Metabolomics-Guided Analysis of the Biocatalytic Conversion of Sclareol to Ambradiol by Hyphozyma roseoniger

The biocatalytic conversion of sclareol to ambradiol, a valuable component in the fragrance industry, using whole-cell biotransformation by the dimorphic yeast Hyphozyma roseoniger, was investigated using metabolomics tools. An integrated approach was used to identify and quantify the participating intermediates in this bioconversion using both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled to mass spectrometry (LC–MS). This study entailed growth stage-dependent analysis of H. roseoniger suspensions grown in batch culture over a 14-day period, beginning with a three-day induction period using 20 mg/200 mL sclareol, followed by a further 1 g/200 mL sclareol dose to enable ambradiol production. The progress of the bioconversion and the resulting dynamic changes to the metabolome were monitored using NMR analysis and semi-targeted LC–MS metabolomics. This outlined the molecular conversions occurring within the matrix and no novel intermediates participating in the sclareol to ambradiol conversion could be identified. This study presents new findings about the transformative capabilities of H. roseoniger as a whole cell biocatalyst, highlighting its potential utility in similar applications.

A One-Pot Biocatalytic and Organocatalytic Cascade Delivers High Titers of 2-Ethyl-2-Hexenal from n-Butanol

Biocatalysis provides facile access to selective chemical transformations and helps satisfy sustainable chemical production criteria. However, the reaction scope of biocatalysts is significantly narrower compared to synthetic chemical transformations. Hybrid biocatalytic-chemocatalytic cascades expand the scope of products while maintaining many of the benefits associated with biocatalysis. Here, we report that single-pot systems with whole cell K. pastoris (ATCC® 28485™) or isolated enzyme alcohol oxidase (E 1.1.3.13) as oxidative biocatalysts with a lysine organocatalyst yields the commercial target, 2-ethyl-2-hexenal (2-EH) from n-butanol in a two-step hybrid cascade. Peak yields for both biocatalysts were achieved with 100 mM n-butanol at pH 8 and 30°C. The isolated enzyme slightly outperformed whole cell K. pastoris, reaching 73% conversion (4.7 g/L titers) compared to 61% (3.9 g/L titers) in whole cells systems. Titers could be improved for both biocatalysts (5.7 – 6.7 g/L) at increased butanol loading; however, this came at the expense of decreased yields. Compared to our initial results with a Gluconobactor oxidans whole cell biocatalyst, the reported system improves upon 2-EH titers by 2.8-3.3-fold at maximal yields.

Efficient 2,3-Butanediol/Acetoin Production Using Whole-Cell Biocatalyst with a New Nadh/Nad(+) Regeneration System

An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.

Cutaneotrichosporon oleaginosus: A Versatile Whole-Cell Biocatalyst for the Production of Single-Cell Oil from Agro-Industrial Wastes

Cutaneotrichosporon oleaginosus is an oleaginous yeast with several favourable qualities: It is fast growing, accumulates high amounts of lipids and has a very broad substrate spectrum. Its resistance to hydrolysis by-products makes it a promising biocatalyst for custom tailored microbial oils. C. oleaginosus can accumulate up to 60 wt.% of its biomass as lipids. This species is able to grow by using several compounds as a substrate, such as acetic acid, biodiesel-derived glycerol, N-acetylglucosamine, lignocellulosic hydrolysates, wastepaper and other agro-industrial wastes. This review is focused on state-of-the-art innovative and sustainable biorefinery schemes involving this promising yeast and second- and third-generation biomasses. Moreover, this review offers a comprehensive and updated summary of process strategies, biomass pretreatments and fermentation conditions for enhancing lipid production by C. oleaginosus as a whole-cell biocatalyst. Finally, an overview of the main industrial applications of single-cell oil is reported together with future perspectives.