Abstract
Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiae BIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiae IMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.
Highlights
Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life
In biotin-free SMG, the abundance of the auxotroph was reduced to 2% of the total fluorescent population after three batch cultivation cycles (Fig. 7, bottom). These results indicated that while the expression of C. fabianii BIO1 (CfBIO1) (IMX2238) provided a significant growth advantage in biotinfree SMG, biotin is not released into the medium in amounts sufficient to support the growth of a complete biotin auxotroph or of a strain with a very low biosynthesis capacity, such as CEN.PK113-7D
We report a novel and simple metabolic engineering strategy to convert biotin-auxotrophic S. cerevisiae strains into biotin prototrophs
Summary
An important cofactor for carboxylases, is essential for all kingdoms of life. IMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae. The omission of biotin from medium formulations would enable the design of less expensive, easier-to-handle media with a longer shelf life Such advantages have been reported for heterologous protein production with a Komagatella phaffi (formerly known as Pichia pastoris) strain expressing the S. cerevisiae biotin biosynthesis genes BIO1 and BIO6. This biotin-prototrophic strain exhibited slower growth in the absence of biotin than in its presence [7]. The use of fully biotinprototrophic strains of yeast in fermentation processes that do not contain biotin may reduce the impact of contamination with wild yeast or even bacterial strains that grow slower or not at all in the absence of this cofactor [8,9,10]
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