iGEM 2023 — Team hubu-hubei-china


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{% block title %}engineering{% endblock %}
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* [Cycle1](#Part1)
* [Cycle2](#Part2)
* [Cycle3](#Part3)
* [Cycle4](#Part4)
* [Cycle5](#Part5)
* [Cycle6](#Part6)
* [Cycle7](#Part7)
* [Cycle8](#Part8)
* [Cycle9](#Part9)
* [Cycle10](#Part10)
* [Cycle11](#Part11)
* [Summary](#Part12)

EXPERIMENT

ENGINEERING

Engineering is the art of crafting solutions, where imagination meets functionality to shape the future.

# Introduction

When designing and constructing our biological systems, the DBTL (Design-

Build-Test-Learn) cycle stands as a cornerstone. We improve the initial design by testing biological systems and learning from the results.

 The engineering cycle is punctuated by four main phases: Design, where concepts take shape; Build, where these concepts manifest into tangible forms; Test, where each construct undergoes rigorous scrutiny; and Learn, the phase of introspection and adaptation. It 's the iterative spirit of this cycle that ensures our biological designs evolve, inching closer towards perfection with each iteration, and ultimately resonating with our envisioned objectives.

For the torchbearers of iGEM who will walk this path in the future, we 've streamlined your journey. Every strain, plasmid, and primer meticulously employed in our engineering odyssey is catalogued under

[Materials](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27materials%27%29%20%7D%7D)
.
Meanwhile, a detailed repository of reagents, enzymes, and an array of methodologies awaits your perusal under

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
.

For a deeper understanding, please refer to the

[Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
.

In the early stages, Dr. Chen Shouwen 's laboratory took
Bacillus licheniformis
 DWc9n as the research subject. Through a series of synthetic biology  approaches such as introducing the hydroxytyrosol synthesis pathway, inhibiting byproduct synthesis, and enhancing precursor supply, they obtained the strain DHT11 (
Bacillus licheniformis
 DWc9n Δpyk-xkdE::yugJ-ldh::tyrA
fbr
-xkdG::aroG
fbr
-PbacA

-aroK ΔpheA ΔhisC ΔdhaS ΔdhbC ΔadhA/pHY-PbacA-kivD-P43-haBC). When cultured in LBG medium containing 40 g/L glucose, this strain produced hydroxytyrosol at a concentration of 1900.00 ±126.02g/L. Its conversion efficiency was 0.03 mol/mol glucose, a figure that still has significant scope for improvement compared to the theoretical conversion rate of 0.5mol/mol glucose.

![](https://static.igem.wiki/teams/4912/wiki/description/image10.png)

Fig. A1

Metabolic engineering
B. licheniformis
 for
de novo
 synthesis of

hydroxytyrosol of
 DHT1
1

Heterologous introduced and overexpressed genes are highlighted in red, deleted genes are annotated in red cross, and knock-down genes are annotated in green.
Glc
: glucose,
2,3-BD
: 2,3-butanediol,
AC
: acetoin,
 AHAS
: acetolactate,
 Ac-CoA:
 acetyl-CoA,
 PYR
: pyruvate,
PEP:
 phosphoenolpyruvate,
 G6P
: glucose-6-phosphate,
 F6P
:

fructose 6-phosphate ,
G3P
: glyceraldehyde 3-phosphate,
Ru5P
:

ribulose 5-phosphate,
X5P
: xylulose 5-phosphate,
 R5P
: ribose 5-phosphate,
S7P
:sedoheptulose 7-phosphate,
E4P
: erythose-4-phosphate,
DAHP
: 3-deoxy-D-arabino-heptulosonate-7-phosphate,
DHQ
: 3-dehydroquinic acid,
DHS
: 3-dehydroshikimic acid,
SHIK:
 shikimate,
S3P
: Shikimate-3-phosphate,
EPSP
: 5-enolpyruvylshikimate 3-phosphate,
CHA
: chorismate,
 PPA
: prephenate, 4-
HPP
: 4-hydroxyphenylpyruvate,
4-HPAA
: 4-hydroxyphenylacetaldehyde,
 HT
: hydroxytyrosol,
ICHA
: isochorismate,
 OPP
: phenylpyruvate,
PHE
: L-phenylalanine,
TYR
: L-tyrosine,
 EMP
: Embden-Meyerhof-Parnas pathway,
PPP:
 pentose phosphate pathway,
 TCA cycle
: tricarboxylic acid cycle.

We hypothesize that the observed results might be due to lower enzymatic activity of key enzymes in the synthetic pathway, metabolic imbalances, the presence of unknown rate-limiting steps within the synthesis route, or potential oxidative degradation of hydroxytyrosol during fermentation.

Therefore, building on the DHT11 strain, we intend to employ protein engineering of the phenylpyruvate decarboxylase KivD, balance the metabolism of

phenylpyruvate decarboxylase KivD and hydroxylase HpaBC, investigate limiting factors in the shikimic acid pathway and the pentose phosphate pathway, and utilize oxygen-responsive switches. We aim is to identify new rate-limiting steps and balance the expression levels of critical enzymes to divert more carbon flux towards the production of HT (hydroxytyrosol).

## Cycle 1: Testing the catalytic ability of Heterologous Introduced phenylpyruvate decarboxylase KivD on 4-HPP

Design 1:

The Ehrlich pathway consists of two enzymes: keto acid decarboxylase and alcohol dehydrogenase, facilitating the synthesis of various branched-chain alcohols and aromatic alcohol compounds.
Bacillus licheniformis
 intrinsically metabolizes 4-hydroxyphenylpyruvate (4-HPP), which can be converted into the precursor of hydroxytyrosol via the Ehrlich pathway.

Bacillus licheniformis
 possesses an endogenous alcohol dehydrogenase, YugJ, but lacks keto acid decarboxylase, necessitating heterologous introduction.

As reported in the literature by Zhan Y. (2022), overexpression of α-keto acid decarboxylase KivD from
Lactococcus lactis
 was achieved in
Bacillus licheniformis
, successfully establishing a hybrid Ehrlich pathway in the bacterium. Our initial step is to construct an overexpression strain to better evaluate the catalytic ability of the phenylpyruvate decarboxylase KivD on 4-HPP. The co-expression of phenylpyruvate decarboxylase KivD and the endogenous alcohol dehydrogenase YugJ present in
Bacillus licheniformis
 can catalyze 4-HPP into tyrosol. Hence, during our evaluation, we 'll employ HPLC to quantify tyrosol production, validating the catalytic efficiency of the heterologously introduced keto acid decarboxylase KivD on 4-HPP.

Build 1:

The pHY-kivD was electroporated into the host bacterium
Bacillus licheniformis
DWc9n. Subsequent transformation verification was performed, and the successfully constructed positive strain was named DWc9n/pHY-kivD.

![2700bp](https://static.igem.wiki/teams/4912/wiki/description/image12.png)

Fig. B1 Agarose gel map for verification of 2700bp target DNA bands

Test 1:

Using LBG medium as a foundation, we added 0.5 g/L of 4-hydroxyphenylpyruvate. The strain DWc9n/pHY-kivD was inoculated and subjected to a shake flask fermentation experiment under conditions of 230 rpm and 37 °C for 24 hours. The tyrosol yield, as detected by HPLC, was 61.98 ±3.23 mg/L.

Learn 1:

In LBG medium supplemented with 0.5 g/L of tyrosine, the strain DWc9n/pHY-kivD produced a tyrosol yield of 61.98 ±3.23 mg/L, translating to a low conversion rate of just 12.4%. This indicates that the enzyme kivD has relatively low catalytic activity towards 4-hydroxyphenylpyruvate. To address this, we plan to undertake protein engineering in Cycle 2.

Cycle 2: Protein Engineering to Enhance KivD 's Catalytic Activity Towards 4-Hydroxyphenylpyruvate

Design 2:

The catalytic activity of phenylpyruvate decarboxylase KivD towards 4-hydroxyphenylpyruvate is suboptimal. This can be augmented through protein engineering. We identified that KivD shares a high sequence homology, up to 88%, with the 2-keto acid decarboxylase (KdcA) from
L. lactis
 B1157 (PDB: 2VBG) as mentioned (Yep, Alejandra et al., 2006). The key amino acids in the active site of KivD (S286, E462, V461, M538, and F542) are also conserved with KdcA. To gain preliminary insights into the interaction between kivD enzyme and its substrate 4-HPP, and subsequently design experimental mutations more effectively, we carried out molecular docking using Discovery Studio. We eventually

selected four key amino acid residues that exhibit tighter binding with 4-HPP and mutated them to the smallest neutral amino acid - glycine (G). These amino acid residues are serine (S) at position 286, glutamic acid (E) at position 462, phenylalanine (F) at position 542, and valine (V) at position 461.

![](https://static.igem.wiki/teams/4912/wiki/description/image11.png)

![](https://static.igem.wiki/teams/4912/wiki/description/image14.png)

Fig. D2 The molecular docking diagram of the key amino acid residues of 4-HPP catalyzed by KivD

Build 2:

Following the experimental

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
 constructing point mutation plasmids
, the pHY-kivD was utilized as a template for inverse PCR amplification. The linearized PCR product was treated with DpnI enzyme to digest and remove the original plasmid template. After the digestion, the products were recovered and mixed as per the stipulated ratios detailed in the one-step cloning kit manual. This mixture was then transformed into E. coli DH5 αcompetent cells. PCR validation was executed on the transformants to confirm successful incorporation. Plasmids from the positive transformants were then extracted and subjected to DNA sequencing.

Consequently, we successfully constructed four mutant vectors of kivD: pHY-kivD
S286G
, pHY-kivD
E462G
, pHY-kivD
F542G
, and pHY-kivD
V461G
, with sequencing confirming their correctness. These mutated vectors were then electroporated into
Bacillus licheniformis
DWc9n. The successfully constructed strains were respectively named DWc9n/pHY-kivDS286G, DWc9n/pHY-kivDE462G, DWc9n/pHY-kivDF542G and DWc9n/pHY-kivDV461G.

Test 2:

Using LBG medium as a foundation, we added 0.5 g/L of 4-HPP,

four strains were inoculated: DWc9n/pHY-kivD, DWc9n/pHY-kivD
S286G
, DWc9n/pHY-kivD
E462G
, DWc9n/pHY-kivD
F542G
, and DWc9n/pHY-kivD
V461G
.

These strains underwent shake flask fermentation for 24 hours under conditions of 230 rpm and 37 °C. The catalytic activity of different KivD and YugJ co-expressed enzymes in converting 4-HPP to tyrosol was assessed using HPLC to determine tyrosol production levels.

The results were as follows:

DWc9n/pHY-kivD, used as the control group,

produced a tyrosol yield of 63.65 ±3.46 mg/L.

DWc9n/pHY-kivDS286G produced a tyrosol yield of 52.44 ±7.60 mg/L.

DWc9n/pHY-kivDE462G produced a tyrosol yield of 69.05 ±0.30 mg/L.

DWc9n/pHY-kivDF542G did not produce any detectable levels of tyrosol, registering 0.00 mg/L.

DWc9n/pHY-kivDV461G showcased the highest yield with 91.73 ±7.03 mg/L of tyrosol.

![四个位点突变](https://static.igem.wiki/teams/4912/wiki/description/image13.png)

Fig. 2T: Comparison of the catalytic ability of wild-type KivD and four mutants for 4-HPP

Learn 2:

The experimental results indicate that the KivD enzyme, after mutating phenylalanine (F) at position 542 to glycine, became inactive. The catalytic activities of the enzyme, when serine (S) at position 286 and glutamic acid (E) at position 462 were mutated to glycine, did not change significantly. However, mutating valine (V) at position 461 to glycine resulted in a significant 44% increase in tyrosol yield, reaching 91.73 ±7.03 mg/L. The valine (V) at position 461 may play a crucial role in catalyzing 4-HPP.

## Cycle 3: Saturation Mutagenesis of Valine at Position 461

Design 3:

Results from the previous cycle suggest that the valine (V) at position 461 likely plays a pivotal role in catalyzing 4-hydroxyphenylpyruvate. Point saturation mutagenesis is an invaluable technique for identifying protein functional sites, enhancing enzyme activity, improving enzyme thermostability, and enhancing substrate binding specificity. Therefore, we decided to conduct point saturation mutagenesis on the KivD at position 461.

Build 3:

Following

the experimental

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
constructing point mutation plasmids,
 the pHY-kivD was utilized as a template for inverse PCR amplification. The linearized PCR product was treated with DpnI enzyme to digest and remove the original plasmid template. After the digestion, the products were recovered and mixed as per the stipulated ratios detailed in the one-step cloning kit manual. This mixture was then transformed into E. coli DH5 αcompetent cells. PCR validation was executed on the transformants to confirm successful incorporation. Plasmids from the positive transformants were then extracted and subjected to DNA sequencing. We successfully constructed 18 mutant vectors of KivD, including:
pHY
-
kivD
V461I
、
pHY
-
kivD
V461L
、
pHY
-
kivD
V461F
、
pHY
-
kivD
V461A
、
pHY
-
kivD
V461W
、
pHY
-
kivD
V461P
、
pHY
-
kivD
V461M
、
pHY
-
kivD
V461H
、
pHY
-
kivD
V461R
、
pHY
-
kivD
V461K
、
pHY
-
kivD
V461D
、
pHY
-
kivD
V461E
、
pHY
-
kivD
V461Y
、
pHY
-
kivD
V461T
、
pHY
-
kivD
V461C
、
pHY
-
kivD
V461Q
、
pHY
-
kivD
V461S
、
pHY
-
kivD
V461N
.

These were further electroporated into the host bacterium DWc9n, resulting in the successful construction of strains named:
DWc9n /pHY-kivD
V461I
、DWc9n /pHY-kivD
V461L
、DWc9n /pHY-kivD
V461F
、DWc9n /pHY-kivD
V461A
、DWc9n /pHY-kivD
V461W
、DWc9n /pHY-kivD
V461P
、DWc9n /pHY-kivD
V461M
、DWc9n /pHY-kivD
V461H
、DWc9n /pHY-kivD
V461R
、DWc9n /pHY-kivD
V461K
、DWc9n /pHY-kivD
V461D
、DWc9n /pHY-kivD
V461E
、DWc9n /pHY-kivD
V461Y
、DWc9n /pHY-kivD
V461T
、DWc9n /pHY-kivD
V461C
、DWc9n /pHY-kivD
V461Q
、DWc9n /pHY-kivD
V461S
、DWc9n /pHY-kivD
V461N
.

Test 3:

Using LBG medium supplemented with 0.5 g/L tyrosine, 20 strains were inoculated: and subjected to shake flask fermentation for 24 hours at 230 rpm and 37 °C. Tyrosol production levels were determined using HPLC. The results are as follows:
DWc9n /pHY-kivD 、DWc9n /pHY-kivD
V461I
、DWc9n /pHY-kivD
V461L
、DWc9n /pHY-kivD
V461F
、DWc9n /pHY-kivD
V461A
、DWc9n /pHY-kivD
V461W
、DWc9n /pHY-kivD
V461P
、DWc9n /pHY-kivD
V461M
、DWc9n /pHY-kivD
V461H
、DWc9n /pHY-kivD
V461R
、DWc9n /pHY-kivD
V461K
、DWc9n /pHY-kivD
V461D
、DWc9n /pHY-kivD
V461E
、DWc9n /pHY-kivD
V461Y
、DWc9n /pHY-kivD
V461T
、DWc9n /pHY-kivD
V461C
、DWc9n/pHY-kivD
V461G
、DWc9n /pHY-kivD
V461Q
、DWc9n /pHY-kivD
V461S
、DWc9n /pHY-kivD
V461N
.
These strains underwent shake flask fermentation for 24 hours under conditions of 230 rpm and 37 °C.

Tyrosol production levels were determined using HPLC. The results are as follows:

Table T3 Results of catalytic activity of different mutants at 461 of KivD on 4-HPP

|  |  |  |
| --- | --- | --- |
| Mutant type | Tyrosol ( mg/L) | Error (mg/L) |
| wild-type | 61.98 | 7.29 |
| kivD V461L | 52.06 | 7.74 |
| kivD V461F | 0.00 | 0.00 |
| kivD V461A | 0.00 | 0.00 |
| kivD V461W | 0.00 | 0.00 |
| kivD V461P | 0.00 | 0.00 |
| kivD V461M | 33.39 | 4.83 |
| kivD V461H | 0.00 | 0.00 |
| kivD V461R | 0.00 | 0.00 |
| kivD V461K | 0.00 | 0.00 |
| kivD V461D | 0.00 | 0.00 |
| kivD V461E | 57.23 | 2.24 |
| kivD V461Y | 0.00 | 0.00 |
| kivD V461T | 20.24 | 3.46 |
| kivD V461C | 31.10 | 1.08 |
| kivD V461G | 92.61 | 6.98 |
| kivD V461Q | 23.67 | 4.10 |
| kivD V461S | 25.08 | 0.39 |
| kivD V461N | 23.73 | 1.08 |

![20个位点突变](https://static.igem.wiki/teams/4912/wiki/description/image16.png)

Fig. T3 Results of catalytic activity of different mutants at 461 of KivD on 4-HPP

Study 3:

When the valine (V) at position 461 of KivD is mutated to polar but uncharged residues, the impact on its decarboxylation activity towards 4-HPP is relatively minor. When mutated to charged or nonpolar amino acids (A and M), tyrosol is almost undetectable. Additionally, substituting V461 with bulky side chains with aromatic or cyclic structures (F, W, P) has a severely detrimental effect on KivD 's decarboxylation activity towards 4-HPP. However, when mutated to isoleucine (I) at position 461 (yielding 94.08 ±8.53 mg/L), there 's a significant 46.7% increase in tyrosol production, indicating enhanced catalytic efficiency. To ascertain the enhanced catalytic efficiency of the KivD
V461I
 mutation and to determine its magnitude, our next phase will involve further enzyme activity testing and molecular modeling analysis of kivD
V461I
.

## Cycle 4: Verification of Enhanced Catalytic Efficiency of KivD V461I

Design 4:

To verify the enhanced catalytic efficiency of KivD towards 4-HPP when valine at position 461 is mutated to isoleucine and to determine the extent of this enhancement, we 'll use 4-HPP as the substrate, adding alcohol dehydrogenase YugJ and using NADH as a cofactor. This will enable us to compare the enzyme activities and kinetics of KivD and mutant KivD
V461I
 in catalyzing the synthesis of tyrosol from 4-HPP.

Build 4:

Following the experimental

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
Construction of Protein Induction Expression Strain,
 we amplified the kivD fragment using pHY-kivD as the template and the kivD
V461I
 fragment using pHY-kivD
V461I
. The backbone was amplified using pET-28a(+). After calcium transformation and verification, we successfully constructed the vectors pET28a-kivD and pET28a-kivD
V461I
. Sequenced correctly, these plasmids were transformed into
E. coli
 BL21(DE3), resulting in inducible expression strains named BL21(DE3)/pET28-kivD and BL21(DE3)/pET28-kivD
V461I
.

Test 4:

Following the experimental

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
 Induction of Protein Expression
and
Protein Purification
, we induced and purified the KivD, KivD
V461I
, and YugJ proteins. A band appeared around 60 kDa post-induction, consistent with the calculated protein size, indicating successful induction of both KivD and its mutant KivD
V461I
. Ni-NTA affinity chromatography was used for purification, with KivD and KivD
V461I
 being eluted entirely with 250 mM imidazole. Following the experimental

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
 Protein Desalting,
the purified proteins underwent desalting, and with 4-HPP as the substrate, and YugJ addition, the enzyme activities of KivD and KivD
V461I
 were evaluated using NADH as the cofactor.

![](https://static.igem.wiki/teams/4912/wiki/description/image15.png)

Fig. T41 Protein gel images of KivD, KivDV461I, and YugJ

M: Protein marker (170 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, and 14 kDa).

Lanes 1 and 2 represent the protein electrophoresis results of KivD before induction and after desalting, lanes 3 and 4 show the protein electrophoresis results of KivD
V461I
before induction and after desalting, and lanes 5 and 6 display the protein electrophoresis results of YugJ before induction and after desalting

![](https://static.igem.wiki/teams/4912/wiki/description/image18.jpg)

Fig. T42 Enzyme Kinetic Analysis of KivD and KivD
V461I
 with 4-HPP

Learn 4:

The mutant KivD
V461I
 exhibited an increase in both Km and Kcat when compared to the

wild-type KivD. Its Km/Kcat ratio improved by 33.33%, indicating a heightened catalytic efficiency per unit time. Mutating the valine at position 461 to isoleucine significantly enhanced KivD 's catalytic efficiency towards 4-HPP. To better understand the implications of this mutation on the enzyme 's structure and function, we proceeded with additional

[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
.

With four cycles completed, we successfully enhanced the key enzyme in the HT biosynthesis pathway: phenylpyruvate decarboxylase KivD. By improving the combined catalytic efficiency of KivD and the endogenous alcohol dehydrogenase YugJ in
Bacillus licheniformis
, more 4-HPP is converted to tyrosol. In the subsequent cycle, to understand the exact boost this protein engineering imparts to hydroxytyrosol synthesis, we will co-express kivD
V461I
 and hpaBC, testing the engineered strain 's efficiency in synthesizing hydroxytyrosol from glucose.

## Cycle 5: Co-expression of hpaBC and kivD V461I  to Test the Efficiency of Bacillus licheniformis  in de Novo Synthesis of Hydroxytyrosol from Glucose

Design 5:

Having enhanced the KivD 's efficiency towards 4-HPP over the previous four cycles and optimized the Ehrlich pathway, it remains uncertain what effect this will have on hydroxytyrosol production efficiency. This cycle aims to test if KivD
V461I
 can effectively synthesize hydroxytyrosol in
Bacillus licheniformis
 using glucose by constructing a co-expression vector of kivD and hpaBC. We will perform a comparative experiment using the previously engineered strain DHT11.

Build 5:

Following the

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
Constructing of the Co-expression Plasmid
and using pHY-kivD
V461I
 as the template, we successfully constructed the co-expression vector pHY-PbacA-kivD
V461I
-P43-hpaBC. This vector was then transformed into the DH10 strain, resulting in the engineered strain named DHT12 (DH10/pHY-PbacA-kivD
V461I
-P43-hpaBC).

![](https://static.igem.wiki/teams/4912/wiki/description/image17.png)

Fig. 5B1 plasmid profile of pHY-PbacA-kivD
V461I
-P43-hpaBC

![2400bp](https://static.igem.wiki/teams/4912/wiki/description/image21.png)

Fig. 5B2
Agarose gel map for verification of 2463bp target DNA bands

Test 5:

Both strains DHT11 and DHT12 were inoculated into LBG medium with 40g of glucose as the substrate. They were fermented in a rotary shaker at 230 rpm and 37 ℃for 48 hours. The content of hydroxytyrosol was detected by HPLC. The production of hydroxytyrosol by strain DHT11 was 1898.73 ±134.24mg/L, while that by strain DHT12 was 3298.85 ±150.28mg/L.

![DHT11和DHT12](https://static.igem.wiki/teams/4912/wiki/description/image19.png)

Fig. 5T Comparison of hydroxytyrosol yield between DHT11 and DHT12

Study 5:

Strain DHT12 was able to produce 3298.85 ±150.28mg/L of hydroxytyrosol (HT) using 40g of glucose. Compared to DHT11, which produced 1898.73 ±134.24mg/L of HT, this is a significant increase of 73.74%. This result shows that the protein engineering modifications made to phenylpyruvate decarboxylase KivD had a notable impact on hydroxytyrosol production.

## Cycle 6: Stoichiometric Optimization of kivD V461I  and hpaBC for Enhanced Hydroxytyrosol Production

Design 6:

![](https://static.igem.wiki/teams/4912/wiki/description/image20.png)

The two enzymes kivD
V461I
 and hpaBC are pivotal in the HT synthesis pathway. Overexpression of kivD
V461I
 could result in the accumulation of the toxic compound phenylacetaldehyde, intensifying the metabolic burden on the cells. On the other hand, inadequate expression of hpaBC would lead to tyrosol accumulation, making HT production inefficient. It is evident that a balanced expression of kivD
V461I
 and hpaBC is critical for optimal HT yield. To reduce the accumulation of tyrosol and further boost the production of HT, we aim to build on the foundational research of DHT12 by optimizing the stoichiometric balance of kivD
V461I
 and hpaBC in future experiments.

![](https://static.igem.wiki/teams/4912/wiki/description/image22.png)

Fig. D6 Schematic Representation of the Expression Intensity of Three Promoters: Pbay, PbacA, and P43

Using promoters of different intensities can regulate the expression of two genes. The intensity comparison of strong promoters from lichen bacillus PbacA, Pbay, and the promoter from Bacillus subtilis P43 is

Pbay >PbacA >P43 (Rao Y., 2021). We will use these three promoters of different intensities to optimize the expression strength of kivD
V461I
 and hpaBC.

Build 6:

Following the

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
Constructing of the Co-expression Plasmid
 , using pHY-kivD
V461I
 and pHY-hpaBC as templates, we respectively used PbacA, Pbay, and P43 as promoters, replacing the original promoters in kivD
V461I
 and hpaBC. We further constructed the other 8 combination expression vectors:
pHY-PbacA
-kivD
V461I-
PbacA-hpaBC 、pHY-PbacA-kivD
V461I
-Pbay-hpaBC 、pHY-P43-kivD
V461
I-P43-hpaBC 、
pHY
-
P43-
kivD
V461I
-PbacA-hpaBC 、
pHY-P43-kivD
V461I
-Pbay-hpaBC 、pHY-Pbay-kivD
V461I
-P43-hpaBC 、pHY-Pbay-kivD
V461I
-PbacA-hpaBC 、pHY-Pbay-kivD
V461I
-Pbay-hpaBC.
Subsequently, we electroporated these 8 vectors into DH10, resulting in strains DHT13 to DHT20.

![屏幕截图 2023-10-10 185024](https://static.igem.wiki/teams/4912/wiki/description/image23.png)

Fig. 6B Different Recombination Part Combinations from DHT13 to DHT20

Test 6:

Each of the strains DHT12 to DHT20 was inoculated into LBG medium and fermented in shaker at 230 rpm and 37 ℃for 48 hours. Afterward, we used HPLC to measure the HT yield.

DHT12 produced 3296.58 ±140.82mg/L of hydroxytyrosol, DHT13 produced 3484.10 ±215.64mg/L, DHT14 produced 3504.34 ±156.34mg/L, DHT15 produced 2543.66 ±140.45mg/L, DHT16 produced 4036.03 ±289.20mg/L, DHT17 produced 4650.12 ±228.92mg/L, DHT18 produced 3432.45 ±103.21mg/L, DHT19 produced 3671.83 ±273.71mg/L, and DHT20 produced 2820.30 ±235.56mg/L.

![启动子工程1](https://static.igem.wiki/teams/4912/wiki/description/image24.png)

![启动子工程2](https://static.igem.wiki/teams/4912/wiki/description/image25.png)

Fig. 6T: Comparison of Hydroxytyrosol Yield from DHT12 to DHT20

Learn 6:

Compared with DHT12 (3296.58 ±140.82mg/L), DHT17 (4650.12 ±228.92mg/L) saw a substantial increase in HT production by 41.06%. The promoter combination of Pbay-kivD
V461I
-Pbay-hpaBC can optimize the expression of kivD
V461I
 and hpaBC to the greatest extent. Furthermore, the biomass also increased by 33.97%. Therefore, we speculate that optimizing the expression of kivD
V461I
 and hpaBC can reduce the production of certain secondary metabolites (such as aldehydes or alcohols) in the cell, thereby alleviating the metabolic burden of the cell and increasing the yield of the target product HT.

## Cycle 7: Screening Limiting Factors in the Shikimic Acid Pathway

Design 7:

The presence of rate-limiting steps in metabolic

pathways exists. To explore whether important precursors in the HT synthetic pathway are rate-limiting factors, we will add 0.5 g/L of two important precursors, 4-HPP and shikimic acid, separately to the medium to verify whether the supply of these precursors affects the synthesis of HT.

Test 7:

Based on the LBG medium, we separately added 0.5 g/L of 4-HPP and shikimic acid. The medium without added precursors served as the control group. The DHT17 strain was then inoculated and fermented in shaker at 230 rpm and 37 ℃for 48 hours; HPLC chromatography was used to analyze the HT yield. After adding 0.5 g/L of shikimic acid to the medium, the HT yield of DHT17 was 4232.41 ±59.44 mg/L. After adding 0.5 g/L of 4-HPP, the HT yield of the DHT17 strain was 5043.39 ±48.18 mg/L. The DHT17 strain without any added precursors had an HT yield of 4648.12 ±228.82 mg/L.

![限速步骤挖掘1](https://static.igem.wiki/teams/4912/wiki/description/image26.png)

Fig. T7 Comparison of Hydroxytyrosol Production with Added Precursor Substances

Learn 7:

When 0.5 g/L of shikimic acid was added to the culture medium, the HT yield (4232.41 ±59.44 mg/L) was reduced by 8.94% compared to the yield of the strain without addition (4648.12 ±228.82 mg/L). However, when we added 0.5 g/L of 4-HPP, the HT yield reached 5043.39 ±48.18 mg/L, which is 8.50% higher than the yield of the strain without addition (4648.12 ±228.82 mg/L). The insufficient supply of HT 's precursor, 4-HPP, is very likely the critical step restricting the HT synthesis pathway.

## Cycle 8: Increasing 4-HPP Supply with Multiple Copies of tyrA fbr

Design 8:

The insufficient supply of HT 's precursor 4-HPP is likely the critical step restricting the HT synthesis pathway. The bifunctional branched-chain amino acid transaminase/prephenate dehydrogenase TyrA, originating from
E. coli
, is a key enzyme in the synthesis of 4-HPP. The inadequate expression of the Tyr mutant gene, tyrA
fbr
, is very likely the key rate-limiting factor causing the insufficient supply of the precursor 4-HPP.

Hence, in this cycle, we plan to address this by increasing the copy number of tyrA
fbr
. We select the non-essential gene yjic locus in the DWc9n genome and the PbacA promoter to construct the tyrA
fbr
 integration expression vector based on the T2-yjiC-pheA plasmid.

Build 8:

Following the

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
Construction of Gene Integration and Promoter Replacement Plasmids
, using pHY-tyrA
fbr
 and T
2
(2)-yjiC-pheA as templates, we successfully constructed the tyrA
fbr
 integration vector T
2
(2)-yjiC-tyrA
fbr
. The multi-copy integration plasmid constructed is electroporated into the competent DH10  and acquired the multiple-copy strain DH11 through single-double exchanges. DH11
(
Bacillus licheniformis

DWc9n Δpyk-xkdE::yugJ-ldh::tyrA
fbr-
yvnA::tyrA
fbr
-xkdG::aroG
fbr
-PbacA-aroK ΔpheA ΔhisC ΔdhaS ΔdhbC ΔadhA-PbacA-glcU-PbacA-glcK-ParoK-ptsG)
Subsequently, the plasmid pHY-P43-kivD
V461I
-Pbay-hpaBC is electroporated into the DH11 strain, and the successfully constructed strain is named DHT21 (DH11/pHY-P43-kivD
V461I
-Pbay-hpaBC).

![](https://static.igem.wiki/teams/4912/wiki/description/image27.png)

Fig. B81 Plasmid profile of T
2
(2)-yjiC-tyrA
fbr

![1300BP](https://static.igem.wiki/teams/4912/wiki/description/image28.png)

Fig. B82 Agarose gel map for verification of 1300bp target DNA bands

Test 8:

Both DHT21 and DHT17 are inoculated into the LBG medium and fermented at 230 rpm, 37 ℃for 48 hours. HPLC chromatography is then employed to analyze the HT yield. The HT yield of DHT17 is 4650.43 ±28.92 mg/L, while that of DHT21 is 5907.44 ±68.54 mg/L.

![多拷贝](https://static.igem.wiki/teams/4912/wiki/description/image29.png)

Fig. T8 Comparison of Hydroxytyrosol Yield between DHT17 and DHT21

Learn 8:

The HT production of strain DHT21 (5907.44 ±68.54 mg/L) is 27.03% higher than that of the control strain DHT17 (4650.43 ±28.92 mg/L). It is evident that multiple copies of tyrA
fbr
 help address the deficiency in the 4-HPP supply, thereby enhancing the yield of hydroxytyrosol.

# Cycle 9: Screening Limiting Factors in the Pentose Phosphate Pathway

Design 9:

The pentose phosphate (E4P) pathway plays a crucial role in the synthesis of HT when glucose is used as a substrate by the licheniformis Bacillus. Enhancing the E4P pathway to increase carbon flux can further improve the synthesis efficiency of HT. Transketolase is the key enzyme in the E4P pathway, so we decided to overexpress the gene encoding transketolase, tkt.

Build 9:

Following the

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
Construction of Gene Integration and Promoter Replacement Plasmids,
 we used the strong promoter PbacA to replace the tkt promoter in the DH11 strain 's genome. Using the pHY-PbacA-hpaBC as a template to amplify the PbacA fragment and using the T2-Pbay-hisC as a template to separately amplify the homology arm fragments, we constructed the

pHY-PbacA-tkt by the SOE-PCR method. After electroporating the plasmid pHY-PbacA-tkt into the DH11 strain, the successfully constructed strain was named DHT12 (DWc9n Δpyk-xkdE::yugJ-ldh::tyrA
fbr
-yvnA::tyrA
fbr
-xkdG::aroG
fbr
-PbacA-aroK ΔpheA ΔhisC ΔdhaS ΔdhbC ΔadhA-PbacA-glcU-PbacA-glcK-ParoK-ptsG-PbacA-tkt). Following the electroporation of the co-expression plasmid pHY-P43-kivD
V461I
-Pbay-hpaBC, the constructed strain was named DHT22.

![](https://static.igem.wiki/teams/4912/wiki/description/image1.png)

Fig. 9B1 Plasmid profile of pHY-PbacA-tkt

![2000BP](https://static.igem.wiki/teams/4912/wiki/description/image2.png)

Fig. 9B2 Agarose gel map for verification of 2000bp target DNA bands (Lanes 1, 2, 7 and 8 are the target bands)

Test 9:

Both strains, DHT21 and DHT22, were inoculated in LBG culture medium and fermented at 230 rpm and 37 °C for 48 hours. The HT content was detected by HPLC. The hydroxytyrosol yield of DHT21 was 5907.77 ±268.45mg/L, and that of DHT22 was 5890.42 ±142.35mg/L.

![只增强tkt](https://static.igem.wiki/teams/4912/wiki/description/image3.png)

Fig. T9 Comparison of Hydroxytyrosol Yield between DHT21 and DHT22

Learn 9:

Compared to the control strain DHT21, the HT yield of DHT22 did not show a significant change. Merely overexpressing tkt did not break the limitation. We must seek other methods.

## Cycle 10: Enhancement of the tkt and zwf Gene Expression to Increase the accumulation of E4P

Design 10:

In the previous cycle, we found that enhancing only tkt expression showed no significant changes. We hypothesize that this might be due to insufficient expression levels of the initial enzyme in the pentose phosphate pathway. Since glucose 6-phosphate dehydrogenase, encoded by the gene zwf, is the key rate-limiting enzyme of the pentose phosphate pathway, we decided to further enhance the expression of  zwf .

Build 10:

Following the

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
Construction of Gene Integration and Promoter Replacement Plasmids
, the strong promoter PbacA was used to replace the zwf promoter in the DH12 strain genome. Using PHY-PbacA-hpaBC as a template, the PbacAu12 fragment was amplified. Using T2-Pbay-hisC as a template, the upstream and downstream homologous arms were separately amplified. Using T2-ΔyjiC-PyjiC ∷yddG as a template, the skeleton was amplified. Through SOE-PCR, in the order of the upstream homologous arm + PbacAU12 + downstream homologous arm, they were fused together. After transforming into E.coli DH5 αand validating the transformant, the plasmid pHY-PbacAu12-zwf was successfully constructed. After electroporating pHY-PbacAu12-zwf into DH12, the strain DH13 was successfully constructed (
Bacillus licheniformis
DWc9n
Δpyk-xkdE::yugJ-ldh::tyrA
fbr
-yvnA::tyrA
fbr
-xkdG::aroG
fbr
-PbacA-aroK ΔpheA ΔhisC ΔdhaS ΔdhbC ΔadhA-PbacA-glcU-PbacA-glcK-ParoK-ptsG-PbacA-tkt-PbacA-zwf). Then, the co-expression plasmid pHY-P43-kivD
V461I
-Pbay-hpaBC was electroporated, and the constructed strain was named DHT23.

![](https://static.igem.wiki/teams/4912/wiki/description/image4.png)

Fig. 10B1 Plasmid profile of pHY-PbacAu12-zwf

![1500bp](https://static.igem.wiki/teams/4912/wiki/description/image5.png)

Fig. 10B2 Agarose gel map for verification of 1600bp target DNA bands

Test 10:

Both strains DHT21 and DHT23 were inoculated and fermented in LBG medium at 230 rpm and 37 °C for 48 hours. The HT content was detected using HPLC. The hydroxytyrosol yield of DHT21 was 5907.77 ±268.45mg/L, DHT22 produced 5890.42 ±142.35mg/L, and DHT23 yielded 6298.54 ±270.5mg/L.

![叠加强化](https://static.igem.wiki/teams/4912/wiki/description/image6.png)

Fig. T10 Comparison of Hydroxytyrosol Yield between DHT21, DHT22 and DHT23

Learn 10:

By co-enhancing the expression of tkt and zwf, the DHT23 strain fermented for 48h synthesized 6298.05 ±70.52 mg/L of HT, which is a 6.61% increase compared to the control strain DHT21 (5907.44 ±34.18 mg/L). Simultaneously enhancing the expression of tkt and zwf can strengthen the E4P pathway, allowing more carbon flux to flow to the HT synthesis pathway, further promoting HT synthesis.

## Cycle 11: Utilizing Oxygen-responsive Regulatory Switch to Regulate hpaBC Expression

Design 11:

During our fermentation tests, we observed the fermentation liquid turning black. Given HT 's strong antioxidative properties, we hypothesized that it might be getting oxidized by the air during fermentation.

The regulatory network behind anaerobic adaptation has been widely studied in recent years. The FNR transcription factor plays a crucial regulatory role in bacteria, primarily functioning under anaerobic or low-oxygen conditions, assisting bacteria in adapting to fluctuations in oxygen levels. In environments with abundant oxygen, FNR is in its oxidized state, preventing it from binding to DNA, which provides favorable conditions for cell growth and metabolism. However, in oxygen-deficient environments, FNR undergoes reduction, binds to DNA, and regulates the expression of genes related to oxygen metabolism (Bates, D M et al., 1995).

Using the DBTBS database, we identified the promoter Pnark with a conserved Fnr binding site. We planned to replace the original promoter Pbay of hpaBC with Pnark, allowing the transcriptional regulator Fnr to regulate the expression of the key enzyme HpaBC, which catalyzes the conversion of tyrosol to hydroxytyrosol (HT).         This will enable the engineered bacteria to grow optimally in the initial phase when oxygen is abundant, accumulating tyrosol, and express HpaBC to transform tyrosol into HT when oxygen becomes limited, thereby avoiding the oxidation of HT.

Build 11:

Following the

[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
 for
 Constructing of the Co-expression Plasmid,
 we amplified the Pnark fragment from the
Bacillus licheniformis
 DWc9n total genome and the backbone from the pHY-Pbay-hpaBC template. After calcium transformation and transformant verification, we successfully constructed pHY-Pnark-hpaBC, which was sequenced and confirmed. This was then electrotransformed into DH13, resulting in the construction of strain DHT24 (DH13/PHY-Pnark-hpaBC).

![](https://static.igem.wiki/teams/4912/wiki/description/image7.png)

Fig. 11B1 Plasmid profile of pHY-PbacA-tkt

![400bp](https://static.igem.wiki/teams/4912/wiki/description/image8.png)

Fig. 11B2 Agarose gel map for verification of 400bp target DNA bands

Test 11:

The constructed strains DHT24 and DHT23 were separately fermented in 40 g/L glucose LBG with three different filling volumes (50ml, 75ml, 100ml) under the conditions of 230rpm and 37 ℃for 48h. Subsequently, the production of hydroxytyrosol and tyrosol was determined using HPLC technology. It 's important to note that at the same rotation speed, different filling volumes imply different dissolved oxygen levels in the fermentation liquid inside the flask. By adjusting the filling volume, we can conduct a preliminary oxygen-responsive test.

![fnr](https://static.igem.wiki/teams/4912/wiki/description/image9.png)

Fig. 11T Comparison of Tyrosol and Hydroxytyrosol Production for DHT23 and DHT24 at Different Volumes of Solution (50ml, 75ml, 100ml)

Learn 11:

At 50ml and 75ml volumes, a large amount of tyrosol accumulated in DHT24 and was not effectively converted into HT. However, at 100ml volume, the accumulated tyrosol in DHT24 was transformed into HT. This indicates that when the fermentation liquid is oxygen-rich, the Fnr transcription factor inhibits the expression of the downstream gene hpaBC, allowing the engineered bacteria to grow and accumulate tyrosol. When the fermentation liquid is oxygen-limited, Fnr regulation increases the expression of the downstream gene hpaBC, catalyzing the conversion of tyrosol to HT.

This suggests that the bacterium can utilize the Fnr transcription factor to regulate the synthesis of HT based on oxygen concentration, and the Fnr oxygen regulatory switch is quite successful. However, due to lab constraints, we struggle to provide a genuinely low-oxygen environment for the bacteria, allowing the oxygen-responsive regulatory switch to be fully "turned on "for more in-depth experimental research. To address the oxidation of HT during fermentation testing, we will employ fermentation tanks to control oxygen concentration more precisely. This will provide a more stable, accurate, and controllable experimental environment for our subsequent research.

Summary

Throughout the engineering cycle, we first conducted protein engineering modifications on the heterologously introduced phenylacetate decarboxylase, KivD. This yielded a mutant, KivD
V461I
, which displayed a significantly enhanced catalytic efficiency for 4-hydroxyphenylpyruvate. By co-expressing this mutant with hydroxylase HpaBC from Escherichia coli, KivD
V461I
, and endogenous alcohol dehydrogenase YugJ, our recombinant strain DHT12 achieved an HT production of 3298.85 ±150.28 mg/L.

Further, we identified and eliminated rate-limiting steps in the hydroxytyrosol synthesis process. Firstly, by utilizing promoter engineering, we optimized the expression intensity of key genes hpaBC and kivD
V461I
 in the hydroxytyrosol synthesis module, with the P43-kivD
V461I
-Pbay-hpaBC combination strain achieving the highest HT production. Subsequently, by overexpressing tyrA
fbr
, we addressed the limitation arising from insufficient 4-HPP supply, further elevating HT yield. Next, by boosting the expression of tkt and zwf, we aimed to enhance

 the accumulation of the precursor E4P, resulting in the best recombinant strain, DHT23. In laboratory shake flask fermentation experiments, this strain achieved an HT yield of 6298.05 ±70.52 mg/L, which is 3.31 times greater than the starting strain DHT11 (1900.00 ±126.02mg/L).

Notably, our optimal engineered strain, DHT23, remarkably achieved an HT production of 9475 mg/L in a 5L fermenter under fed-batch conditions, reaching a productivity rate of 395 mg/L/h, marking the highest ever reported in microbial fermentation of HT. The DBTL engineering phase solely revolved around iterative optimizations within the biological system. We cordially invite you to view our definitive results in

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

# References

* [1] Yep, Alejandra et al. “Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase.” Bioorganic chemistry vol. 34,6 (2006): 325-36. doi:10.1016/j.bioorg.2006.08.005
* [2] Berthold, Catrine L et al. “Structure of the branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction.” Acta crystallographica. Section D, Biological crystallography vol. 63,Pt 12 (2007): 1217-24. doi:10.1107/S0907444907050433
* [3] Chen, Grey S et al. “Saturated mutagenesis of ketoisovalerate decarboxylase V461 enabled specific synthesis of 1-pentanol via the ketoacid elongation cycle.” Scientific reports vol. 7,1 11284. 12 Sep. 2017, doi:10.1038/s41598-017-11624-z
* [4] Milne, N et al. “Comparative assessment of native and heterologous 2-oxo acid decarboxylases for application in isobutanol production by Saccharomyces cerevisiae.” Biotechnology for biofuels vol. 8 204. 1 Dec. 2015, doi:10.1186/s13068-015-0374-0
* [5] Rao Y, Li P, Xie X, et al. Construction and characterization of a gradient strength promoter library for fine-tuned gene expression in Bacillus licheniformis[J]. ACS Synthetic Biology, 2021, 10(9): 2331-2339.
* [6] Bates, D M et al. “Characterization of FNR\* mutant proteins indicates two distinct mechanisms for altering oxygen regulation of the Escherichia coli transcription factor FNR.” Journal of bacteriology vol. 177,14 (1995): 3972-8. doi:10.1128/jb.177.14.3972-3978.1995

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

* [Introduction](#Part1)
* [Scale-up Production](#Part2)
* [Conclusions and Outlook](#Part3)

EXPERIMENT

RESULTS

In our results, we embrace the principle of 'reviewing the old to understand the new',combining past research with
 current findings to pave the way for future explorations.

# Introduction

After 11 cycles of engineering, we successfully developed the optimized engineered strain DHT23(
Bacillus licheniformis
DWc9n Δpyk-xkdE::yugJ-ldh::tyrA
fbr
-yvnA::tyrA
fbr
-xkdG::aroG
fbr
-PbacA-aroK ΔpheA ΔhisC ΔdhaS
ΔdhbC ΔadhA-PbacA-glcU-PbacA-glcK-ParoK-ptsG-PbacA-tkt-PbacA-zwf/
pHY-P43-kivD
V461I
-Pbay-hpaBC). Under laboratory shake flask fermentation conditions, this strain produced 6298.05 ±70.52 mg/L of HT.

The scale-up of cultivation is pivotal for translating laboratory research into practical applications and commercial production. In biotechnology, a cell culture might exhibit excellent performance in a laboratory shake flask; however, when scaled up to large fermenters, aspects like oxygen transfer, nutrient supply, temperature, and pH control can become increasingly complex. Therefore, a detailed consideration and optimization of these parameters are essential to ensure the success of the production process.

In the following sections of our results, we will present the HT yield from the scaled-up production using Bacillus licheniformis and summarize the strategies employed. A deeper discussion and future perspectives will also be provided.

* Scale-up Production
* Conclusions and Outlook

## Scale-up Production

To genuinely harness synthetic biology in addressing the existing challenges of low HT yield, which consequently leads to incomplete industry standards and limited applications, we aim to transition the microbial production of HT from lab research to real-world applications and commercial outputs. Using our best-performing strain, DHT23, we conducted fed-batch fermentation in a 5L fermenter and explored the effects of stirring speed on HT production.

To investigate the potential of HT production at the scale-up level, fed-batch fermentation was performed in 5L-fermentors using the best strain DHT23. The impact of agitation speed on HT production was studied. The culture of DHT23 was performed with agitation speed maintained at 600 rpm during the entire cultivation. The biomass reached the highest at 18 h and then fluctuated around 40 (OD600). As shown in Fig. 1A, the amount of HT produced by DHT23 reached 9475 mg/L with a yield of 135.4 mg/g glucose after 24 h of cultivation. The dissolved oxygen (DO) was decreased to 2% after 21 h of cultivation, and glucose concentration was maintained between 10 g/L and 20 g/L. In another scenario, the culture of HT was performed with agitation speed shifted from 600 to 400 rpm after 12 h of cultivation. The maximal cell growth (OD
600
) reached 40 at 15 h and decreased after glucose content was below 10 g/L. Under this condition, the highest titer of HT was 8887 g/L at 36 h with a yield of 127 mg/g (Fig. 1B). These results showed that high agitation speed contributed to the improvement of cell growth and HT synthesis due to the increase of the transfer efficiency of oxygen and nutrients.

![](https://static.igem.wiki/teams/4912/wiki/results/results/image1.jpg)

Fig. 1 Fed-batch production of HT in 5-L bioreactor.

(1A) The culture of DHT23 was performed with agitation speed maintained at 600 rpm during the entire cultivation and 15 g/L glucose was added one time at 12, 18 and 30 h; (1B)The culture of HT was performed with agitation speed shifted from 600 to 400 rpm after 12 h of cultivation and 15 g/L glucose was added one time at 12, 24 and 36 h.

Red arrows mean feeding time of glucose and Green arrows mean the time of maximum HT titer. The results were one of the three times, which was given as an example to show the fermentation results.

 To explore the distribution of carbon flux, the extracellular metabolites were analyzed using the GC-MS method. Previous research has reported that HT may be further oxidized to generate o-quinone-3,4-quinonephenylethenol.34 The o-quinone-3,4-quinonephenylethenol was not found. However, 2, 5-dihydroxymandelic acid, galactose, N-acetylgalactosamine and 3, 4-dihydroxyphenyllactic acid were accumulated in the fermentation media, which needs to be solved in the future.

![Fig.S2](https://static.igem.wiki/teams/4912/wiki/results/results/image3.jpg)

Fig. 2 GC-MS analysis results of the main extracellular metabolites of DHT23 strain at 24 h.

1: HT, 2: glucose, 3: 2, 5-dihydroxymandelic acid, 4: galactose, 5: 3,4-Dihydroxyphenylacetic acid, 6: N-Acetyl-D-galactosamine

# Conclusions and Outlook

HT is a potent antioxidant with a myriad of health-related functions. Microbial production of HT serves as an alternative to traditional methods. Building upon the DHT11, we successfully established an efficient HT production platform primarily by employing four strategies: protein engineering of phenylpyruvate decarboxylase KivD, metabolic balance between KivD and hydroxylase HpaBC, amplification of tyrA
fbr
 to enhance the supply of 4-HPP, and overexpression of tkt and zwf to bolster the supply of E4P.

![](https://static.igem.wiki/teams/4912/wiki/results/results/image2.png)

Fig. 3
Metabolic engineering
B. licheniformis
 for
de novo
 synthesis of

hydroxytyrosol

Heterologous introduced and overexpressed genes are highlighted in red, deleted genes are annotated in red cross, and knock-down genes are annotated in green.
Glc
: glucose,
2,3-BD
: 2,3-butanediol,
AC
: acetoin,
 AHAS
: acetolactate,
 Ac-CoA:
 acetyl-CoA,
 PYR
: pyruvate,
PEP:
 phosphoenolpyruvate,
 G6P
: glucose-6-phosphate,
 F6P
:

fructose 6-phosphate ,
G3P
: glyceraldehyde 3-phosphate,
Ru5P
:

ribulose 5-phosphate,
X5P
: xylulose 5-phosphate,
 R5P
: ribose 5-phosphate,
S7P
:sedoheptulose 7-phosphate,
E4P
: erythose-4-phosphate,
DAHP
: 3-deoxy-D-arabino-heptulosonate-7-phosphate,
DHQ
: 3-dehydroquinic acid,
DHS
: 3-dehydroshikimic acid,
SHIK:
 shikimate,
S3P
: Shikimate-3-phosphate,
EPSP
: 5-enolpyruvylshikimate 3-phosphate,
CHA
: chorismate,
 PPA
: prephenate, 4-
HPP
: 4-hydroxyphenylpyruvate,
4-HPAA
: 4-hydroxyphenylacetaldehyde,
 HT
: hydroxytyrosol,
ICHA
: isochorismate,
 OPP
: phenylpyruvate,
PHE
: L-phenylalanine,
TYR
: L-tyrosine,
 EMP
: Embden-Meyerhof-Parnas pathway,
PPP:
 pentose phosphate pathway,
 TCA cycle
: tricarboxylic acid cycle.

In the Ehrlich pathway, the activity of keto acid decarboxylases is crucial for the efficient biosynthesis of aromatic and branched-chain alcohols (Miao, Rui et al., 2018). This is due to KivD 's lower catalytic activity towards 4-HPP compared to 2-keto acids and phenylpyruvate (Yep, Alejandra et al., 2006). Numerous studies have utilized techniques like homology modeling, site-directed mutagenesis, and molecular dynamics simulations to alter KivD 's decarboxylation activity towards 2-keto acids for producing various alcohols. Significantly, the V461 residue in KivD 's active site plays a pivotal role in its catalytic activity towards 2-keto acids. Research indicates that by mutating V461, the specificity and catalytic efficiency towards the precursors of isobutanol and 1-pentanol, namely 2-ketovalerate and 2-ketohexanoate, are enhanced (Miao, Rui et al., 2018; Chen, Grey S et al., 2017). For instance, the V461G mutant exhibited an evident improvement in specificity towards long-chain 2-keto acids and an enhanced yield of 3-methyl-1-butanol (Yep, Alejandra et al., 2006). We screened variants of the V461 residue to elevate KivD 's catalytic activity towards 4-HPP. By employing this strategy, the tyrosol titer in the V461I mutant showed a substantial boost compared to the wild-type, effectively overcoming the rate-limiting step in HT production.

We further investigated several ambiguous factors limiting HT production with the aim of directing more carbon flux towards the synthesis of HT, thereby increasing its yield.

Initially, an imbalance in the expression of KivDV461I and hpaBC was identified as a potential cause for inadequate accumulation of tyrosol. By fine-tuning the expression levels of kivDV461I and HpaBC, we cultivated the optimized strain DHT17, which exhibited an impressive HT titer of 4680mg/L in shake flasks. Remarkably, this HT titer surpasses any other reported titers to date by at least 2.6-fold (Table 1). This identified bottleneck could serve as a target for metabolic engineering in other microbial cell factories aiming to boost HT production.

Additionally, we delved deeper into potential limiting factors within the cinnamic acid and E4P pathways. Importantly, we discerned that a deficiency in 4-HPP availability acted as a rate-limiting factor, corroborating previously published findings (Liu, Huayi et al., 2022). By amplifying the expression of tyrA
fbr
, we achieved a notable surge in HT yield. Given that E4P is a pivotal precursor for the synthesis of aromatic amino acid derivatives, we posited that its availability in the DHT21 strain could be a limiting factor for HT synthesis. Past research has indicated that the overexpression of transketolase Tkt and glucose-6-phosphate dehydrogenase Zwf enhanced the production of aromatic compounds (Zhang, Chuanzhi et al., 2015). In our study, we substantiated that elevating the expression of tkt and zwf serves as an efficacious strategy to augment the supply of E4P, consequently boosting HT production.

We further delved into the application of the Oxygen-responsive Regulatory Switch to implement a "two-phase "precision fermentation strategy. In the initial phase of this strategy, nutrients predominantly support cell growth, while the subsequent phase primarily focuses on the accumulation of HT. Although constraints in our laboratory conditions prevented us from achieving optimal oxygen dissolution control, this approach still offers invaluable insights for the production of other aromatic compounds susceptible to oxidation. Our enthusiasm for research did not wane with the conclusion of the competition, and continuous experimentation and innovative endeavors remain ongoing. Currently, a promising strategy we 're exploring involves integrating the conservative binding sequence of FNR with other promoters (like Pbay) to optimize promoter design.

Table 1 Summary of hydroxytyrosol (HT) production from simple carbon sources by engineered microorganisms

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Strains | Carbon source | Characteristics | HT titer  (mg/L) | HT yield  mg/g | Produc  -tivity (mg/L/h) | Refer-ences |
| E. coli | Glucose | TH, PCD, DHPR, DDC  and  TYO ,  ∆feaB | 12.3  shake flasks | 1.2 | 0.17 | Satoh, Yasuharu et al., 2012 |
| E. coli | Glucose | aroG fbr ,tyrA fbr , TDC, TYO, and hpaBC, ∆tyrR ∆pheA ∆feaB | 268.3  shake flasks | Not shown | 8.9 | Choo, H.J. et al., 2018 |
| 5. coli | Glucose | aroE, aroD,aroB OPT ,aroG fbr , ppsA, tktA, tyrB, tyrA fbr  ,aroC, aroA,aroL, PcAAS, RsTYR, yqhD | 270.8  shake flasks | 27.1 | 6.0 | Trantas, Emmanouil et al., 2019 |
| E. coli | Glucose glycerol | tyrA fbr , ppsA, tktA, aroG fbr , ARO10, ADH6, hpaBC ∆feaB ∆pheA ∆tyrA | 647.0  shake flasks | 34.2 | 13.5 | Li, Xianglai et al., 2018 |
| E. coli | Glucose | ΔfeaB ΔpheA ΔtyrB ΔtyrR ΔtrpE ΔpabB ΔpabA ΔpykF ΔpoxB, 5\*ARO10\*, EchpaBC | 1810.0  shake flasks    2950.0  5L bioreactor | 90.5    Not shown | 37.7    122.9 | Liu, Chunxiao et al., 2021 |
| S. cerevisiae | Sucrose glycerol | ARO4 K229L , ARO7 G141S ,  Bbxfpk opt ,  Pcaas opt , PahpaBC opt , ∆ GAL80 | 308.7  shake flasks | Not shown | 3.2 | Liu, Yingjie et al.，2022 |
| S. cerevisiae | Glucose | ARO3 D154N , ARO4 K229L , ARO7 G141S , ARO10, EchpaBC, | 375.0  shake flasks | 2.2 | 1.5 | Bisquert, Ricardo et al.，2022 |
| S. cerevisiae | Glucose | ARO2, ARO3 D154N , ARO4 K229L , ARO7 G141S , ARO10, TKL1, RKI1, PahpaB, EchpaC, TRP2 ↓, ∆TRP1 | 1120.0  shake flasks  6970.0  5L bioreactor | 56    36.5 | 13.3    79.2 | Liu, Huayi et al.，2022 |
| B. licheniformis | Glucose | aroG fbr ,2\*tyrA fbr , aroK, EchpaBC, yugJ, kivD VI , glcU, glcK, tkt, zwf ∆pyk ∆hisC ∆pheA ∆dhaS ∆dhbC ∆adhA,ptsG ↓ | 6299.0  shake flasks  9475.0  5L bioreactor | 157.3    135.4 | 131.2    395.0 | This study |

Numerous researchers have dedicated significant efforts to achieve the de novo production of HT from basic carbon sources using various microorganisms. Compared to engineered strains of Escherichia coli and Saccharomyces cerevisiae, Bacillus licheniformis presents as a more apt host for HT production. In this study, our engineered strain DHT23 achieved a peak HT titer of 9475 mg/L from glucose through fed-batch fermentation, marking a fivefold enhancement from the initial strain DHT11, which stood at 1900.00 ±126.02mg/L. To the best of our knowledge, the titer, yield, and productivity of HT documented in this study are unparalleled in microbial fermentation literature (as cited in Table 1).

In contrast to chemical synthesis, our devised procedure is distinctly eco-friendly and cleaner, leaning heavily on cost-effective raw materials. Moreover, our process sidesteps wastewater generation (by eschewing acid/base consumption) and operates independently of olive-derived resources, which casts it as a markedly cleaner and more sustainable method compared to plant extraction techniques. Hence, when juxtaposed with chemical synthesis and plant extraction routes, our established protocol emerges as a green, sustainable, and environmentally benevolent alternative.

However, the yield, at 0.155mol/mol of glucose, taps merely 31% of the maximum theoretical yield of HT from glucose, which stands at 0.5mol/mol. To catapult HT into commercial production, an elevation in its yield and titer is imperative, achievable through nuanced metabolic engineering strategies and refined fermentation optimization. It 's well-recognized that aromatic alcohols can hinder cell growth by disrupting cell membrane permeability, impeding primary metabolic pathways, and inducing respiratory deficiencies (Britton, James et al., 2019). Consequently, the inherent toxicity of HT to microbial cells might pose a significant challenge to its large-scale fermentation. As a remedy, balancing cytotoxic product accumulation and cellular growth through the identification and engineering of membrane proteins to expel HT out of cells holds promise as a strategic solution. The ambiguous expression levels of pivotal enzymes in the HT biosynthetic pathway, such as GndA, Tal, AroB, AroC, AroD, AroE, AroF, and Pps, might be acting as bottlenecks. The consequent effects of these enzyme expression levels on HT production warrant thorough exploration. It 's noteworthy that the synthesis of 1 mol of HT from glucose siphons 3 mol of ATP; thus, the ATP regeneration and synthesis pathways need meticulous modulation. According to extracellular metabolite analysis, the production pathways of certain by-products, like 2,5-dihydroxymandelic acid, galactose, N-acetylgalactosamine, and 3,4-dihydroxyphenyllactic acid, need to be curtailed. Under fed-batch conditions, a substantial chunk of carbon flux fuels cellular growth. Hence, the fed-batch procedure needs recalibration, coupled with a repeated batch fermentation approach, to maximize the HT titer and yield.

![68cb8d64a6a4e880739ccad7bddc4e5](https://static.igem.wiki/teams/4912/wiki/results/results/image4.jpg)

Fig. 4  Metabolic engineering
B. licheniformis
 for
de novo
 synthesis of

hydroxytyrosol is a green and sustainable process

In summary, we have pioneered

a green and sustainable process for the
de novo
 production of HT from glucose. The augmented production of HT in
Bacillus licheniformis
 was realized through the modification of the HT biosynthetic pathway and the enhancement of precursor supply, as illustrated. Notably, by engineering the protein structure of KivD, fine-tuning the expression levels of kivD
V461I
 and hpaBC, amplifying the copies of tyrA
fbr
, and overexpressing tkt and zwf, we identified and eliminated several novel rate-limiting steps. Ultimately, our optimal engineered strain, DHT23, under fed-batch conditions, achieved an impressive HT titer of 9475 mg/L, with a yield of 0.155mol/mol of glucose and a productivity rate of 395 mg/L/h. This represents the highest reported titer, yield, and productivity of HT in microbes to date. Our work paves the way for further strain improvements and fermentation optimizations, positioning us closer to the industrial-scale production and widespread market application of HT. Furthermore, the strategies deployed in this study can be extrapolated to the production of other aromatic compounds.

# References

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* [2]Yep, Alejandra et al. “Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase.” Bioorganic chemistry vol. 34,6 (2006): 325-36. doi:10.1016/j.bioorg.2006.08.005
* [3]Chen, Grey S et al. “Saturated mutagenesis of ketoisovalerate decarboxylase V461 enabled specific synthesis of 1-pentanol via the ketoacid elongation cycle.” Scientific reports vol. 7,1 11284. 12 Sep. 2017, doi:10.1038/s41598-017-11624-z
* [4]Mak, Wai Shun et al. “Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway.” Nature communications vol. 6 10005. 24 Nov. 2015, doi:10.1038/ncomms10005
* [5]Zhang, Chuanzhi et al. “Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum.” Journal of industrial microbiology & biotechnology vol. 42,5 (2015): 787-97. doi:10.1007/s10295-015-1593-x
* [6]Satoh, Yasuharu et al. “Engineering of L-tyrosine oxidation in Escherichia coli and microbial production of hydroxytyrosol.” Metabolic engineering vol. 14,6 (2012): 603-10. doi:10.1016/j.ymben.2012.08.002
* [8]Trantas, Emmanouil et al. “Dual pathway for metabolic engineering of Escherichia coli to produce the highly valuable hydroxytyrosol.” PloS one vol. 14,11 e0212243. 4 Nov. 2019, doi:10.1371/journal.pone.0212243
* [9]Li, Xianglai et al. “Establishing an Artificial Pathway for Efficient Biosynthesis of Hydroxytyrosol.” ACS synthetic biology vol. 7,2 (2018): 647-654. doi:10.1021/acssynbio.7b00385
* [10]Liu, Chunxiao et al. Sheng wu gong cheng xue bao = Chinese journal of biotechnology vol. 37,12 (2021): 4243-4253. doi:10.13345/j.cjb.210046
* [11]Liu, Yingjie et al. “De Novo Production of Hydroxytyrosol by Metabolic Engineering of Saccharomyces cerevisiae.” Journal of agricultural and food chemistry vol. 70,24 (2022): 7490-7499. doi:10.1021/acs.jafc.2c02137
* [12]Bisquert, Ricardo et al. “Metabolic engineering of Saccharomyces cerevisiae for hydroxytyrosol overproduction directly from glucose.” Microbial biotechnology vol. 15,5 (2022): 1499-1510. doi:10.1111/1751-7915.13957
* [13]Liu, Huayi et al. “High-Level Production of Hydroxytyrosol in Engineered Saccharomyces cerevisiae.” ACS synthetic biology vol. 11,11 (2022): 3706-3713. doi:10.1021/acssynbio.2c00316
* [14]Britton, James et al. “Chemical, physical and biotechnological approaches to the production of the potent antioxidant hydroxytyrosol.” Applied microbiology and biotechnology vol. 103,15 (2019): 5957-5974. doi:10.1007/s00253-019-09914-9
* [15]Zhan, Yangyang et al. “Multilevel metabolic engineering of Bacillus licheniformis for de novo biosynthesis of 2-phenylethanol.” Metabolic engineering vol. 70 (2022): 43-54. doi:10.1016/j.ymben.2022.01.007

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