Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts

Abstract

BackgroundLipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility.ResultInspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts.ConclusionWe have proposed a new concept of integrated biodiesel production. This integrated system couples lipase production to lipase-catalyzed biodiesel synthesis in one pot. The proof-of-concept was established through construction of a recombinant P. pastoris yeast strain that was able to grow, overexpress T. lanuginosus lipase, and efficiently catalyze biodiesel production from fed waste cooking oils and methanol simultaneously. This simplified single-step process represents a significant advance toward achieving economical production of biodiesel at industrial scale via a ‘green’ biocatalytic route.

Production and Characterization of Biodiesel Using Nonedible Castor Oil by Immobilized Lipase fromBacillus aerius

“…Thus transesterification of vegetable oils can be used for the synthesis of fatty acid methyl esters with properties similar to petroleum-based diesel fuel which is renewable source of energy and this process is increasingly researched as a means of producing an environmentally acceptable alternative fuel [ 2 ]. The use of lipases as biocatalysts in the transesterification of triacylglycerides allows mild reaction conditions and easy recovery of glycerol, without need for further purification or chemical waste production [ 3 ]. Enzymatic production of biodiesel by methanolysis of triglycerides using lipase as the catalyst offers several advantages compared to chemical processes.…”

Metabolic engineering of microbes for branched-chain biodiesel production with low-temperature property

“…However, it has not been reported to produce FABCEs. Recently, P. pastoris yeast whole cell catalysts were constructed for biodiesel synthesis with intracellular expression of a lipase [ 52 ]. Therefore, we tested our hypothesis by developing a FABCE biosynthetic pathway in P. pastoris .…”

Assembly of lipase and P450 fatty acid decarboxylase to constitute a novel biosynthetic pathway for production of 1-alkenes from renewable triacylglycerols and oils

“…We previously developed an integrated bioprocess for in situ biodiesel production that occurs simultaneously with lipase generation in a Pichia pastoris yeast system [25]. Inspired by this strategy, in the present study, in parallel with combining hydrolysis of TAGs and decarboxylation of FFAs into a single E. coli host, we tested the strategy of developing E. coli in situ catalytic system for biosynthesis of 1-alkenes from TAGs.…”