Expression of Vitreoscilla hemoglobin enhances growth and levels of α-amylase in Schwanniomyces occidentalis

Abstract

A metabolic engineering approach was exploited to improve growth and protein secretion in the non-conventional yeast, Schwanniomyces occidentalis. Vitreoscilla hemoglobin (VHb) gene was expressed in S. occidentalis under the control of the native alpha-amylase (AMY1) promoter. Expression of VHb was confirmed by reverse transcriptase polymerase chain reaction and Western blot hybridization analysis. Effect of VHb on growth and protein secretion was studied in synthetic medium under both limiting and non-limiting dissolved oxygen conditions. Under both conditions, VHb-expressing cells exhibited higher oxygen uptake and higher specific growth rates. Levels of extracellular alpha-amylase were also elevated in the VHb-transformed strain relative to the control strain. In amylase production medium, VHb-expressing cells showed 3-fold elevated levels of alpha-amylase and a 31% increase in the total secreted protein under oxygen-limiting environment. VHb was found to localize in the mitochondria in addition to its cytoplasmic location. Inhibition of respiration by antimycin A resulted in the loss of the growth-enhancing effects of VHb. A 2.5-fold increase in the cytochrome c oxidase (COX) activity was observed in VHb-expressing cells relative to the control. In addition to this, exogenously added VHb in the assay mixture augmented COX activity.

Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccharopolyspora spinosa

“…As P vgb is not functional in most gram-positive bacteria or eukaryotic organisms, various promoter strategies have been adopted by researchers to induce expression of VHb in heterologous hosts. Suthar et al [18] used an α-amylase promoter P AMY1 in Schwanniomyces occidentalis; Horng et al [22] applied P BAD in E. coli; Feng et al [20] used P luxs in Bacillus thuringiensis; and Xiong et al [16] employed P dsz in Rhodococcus erythropolis. In this study, the first and second codons of vgb were mutated to GTG and CTG respectively by site-directed mutagenesis and SOE-PCR.…”

“…VHb has become a versatile tool in high-density fermentation of micro-organisms susceptible to oxygen supply [10,11]. Recent studies of VHb applications include: (i) promoting the biosyntheses of biochemical precursors (such as Dihydroxyacetone) [1215]; (ii) improving bioremediation (improving desulfidations in Rhodococcus erythropolis) [16]; (iii) enhancing the production of important enzymes (such as D-amino acid oxidase) [17,18]; (iv) improving the biosyntheses of antibiotics and biopesticides [19,20], and (v) promoting the production of biopolymer materials (such as poly-γ-glutamic acid) [21,22].…”

Recent advances in understanding the structure, function, and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla

“…In most cases bacteria have been used as the host cells. Enzymes for which production has been increased include D-amino oxidase (an increase of about 90%; Yu et al 2008), L-asparaginase (an increase of up to 70%;Geckil et al 2006), and a-amylase (3-fold increase; Suthar and Chattoo 2006). Production of mussel adhesive protein (90% increase; Kim et al 2008) and Bacillus thuringiensis Li et al 2010).…”

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

“…It has been reported that the overexpression of the gene encoding a bacterial hemoglobin from Vitreoscilla in Acremonium chrysogenum or in A. niger leads to a relief of stress when the transformants were exposed to oxygen limitation [44,45]. These hemoglobin proteins enhanced the production of alpha-amylase by the yeast Schwanniomyces occidentalis [46]. CPPs may produce a similar effect by increasing the concentration of the endogenous A. awamori hemoglobin-like protein.…”