Metabolic Engineering of Cyanobacteria for Direct Conversion of CO2 to Hydrocarbon Biofuels

Christer Jansson

Contents

1 Introduction

2 Fatty Acid Metabolism in Cyanobacteria

3 Biosynthesis of Hydrocarbons

82 83 85 85 87

3.1 Alkanes

3.2 Isoprenoids

4 Conclusions and Perspectives References

Abstract Cyanobacteria are oxygenic photosynthesizers like plant and algae and hence can capture CO2 via the Calvin cycle and convert it to a suite of organic compounds. They are Gram-negative bacteria and are well suited for synthetic biology and metabolic engineering approaches for the phototrophic production of various desirable biomolecules, including ethanol, butanol, biodiesel, and hydrocarbon biofuels. Phototrophic biosynthesis of high-density liquid biofuels in cyanobacteria would serve as a good complement to the microbial production of biodiesel and hydrocarbons in heterotrophic bacteria such as Escherichia coli. Two groups of hydrocarbon biofuels that are being considered in microbial production systems are alkanes and isoprenoids. Alkanes of defined chain lengths can be used as drop-in fuel similar to gasoline and jet fuel. Many cyanobacteria synthesize alkanes, albeit in minute quantities. Optimizing the expression of the alkane biosynthesis genes and enhancing the carbon flux through the fatty acid and alkane biosynthesis pathways should lead to the accumulation and/or secretion of notable amounts of alkanes. It also becomes important to understand how to control the chain lengths of the produced alkane molecules. Isoprenoids, e.g., the monoterpene pinene and the sesquiterpene farnesene, are considered precursors for future

Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA e-mail: [email protected]

U. Luttge et al. (eds.), Progress in Botany Vol. 73, Progress in Botany 73, 81

DOI 10.1007/978-3-642-22746-2_3, © Springer-Verlag Berlin Heidelberg 2012

biodiesel or next-generation jet fuel. Cyanobacteria produce carotenoids and extending the carotenoid biosynthetic pathways by the introduction of constructs for appropriate terpene synthases should allow the biosynthesis of selected mono-and sesquiterpenes.

1 Introduction

Photosynthetic organisms offer the potential to convert sunlight and CO2 directly to transportation fuels, bypassing the need for biomass deconstruction. This is a contrast to approaches for biofuel production that require either conversion of resistant feedstock (lignocellulose) to sugars for fermentation, or lipid extraction, isolation, and transesterification. Cyanobacteria and eukaryotic microalgae (often collectively referred to as "algae" or "microalgae") hold particular interest in the biofuel sector since they can tolerate high CO2 levels such as flue gas streams (Ono and Cuello 2007; Li et al. 2008; Jansson and Northen 2010), and may be superior to higher plants in energy efficiency, biomass and oil productivity, and land and water usage (Chisti 2007, 2008; Gressel 2008; Hu et al. 2008; Packer 2009; Sheehan 2009; Costa and de Morais 2011; Lu 2010; Radakovits et al. 2010; Scott et al. 2010; Ono and Cuello 2007; Li et al. 2008; Clarens et al. 2010). Since a large number of cyanobacterial and microalgal species are halophilic, they can be grown in seawa-ter, saline drainage water, or brine from the petroleum production and refining industry or CO2 injection sites, thereby sparing freshwater supplies. The use of potable water for cultivation can also be avoided by utilizing municipal wastewater as a nutrient source. Additionally, cyanobacteria occupy a wide range of extreme environments such as hot springs, deserts, bare rocks, and permafrost zones, and they are exposed to the highest rates of UV irradiance known on the Earth. The thermophilic nature of many cyanobacteria, as well as microalgae, allows them to tolerate high temperatures characteristic of flue gas.

Cyanobacteria are photosynthetic Gram-negative bacteria that perform oxygenic photosynthesis similar to plants and algae. As opposed to microalgae that can accumulate large amounts of triacylglycerols (TAGs) as storage lipids, the cyanobacteria studied to date produce little or no TAGs, but their fatty acids (FAs) are directly shuttled to membrane lipid synthesis (see further below). Conversely, cyanobacteria are well suited for approaches aimed at redirecting carbon flux in lipid metabolism to specific biofuel molecules. First, whereas in plants and algae, including microalgae, lipid metabolism involves several different cellular compartments, in cyanobacteria, all metabolism occurs via soluble or membrane-bound enzymes in the cytoplasm. Second, being bacteria, cyanobacteria are amenable to homologous recombination, which allows rapid site-directed mutagenesis, gene insertions, replacements, and deletions in a precise, targeted and predictable manner.

Two major groups of hydrocarbon biofuels being considered in microbial production systems are FA-based products like alkanes and alkenes, and isoprenoids such as monoterpenes and sesquiterpenes(Fortman et al. 2008; Keasling and Chou 2008; Kirby and Keasling 2008; Lennen et al. 2010; Lu 2010; Steen et al. 2010). In the following, we will focus on pathway engineering in cyanobacteria for the biosynthesis of alkanes from free FAs and of selected terpenes.

2 Fatty Acid Metabolism in Cyanobacteria

Fatty acid (FA) synthesis in cyanobacteria and other bacteria is accomplished by a type II FAS (FASII), a multienzyme system, utilizing a freely dissociable acyl carrier protein ACP (Campbell and Cronan 2001) (Fig. 1). The FASII enzymes comprise acetyl-CoA carboxylase (ACC; EC 6.4.1.2); malonyl-CoA:ACP transacylase (FabD; 2.3.1.39); three condensing enzymes, B-ketoacyl-ACP synthase I (FabB; EC 2.3.1.41), B-ketoacyl-ACP synthase II (FabF; EC 2.3.1.41), and B-ketoacyl-ACP synthase III (FabH; EC 2.3.1.180) required for initiation of FA synthesis; B-ketoacyl-ACP reductase (FaBG; EC 1.1.1.100); B-hydroxyacyl-ACP dehydratase/isomerase (FabA and FabZ); and enoyl-ACP reductase I (FabI; EC 1.3.1.9). The products of FASII are released as acyl-ACPs and may be directly incorporated into membrane lipids by two acyltransferases, glycerol-3-phosphate O-acyltransferase (GPAT or PlsB; EC 2.3.1.15) and 1-acylglycerol-3-phosphate O-acyltransferase, also called

Membrane lipids

FA acyl-ACP

FA acyl-ACP

I 11 ,CoA Malonyl-CoA

ACP-v

FabD

NADH

FabD

NADH

lbZ FabG / NADPH

Enoyl-ACP

Enoyl-ACP

Fig. 1 Fatty acid synthesis in cyanobacteria. See text for abbreviations lbZ FabG / NADPH

Acetoacetyl-ACP (a p-Ketoacyl-ACP)

Fig. 1 Fatty acid synthesis in cyanobacteria. See text for abbreviations lysophosphatidic acid acyltransferase (AGPAT, LPAT, or PlsC; EC 2.3.1.51) that each attaches an FA to the glycerol 3-phosphate (G3P) backbone to form the key intermediate, phosphatidic acid (PA) (Voelker and Kinney 2001; Cronan 2003).

In plants and algae, de novo FA synthesis occurs in the plastids, which also exhibit the FASII machinery (Jones et al. 1995; Voelker and Kinney 2001; Thelen and Ohlrogge 2002). In the plastids, acyl-ACPs are hydrolyzed by acyl-ACP thioesterases (TE; EC 3.1.2.14, e.g., FatB in Arabidopsis) to yield free FAs for transport across the plastid envelope (Jones et al. 1995; Bonaventure et al. 2003). Upon arrival at the outer plastid surface, the free FAs are re-activated by acyl-CoA synthetase or long-chain-FA-CoA ligase (FadD; EC 6.2.1.3) to form acyl-CoA. Acyl-CoA is the starting substrate for synthesis of TAGs but can also be used for B-oxidation and for synthesis of membrane lipids (Bonaventure et al. 2003). Most bacteria lack intracellular TEs that act on FA-ACPs, and formation of free FAs mainly occurs during recycling of membrane lipids or degradation of acylated proteins. Escherichia coli and other bacteria that can take up and metabolize exogenous FAs possess periplasmic TEs, e.g., TesA in E. coli (Cho and Cronan 1994) that liberate FAs for import. Heterologous expression of plant TEs in bacteria can yield high production of free FAs (Voelker and Davies 1994; Jones et al. 1995; Yuan et al. 1995; Jha et al. 2006; Lu et al. 2008). The concomitant decrease in acyl-ACP levels also relieves the rigorous feedback inhibition of ACC exerted by this end product. Thus, expression of plant TEs in bacteria has the dual effect of producing free FA and enhancing FA synthesis [greater than fivefold; Khosla 2008].

Cyanobacteria seem to lack TE enzymes and the generation of free FAs occurs during recycling of degraded membrane lipids (Figs. 1 and 2). In E. coli, which can

Cyanobacteria Fig
Fig. 2 Proposed pathway(s) for alkane synthesis in cyanobacteria. FadR (ACP), FadR accepting FA-ACP rather than FA-Acyl-CoA as a substrate. See text for other abbreviations

take up and metabolize exogenous FAs, the incoming FAs are activated to acyl-CoA by the soluble but plasma membrane-associated FadD for subsequent utilization in ß-oxidation (Yoo et al. 2001; Kaczmarzyk and Fulda 2010). Uptake of FAs is not a normal modus operandi for cyanobacteria but they are able to import both free FAs and complex lipids and incorporate the exogenous FAs in their membranes (Hagio et al. 2000; Kaczmarzyk and Fulda 2010). Together with their capacity for alkane biosynthesis, this indicates the presence of an activating enzyme like FadD in cyanobacteria. Cyanobacteria most likely lack FadD but the FA-activating process is instead provided by an Acyl-ACP synthetase or long-chain-FA-ACP ligase (AAS; EC 6.2.1.20) (Kaczmarzyk and Fulda 2010; Lu 2010; Schirmer et al. 2010).

Biosynthesis of free FAs is of interest since they can be used for downstream chemical processing to biofuels (Lennen et al. 2010) and also because cyanobacterial biosynthesis of alkanes with specified chain lengths may require free FAs as an intermediate metabolite (Fig. 2).

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