Processes and host cells for genome, pathway, and biomolecular engineering

A method for multiplexed genome engineering of a yeast cell for a desired phenotype, wherein the desired phenotype is one or more of a change in carbon substrate utilization, increased cell growth rate, increased production of a desired chemical, redox cofactor balance, reduced production of one or more undesired byproducts, increased resistance to industrial fermentation, and increased recombinant protein production,

• the method comprising:
• introducing a first oligonucleotide library into a population of yeast cells, the yeast cells expressing a single-stranded annealing protein (SSAP) and recombinase system wherein said system comprises a member selected from the group consisting of:
• RecT protein of RecET system, Rad52-like protein, λ Red Beta protein, Sak protein, Erf protein, Rad51-like protein, Sak4 protein, and Gp2.5-like protein, wherein said system further comprises a deletion, inactivation, reduction, or inhibition of a mismatch repair protein,
• and the first oligonucleotide library targeting regulatory sequences that control gene expression in the cell wherein said regulatory sequences are selected from the group consisting of promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns;
• targeting at least one gene altered by the system with a second oligonucleotide library, or targeting at least one gene with a gene editing system;
• and
• selecting for yeast cells having an improvement in the desired phenotype as a result of oligonucleotide incorporation, wherein the method comprises a co-selection.