Stephen B. del Cardayre

Microbial production of fatty-acid-derived fuels and chemicals from plant biomass.

Increasing energy costs and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals. Major efforts to this end are focused on the microbial production of high-energy fuels by cost-effective ‘consolidated bioprocesses’. Fatty acids are composed of long alkyl chains and represent nature’s ‘petroleum’, being a primary metabolite used by cells for both chemical and energy storage functions. These energy-rich molecules are today isolated from plant and animal oils for a diverse set of products ranging from fuels to oleochemicals. A more scalable, controllable and economic route to this important class of chemicals would be through the microbial conversion of renewable feedstocks, such as biomass-derived carbohydrates. Here we demonstrate the engineering of Escherichia coli to produce structurally tailored fatty esters (biodiesel), fatty alcohols, and waxes directly from simple sugars. Furthermore, we show engineering of the biodiesel-producing cells to express hemicellulases, a step towards producing these compounds directly from hemicellulose, a major component of plant-derived biomass.

Microbial Biosynthesis of Alkanes

Toward Alkane Synthesis Alkanes are major components of fossil fuels, and synthesis of alkanes remains a challenge in the conversion of renewable raw materials to fuels. Even though diverse organisms synthesize alkanes, synthesis pathways have remained elusive. Now Schirmer et al. (p. 559) describe an alkane biosynthesis pathway in cyanobacteria that converts intermediates of fatty acid metabolism to alkanes and alkenes. Heterologous expression of the biosynthetic genes resulted in production of alkanes in Escherichia coli. This pathway is likely to be a valuable tool in the production of biofuels. Alkane biosynthesis genes were identified in cyanobacteria and engineered into Escherichia coli for recombinant hydrocarbon production. Alkanes, the major constituents of gasoline, diesel, and jet fuel, are naturally produced by diverse species; however, the genetics and biochemistry behind this biology have remained elusive. Here we describe the discovery of an alkane biosynthesis pathway from cyanobacteria. The pathway consists of an acyl–acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes. The aldehyde decarbonylase is related to the broadly functional nonheme diiron enzymes. Heterologous expression of the alkane operon in Escherichia coli leads to the production and secretion of C13 to C17 mixtures of alkanes and alkenes. These genes and enzymes can now be leveraged for the simple and direct conversion of renewable raw materials to fungible hydrocarbon fuels.

Genome shuffling leads to rapid phenotypic improvement in bacteria.

For millennia, selective breeding, on the basis of biparental mating, has led to the successful improvement of plants and animals to meet societal needs. At a molecular level, DNA shuffling mimics, yet accelerates, evolutionary processes, and allows the breeding and improvement of individual genes and subgenomic DNA fragments. We describe here whole-genome shuffling; a process that combines the advantage of multi-parental crossing allowed by DNA shuffling with the recombination of entire genomes normally associated with conventional breeding. We show that recursive genomic recombination within a population of bacteria can efficiently generate combinatorial libraries of new strains. When applied to a population of phenotypically selected bacteria, many of these new strains show marked improvements in the selected phenotype. We demonstrate the use of this approach through the rapid improvement of tylosin production from Streptomyces fradiae. This approach has the potential to facilitate cell and metabolic engineering and provide a non-recombinant alternative to the rapid production of improved organisms.

Genome shuffling of Lactobacillus for improved acid tolerance.

Fermentation-based bioprocesses rely extensively on strain improvement for commercialization. Whole-cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as temperature, pH, and solute concentration. Rational approaches to improving such complex phenotypes lack good models and are especially difficult to implement without genetic tools. Here we describe the use of genome shuffling to improve the acid tolerance of a poorly characterized industrial strain of Lactobacillus. We used classical strain-improvement methods to generate populations with subtle improvements in pH tolerance, and then shuffled these populations by recursive pool-wise protoplast fusion. We identified new shuffled lactobacilli that grow at substantially lower pH than does the wild-type strain on both liquid and solid media. In addition, we identified shuffled strains that produced threefold more lactic acid than the wild type at pH 4.0. Genome shuffling seems broadly useful for the rapid evolution of tolerance and other complex phenotypes in industrial microorganisms.

Terminal olefin (1-alkene) biosynthesis by a novel p450 fatty acid decarboxylase from Jeotgalicoccus species.

ABSTRACT Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleTJE), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleTJE is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in α- and/or β-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family.

Directed Evolution and Biocatalysis

This review describes the current state of biocatalysis in the chemical industry. Although we recognize the advantages of chemical approaches, we suggest that the use of biological catalysis is about to expand dramatically because of the recent developments in the artificial evolution of genes that code for enzymes. For the first time it is possible to consider the rapid development of an enzyme that is designed for a specific chemical reaction. This technology offers the opportunity to adapt the enzyme to the needs of the process. We describe herein the development of enzyme evolution technology and particularly DNA shuffling. We also consider several classes of enzymes, their current applications, and the limitations that should be addressed. In a review of this length it is impossible to describe all the enzymes with potential for industrial exploitation; there are other classes, which given appropriate activity, selectivity, and robustness, could become useful tools for the industrial chemist. This is an exciting era for biocatalysis and we expect great progress in the future.

Molecular breeding: the natural approach to protein design.

Publisher Summary This chapter reveals the advances in protein design termed “molecular breeding,” allows protein engineers to homologously recombine multiple related genes by a process that closely mimics sexual recombination to generate functionally diverse libraries of chimeric proteins from which improved variants can be selected. Molecular breeding effects the permutation of diversity within a pool of related sequences and has proven to be an extraordinarily effective method to evolve proteins and pathways for better function. The most widely used format for molecular breeding is in vitro fragmentation and reassembly of DNA. The highly active, functionally diverse gene libraries generated by molecular breeding have extended directed evolution to a plethora of proteins for which only limited throughput screens are feasible. The chapter discusses the method for molecular breeding involves recombination of homologous genes obtained from nature, in order to permutate the proven diversity. Molecular breeding (also called DNA shuffling) was developed to mimic this essential feature of natural evolution.

Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor

ABSTRACT Spent sulfite liquor (SSL) is a waste effluent from sulfite pulping that contains monomeric sugars which can be fermented to ethanol. However, fermentative yeasts used for the fermentation of the sugars in SSL are adversely affected by the inhibitory substances in this complex feedstock. To overcome this limitation, evolutionary engineering of Saccharomyces cerevisiae was carried out using genome-shuffling technology based on large-scale population cross mating. Populations of UV-light-induced yeast mutants more tolerant than the wild type to hardwood spent sulfite liquor (HWSSL) were first isolated and then recursively mated and enriched for more-tolerant populations. After five rounds of genome shuffling, three strains were isolated that were able to grow on undiluted HWSSL and to support efficient ethanol production from the sugars therein for prolonged fermentation of HWSSL. Analyses showed that greater HWSSL tolerance is associated with improved viability in the presence of salt, sorbitol, peroxide, and acetic acid. Our results showed that evolutionary engineering through genome shuffling will yield robust yeasts capable of fermenting the sugars present in HWSSL, which is a complex substrate containing multiple sources of inhibitors. These strains may not be obtainable through classical evolutionary engineering and can serve as a model for further understanding of the mechanism behind simultaneous tolerance to multiple inhibitors.