BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the development of metabolic engineering of yeast. More specifically this invention relates to a non-ethanol producing strain of the yeast Saccharomyces cerevisiae that can convert glucose to acetyl-Coenzyme A (CoA) in the cytosol.

Background of the Invention

With the requirements for development of sustainable solutions for provision of fuels, chemicals and materials, there is much focus on biotechnology, as this may serve as one of the pillars underlying a modern, sustainable society. Biotechnology has been used for generations in the production of fermented beverages and food products, and in the last 60 years for the production of antibiotics, food ingredients and feed additives. In recent years, several new processes for production of chemicals that can be used for polymer production have been introduced, and the production of bioethanol for fuel use has increased rapidly. Currently, there is extensive research on the development of novel cell factories for the production of chemicals and novel fuels, and it is expected that this will lead to implementation of several new biotech processes in the coming years.

The core of this development is the design and construction of cell factories that can ensure efficient conversion of the raw material to the product of interest. Traditionally, microorganisms that naturally produce a desired molecule were identified and then improved through classical strain engineering based on

mutagenesis and screening. This has been a very effective approach and has resulted in low-cost production processes for many different chemicals, e.g. penicillin, citric acid and lysine. With the introduction of genetic engineering and methods for detailed analysis of cellular metabolism it became possible to use a more directed approach to improve cell factories, generally referred to as metabolic engineering.' Today metabolic engineering has evolved into a research field that encompasses detailed metabolic analysis with the objective to identify targets for metabolic engineering and the implementation of metabolic engineering strategies for improvement and/or design of novel cell factories. In recent years, synthetic biology has emerged as another research field that originally aimed at reconstruction of small, artificial biological systems, e.g. assembling a new biological regulon or oscillators that can be used to regulate gene expression in response to a specific input. But synthetic biology also includes the synthesis of DNA and complete chromosomes as illustrated in a recent work on reconstruction of a complete bacterial chromosome. Summary of the Invention

A primary object of the present invention is to generate a yeast platform cell factory with increased cytosolic acetyl-CoA (AcCoA) supply, by introduction of steps for the direct conversion of pyruvate to AcCoA. The yeast platform cell factory of the present invention can efficiently convert pyruvate to acetyl-CoA and this feature is combined with elimination of pyruvate decarboxylase activity, resulting in an efficient cytosolic acetyl-CoA producer that cannot produce ethanol. This will result in high cytosolic levels of the important precursor acetyl-CoA.

Acetyl-CoA metabolism is highly compartmentalized in eukaryotic cells as this metabolite is used for metabolism in the cytosol, mitochondria, peroxisomes and the nucleus. Acetyl-CoA serves as a key precursor metabolite for the production of important cellular constituents such as fatty acids, sterols, and amino acids as well as it is used for acetylation of proteins. Besides these important functions it is also precursor metabolite for many other biomolecules, such as polyketides, isoprenoids, 3-hydroxypropionic acid, 1 -butanol and polyhydroxyalkanoids, which encompass many industrially relevant chemicals. The yeast S. cerevisiae is a very important cell factory as it is already widely used for production of biofuels, chemicals and pharmaceuticals, and there is therefore much interest in developing platform strains of this yeast that can be used for production of a whole range of different products. It is however a problem that such a platform cell factory for efficient production of cytosolic acetyl-CoA is not as efficient as needed for good industrial application. Our invention is a multiple gene modification approach of the yeast generating higher yield of acetyl-CoA, by combining pathways for direct conversion from pyruvate to acetyl-CoA together with elimination of ethanol production. Brief Description of the Drawings

Figure 1 provides a simplified overview of acetyl-CoA metabolism in S.

cerevisiae. Acetyl-CoA is key metabolite in three different compartments: the cytosol

(marked as ), the mitochondria (marked as · · ·), and the peroxisomes {marked as— ). There is no direct transport of the acetyl-CoA between the three

compartments, and the biosynthesis of acetyl-CoA in the three compartments involves different metabolic pathways. In the mitochondria, acetyl-CoA is formed from pyruvate by the pyruvate dehydrogenase complex. In the cytosol, acetyl-CoA is formed from acetate by acetyl-CoA synthase. In the peroxisomes, acetyl-CoA can be formed from both acetate (also by acetyl-CoA synthase, not shown) and from fatty acids by beta-oxidation. In the mitochondria, the primary fate of acetyl-CoA is oxidation via the tricarboxylic acid (TCA) cycle. Acetyl-CoA in the peroxisomes can via the glyoxylate cycle (GYC) be converted to C4 organic acids (malic and succinic acid) that can be transferred to the mitochondria for oxidation via malic enzyme and the TCA cycle. The primary fate of acetyl-CoA in the cytosol is to serve as precursor for cellular lipids (fatty acids and ergosterol). Many industrially interesting

biotechnological products are derived from acetyl-CoA and the biosynthesis of most of these occurs in the cytosol. A platform yeast cell factory for all these products should therefore redirect carbon towards the acetyl-CoA in the cytosol.

Figure 2 provides an overview of the strategy that can be used in the invention. The normal route for conversion of pyruvate to acetyl-CoA in the cytosol is blocked through deletion of the three structural genes encoding pyruvate

decarboxylase activity in yeast (PDC1, PDC5 and PDC6). This strain is auxotrophic for C ₂ carbon sources such as acetate or ethanol, and it has been shown that this requirement is solely to fulfil the need for acetyl-CoA in the cytosol (required for production of cellular lipids). By removing pyruvate decarboxylase activity the yeast cells also cannot produce ethanol from glucose. To re-install a cytosoiic route for production of acetyl-CoA pyruvate formate lyase (PFL) can be expressed in the cytosol. The strains with the novel pathway for production of cytosoiic acetyl-CoA can be used for production of fatty acids, 3-hydroxypropionic acid, isoprenoids, polyhydroxyalkanoates and 1 -butanol.

Figure 3 shows the plasmid pYZ01 described in Example 2. Figure 4 shows the plasmid pKB01 described in Example 2. Figure 5 shows the plasmid pKB02 described in Example 2

Figure 6 shows growth curves on minimal media for strains evolved for increased growth on glucose described in Example 3. Data from 3-4 independently evolved replicates is shown. Detailed Description of the Invention

The invention herein relies, unless otherwise indicated, on the use of conventional techniques of biochemistry, molecular biology, microbiology, cell biology, genomics and recombinant technology.

To facilitate understanding of the invention, a number of terms are defined below.

As used herein the term "recombinant" when used means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.

As used herein, the term "overproducing" is used in reference to the

production of acetyl-CoA in a host cell and indicates that the host cell is producing more acetyl-CoA by virtue of the introduction of recombinant/heterologous nucleic acid sequences encoding polypeptides that alter the host cell's normal metabolic pathways or as a result of other modifications (e.g., altering the expression of one or more endogenous polynucleotides) as compared with, for example, the host cell that is not modified/transformed with the recombinant polynucleotides as described herein.

As used herein, the terms "protein" and "polypeptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.

The terms "increase," "increasing," "increased," "enhance," "enhanced," "enhancing," and "enhancement" (and grammatical variations thereof), as used herein, describe an elevation in, for example, the production of acetyl-CoA in a yeast. This increase can be observed by comparing said increase in a yeast transformed with, for example, recombinant polynucleotides encoding a polypeptide having the enzyme activity of pyruvate formate lyase (PFL) and a polypeptide having the enzyme activity of pyruvate formate lyase-activating enzyme and a recombinant polynucleotide encoding a polypeptide having the enzyme activity of formate dehydrogenase and one or more recombinant polynucleotides encoding a ferredoxin or a flavodoxin polypeptide and a ferredoxin reductase polypeptide compared to the yeast not transformed with the recombinant polynucleotides. Thus, as used herein, the terms "increase," "increasing," "increased," "enhance," "enhanced," "enhancing," and "enhancement" (and grammatical variations thereof), and similar terms indicate an elevation of at least about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 0%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more, or any range therein, as compared to a control.

As used herein, the terms "reduce," "reduced," "reducing," "reduction,"

"diminish," "suppress," and "decrease" (and grammatical variations thereof), describe, for example, a decrease in the pyruvate decarboxylase activity in a yeast (e.g., a yeast having deletions in the polynucleotides PDC1, PDC5 and PDC6) as compared to a control (e.g., a yeast not having said deletions in the polynucleotides PDC1, PDC5 and PDC6). Thus, as used herein, the terms "reduce," "reduces," "reduced," "reduction," "diminish," "suppress," and "decrease" and similar terms mean a decrease of at least about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more, or any range therein, as compared to a control.

The term "overexpress," "overexpresses" or "overexpression" as used herein refers to higher levels of activity of a gene (e.g. transcription of the gene); higher levels of translation of mRNA into protein; and/or higher levels of production of a gene product (e.g., polypeptide) than would be in the cell in its native (or control (e.g., not transformed with the particular heterologous or recombinant polypeptides being overexpressed)) state. These terms can also refer to an increase in the number of copies of a gene and/or an increase in the amount of mRNA and/or gene product in the cell. Overexpression can result in levels that are 25%, 50%, 100%, 200%, 500%, 1000%, 2000% or higher in the cell, as compared to control levels.

In some embodiments, Saccharomyces cerevisiae can be a host for carrying out the invention, as it is a popular host in basic and applied research apart from being a good ethanol producer, a precursor of esters and specifically of fatty acid ethyl esters. In addition, other yeast cells useful with the present invention include, but are not limited to, other Saccharomyces species, Hansenula polymorpha, Kluyveromyces species, Pichia species, Candida species, Trichoderma species, Yarrowia lipolytica, etc.

In industry, there is much interest in applying a limited number of platform cell factories for production of a wide range of fuels and chemicals as this allows for flexible use of production facilities, which are very capital intensive. One of these platform cell factories is the yeast Saccharomyces cerevisiae, which is widely used for the production of beer, bread, wine, bioethanol, nutraceuticals, chemicals and pharmaceuticals. These platform cell factories can efficiently convert raw materials, today typically glucose/fructose derived from starch or sucrose, but in the future also pentoses derived from lignocellulose, into so-called precursor metabolites can then be further converted into a product of interest. One of these precursor metabolites is acetyl-CoA, that is used as precursor for the production of a wide range of industrially very interesting products (see Fig. 1 ). Several of these products are produced by pathways that drain acetyl-CoA from the cytosol and in connection with

reconstruction of synthetic pathways it is generally desirable to position these pathways in this compartment as this will minimize issues related to product secretion. As illustrated in Fig. 1 , acetyl-CoA is, however, produced and used in several different cellular compartments, i.e. the cytosol, mitochondria and the peroxisomes, and in S. cerevisiae it cannot be transported directly between the different compartments (S. cerevisiae holds all the components of the carnitine transport system, but it cannot synthesize carnitine and in industrial fermentations it would be too expensive to add this component to the medium). In yeast, acetyl-CoA in the cytosol is produced from acetate that is derived from acetaldehyde, that is formed by de-carboxyiation of pyruvate. Acetaldehyde can also be converted to ethanol by alcohol dehydrogenase, and during growth on glucose the majority of the glycolytic flux is directed towards ethanol due to the so-called Crabtree effect in yeast. Besides the main alcohol dehydrogenase (Adhl p) there are several alcohol dehydrogenases in S. cerevisiae that can catalyze the conversion of acetaldehyde to ethanol, and it is therefore inherently difficult to eliminate ethanol production in yeast. The only strategy that has worked so far is removing pyruvate decarboxylase activity through deletion of all three genes that encode for this activity, but this results in a strain that will require supplementation of acetate to the medium in order to meet the requirement for acetyl-CoA in the cytosol (needed for biosynthesis of fatty acids and ergosterol). Obviously, such a strain would be problematic to serve as a platform cell factory for acetyl-CoA derived products.

In the invention herein the normal route for conversion of pyruvate to acetyl- CoA in the cytosol is blocked through deletion of the three structural genes encoding pyruvate decarboxylase activity in yeast {PDC1, PDC5 and PDC6), This strain is auxotrophic for C ₂ carbon sources such as acetate or ethanol, and it has been shown that this requirement is solely to fulfil the need for acetyl-CoA in the cytosol (required for production of cellular lipids). By removing pyruvate decarboxylase activity, the yeast cells can also not produce ethanol from glucose. This has been shown by MT Flikweert et. al ((1999) Growth requirements of pyruvate decarboxylase-negative Saccharomyces cerevisiae. FEMS Micorobio. Lett. 174, 73-79).

However, in the invention herein we have generated a yeast platform cell factory that can efficiently convert pyruvate to acetyl-CoA in one step and combine this feature with elimination of pyruvate de-carboxylase activity, thereby establishing an efficient cytosolic acetyl-CoA producer that overproduces acetyl-CoA and at the same time cannot produce ethanol.

It is difficult to introduce direct pathways from pyruvate to acetyl-CoA efficiently into non-ethanol producing strains. However, the inventors of the present invention have identified a possible route for this introduction that enable efficient introduction of a direct route from pyruvate to acetyl-CoA in a non-ethanol producing yeast strain. The strategy for reconstructing a synthetic pathway from pyruvate to acetyl-CoA, leading to a cell factory for overproducing acetyl-CoA, is described below:

The present invention also relates to strain cultivation and evolution. The strains containing introduced pyruvate to AcCoA conversion system can be cultivated, for example on yeast extract peptone dextrose liquid media. The strains can then be evolved to increase growth, for example on glucose as the sole carbon source. This step involves two phases. In the first phase strains are cultivated in a medium, e.g. YP medium. The strains can be transferred with ethanol concentration being gradually decreased until the only carbon source in the media is glucose. When the fast-growing, glucose tolerant strains are obtained they can be further evolved for growth on minimal media.

Pyruvate formate lyase (PFL)

Expressing pyruvate formate lyase (PFL). PFL (encoded by pfIB) is a homodimer and catalyzes the non-oxidative conversion of pyruvate to acetyl-CoA and formate under anaerobic conditions. The catalytic mechanism involves a glycyl radical, which is part of the enzyme and which is sensitive to oxygen. Thus, the enzyme is not active under aerobic conditions. The radical is formed by pyruvate formate lyase-activating enzyme (PFL-AE) (encoded by pfIA), a monomeric iron- sulfur cluster protein, through cleavage of S-adenosylmethionine. In addition to introducing PFL and PFL-AE from £ coli, it is beneficial to over-express one of the two yeast formate dehydrogenase genes {FDH1IFDH2), which are responsible for the oxidation of formate (produced by PFL) to COs and H ₂ 0.

Since PFL-AE receives electrons from reduced ferredoxin or flavodoxin, fdx

(encoding ferredoxin) or fldA (encoding flavodoxin) together with fpr (encoding ferredoxin reductase) are co-expressed. Another alternative is the use of a PFL system derived from a eukaryotic microorganism such as Chlamydomonas

rheinhardtii or Neocallmastix frontalis. Examples of bacterial PFL sources are E. coli, Lactobacillus plantarum, Bifidobacterium longum, Bacteroides thetaiotaomicron, Clostridium pasteurianum, Staphylococcus aureus, Zymomonas mobilis. Examples of archaeal PFL sources are Archaeoglobus fulgidus, Methanobacterium

thermoautotrophicus.

WO09143495 provides a method of increasing production of formate in a modified yeast comprising inserting genes encoding the E. coli pyruvate formate lyase enzyme complex (PFL) into a 58Oa triple auxotrophic strain of Saccharomyces cerevisiae yeast. Unlike the present invention, WO09143495 provides a S. cerevisiae in which FDH1 and FDH2 are deleted, in contrast in the present invention it is beneficial to over-express FDH1 and FDH2. Further, neither pfIA and pfIB

coexpressed with fdx, fldA and fpr in the yeast strains of WO09143495. The modified S. cerevisiae of WO09143495 is intended to increase ethanol production, unlike the present invention where ethanol production is eliminated.

Accordingly, a further embodiment of the invention provides a method of producing a yeast having increased production of cytosolic acetyl-CoA, comprising introducing into a yeast: (a) a deletion of the endogenous polynucleotides encoding pyruvate decarboxylase (PDC) (e.g., a deletion of PDC1, PDC5 and PDC6); (b) one or more recombinant polynucleotides encodes a polypeptide having the enzyme activity of pyruvate formate lyase (PFL) which is selected from the group of polypeptides consisting of: E. coli PfIB Chlamydomonas rheinhardtii Pfl;

Neocallimastix frontalis PFL; Lactobacillus plantarum PFL, Bifidobacterium longum PFL; Bacteroides thetaiotaomicron PFL; Clostridium pasteurianum PFL;

Staphylococcus aureus PFL; Zymomonas mobilis PFL; Archaeoglobus fulgidus PFL; or Methanobacterium thermoautotrophicus PFL or a polypeptide having at least 50 % identity to any of the polypeptides mentioned above and (ii) a polypeptide having the enzyme activity of pyruvate formate lyase-activating enzyme (PFL-AE), which is selected from the group of polypeptides consisting of: E. coli PfIA; Chlamydomonas rheinhardtii PfIA; Neocallimastix frontalis PFL-AE; Lactobacillus plantarum PFL-AE; Bifidobacterium longum PFL-AE; Bacteroides thetaiotaomicron PFL-AE; Clostridium pasteurianum PFL-AE; Staphylococcus aureus PFL-AE; Zymomonas mobilis PFL- AE; Archaeoglobus fulgidus PFL-AE; or Methanobacterium thermoautotrophicus PFL-AE or a polypeptide having at least 50 % identity to any of the polypeptides mentioned above; (c) a polynucleotide encoding a polypeptide having the enzyme activity of formate dehydrogenase (e.g., S. cerevisiae FDH1 or FDH2); and (d) one or more recombinant polynucleotides encoding a ferredoxin or a flavodoxin polypeptide and a ferredoxin reductase polypeptide (e.g., fdx and fpr from E. coli or fldA and fpr from E. coli), thereby producing a stably transformed yeast having increased cytosolic acetyl-CoA production. In some embodiments, the polynucleotide encoding a polypeptide having the enzyme activity of formate dehydrogenase is overexpressed as compared to the yeast's endogenous formate dehydrogenase.

In further embodiments, the invention provides a yeast having reduced pyruvate decarboxylase activity and increased acetyl-CoA production, comprising (a) a deletion of the endogenous polynucleotide sequences encoding pyruvate

decarboxylase (PDC) (e.g., a deletion of PDC1, PDC5 and PDC6); (b) one or more recombinant polynucleotides encodes a (i) a polypeptide having the enzyme activity of pyruvate formate lyase (PFL), which is selected from the group of polypeptides consisting of: E. coli PfIB; Chlamydomonas rheinhardtii Pfl; Neocallimastix frontalis PFL; Lactobacillus plantarum PFL, Bifidobacterium longum PFL; Bacteroides thetaiotaomicron PFL; Clostridium pasteurianum PFL; Staphylococcus aureus PFL; Zymomonas mobilis PFL; Archaeoglobus fulgidus PFL; or Methanobacterium thermoautotrophicus PFL or a polypeptide having at least 50 % identity to any of the polypeptides mentioned above and (ii) a polypeptide having the enzyme activity of pyruvate formate lyase-activating enzyme (PFL-AE), which is selected from the group of polypeptides consisting of: E. coli PfIA; Chlamydomonas rheinhardtii PfIA; Neocallimastix frontalis PFL-AE; Lactobacillus plantarum PFL-AE; Bifidobacterium longum PFL-AE; Bacteroides thetaiotaomicron PFL-AE; Clostridium pasteurianum PFL-AE; Staphylococcus aureus PFL-AE; Zymomonas mobilis PFL-AE;

Archaeoglobus fulgidus PFL-AE; or Methanobacterium thermoautotrophicus PFL-AE or a polypeptide having at least 50 % identity to any of the polypeptides mentioned above; (c) a polynucleotide encoding a polypeptide having the enzyme activity of formate dehydrogenase (e.g., S. cerevisiae FDH1 or FDH2); and (d) one or more recombinant polynucleotides encoding a ferredoxin or a f lavodoxin polypeptide and a ferredoxin reductase polypeptide {e.g., fc/x and fpr from E. coli or fldA and fpr from E. coli).

EXAMPLES

In the examples below references are made to several primers, these primers are to be found in Table 1.

EXAMPLE 1 .

Deletion of the three structural genes encoding pyruvate decarboxylase activity in yeast (PDC1, PDC5 and PDC6).

PDC1, PDC5 and PDC6 were deleted using a bipartite strategy (Erdeniz et al., 1997). Two overlapping fragments of the kanMX resistance marker cassette flanked by loxP sites were amplified via PCR from plasmid pUG6 (Guldener et al., 1996) using primers 13-16. Sequences upstream and downstream of the individual genes were amplified using primers 1 -12. Due to overlapping ends (introduced through the primer sequences) the PDC-upstream fragments could be fused to the 5 ^' kanMX fragment and the 3 ^' kanMX fragment to the individual PDC-downstream fragments by fusion PCR using the outer primers for amplification. The two overlapping PCR fragments thus generated for each gene deletion were transformed into yeast using the lithium acetate method (Gietz and Woods, 2002). After each gene deletion, the kanMX marker cassette was looped out via Cre recombinase mediated recombination between the two flanking loxP sites using plasmid pUC47 or pUG62 as described previously (Guldener et al., 1996).

PDC1, PDC5, and PDC6 were consecutively deleted in two different background strains: CEN.PK 113-5D (MATa ura3-52) and CEN.PK 1 10-10C (MATa his3-A 1). This resulted in construction of strains YMZ-C1 (MATa ura3-52 pdc1A pdcSA pdc6A), and YMZ-A3 (MATa his3-A 1 pdc6A). Strains YMZ-C1 and YMZ-A3 were crossed to generate YMZ-E1 (MATa ura3-52 his3-A 1 pddA pdcSA pdc6A).

EXAMPLE 2

Expressing pyruvate formate lyase (PFL)

Genes pfIA (encoding PFL-AE) and pfIB (encoding PFL) were codon optimised for expression in yeast and synthesized by GenScript. The gene sequences including introduced restriction sites can be found in table 2. For expression from episomal plasmids, pfIB was restricted with Sad/NoA and cloned into vector pSP-GM1 . PfIA was cut with Xma\/Xho\ and cloned into the same vector generating plasmid pYZ01 (Fig. 3). For integration into the genome, the pflA-pflB cassette including the bidirectional PTEFI-PPGKI promoter was PCR amplified from pYZ01 using primers 21/22 and cloned into vector pXII-5 (Mikkelsen et al., 2012) using USER cloning. The integration constructs was separated from the vector backbone by Xba\ restriction and integrated into YMZ-E1 from example 1 yielding YMZ-E1 -PFL.

The genes fldA and fprwere amplified by PCR using E. coli DH5a genomic DNA as a template and primers 17-20. The NoAISad restricted fldA fragment and Bam VXhol restricted fpr fragment were cloned into plYC04 generating pKB01 (Fig. 4).

The E. coli gene coding for fc/ was PCR-amplified using E. coli DH5pgenomic DNA as a template and primers 60/61 . This fragment was then restricted with

/Vofl/Sacl and cloned into pKB01 restricted with the same enzymes instead of fldA, yielding pKB02 (Fig. 5).

To integrate fldA, fof and fpr, a fragment containing fpr and either fdxox fldA under P TEFI-PPGKI was PCR-amplified from pKB02 or pKB01 using primers 62/64 or 63/64, respectively, and cloned via CPEC into integrative vector pXI-5HIS (vector pXI-5 described in Mikkelsen et al [2012], in which the KIURA3 marker was replaced by the loxP flanked SphisS cassette of plasmid pUG27 described in Guldener et al [2002]; Jensen et al., submitted). The integration constructs were then PCR-amplified using primers 65/66 and integrated into YMZ-E1 -PFL generating strains YMZ-E1 - PFLfld and YMZ-E1 -PFLfdx, respectively.

FDH1 encoding formate dehydrogenase is amplified from yeast genomic DNA using primers 54/55. The P _T EFI promoter is amplified from pSP-GM1 using primers 33/53, Both fragments are cloned into pX-2 by USER cloning. The integration construct is separated from the vector backbone by Λ οίΙ restriction and integrated in to the genome of YMZ-E1 -PFL, YMZ-E -PFLfld and YMZ-E1 -PFLfdx, respectively, to generate YMZ-E1 -PFLF, YMZ-E1 -PFLfldF and YMZ-E1-PFLfdxF.

To introduce the C. rheinhardtii PFL system, the coding sequences of pfl (encoding PFL) and pfIA (encoding PFL-AE) are amplified from C. rheinhardtii cDNA (using primers 56-59) and together with the P _T EFI-PPGKI promoter (amplified from pSP-GM1 using primers 33/34) cloned into pXII-5 by USER cloning. The integration construct is used to replace the E. coli pflA-pfIB cassette in the above mentioned strains leading to construction of YMZ-E1 -crPFL, YMZ-E1-crPFLfld YMZ-E1 - crPFLfdx, YMZ-E1 -crPFLF, YMZ-E1 -crPFLfldF and YMZ-E1 -crPFLfdxF, respectively.

EXAMPLE 3

Strain cultivation and evolution

PDC deletion strains containing the introduced pyruvate to AcCoA conversion system (YMZ-E1 -PFL, YMZ-E1 -PFLfld and YMZ-E1 -PFLfdx), according to example 2 above, were initially cultivated on yeast extract peptone dextrose (YPD) liquid media in either shake-flasks or tubes and their growth was compared to strains containing the PDC deletion alone to evaluate system function.

Next, strains were evolved to increase growth on glucose as the sole carbon source. This involved two phases. In the first phase, strains were cultivated in shake flasks in YP medium (10 g/L yeast extract, 20 g/L peptone)containing 1.4 % glucose and 0.6 % ethanol. Strains were serially transferred every 48 hours or 24 hours, and the ethanol concentration was gradually decreased until glucose became the sole carbon source in the media. The growth rate of the strains was occasionally determined to evaluatetheir adaptation level of glucose tolerance.

Once fast-growing, glucose tolerant strains were obtained in YPD media, they were further evolved for growth on minimal media. The strains were cultivated in minimal medium (Verduyn et al., 1992) containing 2 % glucose and serially transferred every 24 hours. The growth rate of strains was occasionally determined to evaluate their adaptation level. Growth curves for the evolved strains are shown in figure 6.

References:

Chen, Y., Partow, S., Scalcinati, G., Siewers, V., Nielsen, J., 2012a. Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production. FEMS Yeast Res. 12, 598-607. Chen, Y., Daviet, L, Schalk, M, Siewers, V, Nielsen, J., 2012b. Establishing a platform cell factory through engineering of yeast Acetyl-CoA metabolism, under revision

Erdeniz, N., Mortensen, U.H., Rothstein, R., 1997. Cloning-free PCRbased allele replacement methods. Genome Res. 7, 1 174-1 183. Gietz, R.D., Woods, R.A., 2002. Transformation of yeast by lithium acetate/single- stranded carrier DN A/polyethylene glycol method. Meth. Enzymol. 350, 87-96.

Giildener, U., Heck, S., Fiedler, T., Beinhauer, J., Hegemann, J.H., 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519-2524. Giildener, U., Heinisch, J., Kohler G.J., Voss, D., Hegemann, J.H., 2002. A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res. 30, e23

Mikkelsen, M.D, Buron, L.D., Salomonsen, B., Olsen, C.E., Hansen, B.G.,

Mortensen, U.H., Halkier, B.A., 2012. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform. Metab Eng. 14, 104-1 1 .

Nour-Eldin, H., Hansen, B., Norholm, M., Jensen, J., Halkier, B., 2006. Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res. 34, E122.

Quan, J., Tian, J., 201 1 . Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc. 6, 242-51 .

Verduyn, V., Postma, E., Scheffers, W.A., Van Dijken, J.P,. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: A continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501 -517.

Table 1 . Primer sequences

17 fpr-BamHI- GTTGTTGGATCCCAGGAGAAAAACATGGCTGA 17 fw

18 fpr -Xhol-rev GTTGTTCTCGAGCGTTTATCGATAAGTAACCGCT 18

19 fldA-Not1 -fw GTTGTTGCGGCCGCGAGGTTATTTCACTCATGG 19

CT

20 fldA-Sacl- GTTGTTGAGCTCCATCACATCAGGCATTGAGA 20 rev

21 pflAB_pfu_f CGTGCGAUCTATTACGCCAGCTGGAT 21 w

22 pflAB_pfu_r C ACGCG AUCATTAATG C AG CTG G ATAAACG 22 ev

33 P1 -TEF1 ACGTATCGCUTTGTAATTAAAACTTAGATTAGAT 23

TGCTATG

34 P2-PGK1 ACCCGTTGAUTTGTTTTATATTTGTTGTAAAAAG 24

TAGATAATTAC

53 TEF1-fw CGTGCGAUGCACACACCATAGCTTCAAAATG 25

54 G1 -FDH1 - AGCGATACGUATGTCGAAGGGAAAGGTTTTG 26 fw

55 G1 -FDH1 -rv CACGCGAUTTATTTCTTCTGTCCATAAGCTCTGG 27

56 G1 -Crpfl-fw AGCGATACGUATGTTAACACCCTTAAGCTATCCT 28

ATC

57 G1 -Crpfl-rv CACGCGAUTTACATGGTGTCGTGGAAGGTG 29

58 G2-CrpflA- ATC AACGG G U ATGTTG AAG GCTGCGTTGC 30 fw

59 G2-CrpflA- CGTGCGAUTCACTCGGCGCAGATGACG 31 rv

60 fdx-Not1 -fw ATCGAAGCGGCCGCAAAACAATGCCAAAGATTG 32

TTA I I I I GC

61 fdx-Sacl-rev ATCGTCGAGCTCTTAATGCTCACGCGCATG 33

62 fdx-F1 CAACAACGTATCTACCAACGGAATGCGTGCGAT 34

TTAATGCTCACGCGCATGGTTGATAG

63 fldA-F1 CAACAACGTATCTACCAACGGAATGCGTGCGAT 35

TCAGGCATTG AGAATTTCGTCGAGATG

64 FdR-R1 CTTTTCGGTTAGAGCGGATGAATGCACGCGTTA 36

CCAGTAATGCTCCGCTGTCATA

65 IVF3 ATTGTCTCATGAGCGGATAC 37

66 IVR3 CCTGGCCTTTTGCTGGCCTT 38

Table 2

Codon optimized gene sequences (start and stop codons are underlined)

pfIA GAATTCGATCCGTAATACGACTCACTATAGGGCCCGGGAAA

ACA SEQ ID

(Ε. coli) ATGTCAGTAATAGGTAGAATACACTCATTTGAATCCTGCGGT NO: 39

ACAGTAGATGGTCCTGGTATCAGATTCATAACTTTCTTCCAA

GGTTGTTTGATGAGATGTTTATATTGCCATAACAGAGATACTT

GGGACACACACGGTGGTAAAGAAGTCACAGTTGAAGATTTG

ATGAAGGAAGTTGTAACCTACAGACATTTTATGAATGCATCT

GGTGGTGGTGTTACCGCCAGTGGTGGTGAAGCTATATTGCA AGCAGAATTTGTTAGAGATTGGTTCAGAGCTTGTAAAAAGGA

AGGTATTCACACCTGCTTAGACACTAACGGTTTCGTAAGAAG

ATATGATCCAGTCATAGACGAATTGTTAGAAGTCACTGATTT

GGTTATGTTGGACTTAAAGCAAATGAACGATGAAATCCATCA

AAACTTAGTAGGTGTCTCTAATCACAGAACATTGGAATTTGC

CAAGTATTTGGCTAATAAGAACGTTAAAGTATGGATTAGATAT

GTCGTTGTACCAGGTTGGTCTGATGACGATGACTCAGCTCAT

AGATTAGGTGAGTTTACTAGAGATATGGGTAACGTTGAAAAG

ATTGAATTGTTGCCTTATCATGAATTGGGTAAACACAAGTGG

GTCGCAATGGGTGAAGAATACAAATTGGATGGTGTAAAGCC

ACCTAAAAAGGAAACTATGGAAAGAGTAAAGGGTATTTTGGA

ACAATATGGTCACAAGGTTATG 1 1 1 1 GA

CTCGAGTAAGCTTGGTACCGCGGCTAGCTAAGAATTC pfiB GAATTCGCATAGCAATCTAATCTAAGTTTTAATTACAAGCGGC

CGCAAAACA SEQ ID

(E. coli) ATGTCCGAATTGAATGAAAAGTTGGCTACCGCCTGGGAAGG NO: 40

1 1 1 1 ACTAAGGGTGACTGGCAAAATGAAGTTAATGTCAGAGA

C I 1 1 ATTCAAAAGAATTATACCCCTTACGAAGGTGACGAATCA

TTTTTGGCTGGTGCTACTGAAGCAACTACAACCTTATGGGAC

AAAGTCATGGAAGGTGTTAAGTTGGAAAATAGAACACATGCA

CCAGTAGA 1 1 1 CGACACCGCTGTCGCATCCACTATAACAAGT

CACGATGCTGGTTACATCAACAAACAATTGGAAAAGATCGTT

GGTTTACAAACTGAAGCTCCTTTGAAAAGAGCATTAATCCCA

TTCGGTGGTATAAAAATGATCGAAGGTTCATGCAAGGCTTAC

AATAGAGAATTAGATCCAATGATTAAGAAAATTTTTACTGAAT

ACAGAAAGACACATAACCAAGGTGTATTCGATGTCTACACAC

CTGACA 1 1 1 1 GAGATGCAGAAAGTCCGGTGTATTGACCGGTT

TACCAGATGCCTATGGTAGAGGTAGAATTATTGGTGACTACA

GAAGAGTTGC 1 1 1 GTAC G GTATCG ATTACTTG ATG AAG G ACA

AGTTAGCCCAATTCACTTCTTTGCAAGCTGATTTGGAAAACG

GTGTTAACTTGGAACAAACAATCAGATTGAGAGAAGAAATCG

CCGAACAACATAGAGCTTTAGGTCAAATGAAAGAAATGGCTG

CAAAGTATGGTTACGATATATCTGGTCCAGCTACTAATGCAC

AAGAAGCCATCCAATGGACATA 1 1 1 CGGTTACTTGGCCGCTG

TCAAATCACAAAACGGTGCAGCCATGTC 1 1 1 1 GGTAGAACCT

CAACTTTCTTGGATGTTTACATCGAAAGAGACTTAAAGGCAG

GTAAAATAACTGAACAAGAAGCCCAAGAAATGGTAGATCACT

TGGTCATGAAATTAAGAATGGTTAGATTTTTGAGAACCCCTG

AATATGATGAATTATTCTCTGGTGACCCAATTTGGGCTACTG

AATCAATAGGTGGTATGGGTTTGGATGGTAGAACTTTGGTTA

CTAAAAATTCTTTTAGATTTTTGAACACCTTATATACTATGGGT

CCATCCCCTGAACCAAACATGACTATCTTGTGGAGTGAAAAA

TTGCCATTGAACTTCAAAAAGTTCGCTGCAAAGGTTTCCATC

GATACATCTTCATTGCAATACGAAAACGATGACTTAATGAGA

CCTGACTTTAATAACGATGACTACGCCATCGCTTGTTGCGTT

AGTCCAATGATAGTAGGTAAACAAATGCAATTTTTCGGTGCA

AGAGCCAATTTGGCAAAGACAATGTTATATGCCATCAACGGT

G GTG TAG ATG AA AA ATTG A AG ATG C AAGTC G GTCCTAAATC A

GAACCAATTAAGGGTGACG 1 1 1 1 GAATTACGACGAAGTAATG GAAAGAATGGATCATTTTATGGACTGGTTGGCTAAGCAATAC

ATAACTGCATTGAACATCATACATTACATGCACGATAAGTATT

CTTACGAAGCATCTTTGATGGCATTACACGATAGAGACGTCA

TTAGAACAATGGCCTGTGGTATAGCTGGTTTGTCTGTTGCCG

CTGATTCCTTGAGTGCTATTAAATACGCCAAAGTAAAGCCTA

TCAGAGATGAAGACGGTTTAGCTATCGATTTTGAAATTGAGG

GTGAATACCCTCAATTCGGTAATAACGATCCAAGAGTTGATG

ACTTGGCAGTTGACTTAGTAGAAAGATTCATGAAAAAGATTC

AAAAATTGCATACCTATAGAGATGCTATTCCAACTCAATCCGT

TTTGACAATCACCAGTAACGTTGTATACGGTAAAAAGACAGG

CAACACCCCTGATGGTAGAAGAGCTGGTGCACCTTTCGGTC

CAGGTGCAAATCCAATGCACGGTAGAGACCAAAAAGGTGCC

GTCGCTTCTTTGACATCAGTTGCTAAATTGCCTTTTGCATATG

CCAAGGATGGTATCTCCTACACCTTCAGTATTGTTCCAAATG

CTTTGGGTAAAGATGACGAAGTAAGAAAGACAAACTTGGCTG

GTTTAATGGATGGTTATTTTCATCACGAAGCATCTATTGAAGG

TGGTCAACATTTGAACGTCAACGTTATGAACAGAGAAATGTT

GTTGGATGCAATGGAAAACCCTGAAAAGTACCCACAATTGAC

TATCAGAGTCTCAGGTTACGCTGTTAGATTCAATAGTTTGAC

CAAGGAACAACAACAAGACGTAATCACCAGAACTTTCACCCA

ATCTATGTAG

GAGCTCTTAATTAACAATTCTTCGCCAGAGGAATTC