1. Construction of the mutation system

(1) Purpose of the experiment

As depicted in Fig.1, there are at least 5 kinds of by-products during the patchoulol synthesis procedure. Thus, we planned to do patchoulol synthase (PTS) mutation to increase the specificity of the enzyme, as well as a supplement of static control system.

Fig.1 Postulated reaction pathways to account for the different products generated by the patchoulol synthase from farnesyl diphosphate (adapted from Croteau et al.).

(2) Experiments

• Error-prone PCR

Generation of a maximally diverse gene library is particularly important when employing non-targeted mutagenesis strategies. The method most often used to generate variants with random mutations is error-prone PCR (epPCR). Here we use the basic method for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants.

We chose manganese ions (Mn²⁺) as mutagen for patchouli alcohol synthase gene to produce random mutations. The epPCR system are showing as follows. (Tbl.1)

Fame	Volume
PCR Mix	25μL
Top strand primers	2μL
Bottom strand primers	2μL
Mn2+	-
dd water	~50μL

Tbl.1 System of epPCR.

(3) Results and discussion

We chose different kinds of Mn²⁺ and tried different concertation and volume of it to construct the best mutation system. Result are showing in the table below. (Tbl.2)

Cycle	Types of salt	Concentration	Volume	Result
1	MnSO4	2mM	0.4μL	Agarose gel electrophoresis showed no bands or the wrong bands
			0.8μL
			1μL
			2μL
2	MnSO4	8mM	1μL	The band are right through the agarose gel electrophoresis, but the sequencing results are all wrong.
			2μL
			3μL
			4μL
3	MnCl2	8mM	2μL	There were much more mutations than we expected.
			4μL
			6μL	The band are right through the agarose gel electrophoresis, but the sequencing results show no sign.
			8μL
4	MnCl2	8mM	0.5μL	There were less mutations than we expected.
			0.75μL	3-5 bp mutation per 1000bp
			1.25μL	There were much more mutations

Tbl.2 Result of PTS mutation.il

From the results above, we finally chose 0.75μL 8mM MnCl₂ as our mutants to built our mutation system.

2. Construction of a rapid selection system of mutants

(1) Purpose of the experiment

As we know, traditional method to text the titer of terpenoids we use either GC or HPLC, which are not only complex but time consuming. Thus, based on the relationship between lycopene and patchoulol production in P.pastoris we have already mentioned, we developed a visual method to screen for efficient mutations in PTS.

(2) Experiments & Result

As mentioned before, we selected two promoters, P₀₅₄₇ and P_PGK1, from the promoter library as the promoters of the mutant PTS, and transferred the mutant PTS into P.pastoris strain 2OZPP by homologous recombination.

P.pastoris strain 2OZPP and strains containing unmutated PTS with P₀₅₄₇ and P_PGK1 as promoters were used as controls, and strains with mutated PTS were inoculated on BMMY solid plates at 30℃ and given 1% methanol daily. After 96h of incubation, we observed colonies of different colors (Fig.2).

Fig.2 First round of PTS visualization screening.

We observed that colonies growing on the edges of the plates were generally lighter in color, and we believe that this situation is due to the edge effect caused by nutrient limitation at the edges of the plates, which can mislead the judgment of the PTS mutation effect. To reduce the interference of edge effects, we replaced the smaller plates and re-cultured and re-screened for the lighter colored strains.

In the second round of screening, we found several strains that were lighter in color and might be good mutants (Fig.3).

Fig.3 Second round of PTS visualization screening. The left one is the result of pPICZA-P_GAP-mut PTS-His. The left one is the result of pPICZA-P₀₅₄₇-mut PTS-His.

Due to time constraints, we did not obtain enough positive strains, and we plan to continue to expand the capacity of the PTS library to screen for more positive strains.

Considering that the issue of nutrient limitation may interfere with the judgment of strain color when cultured on solid plates, we will select strains with lighter color and transfer them to BMMY liquid medium to be cultured in 96-well plates for screening.

Strains that are lighter in color in the 96-well plates will be transferred to shake flasks for fermentation and finally for quantitative validation of patchoulol yield. The best mutant PTS will thus be selected.

Screening in 96-well plates and shake flasks was not carried out due to time constraints, and we will continue to do the screening in future experiments.