Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria

Abstract

BackgroundThere is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.ResultsHere we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.ConclusionsWe present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.Electronic supplementary materialThe online version of this article (10.1186/s12896-017-0393-y) contains supplementary material, which is available to authorized users.

Establishing BARA: Biological Nitrogen Fixation for Future Agriculture

“…Contemporaneous with that report, the Nfixing genes (nif) of Klebsiella pneumoniae had been inserted into Escherichia coli, causing the nonfixing bacterium capable of growing in the absence of combined N (Dixon & Postgate, 1972). Since then, there has been some research and attempts on introducing N fixation in nonfixing plants and transferring nif genes into heterologous hosts (Yang et al, 2018;Good, 2018;Burén, 2018;Allen et al, 2107;Pérez-González, 2017;López-Torrejón, 2016); however, scientists still lack a comprehensive understanding of the composition of the microorganisms that carry out N fixation processes. To better understand N fixation processes and other biological functions that microorganisms provide plants or their hosts, it is first essential to determine why a microorganism or community evolved, why it persists, and what benefits it imparts to the host itself, other organisms, and the community.…”

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond

“…The third strategy was designed for Saccharomyces cerevisiae , which has well‐characterized loci conferring high‐level expression of transgenes (Flagfeldt, Siewers, Huang, & Nielsen, 2009). This latter strategy involves backbone adaptation in order to create two sets of GB destination plasmids, each of them containing a pair of widely used homology arms adjacent to the GB cassette (Pérez‐González et al., 2017). This variety of strategies and some of their published applications are summarized in Table 1, and show the suitability of GB to accommodate the specificities of organisms other than plants.…”

“…Although GB was initially developed for nuclear transformation in plants (Sarrion-Perdigones et al, 2011), it has been adapted since then for its use in plastids (Vafaee, Staniek, Mancheno-Solano, & Warzecha, 2014), yeast (Pérez-González et al, 2017), filamentous fungi (Hernanz-Koers et al, 2018), and human cells (Sarrion-Perdigones et al, 2019). The creation of new parts (i.e., promoters, new codon-optimized CDSs…) is usually enough to make GB fully functional for transfection in any other organisms in addition to plants, fungi, and human cells.…”

Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering

“…However, these non-modular approaches depend on sequence overlaps to direct assembly, so they have a greater need for custom oligonucleotide primers (typically one pair per junction between parts) and sequence verification (due to PCR steps) and are less suitable for combinatorial assembly due to lower efficiency (meaning fewer clones are obtained following transformation so smaller libraries are generated) and greater potential for bias particularly due to repetitive sequences. Modular multi-part DNA assembly methods include Golden Gate assembly (2)(3)(4) (and variants (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) ), BASIC (15) , BioBrick assembly (16) (and variants such as BglBrick (17,18) ) and Gateway cloning (19)(20)(21) . Bespoke multi-part DNA assembly methods include Gibson Assembly (22) , AQUA cloning (23) , Twin Primer Assembly (24) , ligase cycling reaction (25) , SLIC (26) , SLiCE (27) , overlap extension PCR (28) and CPEC (29) .…”

“…Of the modular DNA assembly methods, Golden Gate assembly (2, 3) and variants (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) are particularly widely used, including for construction of metabolic pathway-encoding constructs (10,14,(40)(41)(42) . Golden Gate uses type IIS restriction endonucleases, which are similar to classical type II restriction endonucleases, except their restriction site is offset from their recognition site, rather than within it.…”