Yeast systems biotechnology for the production of heterologous proteins

Systems biotechnology has been established as a highly potent tool for bioprocess development in recent years. The applicability to complex metabolic processes such as protein synthesis and secretion, however, is still in its infancy. While yeasts are frequently applied for heterologous protein production, more progress in this field has been achieved for bacterial and mammalian cell culture systems than for yeasts. A critical comparison between different protein production systems, as provided in this review, can aid in assessing the potentials and pitfalls of applying systems biotechnology concepts to heterologous protein producing yeasts. Apart from modelling, the methodological basis of systems biology strongly relies on postgenomic methods. However, this methodology is rapidly moving so that more global data with much higher sensitivity will be achieved in near future. The development of next generation sequencing technology enables an unexpected revival of genomic approaches, providing new potential for evolutionary engineering and inverse metabolic engineering.     

Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris

The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis‐sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography‐mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.     

Stratigraphic architecture of a Santonian mixed siliciclastic-carbonate succession (Catalonian Pyrenees, Spain)

Summary At Collades de Bastus, Catalonian Pyrences, a Santonian mixed siliciclastic-carbonate succession indicates two proximal-distal gradients, and records two styles of stratigraphical development upon relative sea-level change. The succession consists of four small-scale sequences (5.1 to 5.4) within the highstand systems tract of the. “Valicarca-5” depositional sequence of Simo (1993), and is topped by a drowning sequence (small-scale sequence 5.5). The investigated succession (Collades Member) accumulated near the margin of the south-Pyrenean shelf, shortly before development of the south-vergent Boixols thrust system. Deposition of the Collades Member commenced with moderate sea-level rise accompanied by increased siliciclastic input. In the larger, eastern outcrop sector the Collades Member consists of a succession of neritic marls with four intercalated intervals each deposited from a carbonate shelf. Each carbonate interval consists of stacked upward-shoaling cycles interpreted as parasequences. From bottom to top, most parasequences consist of a coral-sponge-rudist bioconstruction, a rudist biostrome, and bioclastic limestones. Depositional sequences 5.1 to 5.4 developed by overstep of shelf carbonates with neritic marls, corresponding to the transgressive systems tract (TST) and to part of the highstand systems tract(HST) The carbonate facies tract of the HST consists of stacked parasequences that become thinner up-section and record a westward component of progradation. Each highstand carbonate interval is overlain by a stack of carbonate parasequences that become thicker up-section and, down depositional dip, by neritic marls. Together, the upward-thickening parasequence stack and the laterally adjacent overlying succession of neritic marls comprise the TST and part of the HST of the successive sequence. The sequence boundary is the level of maximum shoaling within each carbonate shelf interval. The uppermost sequence 5.5 is a drowning sequence (cf. Simo 1993). In the western outcrop sector, the Collades Member consists of hummocky cross-laminated to bioturbated sandy calcarenites, of neritic marls and of relatively thin intervals of coral-sponge-rudist limestones. Sequence development may have started with deposition of sharp-based bedsets of sandy calcarenites that both eastward and up-section become thinner and grade into neritic marls. Together, the succession of sandy calcarenites and neritic marls may comprise the TST and, possibly, part of the HST. In the HST neritic marls and, locally, coral-sponge-rudist bioconstructions accumulated. Deposition of some calcarenite bedsets seems to have started near or closely after maximum progradation of each carbonate shelf in the eastern part of outcrop. The stratigraphic architecture of the Collades Member indicates, for the eastern outcrop sector, an east-west proximal-distal gradient, whereas the western sector records a west-east gradient. The opposite gradients result from outcrop intersection subparallel to oblique to general northward depositional dip, across two distinct shelf depositional systems.     

Segmentation gene expression in the mothmidge Clogmia albipunctata (Diptera, Psychodidae) and other primitive dipterans

 To obtain a clearer understanding of the evolutionary transition between short- and long-germ modes of embryogenesis in insects, we studied the expression of two gap genes hunchback (hb) and Krüppel (Kr) as well as the pair-rule gene even-skipped (eve) in the dipteran Clogmia albipunctata (Nematocera, Psychodidae). This species has features of both short- and long-germ mode of embryogenesis. In Clogmia hb expression deviates from that known in Drosophila in two main respects: (1) it shows an extended dorsal domain that is linked to the large serosa anlage, and (2) it shows a terminal expression in the proctodeal region. These expression patterns are reminiscent of the hb expression pattern in the beetle Tribolium, which has a short germ mode of embryogenesis. Krüppel expression, on the other hand, was found to be rather similar to the Drosophila expression, both at early and late stages. eve expression starts with six stripes formed at blastoderm stage, while the seventh is only formed after the onset of gastrulation and germband extension. Surprisingly, no segmental secondary Eve stripes could be observed in Clogmia although such segmental stripes are known from higher dipterans, beetles and hymenopterans. We therefore also studied another nematoceran, Coboldia, to address this question and found that some segmental stripes form by intercalation as in Drosophila, although belatedly. Our results suggest that Clogmia embryogenesis, both with respect to morphological and molecular characteristics represents an intermediate between the long-germ mode known from higher dipterans such as Drosophila, and the short-germ mode found in more ancestral insects. Received: 24 August 1998 / Accepted: 29 October 1998     

Evo-devo graduates to new levels

The second symposium on 'The Developmental Basis of Evolutionary Change' was held at the University of Chicago, IL, USA, from 25 to 28 October 2001.