The work of Konrad Bloch's laboratory on unsaturated fatty acid biosynthesis in bacteria

Lipids are ubiquitous molecules, serving as the structural building blocks for membranes, messengers in cell signaling, or compounds for energy storage. Their functional versatility relies in part on the presence of one or more double bonds in the long lipid carbon chains. The more double bonds there are in a lipid, the more unsaturated it is; and the more unsaturated lipids there are in a membrane, the more flexible the membrane tends to be. Cells take advantage of this property by fine-tuning the levels of saturated and unsaturated fatty acids in membranes to external conditions and required membrane function.

Many organisms, including humans, produce unsaturated fatty acids by desaturating saturated fatty acids, as Rudolf Schoenheimer and David Rittenberg reported in the 1930s working with mammalian cells (1) and Konrad Bloch and Daniel Bloomfield showed in the 1960s studying yeast (2). When it comes to making unsaturated fatty acids, cells growing in the presence of molecular oxygen have an advantage; they can use this powerful oxidant to insert a double bond into a fatty acid chain via a dehydrogenation reaction. But what about organisms that cannot grow in the presence of molecular oxygen?

This question piqued the curiosity of Bloch, who had a longstanding interest in oxygen as a biosynthetic reagent and was also aware that molecular oxygen is toxic to some microorganisms. In the 1950s, he had taken a summer course taught by the eminent microbiologist Cornelius van Niel. The course acquainted Bloch with many microorganisms, including obligately anaerobic bacteria that thrive only in the absence of oxygen (3).

Howard Goldfine worked with Bloch on bacterial fatty acid biosynthesis and is now emeritus professor of microbiology at the University of Pennsylvania.

“[The course] caused him to wonder what goes on in anaerobes, because obviously, they could not use molecular oxygen [for making unsaturated fatty acids],” Goldfine says.

Bloch sought to answer this question by studying fatty acid biosynthesis in two species of anaerobic bacteria, Clostridium kluyveri and Clostridium butyricum (strain 6015, now known as Clostridium beijerinckii).

“Clostridia are present anywhere where there's no oxygen,” Goldfine says. “If you look in mud several inches below the surface, you'll find clostridia, and they're happily growing.”

The work of Bloch, Goldfine, and colleagues on unsaturated fatty acid biosynthesis in clostridia resulted in two JBC papers, recognized as Classics here (4, 5). The studies reported that C. kluyveri and C. butyricum 6015 (C. beijerinckii) produce unsaturated fatty acids via a biosynthetic route distinct from that present in aerobic cells.

Goldfine's association with Bloch began through their shared interest in anaerobic metabolism. Goldfine had worked on anaerobic bacteria in Earl Stadtman's lab at the National Heart Institute of the National Institutes of Health, prompting Bloch to explore whether Goldfine might be interested in working with him.

“He was coming down to NIH for a study section, and he asked if we could have lunch together, and then he asked, would I be interested in coming to visit his lab?” Goldfine remembers.

Goldfine was familiar with Bloch's work on cholesterol and fatty acids, for which Bloch won the Nobel prize in physiology and medicine with Feodor Lynen in 1964 (6). So Goldfine enthusiastically jumped at the opportunity, resulting in a 3-year stay in the Bloch laboratory.

“I was really very, very fortunate to have had the opportunity to work with Bloch, because he was a man of really great depth and understanding,” Goldfine says. “It was a turning point in my career.”

Goldfine's expertise was soon tested: removing all oxygen from the experimental system to study unsaturated fatty acid formation in clostridia posed a challenge all its own. Initially, Goldfine tried to grow the bacteria in a helium atmosphere in a vacuum desiccator, “but that did not completely remove oxygen, because there could be a trace of oxygen in the helium,” he recalls.

So Goldfine resorted to a chemical trick and skillful lab acrobatics. He poured a 5% potassium carbonate solution into the desiccator base, above which he placed the flasks holding the bacteria in growth media, along with a beaker containing pyrogallic acid. After sealing and helium-purging the desiccator, Goldfine gingerly tilted it to tip the pyrogallol into the potassium carbonate solution. The resulting mixture then removed any traces of oxygen from the helium-filled desiccator.

Tools such as mass spectrometry to analyze the bacterial lipids were not readily available at the time. Instead, the researchers relied on Bloch's longstanding expertise in using ¹⁴C labeling and detection. After feeding the ¹⁴C-labeled lipid precursor acetate to the bacterial cultures, Bloch and Goldfine extracted the lipids from the cells, used gas chromatography to separate fatty acids, identified them with standards, and assessed them for ¹⁴C labeling in a scintillation counter.

In the first study (4), Goldfine and Bloch reported incorporation of two-thirds of the ¹⁴C-labeled acetate into saturated lipids and one-third into monounsaturated lipids, showing that bacteria can produce unsaturated fatty acids in the absence of molecular oxygen.

This finding raised the question how the clostridia could create double bonds in fatty acids. One possibility was that the bacterial cells possessed the means to desaturate a bond in an existing saturated fatty acid, as previously found in other organisms, but by using an oxidant other than molecular oxygen. Or the clostridia might deploy a different, yet unknown mechanism to generate a lipid double bond.

To test these possibilities, Goldfine and Bloch fed a series of ¹⁴C-labeled saturated fatty acids ranging in length from 8 to 18 carbons to the cells. They found that the clostridia do not make unsaturated fatty acids by desaturating bonds in their saturated counterparts. Instead, they produce a single double bond during elongation of the saturated fatty acids octanoic acid (C8) and decanoic acid (C10), resulting in long-chain (C12 to C18) monounsaturated fatty acids.

Interestingly, the clostridia did not use saturated fatty acid chains longer than 10 carbons for making unsaturated fatty acids. This indicated that these bacteria use short- and medium-chain fatty acids (up to 8 or 10 carbons long) as precursors of both saturated and unsaturated fatty acids through the same biochemical route, but make longer-chain saturated and unsaturated fatty acids via two distinct pathways.

Peering into the clostridial lipid products a little more, Bloch and colleagues saw something odd. “C. beijerinckii [presented] an additional problem because the unsaturated fatty acids have two isomers,” says Goldfine. The unsaturated fatty acid hexadecenoic acid isomers had a double bond either at the C7 or C9 (Δ⁷ or Δ⁹) position, and octadecenoic acid isomers either at C9 or C11 (Δ⁹ or Δ¹¹).

To find out how these isomers are made, and as described in the second JBC paper (5), Bloch, Goldfine, and another member of the Bloch lab, Günter Scheuerbrandt, fed ¹⁴C-labeled octanoic and decanoic acids to C. beijerinckii and again measured ¹⁴C incorporation into hexadecenoic and octadecenoic acids. They tweaked their procedure to include exposure of the unsaturated fatty acids to potassium metaperiodate-potassium permanganate (KIO₄–KMnO₄), a strong oxidant that breaks lipid double bonds. This enabled them to trace the origins of both ends of the unsaturated products and home in on the mechanism that creates the different fatty acid isomers.

“I distinctly remember, Paul Baronowsky—he was a graduate student at the time—standing at the blackboard at the end of the lab writing that pathway,” Goldfine says. “'This is how you get the double bond here. And if you do it with another precursor, you get the double bond there.' He drew it all out beautifully.”

Their carefully designed experiments and analyses revealed that the individual isomers do not arise from interconversions among them. Instead, the researchers found that octanoic acid is the precursor to the long-chain fatty acids having a double bond at the C9 or C11 position and decanoic acid is the precursor to those with a double bond at the C7 or C9 position (Fig. 1).

Figure 1.

Medium-chain saturated fatty acids are precursors to unsaturated long-chain fatty acids in clostridia. Bloch and colleagues fed ¹⁴C-labeled octanoic acid (the ¹⁴C-labeled carbon of the carboxyl group is marked by a blue asterisk) to C. beijerinckii cultures. They treated the unsaturated fatty acids extracted from the cultures with a strong oxidant to break their double bonds; the lengths of the resulting dicarboxylic acid (C9 dicarboxylic acid in this case) and monocarboxylic acid (C7 monocarboxylic acid) revealed the location of the double bond (i.e. at the C9 position of hexadecanoic acid, indicating that the double bond was created immediately after a single chain-elongation step). Adapted from Fig. 1 in Ref. 5.

The authors' findings supported an earlier proposal by famed chemist Klaus Hofmann at the University of Pittsburgh. Working with lactobacilli, Hofmann and colleagues had speculated that bacteria may produce long-chain unsaturated fatty acids by chain elongation from a short-chain precursor (7).

On the basis of their published and unpublished findings, Bloch and colleagues proposed that in clostridia and several other bacteria, including lactobacilli and Escherichia coli, the long-chain unsaturated fatty acids are produced by an anaerobic pathway involving elongation of octanoic acid or decanoic acid, immediately followed by a β,γ elimination of water from a hydroxy acid intermediate and further elongation of the lipid chain without reduction of the double bond (5).

Further studies in the Bloch lab, done with E. coli (8–10), focused on the bacterial enzymes involved in fatty acid chain elongation and double bond formation. This work culminated in two papers confirming that the anaerobic bacterial pathway indeed involves enzymatic dehydration of medium-chain β-hydroxy acids rather than oxidative dehydrogenation of a saturated acid (11, 12).

Thus, innovative rejiggering of lab equipment, foundational biochemistry methods, and several incisive blackboard sketches yielded results that uncovered how anaerobes make unsaturated fatty acids and that have stood the test of time to this day.

“The pathway that is in the second JBC [Classics] paper has held up for 60 years, it's pretty astonishing,” concludes Goldfine.