Biological Parts for Kluyveromyces marxianus Synthetic Biology

Abstract

Kluyveromyces marxianus is a non-conventional yeast whose physiology and metabolism lends itself to diverse biotechnological applications. While the wild-type yeast is already in use for producing fragrances and fermented products, the lack of standardised tools for its genetic and metabolic engineering prevent it from being used as a next-generation cell factory for bio-based chemicals. In this paper, we bring together and characterise a set of native K. marxianus parts for the expression of multiple genes for metabolic engineering and synthetic biology. All parts are cloned and stored according to the MoClo/Yeast Tool Kit standard for quick sharing and rapid construction. Using available genomic and transcriptomic data, we have selected promoters and terminators to fine-tune constitutive and inducible gene expression. The collection includes a number of known centromeres and autonomously replication sequences (ARS). We also provide a number of chromosomal integration sites selected for efficiency or visible phenotypes for rapid screening. Finally, we provide a single-plasmid CRISPR/Cas9 platform for genome engineering and facilitated gene targeting, and rationally create auxotrophic strains to expand the common range of selection markers available to K. marxianus . The curated and characterised tools we have provided in this kit will serve as a base to efficiently build next-generation cell factories from this alternative yeast. Plasmids containing all parts are available at Addgene for public distribution.

Rational engineering ofKluyveromyces marxianusto create a chassis for the production of aromatic products

“…Level I plasmids for each gene and the desired promoter and terminator were then assembled into transcriptional units (TUs) by Golden Gate assembly with BsaI/T7 DNA ligase, which were then transformed into E.coli and verified by colony PCR using OneTaq Quick-Load Polymerase (M0486L, NEB) and restriction digestion by NotI (FD0596, Fisher Scientific). The K. marxianus promoters and terminators used for these plasmids have been described elsewhere (Rajkumar et al, 2019).Depending on their intended positions in a multi-TU plasmid, TUs in level II plasmids were cloned with directionspecific connectors flanked by BsmBI sites provided in the Yeast Toolkit to ensure directional assembly. Depending on the planned constructs, some TUs were cloned multiple times, with different connectors.…”

“…All level I and level II plasmids constructed for this study are listed in Table S1, and the primers used to create them in Table S2. More detailed information on Golden Gate assembly of the plasmids can be found in refs (Lee et al, 2015;Rajkumar et al, 2019).…”

“…The subsequent overexpression of genes in the resulting strains (Table 2, Fig. S1) were done using the cloning vectors pI2-MTU-DO-G418 and pI3-MTU-DO-G418 that target integration sites on chromosomes V and IV respectively and previously characterized by us (Rajkumar et al, 2019). Primer sequences for colony PCR verification are listed in Table S2.…”

“…This enzyme converts prephenate to 4-hydroxphenylpyruvate, committing it to tyrosine synthesis: by reducing its expression as low as possible without creating a tyrosine auxotroph, more prephenate from Aro7p should be available for conversion to phenylpyruvate and phenylalanine. To achieve this, we substituted the native KmTYR1 promoter (TYR1pr) with one of two weaker promoters, GDH2pr and REV1pr, that we had previously characterized (Rajkumar et al, 2019). Both strains, ASR.047kd and ASR.047.kd2 increased the relative proportion of 2-PE to tyrosol, with the strongest effect seen when REV1pr (strain KmASR.047kd2) was used Fig.…”

“…AaTEF1pr-SpCas9-ScPHO5t ScTDH3pr-HH-gRNA empty -HDV-ScCYC1t hphMX (Varela et al, 2019) This work pI2-MTU-DO-G418 Integrative plasmid with GFP drop-out, kanMX, targeting integration site I2 (Rajkumar et al, 2019) This work pI4-MTU-DO-G418 Integrative plasmid with GFP drop-out, kanMX, targeting integration site I4 (Rajkumar et al, 2019) This are functional, with their episomal overexpression leading to the production of Ehrlich alcohols of phenylalanine (white) and tyrosine (grey) from the overflow of their increased synthesis, as well as excess shikimate (dark grey). All compounds were measured by HPLC in culture medium after 48h growth at 30°C as described in the Methods.…”