iGEM 2021 — Team NJTech_China

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# Team:NJTech China/Engineering

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**Engineering Success**

In short, the purpose of our project this year is to establish a robust, stable,
and controllable artificial microbial consortia system for the green and efficiency
phenylethanol biosynthetic. We have designed and characterized plant-derived phenylacetaldehyde
synthase (PAAS) according to the phenethylamine pathway of plants. Phenylethylamine pathway is
artificially delivered to *S. cerevisiae* for phenylethanol biosynthesis.
Well-characterized
promoters are essential for metabolic engineering and synthetic biology efforts in the model
eukaryotic yeast *Saccharomyces cerevisiae*. However, the expression level of allogenic
genes is
not satisfactory. To enable fine-tuned control and amplification of gene expression, we designed
the hybrid promoters pTEF, trying to entails combining core promoters pTEF with upstream
activation sequences (UAS elements). By reading literature, we learned that upstream activating
sequences (UAS elements) can serve as synthetic transcriptional amplifiers and can modulate the
expression of any promoter element. We constructed the UASCLB-pTEF-GFP strain and
UASCIT-pTEF-GFP strain to increase the expression of phenylacetaldehyde synthase by high
activity engineering promoter. As a result, the hybrid promoters can be used for improving the
expression of PAAS. In addition, to reduce carbon dioxide, we proposed to establish a
yeast-microalgae microbial consortia system based on nutritional complementation and metabolites
exchange. We tried to use 3D printing technology to combine microalgae on bioactive materials
and mixed cultured with yeast. The "living material", PVA-SA hydrogel, we used, offer
significant advantages as a scaffold for living materials because it provides a cellular
environment that is similar to natural biofilms.

**Characterization and optimization of the PAAS in recombinant yeast
strains**

**1.1 Inspiration and design**

After reviewing the literature, we found that there is an Ehrlich pathway in
*S. cerevisiae*, which can produce 2-phenylethanol (2-PE) from glucose. In addition, there is
phenylethylamine
pathway in petunia and vanda for de novo efficient synthesis of 2-PE, and it is
simpler and more convenient than the Ehrlich pathway. More importantly, it can be uncoupled with
cell growth for fractional optimization.

In our project, the key gene *paas* is heterologously expressed in yeasts to
produce 2-PE from L-phenylalanine. Considering the compatibility between
different plant genes and yeast genes, three different *paas* genes were obtained from rose,
petunia and vanda, respectively and integrated into *S. cerevisiae* after codon
optimization[1].
This constructed pathway significantly simplified the route from phenylalanine to 2-PE, which
will be more convenient in terms of scaling up and with fewer variables (gene expression,
exterior co-factors, etc.). Moreover, this enzyme can be further subjected to protein
engineering for improvement of its activity.

**1.2 Construction of the recombinant yeast strains: pRS426-Petunia,
pRS426-Vanda, pRS426-Rosa**

We first constructed the recombinant plasmids carrying the exogenous gene *paas*
from petunia, vanda and rose, respectively. The plasmid skeletons were the integrated plasmid
pRS406 and the high copy plasmid pRS426. The recombinant plasmid was introduced into
*Escherichia
coli* DH5α by transformation. Figure 1 is the nucleic acid electrophoresis verification
of the
plasmids PCR, from which it can be seen that all the recombinant plasmids have been successfully
introduced into *E.coli* DH5α.

![](https://static.igem.org/mediawiki/2021/e/e0/T--NJTech_China--eng1.png)

**Fig. 1
Nucleic acid electrophoresis verification of the plasmids PCR**

M: Trans2K Plus DNA Marker

1: pRS426-Petunia

2: pRS426-Vanda

3: pRS426-Rosa

4: pRS406-Petunia

5: pRS406-Vanda

6: pRS406-Rosa

Then, we transformed the recombinant plasmids into *Saccharomyces
cerevisiae*
BY4741. Figure 2 shows the colony PCR verification diagram of recombinant yeast strains. After
that, through gene sequencing, we confirmed that we successfully constructed three recombinant
yeast strains: pRS426-Petunia, pRS426-Vanda and pRS426-Rose.

![](https://static.igem.org/mediawiki/2021/3/34/T--NJTech_China--eng2.png)

**Fig. 2
Nucleic acid electrophoresis verification of the plasmids PCR**

M: Trans2K Plus DNA Marker

1-8: pRS406-Petunia

9-16: pRS406-Vanda

17-24: pRS406-Rose

25-32: pRS426-Petunia

33-40: pRS426-Vanda

41-48: pRS426-Rose

**1.3 Characterization of the PAAS by
high performance liquid chromatography (HPLC)**

![](https://static.igem.org/mediawiki/2021/2/2f/T--NJTech_China--eng3.png)

**Fig. 3 The growth curve of the wild type, pRS426-Petunia,
pRS426-Vanda, pRS426-Rosa**

The figure above shows the changes of biomass concentration in WT,
BY4741-pRS426-Petunia, BY4741-pRS426-Vanda, BY4741-pRS426-Rosa yeast over time. The OD600 of
different strain cultures is measured at the designated time points (0h, 24h, 48h, 72h). The
result shows that the growth trend of the recombinant strain is basically the same as the
wild-type strain, indicating that the introduction of heterogeneous gene has no significant
effect on the growth of yeast.

![](https://static.igem.org/mediawiki/2021/9/90/T--NJTech_China--eng4.png)

**Fig. 4 The 2-PE production of the recombinant strains
After 72 hours of fermentation, 2-PE production of the wild-type of BY4741, pRS426-Petunia,
pRS426-Vanda and pRS426-Rosa was tested by HPLC. Data represent means of triplicate culture
± standard error.**

*Saccharomyces cerevisiae* BY4741 contains Ehrlich pathway and other
metabolic
pathways to operate simultaneously to produce 2-PE, so the wild-type of BY4741 has a certain
amount of 2-PE production (1.205g/L). After the introduction of heterogeneous *paas* gene, the
2-PE production has remarkably increased. Among them, the production of 2-PE produced by the
yeast strain which was introduced Petunia-PAAS increased the most (1.570g/L), followed by the
strain which was introduced Vanda-PAAS (1.514g/L) and Rosa-PAAS (1.341g/L).

**Research on UAS**

**2.1 Inspiration and design**

Promoters are considered as basic regulatory elements responsible for
transcription initiation. Well-characterized promoters are essential for metabolic engineering
and synthetic biology efforts in the *Saccharomyces cerevisiae*. Promoter engineering
provides
sophisticated and diverse expression strategies for metabolic engineering. Upstream activating
sequences (UAS elements) can serve as synthetic transcriptional amplifiers and can modulate the
expression of any promoter element. Several upstream promoter sequences have previously been
identified as transcriptional enhancing elements in *S. cerevisiae*, including a 240 bp
sequence
5' of the mitotic cyclin *clb2* gene , a 203 bp sequence 5' of the *tef1* gene and a 275 bp sequence
5' of the mitochondrial citrate synthase *cit1* gene referred to henceforth as UASCLB,
UASTEF and
UASCIT, respectively. So, overall this year our team makes efforts to combine core
promoter with
upstream activating sequences (UAS elements) to enhance transcription efficiency and tune the
expression of the target gene[2].

![](https://static.igem.org/mediawiki/2021/7/79/T--NJTech_China--eng5.png)

**Fig. 5 Improvement of pTEF**

**2.2 Modification and Characterization of three hybrid promoters**

The plasmid of pRS426 was extracted and single digested by *Bam*HⅠ and
*Eco*RⅠ. The
enzyme digestion result was verified by agarose gel electrophoresis. Using BY4741 genome as a
template, pTEF-Primer-F, pTEF-Primer-R as primers to amplify the TEF promoter; using BY4741
genome as a template, USATEF-Primer-F, USATEF-Primer-R/ USACLB-Primer-F, USACLB-Primer-R/
UASCIT-Primer-F, UASCIT-Primer-R as primers to amplify the USATEF/ USACLB/ UASCIT fragments.
Using pRS415 genome as a template, GFP-Primer-F and CYC1-Primer-R are primers to amplify
GFP-CYC1 fragments. After one-step cloning, the yeast is transformed by the recombinant plasmid.
The yeast genome was extracted using the Zoman Yeast Genomic DNA Extraction Kit. Using the
proposed genome as a template and M13-F and M13-R as primers, genomic PCR was performed to
verify whether our target gene was successfully transformed the strains. As shown in figure 6,
it has been verified that the recombinant yeast containing the plasmid pRS426-pTEF-GFP-CYC1,
pRS426-USATEF-pTEF-GFP-CYC1, pRS426-USACLB-pTEF-GFP-CYC1, pRS426-UASCIT-pTEF-GFP-CYC1 have been
obtained.

![](https://static.igem.org/mediawiki/2021/1/1d/T--NJTech_China--eng6.png)

**Fig. 6 Electrophoresis verification of PCR products of recombinant
yeast**

A: M: DL5000 ( Vazyme ); 1-6: pRS425-pTEF-GFP-CYC1 ( 2140bp )

B: M: DL5000 ( Vazyme )

1-2: pRS426-USATEF-pTEF-GFP-CYC1 (3079bp)

3-4: pRS426-USACLB-pTEF-GFP-CYC1 (2981bp)

5-6: pRS426-UASCIT-pTEF-GFP-CYC1 (2863bp)

The yeast culture with recombinant plasmid was normalized to OD=0.01 after 48h
culture and shaking for 15h shaking. The results measured by flow cytometry are shown in figure
7. It's shown that the function of promoter can be improved by adding tandem UAS elements to the
upstream of pTEF. The UASCIT-pTEF promoter strength was expanded beyond the pTEF by 1.44-fold in
terms of mean fluorescence intensity. USACLB-pTEF and USATEF-pTEF promoter resulted 1.2-fold
improvement.

![](https://static.igem.org/mediawiki/2021/e/e5/T--NJTech_China--eng7.png)

**Fig. 7 The fluorescence intensity of GFP expressed by different
promoters**

The addition of a tandem UAS element upstream of the pTEF improves the promoter
function. In contrast, the average fluorescence intensity of promoters of USACLB-pTEF,
USATEF-pTEF and UASCIT-pTEF was higher than that of pTEF.

**2.3 The application of Mathematical models in our project**

Mathematical models are another important tool for characterizing promoters. In
our project, neural network algorithms can predict the expression level of hybrid
promoters.( [https://2021.igem.org/Team:NJTech\_China/Model](https://2021.igem.org/Team%3ANJTech_China/Model) )

**Construction of the yeast-microalgae interaction system by
3D printing technology**

**3.1 Inspiration and design**

In March 2021, China released the "Peak Carbon 2030 Study" and the "Carbon
Neutral 2060 Study", explaining the significance of carbon peaking and carbon neutrality. Due to
the higher carbon dioxide available for microalgae use in photosynthesis and higher oxygen
availability for heterotrophy of yeast, we got the idea to design a yeast-microalgae system to
reduce carbon dioxide while promoting yeast growth and 2-PE production as well. This
yeast-microalgae interaction system is potential to address three important societal needs: (i)
New mode production of 2-PE; (ii) The protection of aquatic environments; and (iii) The
reduction of the global anthropogenic greenhouse effect[6]. In the mixed culture,
microalgae
could generate oxygen for yeast while yeast provided CO2 to microalgae and produce
2-PE. To
achieve the stable co-cultivation, the growth conditions of the two organisms should be
optimized to improve the stability of our system[3].

We also used 3D printing technology to combine microalgae on bioactive materials
and mixed cultured with yeast. The "living material", PVA-SA hydrogel, offer significant
advantages as a scaffold for living materials because it provides a cellular environment that is
similar to natural biofilms[4]. Notably, the additive manufacturing of living whole
cells can be
used not only as a new bioreactor with high volumetric productivity and long lifetime, but also
as a versatile platform for fundamental studies of microbial behaviors, communications, and
interaction with the microenvironment[5].

**3.2 Mixed culture of the yeast-microalgae interaction system by 3D
printing technology for production**

To further increase production of 2-PE, the improvements we made is to design a
yeast-microalgae system. The mixed cultures performed better due to the higher carbon dioxide
available for microalgae use in photosynthesis and higher oxygen availability for heterotrophy
of yeast, leading to reduced microalgae production costs while maintaining alga production
reliability.

During yeast cultivation, organic acids were synthesized and the pH of the
culture dropped slightly. In addition, the media acidified with organic acids (e.g. acetic,
lactic acids) are more inhibitor to yeast growth compared with those acidified with mineral
acids.
When CO2 dissolves in water at neutral pH, bicarbonate (HCO3-) is formed.
During microalgal
photosynthesis activity HCO3- is converted to CO2 and hydroxide ion (OH-).
Hence, when CO2 is
consumed by microalgae, the OH- is formed, and the pH becomes more alkaline. So
microalgae can
improve the stability of the mixed culture and promote the production of 2-PE.

![](https://static.igem.org/mediawiki/2021/2/21/T--NJTech_China--eng8.png)

**Fig. 8 The 2-PE production in the yeast-microalgae microbial consortia
system**

However, the results do not meet our expectations when our recombinant yeast was
directly co-cultured with microalgae. The decline in the production of 2-PE may be due to the
insufficient nutrients resulted by substrate competition and different nutritional conditions of
yeasts and microalgae, posing great challenges to the stability of the artificial microbial
consortia system.

To solve the problems above, we have tried immobilized cell technique to realize
the spatial compartmentalization of microbial coculture, using polyvinyl alcohol-sodium alginate
(PVA-SA) as supporter material. The composite supporter material has the benefits of superior
biocompatibility, better processibility, stronger mechanic stiffness, and chemical inertia. In
our project, immobilized cell technique is combined with 3D printing technology. Thus, mass
exchange at the medium (PVA-SA) interface can be improved by optimizing 3D geometries, resulting
in high catalytic efficiency. Finally, the production of 2-PE is increased from 1.585 g/L to
1.755 g/L (Fig. 8).

Due to limited time, we are unable to make further improvements to our
artificial microbial consortia system, including adding time of microalgae and materials used in
3D printed. We believe that the yeast-microalgae interaction system combined with immobilized
cell technique and 3D printing technology has a promising future.

**References**

1. Kaminaga, Y.; Schnepp, J.; Peel, G.; Kish, C. M.; Ben-Nissan, G.; Weiss, D.;
Orlova, I.; Lavie, O.; Rhodes, D.; Wood, K.; Porterfield, D. M.; Cooper, A. J.; Schloss, J. V.;
Pichersky, E.; Vainstein, A.; Dudareva, N., Plant phenylacetaldehyde synthase is a bifunctional
homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation. J Biol Chem
2006, 281 (33), 23357-66.

2. Keren, L.; Zackay, O.; Lotan-Pompan, M.; Barenholz, U.; Dekel, E.; Sasson,
V.; Aidelberg, G.; Bren, A.; Zeevi, D.; Weinberger, A.; Alon, U.; Milo, R.; Segal, E., Promoters
maintain their relative activity levels under different growth conditions. Mol Syst Biol 2013,
9, 701.

3. Cheirsilp, B.; Suwannarat, W.; Niyomdecha, R., Mixed culture of oleaginous
yeast Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from industrial
wastes and its use as biodiesel feedstock. N Biotechnol 2011, 28 (4), 362-8.

4. Saha, A.; Johnston, T. G.; Shafranek, R. T.; Goodman, C. J.; Zalatan, J. G.;
Storti, D. W.; Ganter, M. A.; Nelson, A., Additive Manufacturing of Catalytically Active Living
Materials. ACS Appl Mater Interfaces 2018, 10 (16), 13373-13380.

5. Qian, F.; Zhu, C.; Knipe, J. M.; Ruelas, S.; Stolaroff, J. K.; DeOtte, J. R.;
Duoss, E. B.; Spadaccini, C. M.; Henard, C. A.; Guarnieri, M. T.; Baker, S. E., Direct Writing
of Tunable Living Inks for Bioprocess Intensification. Nano Lett 2019, 19 (9), 5829-5835.

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# Team:NJTech China/Results

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**Results**

**1. Construction of engineered *E. coli* DH5α and
*Saccharomyces
cerevisiae*
BY4741**

The designed plasmids (pRS406-Petunia, pRS406-Vanda, pRS406-Rosa, pRS426-Petunia,
pRS426-Vanda, pRS426-Rosa) were synthesized by GenScript.

![](https://static.igem.org/mediawiki/2021/thumb/a/a7/T--NJTech_China--results1.png/800px-T--NJTech_China--results1.png)

**Fig. 1 Plasmid maps of the recombinant plasmids**

**A: pRS406-Petunia; B: pRS406-Vanda; C: pRS406-Rosa**

**D: pRS426-Petunia; E: pRS426-Vanda; F: pRS426-Rosa**

The transformation of the recombinant plasmids into the *E. coli* DH5α.

The plasmids were extracted using plasmids extraction kit and verified by plasmids PCR. The target
fragment was about 2300bp.

![](https://static.igem.org/mediawiki/2021/9/9d/T--NJTech_China--results2.png)

**Fig. 2 Nucleic acid electrophoresis verification of the plasmids
PCR**

M:Trans2K Plus DNA Marker

1:pRS426-Petunia

2: pRS426-Vanda

3: pRS426-Rosa

4:pRS406-Petunia

5: pRS406-Vanda

6: pRS406-Rosa