Protein design for a de novo synthetic pathway of microbial production of 1,3-propanediol from sugar

Feng Geng, Zhen Chen and An-Ping Zeng

Institute for Bioprocess and Biosystems Engineering, Hamburg University of

Technology (IBB/TUHH), Denickestrasse 15, D-21073, Hamburg, Germany;

Low energy-cost and environmentally friend bioproduction processes are a major goal of sustainable bio-based economy. The limitation of natural biosynthetic pathways hints the development of efficient bioprocesses for the production of certain desired products. Resent years, protein engineering and synthetic biology opens doors to efficient or completely new bioprocesses by De novo design of non-natural pathways. This is demonstrated in this work for the realization of a non-natural pathway and reengineered enzyme for the production of 1,3-propanediol (PDO) from sugar.
PDO is an important chemical monomer that can be utilized for the synthesis of high value polymers and coating materials. However, no natural microorganisms have been found which can directly use sugars to produce PDO. The Dupont Bio-PDO process was considered as a major achievement of metabolic engineering and industrial biotechnology in the last years. In the Dupont-PDO a synthetic pathway was successfully developed to produce PDO from glucose, in which the glycerol synthesis pathway from Saccharomyces cerevisiae and the metabolic pathway of converting glycerol to PDO from Klebsiella pneumonia were integrated into E. coli. However, several major burdens have to be overcome in designing such a synthetic metabolic pathway such as (1) the need of coenzyme B12 by one of the key enzymes glycerol dehydratase (GDHt) and (2) substrate-suicide of GDHt which could limit the productivity. Furthermore, E. coli can normally only use C6 sugar
In our work, protein engineering is used to design a new and de novo route for the biosynthesis of PDO from sugars (Fig.1). It is based on pathways of amino acid biosynthesis and uses homoserine as a key intermediate. Homoserine is converted into PDO in an unnatural pathway involving three enzymatic steps: (1) the deamination of homoserine to 4-hydroxy-2-ketobutyrate; (2) the decarboxylation of 4- hydroxy-2-ketobutyrate to 3-hydroxypropionaldehyde; and (3) the reduction of 3- hydroxypropionaldehyde to PDO. The theoretical maximum yield (1.5 mol PDO/
glucose) of the new PDO pathway is the same as that of the Dupont route. Since homoserine synthesis is a common pathway in most of the bacteria, the proposed route can be engineered into selected hosts with the more favorable ability to utilize different and cheap sugars. Moreover, the proposed pathway does not utilize GDHt and thus can avoid the serious problems associated with vitamin B12 and substrate
suicide. This non-natural pathway is thus very appealing for PDO production.

Fructose

Glucose

Sucrose

Pyruvate

pyc

L-Aspartate

lysc

aspc

Oxaloacetate

TCA cycle

L-Ã?-Aspartyl phophate

asd

L-Aspartyl-Ã?-semialdehyde

hom L-Homoserine

Deamination

4-Hydroxy-2-ketobutyrate

Decarboxylation

L-Lysine L-Threonine

3-Hydroxypropionaldehyde

yqhD

1, 3-Propanediol

Figure 1. Simplified scheme of a de novo pathway for biosynthesis of 1,3-propanediol from sugars which uses homoserine as a key intermediate.

Since no naturally efficient enzymes are known for the first reaction starting from homoserine, the engineering of a suitable enzyme for an altered substrate specificity is the most important step towards the new PDO biosynthesis route. We chose glutamate dehydrogenase (GDH) as a candidate. The wildtype GDH had a relatively low activity towards homoserine. Several mutants were designed by a structure- based approach which showed significantly increased activities. For a proof of concept we used an in vitro enzyme cascade consisting of mutated GDH, pyruvate decarboxylase (PDC) from Zymomonas mobilis DSM 3580 and alcohol
dehydrogenase (yqhD) from E. coli MG1655 to demonstrate the production of PDO rom homoresine. Initial test resulted in the production of 35 mg/L PDO from homoserine.
Subsequently, an E. coli strain was constructed to test the new pathway in vivo. Genes for the three key enzymes (gdhA or its mutant M1, kdcA, yqhD) were integrated into plasmids and overexpressed in E. coli MG1655 â??ThrB. E. coli MG1655â??ThrB (pZA-gdhA-M1-kdcA-yqhD) with the mutated GDH produced 51.5±4.9 mg/L PDO which was 110% higher than the strain MG1655â??ThrB (pZA gdhA-kdcA- yqhD) with the widetype GDH whereas the blank control MG1655â??ThrB did not produce any PDO, demonstrating the possibility of producing PDO from sugar with the new pathway.