Metabolic Engineering of Zymomonas mobilis for Acetoin Production by Carbon Redistribution and Cofactor Balance

Abstract

Biorefinery to produce value-added biochemicals offers a promising alternative to meet our sustainable energy and environmental goals. Acetoin is widely used in the food and cosmetic industries as taste and fragrance enhancer. The generally regarded as safe (GRAS) bacterium Zymomonas mobilis produces acetoin as an extracellular product under aerobic conditions. In this study, metabolic engineering strategies were applied including redistributing the carbon flux to acetoin and manipulating the NADH levels. To improve the acetoin level, a heterologous acetoin pathway was first introduced into Z. mobilis, which contained genes encoding acetolactate synthase (Als) and acetolactate decarboxylase (AldC) driven by a strong native promoter Pgap. Then a gene encoding water-forming NADH oxidase (NoxE) was introduced for NADH cofactor balance. The recombinant Z. mobilis strain containing both an artificial acetoin operon and the noxE greatly enhanced acetoin production with maximum titer reaching 8.8 g/L and the productivity of 0.34 g∙L−1∙h−1. In addition, the strategies to delete ndh gene for redox balance by native I-F CRISPR-Cas system and to redirect carbon from ethanol production to acetoin biosynthesis through a dcas12a-based CRISPRi system targeting pdc gene laid a foundation to help construct an acetoin producer in the future. This study thus provides an informative strategy and method to harness the NADH levels for biorefinery and synthetic biology studies in Z. mobilis.

A new Zymomonas mobilis platform strain for the efficient production of chemicals

“…5). Data are derived from or partial knockout however already shows beneficial effects on the yields of products other than ethanol [8,[14][15][16][17]. The most promising approach was presented by Liu et al [15] who expressed pdc of Z. mobilis from a plasmid and subsequently deleted the original pdc locus (ZMO1360) from the chromosome.…”

“…However, to our knowledge, this approach has not been reported so far in peer review publications and it does not easily allow a switch to another product pathway as is possible with our platform strain sGB027. A knockdown or partial knockout however already shows beneficial effects on the yields of products other than ethanol [ 8 , 14 – 17 ]. The most promising approach was presented by Liu et al [ 15 ] who expressed pdc of Z. mobilis from a plasmid and subsequently deleted the original pdc locus (ZMO1360) from the chromosome.…”

“…While the wild type only has a narrow substrate spectrum, being able to metabolize glucose, fructose and sucrose, it has many desirable for high ethanol yield and productivity but hampers the production of other chemicals of interest. There is an increasing spectrum of products synthesized by genetically modified Z. mobilis strains, including malate, lactate, alanine, acetoin or the heterologous products 2,3-butanediol and isobutanol, but ethanol remains an essential by-product of all those strains, thereby reducing the yields for the respective product to varying degrees [8][9][10][11][12][13][14][15].…”

“…To reduce the amount of ethanol as a by-product, the activity of the enzyme pyruvate decarboxylase (PDC; GenBank: AAV89984.1), which converts pyruvate to acetaldehyde, has to be eliminated or strongly reduced. However, a complete knock-out of pdc (ZMO1360) in the wild type (WT) background proved impossible leading to different strategies to reduce its activity [8,[14][15][16][17]. Most studies were only able to generate heterogeneous knockout strains, which retained significant PDC activity.…”

Metabolic engineering of an industrial bacterium Zymomonas mobilis for anaerobic l-serine production

“…With the rapid technology advancement in systems and synthetic biology, native and exogenous CRISPR-Cas genome editing toolkits [ 21 , 22 ] as well as systems for biological parts identification and characterization [ 23 ] have been established in Z. mobilis . Although various recombinant strains have been constructed to produce platform biochemicals such as cellulosic ethanol, lactate, acetoin, isobutanol, 2,3-butanediol (2,3-BDO ) , and poly-3-hydroxybutyrate (PHB ) [ 21 , [24] , [25] , [26] , [27] , [28] ], there are limited reports on engineering Z. mobilis for the production of biochemicals involved in sophisticated regulations such as amino acids.…”