Fermentation process for producing CFAs using Yarrowia lipolytica.

Past research has sought to improve the production of cyclopropane fatty acids by the oleaginous yeast Yarrowia lipolytica by heterologously expressing the E. coli fatty acid synthase gene and improving cultivation processes. Cyclopropane fatty acids display properties that hold promise for biofuel applications. The E. coli fatty acid synthase gene was introduced into several genetic backgrounds of the yeast Y. lipolytica to optimize lipid synthesis; the mean cyclopropane fatty acid productivity was 43 mg L-1 h-1 on glucose, and the production rate reached its maximum (3.06 g L-1) after 72 h of cultivation in a bioreactor. The best strain (JMY6851) overexpressed simultaneously the E. coli cyclopropane fatty acid synthase gene under a hybrid promoter (hp8d) and Y. lipolytica LRO1 gene. In fed-batch process using crude glycerol as carbon source, JMY6851 strain displayed high lipid accumulation (78% of dry cell weight) and high biomass production (56 g L-1). After 165 h of cultivation, cyclopropane fatty acids represented 22% of the lipids produced; cyclopropane fatty acid productivity (103.3 mg L-1 h-1) was maximal at 72.5 h of cultivation.

A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of ß-carotene.

The increasing market demands of ß-carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl-CoA. We first found that a lipid overproducer strain was capable of producing more ß-carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter-gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. ß-carotene production was further increased in controlled conditions using a fed-batch fermentation. A total production of ß-carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of ß-carotene.

High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production.

BACKGROUND: Because the model yeast Yarrowia lipolytica can synthesize and store lipids in quantities up to 20 % of its dry weight, it is a promising microorganism for oil production at an industrial scale. Typically, optimization of the lipid production process is performed in the laboratory and later scaled up for industrial production. However, the scale-up process can be complicated by genetic modifications that are optimized for one set of growing conditions can confer a less-than-optimal phenotype in a different environment. To address this issue, small cultivation systems have been developed that mimic the conditions in benchtop bioreactors. In this work, we used one such microbioreactor system, the BioLector, to develop high-throughput fermentation procedures that optimize growth and lipid accumulation in Y. lipolytica. Using this system, we were able to monitor lipid and biomass production in real time throughout the culture duration. RESULTS: The BioLector can monitor the growth of Y. lipolytica in real time by evaluating scattered light; this produced accurate measurements until cultures reached an equivalent of OD600nm = 115 and a cell dry weight of 100 g L(-1). In addition, a lipid-specific fluorescent probe was applied which reliably monitored lipid production up to a concentration of 12 g L(-1). Through screening various growing conditions, we determined that a carbon/nitrogen ratio of 35 was the most efficient for lipid production. Further screening showed that ammonium chloride and glycerol were the most valuable nitrogen and carbon sources, respectively, for growth and lipid production. Moreover, a carbon concentration above 1 M appeared to impair growth and lipid accumulation. Finally, we used these optimized conditions to screen engineered strains of Y. lipolytica with high lipid-accumulation capability. The growth and lipid content of the strains cultivated in the BioLector were compared to those grown in benchtop bioreactors. CONCLUSION: To our knowledge, this is the first time that the BioLector has been used to track lipid production in real time and to monitor the growth of Y. lipolytica. The present study also showed the efficacy of the BioLector in screening growing conditions and engineered strains prior to scale-up. The method described here could be applied to other oleaginous microorganisms.

Repeat-based Sequence Typing of Carnobacterium maltaromaticum.

Carnobacterium maltaromaticum is a Lactic Acid Bacterium (LAB) of technological interest for the food industry, especially the dairy as bioprotection and ripening flora. The industrial use of this LAB requires accurate and resolutive typing tools. A new typing method for C. maltaromaticum inspired from MLVA analysis and called Repeat-based Sequence Typing (RST) is described. Rather than electrophoresis analysis, our RST method is based on sequence analysis of multiple loci containing Variable-Number Tandem-Repeats (VNTRs). The method described here for C. maltaromaticum relies on the analysis of three VNTR loci, and was applied to a collection of 24 strains. For each strain, a PCR product corresponding to the amplification of each VNTR loci was sequenced. Sequence analysis allowed delineating 11, 11, and 12 alleles for loci VNTR-A, VNTR-B, and VNTR-C, respectively. Considering the allele combination exhibited by each strain allowed defining 15 genotypes, ending in a discriminatory index of 0.94. Comparison with MLST revealed that both methods were complementary for strain typing in C. maltaromaticum.

Recombinant pediocin in Lactococcus lactis: increased production by propeptide fusion and improved potency by co-production with PedC.

We describe the impact of two propeptides and PedC on the production yield and the potency of recombinant pediocins produced in Lactococcus lactis. On the one hand, the sequences encoding the propeptides SD or LEISSTCDA were inserted between the sequence encoding the signal peptide of Usp45 and the structural gene of the mature pediocin PA-1. On the other hand, the putative thiol-disulfide oxidoreductase PedC was coexpressed with pediocin. The concentration of recombinant pediocins produced in supernatants was determined by enzyme-linked immunosorbent assay. The potency of recombinant pediocins was investigated by measuring the minimal inhibitory concentration by agar well diffusion assay. The results show that propeptides SD or LEISSTCDA lead to an improved secretion of recombinant pediocins with apparently no effect on the antibacterial potency and that PedC increases the potency of recombinant pediocin. To our knowledge, this study reveals for the first time that pediocin tolerates fusions at the N-terminal end. Furthermore, it reveals that only expressing the pediocin structural gene in a heterologous host is not sufficient to get an optimal potency and requires the accessory protein PedC. In addition, it can be speculated that PedC catalyses the correct formation of disulfide bonds in pediocin.

Growth of aerobic ripening bacteria at the cheese surface is limited by the availability of iron.

The microflora on the surface of smear-ripened cheeses is composed of various species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. The objective of the present study was to show that iron availability is a limiting factor in the growth of typical aerobic ripening bacteria in cheese. For that purpose, we investigated the effect of iron or siderophore addition in model cheeses that were coinoculated with a yeast and a ripening bacterium. Both iron and the siderophore desferrioxamine B stimulated the growth of ripening bacteria belonging to the genera Arthrobacter, Corynebacterium, and Brevibacterium. The extent of stimulation was strain dependent, and generally, the effect of desferrioxamine B was greater than that of iron. Measurements of the expression of genes related to the metabolism of iron by Arthrobacter arilaitensis Re117 by real-time reverse transcription-PCR showed that these genes were transcribed during growth in cheese. The addition of desferrioxamine B increased the expression of two genes encoding iron-siderophore ABC transport binding proteins. The addition of iron decreased the expression of siderophore biosynthesis genes and of part of the genes encoding iron-siderophore ABC transport components. It was concluded that iron availability is a limiting factor in the growth of typical cheese surface bacteria. The selection of strains with efficient iron acquisition systems may be useful for the development of defined-strain surface cultures. Furthermore, the importance of iron metabolism in the microbial ecology of cheeses should be investigated since it may result in positive or negative microbial interactions.