Inverse metabolic engineering for improving protein content in Saccharomyces cerevisiae.

Production of Saccharomyces cerevisiae-based single cell protein (SCP) has recently received great attention due to the steady increase in the world's population and environmental issues. In this study, an inverse metabolic engineering approach was applied to improve the production of yeast SCP. Specifically, an S. cerevisiae mutant library, generated using UV-random mutagenesis, was screened for three rounds to isolate mutants with improved protein content and/or concentration. The #1021 mutant strain exhibited a respective 31% and 23% higher amino acid content and concentration than the parental S. cerevisiae D452-2 strain. Notably, the content, concentration, and composition of amino acids produced by the PAN2* strain, with a single nucleotide polymorphism in PAN2 coding for a catalytic subunit of the poly(A)-nuclease (PAN) deadenylation complex, were virtually identical to those produced by the #1021 mutant strain. In a glucose-limited fed-batch fermentation, the PAN2* strain produced 19.5 g L-1 amino acids in 89 h, which was 16% higher than that produced by the parental D452-2 strain. This study highlights the benefits of inverse metabolic engineering for enhancing the production titer and yield of target molecules without prior knowledge of rate-limiting steps involved in their biosynthetic pathways.

Enhancing acid tolerance of Escherichia coli via viroporin-mediated export of protons and its application for efficient whole-cell biotransformation.

Escherichia coli-based whole-cell biocatalysts are widely used for the sustainable production of value-added chemicals. However, weak acids present as substrates and/or products obstruct the growth and fermentation capability of E. coli. Here, we show that a viroporin consisting of the influenza A matrix-2 (M2) protein, is activated by low pH and has proton channel activity in E. coli. The heterologous expression of the M2 protein in E. coli resulted in a significant increase in the intracellular pH and cell viability in the presence of various weak acids with different lengths of carbon chains. In addition, the feasibility of developing a robust and efficient E. coli-based whole-cell biocatalyst via introduction of the proton-selective viroporin was explored by employing (Z)-11-(heptanolyoxy)undec-9-enoic acid (ester) and 2-fucosyllactose (2'-FL) as model products, whose production is hampered by cytosolic acidification. The engineered E. coli strains containing the proton-selective viroporin exhibited approximately 80% and 230% higher concentrations of the ester and 2'-FL, respectively, than the control strains without the M2 protein. The simple and powerful strategy developed in this study can be applied to produce other valuable chemicals whose production involves substrates and/or products that cause cytosolic acidification.