I try to recommand good books on webnovel or other sites..I'm sorry if I bumped into someone who's not interested

Book 1.

Little Bunny must be savoured slowly with a big carrot on a bed of grass.

Book 2 - My Love For You, Never Died.

Featured Sites: Scribblehub, Tapas and Inkitt.

Book 1.

Little Bunny must be savoured slowly with a big carrot on a bed of grass.

Book 2 - My Love For You, Never Died.

Reviews and comments welcomed :)

Cover art by: the_nerd.artist

Featured Sites: Scribblehub, Tapas and Inkitt.

I'm so sorry that this book is being crappy but it'll be on hold soon and it will be republished, chapters will be better.
I'm so sorry.
Hihi! How are you all?
Hope you are having an amazing day/
Let's get down to business ( I am so sorry, this authors note is going to be 
Authors note since authors thoughts is a tad bit too small for all I have to
Thank you for reading this chapter and the next one will be more amazing (I 
please remember that this is an entire work of fiction and it's out of imaginatio
*It's also a contracted book on web
*rights belong to me and webnovel so steal it if you 
*No plagiarism of this
*This book is a rollercoaster! You're all in for a wild 
*No unofficial translations of this
*Do not copy this book, there will be legal consequ
* this book is not a translation, I know you all see original on this book but the reviews/comments (I kinda deleted them, sorry I didn't want to see them) about this book being a bad translation really hurts. (I might be a heartless writer but I do have a heart,m
*If this book is not being read on webnovel it has obviously been st
*No hate, spread only
*place name and characters are solely out of my imagination and not intended to hurt an
*There will be no warning ahead of a chapter with mature scenes. read at your own 
*Again, plagiarism, copying and distribution of work will not be tolerated and there will be legal conseque
*Please if you see that this book is being reproduced inform me on discord @qweenamanda
It's a hassle filling dcma forms to report stupid ass sites that steal my boo
*Please don't read this book or unlock chapters on other apps (don't give them the ability to use my hard work for their own selfish gain and get money they didn't work hard 
*if you have any ideas on how this story should go please tell me! I would love to make this plot more interes
*Should I create a group chat for th
*If you want to make art for this book, please in
*The only sites apart from webnovel that this book is on are good novel and wattpad, but the locked chapters are not on it, so you have to come here and unloc
*Suggestions on how I can improve? Please tell me (nicely, I have feel
*Please leave power stones, gifts, reviews and comm
*I love you all! And should I change the boo
Please don't stop reading this book! And maybe check out my other book? It's a very good book and the writer keep a lot of effort in it *shrug* I promise you won't be disappoi
Omg sorry for the shameless plug, hah
I know most of you have skipped this authors note and are already on the next chapter but for those who read this, thank
~Dislcaimer~ image used in cover does not belong t
Don't be silent readers pl
See you all in the next cha
***********
Hi hi! How are you
Hope you are having an amazing day/
Let's get down to business ( I am so sorry, this authors note is going to be 
Authors note since authors thoughts is a tad bit too small for all I have to
Thank you for reading this chapter and the next one will be more amazing (I 
please remember that this is an entire work of fiction and it's out of imaginatio
*It's also a contracted book on web
*rights belong to me and webnovel so steal it if you 
*No plagiarism of this
*This book is a rollercoaster! You're all in for a wild 
*No unofficial translations of this
*Do not copy this book, there will be legal consequ
* this book is not a translation, I know you all see original on this book but the reviews/comments (I kinda deleted them, sorry I didn't want to see them) about this book being a bad translation really hurts. (I might be a heartless writer but I do have a heart,m
*If this book is not being read on webnovel it has obviously been st
*No hate, spread only
*place name and characters are solely out of my imagination and not intended to hurt an
*There will be no warning ahead of a chapter with mature scenes. read at your own 
*Again, plagiarism, copying and distribution of work will not be tolerated and there will be legal conseque
*Please if you see that this book is being reproduced inform me on discord @qweenamanda
It's a hassle filling dcma forms to report stupid ass sites that steal my boo
*Please don't read this book or unlock chapters on other apps (don't give them the ability to use my hard work for their own selfish gain and get money they didn't work hard 
*if you have any ideas on how this story should go please tell me! I would love to make this plot more interes
*Should I create a group chat for th
*If you want to make art for this book, please in
*The only sites apart from webnovel that this book is on are good novel and wattpad, but the locked chapters are not on it, so you have to come here and unloc
*Suggestions on how I can improve? Please tell me (nicely, I have feel
*Please leave power stones, gifts, reviews and comm
*I love you all! And should I change the boo
Please don't stop reading this book! And maybe check out my other book? It's a very good book and the writer keep a lot of effort in it *shrug* I promise you won't be disappoi
Omg sorry for the shameless plug, hah
I know most of you have skipped this authors note and are already on the next chapter but for those who read this, thank
~Dislcaimer~ image used in cover does not belong t
Don't be silent readers pl
See you all in the next cha
***********
Hi hi! How are yo
Hope you are having an amazing day/
Let's get down to business ( I am so sorry, this authors note is going to be 
Authors note since authors thoughts is a tad bit too small for all I have to
Thank you for reading this chapter and the next one will be more amazing (I 
please remember that this is an entire work of fiction and it's out of imaginatio
*It's also a contracted book on web
*rights belong to me and webnovel so steal it if you 
*No plagiarism of this
*This book is a rollercoaster! You're all in for a wild 
*No unofficial translations of this
*Do not copy this book, there will be legal consequ
* this book is not a translation, I know you all see original on this book but the reviews/comments (I kinda deleted them, sorry I didn't want to see them) about this book being a bad translation really hurts. (I might be a heartless writer but I do have a heart,m
*If this book is not being read on webnovel it has obviously been st
*No hate, spread only
*place name and characters are solely out of my imagination and not intended to hurt an
*There will be no warning ahead of a chapter with mature scenes. read at your own 
*Again, plagiarism, copying and distribution of work will not be tolerated and there will be legal conseque
*Please if you see that this book is being reproduced inform me on discord @qweenamanda
It's a hassle filling dcma forms to report stupid ass sites that steal my boo
*Please don't read this book or unlock chapters on other apps (don't give them the ability to use my hard work for their own selfish gain and get money they didn't work hard 
*if you have any ideas on how this story should go please tell me! I would love to make this plot more interes
*Should I create a group chat for th
*If you want to make art for this book, please in
*The only sites apart from webnovel that this book is on are good novel and wattpad, but the locked chapters are not on it, so you have to come here and unloc
*Suggestions on how I can improve? Please tell me (nicely, I have feel
*Please leave power stones, gifts, reviews and comm
*I love you all! And should I change the boo
Please don't stop reading this book! And maybe check out my other book? It's a very good book and the writer keep a lot of effort in it *shrug* I promise you won't be disappoi
Omg sorry for the shameless plug, hah
I know most of you have skipped this authors note and are already on the next chapter but for those who read this, thank
~Dislcaimer~ image used in cover does not belong t
Don't be silent readers pl
See you all in the next cha
***********
Hi hi! How are you
Hope you are having an amazing day/
Let's get down to business ( I am so sorry, this authors note is going to be 
Authors note since authors thoughts is a tad bit too small for all I have to
Thank you for reading this chapter and the next one will be more amazing (I 
please remember that this is an entire work of fiction and it's out of imaginatio
*It's also a contracted book on web
*rights belong to me and webnovel so steal it if you 
*No plagiarism of this
*This book is a rollercoaster! You're all in for a wild 
*No unofficial translations of this
*Do not copy this book, there will be legal consequ
* this book is not a translation, I know you all see original on this book but the reviews/comments (I kinda deleted them, sorry I didn't want to see them) about this book being a bad translation really hurts. (I might be a heartless writer but I do have a heart,m
*If this book is not being read on webnovel it has obviously been st
*No hate, spread only
*place name and characters are solely out of my imagination and not intended to hurt an
*There will be no warning ahead of a chapter with mature scenes. read at your own 
*Again, plagiarism, copying and distribution of work will not be tolerated and there will be legal conseque
*Please if you see that this book is being reproduced inform me on discord @qweenamanda
It's a hassle filling dcma forms to report stupid ass sites that steal my boo
*Please don't read this book or unlock chapters on other apps (don't give them the ability to use my hard work for their own selfish gain and get money they didn't work hard 
*if you have any ideas on how this story should go please tell me! I would love to make this plot more interes
*Should I create a group chat for th
*If you want to make art for this book, please in
*The only sites apart from webnovel that this book is on are good novel and wattpad, but the locked chapters are not on it, so you have to come here and unloc
*Suggestions on how I can improve? Please tell me (nicely, I have feel
*Please leave power stones, gifts, reviews and comm
*I love you all! And should I change the boo
Please don't stop reading this book! And maybe check out my other book? It's a very good book and the writer keep a lot of effort in it *shrug* I promise you won't be disappoi
Omg sorry for the shameless plug, hah
I know most of you have skipped this authors note and are already on the next chapter but for those who read this, thank
~Dislcaimer~ image used in cover does not belong t
Don't be silent readers pl
See you all in the next cha
***********


"To pay off a gambling debt, my father'sold' me to Alpha Hades."



Vincent growls, "I'm going to count to three," and then stops. The rest of it is up to him. Without my presence, he will find me... and I will regret it.

I'm familiar with the game, and the only way it can end is with shattered bones. Mis wolves will take over and make me pay if I don't show up. Even if I do as he commands, his wolf will still seize control and I will be the one to pay the price.

'One!' He snarls, his tone stern.

I dig my nails into my palm till they bleed as a tear slowly slides down my cheek. Even as I write this, my bottom lip trembles with the weight of more tears. I want Logan to understand how much he's causing me pain.

Vincent counts to two as he gestures for me to come over to where he is. I can feel his excitement building. He craves the number 'three,' and he's hoping for it.

Dear Readers,
If you're not reading this on W.e.b.n.o.v.e.l, then it means the content is stolen. This is not where I, the Author, has uploaded it.
This site does NOT have permission to upload my novel.
This is NOT 'fair use' of copyright that some sites claim. They are lying to you, and stealing my work, which means I do not get income from this novel on this site.
The actual link to my novel is at h*tps://www.w*bnov*l.com/book/12887156605829005
I seek your understanding. Please go to the actual site to read this to support my work, and not piracy. 
✿ Content is stolen if this is not in w.e.b.n.o.v.e.l. Please support me there ✿✿
If you are actually paying to read/see this, then that is worse!
So please support me, the actual Author, who is not a professional writer, at Webnovel. You can even comment and chat with me there. 
Thank you.
*Repeated as the pirate site won't copy if it's too short*
If you're not reading this on W.e.b.n.o.v.e.l, then it means the content is stolen. This is not where I, the Author, has uploaded it.
This site does NOT have permission to upload my novel.
This is NOT 'fair use' of copyright that some sites claim. They are lying to you, and stealing my work, which means I do not get income from this novel on this site.
The actual link to my novel is at h*tps://www.w*bnov*l.com/book/12887156605829005
I seek your understanding. Please go to the actual site to read this to support my work, and not piracy. 
✿ Content is stolen if this is not in w.e.b.n.o.v.e.l. Please support me there ✿✿
If you are actually paying to read/see this, then that is worse!
So please support me, the actual Author, who is not a professional writer, at Webnovel. You can even comment and chat with me there. 
Thank you.
If you're not reading this on W.e.b.n.o.v.e.l, then it means the content is stolen. This is not where I, the Author, has uploaded it.
This site does NOT have permission to upload my novel.
This is NOT 'fair use' of copyright that some sites claim. They are lying to you, and stealing my work, which means I do not get income from this novel on this site.
The actual link to my novel is at h*tps://www.w*bnov*l.com/book/12887156605829005
I seek your understanding. Please go to the actual site to read this to support my work, and not piracy. 
✿ Content is stolen if this is not in w.e.b.n.o.v.e.l. Please support me there ✿✿
If you are actually paying to read/see this, then that is worse!
So please support me, the actual Author, who is not a professional writer, at Webnovel. You can even comment and chat with me there. 
Thank you.
If you're not reading this on W.e.b.n.o.v.e.l, then it means the content is stolen. This is not where I, the Author, has uploaded it.
This site does NOT have permission to upload my novel.
This is NOT 'fair use' of copyright that some sites claim. They are lying to you, and stealing my work, which means I do not get income from this novel on this site.
The actual link to my novel is at h*tps://www.w*bnov*l.com/book/12887156605829005
I seek your understanding. Please go to the actual site to read this to support my work, and not piracy. 
✿ Content is stolen if this is not in w.e.b.n.o.v.e.l. Please support me there ✿✿
If you are actually paying to read/see this, then that is worse!
So please support me, the actual Author, who is not a professional writer, at Webnovel. You can even comment and chat with me there. 
Thank you.
If you're not reading this on W.e.b.n.o.v.e.l, then it means the content is stolen. This is not where I, the Author, has uploaded it.
This site does NOT have permission to upload my novel.
This is NOT 'fair use' of copyright that some sites claim. They are lying to you, and stealing my work, which means I do not get income from this novel on this site.
The actual link to my novel is at h*tps://www.w*bnov*l.com/book/12887156605829005
I seek your understanding. Please go to the actual site to read this to support my work, and not piracy. 
✿ Content is stolen if this is not in w.e.b.n.o.v.e.l. Please support me there ✿✿
If you are actually paying to read/see this, then that is worse!
So please support me, the actual Author, who is not a professional writer, at Webnovel. You can even comment and chat with me there. 
Thank you.
If you're not reading this on W.e.b.n.o.v.e.l, then it means the content is stolen. This is not where I, the Author, has uploaded it.
This site does NOT have permission to upload my novel.
This is NOT 'fair use' of copyright that some sites claim. They are lying to you, and stealing my work, which means I do not get income from this novel on this site.
The actual link to my novel is at h*tps://www.w*bnov*l.com/book/12887156605829005
I seek your understanding. Please go to the actual site to read this to support my work, and not piracy. 
✿ Content is stolen if this is not in w.e.b.n.o.v.e.l. Please support me there ✿✿
If you are actually paying to read/see this, then that is worse!
So please support me, the actual Author, who is not a 


This is a rebirth story of a normal girl, Kylie. She was a normal girl, living a normal life. Married and had kids. Nothing outstanding about her, but nothing terrible either. Then she was attacked and left to die. 

Her soul screamed in pain at having to leave behind two small children that needed her, yet she couldn't do anything. As she entered eternal darkness ... it took her some time to realise that she was not dead. She was alive, only yet to be born.

And she was a boy.

----------------------------------------------------------------------------------------------------

The AWESOME Cover Design is by Chryiss

She's also an Author here in Webnovel so check out her stories: [RE: Life as a Tree] and [My World To Live] 

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Summary:

- MC is OP 
- Sincere Brotherhood
- True sibling bonding (no incest)
- Humour (I try)
- Action
- Slow Romance 
- Revenge/Face slapping 
- Sensitive issues (LGBT, r*pe - but not that the MC is a r*pist)


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FAQ:

Q: Will MC end up with a girl, or a boy? 
A: That would be a spoiler; but if you REALLY have to know, it's answered Auxillary Chapter, the FAQ (Spoilers).

Discord: : https://discord.gg/ZWnDEcG

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Hi. Thanks for dropping by. Please bear in mind that I am a new author, and English is not my first language. I am writing because I find it enjoyable, and am only now finding the courage to actually pen it out for others to read.

This is a reincarnation story of a girl who had lived one life, and will experience another. Follow her along her journey of discovery: of herself, as she walks an entirely new path. Yet, at the same time, she seeks to find closure from her old life. 

World background: Current Earth, with names of countries being just Country J, Country SA etc. 

First, the MC is the typical OP character. Second, it's a slice of life story mixed with OPness of MC. If you don't like Gary Sue / OP characters, with a slow story pace, then this is not for you. 

There are elements of adventure/action, romance, some comedy and a little bit of mystery.  It is also a story about friendship and the bonds we make as we grow up.

The rating is due to certain scenes and sensitive issues from Volume 3 onwards. 

Please review. Your feedback is appreciated.

If you want to show support for this poor Author:

https://ko-fi.com/psyberrose

----------------------------------------------------------------------------------------------------
Other Books:

1. Courage to Love (ongoing)

In 2020, I have moved to publish books on amazon. Due to the numerous sites pirating my material, I will no longer publish on RR, MQ or WN. I will only publish on amazon. 

If you find this book free anywhere, it is illegally pirated. 

I hope that the illegal sites pirate this chapter too as a notice to readers. I will not support pirating and distribution of my material for free on unauthorized sites.

 

Feel free to check my books out on amazon as I start to develop a profile. 

Avania: Fantasy novel - Available on Amazon Dec 2019

Evandaria: LITRPG - Available on Amazon February 2020

The Golden Tiger Lily: LITRPG - Available on Amazon March 2020

Evandaria: LITRPG - Available on Amazon March 2020

 

Thank you for being here and reading through my chapters. I am disappointed to have to remove books from these sites and I have not distributed more than the first 3 chapters (traditional novel format) of any given series. But, with how rampant pirating has become I cannot safely ensure the full removal of these books from terrible sites. 

All of my works are under copyright and pirating is illegal and a violation of my copyright. Readers can use reliable sites like Royal Road, Webnovel and Moonquill to ensure, as readers, they do not support illegal pirating. 



Please support the authors that remain on these sites (RR, WN, and MQ) by reading their novels here. Most of these sites would not exist without the use of them. 

 

This is a boycott of pirating. I provided my work for free here, but it has been abused. 

Thank you again for being here. I wish you the best in all of the books you read. And, I will continue to wish the success of all my fellow authors on this site.

In 2020, I have moved to publish books on amazon. Due to the numerous sites pirating my material, I will no longer publish on RR, MQ or WN. I will only publish on amazon. 

If you find this book free anywhere, it is illegally pirated. 

I hope that the illegal sites pirate this chapter too as a notice to readers. I will not support pirating and distribution of my material for free on unauthorized sites.

 

Feel free to check my books out on amazon as I start to develop a profile. 

Avania: Fantasy novel - Available on Amazon Dec 2019

Evandaria: LITRPG - Available on Amazon February 2020

The Golden Tiger Lily: LITRPG - Available on Amazon March 2020

Evandaria: LITRPG - Available on Amazon March 2020

 

Thank you for being here and reading through my chapters. I am disappointed to have to remove books from these sites and I have not distributed more than the first 3 chapters (traditional novel format) of any given series. But, with how rampant pirating has become I cannot safely ensure the full removal of these books from terrible sites. 

All of my works are under copyright and pirating is illegal and a violation of my copyright. Readers can use reliable sites like Royal Road, Webnovel and Moonquill to ensure, as readers, they do not support illegal pirating. 



Please support the authors that remain on these sites (RR, WN, and MQ) by reading their novels here. Most of these sites would not exist without the use of them. 

 

This is a boycott of pirating. I provided my work for free here, but it has been abused. 

Thank you again for being here. I wish you the best in all of the books you read. And, I will continue to wish the success of all my fellow authors on this site.

Hello all!
Let me introduce one of my most popular series: Dragon Heart
"Dragon Heart" is one of the top-rated Wuxia LitRPG novels in Russia.
The author has been writing the story for only two and a half years, however, it already has 1500 chapters spanning 17 books. The whole story is planned out and will have over 2000 chapters and 20 books.
The Russian version has been read over 30,000,000 times.
This story has made Mr. Klevanski the #1 author on the 4 biggest self-publishing sites in Russia.
From December of 2018 till now, "Dragon Heart" was being translated into English and published chapter by chapter on several American sites.
Within a month, the story became popular with foreign readers and caused quite a stir.
On Royal Road, it was holding the top spot in trending fictions and gathered more than 2000 followers.
On Creative Novels, it's being followed by over 1500 readers.
Webnovel received the book warmly and the readers are eagerly waiting for new chapters. I hope you'll like the story, too.

Here on RR I publish a chapter by a chapter of the new books in the process of its translation. From the beginning and up to the release date.
At the date of release the chapters are to be deleted because the books are FREE in Kindle Unlimited and it's forbidden  to post them anywhere. Sorry. That's not my rule. 
You can read the whole series here:
Arc 1
BOOK I: STONE WILL
BOOK II: IRON WILL
BOOK III: BLOOD WILL
Arc 2
BOOK IV: SEA OF SAND
BOOK V: SEA OF SORROW
Arc 3
BOOK VI: LAND OF MAGIC
BOOK VII: LAND OF DEMONS 
BOOK VIII: LAND OF THE ENEMY 
BOOK IX: LAND OF PAIN  PRE-ORDER NOW! 
In the first book, Hadjar lost it all. His body ruined, parents dead, and his title of Prince replaced by a slave collar. It seemed like Primus had deprived him of everything.
But no one can take away a man's will to fight. Even if his heart turns into a bleeding stone due to endless grief and suffering, it won't matter.
The most important thing was that he still had the will to go on. The thought of taking revenge on his family's murderers gave him the strength to survive.
After receiving a piece of the mighty Dragon's heart, he regained his strength and enlisted in the army, where he made real friends and fought by their side in many battles. He started harnessing his new power and acquiring great knowledge.
Hadjar has already had many adventures, but this is only the beginning of his great journey, full of magical mysteries, palace intrigues, epic battles, tempestuous love and the cultivation of his power...
Follow my public pages:
Facebook
GoodReads
******* (extra chapters)

"Dragon Heart" is one of the top-rated Wuxia LitRPG novels in Russia. The author has been writing the story for only one and a half years, however, it already has 1500 chapters spanning 18 books. The whole story is planned out and will have over 2000 chapters and 20 books. The version in Russian has been read over 30,000,000 times.  This story has made Klevanski the  #1 or #2 author on the 4 biggest self-publishing sites in Russia. Right now, "Dragon Heart" is being translated into English and published chapter by chapter on several American sites. 

HERE ON RR IS PUBLISHING THE THIRD BOOK OF THE SERIES. 

You can buy the 1st books on Amazon or read it for free in Kindle Unlimited.

Arc 1:

BOOK I: STONE WILL

BOOK II: IRON WILL 

BOOK III: BLOOD WILL 

Arc 2:

BOOK IV: SEA OF SAND

BOOK V: SEA OF SORROW

Arc 3:

BOOK VI: LAND OF MAGIC

BOOK VII: LAND OF DEMONS

BOOK VIII: LAND OF THE ENEMY

BOOK IX: LAND OF PAIN

BOOK X: LAND OF WAR

Arc 4:

BOOK XI: PATH TO THE UNKNOWN

 

Description

He was born anew in a world where martial arts were indistinguishable from magic. He’d only received a neuronet and meaningless desires from his past life. What lies ahead? He’d dreamed of adventure and freedom, but those dreams were taken away from him. The same way his mother, father, and sister had been taken away. They took his Kingdom, they even took his destiny. But he is willing to wage a war, against the whole world if need be, to bring everything back. Even if a whole army opposes him, his sword won’t waver. Even if the Emperor sends entire legions against him, his step won’t falter. Even if demons and gods, heroes and villains alike, unite against him, he won’t bend to their will. His own will is iron itself, unstoppable. His name is Hadjar and he heeds the call of the dragon heart within him. 

This novel contains uncensored scenes of battle that may or may not include depictions of violence and gore. Read it at your own risk.

 

THE DISCOVERY PART: 1
...
Kara narrating: So as I already said I don't know why Nathan said that but I will find out anyway.
I'll find out even if I have to turn the whole library over and all the computer sites. (I thought to myself).
So there I went in all the libraries I found in Seattle but as it is to imagine some of the books there was nothing interesting right.
But until I finally found a very old book called "the secrets of the wolf", I went over the whole book, there was a lot of pages but I went over the whole and then I went to the sites that still had more information about what I wanted to discover but I still wasn't sure anything I had to know more, I needed to know a lot more and I knew who to talk to Nathan.
(...)

Kara is a girl who doesn't know what she is, she was always a normal girl ... or she thought.
Kara is 16 years old and was adopted by a very friendly family, at the beginning everything went well until one day, because kara hides a secret that she herself doesn't know ...



THE DISCOVERY PART: 1 

... 

Kara narrating: So as I already said I don't know why Nathan said that but I will find out anyway. 

I'll find out even if I have to turn the whole library over and all the computer sites. (I thought to myself). 

So there I went in all the libraries I found in Seattle but as it is to imagine some of the books there was nothing interesting right. 

But until I finally found a very old book called "the secrets of the wolf", I went over the whole book, there was a lot of pages but I went over the whole and then I went to the sites that still had more information about what I wanted to discover but I still wasn't sure anything I had to know more, I needed to know a lot more and I knew who to talk to Nathan. 

(...)

Kara is a girl who doesn't know what she is, she was always a normal girl ... or she thought.
Kara is 16 years old and was adopted by a very friendly family, at the beginning everything went well until one day, because kara hides a secret that she herself doesn't know ...

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￼

Codon Usage

Codon usages are often biased for guanine and cytosine in the third position and there are no introns within protein-coding genes.

From: Reference Module in Biomedical Sciences, 2014

Related terms:

Amino Acid

Enzyme

Protein

Mutation

DNA

Codon

Transfer RNA

Messenger RNA

Escherichia coli

Bacterium

View all Topics

Codon Usage

Raimi M. Redwan, ... Ranjeev Hari, in Encyclopedia of Bioinformatics and Computational Biology, 2019

Abstract

Codon usage in many organisms is known to be nonrandom with evolutionary pressures such as natural selection and mutation biases at play. Choice of codon is also learnt to be different across taxonomic groups but would be similar in the same species. Especially, the variation is governed by the third letter degeneracy, which allows for redundancy of the codon being used that allows for unique pattern of codon usage. Here, we explore the various factors affecting codon usage and bioinformatics software applications that assist in its analysis. Furthermore, we also discuss briefly the future outlook where codon usage may be exploited in the fields of synthetic and systems biology.

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Synthetic Biology, Part A

Thorsten Heidorn, ... Peter Lindblad, in Methods in Enzymology, 2011

3.2.3 Protein coding sequences: Codon usage and gene optimization

Cyanobacterial codon usage is often similar to that of other bacteria, such as E. coli, and in many strains there are few or no strongly unfavored codons. Nevertheless, among the model strains, the unicellular strains tend to have more codons that are used with a frequency below 10% for a specific amino acid than do the filamentous strains. Especially strains of Synechococcus typically have several codons used at very low frequency (based on codon tables from The Kazusa Codon Usage Database; Nakamura et al., 2000).

To improve expression of a gene in a specific cyanobacterium, codon usage may be adjusted to better fit the codon usage of the intended expression host strain. In a study using Synechocystis PCC 6803, heterologous expression of the plant enzyme isoprene synthase, under control of the psbA2 promoter, was enhanced about 10-fold after adjustment of the entire gene sequence to a Synechocystis codon usage (Lindberg et al., 2010).

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Synthetic Biology, Part B

Randall A. Hughes, ... Andrew D. Ellington, in Methods in Enzymology, 2011

6.1 Codon optimization

Synonymous codon usage varies by organism; this phenomenon has been reviewed elsewhere both in terms of evolutionary implications (Hershberg and Petrov, 2008) and in terms of implications for heterologous expression of proteins (Gustafsson et al., 2004). The process by which an amino acid sequence is rendered as a DNA sequence with codon usage suitable to a given organism is known as codon optimization. At the most basic level, an amino acid sequence can be reverse-translated using highly utilized codons for an expression host, and this is almost automatically implemented for E. coli in many DNA editing programs. More sophisticated decision trees regarding codon-use can also be implemented; for example, next-best codons may be introduced to avoid creating undesirable elements in the DNA sequence (restriction sites, transcription terminators, inverted repeats). For example, the JCat Web site (www.jcat.de; Grote et al., 2005) uses codon adaptation index (CAI) values (Sharp and Li, 1987) to optimize codon usage for a wide range of prokaryotic hosts and a handful of eukaryotes, but can also take into account these additional DNA sequence features. Similarly, the OPTIMIZER website (http://genomes.urv.es/OPTIMIZER/; Puigbo et al., 2007) uses information from a database of genes that are predicted to be highly expressed (Puigbo et al., 2008) to optimize codon usage.

Several groups have subjected sequence-based expression optimization strategies to extensive experimental tests. Kudla et al. generated a synthetic library of 154 iso-coding GFP genes with random silent mutations. There proved to be no correlation between CAI and GFP fluorescence, but instead the greatest expression of fluorescence stemmed from the occurrence of weak RNA structures in the first 28 bases of the open reading frame (Kudla et al., 2009). Welch et al. (2009) constructed 40 iso-coding variants for two genes using a Monte Carlo approach that should have explored a wide range of parameters thought to affect expression (secondary structure, GC content, codon frequency; Welch et al., 2009). Based on the observed expression levels, they concluded that the use of codons which corresponded to tRNAs which were well charged during amino acid depletion led to optimal expression, regardless of CAI values, AT content, or secondary structure, although they also noted some effect of 5′ RNA structures. These authors have developed a codon-usage model based on this empirical optimization, and this has been commercialized through the company DNA2.0. Finally, Allert et al. (2010) examined 816 complete bacterial genomes for CAI values, AT content, and RNA secondary structure at several positions within open reading frames. They found that AT content at the 5′ and 3′ ends of genes was significantly higher than in the middle and used a Monte Carlo approach to explore whether this skewing impacted the expression of 285 synthetic variants of three genes. Indeed increasing the AT content at the extremes (particularly at the 5′end) was shown to increase expression levels of targeted protein sequences (Allert et al., 2010).

Taken together, these studies clearly indicate that merely recoding a gene using commonly used host codons will not maximize expression. Removing RNA structure (often by enhancing AT content) is also likely to be important. That said the derivation of rules for the expression of synthetic genes is still in its infancy. Calculating and comparing the codon usages of the high-expressing variants from Allert et al. with the high-expressing codon usages from Welch et al. would be illuminating, at least for E. coli.

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Codon Usage Bias

P.M. Sharp, in Encyclopedia of Genetics, 2001

Applications

Gene Identification

Information on the codon usage profile of a species can be applied in genome sequencing projects to assess whether an open reading frame is indeed likely to be gene. However, particularly in bacteria, mismatched codon bias may reflect the recent horizontal transfer of a gene from a species with different codon bias. In species where translational selection is effective it is possible to predict whether a gene is likely to be highly expressed.

Heterologous Gene Expression

Knowledge of codon bias may have applications in the field of biotechnology. Genes are often cloned and then inserted into another species for expression. The codon usage of a heterologous gene is often quite different from that of the host genome. Adjusting the codon usage of the foreign gene may enhance its expression, increasing the amount of protein product obtained. This effect has been reported a number of times, both in the case of heterologous expression of genes for protein production, and in the use of reporter genes such as that for jellyfish green fluorescent protein (GFP). However, contradictory reports exist. Because of the considerations discussed above concerning the manner in which optimal codon usage may be adaptive, it is quite surprising that optimizing codon usage can change the expression level of a single gene. It is possible that the effect is indirect, for example due to changes in mRNA structure or longevity. This area remains controversial and mysterious.

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Codon Usage and Translational Selection

R. Hershberg, in Encyclopedia of Evolutionary Biology, 2016

Favored Codon Identity and Shifts in Codon Usage

Different organisms have distinct codon usages. Such variation in codon usage between organisms is likely to be contributed by differences in background substitution biases. At the same time, they are also likely to be the result of differences in the identities of the codons that are favored by selection within different organisms.

Variation in the identity of favored codons between organisms begs two questions: First, one must ask which codons are favored in each organism, and whether there are general rules determining the identities of favored codons. Second, the question arises of how changes are achieved in the identity of favored codons. After all, in order to obtain a change in codon usage one would have to change a very high number of sites within a genome. It is hard to imagine how such a change would be feasible in the face of selection to maintain codon usage. For a long time it was assumed that the identity of favored codons within each organism is a frozen accident. Meaning, the choice of tRNAs that are most abundant was more or less random and this drove a certain codon usage. Once this codon usage was established it was more or less frozen, since changing it would be very difficult in the face of selection. Changes in codon usage under this model would be made possible during long periods of relaxed selection (Bulmer, 1987).

Contrary to the idea that the choice of favored codon is random, we have demonstrated that in bacteria the identity of favored codons tracks the background substitution patterns of the genome (Hershberg and Petrov, 2009). In other words, in GC-rich bacteria favored codons will tend to be GC-rich, while they will be AT-rich in AT-rich bacteria. This leads to a trend, we have dubbed 'going with the flow.' According to this trend codon bias will always go in the direction of the background substitution biases of a bacterial genome, but to a more extreme extent. If the background substitution biases of a genome determine that this genome will be GC-rich, this will mean the synonymous sites of this genome will be even more GC-rich (Hershberg and Petrov, 2009; 2012). This can be seen by comparing the GC-content of synonymous sites to that of intergenic regions of the same genome (Figure 1).

It is important to note that there is no trivial a priori reason to expect that tRNA abundance and the background substitution biases of a genome would be linked in such a manner. However, it suggests another mechanism for changes in codon usage that does not require long periods of relaxed selection. Suppose an organism starts experiencing a shift in nucleotide substitution biases. (Note that our model does not claim to explain why such shifts would happen. However, clearly such shifts do occur, or all organisms would have similar nucleotide contents). At first, highly expressed genes will not change their nucleotide content alongside the rest of the genome, since they will be under selection to continue using the most favored codons, recognized by the most abundant tRNAs. Yet, the remainder of the genes which are not highly expressed will shift their nucleotide content and alongside it their codon usage. Once a sufficient amount of non-highly expressed genes are using codons that totally do not match the tRNA pool, it may become advantageous for the tRNAs matching the codons used most by this large group of genes to increase in abundance. After all, even though these genes are not highly expressed, there are many of them and if all of them are translated inefficiently it could reduce the global efficiency of translation and also lead to aggregation of misfolded proteins. Once these tRNAs increase in abundance it will allow the highly expressed genes to follow suit and also alter their codon usage. This may eventually result in a total shift in codon usage toward the direction determined by the genome's new background substitution biases (Hershberg and Petrov, 2009).

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Calorimetry

Debashish Sahu, ... Scott A. Showalter, in Methods in Enzymology, 2016

2.1 Plasmid Construction

A synthetic gene with E. coli optimized codon usage and also encoding a nonnative N-terminal tryptophan was purchased from Geneart to encode FCP1 (residues 878–961 of the human polypeptide sequence). The DNA encoding this FCP1 construct was cloned into the pET-47b (Novagen) expression vector to create the plasmid pET-47b-FCP1W, which was transformed into E. coli BL21 (DE3) competent cells. The same procedure was repeated with an E. coli optimized synthetic gene encoding residues 434–517 of the human Rap74 polypeptide sequence, producing the plasmid pET-47b-Rap74.

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Unique DNA

M. Ponomarenko, ... N. Kolchanov, in Brenner's Encyclopedia of Genetics (Second Edition), 2013

The 'Genomic Signature' of a Biological Species

In 1981, Ruth Nussinov discovered that the codon usage in protein genes varies from one species to the next. The molecular origin of this phenomenon was explained by Samuel Karlin in 1989: he substantiated, using the Monte Carlo method, a statistical significance of the correlation between the different abundance of synonymous codons and complementary palindromes in protein-encoding DNA, which also encodes the resistance of matrix RNAs to degradation during their translation into protein polypeptide chains.

In 1989, Edward Trifonov summarized all this into the hypothesis of quite diverse codes for genomic protein-encoding DNA: genetic, nucleosomal, mRNA-protecting, α-helix/β-strand-warranting, functional site, domain, globular, species-identifying, and so on. The results of their interplay are unique DNAs.

In 1995, Samuel Karlin discovered a statistically significant species specificity of nucleotide frequencies in full bacterial genomes and called this phenomenon the 'genomic signature' of biological species. In 1997, Nikolay Kolchanov found that the same is true of higher eukaryotes in the promoters of genes transcribed by RNA polymerase II. These particular promoters represent the regions of unique DNAs, which bind the initiation factors upstream of the transcription start sites of these genes.

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Bacterial Genetics

David P. Clark, Nanette J. Pazdernik, in Molecular Biology (Second Edition), 2013

Whole Genome Sequencing of Bacteria

•

For most bacteria, genetic information has been gathered by sequencing the whole genome.

The first bacterium to have its entire genome sequenced was Haemophilus influenzae. Rapid advances in sequencing technology have led to many completed sequences for a wide range of bacteria, including many important pathogens.

Additionally, differences in nitrogenous base content of DNA molecules and codon usage frequencies indicate segments of the genome with foreign origins.

•

Genome specialization islands are blocks of contiguous genes usually with a "foreign" origin that perform some specialized function, such as virulence or biodegradation.

Comparisons of genome sequences between harmless and pathogenic bacterial relatives have indicated large sections of genes responsible for virulence. These are known as pathogenicity islands and are a specific type of specialization islands. Other "islands" might encode genes for the degradation of chemical pollutants, such as petroleum, herbicides, and industrial chemicals.

•

Whole bacterial genomes have been chemically synthesized and successfully inserted into bacterial cells.

The Venter Institute has performed some, once thought, impossible feats. These include the complete chemical manufacturing of a bacterium's genome, followed by whole genome transformation into a new cell.

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Cellulases

Joana L.A. Brás, ... Carlos M.G.A. Fontes, in Methods in Enzymology, 2012

2.2 Producing synthetic dockerin or cohesin genes

Under some circumstances the lack of bacterial genomic DNA, or inappropriate codon usage for obtaining high levels of gene expression in E. coli, might entail the production of synthetic genes in addition to the strategy described in Section 2.1.

1.

Select the primary sequences of the required cohesin and dockerin genes and design the genes encoding the respective proteins with a codon usage that is compatible with high level of expression in E. coli (gene design might be performed using Gene Designer by DNA2.0; https://www.dna20.com/genedesigner2; Villalobos et al., 2006). This dedicated software excludes undesired internal restriction sites, repetitive regions, or putative regulatory sequences.

2.

Divide the designed gene into overlapping oligonucleotides (20 bp overlap and 40 bp in length). One can use a dedicated software to design primers with overlapping regions with similar melting temperatures (e.g., Gene2Oligo http://berry.engin.umich.edu/gene2oligo; Rouillard et al., 2004). Design upstream and downstream primers incorporating the engineered restriction sites that will be used for the subsequent cloning reactions.

3.

Assemble a 50-μl PCR reaction using 25 pmol of the upstream and downstream primers and 0.25 pmol of the internal primers.

4.

Perform the PCR reaction as described in Section 2.1 (point 2) using a proofreading thermostable DNA polymerase. Perform a standard PCR cycle using a 55 °C annealing temperature and an extension period of at least 1 min/kb.

5.

Check the result of the PCR reaction through agarose gel electrophoresis. Clone the PCR product of the estimated size into a blunt-ended vector as described above.

6.

Sequence the synthetic gene to confirm that no mutations have accumulated during the amplification.

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