Randor Radakovits

Genetic Engineering of Algae for Enhanced Biofuel Production

ABSTRACT There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H2 yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H2 production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana

The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga.

Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains

ABSTRACT The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O2 evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O2 evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.

Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum.

Renewable diesel surrogates made from shorter chain length fatty acids have improved cold flow properties. Acyl-ACP thioesterases specific for shorter chain length fatty acids are therefore of considerable interest in the genetic engineering of biofuel producing organisms, both for their ability to increase the production of shorter fatty acids, and for their involvement in fatty acid secretion in bacterial systems. Here we show that the heterologous expression of two thioesterases, biased towards the production of lauric (C12:0) and myristic acid (C14:0), causes increased accumulation of shorter chain length fatty acids in the eukaryotic microalga Phaeodactylum tricornutum. Accumulation of shorter chain length fatty acids corresponds to transgene transcript levels. We achieved levels of C12:0 of up to 6.2% of total fatty acids and C14:0 of up to 15% by weight. Unlike observations in cyanobacteria, no significant secretion of fatty acids was observed. Instead, we found that 75-90% of the shorter chain length fatty acids produced was incorporated into triacylglycerols. Our results demonstrate that overexpression of thioesterases is a valid way to improve the biofuel production phenotype of eukaryotic microalgae.

Improving photosynthesis and metabolic networks for the competitive production of phototroph-derived biofuels.

To improve bioenergy production from photosynthetic microorganisms it is necessary to optimize an extensive network of highly integrated biological processes. Systematic advances in pathway engineering and culture modification have resulted in strains with increased yields of biohydrogen, lipids, and carbohydrates, three bioenergy foci. However, additional improvements in photosynthetic efficiency are necessary to establish a viable system for biofuel production. Advances in optimizing light capture, energy transfer, and carbon fixation are essential, as the efficiencies of these processes are the principal determinants of productivity. However, owing to their regulatory, catalytic, and structural complexities, manipulating these pathways poses considerable challenges. This review covers novel developments in the optimization of photosynthesis, carbon fixation, and metabolic pathways for the synthesis of targeted bioenergy carriers.

Genomic insights from the oleaginous model alga Nannochloropsis gaditana.

Nannochloropsis species have emerged as leading phototrophic microorganisms for the production of biofuels. Several isolates produce large quantities of triacylglycerols, grow rapidly, and can be cultivated at industrial scales. Recently, the mitochondrial, plastid and nuclear genomes of Nannochloropsis gaditana were sequenced. Genomic interrogation revealed several key features that likely facilitate the oleaginous phenotype observed in Nannochloropsis, including an over-representation of genes involved in lipid biosynthesis. Here we present additional analyses on gene orientation, vitamin B12 requiring enzymes, the acetyl-CoA metabolic node, and codon usage in N. gaditana. Nuclear genome transformation methods are established with exogenous DNA integration occurring via either random incorporation or by homologous recombination, making Nannochloropsis amenable to both forward and reverse genetic engineering. Completion of a draft genomic sequence, establishment of transformation techniques, and robust outdoor growth properties have positioned Nannochloropsis as a new model alga with significant potential for further development into an integrated photons-to-fuel production platform.

The production of the sesquiterpene β-caryophyllene in a transgenic strain of the cyanobacterium Synechocystis.

The plant secondary metabolite, β-caryophyllene, is a ubiquitous component of many plant resins that has traditionally been used in the cosmetics industry to provide a woody, spicy aroma to cosmetics and perfumes. Clinical studies have shown it to be potentially effective as an antibiotic, anesthetic, and anti-inflammatory agent. Additionally, there is significant interest in engineering phototrophic microorganisms with sesquiterpene synthase genes for the production of biofuels. Currently, the isolation of β-caryophyllene relies on purification methods from oleoresins extracted from large amounts of plant material. An engineered cyanobacterium platform that produces β-caryophyllene may provide a more sustainable and controllable means of production. To this end, the β-caryophyllene synthase gene (QHS1) from Artemisia annua was stably inserted, via double homologous recombination, into the genome of the cyanobacterium Synechocystis sp. strain PCC6803. Gene insertion into Synechocystis was confirmed through PCR assays and sequencing reactions. Transcription and expression of QHS1 were confirmed using RT-PCR, and synthesis of β-caryophyllene was confirmed in the transgenic strain using GC-FID and GC-MS analysis.

Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A.

Abstract[FeFe]-hydrogenases (HYDA) link the production of molecular H2 to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H2-photoproduction activity observed is as O2 sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.

Corrigendum: Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana.

Corrigendum: Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana