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The dilemma for lipid productivity in green microalgae: importance of substrate provision in improving oil yield without sacrificing growth

View Article: PubMed Central - PubMed

ABSTRACT

Rising oil prices and concerns over climate change have resulted in more emphasis on research into renewable biofuels from microalgae. Unlike plants, green microalgae have higher biomass productivity, will not compete with food and agriculture, and do not require fertile land for cultivation. However, microalgae biofuels currently suffer from high capital and operating costs due to low yields and costly extraction methods. Microalgae grown under optimal conditions produce large amounts of biomass but with low neutral lipid content, while microalgae grown in nutrient starvation accumulate high levels of neutral lipids but are slow growing. Producing lipids while maintaining high growth rates is vital for biofuel production because high biomass productivity increases yield per harvest volume while high lipid content decreases the cost of extraction per unit product. Therefore, there is a need for metabolic engineering of microalgae to constitutively produce high amounts of lipids without sacrificing growth. Substrate availability is a rate-limiting step in balancing growth and fatty acid (FA) production because both biomass and FA synthesis pathways compete for the same substrates, namely acetyl-CoA and NADPH. In this review, we discuss the efforts made for improving biofuel production in plants and microorganisms, the challenges faced in achieving lipid productivity, and the important role of precursor supply for FA synthesis. The main focus is placed on the enzymes which catalyzed the reactions supplying acetyl-CoA and NADPH.

No MeSH data available.


Related in: MedlinePlus

Simplified scheme of central carbon metabolism in microalgae. Arrows represent potential carbon fluxes. Enzymes are in bold italics. Blue arrows represent reducing power (NADPH). Red arrows represent acetyl-CoA. Black boxes denote pathway names. Neutral lipid droplets found in microalgae consist mostly of triacylglycerols (TAGs), formed by combining FAs and glycerol. ACCase acetyl-CoA carboxylase; ACD acyl-CoA dehydrogenase; ACL ATP-citrate lyase; ACS acyl-CoA synthetase; AGPP ADP-glucose pyrophosphorylase; AMY amylase; CA carbonic anhydrase; DGAT diacylglycerol acyltransferase; DHAP dihydroxyacetone phosphate; F1,6P fructose 1,6-bisphosphate; F6P fructose 6-phosphate; FAT fatty acyl–acyl carrier protein (ACP) thioesterase; G1P glucose 1-phosphate; G6P glucose 6-phosphate; G6PDH G6P dehydrogenase; GAP glyceraldehyde 3-phosphate; GPAT glycerol-3-phosphate acyltransferase; MAL malate; MDH malate dehydrogenase; MME NADP-malic enzyme; OAA oxaloacetate; PDC pyruvate dehydrogenase complex; PEP phosphoenolpyruvate; PEPC PEP carboxylase; PK pyruvate kinase; Ru5P ribulose 5-phosphate; Ru1,5BP ribulose 1,5-bisphosphate; RuBisCO Ru1,5BP carboxylase/oxygenase; 3-PGA 3-phosphoglycerate; 6PGDH 6-phosphogluconate dehydrogenase
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Fig1: Simplified scheme of central carbon metabolism in microalgae. Arrows represent potential carbon fluxes. Enzymes are in bold italics. Blue arrows represent reducing power (NADPH). Red arrows represent acetyl-CoA. Black boxes denote pathway names. Neutral lipid droplets found in microalgae consist mostly of triacylglycerols (TAGs), formed by combining FAs and glycerol. ACCase acetyl-CoA carboxylase; ACD acyl-CoA dehydrogenase; ACL ATP-citrate lyase; ACS acyl-CoA synthetase; AGPP ADP-glucose pyrophosphorylase; AMY amylase; CA carbonic anhydrase; DGAT diacylglycerol acyltransferase; DHAP dihydroxyacetone phosphate; F1,6P fructose 1,6-bisphosphate; F6P fructose 6-phosphate; FAT fatty acyl–acyl carrier protein (ACP) thioesterase; G1P glucose 1-phosphate; G6P glucose 6-phosphate; G6PDH G6P dehydrogenase; GAP glyceraldehyde 3-phosphate; GPAT glycerol-3-phosphate acyltransferase; MAL malate; MDH malate dehydrogenase; MME NADP-malic enzyme; OAA oxaloacetate; PDC pyruvate dehydrogenase complex; PEP phosphoenolpyruvate; PEPC PEP carboxylase; PK pyruvate kinase; Ru5P ribulose 5-phosphate; Ru1,5BP ribulose 1,5-bisphosphate; RuBisCO Ru1,5BP carboxylase/oxygenase; 3-PGA 3-phosphoglycerate; 6PGDH 6-phosphogluconate dehydrogenase

Mentions: Acetyl-CoA can also be produced from sugars via citrate through ACL which cleaves cytosolic citrate to form acetyl-CoA and oxaloacetate (Fig. 1). Compared to acetate, citrate is produced continuously from the TCA cycle, thus representing a more sustained source of acetyl-CoA for FA synthesis. ACL has often been proposed to be a major rate-limiting enzyme in oleaginous heterotrophs [70, 84], and overexpression of ACL and ME in the yeast Yarrowia lipolytica reportedly resulted in a 60-fold increase in lipid yields by providing substrates for the induction of FA synthesis [85] (Table 1). In plants, ACL activity is correlated with lipid accumulation [86] and its mRNA levels coincide with peak cytosolic ACCase expression [87]. In addition, negative regulation of ACL subunit A (ACLA) reduced cuticular wax synthesis in epidermal cells of Arabidopsis [88], leading to the notion that increased acetyl-CoA supply in the cytosol may be to support the synthesis and elongation of long-chain FAs. Two species of microalgae, Nannochloropsis salina and Chlorella sp., were found to exhibit ACL activity comparable to those in oleaginous heterotrophs [89], indicating that microalgae possess ACL required to utilize citrate as a carbon source for generating acetyl-CoA. Gene expression of cytoplasmic ACL was also upregulated prior to TAG accumulation in C. desiccata, but not in low TAG accumulators such as D. tertiolecta and C. reinhardtii [83]. However, ACL activity would require an efflux of citrate from the mitochondria to the cytoplasm, effectively draining the TCA cycle of its intermediates. The contribution of cytoplasmic acetyl-CoA to FA synthesis in oleaginous microalgae also requires further validation [83]. As mitochondria functions in the light to export citrate via the citrate-oxaloacetate shuttle [90], the exported citrate could thus be exploited to produce acetyl-CoA. However, unlike yeasts where acetyl-CoA can be used directly for fatty acid synthesis in the cytosol [91], microalgae may require the import of carbon substrates into the chloroplast where FA synthesis takes place. Targeting ACL to the plastids of tobacco leaves was previously found to increase fatty acid content by 16% [92], but as the source of plastidial citrate was not identified, it is doubtful that ACL can provide acetyl-CoA for FA synthesis in the chloroplast. Subsequent biochemical and bioinformatics studies in Arabidopsis did not reveal the presence of ACL in chloroplasts [87, 93]. Hence, its cytosolic nature means that the supply of acetyl-CoA by ACL is likely to be restricted to FA synthesis within the cytosol.Fig. 1


The dilemma for lipid productivity in green microalgae: importance of substrate provision in improving oil yield without sacrificing growth
Simplified scheme of central carbon metabolism in microalgae. Arrows represent potential carbon fluxes. Enzymes are in bold italics. Blue arrows represent reducing power (NADPH). Red arrows represent acetyl-CoA. Black boxes denote pathway names. Neutral lipid droplets found in microalgae consist mostly of triacylglycerols (TAGs), formed by combining FAs and glycerol. ACCase acetyl-CoA carboxylase; ACD acyl-CoA dehydrogenase; ACL ATP-citrate lyase; ACS acyl-CoA synthetase; AGPP ADP-glucose pyrophosphorylase; AMY amylase; CA carbonic anhydrase; DGAT diacylglycerol acyltransferase; DHAP dihydroxyacetone phosphate; F1,6P fructose 1,6-bisphosphate; F6P fructose 6-phosphate; FAT fatty acyl–acyl carrier protein (ACP) thioesterase; G1P glucose 1-phosphate; G6P glucose 6-phosphate; G6PDH G6P dehydrogenase; GAP glyceraldehyde 3-phosphate; GPAT glycerol-3-phosphate acyltransferase; MAL malate; MDH malate dehydrogenase; MME NADP-malic enzyme; OAA oxaloacetate; PDC pyruvate dehydrogenase complex; PEP phosphoenolpyruvate; PEPC PEP carboxylase; PK pyruvate kinase; Ru5P ribulose 5-phosphate; Ru1,5BP ribulose 1,5-bisphosphate; RuBisCO Ru1,5BP carboxylase/oxygenase; 3-PGA 3-phosphoglycerate; 6PGDH 6-phosphogluconate dehydrogenase
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Fig1: Simplified scheme of central carbon metabolism in microalgae. Arrows represent potential carbon fluxes. Enzymes are in bold italics. Blue arrows represent reducing power (NADPH). Red arrows represent acetyl-CoA. Black boxes denote pathway names. Neutral lipid droplets found in microalgae consist mostly of triacylglycerols (TAGs), formed by combining FAs and glycerol. ACCase acetyl-CoA carboxylase; ACD acyl-CoA dehydrogenase; ACL ATP-citrate lyase; ACS acyl-CoA synthetase; AGPP ADP-glucose pyrophosphorylase; AMY amylase; CA carbonic anhydrase; DGAT diacylglycerol acyltransferase; DHAP dihydroxyacetone phosphate; F1,6P fructose 1,6-bisphosphate; F6P fructose 6-phosphate; FAT fatty acyl–acyl carrier protein (ACP) thioesterase; G1P glucose 1-phosphate; G6P glucose 6-phosphate; G6PDH G6P dehydrogenase; GAP glyceraldehyde 3-phosphate; GPAT glycerol-3-phosphate acyltransferase; MAL malate; MDH malate dehydrogenase; MME NADP-malic enzyme; OAA oxaloacetate; PDC pyruvate dehydrogenase complex; PEP phosphoenolpyruvate; PEPC PEP carboxylase; PK pyruvate kinase; Ru5P ribulose 5-phosphate; Ru1,5BP ribulose 1,5-bisphosphate; RuBisCO Ru1,5BP carboxylase/oxygenase; 3-PGA 3-phosphoglycerate; 6PGDH 6-phosphogluconate dehydrogenase
Mentions: Acetyl-CoA can also be produced from sugars via citrate through ACL which cleaves cytosolic citrate to form acetyl-CoA and oxaloacetate (Fig. 1). Compared to acetate, citrate is produced continuously from the TCA cycle, thus representing a more sustained source of acetyl-CoA for FA synthesis. ACL has often been proposed to be a major rate-limiting enzyme in oleaginous heterotrophs [70, 84], and overexpression of ACL and ME in the yeast Yarrowia lipolytica reportedly resulted in a 60-fold increase in lipid yields by providing substrates for the induction of FA synthesis [85] (Table 1). In plants, ACL activity is correlated with lipid accumulation [86] and its mRNA levels coincide with peak cytosolic ACCase expression [87]. In addition, negative regulation of ACL subunit A (ACLA) reduced cuticular wax synthesis in epidermal cells of Arabidopsis [88], leading to the notion that increased acetyl-CoA supply in the cytosol may be to support the synthesis and elongation of long-chain FAs. Two species of microalgae, Nannochloropsis salina and Chlorella sp., were found to exhibit ACL activity comparable to those in oleaginous heterotrophs [89], indicating that microalgae possess ACL required to utilize citrate as a carbon source for generating acetyl-CoA. Gene expression of cytoplasmic ACL was also upregulated prior to TAG accumulation in C. desiccata, but not in low TAG accumulators such as D. tertiolecta and C. reinhardtii [83]. However, ACL activity would require an efflux of citrate from the mitochondria to the cytoplasm, effectively draining the TCA cycle of its intermediates. The contribution of cytoplasmic acetyl-CoA to FA synthesis in oleaginous microalgae also requires further validation [83]. As mitochondria functions in the light to export citrate via the citrate-oxaloacetate shuttle [90], the exported citrate could thus be exploited to produce acetyl-CoA. However, unlike yeasts where acetyl-CoA can be used directly for fatty acid synthesis in the cytosol [91], microalgae may require the import of carbon substrates into the chloroplast where FA synthesis takes place. Targeting ACL to the plastids of tobacco leaves was previously found to increase fatty acid content by 16% [92], but as the source of plastidial citrate was not identified, it is doubtful that ACL can provide acetyl-CoA for FA synthesis in the chloroplast. Subsequent biochemical and bioinformatics studies in Arabidopsis did not reveal the presence of ACL in chloroplasts [87, 93]. Hence, its cytosolic nature means that the supply of acetyl-CoA by ACL is likely to be restricted to FA synthesis within the cytosol.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Rising oil prices and concerns over climate change have resulted in more emphasis on research into renewable biofuels from microalgae. Unlike plants, green microalgae have higher biomass productivity, will not compete with food and agriculture, and do not require fertile land for cultivation. However, microalgae biofuels currently suffer from high capital and operating costs due to low yields and costly extraction methods. Microalgae grown under optimal conditions produce large amounts of biomass but with low neutral lipid content, while microalgae grown in nutrient starvation accumulate high levels of neutral lipids but are slow growing. Producing lipids while maintaining high growth rates is vital for biofuel production because high biomass productivity increases yield per harvest volume while high lipid content decreases the cost of extraction per unit product. Therefore, there is a need for metabolic engineering of microalgae to constitutively produce high amounts of lipids without sacrificing growth. Substrate availability is a rate-limiting step in balancing growth and fatty acid (FA) production because both biomass and FA synthesis pathways compete for the same substrates, namely acetyl-CoA and NADPH. In this review, we discuss the efforts made for improving biofuel production in plants and microorganisms, the challenges faced in achieving lipid productivity, and the important role of precursor supply for FA synthesis. The main focus is placed on the enzymes which catalyzed the reactions supplying acetyl-CoA and NADPH.

No MeSH data available.


Related in: MedlinePlus