Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

<p>Abstract</p> <p>Background</p> <p>Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory <it>Saccharomyces cerevisiae </it>strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.</p> <p>Results</p> <p>Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory.</p> <p>Conclusions</p> <p>The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory <it>S. cerevisiae </it>strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.</p

Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

BACKGROUND: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. RESULTS: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. CONCLUSION: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations

A phenotypic study of a unique respiratory strain of Saccharomyces cerevisiae and its application in protein production

During aerobic growth at high glucose concentrations, the yeast Saccharomyces cerevisiae prefers to catabolise glucose through fermentation rather than through respiration. Previously fully respiratory growth could only be achieved when a low external glucose concentration was maintained, in fed-batch and continuous cultures, but since the development of the respiratory strain, TM6*, in our laboratory this can now be achieved even at high external glucose concentration.In this thesis the transferability of the respiratory phenotype has been confirmed by integration of the chimeric TM6* construct into a hxt1-hxt7 wine strain. The genomic profile of the resulting V5.TM6*P strain was investigatedby miniarray and the results showed that genes involved in the TCA cycle, glyoxylate cycle, gluconeogenesis pathway and the respiratory chain were upregulated compared to the parental strain. The transcriptional responses in therespiratory strain were not altered when the external glucose concentration changed in contrast to the wild type strain where a change in transcription (both up and down regulated) was observed when glucose was changed from high to low concentration in a batch culture. It was further observed that the respiratory strain had a lower concentration of fructose-1, 6-bisphosphate compared to its parent strain. Modelling of regulation of glycolysis showed that there are differences in the kinetics of the phosphofructokinase between the TM6* strain and the wild type; suggesting a key role of this enzyme in the shiftbetween a respiro-fermentative and a respiratory metabolism.As a result of its respiratory metabolism, the TM6* strain is able to produce more biomass, making the TM6* strain an excellent candidate as a protein production host. In this thesis a two-fold yield improvement, in overproductionof a membrane protein, Fps1, is demonstrated, compared to two wild type strains

A phenotypic study of a unique respiratory strain of Saccharomyces cerevisiae and its application in protein production

During aerobic growth at high glucose concentrations, the yeast Saccharomyces cerevisiae prefers to catabolise glucose through fermentation rather than through respiration. Previously fully respiratory growth could only be achieved when a low external glucose concentration was maintained, in fed-batch and continuous cultures, but since the development of the respiratory strain, TM6*, in our laboratory this can now be achieved even at high external glucose concentration. In this thesis the transferability of the respiratory phenotype has been confirmed by integration of the chimeric TM6* construct into a hxt1-hxt7 wine strain. The genomic profile of the resulting V5.TM6*P strain was investigated by miniarray and the results showed that genes involved in the TCA cycle, glyoxylate cycle, gluconeogenesis pathway and the respiratory chain were upregulated compared to the parental strain. The transcriptional responses in the respiratory strain were not altered when the external glucose concentration changed in contrast to the wild type strain where a change in transcription (both up and down regulated) was observed when glucose was changed from high to low concentration in a batch culture. It was further observed that the respiratory strain had a lower concentration of fructose-1, 6-bisphosphate compared to its parent strain. Modelling of regulation of glycolysis showed that there are differences in the kinetics of the phosphofructokinase between the TM6* strain and the wild type; suggesting a key role of this enzyme in the shift between a respiro-fermentative and a respiratory metabolism. As a result of its respiratory metabolism, the TM6* strain is able to produce more biomass, making the TM6* strain an excellent candidate as a protein production host. In this thesis a two-fold yield improvement, in overproduction of a membrane protein, Fps1, is demonstrated, compared to two wild type strains