Metabolic engineering and classic selection of the yeast Candida famata (Candida flareri) for construction of strains with enhanced riboflavin production
Introduction
Riboflavin (vitamin B2, RF) is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are coenzymes of flavoproteins involved in numerous redox reactions, and more rarely in reactions with no net change in redox state (Massey, 2000, Bornemann, 2002). Most microorganisms as well as all plants and fungi synthesize RF, whereas it is not produced by animals and rare pro- and eukaryotic microorganisms. Thus, human and domestic animals must obtain RF through nutrition. Currently, vitamin B2 is produced on a large scale by both chemical and biotechnological synthesis, with the latter gaining more significance because of the advantages of biotechnological processes such as cost effectiveness, reduction in waste and energy requirements, and the use of renewable feedstocks (Vandamme, 1992, Stahmann et al., 2000, Lim et al., 2001).
Industrial production of RF is facilitated mainly through the use of constructed strains of the bacterium Bacillus subtilis, the filamentous fungus Ashbya gossypii and the yeast Candida famata (Lim et al., 2001, Lim et al., 2003, Burgess et al., 2009). All accumulate large amounts of RF (15–20 g RF/l), however, each has its own advantages and disadvantages (Stahmann et al., 2000, Wu et al., 2007). While engineered B. subtilis is certainly the fastest-growing organism, it is subject to phagolysis and its growth-linked RF production bears the risk of selecting non-producing mutants. In A. gossypii this risk is absent because RF production occurs in stationary phase and is not growth-linked; however, growth time (the trophic phase) is lost for production (Stahmann et al., 2000). C. famata, as the unicellular yeast, is easier to distribute in a large-scale fermentor than a filamentous fungus growing in mycelial pellets. But C. famata RF overproducing industrial strain dep8 is quite unstable (Dmytruk and Sibirny, unpublished).
Biosynthesis of RF starts with guanosine triphosphate and ribulose-5-phosphate and is completed in six enzymatic steps (Fischer and Bacher, 2005). Numerous successful studies on the construction of RF overproducing strains of B. subtilis and A. gossypii using rational approaches of metabolic engineering have been described. This involved overexpression or disruption of genes encoding enzymes, which either limit important steps in the flux or catalyze undesirable reactions. For B. subtilis, these include enhancement of both gene dosages and transcription efficiency of the RF operon (Perkins et al., 1999), constitutive expression of the key gene (ribA) in RF biosynthetic pathway (Hümbelin et al., 1999), enhancing energy generation and reducing maintenance metabolism (Zamboni et al., 2003), increasing precursor supply by modulating carbon flow through the pentose phosphate pathway (Zamboni et al., 2004, Zhu et al., 2006), deregulating gapB expression by ccpN knockout based on screening B. subtilis transposon mutants (Tännler et al., 2008), and elevating the phosphoribosylpyrophosphate (PRPP) pool, thus the increasing transcript abundances of PurR-regulated genes participating in RF precursor biosynthesis (Shi et al., 2009). For A. gossypii, metabolic engineering approaches for improvement of RF production involved: increasing the flux of glycine, an early precursor of purine nucleotide synthesis, by overexpression of the glycine biosynthetic enzyme threonine aldolase (Monschau et al., 1998); reducing the flux from glycine to serine by disruption of the SHM2 gene, encoding serine hydroxymethyltransferase (Schlupen et al., 2003); conversion of glyoxylate into glycine via expression of the alanine:glyoxylate aminotransferase gene from Saccharomyces cerevisiae (Kato and Park, 2005); deregulation of genes involved in the biosynthesis of purines and glycine by C-terminal deletion in the AgBAS1 gene encoding a transcription factor (Mateos et al., 2006) and enhancing metabolic flow through the purine pathway by modification of key enzymes PRPP amidotransferase (Jiménez et al., 2005) and PRPP synthetase (Jiménez et al., 2008). Previous research on metabolic engineering of C. famata for construction of efficient yeast RF producers has not been published most likely because the molecular tools for research of C. famata have only recently been developed (Stahmann et al., 2000). Since then, methods of transformation (Voronovsky et al., 2002) and insertional mutagenesis (Dmytruk et al., 2006) have been developed and structural and regulatory genes of the RF biosynthesis pathway have been cloned from C. famata (Dmytruk et al., 2004, Dmytruk et al., 2006, Voronovsky et al., 2004). Overproducers of FMN in C. famata have been isolated earlier in our laboratory as RF overproducing strains (Sibirny et al., 2008) overexpressing the FMN1 gene coding RF kinase (Yatsyshyn et al., 2009).
In this study, we report on construction of the C. famata RF overproducing strain AF-4 using methods of classic mutagenesis and selection for resistance to several antimetabolites, guanosine and for RF overproduction at high pH (Sibirny et al., 2008). We also describe the construction of the recombinant C. famata strain overexpressing SEF1 (putative transcriptional factor) and IMH3 (IMP dehydrogenase) orthologs from D. hansenii together with native RIB1 (GTP cyclohydrolase II) and RIB7 (RF synthase) genes in RF overproducing strain isolated by conventional mutagenesis. Constructed recombinants showed significant enhancement in RF synthesis as compared to the parental RF overproducer obtained via conventional mutagenesis.
Section snippets
Strains, media, cultivation conditions
Candida famata wild-type VKM Y-9 (All-Russian Collection of Microorganisms, Pushchino, Russia) and RF-overproducing ATCC 20849 (dep8) strains were used throughout this work. Yeast cells were cultured at 30 °C in YPD media (0.5% yeast extract, 1% peptone and 2% glucose), SD (0.67%, yeast nitrogen base without amino acids, DIFCO) plus 2% glucose or modified Burkholder medium (Yatsyshyn et al., 2009) supplemented with 0.5% yeast extract. For selection of yeast transformants, 3 mg/l phleomycin, 15
Isolation of RF overproducing strain AF-4 using methods of conventional mutagenesis and selection
The RF overproducing strain AF-4 was isolated from the wild-type strain C. famata VKM Y-9 in six consecutive steps of conventional mutagenesis (Sibirny et al., 2008). The performed selection is summarized in Table 1. At the first step, mutants resistant to RF structural analog, 7-methyl-8-trifluoromethyl-10-(1'-d-ribityl)isoalloxazine, were selected. Isolation of the resistant mutants was carried out on solid medium supplemented with RF analog (200 mg/l) and 0.6 M K2SO4. RF synthesis of the
Discussion
The yeast strain C. famata dep8 (Foster et al., 1992) is currently used in USA for industrial production of RF despite its low genetic stability (Dmytruk and Sibirny, unpublished). Though selected by conventional mutagenesis, strain AF-4 produced less RF as compared to dep8 (Table 2), but is very stable with respect to RF overproducing capability. However, both these strains produce rather small amounts of RF, far from the theoretical maximum—only 4% of carbon substrate, glucose, is converted
Conclusion
It was demonstrated that an increase in expression of positive regulator of RF synthesis SEF1 together with the first and last genes of the RF pathway enhance the production of RF to a considerable extent. Moreover, we constructed a strain considering its level of RF synthesis, may now be employed in biotechnological production of vitamin B2.
Acknowledgments
This work was partially supported by CRDF STEP BPG Grant UKB1-9047-LV-10. We are grateful to John Santa Maria (from Harvard Medical School, Boston, MA) for a critical reading of the manuscript.
References (41)
- et al.
Bacterial vitamin B2, B11 and B12 overproduction: an overview
Int. J. Food Microbiol.
(2009) - et al.
Functional distinctions between IMP dehydrogenase genes in providing mycophenolate resistance and guanine prototrophy to yeast
J. Biol. Chem.
(2003) - et al.
Development of a promoter assay system for the flavinogenic yeast Candida famata based on the Kluyveromyces lactis β-galactosidase LAC4 reporter gene
Enzyme Microb. Technol.
(2008) - et al.
The functional basis of mycophenolic acid resistance in Candida albicans IMP dehydrogenase
J. Biol. Chem.
(2005) - et al.
Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production
Metab. Eng.
(2009) - et al.
Screening of Bacillus subtilis transposon mutants with altered riboflavin production
Metab. Eng.
(2008) - et al.
Development of a transformation system for the flavinogenic yeast Candida famata
FEMS Yeast Res.
(2002) - et al.
Production of flavin mononucleotide by metabolically engineered yeast Candida famata
Metab. Eng.
(2009) - et al.
The phosphoenolpyruvate carboxykinase also catalyzes C3 carboxylation at the interface of glycolysis and the TCA cycle of Bacillus subtilis
Metab. Eng.
(2004) - et al.
Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis
Metab. Eng.
(2003)
Flavoenzymes that catalyse reactions with no net redox change
Nat. Prod. Rep.
Comparison of two alternative dominant selectable markers for wine yeast transformation
Appl. Environ. Microbiol.
Inhibition of the phosphoribosylformylglycineamide synthetase of Ehilich ascites tumor cells by glutamine analogues
Biochem. Pharmacol.
Functional analysis of the PUT3 transcriptional activator of the proline utilization pathway in Saccharomyces cerevisiae
Genetics
Cloning of structural genes involved in riboflavin synthesis of the yeast Candida famata
Ukr. Biokhim. Zh.
Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments
Curr. Genet.
6-Azauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae
Curr. Genet.
Biosynthesis of flavocoenzymes
Nat. Prod. Rep.
A mutated ARO4 gene for feedback-resistant DAHP synthase which causes both o-fluoro-DL-phenylalanine resistance and beta-phenethyl-alcohol overproduction in Saccharomyces cerevisiae
Curr. Genet.
Cited by (61)
Metabolic engineering of non-conventional yeasts for construction of the advanced producers of biofuels and high-value chemicals
2023, BBA AdvancesCitation Excerpt :Such work was hampered by the fact that C. famata belongs to CUG yeast clade which decode this codon as serine rather than leucine [88], so standard genes coding for antibiotic resistance could not be used without codon adjusting. Nevertheless, the ble gene from Staphylococcus aureus (which does not contain a CUG codon) conferring resistance to the antibiotic phleomycin; modified D. hansenii IMH3 and ARO4 genes conferring resistance to mycophenolic acid and fluorophenylalanine, respectively, and BSD gene from Aspergillus terreus coding resistance to blasticidin, were successfully used as dominant markers [35,86,89,90]. Methods of insertion mutagenesis have been developed for C. famata and genes SEF1, MET2 have been tagged which disruptions led to inability to overproduce riboflavin [89].
Microbial production of riboflavin: Biotechnological advances and perspectives
2021, Metabolic EngineeringEfficient production of bacterial antibiotics aminoriboflavin and roseoflavin in eukaryotic microorganisms, yeasts
2023, Microbial Cell FactoriesEnhanced Ribonucleic Acid Production by High-Throughput Screening Based on Fluorescence Activation and Transcriptomic-Guided Fermentation Optimization in Saccharomyces cerevisiae
2023, Journal of Agricultural and Food Chemistry