Abstract
An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds.
Highlights
Typical industrial substrates derived from plant biomass such as sugarcane juice, starch, and ligno-cellulosic hydrolysates are subjected to harsh physical-chemical treatments that result in lowering nutritional properties (Basso et al, 2008) by affecting stability of vitamins (Brown and Du Vigneaud, 1941; Mauri et al, 1989; Saidi and Warthesen, 1983; Schnellbaecher et al, 2019)
In absence of oxygen, the specific growth rate of strain IMX2035 on biotin-supplement medium was lower than observed in cultures of reference strains (Fig. 2C). These results demonstrated that expression of the E. coli keto-8-aminopelargonic acid (KAPA) pathway in S. cerevisiae supports conversion of malonyl-coenzyme A (CoA) into KAPA and pro motes biotin-independent anoxic growth of S. cerevisiae
This study shows that functional expression of the E. coli KAPA pathway yields S. cerevisiae strains that are biotin prototrophic irrespective of the applied oxygen regime and whose spe cific growth rates can be further improved by tuning of the expression levels of specific KAPA-pathway enzymes
Summary
Typical industrial substrates derived from plant biomass such as sugarcane juice, starch, and ligno-cellulosic hydrolysates are subjected to harsh physical-chemical treatments that result in lowering nutritional properties (Basso et al, 2008) by affecting stability of vitamins (Brown and Du Vigneaud, 1941; Mauri et al, 1989; Saidi and Warthesen, 1983; Schnellbaecher et al, 2019). Evolutionary engineering and rational metabolic en gineering strategies led to the selection of yeast strains whose growth in biotin-free medium was as fast as the growth of the reference strain in the presence of biotin (Bracher et al, 2017; Wronska et al, 2020) In both cases, acquisition of the biotin prototroph phenotype was restricted to the presence of oxygen (Wronska et al, 2020)
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