Engineering of CYP153A33 With Enhanced Ratio of Hydroxylation to Overoxidation Activity in Whole-Cell Biotransformation of Medium-Chain 1-Alkanols

α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.

Evaluation of two thioesterases from Marinobacter aquaeolei VT8: Relationship to wax ester production

The biosynthesis of lipid-based biofuels is an important aspect of developing sustainable alternatives to conventional oils derived from fossil fuel reserves. Many biosynthetic approaches to biodiesel fuels and oils involve fatty acid derivatives as a precursor, and thioesterases have been employed in various strategies to increase fatty acid pools. Thioesterases liberate fatty acids from fatty acyl-coenzyme A or fatty acyl-acyl carrier protein substrates. The role played by thioesterases has not been extensively studied in model bacteria that accumulate elevated levels of biological oils based on fatty acid precursors. In this report, two primary thioesterases from the wax ester accumulating bacterium Marinobacter aquaeolei VT8 were heterologously expressed, isolated and characterized. These genes were further analyzed at the transcriptional level in the native bacterium during wax ester accumulation, and their genes were disrupted to determine the effect these changes had on wax ester levels. Combined, these results indicate that these two thioesterases do not play an integral role in wax ester accumulation in this natural lipid-accumulating model bacterium.

Production of 1-Dodecanol, 1-Tetradecanol, and 1,12-Dodecanediol through Whole-Cell Biotransformation inEscherichia coli

ABSTRACTMedium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A fromMarinobacter aquaeoleiVT8 (CYP153AM.aq; the strain is also known asMarinobacter hydrocarbonoclasticusVT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153AM.aqis limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkRM.aqis functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153AM.aqoperon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) fromPseudomonas putidaGPo1. Using a recombinantEscherichia colistrain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane–1-dodecanol substrate mixture. This study reports a very efficient method of producing C12/C14alkanols and C121,12-alkanediol by whole-cell biotransformation.IMPORTANCETo produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.

Molecular cloning, overexpression and characterization of a new thiol-dependent, alkaline serine protease with destaining function and fibrinolytic potential from Marinobacter aquaeolei MS2-1

Proteolytic marine bacterium designated as strain MS2-1 was isolated from the deep-sea sediment of Bay of Bengal. Strain MS2-1 was taxonomically identified as Marinobacter aquaeolei on the basis of 16S rRNA gene sequence homology analysis. A new alkaline serine protease gene (1,086 bp) was delineated, cloned into pET-28a-(+) vector and overexpressed in Escherichia coli. The enzyme showed 90% amino acid sequence identity towards subtilisin-like protease from a Pseudoalteromonas sp. AS-11. The three-dimensional homology model predicted the active site residues that may be responsible for the proteolytic activity. Molecular weight of the purified protein was calculated as 39 kDa. The enzyme exhibited the stability within a wide range of pH (7.0-12.0) and temperature (40-70°C). Maximum enzyme activity was observed at pH 8.0 and 50°C. The purified enzyme showed stability in the presence of metal ions, solvents, surfactants and detergents. The loss of activity with PMSF and the 3-fold increase of activity with DTT suggested the thiol-dependent nature of this serine protease enzyme. Results reported in this study also suggested that the new alkaline serine protease produced by the strain MS2-1 can be used as an efficient blood stain remover in detergent industries and as a thrombolytic agent in biomedical applications.

Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway

ABSTRACTThe biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previousin vitroenzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol inMarinobacter aquaeoleiVT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesisin vivo, but evolution has clearly selected one of these, a previously characterized fatty aldehyde reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previousin vitrostudies and provide the first glimpse into the role of each enzymein vivoin the native organism.