Xylitol production is increased by expression of codon-optimized Neurospora crassa xylose reductase gene in Candida tropicalis

Xylose reductase (XR) is the first enzyme in d-xylose metabolism, catalyzing the reduction of d-xylose to xylitol. Formation of XR in the yeast Candida tropicalis is significantly repressed in cells grown on medium that contains glucose as carbon and energy source, because of the repressive effect of glucose. This is one reason why glucose is not a suitable co-substrate for cell growth in industrial xylitol production. XR from the ascomycete Neurospora crassa (NcXR) has high catalytic efficiency; however, NcXR is not expressed in C. tropicalis because of difference in codon usage between the two species. In this study, NcXR codons were changed to those preferred in C. tropicalis. This codon-optimized NcXR gene (termed NXRG) was placed under control of a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter derived from C. tropicalis, and integrated into the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis. High expression level of NXRG was confirmed by determining XR activity in cells grown on glucose medium. The resulting recombinant strain, LNG2, showed high XR activity (2.86 U (mg of protein)−1), whereas parent strain BSXDH-3 showed no activity. In xylitol fermentation using glucose as a co-substrate with xylose, LNG2 showed xylitol production rate 1.44 g L−1 h−1 and xylitol yield of 96% at 44 h, which were 73 and 62%, respectively, higher than corresponding values for BSXDH-3 (rate 0.83 g L−1 h−1; yield 59%)

Biotechnological production of γ-decalactone, a peach like aroma, by Yarrowia lipolytica

The request for new flavourings increases every year. Consumer perception that everything natural is better is causing an increase demand for natural aroma additives. Biotechnology has become a way to get natural products. γ-Decalactone is a peach-like aroma widely used in dairy products, beverages and others food industries. In more recent years, more and more studies and industrial processes were endorsed to cost-effect this compound production. One of the best-known methods to produce -decalactone is from ricinoleic acid catalyzed by Yarrowia lipolytica, a generally regarded as safe status yeast. As yet, several factors affecting -decalactone production remain to be fully understood and optimized. In this review, we focus on the aromatic compound -decalactone and its production by Y. lipolytica. The metabolic pathway of lactone production and degradation are addressed. Critical analysis of novel strategies of bioprocess engineering, metabolic and genetic engineering and other strategies for the enhancement of the aroma productivity are presented.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684)

CHARACTERIZATION AND OVER-EXPRESSION OF A CLONED PSEUDOMONAS LIPASE GENE

Triacylglycerollipases (E.C.3.1.1.3) are ubiquitous amongst microbes, plants, and animals and catalyse the hydrolysis of ester bonds of triglycerides to form glycerol and fatty acids. On the other hand, lipases catalyse the reverse esterification reaction in low water conditions, forming glycerides from glycerol and free fatty acids. Moreover, some lipases catalyse transesterification through the exchange ofesterified fatty acids with free fatty acids. These reactions can either be selective toward a specific ester bond in triglycerides or completely non-specific. Besides positional specificity, some lipases demonstrate selectivity toward a particular fatty acid substrate. Since lipases have wide versatility, considerable interest in the industrial uses of lipases has recently developed. Industrial applications of lipases include enzymatic fat splitting, accelerated cheese ripening, production of cocoa butter substitutes, and as a detergent additive. Also, the enanatioselectivity of certain lipases offers an attractive opportunity for the preparation of chiral intermediates for pharmaceutical syntheses. Thelipase from Pseudomonassp. ATCC 21808is of particular interest for potential industrial applications becauseofits high temperature optimum for enzymatic activity (65 deg. C), its thermostability (no activity loss after one week at 50 deg C in a phosphate buffer), and activity over a broad pH range (4-9)