Means and Methods for Increased Protein Expression by Use of a Combination of Transport Proteins and Either Chaperones or Transcription Factors

A method of manufacturing at least one protein of interest in a eukaryotic host cell comprising:

• providing said host cell engineered to overexpress
• a) at least one polynucleotide encoding at least one Sbh1 transport protein, and
• b) (i) at least one polynucleotide encoding at least one Kar2 endoplasmic reticulum (ER) helper protein;
• or
• (ii) at least one polynucleotide encoding at least one Hac1 transcription factor,
• wherein the host cell further comprises at least one polynucleotide encoding the at least one protein of interest,
• culturing said host cell under suitable conditions to overexpress the polynucleotides a) and b) and to express the at least one polynucleotide encoding the at least one protein of interest, optionally
• isolating the at least one protein of interest from the cell culture, optionally
• purifying the at least one protein of interest, optionally
• modifying the at least one protein of interest, and optionally
• formulating the at least one protein of interest.

The method of any one of the preceding claims, wherein the at least one polynucleotide encoding the at least one Sbh1 transport protein and the at least one polynucleotide encoding the at least one Kar2 ER helper protein or the at least one polynucleotide encoding the at least one Hac1 transcription factor encode for the respective proteins according to one of the following:

• a) a heterologous Sbh1 transport protein and a heterologous Kar2 ER helper protein or a heterologous Hac1 transcription factor;
• b) a homologous Sbh1 transport protein and a homologous Kar2 ER helper protein or a homologous Hac1 transcription factor;
• c) a heterologous Sbh1 transport protein and a homologous Kar2 ER helper protein or a homologous Hac1 transcription factor;
• or
• d) a homologous Sbh1 protein transport protein and a heterologous Kar2 ER helper protein or a heterologous Hac1 transcription factor.

The method of any one of the preceding claims, wherein the overexpression of the polynucleotides a) and b) is achieved by

• (i) using a recombinant promoter which drives expression of the polynucleotides;
• (ii) exchanging or modifying a regulatory sequence operably linked to the polynucleotides;
• or
• (iii) introducing one or more copies of the polynucleotides operably linked to a promoter into the host cell.

The method of any one of the preceding claims, wherein the overexpression of the polynucleotides a) and b) is achieved by

• i) exchanging the native promoter operably linked to the endogenous polynucleotides by a stronger recombinant promoter;
• ii) exchanging the coding sequence of said endogenous polynucleotides by a codon-optimized coding sequence, which codon-optimization is done according to the codon-usage of said host cell;
• iii) exchanging a native positive regulatory element of the endogenous polynucleotides for a more efficient positive regulatory element;
• iv) introducing a positive regulatory element for the polynucleotides, which element is not present in the respective endogenous expression cassette of the host cell;
• or
• v) deleting a negative regulatory element of the endogenous polynucleotides, which element is normally present in the respective endogenous expression cassette of the host cell, or a combination thereof.

The method of any one of the preceding claims, wherein the Sbh1 transport protein comprises an amino acid sequence as shown in any one of SEQ ID NOs:

• 1-9 or a functional homolog thereof comprising at least 90% sequence identity to an amino acid sequence as shown in any one of SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 5, SEQ ID NO:
• 6, SEQ ID NO:
• 7, SEQ ID NO:
• 8 or SEQ ID NO:
• 9.

The method of any one of the preceding claims, wherein the Kar2 ER helper protein comprises an amino acid sequence as shown in any one of SEQ ID NOs:

• 10-18 or a functional homolog thereof comprising at least 90% sequence identity to an amino acid sequence as shown in any one of SEQ ID NO:
• 10, SEQ ID NO:
• 11, SEQ ID NO:
• 12, SEQ ID NO:
• 13, SEQ ID NO:
• 14, SEQ ID NO:
• 15, SEQ ID NO:
• 16, SEQ ID NO:
• 17 or SEQ ID NO:
• 18.

The method of any one of the preceding claims, wherein the Hac1 transcription factor comprises at least:

• a) a DNA binding domain comprising:
• i) an amino acid sequence as shown in any one of SEQ ID NOs:
• 19-27, or
• ii) a functional homolog thereof comprising at least 90% sequence identity to an amino acid sequence as shown in any one of SEQ ID NO:
• 19, SEQ ID NO:
• 20, SEQ ID NO:
• 21, SEQ ID NO:
• 22, SEQ ID NO:
• 23, SEQ ID NO:
• 24, SEQ ID NO:
• 25, SEQ ID NO:
• 26 or SEQ ID NO:
• 27, and
• b) an activation domain.

The method of any one of the preceding claims, wherein the Hac1 transcription factor comprises an amino acid sequence as shown in any one of SEQ ID NOs:

• 28-36 or a functional homolog thereof comprising at least 90% sequence identity to an amino acid sequence as shown in any one of SEQ ID NO:
• 28, SEQ ID NO:
• 29, SEQ ID NO:
• 30, SEQ ID NO:
• 31, SEQ ID NO:
• 32, SEQ ID NO:
• 33, SEQ ID NO:
• 34, SEQ ID NO:
• 35 or SEQ ID NO:
• 36.