phosphatidylcholine 
 ( pc ) , phosphatidylethanolamine ( pe ) , or another type of pl .
eventually , 
 dag is generated by cleavage of the pl via phospholipase c. both alternatives 
 are in metabolic interlock with each other .
this creates a positive 
 feedback system that channels pa into the tag pathway if cdp - dag concentration 
 is higher than dag concentration or directs pa to the membrane pool 
 in the opposite situation .
lastly , dag is transformed into tag by esterifying an acyl moiety 
 from acyl coa catalyzed by an acyl coa : dag acyltransferase ( adat ) , 
 encoded by either dga1 or lro1 , depending on whether dag is generated directly 
 from pa via pap or it comes from the er membrane pl pool , respectively .
another me , different from the cytosolic 
 malic enzyme , catalyzes the conversion of malate to pyruvate and the 
 consequent reduction of nadp to nadph .
nadph then couples to a series 
 of electron transfer reactions involving cytochrome b5 reductase and further activating the desaturase , which adds a double 
 bond on the fatty acid chain at the expense of converting 1 mol of 
 oxygen into a mole of water .
, ergosterol 
 is the major sterol present in yeast . for a review in 
 the biosynthesis of ergosterol backbone
yeast se are synthesized 
 via transesterification of ergosterol with acyl - coa . in yeast ,
two acyl - coa cholesterol acyltransferase ( acat ) 
 related enzymes , are1p and are2p ( encoded by are1 and are2 ) , catalyze the reaction .
microscopic localization 
 of green fluorescent protein hybrids and enzyme measurements showed 
 that both are1p and are2p are localized to the er . are1p esterifies ergosterol and its precursors with nearly 
 equal efficiency with a slight preference for lanosterol , whereas 
 are2p uses ergosterol as a preferred substrate .
an acyl - coa - independent pathway for the formation of ses 
 has not been identified in yeast .
lipid droplet biogenesis 
 is an area of intensive current research , and there is emerging scientific 
 evidence showing that in yeast it takes place between the two membrane 
 leaflets of the er . by mechanisms still not clearly understood there are spots in the 
 er membrane where there is a concentration of adats dga1 and lro1 . 

the first one is responsible for tag synthesis in the outer leaflet 
 of the endoplasmic reticulum , whereas the second is responsible for 
 tag synthesis in the inner leaflet of the er . the synthesized tag begin to accumulate , generating a lens - like 
 protrusion and promoting the recruitment of structural proteins . in 
 yeast ,
pat proteins ( from the initials of perilipin , adipocyte differentiation - related 
 protein , and tip47 ) accumulate in the 
 outer leaflet ( see figure 3 ) , whereas in plants 
 this role is played by oleosins .
when there is enough accumulation 
 of tag between the leaflets , the outer buds off and the lipid droplet 
 is formed ( figure 3 ) .
model of lipid droplet formation in the er membrane . adapted 
 from 
 ref ( 11 ) .
there seems to be a functional relationship between 
 the lipid droplet 
 membrane and the er membrane , because several studies have shown that 
 certain functional proteins can migrate between the lipid droplet 
 and er membranes by mechanisms that do not require energy expenditure . in yeast ,
for example , experiments using yeast 
 mutants unable to synthesize tag revealed that ld were not formed . 

however , the proteins that were present in the wild forms in ld were 
 also present in the er of the yeast mutants , suggesting a relationship 
 between er proteins and lipid droplet proteins . in some cases , lipid droplet - localized proteins can relocate 
 back to the er , indicating that some continuity between the two organelles 
 is maintained , even if only transiently , in a way that allows the 
 two - way partitioning of proteins between the two compartments . in describing the functions of proteins 
 embedded in the droplet 
 monolayers , two mechanisms have been proposed .
examples of this strategy 
 are the multifunctional caveolin protein , which has been localized 
 to both the plasma membrane and ld , and dga1 , the major enzyme catalyzing 
 triacylglycerol synthesis .
their long internal hydrophobic stretch may enable 
 them to be embedded in either bilayers or monolayers .
the second mechanism 
 is best represented by the previously mentioned pat protein family . 

they display a four - helix bundle with great similarity to the n - terminal 
 domain of apolipoprotein e. upon binding to lipids , 
 the apoe four - helix bundle opens to expose amphipathic helices that 
 can bind the monolayer surfaces of lipoproteins . in an analogous manner
, 
 pat proteins may bind to lipid droplets by embedding hydrophobic helices 
 into the droplet surface .
pat proteins also share a common structural 
 element : n - terminal 11-mer repeats that have an amphipathic helical 
 structure .
it is still unclear why or how they do it . in the specific case of perilipins ( members of the pat family ) , 

there are five groups , and their abundance has been correlated with 
 the abundance of tag in the lipid droplet as well .
it is important 
 to mention that perilipin - like proteins are not found in plants .
as 
 was already mentioned , functionally similar oleosins and caleosins 
 are present in plants instead .
the accumulation 
 of lipids that promote concavity decreases the energy burden required 
 for budding .
it has been shown that lipid droplet pl contain more 
 lysophospholipids and less sphingomyelin and pa compared to the total 
 membrane . in silico studies
proposed a packing parameter s to quantify the degree of convexity / concavity in pl .
lysophosphatidylcholine and phosphatidylinositol 
 ( pi ) promote a convex shape where their lipid footprint areas are 
 much smaller than their headgroup areas .
lipids 
 with s values < 1 adopt a convex surface , favoring 
 lipid droplet formation .
configurations adopted by different phospholipids , which can affect 
 the curvature during lipid droplet formation .
eukaryotic microalgae are classified into nine divisions : glaucophyta , 
 rhodophyta ( red algae ) , heterokontophyta , haptophyta , cryptophyta , 
 dinophyta ( dinoflagellates ) , euglenophyta , chlorarachniophyta , and 
 chlorophyta ( green algae ) all of these divisions include single - cell strains , which can be 
 either motile , nonmotile , or both . in terms of their nutritional strategies , 
 microalgae can be divided into obligate heterotrophs , obligate photoautotrophs , 
 facultative mixotrophs , and obligate mixotrophs .
most algal divisions 
 contain colorless heterotrophic species that can obtain organic carbon 
 from the external environment either by taking up dissolved substances 
 ( osmotrophy ) or by engulfing bacteria and other cells as particulate 
 prey ( phagotrophy ) . in most cases , lipid 
 accumulation strategies in microalgae and
some single - cell protists 
 ( both primary and secondary endosymbionts ) follow the four - stage lipid 
 accumulation strategy described for yeast .
, further studies are demonstrating 
 that lipid biosynthesis pathways in microalgae are not a simple mirror 
 image of what happens in higher plants , which have been more thoroughly 
 studied .
it seems likely that regulation of triacylglycerol 
 synthesis and breakdown in microalgae tends to obey a stress response 
 phenomenon , whereas in plants it follows a developmental phenomenon . 
 for a review comparing lipid metabolism between microalgae and plants , 
 see liu .
microalgae 
 differ from yeast in the location of the acetyl - coa pools within the 
 cell .
microalgae display plastidial and cytosolic acetyl - coa pools , 
 which are key for lipid accumulation .
in contrast , yeast s 
 main acetyl - coa pool used for lipid accumulation is located in the 
 cytosol .
there is an additional acetyl - coa pool present in mitochondria 
 as well as a mitochondrial lipid biosynthesis pathway employing a 
 type ii fas ( different from plastidial fas ii ) both in algae and in 
 yeasts .
they play important roles in different cell processes , such as rna 
 processing , mitochondrial lipoic acid synthesis , and protein lipoylation . 

however , mitochondrial lipid synthesis is not the main avenue for 
 lipid accumulation and will not be covered in the present work . for 
 a review of mitochondrial lipid biosynthesis and its relevance in 
 cell function ,
plastids present in algae play a key role in 
 de novo lipid biosynthesis ( for a review of plastid evolution and 
 diversification , see the work of keeling ) . in photoautotrophic
microalgae , photosynthesis provides an endogenous 
 source of plastidial acetyl - coa , although more than one pathway may 
 contribute to maintaining the acetyl - coa pool .
for instance , in photoautotrophic microalgae plastidial 
 pyruvate can be sourced via transformation of photosynthesis - derived 
 glyceraldehyde-3-phosphate to phosphoenolpyruvate ( pep ) .
pep is irreversibly 
 converted to pyruvate ( see figure 5 ) by pyruvate 
 kinase ( pk ) .
finally , the plastidial pyruvate 
 dehydrogenase complex ( pdh ) catalyzes the oxidative decarboxylation 
 of pyruvate to produce plastidial ac - coa , co2 , and nadh . 

pdh contains three components : e1 ( pyruvate dehydrogenase , composed 
 of e1 and e1 subunits ) , e2 ( dihydrolipoyl acyltransferase ) , 
 and e3 ( dihydrolipoamide dehydrogenase ) .
it is possible that photosynthesis - derived 
 pyruvate is the major contributor to plastidial ac - coa for de novo 
 fatty acid synthesis .
however , in mixotrophic grown cultures of heterokonts 
 such as nannochloropsis sp . , incorporation of acetate 
 directly into lipids occurs .
the acetyl - coa synthetase ( acsin ) converts acetate to plastidial 
 ac - coa .
additionally , a study showed that under nitrogen deprivation chlamydomonas is capable of changing its metabolism from 
 converting acetate to glucose to a more direct incorporation of acetate 
 into fatty acids by down - regulating glyoxylate cycle activity and 
 gluconeogenesis .
abbreviations : er , endoplasmic reticulum ; accase , 
 acetyl - coa carboxylase ; acp , acyl carrier protein ; dagat , diacylglycerol 
 acyltransferase ; dhap , dihydroxyacetone phosphate ; enr , enoyl - acp 
 reductase ; fat , fatty acyl - acp thioesterase ; g3pdh , glycerol-3-phosphate 
 dehydrogenase ; gpat , glycerol-3-phosphate acyltransferase ; hd , 3-hydroxyacyl - acp 
 dehydratase ; kar , 3-ketoacyl - acp reductase ; kas , 3-ketoacyl - acp synthase ; 
 lpaat , lysophosphatidic acid acyltransferase ; lpat , lysophosphatidylcholine 
 acyltransferase ; mat , malonyl - coa : acp transacylase ; pdh , pyruvate 
 dehydrogenase complex . in microalgae , the cytosolic ac - coa pool is mainly fueled 
 by the 
 release of mitochondrial cit to the cytosol and further cleaved into 
 oxaloacetate and ac - coa by acl . cytosolic ac - coa is the building block 
 used for lc - pufa elongation in the er .
concomitant production of nadph 
 by me generates the reductive power necessary to drive plastidial 
 de novo fatty acid synthesis forward .
availability of nadph can increase 
 the reaction velocity of acc 2 ( see stage 2 ) and acl .
fatty acid synthesis is an energy - demanding process due 
 to the activity of elongases and desaturases .
for instance , the formation 
 of a c18 fa requires 54 nadph from oxygenic photosynthesis .
other functions of malic enzyme in algae may 
 include delivery of co2 from the tca for the plastidial 
 ribulose-1,5-bisphosphate carboxylase ( rubisco ) .
studies also suggest 
 the existence of a plastidial me ( absent in yeast ) , which can provide 
 electrons for plastidial fa synthesis . for a review about
some microalgae are capable of accumulating 
 intracellular starch , 
 such as the thoroughly studied microalgal model chlamydomonas .
starch synthesis is an example of how carbon partitioning might 
 play a key role in lipid accumulation .
one study showed that in wild - type chlamydomonas , tag accumulated only after the maximum amount 
 of starch was reached , whereas starchless chlamydomonas mutants initiated tag accumulation earlier and reached a higher 
 level than wild - type strain .
therefore , it is possible 
 to engineer high tag algal strains by eliminating competing carbon 
 utilization pathways such as starch synthesis to maximize lipid biosynthesis . in algae , 

the 
 committed step in plastidial fatty acid synthesis is the conversion 
 of plastidial ac - coa to mal - coa by acc1 .
the three domains of the 
 homomeric acc1 are located on a multifunctional polypeptide encoded 
 by a nuclear gene .
others , such as t. pseudonana and p. tricornutum , contain two homomeric accases , acc1 ( described above ) and a cytosolic 
 acc ( acc2 ) , which uses cytosolic ac - coa to generate mal - coa .
the latter 
 plays a role in lc - pufa elongation in the er membrane .
fas are synthesized in microalgae s plastid via a dissociated 
 type ii fas , containing discrete , monofunctional enzymes encoded by 
 distinct genes .
in plastidial fas ii 
 mal - coa is loaded to acp via malonyl - coa : acp transacylase encoded 
 by fabd .
malonyl - acp is used in the cyclic condensation 
 reactions to extend the acyl group to palmitoyl acp or stearoyl acp . 

the acyl acp can be released from fas ii in several ways : it can be 
 hydrolyzed by a fatty acyl - acp thioesterase located in the chloroplast 
 envelope , forming a free fatty acid , or it can be transesterified 
 from acp to coa via ( acs ) , or it can even be coupled to either g3p 
 or mag3p through an at in the chloroplast .
there are some heterotrophic microalgal species that contain a 
 cytosolic type i fas , synthesized from one or two polypeptides , different from plastidial type ii fas .
for example , aurantiochytrium(100 ) contains a type i fas , which synthesizes 
 saturated c14:0 and c16:0 .
the synthesized free fas and the absence 
 of genes homologous to a type ii te may indicate integration of te 
 activity into the synthase .
several 
 attempts have been made to overexpress specific enzymes 
 in the lipid biosynthetic pathways . in the cases of acc and ks ( ks 
 iii ) , overexpression failed to increase lipid accumulation .
the released acyl moieties as free fatty acids 
 destined to stay within the plastid may be further desaturated ( typically 
 to hexadecatrienoic acid hdt , c16:3n-4 ) and coupled 
 to a monogalactosyldiacylglycerol ( mgdg ) backbone .
mgdg , along with 
 other types of galactosylglycerides ( gg ) , is a major component of 
 photosynthetic membranes in microalgae and in some cases may be even 
 more abundant than pl . in plants ,
the fa combination 
 of gg can be traced back to their biosynthetic pathways ; the so - called 
 eukaryotic molecular species ( c18/c18 ) of gg are synthesized outside 
 the chloroplast in the eukaryotic pathway , and the prokaryotic molecular 
 species ( c18/c16 ) are synthesized in the plastid via the prokaryotic 
 pathway .
microalgae differ from plants in that most 
 c20 fas are synthesized outside the chloroplast and are present in 
 both the eukaryotic - like ( c20/c20 , c18/c18 ) and prokaryotic - like ( c18/c16 , 
 c20/c16 ) molecular species .
c20/mlc  
 ( where mlc means medium to long chain ) rather than eukaryotic- and 
 prokaryotic - like gg .
when acyl acp s are esterified to 
 either g3p or mag3p , they can join the plastidial pl pool . in plants 
 two ats
the second one resides on the inner 
 chloroplast envelope membrane and preferentially selects palmitoyl - acp . in some algal species , such as c. reinhardtii and p. lutheri , pc is absent in both plastidial and extraplastidial phospholipid 
 pools .
instead , they contain the non - phosphorus betaine lipid diacylglyceryl - n , n , n - trimethylhomoserine 
 ( dgts ) , which has similar physicochemical properties , as a major membrane 
 component .
it has been 
 suggested that dgts may have a role in lipid droplet formation similar 
 to that of pc in higher plants .
however , 
 they contain other types of phospholipids , such as pa , pe , and pi .
acyl acps synthesized in plastidial type ii fas can be transesterified 
 with coa via acs .
thus , stearoyl acp can leave the fas ii complex 
 via transesterification by a series of acs responsible for maintaining 
 both intraplastidial and cytosolic acyl - coa pools .
in both cases , acyl - coa is destined to leave the plastid and enter 
 the er membrane for further oxygen - dependent elongation and desaturation , 
 to become long - chain polyunsaturated fatty acids ( lc - pufa ) , such as 
 eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) .
the first are front - end 
 desaturases containing an n - terminal cytochrome b5 domain and insert the new double bond between the fa carboxyl group 
 and a possible existing double bond .
one example is the high substrate 
 specific plastidial - located 12 desaturase , identified in p. tricornutum , which desaturates palmitoleic acid 
 c16:1n-7 to hexadecadienoic acid 16:2n-4 .
the second group comprises the less 
 common 6/3 desaturases capable of inserting a new double 
 bond between the fa methyl end and a pre - existing double bond .
stearoyl coa can be desaturated by an er membrane - bound 9 
 desaturase to oleyl - coa and then linked to a glycerol backbone for 
 further processing .
some microalgae contain a plastidial 9 
 desaturase , capable of direct desaturation of stearoyl acp to be then 
 transesterified to coa and sent to the er for elongation and desaturation . 
 in most microalgae , n-3 lc - pufa are more abundant 
 than n-6 lc - pufa , whereas 
 in filamentous fungi , such as mortierella and mucor , n-6 lc - pufa are more common . in microalgae the most common pathways for epa and
dha synthesis 
 are the n-3 and n-6 pathways , but 
 variations of the theme occur . in the n-3 pathway , 

oleic acid already linked to a glycerol backbone in the er membrane 
 is desaturated to linoleic acid ( la ) , via a 12 desaturase ( encoded 
 by fad2 ) .
a third double bond inserted by an n-3 
 15 desaturase gives -linolenic acid ( lna ) , which is 
 further desaturated to produce stearidonic acid ( sa ) by a 6 
 desaturase .
sa is then elongated to c20:4 n-3 ( eicosatetraenoic 
 acid , ea ) and finally desaturated by a 5 desaturase to produce 
 epa . in most dha - producing microalgae
this is a different , less complicated strategy 
 for dha synthesis from epa , compared to the sprecher pathway , present 
 in mammals , which involves an additional elongation and a -oxidation . 

in contrast to the n-3 pathway , some microalgae synthesize 
 epa via the n-6 pathway , following 6 desaturation 
 of la to -linolenic acid ( gla ) , elongation to dihomo--linolenic 
 acid ( dgla ) , creation of a fourth double bond by 5 desaturase 
 to produce arachidonic acid ( ara ) , and a final desaturation to create 
 epa via a n-3 17 desaturase . some microalgae express 9 elongase and 8 
 desaturase , allowing them to synthesize epa in a different way compared 
 to the n-3 and n-6 pathways . in a variation of the n-3 
 pathway
, lna is elongated by a 9 elongase to eicosatrienoic 
 acid ( eta ) and desaturated by the 8 desaturase , creating ea . 
 on the other hand , a variation of the n-6 pathway 
 using this pair of enzymes involves 9 elongation of la to eicosadienoic 
 acid ( eda ) and 8 desaturation to dgla .
thus , microalgae can switch from n-6 fatty acids 
 to n-3 fatty acids via the n-3 15 
 desaturase and the n-3 17 desaturase . in some 
 species of the thraustochytrids , it is also possible to switch from n-6 docosapentaenoic acid ( dpa ) to dha , via an n-3 4 desaturase .
in addition to these pathways , there 
 is a different n-3 lc pufa biosynthetic pathway present 
 in one of the three genera 
 of heterotrophic thraustochytrids , namely auranthiochytrium , based on an anaerobic polyketide synthase pathway ( pks ) . a large multifunctional enzyme complex carries out the multitude 
 of individual reactions , utilizing mal - coa and producing free n-3 lc - pufas . the major free fas dpa ( 22:5n-6 ) and dha ( 22:6n-3 )
are then activated to acyl - coa 
 and incorporated into tags . because pks does not 
 require aerobic desaturation , the pathway is energetically favorable 
 compared to the membrane - bound desaturases and elongases .
tag synthesis in microalgae follows the 
 kennedy pathway in the 
 er in a similar fashion as yeast ( see figure 5 ) .
however , an acyl - coa - independent mechanism for triacylglycerol 
 synthesis in some plants and yeast has been reported .
this pathway uses pl as acyl donors and dag as the acceptor , and 
 the reaction is catalyzed by the enzyme phospholipid : dag acyltransferase .
there are two diacylglycerol acyltransferase families identified 
 in chlamydomonas , involved in the final step of triacylglycerol 
 synthesis : type one ( dgat ) , encoded by dgat1 , and type two ( dgtt ) , 
 encoded by five dgtt genes , which do not share sequence similarity . 

two independent studies showed the expression levels of dgat1 and 
 dgtt1 increased considerably following nitrogen deprivation .
however , it remains unclear 
 which of these dgats are primarily responsible for the accumulation 
 of tags under this condition or whether individual isoforms have specific 
 roles .
triacylglycerol synthesis can also occur within the plastid , 
 through 
 a series of acyl - acp esterifications to plastidial g3p , catalyzed 
 by plastidial ats similar to the kennedy pathway in the er . the involvement of gg ( the major polar lipid 
 family in plastidial membranes in several microalgae ) in this process 
 remains to be elucidated . during light 
dark cycles , many 
 microalgae initiate triacylglycerol 
 storage during the day and deplete those stores at night to support 
 cellular atp demands and/or cell division .
this cycling has to be taken into account when scaling up processes 
 for production of lipids from algae .
this variable may be key to the 
 overall success of an open pond process or a closed photoreactor process .
they can grow facing the 
 cytosol or toward the inside of the plastid , facing the stroma , which 
 is the major aqueous fluid surrounding the thylakoids inside the chloroplast .
the mechanisms underlying the orientation of lipid droplet growth 
 in plastid membranes are not well understood . when ld grow toward 
 the inside of the plastid ( facing the stroma ) , they can also be called 
 plastoglobules ( ptg ) .
ptg can be considered to be functionally equivalent 
 to cytosolic ld , but differ in three major respects .
second , they can assume several different 
 forms including rods , fibers , and globules ; and third , they are bound 
 by a specific family of proteins , variously termed plastoglobulins , 
 plastid lipid - associated proteins , and fibrillins .
oleosins ( the major lipid droplet proteins present in plants ) 
 are not present in green algae , but a major lipid droplet protein 
 was identified by applying proteomics in chlamydomonas and shown to modulate lipid droplet size .
a wide variety of parameters affect the abundance of 
 ld in chlamydomonas , and there is ample evidence 
 that turnover of ld plays crucial roles in cellular lipid or carbon 
 homeostasis .
in bacteria , the most frequent types of neutral and polymeric lipids 
 synthesized and accumulated are pha , tag , wax esters ( we ) , and , to 
 a lesser extent , se .
however , a special 
 situation occurs in bacteria such as rhodococcus ruber , and other related bacteria , capable of accumulating both types 
 of lipids from unrelated carbon sources such as glucose .
the mechanisms of wax ester accumulation have been reviewed 
 elsewhere . in a nutshell ,
an acyl - coa is 
 transformed to an aldehyde via an 
 nadph - dependent acyl - coa reductase ( encoded by acr1 ) , which is then reduced to an alcohol via an also nadph - dependent 
 fatty aldehyde reductase .
the alcohol is then transesterified to an 
 acyl - coa via an enzyme displaying both wax ester synthase ( ws ) and 
 acyl - coa : diacylglycerol acyltransferase ( dgat ) activities ( abbreviated 
 ws / dgat )
. the only report of the 
 presence of an sterol ester - synthesizing 
 enzyme in prokaryotes was provided by thornton et al . to date , triacylglycerol 
 biosynthesis has been detected only in aerobic heterotrophic bacteria 
 and in cyanobacteria . in most bacteria , 

accumulation of tag and other neutral lipids , such as we , is stimulated 
 by a carbon source present in excess , together with limited nitrogen 
 in the medium .
structurally , fluorescence 
 staining experiments showed that lipid biosynthesis starts at peripheral 
 lipid domains close to the cytoplasm membrane .
biochemically , fatty acid biosynthesis begins in bacteria with acc , 
 a heterotetrameric enzyme encoded by four genes , acca , accb , accc , and accd .
ac - coa is converted to mal - coa and transferred 
 to acp by malonyl - coa : acp transacylase ( fabd in bacteria ) to form 
 malonyl - acp .
a first condensation of malonyl - acp with acetyl - coa by 
 -ketoacyl - acp synthase iii ( fabh ) to form -ketobutyryl - acp 
 and co2 initiates a cycle that can elongate the fatty acyl - acp 
 by two carbon units for each cycle until a saturated fatty acid of 
 16 or 18 carbons is made .
ksi ( fabb ) and ksii ( fabf ) are responsible 
 for the subsequent elongation cycles of the growing acyl - acp chain .
to balance chain initiation with growth and utilization in bacterial 
 fasii , ksiii , enoyl - acp reductase ( fabi in bacteria ) , and -ketoacyl - acp 
 reductase ( fab g in bacteria ) are under negative feedback control 
 by long - chain acyl - acps .
long - chain acyl - acps 
 directly control reductase activities ; consequently , fasii is biased 
 to catalyze forward if these products are withdrawn from the system 
 by any conversion , including phospholipid or triacylglycerol synthesis . in some bacteria , fas ii yields unsaturated fatty acids , which 
 play a key role in bacterial membrane fluidity and function . unlike er desaturases present in microalgae or yeast , which introduce 
 double bonds into the completed fatty acid chains at the expense of 
 oxygen , the bacterial fas ii system can also desaturate fatty acids 
 anaerobically , because it does not require molecular oxygen .
faba introduces the double bond at the 10-carbon 
 intermediate , forming cis-2-decenoyl - acp .
it additionally 
 isomerizes it into trans-3-decenoyl - acp , which is 
 further elongated by fabb .
two genes , faba and fabb , are the key players in this 
 pathway and occur together in bacteria that produce unsaturated fatty 
 acids . some rhodococcus and nocardia bacteria are capable of incorporating branched or phenylic groups 
 into their intracellular tag if the corresponding substrate is fed 
 to the medium .
for example , nocardia globerula strain 432 accumulated tag containing the branched fatty acid 4,8,12-trimethyltridecanoic 
 acid after cells were fed pristane ( a branched alkane ) , and tag with a subfraction containing phenyldecanoic 
 acid residues were detected in cells of r. opacus pd630 after phenyldecane was fed as sole carbon source .
the enzymes involved in the esterification of the glycerol 
 moiety probably act via sequential acylation of the sn-1 , -2 , and 
 -3 positions of g3p , with the removal of the phosphate group occurring 
 before the final acylation step .
the first 
 esterification is catalyzed by a g3pat using either acyl - coa or acyl 
 acp to form lysophosphatidic acid .
a second acylation to lysophosphatidic 
 acid gives pa , which is the first branchpoint for the synthesis of 
 tag and pl , because it can be converted to cdp - dag , the precursor 
 of the different pl species in bacterial membranes .
dag itself is 
 also at a metabolic branchpoint that divides phospholipid and triacylglycerol 
 formations , because it acts as a precursor for tag , pc , and pe biosynthesis . 

in addition , dag can also be derived from pl by the action of phospholipase 
 c the distribution of acyl groups on the hydroxyl groups 
 of the glycerol backbone is nonrandom , as has been demonstrated for r. opacus pd630 .
the shorter and saturated 
 fatty acids were esterified to the hydroxyl group at position 2 , whereas 
 unsaturated fatty acids were preferentially found at position 3 .
this 
 distribution in bacterial tag is different from the tag of mammals 
 and plants , where the longer unsaturated fatty acids are found at 
 position sn-2 .
the final step 
 in wax ester and triacylglycerol biosyntheses in bacteria is catalyzed 
 by ws / dgat .
ws / dgat is encoded by atfa and is not related to any known at involved in the 
 formation of tag and we in eukaryotes .
it has also been shown that 
 ws / dgat is localized at the bacterial cytoplasmic membrane , presumably 
 attached to the inner leaflet of the membrane , probably via ionic 
 interactions .
for a detailed review about 
 ws / dgat , the reader is referred to the work of waltermann et al .
alternative pathways for triacylglycerol synthesis 
 that do not involve dag in bacteria have also been reported and may 
 involve an enzyme similar to that reported by dalquist in yeast , which catalyzes the formation of tag from the 
 transesterification of an acyl donor ( e.g. , acyl - coa ) to pl .
this follows observations that following a double 
 knockout of ws / dgat genes in a. borkumensis , cells were still capable of substantial triacylglycerol accumulation 
 in ld .
triacylglycerol / wax ester lipid droplet biogenesis presumably begins 
 by allocation of newly formed tag in a hydrophobic zone within ws / dgat . 

as time goes by , more ws / dgat attaches to the membrane , and cumulative 
 synthesis advances , presumably leading to the formation of very small 
 triacylglycerol agglomerates , depicted by wltermann et al . 
 as small lipid droplets ( sld ) .
sld apparently 
 recruit pl from the membrane by a mechanism that is still not well 
 understood , forming a layer toward the cytoplasmic side ( see figure 6 ) .
the agglomeration of phospholipid - coated sld 
 appears in microscopy as an oleaginous layer just parallel to the 
 membrane .
then , accumulation of sld in 
 a given point gives birth to ld , which are coated by pl and detach 
 from the oleaginous layer .
it has been suggested that acquisition of additional tag is 
 by merging with other ld freshly synthesized from the oleaginous layer 
 via coalescence and/or through a protein 
 identified as tada .
an interesting study 
 by ding and co - workers examined the proteome 
 of rhodococcus sp .
rha 1 ( a gram - positive bacteria 
 capable of accumulating tag ) grown both 
 in nitrogen - abundant ( not favoring lipid accumulation ) and nitrogen - depleted 
 conditions ( favoring lipid accumulation ) . using a combination of techniques , 
 including lc - ms , sds - page , and immunoblot assays , they reported 228 
 lipid droplet - associated proteins , which clustered primarily into 
 metabolism - related enzymes , transcriptional regulators , ribosome proteins , 
 and cell division - related proteins .
interestingly , they identified 
 two major proteins , ro02104 and pspa , which constituted about 15% 
 of the total lipid droplet protein . according to their findings ,
the 
 structure predicted for ro02104 resembles that of apolipoproteins , 
 the structural proteins of plasma lipoproteins in mammals . suggested mechanisms 
 of neutral ld formation in bacteria . adapted 
 from ref
pha comprise 
 a complex class of storage polyesters , with almost 150 different hydroxyalkanoic 
 acids known as constituents .
a wide variety 
 of gram - positive as well as gram - negative bacteria synthesize pha . 

examples include pseudomonas , bacillus , ralstonia , aeromonas , and rhodobacter , among others .
pha are divided into two groups 
 on the basis of the number of constituent carbon atoms in their monomer 
 units : short chain length phas ( scl pha ) and medium chain length phas 
 ( mcl pha ) .
monomers of scl pha are 35 carbon atoms long , compared 
 to 614 carbon atoms in mcl pha . in addition , scl pha are stiff 
 and brittle with a high degree of crystallinity , whereas mcl pha are 
 flexible and have low crystallinity , tensile strength , and melting 
 point .
accumulation of 
 pha starts with the creation of hydroxyalkanoate 
 monomers from three different biosynthetic pathways .
the first one 
 involves incorporation of two acetyl - coas to form acetoacetyl - coa , 
 by the enzyme -ketothiolase .
this 
 pathway creates hydroxybutyrate ( hb ) monomers exclusively and is used 
 by bacteria such as cupriavidus necator and azotobacter beijerinckii ( see 
 figure 7 ) . on a second pathway involving de novo fatty acid 
 biosynthesis , monomers of different lengths can be formed by transesterifying 
 3-hydroxyacyl - acp , an intermediate of fas ii to 3-hydroxyacyl - coa , 
 presumably by the enzyme acyl - acp - coa transacylase , encoded by phag . this enzyme is the key link between de novo fatty 
 acid synthesis and polyhydroxyalkanoate biosynthesis .
this pathway is of biotechnological interest because it helps generate 
 monomers for polyhydroxyalkanoate synthesis from structurally unrelated 
 and simple , inexpensive carbon sources such as glucose or related 
 simple sugars . on a third embodiment , monomers of different lengths 

can also be sourced from fatty acid -oxidation pathway either 
 by conversion of 2-enoyl - coa by an r - specific enoyl - coa 
 hydratase , encoded by phaj , or by reduction of 3-ketoacyl - coa , 
 presumably by fabg or phab . in this case
the ability 
 of microorganisms to synthesize a particular form of polyhydroxyalkanoate 
 is mainly due to the substrate specificity of polyhydroxyalkanoate 
 synthases .
these enzymes are divided into four classes , depending 
 on their structure and specificity .
class i enyzmes utilize coa thioesters 
 of 3-hydroxyalkanoates ( 3-has ) , 4-has , and 5-has comprising three 
 to five carbon atoms .
members of class ii display major 
 specificity for monomers ranging from 6 to 14 carbon atoms .
enzymes 
 from both classes consist of a single subunit of an average size of 
 6070 kda and are encoded by phac .
in contrast , 
 class iii and class iv synthases are encoded by two genes , phac / phae , 
 and phac / phr respectively , and consist of two subunits .
class iii 
 members are capable of polymerizing preferably monomers ranging from 
 three to five carbons , yet can utilize monomers from six to eight 
 carbons as well .
species such as allochromatium vinosum contain class iii synthases , 
 whereas class iv has been reported only in bacillus sp . all polyhydroxyalkanoate synthases 
 share a conserved cysteine as a catalytic site to which the growing 
 polyhydroxyalkanoate chain is covalently attached .
the active - site 
 cysteine , histidine , and aspartate constitute a catalytic triad similar 
 to esterases .
two models 
 currently exist that may explain the formation of in vivo polyhydroxyalkanoate 
 ld ( which are also called granules or carbonosomes ) : the micellar and the budding models .
the first one is based on 
 the assumption that the polyhydroxyalkanoate synthase is present in 
 the cell as a soluble enzyme , distributed throughout the cytoplasm . 

once polymerization of substrate molecules ( coa - thioesters of suitable 
 hydroxyalkanoic acids ) starts , the nascent polyester chain converts 
 the initially soluble enzyme into an amphipathic molecule and the 
 increasingly hydrophobic polyhydroxyalkanoate chains aggregate into 
 a micelle - like structure .
polyhydroxyalkanoate synthase remains attached 
 to the surface of the granule and therefore becomes insoluble ( see 
 figure 8) . in this model ,
pl and proteins of 
 the surrounding layer would gradually become incorporated as the self - assembled 
 polyhydroxyalkanoate inclusion increases in size . this model requires the polyhydroxyalkanoate granule to be localized 
 in the cytoplasm at all stages of formation .
in contrast , the budding 
 model assumes that polyhydroxyalkanoate synthase is associated with 
 the inner face of the cytoplasmic membrane , either inherently or as 
 soon as a polyhydroxyalkanoate chain emerges from the enzyme . in this 
 case ,
biosynthesis of the polyester would be directed into the intermembrane 
 space where the extending chains would accumulate until eventually 
 the granules detach from the membrane and polyhydroxyalkanoate - specific 
 surface proteins can be attached to the growing granules .
although the micelle model is supported by the fact that polyhydroxyalkanoate 
 granules can be produced in vivo in the absence of membranes , most 
 of the recently emerging evidence is in favor of the budding model .
phas may constitute approximately 5% 
 ( w / w ) of total cellular proteins , and they 
 play a main structural role in preventing polyhydroxyalkanoate granules 
 from aggregating and in preventing the nonspecific attachment of other 
 proteins to polyhydroxyalkanoate granules .
in addition , phasins are presumably involved in the regulation of 
 polyhydroxyalkanoate synthesis , polyhydroxyalkanoate degradation , 
 polyhydroxyalkanoate granule size control , the formation of networks on the polyhydroxyalkanoate granule surface , and the distribution of polyhydroxyalkanoate 
 granules during cell division .
another important group of proteins 
 present in the external granule layer is the depolymerases , which 
 are responsible of catalyzing polyhydroxyalkanoate breakdown .
this 
 is a relevant step in the role of polyhydroxyalkanoate accumulation 
 as a survival mechanism in the absence of suitable energy / carbon sources , 
 as was demonstrated many years ago in r. eutropha .
phazs have 
 been investigated much less than extracellular depolymerases , and 
 the mechanism by which intracellular native polyhydroxyalkanoate granules 
 can be reutilized is still not well understood .
interestingly , the gene coding for phazs is located between 
 two copies of phac1 and phac2 ( both 
 polyhydroxyalkanoate synthase genes ) in all investigated bacteria 
 that accumulate mcl pha .
in contrast , 
 secreted depolymerases are used by most bacteria to assimilate pha 
 present in the environment from , for example , other nonliving cells .
proposed mechanism of pha containing lipid droplet formation 
 in 
 bacteria ( micelle model ) . adapted from ref ( 22 ) .
polyhydroxyalkanoate granule synthesis 
 and phasin production are 
 tightly regulated by the effectiveness of the transcriptional regulator 
 phar .
genes encoding proteins homologous to phar are widely distributed 
 among scl pha producing bacteria , indicating an important role in 
 the regulation of scl pha biosynthesis .
the discovery 
 of the archaea domain in 1977 revealed a novel class 
 of microorganisms encountered in exceptional ecological niches such 
 as high ( thermophiles and hyperthermophiles ) or low ( psychrophiles ) 
 temperatures , acidic media ( acidophiles an thermoacidophiles ) , anaerobic 
 atmosphere ( methanogens ) , and high salinity ( halophiles ) .
the unique chemical structure of their core membrane lipids is in 
 part responsible for their adaptation to such hostile environments . 

archaeal membrane lipids , in contrast to those of bacteria and eukaryotes , 
 are made up of saturated chains containing methyl branches , attached 
 to glycerol by ether linkages with a stereochemistry in the 2-position 
 of the glycerol opposite that of conventional mesophilic lipids .
( for 
 a review on archaeal ether lipid structures , the reader is referred 
 to the work of jacquemet et al . ) moreover , 
 archaea do not synthesize fatty acyl esters , which are the most common 
 constituents of ld ; instead , their lipids are based on isoprenoid 
 chains . therefore , no accumulation of 
 tag has been reported yet in archaea . despite these differences , however , 
 evidence of polyhydroxyalkanoate accumulation was first reported in 
 haloarchaea back in 1972 .
the strains were called 
 at that time  halobacterium sp . from the dead 
 sea  , but later identified as haloarcula marismortui . since then , strains of several other 
 haloarchaeal genera , including haloferax , halobiforma , and haloquadratum , have been 
 found to accumulate pha . as in bacteria ,
archaea 
 produce pha under conditions of nutrient limitation but where carbon 
 is available in excess .
the mechanisms of polyhydroxyalkanoate accumulation within archaea 
 are beginning to be understood , and work is underway to elucidate 
 the type of proteins involved in archaeal polyhydroxyalkanoate accumulation . 

genes involved in polyhydroxyalkanoate biosynthesis in haloarchaea 
 were not recognized until recently , when the polyhydroxyalkanoate 
 synthase genes were identified and characterized for haloarcula marismortui and haloferax 
 mediterranei .
these archaeal polyhydroxyalkanoate synthases are all composed of 
 two subunits , phae and phac , and they are homologous to class iii 
 bacterial polyhydroxyalkanoate synthases but have a longer c - terminal 
 extension in the phac subunit .
the close 
 similarity of archaeal and bacterial type iii polyhydroxyalkanoate 
 synthase genes and the lack of other polyhydroxyalkanoate gene types 
 in archaea suggest that archaeal pha originated from the horizontal 
 transfer of an ancestral type iii gene from a bacterium .
some authors suggest this transfer to have occurred 
 already before permian times . genome - wide analysis of h. marismortui atcc 43049 revealed eight paralogues of a short - chain dehydrogenase / reductase , 
 responsible for reduction of acetoacetyl - coa to ( r)-3-hydroxybutyryl - coa ( 3-hb coa ) , a monomer used by polyhydroxyalkanoate 
 synthase to produce polyhydroxybutyrate ( phb ) .
another study demonstrated that a similar paralogue in h. hispanica , namely fabg1 , encodes a pha - specific 
 acetoacetyl - coa reductase responsible for providing 3-hb - coa for polyhydroxyalkanoate 
 biosynthesis in haloarcula species .
the authors concluded 
 that the polyhydroxyalkanoate biosynthesis pathway from ac - coa , catalyzed 
 by -ketoacyl thiolase , acetoacetyl - coa reductase , and polyhydroxyalkanoate 
 synthase , as distributed in bacteria , likely also exists in the domain 
 of archaea .
nonetheless , they pointed out that for phb - accumulating 
 haloarchaeal natrialba strain 56 , no enzyme activity of acetoacetyl - coa reductase or -ketoacyl 
 thiolase was detected in the crude extract , indicating that a different 
 metabolic route toward production of phb might be employed .
polyhydroxyalkanoate synthase , putative enoyl - coa hydratase , and 
 two structural phasin - like proteins have been identified in haloarchaeal 
 polyhydroxyalkanoate granule surfaces .
the phasin - like proteins in haloarchaea share some structural features 
 with bacterial phas , such as the presence of hydrophobic domains and 
 a high -helix content . after a genome - wide 
 investigation into the 12 haloarchaea that harbored the phap gene , 

most of these archaea were found to possess a similar pha cluster , with five genes , namely , maoc - gap12-phap - phae - phac , having the same organization as that in h. mediterranei . the extensive existence of this pha gene cluster was suggested as an indication of an evolutionarily 
 conserved pha gene cluster unique to haloarchaea .
a promising approach is to develop archaeal 
 species as industrial 
 scale polyhydroxyalkanoate producers .
in particular , several halophilic 
 archaea have the advantages of utilizing much cheaper carbon sources 
 ( including waste materials ) , as well as having less strict sterilization 
 requirements , plus easier and more efficient methods for polyhydroxyalkanoate 
 extraction .
microorganisms provide an exciting platform for the development 
 of lipid technologies . in the course of evolution , they have developed 
 elegant pathways to synthesize a wide array of lipids , providing a 
 versatile and cost - effective approach for sourcing lipids to virtually 
 all sectors of industry .
the understanding of the biochemical and 
 cellular mechanisms of lipid production , accumulation , and secretion 
 will provide valuable insights on innovations to overcome the hurdles 
 in microbial lipid utilization .
ongoing studies using different 
  omics  approaches 
 will provide a holistic view of flows and interactions between the 
 different metabolic pathways involved in lipid accumulation .
tools 
 such as next - generation sequencing , transcriptome analysis , proteomics , 
 and mass spectrometry are already clearing out the missing links in 
 single - cell lipid biosynthesis . this information will permeate in 
 the creation and scaleup of more efficient processes .
it is 
 thus important to project the potential applications and 
 envision the frontiers to which this valuable toolset can lead the 
 diverse fields of lipid technology .
for example , in the field of biotherapeutics , 
 microbial - based approaches have the flexibility to construct platforms 
 to manufacture personalized lipid therapies starting as simply as 
 lc pufa combinations , appropriate to the metabolic phenotype of each 
 individual and eventually becoming as 
 complex and selective as personalized cancer interventions .
this approach 
 of personal medicine will carry improved benefits in the treatment 
 of a range of conditions : in immunological diseases from infections 
 to autoimmunities ; in metabolic conditions from diabetes to cardiometabolic 
 diseases ; and in microbiota dysbiosis from ibs to ibd .
microbial technologies 
 can also provide an alternative pathway for sourcing promising lipids 
 and fatty acids that are unavailable in the market at present .
for 
 example , oil - producing microbes can be considered as appropriate vehicles 
 in which foreign ( plant ) genes could be cloned for the production 
 of commercially attractive fatty acids such as nervonic acid ( c24:1 
 15 ) obtained from honesty ( lunaria ) , which is used in small amounts in 
 the treatment of particular neuropathies .
another example is sterculic 
 acid { -(2n - octylcycloprop-1-enyl)-octanoic 
 acid } , which has been considered as 
 a treatment for certain cancers of the bowel .
one more promising example 
 is represented by the non - methylene - interrupted fatty acids ( e.g. , 
 c20:3 5 , 11 , 14 ) , which can be obtained from juniperus chinensis seed oil and is known to reduce 
 the amount of arachidonic acid in certain phospholipid pools and , 
 thus , act to alter eicosanoid signaling . in the field of energy and biofuels ,
understanding the mechanisms 
 by which each group of microorganisms generates highly combustible 
 lipids will foster the production of cost - effective fuels through 
 sustainable processing and with improved performance .
it is yet to 
 be tested whether the biochemical mechanisms for amphipathic lipid 
 secretion present in some microorganisms would work for the secretion 
 of neutral lipids .
this concept , if brought to practice , will significantly 
 reduce the processing costs and render high - quality combustible lipids 
 for the production of biofuels . in the case of the food industry
, 
 a microbial - based approach will 
 allow food companies to expand their core businesses by creating new 
 product portfolios out of their current byproducts .
two promising 
 examples are the production of cocoa butter analogues and the production of high - value oils out of spent agricultural 
 materials . in the field of biomaterials , microbial lipids are 
 already being 
 used for producing biosurfactants and bioplastics , replacing synthetic 
 analogues due to their improved biodegradability and reduced cost . 

products such as metabolix pha and biomer are examples 
 on how microbial - derived plastics are gaining momentum in the market .
their specific modes of action , 
 low toxicity , relative ease of preparation , and widespread applicability 
 are increasing their use in applications such as emulsifiers , wetting 
 and foaming agents , functional food ingredients , detergents in petroleum , 
 petrochemicals , environmental management , agrochemicals , cosmetics 
 and pharmaceuticals , commercial laundry detergents , mining and metallurgical 
 industries ( for an overview of microbial surfactant applications , 
 see mukherjee ) .
the scope of applications 
 goes to such an extent that the petroleum extractive industry is already 
 researching the use of microbial lipid - based biosurfactants for increasing 
 oil recovery yields from subterranean depots .
the great era of chemistry culminating in the restructuring of the 
 human condition in the 20th century has been defined by the principles 
 of reductionism and simplicity .
in contrast , the 21st century heralds 
 the era of complexity for which the inherent biological diversity 
 and information content of microorganisms will be key to adding value 
 to all industrial chains . a search performed on july 6 , 2013 , 
 with the terms  microbial 
 lipids  in the u.s .
patent publication since 2008 , showing the 
 increasing interest in the generation of intellectual property around 
 this area of research .
the estimated patents 
 by the end of 2013 according to the current trend will be around 1420 , 
 almost doubling the number registered in 2009 .
the next step 
 in microbial lipid technology is the integration 
 of processes and applications to generate comprehensive , sustainable 
 solutions .
the immediate challenge is to combine desired metabolic 
 pathways present in different species or strains to generate superior 
 microbial species .
benefits will include improved yields , targeted 
 lipidomic profiles , and structural features to synthesize in a sustainable 
 and cost - effective way the lipid structures necessary to satisfy the 
 breadth of needs of 21st century industry .
for example , an appropriate 
 integration of microbial - based lipid technologies will allow delivery 
 of smart biofuels and even personalized edible oils derived from microorganisms 
 and reporter - equipped biodegradable containers also derived from microorganisms . 

currently the most used packaging material for commercial edible oils 
 is polyethylene terephthalate , which is virtually a nondegradable 
 plastic . with the appropriate use of microbial pha
, it could be possible 
 to see in the same facility the production of high - value microbial 
 oils ( rich in a combination of lc pufa ) , along with the production 
 of polyhydroxyalkanoate - based bottles for its commercialization .
furthermore , 
 additional microbial biotechnology platforms will be integrated to 
 transform the byproducts either into biomass nutrients to make the 
 process sustainable or even into another portfolio of high - value products , 
 as a way to increase the business profitability ( figure 10 ) .
conceptual flowchart for an integral , sustainable 
 microbial - based 
 edible oil process .
microbial lipid technologies 
 are a valuable toolset available for 
 the future generation of scientists , which will help them push the 
 boundaries of production solutions ultimately toward a more sustainable 
 society .
 in recent years attention has been 
 focused on the utilization of 
 microorganisms as alternatives for industrial and nutritional applications . 
 
 considerable research has been devoted to techniques for growth , extraction , 
 and purification of high - value lipids for their use as biofuels and 
 biosurfactants as well as high - value metabolites for nutrition and 
 health . 
 these successes argue that the elucidation of the mechanisms 
 underlying the microbial biosynthesis of such molecules , which are 
 far from being completely understood , now will yield spectacular opportunities 
 for industrial scale biomolecular production . 
 there are important 
 additional questions to be solved to optimize the processing strategies 
 to take advantage of the assets of microbial lipids . 
 the present review 
 describes the current state of knowledge regarding lipid biosynthesis , 
 accumulation , and transport mechanisms present in single - cell organisms , 
 specifically yeasts , microalgae , bacteria , and archaea . 
 similarities 
 and differences in biochemical pathways and strategies of different 
 microorganisms provide a diverse toolset to the expansion of biotechnologies 
 for lipid production . 
 this paper is intended to inspire a generation 
 of lipid scientists to insights that will drive the biotechnologies 
 of microbial production as uniquely enabling players of lipid biotherapeutics , 
 biofuels , biomaterials , and other opportunity areas into the 21st 
 century . 
Introduction
Accumulation
of Lipids in Single-Cell Microorganisms: An Overview
The
Case of Yeast
The Case of Microalgae
The Case of Bacteria
The Case of Archaea
Future Perspectives