Yeast Molecular Biology Lecture 2 Applications of molecular biology in yeast research of potential relevance to fermentation industry.
Key things that you might want to improve/ Features of a good brewing yeast. • Rapid fermentation without excessive yeast growth • Efficient utilisation of maltose and maltotriose with good conversion to ethanol • Ability to withstand the stresses imposed by alcohol and osmotic pressures. • Reproducible production of aroma and flavour components. • Ideal flocculation character. • Viability during storage.
Metabolic Engineering of Saccharomyces cerevisiae • See Ostergaard et al. MMBR. • Directed improvements of the cellular properties of yeast, relying on biochemical information, and the application of genetic engineering has been referred to as metabolic engineering. • the analytical side of metabolic engineering, which deals with the analysis of the cells in order to identify the most promising target(s) for genetic manipulation. • (ii) genetic engineering of the cells, where the cell with the genetic modifications is constructed. Possible areas of manipulation: extension of substrate range; improvements of productivity and yield; elimination of by-products; improvement of process performance; improvements of cellular properties; and extension of product range including heterologous protein production.
What is metabolic engineering? Improve yields by changing enzyme activities (up or down) in existing metabolic pathways. metabolic flux. If B is a useful product (eg ethanol) can Inc activity leading to I Inc activity leading to B Decrease activity leading to C
Or may want to extend substrate range by creating new metabolic pathways A simple change can have a lot of metabolic consequences. When you look at some of the papers I have highlighted, what they are changing may be simple and you may expect a simple result. Changing the activity of an enzyme/level of one substrate may have a knock on effect on a number of metabolic pathways.
Key Recombinant approaches to strain improvement • Increase activity of an enzyme • Knock out gene functions • Express foreign genes.
Ostergarrd et al. Key sections. • Extend substrate range: Starch, lactose, melibiose and xylose • Improve productivity and yield and decrease by products. • Improve process performance (flocculation. • Improve metabolic regulatory control • (Other products eg heterologous protein production)
Examples of yeast Improvememt 1. Increase activity of an enzyme.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1999, p. 143–149 Vol. 65, No. 1 Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase REMIZE et al. In this molecular biology techniques were used to simply overexpress one gene, glycerol-3-phosphate dehydrogenase (GDP1) in a multi-copy plasmid (Yep?).
Considerations with expression vectors Y X Coding region on gene of interest. Cloned by PCR. Primers designed to have restriction enzyme sites X and Y and clone the gene in frame (if necessary) Termination sequences Constituitive or inducible promoter
It was Cloned by PCR from a plasmid clone so that it could be cloned in frame into a plasmid, high copy number expression vector. Overexpressing this gene had a large number of effects. You can see why from looking at the figure. Overexpressing GDP1 will shift the flux over to the left, therefore you get more glycerol and less ethanol. There were quite marked strain differences. For all strains tested there was less cell yield, faster CO2 production and the glucose was used up quicker. The redox balance was shifted and thisis reflected in the levels measured of some of the metabolites.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2000 Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates M. LILLY et al. Esters are fruity smelling organic molecules. If inc level of acetyl transferase, the level of acetate esters should increase.
Again altering metabolic fluxes by inc level of an enzyme. ATF1, alcohol acetyl-transferase (AAT). PCR was used to amplify the gene from a wine strain. NB it must be designed to clone the gene in frame in the expression vector. Constituitive yeast PGK promoter. Has a selectable marker, does not replicate, linearised and integrates into the genome (Yip). Integration confirmed by Southern and Northern. PFGE gives the Chromosome id. Northern indicates the inc level expn of the larger recombinant band. The integrative plasmid is being used to put more copies with a stronger promoter. GC used to measure the levels of some of the organic compounds. Ester concs increased significantly, depending on the strain. Decreased methanol and acetic acid. Analysis done on wine before and after aging and after 1st and 2nd distillation. The aroma and taste were modified.
MOLECULAR AND CELLULAR BIOLOGY, Nov. 2000, p. 8093–8102 Vol. 20, No. 21 Tryptophan Permease Gene TAT2 Confers High-Pressure Growth in Saccharomyces cerevisiae FUMIYOSHI AND HORIKOSHI Ability to grow well under high pressure is a useful property of brewing yeast.
Again in this paper they are overexpressing the activity of a yeast gene. The gene which allowed high pressure growth was cloned by transforming a plasmid gene library into yeast cells and looking for growth at high pressure. Plasmid DNA was prepared from colonies that grew at high pressure. Prior to this work, little was known about what genes may have been involved and so the random cloning approach was made. Adding tryptophan enabled the cells to grow at high pressure, 25 MPa (They screened all aa’s). Discovered that the tryptophan porin gene could confer resistance by transforming in a library (Thereby increasing the number of genes) and selecting for high pressure growth.
From a number of clones (overlapping) the TAT2 gene was common to all. They sequenced and identified it. It encodes a tryptophan transporter and elevated levels of the gene product gave it pressure resistance. It elevated the levels of trp in the cell. At elevated pressure the level of trp went down as ability to import is reduced, so getting more of the protein offsets this. This is probably not of relevance to brewing directly but it shows how to find a gene by complementation.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1996, p. 1563–1569 Vol. 62. Disruption of the Yeast ATH1 Gene Confers Better Survival after Dehydration, Freezing, and Ethanol Shock: Potential Commercial Applications KIM et al.
Trehalose a molecule that acts as an osmoticum/protectant. They disrupted the gene encoding the protein that degrades it, ATH1 (Acid trehalase) to increase the cellular levels of trehalose. This strain had been previously made by deleting the ATH1 gene, presumably by cloning it by PCR in Yip vector (maybe not) and cloning in a selectable marker, URA3 to disrupt the protein coding sequence. There were a lot of significant consequences. Greater resistance to freezing, dehydration and toxic levels of ethanol. 2x cell density in rich media.
High affinity glucose transport in Saccharomyces cerevisiae deleted in hexokinase II Diderich et al. At high glucose concentrations the kinetics of glucose transport are predominantly determined by low affinity transporters and when the glucose concentration decreases glucose transport is taken over by high affinity transporters. It was shown that the high affinity glucose transporter is subject to glucose repression. When glucose has entered the cell it is phosphorylated to glucose-6-phosphate. In the yeast S. cerevisiae, there are three enzymes that phosphorylate glucose: glucokinase, hexokinase I and hexokinase II.
Hexookinase II (HXKII) deleted using PCR and a kan selectable marker. Presumably in the mutant the level of phosphorylation is reduced. Mutant has a inc of high affinity glucose transport. In the mutant more glucose is respired rather than fermented under aerobic conditions.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1998, p. 1852–1859 Vol. 64, No. 5 Genetically Engineered Saccharomyces Yeast Capable of Effective Cofermentation of Glucose and Xylose HOet al.
Xylose is the 2nd most important fermentable sugar, but it cannot be fermented by Saccharomyces yeast. Three genes cloned by PCR into high copy number vectors based on 2mm plasmid and antibiotic resistances. It is a shuttle vector (YEp). Xylose reductase XR and Xylitol dehydrogenase XD from the yeast Pichia stipitus and Xylulokinase XK from S. cerevisiae. Each gene without promoter region fused to promoter region of cloned S. cerevisiae pyruvate kinase (PYK) in the vector. Ended up using xylose growth as the selection to maintain the plasmid (in absence of glucose).
To engineer a new metabolic pathway needs a set of genes and care needs to be taken that the enzymes are expressed at a suitable level, hence pyk promoter. Can ferment in presence or absence of glucose. Major fermentation product was xylitol in other peoples strains, but in this one because of the XK gene, they get mostly ethanol. Cofermentation (rather than using an inducible promoter for xylose) allows growth without a 2nd lag phase.
This group have engineered this new metabolic pathway with a high copy number plasmid. • What problems might this cause? • What would they have to do if they wanted to grow this yeast commercially?
JOURNAL OF BACTERIOLOGY, Jan. 1997, p. 157–162 Vol. 179, No. 1 Expression of Bacterial mtlD in Saccharomyces cerevisiae Results in Mannitol Synthesis and Protects a Glycerol-Defective Mutant from High-Salt and Oxidative Stress CHATURVEDI et al.
This is more to investigate the role of mannitol in other fungi (pathogenic) rather than of any direct relevance to brewing. mtlD gene from E. coli encodes mamannitol-1-phosphate. Interconverts fructose-6-phosphate (+NADH) with mannitol-1-phosphate. This gets dephosphorylated to mannitol. PCR used to clone E. coli gene from plasmid into yeast expression vector. The transformed yeast had no obvious phenotype, so the plasmid was transformed into a glycerol deficient strain, which is sensitive to elevated NaCl. Cells were protected from oxidative killing to the same extent as wild type. It showed that mannitol can substitute for glycerol as an osmolyte. The oxidation protection is a new observation, scavenging OH radicals. This is an example of S. cerevisiae as a fungal research tool.
JOURNAL OF BACTERIOLOGY, Dec. 1999. Genome-Wide Transcriptional Analysis of Aerobic and Anaerobic Chemostat Cultures of Saccharomyces cerevisiae Ter LINDEet al. This used DNA chip technology. A robot spots a tiny drop of each gene onto a slide. You prepare RNA from cells grown in different conditions and hybridise with chip. A computer quantifies the level of hybridisation with each gene. It can then look at differences between different growth conditions, to look find genes which are differentially regulated. Do you know what a DNA chip is?
These methods are used for strain management, authentication and verification. Needed when strains are stored and distributed for brewing under licence. Can be used to discriminate between strains with similar physiological properties.
These techniques were used to study the genetic diversity and geographical distribution of wild S. cerevisiae strains from one of the wine producing areas of France. Electrophoretic karyotyping (PFGE), mitochondrial DNA fragment length polymorphisms and PCR of interspersed repeats. Versavaud et al. 1995 App & Env Microbiology.