Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus

Abstract

The emergence of CRISPR-Cas9 for targeted genome editing and regulation has enabled the manipulation of desired traits and enhanced strain development of nonmodel microorganisms. The natural capacity of the yeast Kluyveromyces marxianus to produce volatile esters at high rate and at elevated temperatures make it a potentially valuable production platform for industrial biotechnology. Here, we identify the native localization of ethyl acetate biosynthesis in K. marxianus and use this information to develop a multiplexed CRISPRi system for redirecting carbon flux along central metabolic pathways, increasing ethyl acetate productivity. First, we identified the primary pathways of precursor and acetate ester biosynthesis. A genetic knockout screen revealed that the alcohol acetyltransferase Eat1 is the critical enzyme for ethyl, isoamyl, and phenylethyl acetate production. Truncation studies revealed that high ester biosynthesis is contingent on Eat1 mitochondrial localization. As ethyl acetate is formed from the condensation of ethanol and acetyl-CoA, we modulated expression of the TCA cycle and electron transport chain genes using a highly multiplexed CRISPRi approach. The simultaneous knockdown of ACO2b, SDH2, RIP1, and MSS51 resulted in a 3.8-fold increase in ethyl acetate productivity over the already high natural capacity. This work demonstrates that multiplexed CRISPRi regulation of central carbon flux, supported by a fundamental understanding of pathway biochemistry, is a potent strategy for metabolic engineering in nonconventional microorganisms.

From Eat to trEat: Engineering the mitochondrial Eat1 enzyme for enhanced ethyl acetate production in Escherichia coli

“…This may suggest that similar events occur during the processing of Eat1 in K. marxianus. The fact that Kma trEat1 S-20 is unable to migrate to the mitochondria in K. marxianus (Löbs et al, 2018), but did not show improved performance in E. coli supports this hypothesis. It is likely that Eat1 homologs from other yeasts undergo different processing events as well.…”

“…The strains producing Kma trEat1 variants that were truncated at the predicted positions (Kma trEat1 Y-19 and S-20) did not show substantially improved ethyl acetate production relative to the unprocessed Kma Eat1. However, removing 19 AA from Kma Eat1 (Kma trEat1 S-20) was indeed sufficient to fully prevent Eat1 from being targeted to the mitochondria of K. marxianus (Löbs et al, 2018). The ethyl acetate production only improved when an additional 7-11 AA residues were removed from the N-terminus of Kma Eat1.…”

“…A key difference between Atf1 and Eat1 is their cellular location in yeast. Atf1 localises to lipid particles in the cytosol (Verstrepen et al, 2004;Lin and Wheeldon, 2014), while Eat1 homologs are located in yeast mitochondria (Kruis, Mars, et al, 2018;Löbs et al, 2018). Most mitochondrial enzymes like Eat1 are encoded on the nuclear genome and synthesized in the cytoplasm.…”

From Eat to trEat: engineering the mitochondrial Eat1 enzyme for enhanced ethyl acetate production in Escherichia coli

“…A key difference between Atf1 and Eat1 is their cellular location in yeast. Atf1 localises to lipid particles in the cytosol [13,27], while Eat1 homologs are located in yeast mitochondria [9,15]. Most mitochondrial enzymes like Eat1 are encoded on the nuclear genome and synthesised in the cytoplasm.…”

A CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae

“…CRISPR/Cas9 gene editing is extensively used in metabolic pathway engineering in Saccharomyces cerevisiae and other yeasts, which is particularly relevant to industrial applications (Bracher et al, ; Löbs, Schwartz, Thorwall, & Wheeldon, ; Takayama et al, ). In addition to rationale design, industrial strain improvement is often achieved by directed evolution experiments (reviewed in Steensels et al, ).…”