Team:SCUT-China/Project/Background

1.Antibiotics resistance.

Early in 1945, Alexander Fleming, who discovered penicillin, warned that bacteria would finally be resistant to drugs. An increasing number of antibiotics including penicillin are found to lose their effectiveness because of drug resistance, the result of which is lack of effective ways to defeat bacteria in the future ^[1]. For example, Staphylococcus aureus become less sensitive to penicillin, one of beta-lactam antibiotics, for the acquiring of a penicillin binding-protein. Even worse, all the beta-lactam antibiotics are useless to Staphylococcus aureus infection for the same resistant mechanism. And the strains acquiring the resistance were termed methicillin-resistant Staphylococcus aureus (MRSA). According to the ANTIMICROBIAL RESISTANCE Global Report on Surveillance of World Health Organization in 2014, community-acquired MRSA has increased greatly in several countries over the past decade. The report showed that MRSA proportions had exceeded 20% in all WHO regions, and even 80% in some areas ^[1]. Tuberculosis(TB) is a widespread, fatal, infectious disease. Its resistance appears soon after introduction of antibiotics, and has developed to multi-drug resistance(MDR). It’s estimated that, on global level, there are 3.6% new tuberculosis cases with multi-drug resistance ^[1]. (figure1)'Traditional Arabic'

There are some ways to fight against the antibiotic resistance, such as restricted politics ^[1]. As a researcher, one effective way is to produce more new antibiotics. However, the new antibiotics development is virtually empty ^[2]. Fortunately, some researches showed that antibiotics can be synthesized by using synthetic biology methods. Relationship between antibiotics and polyketide, as well as its mechanism of synthesis show us an approach to solve this problem^[3].

2.Our goal: Programmed synthesis of polyketide.

Polyketides are a large family of compounds that represent a wide range of pharmacological effect ^[4]. And polyketides can be the precursor of many antibiotics. The natural antibiotics such as erythromycin and vancomycin are all the downstream products of polyketides^[5]. Polyketides are synthesized by polykeyide synthase (PKS). They are structurally complicated because of the complication of modular PKS. Combining the modularity of PKS and synthetic biology’s principle, we can standardize the PKS and assemble them just like LEGO blocks ^[6]. In this way, many different assembly lead to various polyketides, which provide a wide range of compounds with biological activities to search for new antibiotics^[7]. Our team tried to standardize domains and “programme” PKSs to synthesize custom polyketide. By synthesizing new polyketides, we can help to discover and screen new antibiotics^[8][9].

3.Introduction of PKS

PKS is the assemble line of polyketide. PKS are very complicated that contain several kinds of domain in one module. Each module catalyst to extend,modified or terminated the polyketide chain. The PKS we choose is type I PKS,which is highly modular. The modularity of type I PKS are reflected in three aspect. First, the gene cluster of type I PKS are modular that each enzyme corresponds to the unique sequence of gene cluster. Second, each enzyme of PKS has only one function. Third, the process of polyketide synthsis are modular that each module modify one unit of polyketide chain^[3].

3.1. PKS: The Domain

PKS is the assemble line of polyketide. PKSs are so complicated that each PKS is composed of a number of modules which contain several kinds of domains^[10]. Each module has catalysed functions for extending, modifying or terminating the polyketide chain. The PKS we choose is type I PKS, which is highly modular. The modularity of type I PKS is reflected in three aspects. First, the gene clusters of type I PKS are modular that each enzyme corresponds to the unique sequence of gene cluster. Second, each enzyme of PKS has only one function. Third, the process of polyketide synthesis are modular that each module modify one unit of polyketide chain^[11].

PKS is comprised of a series of module that contain several domains. Each domain have only one catalyst center with defined function which works well though it’s a part of module. The domains we used are as follows: a ketosynthase (KS), an acyltransferase (AT), a dehydratase (DH), a ketoreductase (KR), an enoylreductase (ER), an acyl-carrier protein (ACP), and a thioesterase (TE).

AT selects an extender unit and charges ACP with it; KS catalyzes a decarboxylative condensation to elongate the polyketide; ACP transfers the newly-formed β-ketoacyl intermediate to processing enzymes; KR can stereoselectively reduce the β-keto group and control the orientation of an a-substituent; DH can catalyzes dehydration to yield a trans-α, β-double bond; ER can stereoselectively reduce the double bond and control the orientation of α -substituent. TE can catalyze the intramolecular cyclization of a polyketide to create a macrolactone^[10][12].(table 1.)

Name of domain	Function
Acyltransfer-ase, AT
Acyl carrier protein, ACP
Ketocaylsynthase, KS
Keto-reductase, KR
Dehydratase, DH
Enoylreductase, ER
Thioesterase, TE

Table 1:Domains and their functions

The domain KS, AT, and ACP are resposible for the elongation of the polyketide chain. And the DH, ER and KR are the processing enzymes that modify the newly-form unit of polyketide chain. TE can terminate the elongation of polyketide. The AT can select the substrate and the processing enzymes can modify the unit, All of that finally determined the structure of polyketide chain^[13].

There are three kinds of module: loading module, elongation and modification module, termination module. Each module condenses and modifies one unit. Basic function of a module is to elongate the polyketide. So a module contains at least the domain KS, AT, ACP. The exist of DH, ER, KR might change the structure of polyketide. Some natural modules may contain unnecessary domain DH, ER, KR without any function. Loading module is the first module of PKS that starts the synthesis. Termination module only contains the domain TE that terminates the elongation of polykeitde. Both modules are connected by short adhesion or docking domains. Docking domain is a kind of connection which can arrange the order of module. It can direct the intermodular transfer of polyketide intermediates ^[13].

The formation of PKS requires for the reaction groups that come from coenzyme A. So that host should afford to synthesis the substrate. Suitable host can be chosen or engineered to provide the substrate. The substrate should be firstly linked to CoA that refers to as CoA-linked PKS extender units, which is building blocks of polyketide. The natural PKS utilize 18 compounds including malonyl-CoA,(2S)-methylmalonyl-CoA, (2S)-ethylmalonyl-CoA, and chloroethymalonyl-CoA and so on^[14].

After the synthesis, the product may enter the steps of post-modification.

6-deoxyerythronolide B (6dEB), the macrocyclic core of the antibiotic erythromycin, is a complex product synthesized through the action of a 6-deoxyerythonolide B synthase (DEBS) by the soil bacterium S.erythraea. DEBS from Saccharopolyspora erythraea has served as a model system for discovering ways to engineer PKSs .The DEBS subunits are encoded by three contiguous erythromycin A (eryA) genes. The gene is divided into three genes, which are organized into six modules that specify how each two-carbon unit of the final product is assembled ^[15].(Figure2)

Reference:

[1]WHO Library Cataloguing-in-Publication Data, Antimicrobial resistance: global report on surveillance[R],France:World Health Organization,(2014): 1; 49; 45.
[2]Kline, Novartis. "Race against time to develop new antibiotics." Bull World Health Organ 89 (2011): 88-89.
[3]Hutchinson, C. Richard, and Isao Fujii. "Polyketide synthase gene manipulation: a structure-function approach in engineering novel antibiotics."Annual Reviews in Microbiology 49.1 (1995): 201-238.
[4]Boghigian, Brett A., Haoran Zhang, and Blaine A. Pfeifer. "Multi‐factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions." Biotechnology and bioengineering 108.6 (2011): 1360-1371.
[5]Williams, Gavin J. "Engineering polyketide synthases and nonribosomal peptide synthetases." Current opinion in structural biology 23.4 (2013): 603-612.
[6]Hernández-Macedo, Maria L., et al. "Antimicrobial potential of Actinomycetes by NRPS and PKS-I pathways." BMC Proceedings. Vol. 8. No. Suppl 4. BioMed Central Ltd, 2014.
[7]Menzella, Hugo G., John R. Carney, and Daniel V. Santi. "Rational design and assembly of synthetic trimodular polyketide synthases." Chemistry & biology 14.2 (2007): 143-151.
[8]Marsden, Andrew FA, et al. "Engineering broader specificity into an antibiotic-producing polyketide synthase." Science 279.5348 (1998): 199-202.
[9]Walsh, Christopher T. "Polyketide and nonribosomal peptide antibiotics: modularity and versatility." Science 303.5665 (2004): 1805-1810.
[10]Yan, John, et al. "Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains." ChemBioChem 10.9 (2009): 1537-1543.
[11]Meier, Jordan L., and Michael D. Burkart. "The chemical biology of modular biosynthetic enzymes." Chemical Society Reviews 38.7 (2009): 2012-2045.
[12]Keatinge-Clay, Adrian T. "The structures of type I polyketide synthases."Natural product reports 29.10 (2012): 1050-1073.
[13]Cane, David E. "Programming of erythromycin biosynthesis by a modular polyketide synthase." Journal of Biological Chemistry 285.36 (2010): 27517-27523.
[14]Chan, Yolande A., et al. "Biosynthesis of polyketide synthase extender units." Natural product reports 26.1 (2009): 90-114.
[15]Hughes, Diarmaid. "Exploiting genomics, genetics and chemistry to combat antibiotic resistance." Nature reviews genetics 4.6 (2003): 432-441.