Enhanced production of biopolymer in Escherichia coli through evolutionary engineering of biopolymer synthase

Abstract

Nowadays, more researchers working on find out alternative of synthetic plastic because of oil exhaustion and increasing of concerning for environment. Petroleum based plastic cause serious problems to ocean, land and air by it’s hard to decomposed property. Bioplastic is one of strong candidate to replace the chemical plastics and it derived from renewable biomass sources. Biopolymers have biodegradable property in certain condition with bacteria, algae, molds and enzyme. With this powerful characteristic and carbon dioxide absorbing cycle, bioplastics can reduce the carbon dioxide emission in minus position comparing with synthetic plastic. Polyhydroxyalkanoates (PHAs) are biomass-derived polyesters consisting of a variety of hydroxycarboxylic acids and, as polymer materials, PHAs are attractive polyesters because they are biocompatible and biodegradable plastics. However, the high production costs of PHAs are hindering their commercialization and there is a strong need to develop microorganisms that can efficiently produce PHAs from renewable carbon sources. There are a lot of attempt to improve the productivity of valuable biopolymers in bacteria in past ten years. Poly(lactic acid), PLA, is a widely used polymer synthesized by two step process of fermentative production of lactic acid followed by its chemical polymerization via lactide. Recently, PLA and lactate-containing polymers have been produced by direct one step fermentative production by employing metabolically engineered bacteria harboring evolved propionyl-CoA transferase (Pct) from Clostridium propionicum and polyhydroxyalkanoate (PHA) synthase. Since natural PhaC’s from various microorganisms have negligible activities towards D-LA-CoA, Pseudomonas sp. MBEL 6-19 PHA synthase ( $PhaC_{Ps6-19}$ ) was engineered to efficiently accept D-LA-CoA as a substrate by site directed mutagenesis. Even though the engineered $PhaC_{Ps6-19}$ could be employed for the production of D-PLA and its copolymers in Escherichia coli and Ralstonia eutropha, the polymer content achievable was rather low. It was reasoned that $PhaC_{Ps6-19}$ has rather low activity towards D-LA-CoA, and is consequently limiting D-PLA production. Here, I report the development of engineered PHA synthases capable of more efficiently polymerizing D-lactyl-CoA through high throughput screening of a large library of PHA synthase variants by fluorescence activated cell sorting. First, I developed a FACS-based analysis technique that can quickly and accurately quantify the biopolymer produced in E. coli. Then, the randomized PHA synthase library was sorted using P (3HB) as a target product, and finally the production amount of P (3HB) was increased 4.5-fold. As a result of introducing engineered PhaC into D-PLA production, it has been doubled. Based on this engineered PHA synthase, another screening was further conducted to select candidates having the best activity for synthesizing D-PLA. An engineered E. coli strain expressing Pct and PHA synthase having V50D and F392S mutations produced D-PLA from glucose to the polymer content of 42.8 wt% of dry cell weight, which is 3.0 times higher than that expressing the control PHA synthase. Finally, I maximized the D-PLA production level by optimizing the two key enzyme expression levels. Randomized RBS libraries of each enzyme were screened using FACS in the same way. Analysis of enzyme expression levels revealed that high amounts of PhaC and low PCT expression carried the most optimal D-PLA production. As a result of applying modified RBS and engineered PHA synthase, it was possible to produce D-PLA with 52.6 wt% of dry cell weight in flask cultivation and $38.7 g l^{-1}$ in fed batch cultivation. These results are the best D-PLA production results reported so far, and they are competitive enough compared to existing commercial methods using two fermentation process. Therefore, it is expected that direct one step fermentation D-PLA using this technology will be produced in the future, and it is expected to solve problems in various modern society including environmental problems.