Characterization and optimization of the LAC4 upstream region for low‐leakage expression in Kluyveromyces marxianus

Abstract

Kluyveromyces marxianus is a promising host for the production of heterologous proteins, chemicals, and bioethanol. One superior feature of this species is its capacity to assimilate lactose, which is rendered by the LAC12–LAC4 gene pair encoding a lactose permease and a β‐galactosidase enzyme. Little is known about the regulation of LAC4 in K. marxianus. In this study, we showed the presence of weak glucose repression in the regulation of LAC4 and that might contribute to the leaky expression of LAC4 in the glucose medium. In a mutagenesis screen of 1000‐bp LAC4 upstream region, one mutant region, named H1, drove low‐leakage expression of a URA3 reporter gene in glucose medium. Two mutations inside a polyadenosine stretch (poly(A)) of 5′ UTR were major contributors to the low‐leakage phenotype of H1. H1 directed low‐leakage expression of GFP on a plasmid and that of LAC4 in situ in the glucose medium, which was not due to the reduction of mRNA levels. Meanwhile, H1 did not affect the induction of GFP or LAC4 by lactose. Cre recombinase expressed by H1 caused lower toxicity in the repressive condition and achieved higher yield after induction, compared with that expressed by a wild‐type LAC4 upstream region or a strong INU1 promoter. Our study suggested that poly(A) inside 5′ UTR played a role in regulating the expression of LAC4 in the repressive condition. Meanwhile, H1 provided a base for the development of a strict inducible system for expressing industrial proteins, especially toxic proteins.

Bioprospecting Kluyveromyces marxianus as a Robust Host for Industrial Biotechnology

“…The expression of β-galactosidases in K. marxianus is regulated by its natural inducers, lactose, and galactose ( Liu et al, 2021 ). Nonetheless, β-galactosidases production depends on the concentration of substrate.…”

Establishment of a Cre-loxP System Based on a Leaky LAC4 Promoter and an Unstable panARS Element in Kluyveromyces marxianus

“…LHP620 plasmid expressing Cre recombinase was described before [18]. ARS1 amplified from the genomic DNA of FIM1 and panARS amplified from the genomic DNA of ATCC8585 were inserted into Pvu II and Sal I sites of LHP620 to obtain LHZ892 and LHZ893, respectively.…”

“…These results indicated that predicted centromeres were functional in K. marxianus. In our previous study, we showed that expression of Cre recombinase by a strong INU1 promoter caused toxicity to K. marxianus cells and the toxicity was alleviated by using a leaky LAC4 promoter in the glucose medium [18]. The LAC4 promoter was induced by lactose or galactose and the activation could be repressed by glucose [18].…”

“…Constitutive overexpression of Cre leads to at least two problems. First, overexpression of Cre recombinase caused toxicity and inhibited the growth of certain hosts, including mammalian cells, Schizosaccharomyces pombe and Kluyveromyces marxianus [16][17][18]. In mammalian cells, the toxicity of Cre depended on the endonuclease activity of Cre recombinase, which might induce DNA damage, such as chromosomal aberrations and sister chromatid exchanges [16].…”

“…marxianus is a natural thermotolerant yeast, which is a promising host for the production of cellulosic ethanol, chemicals and heterologous proteins [31][32][33][34]. In our previous study, the toxicity of Cre recombinase has been proved in K. marxianus [18]. It raised the demand for strict regulation of Cre expression in K. marxianus.…”