Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR-Cas9 cassette or a transient CRISPR-Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

Commercial xylose purification produces xylose mother liquor (XML) as a major byproduct, which has become an inexpensive and abundant carbon source. A portion of this XML has been used to produce low-value-added products such as caramel but the remainder often ends up as an organic pollutant. This has become an issue of industrial concern. In this study, a uracil-deficient Candida tropicalis strain was engineered to efficiently convert XML to the commercially useful product xylitol. The xylitol dehydrogenase gene was deleted to block the conversion of xylitol to xylulose. Then, an NADPH regeneration system was added through heterologous expression of the Yarrowia lipolytica genes encoding 6-phosphate-gluconic acid dehydrogenase and 6-phosphate-glucose dehydrogenase. After process optimization, the engineered strain, C. tropicalis XZX-B4ZG, produced 97.10 g L− 1 xylitol in 120 h from 300 g L− 1 XML in a 5-L fermenter. The xylitol production rate was 0.82 g L− 1 h− 1 and the conversion rate was 92.40 %. In conclusion, this study performed a combination of metabolic engineering and process optimizing in C. tropicalis to enhance xylitol production from XML. The use of C. tropicalis XZX-B4ZG, therefore, provided a convenient method to transform the industrial by-product XML into the useful material xylitol.

/pdf/stepwise-metabolic-engineering-of-candida-tropicalis-for-3uxsm8d9br.pdf

Stepwise metabolic engineering of Candida tropicalis for efficient xylitol production from xylose mother liquor.

α-Humulene is a plant-derived monocyclic sesquiterpenoid with multiple pharmacological activities, and far-reaching potential for the development of new drugs. Currently, the production of α-humulene is typically achieved via plant extraction, which is not sustainable and limited by low yields. The oleaginous yeast Candida tropicalis has recently emerged as a valuable host for producing high-value-added chemicals. However, the potential of C. tropicalis for terpenoid production has not been exploited.In this study, C. tropicalis was engineered for de novo synthesis of α-humulene from glucose. To improve α-humulene production, the codon-optimised α-humulene synthase gene and the entire endogenous farnesyl diphosphate synthesis pathway were co-overexpressed. Furthermore, bottlenecks in the α-humulene synthase pathway were identified and relieved by overexpressing α-humulene synthase, acetoacetyl-CoA thiolase and NADH-dependent HMG-CoA reductase. Combined with fermentation medium optimisation, the engineered strain produced 195.31 mg/L of α-humulene in shake flasks and 4115.42 mg/L in a bioreactor through fed-batch fermentation, a 253- and 5345-fold increase over the initial production, respectively.This study demonstrates the potential of C. tropicalis for α-humulene production, and presents a platform for the biosynthesis of other terpenoids. 

/pdf/engineering-the-oleaginous-yeast-candida-tropicalis-for-a-26ibl3b1.pdf

Engineering the oleaginous yeast Candida tropicalis for α-humulene overproduction

A rapid method of partially purifying carnitine acetyltransferase (CAT) from Candida tropicalis and determination of its enzyme activity was established.The characteristics of CAT-disruption recombinant of Candida tropicalis were also studied.It was confirmed that the specific activity of CAT of this recombinant was approximately half of that of its corresponding wild type. Therefore, the β-oxidation pathway of dicarboxylic acid was effectively suppressed. Correspondingly, the dicarboxylic acid productivity of this recombinant was increased by 11% and its alkane conversion rate was also increased by 12% in average.

Acetyltransferase activity in Candida tropicalis andcharacteristics of CAT disruption recombinant

Candida geneticamente modificata per la produzione di acidi grassi dicarbossilici

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura+ and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10−8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.

Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous GFP genes in Candida tropicalis

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Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous GFP genes in Candida tropicalis