Synthetic Biology for Cellular Remodelling to Elicit Industrially Relevant Microbial Phenotypes

Industrial microbiology is proposing an increasing number of bio-based processes that are ready to move from the validation to the demonstration step, with the industrial world being more open to this opportunity for a change. The challenge is therefore to make such processes viable and competitive. When moving from the lab to the industrial scale, the degree of complexity is increasing, and the engineered cell factories very often display emerging properties that can be explained only from a systems perspective. Unfortunately, cellular rewiring often leads to a lower accumulation of the desired product. Synthetic biology is willing to take advantage from the knowledge on mechanisms involved in cellular homeostasis and, thanks to the principles of abstraction, modularity and standardisation, translate them into more efficient cell factories. Indeed, this novel approach to potentiate the power of metabolic engineering can be applied not only to a specific metabolic pathway but can be extended to networks indirectly connected to the pathway of interest. In this chapter, some of the principal synthetic tools developed to regulate or redirect the remodelling of cell factories, from genomic to metabolic level, with the aim to obtain higher titers, yield and productivity of bio-based products will be described and commented

Metabolically Engineered Yeasts: 'Potential' Industrial Applications

Industrial biotechnology and metabolic engineering can offer an innovative approach to solving energy and pollution problems. The potential industrial applications of yeast are reviewed here

Camelina sativa meal hydrolysate as sustainable biomass for the production of carotenoids by Rhodosporidium toruloides

Background: As the circular economy advocates a near total waste reduction, the industry has shown an increased interest toward the exploitation of various residual biomasses. The origin and availability of biomass used as feedstock strongly affect the sustainability of biorefineries, where it is converted in energy and chemicals. Here, we explored the valorization of Camelina meal, the leftover residue from Camelina sativa oil extraction. In fact, in addition to Camelina meal use as animal feed, there is an increasing interest in further valorizing its macromolecular content or its nutri- tional value.Results: Camelina meal hydrolysates were used as nutrient and energy sources for the fermentation of the carot- enoid-producing yeast Rhodosporidium toruloides in shake flasks. Total acid hydrolysis revealed that carbohydrates accounted for a maximum of 31 \ub1 1.0% of Camelina meal. However, because acid hydrolysis is not optimal for sub- sequent microbial fermentation, an enzymatic hydrolysis protocol was assessed, yielding a maximum sugar recovery of 53.3%. Separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), andSSF preceded by presaccharification of Camelina meal hydrolysate produced 5 \ub1 0.7, 16 \ub1 1.9, and 13 \ub1 2.6 mg/L of carotenoids, respectively. Importantly, the presence of water-insoluble solids, which normally inhibit microbial growth, correlated with a higher titer of carotenoids, suggesting that the latter could act as scavengers.Conclusions: This study paves the way for the exploitation of Camelina meal as feedstock in biorefinery processes. The process under development provides an example of how different final products can be obtained from this side stream, such as pure carotenoids and carotenoid-enriched Camelina meal, can potentially increase the initial value of the source material. The obtained data will help assess the feasibility of using Camelina meal to generate high value- added products

Effect of HXT1 and HXT7 hexose transporter overexpression on wild-type and lactic acid producing Saccharomyces cerevisiae cells

<p>Abstract</p> <p>Background</p> <p>Since about three decades, <it>Saccharomyces cerevisiae </it>can be engineered to efficiently produce proteins and metabolites. Even recognizing that in baker's yeast one determining step for the glucose consumption rate is the sugar uptake, this fact has never been conceived to improve the metabolite(s) productivity.</p> <p>In this work we compared the ethanol and/or the lactic acid production from wild type and metabolically engineered <it>S. cerevisiae </it>cells expressing an additional copy of one hexose transporter.</p> <p>Results</p> <p>Different <it>S. cerevisiae </it>strains (wild type and metabolically engineered for lactic acid production) were transformed with the <it>HXT</it>1 or the <it>HXT</it>7 gene encoding for hexose transporters.</p> <p>Data obtained suggest that the overexpression of an Hxt transporter may lead to an increase in glucose uptake that could result in an increased ethanol and/or lactic acid productivities. As a consequence of the increased productivity and of the reduced process timing, a higher production was measured.</p> <p>Conclusion</p> <p>Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is a well established concept. A high production relies on a multi-factorial system. We showed that by modulating the first step of the pathway leading to lactic acid accumulation an improvement of about 15% in lactic acid production can be obtained in a yeast strain already developed for industrial application.</p

Direct and indirect approaches for the improvement of heterologous proteins secretion levels in Zygosaccharomyces bailii

n/

Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype

<p>Abstract</p> <p>Background</p> <p><it>Zygosaccharomyces bailii </it>is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of <it>Saccharomyces cerevisiae </it>and <it>Pichia pastoris</it>, deleted in the <it>GAS</it>1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the <it>GAS</it>1 homologue of <it>Z. bailii </it>and the evaluation of its deletion on recombinant protein secretion.</p> <p>Results</p> <p>The <it>GAS</it>1 homologue of <it>Z. bailii </it>was cloned by PCR, and when expressed in a <it>S. cerevisiae GAS</it>1 null mutant was able to restore the parental phenotype. The respective <it>Z. bailii</it> Δ<it>gas</it>1 deleted strain was obtained by targeted deletion of both alleles of the <it>ZbGAS</it>1 gene with deletion cassettes having flanking regions of ~400 bp. The morphological and physiological characterization of the <it>Z. bailii </it>null mutant resulted very similar to that of the corresponding <it>S. cerevisiae </it>mutant. As for <it>S. cerevisiae</it>, in the <it>Z. bailii </it>Δ<it>gas</it>1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the <it>Candida rugosa </it>lipase CRL1 and of the <it>Yarrowia lipolytica </it>protease XPR2, while for human IL-1β secretion disruption had no relevant effect.</p> <p>Conclusions</p> <p>The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that <it>Z. bailii </it>is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an "always working" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product.</p

Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation

BACKGROUND: Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. RESULTS: We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified. CONCLUSIONS: We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase

Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export

BACKGROUND: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is becoming a well established concept. For the production of important metabolites, but also for a better understanding of the fundamentals of cell biology, detailed studies are required. In this work we analysed the lactate production from metabolic engineered Saccharomyces cerevisiae cells expressing a heterologous lactate dehydrogenase (LDH) gene. The LDH gene expression in a budding yeast cell introduces a novel and alternative pathway for the NAD(+ )regeneration, allowing a direct reduction of the intracellular pyruvate to lactate, leading to a simultaneous accumulation of lactate and ethanol. RESULTS: Four different S. cerevisiae strains were transformed with six different wild type and one mutagenised LDH genes, in combination or not with the over-expression of a lactate transporter. The resulting yield values (grams of lactate produced per grams of glucose consumed) varied from as low as 0,0008 to as high as 0.52 g g(-1). In this respect, and to the best of our knowledge, higher redirections of the glycolysis flux have never been obtained before without any disruption and/or limitation of the competing biochemical pathways. CONCLUSION: In the present work it is shown that the redirection of the pathway towards the lactate production can be strongly modulated by the genetic background of the host cell, by the source of the heterologous Ldh enzyme, by improving its biochemical properties as well as by modulating the export of lactate in the culture media

The first auxotrophic mutant of Zygosaccharomyces bailii for recombinant productions: a road to practical applications

n/