Whole cell biosynthesis of 1-methyl-3-phenylpropylamine and 2-amino-1,3,4-butanetriol using Komagataella phaffii (Pichia pastoris) strain BG-10 engineered with a transgene encoding Chromobacterium violaceum ω-transaminase

Abstract

We have engineered strain BG-10 of the methylotrophic yeast Komagataella phaffii for use as an effective whole cell biocatalyst. We introduced into the yeast a transgene encoding a Chromobacterium violaceum ω-transaminase for transcription in response to methanol induction. The strain was then assessed with respect to its growth performance and biotransformation of a fed ketoalcohol substrate to an amino-alcohol. In the resultant strain, BG-TAM, methanol induction did not compromise cell growth. Successful bioconversion of fed substrates to the by-product, acetophenone, indicated transaminase activity in shake flask-cultivated BG-TAM cells. We then used bioreactor cultivation to exploit the high levels of biomass achievable by Komagataella phaffii. In a 900 μL reaction the BG-TAM strain at OD₆₀₀ = 1024 achieved up to 0.41 mol mol^-1 (mol_product mol_substrate ^-1) yield on substrate (Yp/s) for production of 1-methyl-3-phenylpropylamine and a space time yield (STY) of 0.29 g L^-1 h^-1 for production of 2-amino-1,3,4-butanetriol. We have shown that transamination, an important step for bespoke synthesis of small molecule medicines, is biologically realisable using enzymes with a broad substrate range, such as ω-transaminases, within living yeast cells that are fed low-cost substrates for bioconversion.

Keywords: Bioengineering; Biotechnology; Chemical engineering; Komagataella phaffii; Pichia pastoris; Transaminase; Whole cell biocatalyst.

Fig. 1

Wildtype BG-10 strain growth performance maintained in BG-TAM strain engineered for overexpression of CV20205 transaminase. Cell growth profile for K. phaffii strains: BG-10 (a) and transformant cell line BG-TAM (b) grown in buffered minimal medium supplemented with different methanol concentrations (as indicated in figure key) in 250 mL shake flasks incubated at 30 °C and 250 rpm. Results are an average of n = 2 cultivations, error bars indicate standard error.

Fig. 2

Transaminase activity in BG-TAM whole cells cultivated in shake flasks. Reaction schemes detail the synthesis of ABT (a) and MPPA (b). Level of ACP generated by strains BG-10 and BG-TAM fed 10 mM MBA and 30 mM ERY as substrates (c). Production of ABT by strain BG-TAM fed 10 mM MBA and 30 mM ERY (d). Production of MPPA by strain BG-TAM fed 10 mM MBA and 10 mM PB (e). Cell concentration normalised to OD₆₀₀ = 39 in all assays. The symbol keys for the graphs obstruct no data points.

Fig. 3

Bioreactor cultivation of BG-TAM to high cell density. Engineered BG-TAM and parental BG-10 strains were cultivated in parallel using a Multifors 1 L bioreactor system. A standard Invitrogen fermentation protocol was used in which an initial glycerol batch phase was applied until 18.5 h post-inoculation, followed by glycerol fed-batch growth until approximately 26 h post-inoculation. From 26.5 h post-inoculation onward methanol fed-batch growth was applied.

Fig. 4

Upper limits of substrate concentration for BG-TAM whole cell biocatalysts at high cell density. BG-TAM was cultivated in a bioreactor to high cell density (OD₆₀₀ = 1024) and biocatalytic performance measured as a function of substrate concentration. Graphs show bioconversion of the fed substrate pair MBA/ERY to ACP (a, black circles) and ABT (b, black squares) and bioconversion of MBA/PB to MPPA (c, triangles – black and grey to indicate different cell densities and substrate concentrations used in the reaction).