High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties

Authors: MENENDEZ-BRAVO, SIMON - National University Of Rosario; ROULET, JULIA - National University Of Rosario; SABATINI, MARTIN - National University Of Rosario; COMBA, SANTIAGO - National University Of Rosario; Dunn, Robert - Bob; GRAMAJO, HUGO - National University Of Rosario; ARABOLAZA, ANA - National University Of Rosario

Submitted to: Biotechnology for Biofuels and Bioproducts
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/28/2016
Publication Date: 10/14/2016
Citation: Menendez-Bravo, S., Roulet, J., Sabatini, M., Comba, S., Dunn, R.O., Gramajo, H., Arabolaza, A. 2016. High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties. Biotechnology for Biofuels. 9:215.

Interpretive Summary: Multimethyl-branched long chain wax esters (MBE) that exhibit good physicochemical properties were developed using microbial synthesis. Native biosynthetic pathways inherent to conventional vegetable oil fatty acids provide only a small variety of linear hydrocarbons with limited structural diversity. This work takes advantage of a biosynthetic system from Mycobacterium tuberculosis to genetically engineer Escherichia coli bacteria to synthesize MBE with more diverse chemical functionality. The genetically engineered E. coli strain increased MBE production by 30.6% in batch cultures. The synthetic MBE had cold flow properties that were significantly lower than those of its linear fatty acid methyl ester analog. Furthermore, the stability of MBE against oxidative degradation was comparable to jojoba wax and better than epoxidized soybean oil. MBE wax esters can be used in many bio-based products including coatings and lubricants. This work will advance the microbial synthesis of oleochemicals by showing that genetically engineered bacteria can be used to produce wax esters with novel chemical structures.

Technical Abstract: Microbial synthesis of oleochemicals derived from native fatty acid (FA) metabolism has presented significant advances in recent years. Even so, native FA biosynthetic pathways often provide a narrow variety of usually linear hydrocarbons, thus yielding end products with limited structural diversity. To overcome this limitation, we took advantage of a polyketide synthase-based system from Mycobacterium tuberculosis and developed an Escherichia coli platform with the capacity to synthesize multimethyl-branched long-chain esters (MBE) with novel chemical structures. With the aim to initiate the characterization of these novel waxy compounds, here, we describe the chassis optimization of the MBE-producer E. coli strain for an up-scaled oil production. By carrying out systematic metabolic engineering, we improved the final titer to 138.1 +/- 5.3 mg MBE-1 in batch cultures. The fed-batch microbial fermentation process was also optimized achieving a maximum yield of 790.2 +/- 6.9 mg MBE-1 with a volumetric productivity of 15.8 +/- 1.1 mg MBE (L h)-1. Purified MBE oil was subjected to various physicochemical analyses, including differential scanning calorimetry (DSC) and pressurized-differential scanning calorimetry (P-DSC) studies. The analysis of the pour point, DSC, and P-DSC data obtained showed that bacterial MBE possess improved cold flow properties than several plant oils and some chemically modified derivatives, while exhibiting high oxidation stability at elevated temperatures. These encouraging data indicate that the presence of multiple methyl branches in these novel esters, indeed, conferred favorable properties which are superior to those of linear esters.