A synthetic biology approach to transform Yarrowia Lypolytica into a competitive biotechnological producer of β-carotene:

I based my assignment on this research: [1]

A SYNTHETIC BIOLOGY APPROACH TO TRANSFORM YARROWIA LYPOLYTICA INTO A COMPETITIVE BIOTECHNOLOGICAL PRODUCER OF β-CAROTENE

1. INTRODUCTION β-carotene is an orange pigment and a well-known precursor of vitamin A. It is a biochemical synthesized terpenoid and is added to the group of carotenoids- β-carotene can be produced either chemically or biotechnologically, extracting it from the natural producer of microorganisms such as Blakeslea trispora. Yarrowia lipolytica is an oleaginous yeast greatly investigated and modified, for the sake of production of biotechnologically relevant compounds.

2. THEORETICAL FRAMEWORK

            2.1 MATERIALS AND METHODS – STRAINS AND MEDIA  

The materials and methods are divided into a series of steps, intended to create a producer of β-carotene out of the aforementioned yeast, Yarrowia lipolytica. The needed steps are: 1) strain construction, 2) construction of the DNA, 3) β-carotene measurement, 4) developing microscopy images, 5) lipid content quantification, 6) biomass, sugar and acid quantification and lastly come the 7) bioreactor procedures.

3. RESULTS AND DISCUSSION

            3.1 Lipid over-producer strain synthesizes higher amount of β-carotene

Previous set of reports documented that the over-expression of three genes (geranylgeranyl diphosphate synthase (GGS1), pytoene synthase/lycopene cyclase and phytoene dehydrogenase) promote and increase the production of β-carotene in Y. lipolytica. The reseachers used an expression cassette (a car-cassette), where the expression of the GGS1 is controlled by the promoter PGMp. The sole expression, within the cassette itself (in the parental strain) allows a production of a substantial amount of β-carotene.

            3.2 Continuous metabolic engineering magnifies carotene content 

In order to enhance the production of caroteonids, the over-expression of HMG1 (hydroxymethylglutaryl-CoA reductase) is well known. In order to enhance the production of β-carotene, they over-expressed the gene YALI0E04807. It was done under the control of the constitutive TEF promoter in the strain of ob-C. The generated strain (ob-CH) enhanced the production β-carotene by a substantial amount. A study was conducted, to analyze if the production β-carotene is rather limited by the levels of expression of the over-expressed genes, a second copy of the car-cassette was introduced in the genome of ob-CH, which resulted in the creation of ob-CHC. The conclusion stated that the generated and produced amount of the desired product increased, more than by using the parental strain ob-CH. The results indicated that the expression of some of the gene encoded in the car-cassette is limiting the production of β-carotene. In order to overcome such a sudden obstacle, they combined the optimization of the promoter strength and the copy number for each gene.

            3.3 Identifying the best promoter set for the production of β-carotene

The increase in promoter strength can enhance the transcription level more than five times. They use the Closing Gate cloning system in promoter shuffling. This strategy consists in a digestion-ligation reaction guided by Bsal defined sites where the three promoters can be introduced and guided within the promoter positions in the car-cassettes. The pool of cassettes were then set for transformation, the wild type parental strains enable the screening of different color intensity. Once they finished the transformation, they obtained 387 colonies. By analyzing the overall combination of promoters, the presence of a strong TEF promoter is favored as it stood in the second position of the car-cassette.

             3.4 The construction of the β-carotene over-producer strain 

Seeing as how an extra copy of the car-cassette in the strain ob-CH increased the production of β-carotene greatly, the cassette was optimized by a set of promoters and as a result there became: carTEF-cassette. By expressing the carTEF-cassette in the strain ob-CH, the production of β-carotene was increased. Moreover, an extra copy of the carTEF-cassette was brought to construct the strain ob-CHCTEFCTEF . Said strain was able to also enhance the production of β-carotene.

             3.5 A trade off between production titer and yield

Two different kinds of media were tested, rich media (YPD) and synthetic media (YNB). A higher concentration of the carbon/nitrogen ratio is known to increase lipid production and by that, it enhances the production of carotenoids. Glucose and glycerol were tested as the first carbon source. The selected media were YPD10, YPD60, YNB20, YNB30, YNB60, and YNBGLY60. They concluded that there wasn't much of an influence on the carbon source since glucose and glycerol showed similar between both titer and yields. The best β-carotene titer so far was found in YPD60, whilst the winning yield is YPD10.

             3.6 Boosting β-carotene production by the engineered strain ob-CHCTEFCTEF

With the perforation of fed-batch fermentation using a rich media (Y10P20D), they added glucose. The glucose concentration began to rise due to the lack of consumption. The strain led to a production of β-carotene and citric acid. Designing a culture media with the double amount of yeast extract and peptone (Y20P40D), they optimized bioreactor conditions. The results show that Y. lipolytica engineered using synthetic biology and metabolic engineering is a potential industrial producer of β-carotene.

4. CONCLUSION With the combination of traditional metabolic engineering strategies and synthetic biology tools, it's been proven that Y. lipolytica serve as a valid production source for β-carotene. The increase of gene copy numbers and lipogenesis with the use of favourable promoters massively enhanced the production of β-carotene and also, the fed-batch fermentation was proven as the best pathway in generating β-carotene. The process can also be further improved not only by engineering strains and bioreactor condition optimization but also by the use of low cost carbon sources. The selection of suitable microorganism and strains plays a great role in electing a more successful source of production of β-carotene. The rapid development of synthetic biology tools for genome editing and DNA assembly is alleviating the manipulation of non-conventional organisms, growing the range of biotechnological „vehicles“ for metabolic engineering.

5. RELATED LITERATURE 1. Larroude, M., Celinska, E., Back, A., Thomas, S., Nicaud, J-M., Amaro, R. N., Wiley, Biotechnology and bioengineering, A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnologist, October 11th, 2017 (data obtained and approved by ResearchGate on March 12th)

2. CSH Protocols (2017) Yeast Extract-Peptone-Dextrose, taken from: http://cshprotocols.cshlp.org/content/2017/8/pdb.rec090563.full?text_only=true (data found on March 18th)

3. Microbial food.org (2014), Microbe guide: Yarrowia lipolytica, taken from: http://microbialfoods.org/microbe-guide-yarrowia-lipolytica/, (data found on March 17th)