Latest revision as of 11:55, 21 October 2021

FMO

Sequence and Features

Description

Flavin-containing monooxygenase, FMO is one kind of microsomal enzyme widely found in ER of most tissue, whose activity relies on flavin adenine dinucleotide (FAD), reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen. FMO could catalyze the oxidation of most exogenous compounds containing Nitrogen, Sulphur, Phosphorus, Selenium and other nucleophilic elements, which is a vital detoxifying process of carcinogen and many other hazardous substances^[1].

Usage and Biology

FMO is a kind of monooxygenase, which could produce C4a peroxyflavin intermediate, which is highly stable due to spectrum observation and could remain unchanged under 4℃ for minutes, even hours. In the first cycle, NADPH reduces FAD into FADH2. This reduced flavin could react with oxygen rapidly and become peroxyflavin, which is the main form of FMO and further react wtih an appropriate nucleophilic substrate. This reaction will form one molecular of water and transfer one oxygen atom to the substrate. The release of NADP+ may be the rate-limiting step of catalytic cycle while the Vmax of substrate binding has little impact on both reactions^[1].

Background related to indigo

The most ancient pigment known to humanity, indigo, now is popular in food, medic and dyeing industries. The pigment application of indigo could date back to at least 2,500 BC. and found on some blue hemp fabrics excavated from the Chinese MaWangDui and Egyptian mummies. One branch of Chinese Yao nationality is named after indigo as LanDianYao due to its unique technology of indigo dyeing. Among the food industry, indigo is used as edible pigment in the form of sodium sulfonate or aluminum, known as "bright blue" and bright blue aluminum lake in China, while being used mainly in its sodium sulfonate in the United States, called as "Indigo element"^[2].

Molecular cloning

AOX1 promoter is the strongest eukaryotic promoter currently known in yeast expression system^[3]. So we choose AOX1 as the primary promoter when we synthesized all these plasmid for the sake of more convenient expression. But noticing that methanol is hazardous, flammable, combustible and therefore, inappropriate to have direct contact with the hair, we need to substrate AOX1 for constitutive promoter Panb1 and xylose induced promoter Pynr071C to realize the projected regulation function as designed.

Fig1. Plasmid construction and colony PCR results of Panb1-α factor-FMO-AOX1 Terminator and Pynr071c-α factor-ROX1-AOX1 Terminator transformed E.coli.

The bands of Panb1-α factor-FMO-AOX1 Terminator (less than 3000bp) and Pynr071c-α factor-ROX1-AOX1 Terminator (3000+bp) from colony PCR are identical to the theoretical lengths of 2676bp and 3321bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.
Not quite to what we expect, after repeated transfection to the yeast, only a few products are expressed inside of eukaryotic system. Because of the large molecular weight and various types of some of our protein, we suspect that the common signal peptide we use, α-factor, is not enough to bring our protein out of the cell. While there is some of the genes without detectable products and we are hoping to get higher expression level, new primers for PCR are designed to ignore α-factor from our target gene in PCR. Then, likewise, we reconstruct this series of plasmid without α-factor through similar double-enzyme digestion and reconnection which insert our target genes right behind Panb1 promoter.

Fig2. Plasmid construction and colony PCR results of Panb1-crtB-AOX1 Terminator, Panb1-crtE-AOX1 Terminator and Panb1-FMO-AOX1 Terminator transformed E.coli.

The bands of Panb1-FMO-AOX1 Terminator (2500bp) from colony PCR is identical to the theoretical length of 2437bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that this target plasmid is successfully constructed.
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.

Fig3.Colony PCR result of yeast after electroporation through electrophoresis

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.

SDS-PAGE

Fig4. SDS-PAGE result of FMO after purification of yeast total protein extraction product through Nickel-affinity chromatography column

Different from impure or permeate bands, the target protein located around 60kDa, bigger than the theoretical 53.96kDa but still within explainable and acceptable range of glycosylation modification. FMO could be confirmed as successfully expressed. The concentration of yeast total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its dark color and consistency among several times of elution, this band could be verified as our target FMO.

Pigment synthesis

For some of our enzymes don’t have standard protocol to estimate their activity at present, we add substrates into culturing medium accordingly to find out whether there exists active target enzymes and do get our indigo and lycopene synthesized.

Fig5. Medium for expression with substrates

From left to right:
GS115 medium with indole and FPP as control; Panb1-FMO-AOX1 Terminator medium with indole; mixture of Panb1-crtE-AOX1 Terminator、Panb1-crtB-AOX1 Terminator、Panb1-crtI-AOX1 Terminator medium with FPP

Hair dyeing experiment

We measured the standard curves of three pigments before using them for hair dyeing experiment. We also found that the amount of melanin contained in hair can have a significant effect on hair dyeing outcomes. Therefore, we define different colors of hair based on bleaching.

We have gained the best dye conditions of three kinds of hair dye(indigo, curcumin and lycopene) at a certain concentration. Under optimal conditions, we dyed 4-9 degrees of hair to get a series of dyeing discs. And we found that as for the three colors selected for the experiment, bleach the hair to 8 degrees could achieve a bright coloring effect.

Under the best conditions, we dyed the hair from 4 degree to 9 degree, and got a series of colors. It is found that it only needed to be bleached to 8 degree so that the hair would show a bright color for all three kinds of dye.
As to indigo hair, 7 to 9 degree hair would become blue. As the dyeing time goes, the color would turn blue from an indigo color; 5 to 6 degree hair would be dyed to celadon, and 4 degree hair was still brown.

The dyeing results of indigo(room temperature,2g/L). From left to right: 9°(0.5,2,6min),8°(0.5,2,6min), 7°(0.5,2,6min)，6°(0.5,2,6min),,5°(0.5,2,6min),4°(0.5,2,6min)

Indigo:
Difficult: we can make indigo paste, but the hair does not dye well.
Solution: Indigo is a water-soluble component, and need to be oxidized to indigo after fixing to the hair. With water-in-oil paste as the matrix, indigo white can not fully enter the interior of the hair, and the oily substances in the matrix and excessive reductant prevent indigo white from oxidation in the hair, resulting in no effective coloring.
So we decided to design a timely manner in which indigo could be produced and used at the same time. Therefore, we consider that indigo dye can be produced and used in time -- the direct production of indigo by yeast, and the production of indigo solution as a dye in time.
For this idea, we dye indigo solution directly on the hair and find that it can be painted, but it can not color the hair evenly. So we designed a hair dye comb to make it possible to evenly smear indigo cryptosomes on the hair. The matching device is a timely fermentation tank, which can meet the needs of users with our engineering bacteria as raw materials, timely production and timely use of indigo white. For detailed information, please refer to Hardware part.

Color fastness is an important aspect to measure the effect of dye, so we design a set of elution scheme and test the color fastness of three kinds of natural pigment dye products and the same color traditional dye paste. The results showed that the color fastness of the natural pigment dyes was better than that of the traditional dyes.

Reference

[1]ESWARAMOORTHY S, BONANNO J B, BURLEY S K, et al. Erratum: Mechanism of action of a flavincontaining monooxygenase (Proceedings of the National Academy of Sciences of the United States of America (2006) 103, (9832-9837) DOI: 10.1073/pnas.0602398103) [J]. Proceedings of the National Academy of Sciences - PNAS, 2007, 104(36): 14543.
[2]HUIMING Z. Study on Dyeing Property and Mechanism of Indigo, Lawsone and their Combination System[D].Wuxi: Jiangnan university,2018.
[3]STALEY C A, HUANG A, NATTESTAD M, et al. Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris [J]. Gene, 2012, 496(2): 118-27.