Innovation trends in industrial biotechnology

Microbial fermentations are used for the sustainable production of a range of products. Due to increasing trends in the food sector toward plant-based foods and meat and dairy product substitutes, microbial fermentation will have an increasing role in this sector, as it will enable a sustainable and scalable production of valuable foods and food ingredients. Microbial fermentation will also be used to advance and expand the production of sustainable chemicals and natural products. Much of this market expansion will come from new start-ups that translate academic research into novel processes and products using state-of-the art technologies. Here, we discuss the trends in innovation and technology and provide recommendations for how to successfully start and grow companies in industrial biotechnology

Moderate expression of SEC16 increases protein secretion by Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous beta-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased beta-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous beta-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-beta-glucosidase, indicating that this mechanism is of general relevance. IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae. This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future

Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

Abstract The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein anterograde trafficking from the endoplasmic reticulum to the Golgi apparatus by the moderate overexpression of SEC16 could increase recombinant protein secretion in S. cerevisiae. In this study, the retrograde trafficking in a strain with moderate overexpression of SEC16 was engineered by overexpression of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of α-amylase but did not induce production of reactive oxygen species. An expanded ER membrane was detected in both the GCS1 and GLO3 overexpression strains. Physiological characterizations during batch fermentation showed that GLO3 overexpression had better effect on recombinant protein secretion than GCS1 overexpression. Additionally, the GLO3 overexpression strain had higher secretion of two other recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-α-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast

Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein anterograde trafficking from the endoplasmic reticulum to the Golgi apparatus by the moderate overexpression of SEC16 could increase recombinant protein secretion in S. cerevisiae. In this study, the retrograde trafficking in a strain with moderate overexpression of SEC16 was engineered by overexpression of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of alpha-amylase but did not induce production of reactive oxygen species. An expanded ER membrane was detected in both the GCS1 and GLO3 overexpression strains. Physiological characterizations during batch fermentation showed that GLO3 overexpression had better effect on recombinant protein secretion than GCS1 overexpression. Additionally, the GLO3 overexpression strain had higher secretion of two other recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-alpha-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase). Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases

UBB+1 reduces amyloid-beta cytotoxicity by activation of autophagy in yeast

UBB+1 is a mutated version of ubiquitin B peptide caused by a transcriptional frameshift due to the RNA polymerase II "slippage". The accumulation of UBB+1 has been linked to ubiquitin-proteasome system (UPS) dysfunction and neurodegeneration. Alzheimer\u27s disease (AD) is defined as a progressive neurodegeneration and aggregation of amyloid-beta peptides (A beta) is a prominent neuropathological feature of AD. In our previous study, we found that yeast cells expressing UBB+1 at lower level display an increased resistance to cellular stresses under conditions of chronological aging. In order to examine the molecular mechanisms behind, here we performed genome-wide transcriptional analyses and molecular/cellular biology assays. We found that low UBB+1 expression activated the autophagy pathway, increased vacuolar activity, and promoted transport of autophagic marker ATG8p into vacuole. Furthermore, we introduced low UBB+1 expression to our humanized yeast AD models, that constitutively express A beta 42 and A beta 40 peptide, respectively. The co-expression of UBB+1 with A beta 42 or A beta 40 peptide led to reduced intracellular A beta levels, ameliorated viability, and increased chronological life span. In an autophagy deficient background strain (atg1 Delta), intracellular A beta levels were not affected by UBB+1 expression. Our findings offer insights for reducing intracellular A beta toxicity via autophagydependent cellular pathways under low level of UBB+1 expression

The impact of respiration and oxidative stress response on recombinant ?-amylase production by Saccharomyces cerevisiae

Studying protein production is important for fundamental research on cell biology and applied research for biotechnology. Yeast Saccharomyces cerevisiae is an attractive workhorse for production of recombinant proteins as it does not secrete many endogenous proteins and it is therefore easy to purify a secreted product. However, recombinant production at high rates represents a significant metabolic burden for the yeast cells, which results in oxidative stress and ultimately affects the protein production capacity. Here we describe a method to reduce the overall oxidative stress by overexpressing the endogenous HAP1 gene in a S. cerevisiae strain overproducing recombinant α-amylase. We demonstrate how Hap1p can activate a set of oxidative stress response genes and meanwhile contribute to increase the metabolic rate of the yeast strains, therefore mitigating the negative effect of the ROS accumulation associated to protein folding and hence increasing the production capacity during batch fermentations

Efficient protein production by yeast requires global tuning of metabolism

The biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. We confirm our findings by inverse engineering and physiological characterization and show that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion

«En kvalitativ studie: Et strategisk møte med millennium generasjonen»

Temaet for denne oppgaven er lojalitet blant de norske ansatte av millennium generasjonen i organisasjoner. For det første har vi funnet ut at den mest sentrale forskningen av lojalitet i organisasjoner som er relevant for vår oppgave, er rundt begrepet organisasjonsforpliktelse. For det andre viser studie at for å kunne besvare problemstillingen, er det viktig å utforske hoved karakteristika av millennium og vi har derfor fokusert på å kunne “definere” millennium så godt som mulig. Det er kritikkverdig at vi ikke kan skjære alle under én kam, og vi har derfor begrenset kriterier for utvalget av respondenter for å vise til tydelige hovedtrekk. For det tredje viser resultatet at organisasjonsforpliktelse handler i stor grad om å imøtekomme forventninger og krav fra de ansatte, samt å få de ansatte til å føle seg komfortable og kompetente i sin egen rolle på arbeidsplassen. Det er også en viktig faktor at millennium føler at de blir sett, hørt og involvert. Dette innebærer blant annet muligheter for utfordrende arbeidsoppgaver, mer ansvar og videre utvikling. Prestasjon og innsats må belønnes og de ansatte trenger å føle seg ivaretatt og inkludert i organisasjonen. Forpliktende ansatte har en effekt på produktiviteten, kulturen og holdningene på arbeidsplassen. Det er dernest viktig for ledelsen å bevisstgjøre de ansatte om organisasjonens verdier, visjoner og mål (Business News Daily). Organisasjonsforpliktelse fører til mer engasjerte og motiverte ansatte som ønsker å bli værende i bedriften, samt at det fører til mindre fravær, lavere turnover, høyere produktivitet og lavere kostnader