Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.     

Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation

Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, does not have a lactose metabolization system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisiae strains, particularly involving the expression of the lactose genes of the phylogenetically related yeast Kluyveromyces lactis, but also the lactose genes from Escherichia coli and Aspergillus niger, as reviewed here. Due to the existing large amounts of whey, the production of bio-ethanol from lactose by engineered S. cerevisiae has been considered as a possible route for whey surplus. Emphasis is given in the present review on strain improvement for lactose-to-ethanol bioprocesses, namely flocculent yeast strains for continuous high-cell-density systems with enhanced ethanol productivity.     

Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae.

Two sets of plasmids, each carrying a Saccharomyces cerevisiae gene and a portion or all of the yeast 2-micron circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. cerevisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-micron circle transform yeast with 2- to 10-fold greater frequency than those carrying the 1.5-megadalton EcoRI fragment of the 2-micron circle. Restriction endonuclease analysis of plasmics retrieved from S. cerevisiae transformed with plasmics carrying the 2.4-megadalton EcoRI fragment showed that in 13 of 96 cases the original plasmic has acquired an additional copy of the 2-mcron circle. These altered plasmids appear to have arisen by means of an interplasmid recombination event while in S. cerevisiae. A clone bank of S. cerevisiae genes based upon one of these composite plasmids has been constructed. By using this bank and selecting directly in S. cerevisiae, the ura3, tyr1, and met2 genes have been cloned.     

Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase.

The TOP3 gene of the yeast Saccharomyces cerevisiae was postulated to encode a DNA topoisomerase, based on its sequence homology to Escherichia coli DNA topoisomerase I and the suppression of the poor growth phenotype of top3 mutants by the expression of the E. coli enzyme (Wallis, J.W., Chrebet, G., Brodsky, G., Golfe, M., and Rothstein, R. (1989) Cell 58, 409-419). We have purified the yeast TOP3 gene product to near homogeneity as a 74-kDA protein from yeast cells lacking DNA topoisomerase I and overexpressing a plasmid-borne TOP3 gene linked to a phosphate-regulated yeast PHO5 gene promoter. The purified protein possesses a distinct DNA topoisomerase activity: similar to E. coli DNA topoisomerases I and III, it partially relaxes negatively but not positively supercoiled DNA. Several experiments, including the use of a negatively supercoiled heteroduplex DNA containing a 29-nucleotide single-stranded loop, indicate that the activity has a strong preference for single-stranded DNA. A protein-DNA covalent complex in which the 74-kDa protein is linked to a 5' DNA phosphoryl group has been identified, and the nucleotide sequences of 30 sites of DNA-protein covalent complex formation have been determined. These sequences differ from those recognized by E. coli DNA topoisomerase I but resemble those recognized by E. coli DNA topoisomerase III. Based on these results, the yeast TOP3 gene product can formally be termed S. cerevisiae DNA topoisomerase III. Analysis of supercoiling of intracellular yeast plasmids in various DNA topoisomerase mutants indicates that yeast DNA topoisomerase III has at most a weak activity in relaxing negatively supercoiled double-stranded DNA in vivo, in accordance with the characteristics of the purified enzyme.     

The Candida albicans myristoyl-CoA:protein N-myristoyltransferase gene. Isolation and expression in Saccharomyces cerevisiae and Escherichia coli.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) has recently been identified as a target for antiviral and antifungal therapy. Candida albicans is a dimorphic, asexual yeast that is a major cause of systemic fungal infections in immunosuppressed humans. Metabolic labeling studies indicate that C. albicans synthesizes one principal 20-kDa N-myristoyl-protein. The single copy C. albicans NMT gene (ca-NMT1) was isolated and encodes a 451-amino acid protein that has 55% identity with Saccharomyces cerevisiae NMT. C. albicans NMT1 is able to complement the lethal phenotype of S. cerevisiae nmt1 null mutants by directing efficient acylation of the approximately 12 endogenous N-myristoylproteins produced by S. cerevisiae. C. albicans NMT was produced in Escherichia coli, a prokaryote with no endogenous NMT activity. In vitro studies of purified E. coli-derived S. cerevisiae and C. albicans NMTs revealed species-specific differences in the kinetic properties of synthetic octapeptide substrates derived from known N-myristoylproteins. Together these data indicate that C. albicans and S. cerevisiae NMTs have similar yet distinct substrate specificities which may be of therapeutic significance.     

Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.     

Genetic complementation of the Saccharomyces cerevisiae leu2 gene by the Escherichia coli leuB gene.

The leucine operon of Escherichia coli was cloned on a plasmid possessing both E. coli and Saccharomyces cerevisiae replication origins. This plasmid, pEH25, transformed leuA, leuB, and leuD auxotrophs of E. coli to prototrophy; it also transformed leu2 auxotrophs of S. cerevisiae to prototrophy. beta-Isopropylmalate dehydrogenase was encoded by the leuB gene of E. coli and the leu2 gene of yeast. Verification that the leuB gene present on pEH26 was responsible for complementing yeast leu2 was obtained by isolating in E. coli several leuB mutations that resided on the plasmid. These mutant leuB- plasmids were no longer capable of complementing leu2 in S. cerevisiae. We conclude that S. cerevisiae is capable of transcribing at least a portion of the polycistronic leu operon of E. coli and can translate a functional protein from at least the second gene of this operon. The yeast Leu+ transformants obtained with pEH25, when cultured in minimal medium lacking leucine, grew with a doubling time three to four times longer than when cultured in medium supplemented with leucine.     

Expression of a synthetic human growth hormone gene in yeast

A synthetic human growth hormone (hGH) gene was efficiently expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae). More than 10(6) molecules of hormone were formed per cell despite the fact that the gene was constructed with codon preference for Escherichia coli.     

Functional analysis of the hemK gene product involvement in protoporphyrinogen oxidase activity in yeast

The Escherichia coli hemK gene has been described as being involved in protoporphyrinogen oxidase activity; however, there is no biochemical evidence for this. In the context of characterizing the mechanisms of protoporphyrinogen oxidation in the yeast Saccharomyces cerevisiae, we investigated the yeast homolog of HemK, which is encoded by the ORF YNL063w, to find out whether it has any protoporphyrinogen oxidase activity and/or whether it modulates protoporphyrinogen oxidase activity. Phenotype analysis and enzyme activity measurements indicated that the yeast HemK homolog is not involved in protoporphyrinogen oxidase activity. Complementation assays in which the yeast HemK homolog is overproduced do not restore wild-type phenotypes in a yeast strain with deficient protoporphyrinogen oxidase activity. Protein sequence analysis of HemK-related proteins revealed consensus motif for S-adenosyl-methionine-dependent methyltransferase.     

Yeast cells allow high-level expression and formation of polyomavirus-like particles

Polyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy. Although VLPs can be generated in E. coli or insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans. We selected the yeast Saccharomyces cerevisiae for expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV). Two entire HaPV VP1-coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels in Saccharomyces cerevisiae. The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid. Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.     

Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae

Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.     

Conserved rules govern genetic interaction degree across species

ABSTRACT: BACKGROUND: Synthetic genetic interactions have recently been mapped on a genome scale in the budding yeast Saccharomyces cerevisiae, providing a functional view of the central processes of eukaryotic life. Currently, comprehensive genetic interaction networks have not been determined for other species, and we therefore sought to model conserved aspects of genetic interaction networks in order to enable the transfer of knowledge between species. RESULTS: Using a combination of physiological and evolutionary properties of genes, we built models that successfully predicted the genetic interaction degree of S. cerevisiae genes. Importantly, a model trained on S. cerevisiae gene features and degree also accurately predicted interaction degree in the fission yeast Schizosaccharomyces pombe, suggesting that many of the predictive relationships discovered in S. cerevisiae also hold in this evolutionarily distant yeast. In both species, high single mutant fitness defect, protein disorder, pleiotropy, protein-protein interaction network degree, and low expression variation were significantly predictive of genetic interaction degree. A comparison of the predicted genetic interaction degrees of S. pombe genes to the degrees of S. cerevisiae orthologs revealed functional rewiring of specific biological processes that distinguish these two species. Finally, predicted differences in genetic interaction degree were independently supported by differences in co-expression relationships of the two species. CONCLUSIONS: Our findings show that there are common relationships between gene properties and genetic interaction network topology in two evolutionarily distant species. This conservation allows use of the extensively mapped S. cerevisiae genetic interaction network as an orthology-independent reference to guide the study of more complex species.     

Synthesis of a gene for human serum albumin and its expression in Saccharomyces cerevisiae.

A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described. Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps. 24 synthetic, 69-85 nucleotides long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks. Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene as large fragments. Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids. The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichia coli-Saccharomyces cerevisiae shuttle vector. This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminal amino acid sequence.     

The regulatory characteristics of yeast fructose-1,6-bisphosphatase confer only a small selective advantage.

The question of how the loss of regulatory mechanisms for a metabolic enzyme would affect the fitness of the corresponding organism has been addressed. For this, the fructose-1,6-bisphosphatase (FbPase) from Saccharomyces cerevisiae has been taken as a model. Yeast strains in which different controls on FbPase (catabolite repression and inactivation; inhibition by fructose-2,6-bisphosphate and AMP) have been removed have been constructed. These strains express during growth on glucose either the native yeast FbPase, the Escherichia coli FbPase which is insensitive to inhibition by fructose-2,6-bisphosphate, or a mutated E. coli FbPase with low sensitivity to AMP. Expression of the heterologous FbPases increases the fermentation rate of the yeast and its generation time, while it decreases its growth yield. In the strain containing high levels of an unregulated bacterial FbPase, cycling between fructose-6-phosphate and fructose-1,6-bisphosphate reaches 14%. It is shown that the regulatory mechanisms of FbPase provide a slight but definite competitive advantage during growth in mixed cultures.     

Ashbya gossypii: a model for fungal developmental biology

Ashbya gossypii is a riboflavin-overproducing filamentous fungus that is closely related to unicellular yeasts such as Saccharomyces cerevisiae. With its close ties to yeast and the ease of genetic manipulation in this fungal species, A. gossypii is well suited as a model to elucidate the regulatory networks that govern the functional differences between filamentous growth and yeast growth, especially now that the A. gossypii genome sequence has been completed. Understanding these networks could be relevant to related dimorphic yeasts such as the human fungal pathogen Candida albicans, in which a switch in morphology from the yeast to the filamentous form in response to specific environmental stimuli is important for virulence.     

Yeast ribosomal protein L12 is a substrate of protein-arginine methyltransferase 2

Type III protein-arginine methyltransferase from the yeast Saccharomyces cerevisiae (RMT2) was expressed in Escherichia coli and purified to apparent homogeneity. The cytosolic, ribosomal, and ribosome salt wash fractions from yeast cells lacking RMT2 were used as substrates for the recombinant RMT2. Using S-adenosyl-l-methionine as co-substrate, RMT2 methylated a protein in the ribosome salt wash fraction. The same protein in the ribosomal fraction was also methylated by RMT2 after pretreating the sample with endonuclease. Amino acid analysis affirmed that the labeling products were delta-N-monomethylarginines. The methylated protein from the ribosomal or the ribosome salt wash fraction was isolated by two-dimensional gel electrophoresis and identified as ribosomal protein L12 by mass spectrometry. Using synthetic peptides, recombinant L12, and its mutant as substrates, we pinpointed Arg(67) on ribosomal protein L12 as the methyl acceptor. L12 was isolated from wild type yeast cells that have been grown in the presence of S-adenosyl-l-[methyl-(3)H]methionine and subjected to amino acid analysis. The results indicate that L12 contains delta-N-monomethylarginines.     

High-level expression of fully active yeast flavocytochrome b2 in Escherichia coli.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from the yeast Saccharomyces cerevisiae and three singly substituted mutant forms (F254, R349 and K376) have been expressed in the bacterium Escherichia coli. The enzyme expressed in E. coli contains the protohaem IX and flavin mononucleotide (FMN) prosthetic groups found in the enzyme isolated from yeast, has an electronic absorption spectrum identical with that of the yeast protein and an identical Mr value of 57,500 estimated by SDS/polyacrylamide-gel electrophoresis. N-Terminal amino-acid-sequence data indicate that the flavocytochrome b2 isolated from E. coli begins at position 6 (methionine) when compared with mature flavocytochrome b2 from yeast. The absence of the first five amino acid residues appears to have no effect on the enzyme-catalysed oxidation of L-lactate, since Km values for the yeast- and E. coli-expressed wild-type enzymes were identical within experimental error. The F254 mutant enzyme expressed in E. coli also showed kinetic parameters essentially the same as those found for the enzyme from yeast. The R349 and K376 mutant enzymes had no activity when expressed in either yeast or E. coli. The yield of flavocytochrome b2 from E. coli is estimated to be between 500- and 1000-fold more than from a similar wet weight of yeast (this high level of expression results in E. coli cells which are pink in colour). The increased yield has allowed us to verify the presence of FMN in the R349 mutant enzyme. The advantages of E. coli as an expression system for flavocytochrome b2 are discussed.