Deletion of hxk1 gene results in derepression of xylose utilization in Scheffersomyces stipitis

Abstract

A major problem in fermenting xylose in lignocellulosic substrates is the presence of glucose and mannose which inhibit xylose utilization. Previous studies showed that catabolite repression in some yeasts is associated with hexokinases and that deletion of one of these gene(s) could result in derepressed mutant strain(s). In this study, the hxk1 encoding hexokinase 1 in Scheffersomyces stipitis was disrupted. The ∆hxk1 SS6 strain retained the ability to utilize the main hexoses and pentoses commonly found in lignocellulosic hydrolysates as efficiently as the wild-type (WT) strain. SS6 also fermented the dominant sugars to ethanol; however, on xylose, the ∆hxk1 strain produced more xylitol and less ethanol than the WT. On mixed sugars, as expected the WT utilized glucose ahead of xylose and xylose utilization did not commence until all the glucose was consumed. In contrast, the ∆hxk1 mutant showed derepression in that it started to utilize xylose even when considerable glucose (about 1.72%, w/v) remained in the medium. Similarly, mannose did not repress xylose utilization by the ∆hxk1 mutant and xylose and mannose were simultaneously utilized. The results are of interest in efforts to engineer yeast strains capable of efficiently utilizing glucose and xylose simultaneously for lignocellulosic biomass conversion.

Genome and metabolic engineering in non-conventional yeasts: Current advances and applications

“… [3] S. stipitis Chemicals Ethanol from lignocellulosic feeds Reviewed in Ref. [21] Fumaric acid [73] Lactic acid [74] Xylitol [75] H. polymorpha Proteins Hepatitis B surface antigen (HBsAg) Insulin IFNα-2a Reviewed in Ref. [76] Hexose oxidase Phytase Chemicals Ethanol from various carbon sources Reviewed in Ref.…”

“…Single gene deletions have also been achieved through genomic integration using a selectable genetic marker. For example, a HR method was used to create a HXK1 deficient strain lacking glucose repression and a XYL2 deficient strain that produces xylitol from xylose [75] . In another example, S. stipitis was engineered to efficiently produce lactic acid through random integration of a heterologous LDH gene, with engineered strains producing up to 58 g L −1 lactate from 100 g L −1 xylose [74] .…”

Construction of advanced producers of first- and second-generation ethanol in Saccharomyces cerevisiae and selected species of non-conventional yeasts (Scheffersomyces stipitis, Ogataea polymorpha)

“…To obtain strains of S. stipitis that can ferment simultaneously glucose and xylose, 2-deoxyglucoseresistant mutants were isolated [173]. This was also accomplished by deletion of the gene HXK1 coding for hexokinase I [26]. The last experiment could be considered as one of very few, in which a metabolic engineering approach was applied for this organism [26].…”

“…This was also accomplished by deletion of the gene HXK1 coding for hexokinase I [26]. The last experiment could be considered as one of very few, in which a metabolic engineering approach was applied for this organism [26]. As a rule, experiments in metabolic engineering of S. stipitis are hampered by our limited knowledge of the limiting steps of the fermentation process.…”

Evolutionary Engineering Improved d-Glucose/Xylose Cofermentation of Yarrowia lipolytica

“…Previous studies have found that strains that can utilize xylose in the presence of D-glucose homologues (structurally similar to D-glucose but not metabolizable), can co-ferment Dglucose/xylose without obvious preference [20][21][22][23][24]. Adaptive laboratory evolution of strains in a medium composing of xylose and the D-glucose analog 2-deoxyglucose (hereafter abbreviated as dG)…”