Cyanotech Will Produce Catalytic Antibodies in Microalgae

Cyanotech Corp. (Kailua-Kona, HI), which manufactures microalgae products for nutritional and immunodiagnostics markets, has agreed to produce patented aldolase catalytic antibody 38C2 in microalgae for The Scripps Institute (La Jolla, CA). The deal puts microalgae on the map: If successful, it will establish algae as viable microorganisms for serious chemical and pharmaceutical manufacturing. Cyanotech believes that Microalgae, a diverse group of over 30,000 species of microscopic plants, represent a largely unexploited, renewable source of chemicals through genetic engineering.

Carbon-Carbon Bond Formation

Aldolases, a group of naturally occurring enzymes, catalyze the aldol reaction, an important carbon-carbon bond-forming process widely used in making pharmaceuticals and fine chemicals. Classically, the aldol reaction joins two aldehydes, two ketones, or an aldehyde and a ketone to form hydroxyaldehydes or hydroxyketones. These products may also be dehydrated non-enzymatically, either spontaneously or by adding acid, to form new carbon-carbon double bonds. Industrially, aldol reactions use catalytic base and heat, and offer little control over the final product. Enzymatic aldol synthesis permits mild reaction conditions and - perhaps most important - control over regiochemistry and stereochemistry.

Figure 1: The simplest aldol reaction, between two molecules of acetaldehyde. More complex structures are available by changing the carbonyl component

Natural aldolase catalysts are highly restricted in the substrates they accept, but Scripps' catalytic antibody accepts hundreds of aldehyde-ketone combinations. The new enzyme also catalyzes the retro-aldol reaction, which forms two carbonyl compounds from a hydroxyketone or hydroxyaldehyde.

Catalytic Antibodies

Catalytic antibodies are artificial, enzyme-like proteins generated by inoculating an animal with a stable, synthetic, transition state analog of the desired reaction. Antibodies generated in this fashion stabilize (lower the energy of) the transition state, thereby accelerating the reaction. Obtaining research quantities of catalytic antibodies is relatively straightforward. Producing them in quantity requires sequencing the protein/gene, inserting the gene in a suitable organism, growing the organism, and purifying the product. For large-scale production, fermentation and plant-based expression systems are much preferred over animals for yield and cost considerations.

Pioneered by Peter Schultz at the University of California, Berkeley, catalytic antibodies often showed low turnover rates compared with natural enzymes. Aldolase 38C2 is the first catalytic antibody whose turnover approaches that of natural aldolases, making it the first industrially significant immunologically-derived enzyme.

Why Microalgae?

The idea of expressing the aldolase gene in microalgae originated at Scripps. "We read in Chemical and Engineering News about Scripps' work in microalgae," said Cyanotech President Gerry Cysewski, "and we immediately saw a fit between the Institute's need for making large quantities of their aldolase and our large-scale production capabilities. We knew that expressing and harvesting proteins from microalgae is similar to techniques used to with mammalian cell culture or fermentations. As usual, the biggest hurdle is getting the right genetic sequence into the cell."

Scripps has supplied Cyanotech with live samples of Chlamydomonas microalgae species in which the aldolase genes are already expressed. The company will grow the algae on its 90-acre "plant," purify the product, and supply it to Scripps for further work or for sale through specialty chemical dealers.

Figure 2: Separation screen for processing microalgae

Because of their need for large quantities of product, Scripps originally looked into producing aldolase in photosynthetic plants. The idea was to insert the aldolase gene into chloroplasts of soybeans or other plants. But this process normally takes at least two years: After generating the transgenic plant the seeds are harvested and, if all goes well, after about two years daughter crops are large enough to meet early production needs.

"With microalgae the same process takes about 2 months," said Cysewski. "Genes are inserted with a particle gun directly into the chloroplasts, which become the production engine for the desired protein. And unlike related techniques using other microorganisms, genes may be used 'as is' in microalgae because these organisms exhibit no codon bias. That is, they expressed implanted genes directly into the desired protein without the need for manipulating the gene beforehand. This makes microalgae a very exciting expression system for humanized antibodies."

Unlike terrestrial plants, which offer at best three growing seasons (generations) per year, microalgae reach the end of their cycle in about one week, so it's possible to go from a culture that fits into a test tube to about 250 liters of cultured microalgae in about one month. Scaleup is also easier in microalgae than in fermentations, according to Cysewski. "Getting to any production target, up to 5000 liters, is as easy as growing the algae," he told Pharmaceutical Online. "For the Scripps project I think we're going to stop at around 500 liters."

Yield and purification from cultured microalgae are comparable to fermentations. Cyanotech has developed "very straightforward" methods for purifying fluorescent pigments from algae using cell lysing, centrifugation, and column chromatography. Microalgae are very good producers, moreover, containing up to 60% by weight of product proteins, a yield Cysewski says is comparable to quantities obtained from yeast. "We look at this as the first of what we hope will be many such deals. We believe microalgae can be sources of a wide array of genetically engineered products," he said.

Microalgae Products

Cysewski describes Cyanotech as "a microalgae company" with three general product lines: a nutritional supplement, Spirulina; phycobiliproteins, fluorescent markers used for medical diagnostics and biotech research; astaxanthin, the pink pigment in salmon used to improve color in aquacultured fish and seafood.

Figure 3: Spirulina growing pond.

Cyanotech was the first producer of natural astaxanthin, which competes against a synthetic material produced by Hoffman LaRoche. Astaxanthin is a $150 million (and growing) annual market.

Figure 4: Cyanotech's astaxanthin growing pond.

For more information: Ronald P. Scott, EVP-Finance & Administration, Cyanotech, 73-4460 Queen Kaahamanu Hwy., Suite 102, Kailua-Kona, HI 96740. Tel: 808-326-1353. Fax: 808-329-4533.

By Angelo DePalma