Methods for Identifying Promoters for Protein Production in Yeast

• Main Authors: MILAN, Aileen Aboy, DODGE, Corey, TAN, Xuqiu, WONG, Kevin Li, ORCHARD, Samantha, CAI, Ruorong
• Format: Patent
• Language: English
• Published: 29.07.2021
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; COMPOSITIONS THEREOF; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CULTURE MEDIA; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROCESSES OF PREPARING SUCH COMPOSITIONS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

Disclosed herein are methods of identifying promoters that drive protein expression independently of methanol and are useful in driving protein expression in yeast. The method may comprise the steps of: fermenting yeast cells under at least one fermentation condition in the absence of methanol, collecting samples at different times during fermentation under the at least one fermentation conditions, determining the relative mRNA levels associated with native yeast genes in the samples, identifying one or more of the native yeast genes associated with higher than average levels of mRNA and determining putative promoters associated with the higher than average levels of mRNA encoding the native yeast genes, making expression constructs, each construct comprising one of the identified putative promoters and a gene encoding a marker protein and introducing the expression constructs into yeast cells, culturing the yeast cells comprising the expression constructs in the absence of methanol, determining marker protein expression by the cultured yeast cells and comparing marker protein expression driven by each of the putative promoters to identify promoters that drive protein expression independently of methanol and are useful in driving protein expression in yeast.