Robert E. Jinkerson

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.

Ultrastructure and Composition of the Nannochloropsis gaditana Cell Wall

ABSTRACT Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.

Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H2 production

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.

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.

Toward a photosynthetic microbial platform for terpenoid engineering.

Plant terpenoids are among the most diverse group of naturally-occurring organic compounds known, and several are used in contemporary consumer products. Terpene synthase enzymes catalyze complex rearrangements of carbon skeleton precursors to yield thousands of unique chemical structures that range in size from the simplest five carbon isoprene unit to the long polymers of rubber. Such chemical diversity has established plant terpenoids as valuable commodity chemicals with applications in the pharmaceutical, neutraceutical, cosmetic, and food industries. More recently, terpenoids have received attention as a renewable alternative to petroleum-derived fuels and as the building blocks of synthetic biopolymers. However, the current plant- and petrochemical-based supplies of commodity terpenoids have major limitations. Photosynthetic microorganisms provide an opportunity to generate terpenoids in a renewable manner, employing a single consolidated host organism that is able to use solar energy, H2O and CO2 as the primary inputs for terpenoid biosynthesis. Advances in synthetic biology have seen important breakthroughs in microbial terpenoid engineering, traditionally via fermentative pathways in yeast and Escherichia coli. This review draws on the knowledge obtained from heterotrophic microbial engineering to propose strategies for the development of microbial photosynthetic platforms for industrial terpenoid production. The importance of utilizing the wealth of genetic information provided by nature to unravel the regulatory mechanisms of terpenoid biosynthesis is highlighted.

Improving biofuel production in phototrophic microorganisms with systems biology

Biofuels derived from algal energy carriers, including lipids, starch and hydrogen, offer a promising, renewable alternative to fossil fuels. Unfortunately, native algal metabolisms are not optimized for the accumulation of these renewable bioenergy carriers. Systems biology, which includes genomics, transcriptomics, proteomics, metabolomics and lipidomics, can inform and provide key insights to advance algal strain development for biotechnological applications. Recent advances in analytical technologies have enabled these sophisticated, high-throughput, holistic ‘omics’ techniques to generate highly accurate and quantitative datasets that can be leveraged to improve biofuel phenotypes in phototrophic microorganisms. The study of algal genomes and transcriptomes allows for the identification of genes, metabolic pathways and regulatory networks. Investigations of algal proteomes reveal protein levels, locations and post-translational modifications, while study of the metabolome reveals metabolite fluxes and intermediates. All of these systems-biology tools are integral for investigating algal metabolism from the whole-cell perspective. This review focuses on how systems biology has been applied to studying metabolic networks in algae and cyanobacteria, and how these technologies can be used to improve bioenergy-carrier accumulation.